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A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

Posted in Cell Biology, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Myocardial adenine nucleotide metabolism, Myocardial Metabolism, Na-K-ATPase, Neurological Diseases, Proteins, Proteomics, tagged , , , on September 6, 2015| Leave a Comment »

A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.5

 

J Clin Invest. 2012 Oct 1; 122(10): 3374–3377.

http://dx.doi.org:/10.1172/JCI66361

The MBI community congratulates Michael Sheetz upon winning the prestigious Albert Lasker Basic Medical Research Award. Michael Sheetz, Director of the Mechanobiology Institute, Singapore, Distinguished Professor of the Department of Biological Sciences, NUS and William R Kenan, Junior Professor at Columbia University, shares this award with two of his collaborators, James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco). The Award was presented at a ceremony on Friday, September 21, 2012, in New York City.

 

The Albert Lasker Basic Medical Research Award was given to Prof Sheetz, Prof Spudich and Prof Vale in honor of seminal contributions made in establishing methods to study cytoskeletal motor proteins. These developments paved the way to study molecular motors and enabled the discovery of the motor protein, kinesin. The landmark achievements in deciphering new components of cellular motors, which helped explain how these motors worked, were pivotal in understanding the basic fundamental process of energy conversion within the cell. These have led to explorations of these physiologically relevant molecules, as potential drug targets in a variety of disease conditions.

 

Many basic cellular functions depend on the directed movement of cytoplasmic organelles, macromolecules, membranes or chromosomes from one place to another within the cell. The transport of this intracellular cargo is achieved by molecular motor proteins, such as myosin and kinesin, which provide force and movement through the conversion of chemical energy (ATP) into mechanical energy. Molecular motor proteins move along scaffolds made of specific protein polymers, with kinesins moving along microtubules and myosins along actin filaments, in order to carry their cargo to the appropriate destination within the cell.
Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out. Michael Sheetz and colleagues, Ronald Vale and Thomas Reese, carried out pivotal experiments that ultimately led to the discovery of kinesins, a novel and hitherto unknown family of motor proteins. These experiments involved the development of an in vitromotility assay, whereby proteins from the cytoplasm of neuronal cells were shown to power the movement of microtubules across the surface of glass coverslips. This technique was found to be a sensitive and rapid assay for testing the activity of kinesin and was adopted by numerous labs following these crucial initial experiments.

For more details of the award winning contribution towards understanding the basics of cellular machinery, please go to http://www.laskerfoundation.org/media/index.htm and also http://www.laskerfoundation.org/media/pdf/2012citation_basic.pdf

 

Michael Sheetz, along with James Spudich and Ronald Vale strongly believe in an open culture of scientific exchange. ‘The most interesting scientific insights result from collaborative, interdisciplinary adventures’, has been the one common theme of Michael Sheetz career. A firm believer of an open laboratory concept where students from different labs and backgrounds, share bench space and often ideas, he emulated the Open Lab model (learn more about MBI’s open labs) at the Mechanobiology Institute, Singapore. This new model of open laboratory environment in interdisciplinary institutes provides an excellent way to encourage fast paced discovery process.

“My greatest excitement comes from considering the puzzle provided by an unexpected result when new technology is applied to an old problem, says Professor Sheetz.„

In his acceptance essay, which can be read here (PDF), Michael Sheetz refers to the importance of collaborations, where the parties are learning from each other and also ‘encourages young scientists to perform speculative experiments whenever they have such an idea, even if most of them fail; since an experiment, even a flawed on, can reveal the solution to an important problem’.

 

The Mechanobiology Institute is delighted to announce that Michael Sheetz has been selected as a Massry Prize Laureate for 2013.

michaelSheetz_WB_9079Shared with James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco), the award to the trio is a recognition of their work defining molecular mechanisms of ‘intracellular motility.’

This process involves the deployment of molecular machines to move cargo on molecular tracks which are a part of the cellular skeleton.

The discovery of a novel family of motor proteins, the kinesins, by Michael Sheetz, Ronald Vale and Thomas Reese and the methodology developed for the same, proved to be pivotal and the technique developed led to many further discoveries by different laboratories.

Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out.

 

 

 

 

 

 

The Lasker Awards: motors take centre stage

Nature Cell Biology | Editorial
Nature Cell Biology 14,1113(2012)  http://dx.doi.org:/10.1038/ncb2618

Michael Sheetz, James Spudich and Ronald Vale have now joined the list of Lasker laureates, having jointly received the 2012 Albert Lasker Basic Medical Research Award for their “discoveries concerning cytoskeletal motor proteins, machines that move cargoes within cells, contract muscles, and enable cell movements”1.

Although the mechanism of action and the cellular functions performed by force-generating cytoskeletal motors, including their roles in intracellular trafficking, cell migration, cell division and muscle contraction, are now a fundamental part of cell biology, in the 1970s and 1980s they were still mostly a mystery. Following a postdoctoral fellowship under the guidance of Hugh Huxley, a pioneer of muscle contraction studies, James Spudich established his independent work at the University of California San Francisco (UCSF) and later at Stanford University on what was, at the time, the largely unchartered territory of myosin activity and function. A fortuitous crossing of paths occurred in 1982, when Michael Sheetz joined the Spudich laboratory on sabbatical from his own independent research at the University of Connecticut Health Center. Working together, Spudich and Sheetz demonstrated myosin movement on actin filaments using the Nitella axillaris alga as a model, and later established an in vitro reconstitution system that demonstrated the ability of purified myosin to move on purified actin filaments in the presence of adenosine triphosphate at rates consistent with those of muscle contraction. This seminal work was published in Nature in 19832 and 19853.

Spurred by the exciting work on myosin-based movement, Ronald Vale, then a graduate student at Stanford University, decided with Michael Sheetz to define the particle movement observed in squid axons. Their experiments at the Marine Biology Laboratory in Woods Hole led to a series of groundbreaking Cell publications in 19854, 5, 6, 7, 8, which determined that axonal movement was not driven by myosin on actin filaments as they had anticipated, but was instead occurring on microtubules and was powered by a then-uncharacterized factor that they purified and named kinesin.

These initial efforts investigating myosin- and kinesin-powered motility, and the in vitro assays developed to characterize cytoskeletal motor activities, opened up new and fascinating avenues of research and have become a corner-stone of cell biology today. Following these key discoveries, Spudich went on to define many other aspects of myosin activity and function. Vale continued his work on molecular motors and their cellular roles in his independent research at UCSF, and Sheetz followed a varied research career ranging from motility studies to work on cell adhesion and mechanosensing at Columbia University and the Mechanobiology Institute in Singapore.

In honouring the early work of Sheetz, Spudich and Vale, the Lasker Foundation recognizes the significance of the cytoskeletal motor field in biology, and also the importance of understanding the principles underlying cellular motor function in human diseases in which such activities are deregulated. Indeed, the characterization of normal myosin and kinesin activity and function has served as the spring-board for studies on their impaired or aberrant action in disease, with the goal of developing therapies for heart conditions in the case of myosins, and neurological disorders and malignancy in the case of kinesins.

It should also be noted that the discoveries acknowledged by the Lasker Award and the subsequent scientific careers of the three awardees were the outcome of an inspired mix of cell and molecular biology, biochemistry and physics, among other disciplines, and are thus a testament to the importance of fostering multidisciplinary science. Moreover, as the three award recipients eloquently noted in their Lasker Award acceptance remarks, the motivating force during the exciting times of their initial research on motors was not only a thirst for discovery and a passion for science, but also a strong collaborative spirit. As a fundamentally creative and adventurous endeavour, science is often seen by outsiders as a solitary pursuit of inquiry and testing one’s own ideas. However, the reality of a bustling laboratory reveals that teamwork, discussion and brainstorming, and a successful combination of different personalities, are just as important as individual intellect and drive. In that respect, the dedication, creativity and collaborative efforts of Sheetz, Spudich and Vale should be an inspiration to scientists everywhere.

References

  1. www.laskerfoundation.org/awards/2012_b_description.htm
  2. Sheetz, M. P. & Spudich, J. A. Nature 303, 31–35 (1983).
  3. Spudish, J. A., Kron, S. J. & Sheetz, M. P. Nature 315, 584–586 (1985).
  4. Vale, R. D., Schnapp, B. J., Reese, T. S. & Sheetz, M. P. Cell 40, 449–454 (1985).

One path to understanding energy transduction in biological systems

James A Spudich

http://www.laskerfoundation.org/awards/pdf/2012_b_spudich.pdf

Who is not fascinated by the myriad biological movements that define life? From cell migration, cell division and a network of translocation activities within cells to highly specialized muscle contraction, molecular motors operate by burning ATP as fuel. Three types of molecular motors—myosin, kinesin and dynein— and nearly 100 different subtypes transduce that chemical energy into mechanical movements to carry out a wide variety of cellular tasks. Understanding the molecular basis of energy transduction by these motors has taken decades. Our understanding of molecular motors could be viewed as beginning with the two 1954 papers in Nature by Hugh Huxley and Jean Hanson and Andrew Huxley and Rolf Niedergerke, respectively, where the authors proposed the sliding-filament theory of muscle contraction. But a good place to start my story is 1969, when Hugh Huxley, on the basis of his remarkable X-ray diffraction experiments on live muscle coupled with electron microscopy, postulated the swinging crossbridge hypothesis of muscle contraction1. Thus, more than 40 years ago, he proposed the basic concepts of how the myosin molecule produces the sliding of actin filaments to produce contraction. Hugh Huxley laid the foundation for the molecular motor field, and we are all indebted to him. My beginnings in myosin research began as a postdoctoral fellow in Hugh’s laboratory at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, coincidentally in 1969. But my fascination with science began much earlier.

Neither of my parents was college educated, but they both had keen intellects, positive and enthusiastic outlooks and profound work ethics. My father was intrigued by how things work and shared that interest with my brother John and me. After the coal mines closed, my father taught himself electrical engineering, founded the Spudich Electric Company and patented one of his inventions. He often told John and me, “do whatever excites you, but do it well and be respectful of people you interact with.”

I was captivated with chemistry from a young age. Beginning at the age of six, I mastered every chemistry set I could get. The myriad chemical reactions that could be created using everyday materials, sometimes with marvelously explosive results, fed my excitement for chemistry. It was a world unfamiliar to my parents, but they respected my preoccupations and cleared the pantry of our modest home for me to set up a lab with discarded equipment given to me by my high school chemistry teacher Robert Brandsmark. My brother John has also followed the allure of science into an exciting and distinguished career in basic research. His work has established the molecular basis of signaling in an important class of rhodopsins that he discovered in 1982 (ref. 2). John was my first collaborator.

A chance encounter with Woody Hastings at the University of Illinois launched my experimental-science career. Throughout my undergraduate years, I worked with Woody on bioluminescence in Vibrio fischeri3. I was inspired by his high-spirited fascination with biology and was fortunate to be invited to help him teach in the physiology course at the Marine Biological Laboratory (MBL) in Woods Hole (Fig. 1). At the MBL, I was introduced to the breadth and potential of many biological systems, including muscle contraction.

In 1963 I joined the PhD program in the new Department of Biochemistry at Stanford University, founded by Arthur Kornberg. One of the many remarkable aspects of the biochemistry department was that, although Arthur was my thesis advisor, all the faculty members were my mentors. This unique environment shaped the way I do research and taught me how to be a responsible colleague and a mentor to others (Fig. 2). I learned how important it is to reduce complex biological systems to their essential components and create quantitative in vitro assays for the function of interest. Those years also made it clear to me that interdisciplinary approaches would be key to understanding complex biological processes. So I decided to do postdoctoral work in both genetics and structural biology. I spent one year at Stanford with another influential role model, Charley Yanofsky, working on the genetics of the Escherichia coli tryptophan operon. I then joined Hugh Huxley’s laboratory in Cambridge.

I chose to study the  unanswered questions in cell biology at the time when I established my own laboratory – how the chemical energy of ATP hydrolysis brings about mechanical movement and what roles a myosin-like motor might have in nonmuscle cells.

The essential first steps were to develop a quantitative in vitro motility assay for myosin movement on actin, which is crucial for understanding the molecular mechanism of energy transduction by this system, and to develop a model organism to unravel the molecular basis of the myriad nonmuscle-cell movements that are apparent by light microscopy. We explored Neurospora crassa, Saccharomyces cerevisiae, Physarum polycephalum, Dictyostelium discoideum, Nitella axillaris and other organisms, all unfamiliar to me at the time. The giant cells of the alga Nitella were particularly intriguing because of their striking intracellular cytoplasmic streaming that was visible under a simple light microscope. Although not suitable for biochemistry or genetics, Nitella would assume an important role in my lab a decade later, after Yolande Kersey in Norm Wessells’s laboratory in the Department of Biological Sciences at Stanford showed oriented actin cables lying along chloroplast rows in these cells 5. The slime mold Dictyostelium proved best for our initial biochemical approach 6. Margaret Clarke, my postdoctoral fellow, identified a myosin in Dictyostelium. We also showed that actin is associated with the cell membrane in this organism, and we isolated membranecoated polystyrene beads with actin filaments emanating from them. We were tremendously excited about the possibilities these results presented as a small step along the way to an in vitro motility assay where these actin-coated particles could move along a myosin-coated surface (Fig. 3).

Figure 3 Dictyostelium has a muscle-like myosin and membrane-associated actin. (a) A possible scheme for pulling two membranes together (redrawn from ref. 6). (b) Margaret Clarke discovered myosin II in Dictyostelium and showed that it forms bipolar thick filaments, similar to muscle myosin. (c) Phagocytized polystyrene beads offered an opportunity to explore one version of an in vitro motility assay where the beads may be pulled along by myosin. Taken from my laboratory notebook, 21 January 1973.

Figure 4 One approach to an in vitro motility assay from a totally defined system. (a) The concept was to observe myosin-coated beads moving along fixed actin filaments oriented by buffer flow. The actin filaments had biotinylated severin bound to their barbed ends; the barbed ends were attached to an avidin-coated surface by way of the tight avidin-biotin link. The filaments were oriented by buffer flow. B, biotin; S, severin. (b) Myosin-coated beads were observed by light microscopy to move upstream toward the barbed end of the surface-attached actin filaments. The position of each of the three bead aggregates is shown as a function of time. This was the first demonstration of quantitative, directed movement of myosin along actin with a totally defined system (taken from ref. 11). ATP binding releases the myosin Myosin binds to actin ATP ADP Pi ADP .

In 1977 I joined the Department of Structural Biology at Stanford. In the next years we extensively characterized the actin-myosin system in Dictyostelium. My student Arturo De Lozanne made the chance discovery that genes in Dictyostelium can be knocked out by homologous recombination and provided the first genetic proof that myosin II is essential for time that myosin II drove the forward movement of cells. Dietmar Manstein, Meg Titus and Arturo then extended these experiments to create a myosin-null cell8, which was crucial to our later work using mutational analysis to define the structure-function relationships of the myosin molecule and for important experiments in support of the swinging cross-bridge hypothesis9. Interestingly, reports from a number of laboratories between 1969 and 1980 did not support the swinging cross-bridge model, and it was more imperative than ever to develop a quantitative in vitro motility system to test the various models under consideration. In 1981 we identified and purified Dictyostelium severin, a protein that tightly binds the ‘barbed ends’ of actin filaments. This provided an opportunity to try another version of an in vitro motility assay. Using biotinylated severin, we attached the actin filament barbed ends to an avidin-coated slide and flowed aqueous solution over them. Long filaments attached to the surface at one end would be expected to orient in the direction of the flowing solution (Fig. 4a). We placed myosin-coated beads on these actin-coated slides and added ATP but saw only sporadic movements. In retrospect, we probably did not have sufficient alignment of filaments; we were not monitoring filament alignment at that time by electron microscopy, as we did later.

A key breakthrough occurred in 1982 when Mike Sheetz came to my laboratory on sabbatical. Not certain what component of our system might be limiting our approach, we took advantage of the known orientation of actin filaments in Nitella5 to overcome the actin filament alignment problem. Peter Sargent, a neurobiologist in the Structural Biology Department at that time, helped us cut open a Nitella cell, and we attached it to a surface to expose the actin fibers. We added myosin-coated beads and eureka! We saw robust ATP-dependent unidirectional movement along chloroplast rows, which mark the actin fibers 10.

Armed with the Nitella results, Mike left my lab and went to the MBL to explore whether myosin-coated vesicles may account for the particle movements observed in squid axons. Ron Vale, then a graduate student at Stanford with Eric Shooter, was fascinated by the movement of organelles in nerve axons and joined Mike at the MBL. To their great surprise, they found that movement in axons is not myosin driven. Instead, they discovered the new molecular motor kinesin, a discovery that completely energized the field and opened up years of exciting work from their laboratories and many others. ..

 

The combination of the in vitro motility assay and the Dictyostelium myosin-null cell provided powerful tools for Kathy Ruppel, Taro Uyeda, Dietmar Manstein, William Shih, Coleen Murphy, Meg Titus, Tom Egelhoff and others in my lab to use mutations along myosin to define the biochemical, biophysical and assembly properties of the molecule. Our results were consistent with the proposed actin-activated myosin chemomechanical cycle derived largely from the elegant biochemical kinetic studies from Edward Taylor’s laboratory in the early 1970’s (ref. 15) (Fig. 5). Then, in 1993, Ivan Rayment and his colleagues16 obtained a high-resolution crystal structure of myosin S1. Ivan’s pivotal work allowed us to place our mutational analyses in a myosin structure-function context.

Figure 5 The actin-activated myosin chemomechanical cycle. This cycle, extensively studied by many researchers over several decades, was derived from kinetic studies of Lymn and Taylor 15. A mechanical stroke only occurs when the myosin is strongly bound to actin. Our mutational analyses of Dictyostelium myosin II probed each of the steps shown and provided structure-function analyses that helped define how the myosin motor works. ADP-Pi , ADP and inorganic phosphate, the products of ATP hydrolysis, remain bound to the active site until actin binds to the myosin.

Figure 6 In vitro motility taken to the single-molecule level using the physics of laser trapping. (a) The Kron in vitro motility assay observing fluorescent actin filaments (yellow) moving on a myosin-coated (red) surface. (b) Two polystyrene beads attached to the ends of a single actin filament are trapped in space by laser beams. The filament is lowered onto a single myosin molecule on a bump on the surface (gray sphere). (c) Jeff Finer building the dual-beam laser trap in around 1990.

Fundamental issues still remained— primarily to establish the step size that the myosin takes for each ATP hydrolysis, which was under considerable debate.

more…

One of my great satisfactions is that the more detailed understanding of energy transduction by myosin has led to potential clinical therapies. A small molecule that binds and activates b-cardiac myosin is now in clinical trials for the treatment of heart failure, and another small molecule currently in clinical trials activates skeletal muscle contraction and may aid patients with amyotropic lateral sclerosis and other diseases.

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Heroes in Basic Medical Research – Robert J. Lefkowitz

Posted in Cardiovascular Pharmacogenomics, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Curation, Disease Biology, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genomic Endocrinology, Genomics Pharmacy, Historical relevance, Innovations in Neurophysiology & Neuropsychology, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Technology Advance Assessment of, tagged , , on September 6, 2015| Leave a Comment »

Heroes in Basic Medical Research – Robert J. Lefkowitz

Author & Curator: Larry H Bernstein, MD, FCAP

Robert J. Lefkowitz, MD

Robert J. Lefkowitz MD, a Howard Hughes Medical Institute investigator who has spent his entire 39-year research career at the Duke University Medical Center, is sharing the 2012 Nobel Prize in Chemistry with Brian K. Kobilka of Stanford University School of Medicine, who was a post-doctoral fellow in Lefkowitz’s lab in the 1980s.

They are being recognized for their work on a class of cell surface receptors that have become the target of prescription drugs, including antihistamines, ulcer drugs and beta blockers to relieve hypertension, angina and coronary disease.

The receptors catch chemical signals from the outside and transmit their messages into the cell, providing the cell with information about changes occurring within the body. These particular receptors are called seven-transmembrane G protein-coupled receptors, or just “G-coupled receptors” for short. Serpentine in appearance, G-coupled receptors weave through the surface of the cell seven times.

The human genome contains code to make at least 1,000 different forms of these trans-membrane receptors, all of which are quite similar. The receptors also bear a strong resemblance to receptors that detect light in the eyes, smells in the nose and taste on the tongue. (See playlist of Lefkowitz science videos here.)

“Bob’s seminal discoveries related to G-protein coupled receptors ultimately became the basis for a great many medications that are in use today across many disease areas,” said Victor J. Dzau, MD, Chancellor for Health Affairs and CEO, Duke University Health System.  “He is an outstanding example of a physician-scientist whose impact can be seen in the lives of the countless patients who have benefited from his scientific discoveries. We are very proud of his magnificent achievements and grateful for his many contributions to Duke Medicine.”

After attending public elementary and junior high schools I entered The Bronx High School of Science (10th grade) in the autumn of 1956, graduating at age 16 in 1959. “Bronx Science” is one of several public high schools in New York City which admits students on the basis of a competitive examination. The student body, representing approximately the top 5% based on the exam, are gifted and interested in science and math. The accomplishments of graduates of this high school are quite remarkable. For example, I am the 8th Nobel Laureate to have graduated from this school, the 7 previous ones having received their prizes in Physics. For me, attending this school was a formative experience. Whereas in elementary and junior high school I was not greatly challenged, here I was among a group of remarkably bright, interesting and stimulating classmates. The curriculum featured many advanced classes at the college level. I was particularly drawn to chemistry and, as a result of taking these college level classes, I was able to receive full credit for two years of chemistry when I entered Columbia College in 1959. Thus I began as a college freshman with organic chemistry, a course generally taken by juniors.

The level of scholarship maintained by the student body was such that even with an average of about 94% my final class rank was about 100th out of 800. A classmate and friend at the time and at present, the famous geneticist David Botstein, had an almost identical average, a fact we tease each other about to this day.

Along with dozens of classmates, I moved on to Columbia University where I enrolled as a pre-medical student majoring in chemistry. The two year core curriculum in “Contemporary Civilization” was required of all students. With an emphasis on reading classic texts in history, philosophy, sociology and the political sciences and discussing these in small seminars, it was for me an opening to a whole new world. In addition, I took courses with and was exposed to, such intellectual giants as the literary critic Lionel Trilling, the cultural historian Jacques Barzun and the sociologist Daniel Bell, among others. I have very fond memories from this period of spending many hours in the public reading room at the 42nd Street New York Public Library, researching papers for those classes.

I also studied advanced Organic Chemistry with Cheves Walling and Physical Chemistry in a department which was strongly influenced by the then recently retired prominent physical organic chemist, Louis Hammett. However, the chemistry professor who had the most profound influence on me was actually a young Assistant Professor of Chemistry, Ronald Breslow. As a college senior I took an advanced seminar in biochemistry which he taught single handedly. This introduction to the chemistry of processes in living organisms really excited me in part, I suspect, because of his very lively teaching style. None of this, however, in any way diverted me from my goal of studying to become a practicing physician.

I greatly enjoyed my four years in medical school. I had dreamed about becoming a physician since grade school and now I was finally doing it. As a freshman immersed in the basic medical sciences I was able to deepen my interest in, and fascination with, biochemistry. Our biochemistry professors included a remarkable array of scholars (not that any of us appreciated that at the time). We heard lectures on metabolism from David Rittenberg, Chair of the Department; from David Shemin on porphyrins; from Irwin Chargaff on nucleic acids; and from David Nachmansohn on cholinergic neurotransmission.

One young professor left a lasting impression on me. Paul Marks was then a young academic hematologist who taught the Introduction to Clinical Medicine course in which we studied clinical problems for the first time, examined case histories, and looked at blood specimens. Not only was he a good clinician but he assigned readings from the basic science literature that were relevant in a very meaningful way to the cases we studied. This showed me how scientific information could be brought to bear on clinical problems. Among my classmates and friends in medical school was Harold Varmus, who was the co-recipient of the 1989 Nobel Prize for the discovery of oncogenes.

On July 1, 1968 I moved my family (now including the recently born Cheryl) to Rockville, Maryland to begin my research career at the NIH in nearby Bethesda, Maryland. I had been assigned, through a matching program, to work with Drs. Jesse Roth and Ira Pastan in the Clinical Endocrinology Branch of the National Institute of Arthritis and Metabolic Diseases (NIAMD), now known as NIDDK, the National Institute of Diabetes and Digestive and Kidney Diseases. I was a Clinical Associate, meaning that in addition to doing full time research ten months out of the year, for two months I also supervised a clinical endocrinology in-patient service. Because of this, I gained a remarkable exposure to unusual endocrine diseases which were under study at the time. An example of this was acromegaly.

It was the heyday of interest in second messenger signaling after the discovery of cAMP by Earl Sutherland. He would receive the Nobel Prize in Medicine and Physiology for this in 1971. One hormone after another was being shown to stimulate the enzyme adenylate cyclase thus increasing intracellular levels of cAMP. The idea that these different hormones might work through distinct receptors was talked about but was controversial. Moreover, at the time there were no direct methods for studying the receptors. I was assigned the challenging task of developing a radioligand binding method to study the putative receptors for adrenocorticotropic hormone (ACTH) in plasma membranes derived from an ACTH responsive adrenocortical carcinoma passaged in nude mice.

Recently, two Nobel Laureates, Mike Brown and Joe Goldstein, published a brief essay discussing the remarkable number of Nobel Laureates (9 so far) who have in common the fact that they came to the NIH as physicians during the brief space between 1964–1972 for postdoctoral research training. (1)

They dissect the unique convergence of circumstances which may have been responsible for this extraordinary result, including the quality of basic science mentors on the full time NIH staff, the competitiveness of “the best and the brightest” to obtain these positions during the Vietnam War years, and the now bygone emphasis on teaching of basic sciences in medical schools in the 1960s.

Lineages among Nobel Laureates are often commented upon. In my case, Jesse Roth had trained with Solomon Berson and Rosalyn Yalow whose development of radioimmunoassay led to the Nobel Prize in Medicine and Physiology to Yalow (1977) after Berson’s untimely death in 1972. Moreover, training in Ira Pastan’s laboratory contemporaneously with me was my medical school and house staff classmate and future Nobel Laureate, Harold Varmus. Ira had himself trained in the lab of another NIH career scientist, Earl Stadtman, who also trained a future Nobel Laureate, Mike Brown.

Dr. Edgar Haber, the Chief of Cardiology and a prominent immunochemist, allowed me to begin working in his lab. I was fascinated by receptors and what I saw as their potential to form the basis for a whole new field of research just waiting to be explored. I spent a great deal of time analyzing which receptor I should attempt to study. As an aspiring academic cardiologist I wanted to work on something related to the cardiovascular system. I also wanted a receptor known to be coupled to adenylate cyclase. I initially focused on two models, the cardiac glucagon and β-adrenergic receptors. However, my attention quickly became focused on the latter, for very practical reasons. Unlike the case for peptide hormones such as glucagon or ACTH, literally dozens, if not hundreds of analogs of adrenaline and noradrenaline, as well as their antagonists were available which could be chemically modified to develop the types of new tools which would need to be developed to study the receptors. These would include radioligands, photoaffinity probes, affinity chromatography matrices and the like. Moreover, the first β-adrenergic receptor blocker (“β-blocker”) had recently been approved for clinical use in the United States, adding further to the attractiveness of this target to me.

So in the early months of 1971 I began the quest to prove the existence of β-adrenergic receptors, to study their properties, to learn about their chemical nature, how they were regulated and how they functioned. This work has consumed me for the past forty years. Over the next several years in Boston, working mostly with membrane fractions derived from canine myocardium, I sought to develop radioligand binding approaches to tag the β-adrenergic receptors. I focused initially on the use of [3H]labeled catecholamines such as norepinephrine, which are agonists for the receptor. Specific saturable binding could be demonstrated, and I thought initially that we had developed a valid approach to label the receptors. However, it became increasingly clear over the next few years that the sites being labeled lacked many of the properties that would be expected for true physiological receptor binding sites. Coming to this realization was difficult.

During this time I also published some of the very first studies demonstrating GTP regulation of β-adrenergic receptor stimulated adenylate cyclase following after the work of Martin Rodbell on GTP regulation of glucagon sensitive adenylate cyclase. I was now a cardiology fellow. As at the NIH, nights on call were often spent in the lab doing experiments while hoping that my on call beeper would remain quiet. During these years, I had many stimulating and profitable discussions with Geoffrey Sharpe, a faculty member in the Nephrology Division with an interest in cell signaling and adenylate cyclase.

In work with postdoc Marc Caron in the spring of 1974, we succeeded in developing [3H]dihydroalprenolol. Contemporaneously, Gerald Aurbach at the NIH, and Alex Levitzki at the Hebrew University in Jerusalem also developed similar approaches using different radioligands. This was a watershed event because it finally opened the door to direct study of the receptors. Together with M.D./Ph.D. student Rusty Williams we developed comparable assays for the α-adrenergic receptors shortly thereafter.

Brian Kent Kobilka is an American physiologist and a corecipient of the 2012 Nobel Prize in Chemistry with Robert Lefkowitz for discoveries that reveal the inner workings of an important family G protein-coupled receptors.

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Heroes in Basic Medical Research – Leroy Hood

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Diagnostics and Lab Tests, Disease Biology, Genome Biology, Genomic Expression, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Innovations, Intellectual Property, Innovations, Commercialization, Investment in technological breakthrough, Mass automation of plasma proteins, Medical Devices R&D Investment, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Patents, Pharmaceutical Discovery, Population Health Management, Genetics & Pharmaceutical, Rapid automation of plasma protein pools, tagged , , , on September 6, 2015| Leave a Comment »

Heroes in Basic Medical Research – Leroy Hood

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 4.5

Leroy Hood, MD, PhD

Dr. Hood created the technological foundation for the sciences of genomics (study of genes) and proteomics (study of proteins) through the invention of five groundbreaking instruments and by explicating the potentialities of genome and proteome research into the future through his pioneering of the fields of systems biology and systems medicine. Hood’s instruments not only pioneered the deciphering of biological information, but also introduced the concept of high throughput data accumulation through automation and parallelization of the protein and DNA chemistries.

The first four instruments were commercialized by Applied Biosystems, Inc., a company founded by Dr. Hood in 1981, and the ink-jet technology was commercialized by Agilent Technologies, thus making these instruments immediately available to the world-community of scientists.

The first two instruments transformed the field of proteomics. The protein sequencer allowed scientists to read and analyze proteins that had not previously been accessible, resulting in the characterization of a series of new proteins whose genes could then be cloned and analyzed. These discoveries led to significant ramifications for biology, medicine, and pharmacology. The second instrument, the protein synthesizer, synthesized proteins and peptides in sufficient quantities to begin characterizing their functions. The DNA synthesizer, the first of three instruments for genomic analyses, was used to synthesize DNA fragments for DNA mapping and gene cloning. The most notable of Hood’s inventions, the automated DNA sequencer developed in 1986, made possible high-speed sequencing of human genomes and was the key technology enabling the Human Genome Project.

In the early 1990s Hood and his colleagues developed the ink-jet DNA synthesis technology for creating DNA arrays with tens of thousands of gene fragments, one of the first of the so-called DNA chips, which enabled measuring the levels of 10,000s of expressed genes. This instrument has also transformed genomics, biology, and medicine.

In 1992, Hood created the first cross-disciplinary biology department, Molecular Biotechnology, at the University of Washington. In 2000, he left the UW to co-found Institute for Systems Biology, the first of its kind. He has pioneered systems medicine the years since ISB’s founding.

In 2000, Hood and two colleagues founded the Institute for Systems Biology (ISB), a nonprofit research institute integrating biology, technology, computation and medicine to take a systems (holistic) approach to studying the complexity of biology and medicine by analyzing all elements in a biological system rather than studying them one gene or protein at a time (an atomistic approach).

Hood has made many seminal discoveries in the fields of immunology, neurobiology and biotechnology and, most recently, has been a leader in the development of systems biology, its applications to cancer, neurodegenerative disease, and the linkage of systems biology to personalized medicine.

Hood’s efforts in a systems approach to disease have led him to pioneer a new approach to medicine that he coined P4 Medicine in 2003. His view is that P4 medicine will transform the practice of medicine over the next decade, moving it from a largely reactive discipline to a proactive one.

Dr. Hood’s outstanding contributions have had a resounding effect on the advancement of science since the 1960s. Throughout his career, he has adhered to the advice of his mentor, Dr. William J. Dreyer: “If you want to practice biology, do it on the leading edge, and if you want to be on the leading edge, invent new tools for deciphering biological information.”

 

Hood is now pioneering new approaches to P4 medicine

Co-founder and Chairman P4 Medicine institute

—predictive, preventive, personalized and participatory, and most recently, has embarked on creating a P4 pilot project on 100,000 well individuals, that is transforming healthcare.

In addition to his ground-breaking research, Hood has published 750 papers, received 36 patents, 17 honorary degrees and more than 100 awards and honors. He is one of only 15 individuals elected to all three National Academies—the National Academy of Science, the National Academy of Engineering, and the Institute of Medicine. Hood has founded or co-founded 15 different biotechnology companies.

 

http://www.youtube.com/watch%3Fv%3D5aE8tgbsl9U Feb 18, 2015 … Dr. Leroy Hood, President and Co-founder, Institute for Systems Biology, gives a talk entitled “Systems Medicine and a Longitudinal, …

http://www.youtube.com/watch%3Fv%3DaYGTLj02sx0  Nov 19, 2014 … … of Healthcare? A Personal View of Biological Complexity, Paradigm Changes, Systems Biology and Systems Medicine .Speaker: Leroy Hood.

http://www.youtube.com/watch%3Fv%3DnT1MvnH6j8Q Sep 26, 2014 … Dr. Leroy Hood discusses how P4 (Predictive, Preventive, … EMBC 2014 Theme Keynote Lecture with Dr. Emery Brown – Duration: 58:49. by …

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Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Posted in Biomarkers & Medical Diagnostics, BioTechnology - Venture Creation, Cancer and Current Therapeutics, Cardiovascular Pharmacogenomics, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, CRISPR/Cas9 & Gene Editing, Curation, Genomic Expression, tagged , , on August 30, 2015| Leave a Comment »

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Author & Curator: Larry H. Bernstein, MD, FCAP

Article 21.4.4- Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

 

Chief Scientific Officer, Leaders in Pharmaceutical Intelligence, Boston, MA

http://pharmaceuticalintelligence.com

Correspondence:
larry.bernstein@gmail.com

2.1.2.2

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Abstract

The recent development of advanced methods for genome engineering has superceded methods already in used in recent years of the 21st century.  The CRISPR-Cas9 application for genome editing has real potential for pharmaceutical development, and perhaps also for diagnostics.  The importance of conjoint development of diagnostics and therapeutics can’t be overstressed. Further, the limitations of the method have to be viewed in the light of the historical development of inborn errors of human metabolism, and current understanding of complex polygenomic and environmental risk factors.

Key words: classic model, CRISPR-Cas9, DNA, genome, genome editing, genetic diseases, Hardy-Weinberg equilibrium, inborn errors of metabolism, polygenetic diseases, RNA, RNAi, translation.

Abbreviations: CRISPR-Cas9; DNA; HWE; RNA.

Introduction.

Genome editing technologies enable the deletion, insertion or correction of DNA at specific targeted sites within an organism’s genome. The power of the technology lies in its ability to specifically target any site in the genome and to alter the DNA sequence at that site. This has opened the door to potentially curing diseases caused by genetic defects, whether inherited or acquired.

Genome editing can be applied across many diverse fields of science. It has allowed researchers to gain a much deeper understanding of the role played by individual genes. Researchers working in the biomedical field use these techniques to address diseases that are known to have a genetic origin.

Early genome-editing research focused on the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs), which laid important foundations in establishing genome engineering as a potential approach for treating human diseases.

The recent discovery of CRISPR-Cas9, followed by work demonstrating its advantages over traditional approaches, promises a step-change in the use of genome editing to develop transformative medicines for serious human diseases.

Cas9* is an endonuclease (an enzyme) that can be easily programmed with RNA to cut DNA at targeted sites within the genome, enabling the deletion, insertion or correction of target genes, including those that cause diseases, with surgical precision. By using CRISPR-Cas9* genome-editing technology, scientists and clinicians are conducting pioneering research with a view to tackling both recessive and dominant genetic defects.

In order to find a place for CRISPR-Cas9 in gene therapy, it becomes necessary to consider inborn errors of metabolism and the evolution of traditional approaches to genetic diseases. Traditional gene therapy approaches to date have only been useful in correcting some recessive genetic disorders. Thanks to its ease of use and broad applicability, CRISPR-Cas9 has truly democratized genome editing and transformed many areas of research. Thousands of academic laboratories across the world are carrying out research using the technology. To this point, the technology known as CRISPR-Cas9 has been a science project, a research tool with enormous potential.

Genetic Disorders

A genetic disorder is a genetic problem caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions.

Genetic disorders may or may not be heritable, i.e., passed down from the parents’ genes. In non-heritable genetic disorders, defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and mainly by environmental causes in still other people. Whether, when and to what extent a person with the genetic defect or abnormality will actually suffer from the disease is almost always affected by the environmental factors and events in the person’s development.

A single-gene disorder is the result of a single mutated gene. Over 4000 human diseases are caused by single-gene defects.[4] Single-gene disorders can be passed on to subsequent generations in several ways. Genomic imprinting and uniparental disomy, however, may affect inheritance patterns. The divisions betweenrecessive and dominant types are not “hard and fast”, although the divisions between autosomal and X-linked types are (since the latter types are distinguished purely based on the chromosomal location of the gene). For example, achondroplasia is typically considered a dominant disorder, but children with two genes for achondroplasia have a severe skeletal disorder of which achondroplasics could be viewed as carriers. Sickle-cell anemia is also considered a recessive condition, but heterozygous carriers have increased resistance to malaria in early childhood, which could be described as a related dominant condition.[5] When a couple where one partner or both are sufferers or carriers of a single-gene disorder wish to have a child, they can do so through in vitro fertilization, which means they can then have a preimplantation genetic diagnosis to check whether the embryo has the genetic disorder.[6]

Prevalence of some single-gene disorders[citation needed]
Disorder prevalence (approximate)
Autosomal dominant
Familial hypercholesterolemia 1 in 500
Polycystic kidney disease 1 in 1250
Neurofibromatosis type I 1 in 2,500
Hereditary spherocytosis 1 in 5,000
Marfan syndrome 1 in 4,000[2]
Huntington’s disease 1 in 15,000[3]
Autosomal recessive
Sickle cell anaemia 1 in 625
Cystic fibrosis 1 in 2,000
Tay-Sachs disease 1 in 3,000
Phenylketonuria 1 in 12,000
Mucopolysaccharidoses 1 in 25,000
Lysosomal acid lipase deficiency 1 in 40,000
Glycogen storage diseases 1 in 50,000
Galactosemia 1 in 57,000

Heritable Diseases and Normal Variants

Identification of Genes for Childhood Heritable Diseases

Annual Review of Medicine Jan 2014; 65: 19-31

Boycott KM, Dyment DA, Sawyer SL, Vanstone MR, and Beaulieu CL.

Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, K1H 8L1 Canada

http://dx.doi.org:/10.1146/annurev-med-101712-122108

Genes causing rare heritable childhood diseases are being discovered at an accelerating pace driven by the decreasing cost and increasing accessibility of next-generation DNA sequencing combined with the maturation of strategies for successful gene identification. The findings are shedding light on the biological mechanisms of childhood disease and broadening the phenotypic spectrum of many clinical syndromes. Still, thousands of childhood disease genes remain to be identified, and given their increasing rarity, this will require large-scale collaboration that includes mechanisms for sharing phenotypic and genotypic data sets. Nonetheless, genomic technologies are poised for widespread translation to clinical practice for the benefit of children and families living with these rare diseases.

Single gene defects result in abnormalities in the synthesis or catabolism of proteins, carbohydrates, fats, or complex molecules. Most are due to a defect in an enzyme or transport protein, which results in a block in a metabolic pathway. Effects are due to toxic accumulations of substrates before the block, intermediates from alternative metabolic pathways, defects in energy production and use caused by a deficiency of products beyond the block, or a combination of these metabolic deviations. Nearly every metabolic disease has several forms that vary in age of onset, clinical severity, and, often, mode of inheritance.

There is a large number of inborn errors of metabolism.

A few examples are:

Fructose intolerance
Galactosemia
Maple sugar urine disease (MSUD)
Phenylketonuria (PKU)

Newborn screening tests can identify some of these disorders

Categories of inborn errors of metabolism, or IEMs, are as follows:

  • Disorders that result in toxic accumulation
    • Disorders of protein metabolism (eg, amino acidopathies, organic acidopathies, urea cycle defects)
    • Disorders of carbohydrate intolerance
    • Lysosomal storage disorders
  • Disorders of energy production, utilization
    • Fatty acid oxidation defects
    • Disorders of carbohydrate utilization, production (ie, glycogen storage disorders, disorders of gluconeogenesis and glycogenolysis)
    • Mitochondrial disorders
    • Peroxisomal disorders

 

 

Giants in the 20th century study of genetic medicine

  1. Victor Almon McKusick

 

 
Victor McKusick 
Known for Mendelian Inheritance in Man,OMIM and McKusick–Kaufman syndrome
Notable awards William Allan Award (1977)
Lasker Award (1997)
Japan Prize (2008)

 

Victor Almon McKusick (October 21, 1921 – July 22, 2008), an internist and medical geneticist, was the University Professor of Medical Genetics and Professor of Medicine at the Johns Hopkins Hospital, Baltimore, MD, USA.[1] He was a proponent of the mapping of the human genome due to its use for studying congenital diseases. He is well known for his studies of the Amish and, what he called, “little people”. He was the original author and, until his death, remained chief editor of Mendelian Inheritance in Man (MIM) and its online counterpart Online Mendelian Inheritance in Man (OMIM). He is widely known as the “father of medical genetics”.[2]

McKusick traveled to Copenhagen to speak about the heritable disorders of connective tissue at the first international congress of human genetics. The meeting looms as the birthplace of the medical genetics field.[2] In the following decades, McKusick created and chaired a new Division of Medical Genetics at Hopkins beginning in 1957. In 1973, he served as Physician-in-Chief, William Osler Professor of Medicine, and Chairman of the Department of Medicine at Johns Hopkins Hospital and School of Medicine.[6]  He held concurrent appointments as University Professor of Medical Genetics at the McKusick–Nathans Institute of Genetic Medicine, Professor of Medicine at the Johns Hopkins School of Medicine, Professor of Epidemiology at the Johns Hopkins Bloomberg School of Public Health, and Professor of Biology at Johns Hopkins University.[5] He co-founded Genomics in 1987 with Dr. Frank Ruddle, and served as an editor.[6] He was a lead investigator in determining if Abraham Lincoln had Marfan syndrome.[8]

  1. Elizabeth F. Neufeld

Born in France, Elizabeth Neufeld immigrated to the United States in 1940. She obtained a BS from Queens College, New York and a Ph.D. from the University of California Berkeley. After postdoctoral training in, she moved to the NIH in Bethesda, MD, where she began her studies of a rare group of genetic diseases. She moved back to California in 1984 as Chair of the Department of Biological Chemistry – a position that she occupied till 2004.

The brain in a mouse model of a genetic lysosomal disorder, Sanfilippo syndrome type B

Our interests have long been the cause, consequences and treatment of human genetic diseases due to deficiency of lysosomal enzymes. The disease currently under investigation is the Sanfilippo syndrome type B (MPS III B). It is caused by mutation in the NAGLU gene, with resulting deficiency of the lysosomal enzyme alpha-N-acetyl-glucosaminidase and accumulation of its substrate (heparan sulfate). The disease manifests itself in childhood by severe mental retardation and intractable behavioral problems. The neurologic deterioration progresses to dementia, with death usually in the second decade. We use a mouse knockout model (Naglu -/-) in order to study the pathophysiology of the disease and to develop therapy. Because of the special cell biology of lysosomal enzymes, which can be taken up by receptor-mediated endocytosis, exogenous administration of the enzyme could theoretically cure the disease. Unfortunately, the blood-brain barrier (BBB) prevents the therapeutic enzyme from reaching the brain. Part of our current research is to develop a novel technology to get lysosomal enzymes across the BBB. We also study changes in gene and protein expression in some specific parts of the brain, in which there is accumulation of certain lipids and proteins which seem unrelated biochemically to each other or to the primary defect. We try to understand the cause and consequences of these accumulations. Although they are secondary defects, they may be relevant to the pathophysiology of the dieease and may have represent targets for pharmacologic intervention.

Neufeld began her scientific studies at a time when few women chose science as a career. The historical bias against women in science, compounded with an influx of men coming back from the Second World War and going to college, made positions for women rare; few women could be found in the science faculties of colleges and universities.

When she first began working on Hurler syndrome in 1967, she initially thought the problem might stem from faulty regulation of the sugars, but experiments showed the problem was in fact the abnormally slow rate at which the sugars were broken down. Working with fellow scientist Joseph Fratantoni, Neufeld attempted to isolate the problem by tagging mucopolysaccharides with radioactive sulfate, as well as mixing normal cells with MPS patient cells. Fratantoni inadvertently mixed cells from a Hurler patient and a Hunter patient—and the result was a nearly normal cell culture. The two cultures had essentially “cured” each other.

In 1973 Neufeld was named chief of NIH’s Section of Human Biochemical Genetics, and in 1979 she was named chief of the Genetics and Biochemistry Branch of the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK). She served as deputy director in NIADDK’s Division of Intramural Research from 1981 to 1983. In 1984 Neufeld went back to the University of California, this time the Los Angeles campus, as chair of the biological chemistry department.

Neufeld’s research opened the way for prenatal diagnosis of such life-threatening fetal disorders as Hurler syndrome. Neufeld chaired the Scientific Advisory Board of the National MPS Society and was president of the American Society for Biochemistry and Molecular Biology from 1992 to 1993. She was elected to both the National Academy of Sciences (USA) and the American Academy of Arts and Sciences in 1977 and named a fellow of the American Association for Advancement in Science in 1988. In 1990 she was named California Scientist of the Year. She was awarded the Wolf Prize, the Albert Lasker Award for Clinical Medical Research, and was awarded the National Medal of Science in 1994 “for her contributions to the understanding of the lysosomal storage diseases, demonstrating the strong linkage between basic and applied scientific investigation.”[3]

  1. Jarvis “Jay” Edwin Seegmiller, M.D.

“Jay Seegmiller was one of the giants of American medicine,” said Edward Holmes, M.D., Vice Chancellor of Health Sciences and dean of the School of Medicine at UCSD. “He and his trainees have made innumerable contributions to our understanding of the pathogenesis of many human disorders. Seegmiller was one of the country’s leading researchers in intermediary metabolism, with a focus on purine metabolism and inherited metabolism.  He worked in the field of human biochemical genetics, with a special interest in the mechanisms by which genetically determined defects of metabolism lead to various forms of arthritis.  His laboratory identified a wide range of primary metabolic defects in metabolism responsible for development of gout.

He is perhaps best known for his discovery of the enzyme defect in Lesch-Nyhan Syndrome, a fatal disorder of the nervous system causing severe mental retardation and self-mutilation impulses.  As Director of the Human Biochemical Genetics Program at UCSD, Seegmiller’s investigations into the translation of genetic research and methods of prevention, detection and treatment of hereditary diseases led to Congressional testimony on the possibility of controlling genetic disease in the United States.  As a result, genetic referral centers have been established throughout the country.

He joined the newly established UCSD School of Medicine in 1969 as head of the Arthritis Division of the Department of Medicine. There, he directed a research program in human biochemical genetics involving senior faculty from five departments within the School of Medicine.  While a professor at UCSD, he served as a Macy Scholar both at Oxford University and at the Basel Institute in Switzerland, as well as a Guggenheim Fellow at the Swiss Institute for Experimental Cancer Research in Lausanne.

In 1983, he became the founding director of what is today UCSD’s Stein Institute for Research on Aging (SIRA). Even after his retirement, he continued to serve as Associate Director of SIRA from 1990 until his death.

“He had the foresight of proposing the formation of and then establishing a new Institute on Aging at UCSD before there was any such Institute in the entire UC system,” said Dilip Jeste, M.D., the Estelle and Edgar Levi Chair in Aging, Professor of Psychiatry and Neurosciences and current Director of SIRA.   “He was himself a role model of successful aging, and continued working in the SIRA till his very last days.

Seegmiller received his Doctor of Medicine with honors from the University of Chicago in 1948.  After he completed his internship at Johns Hopkins Hospital in Baltimore, Maryland, he trained with Bernard Horecker of the National Institute of Arthritis and Metabolic Disease at the National Institutes of Health.

Seegmiller was appointed Senior Investigator of the National Institute of Arthritis and Metabolic Disease in 1954, where he carried out biochemical and clinical studies of human hereditary disease, with a special interest in those causing various forms of arthritis.  He became Assistant Scientific Director of the Institute in 1960, and was appointed Chief of the section on Human Biochemical Genetics in 1966, becoming one of several NIH leaders recruited to help launch UC San Diego’s new medical school.

Seegmiller’s clinical activities included studies in life longevity in South America.  In 1974, he joined a team of notable scientists and traveled to the remote village of Vilcabamba in Ecuador, to find out what role genetic factors played in the population of the Andean villagers who comprised some of the longest-living people in the world.  His later work led to the discovery of free radicals and their damaging effects in the human ability to withstand diseases, bringing forward new investigations on human aging at SIRA.

Seegmiller was a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and was the recipient of numerous prizes and awards in honor of his extraordinary achievements in science and medicine.  He received the United States Public Health Distinguished Service Award in 1969; and was honored as Master of the American College of Rheumatology (ACR) in 1992. He was on the advisory boards for the National Genetics Foundation, the City of Hope Medical Center in Duarte, California, the Task Force on Endocrinology and Metabolism for NIH, the Executive Editorial Board for Analytical Biochemistry, and was President of the Western Association of Physicians in 1979.

What has changed?

The 21st century has seen the mapping of the human genome. The huge focus on the genome came after the Watson and Crick discovery put the genome at the center of the translational network with the central hypothesis. What followed was transcription of RNA into placement of an amino acid into protein. The central hypothesis is DNA           RNA           protein.  However, RNAi and non-translational RNA are now also important.  RNA has a role in suppressing translation, as do proteins by allosteric effects. In addition, the most common diseases involved in age related change are strongly responsive to extracellular matrix effects, ionic fluxes, effects on the cellular matrix, and involve multicentric genome expression. This mode of expression leads one to think hard about the therapeutic target, or targets. The effect of RNA or of protein interacting with the genome is not an element of the classic construct.

Identifying a part of the problem

Type 2 diabetes mellitus, hypertension, arrhythmias, atherosclerotic plaque development, cancer, congestive heart disease, pulmonary hypertension, pulmonary interstitial sclerosis, and renovascular disease are among the common diseases that develop during a lifetime. The phenotypic presentations may have genomic associations, and there may also be population variants.  There is also a cross-talk between these phenotypic expressions. Classically, medical terminology has been based on signs and symptoms of disease.  In the increasingly complex experience, the laboratory has played an increased role in the diagnosis as well as prognostication. The laboratory experience with respect to the practice of medicine has heavily relied of either proteins, enzymes, or the products of chemical reactions.  The use of genomic profiling has rapidly emerged in the laboratory armamentarium, but has had a slow ascent in practice.

Case in Point. Pompe’s disease

William Canfield is a glycobiologist, chief scientific officer and founder of an Oklahoma City-based biotechnology company, Novazyme, which was acquired by Genzyme in August 2001 and developed, among other things, an enzyme that can stabilize (but not cure) Pompe disease, based on Canfield’s ongoing research since 1998.[1][2]   

John Crowley took over a position as a CEO in Novazyme after leaving Bristol-Myers Squibb in March 2000 and together with Dr. Y. T. Chen[4] at Duke University pushed for expedited approval by the U.S. Food and Drug Administration (FDA) of a new drug compound, NZ-1001 under orphan drug designation for the treatment of Glycogen storage disease type II in October 2005. The FDA stated: “We have determined that Novazyme’s recombinant human highly phosphorylated acid alpha-glucosidase (rhHPGAA) qualifies for orphan designation for enzyme replacement therapy in patients with all subtypes of glycogen storage disease type II (Pompe’s disease).” [5][6] Subsequent research at Genzyme on NZ-1001 along with three other potential compounds brought approval of the first enzyme replacement therapy for Pompe’s disease – Alglucosidase alfa (Myozyme or Lumizyme, Genzyme Inc) in 2006.[7]

William Canfields work with Pompes Disease was fictionalized and made the subject of a 2010 movie Extraordinary Measures in which he is called Dr. Robert Stonehill and played by Harrison Ford.[8]

Case in point.  Polymorphisms in the long non-coding RNA

Hypertension (HT) is a complex disorder influenced by both genetic and environmental factors. Recent genome-wide association studies have identified a major risk locus for atherosclerosis on chromosome 9p21.3 (chr9p21.3). SNPs within the coding sequences of CDKN2A/B proteins and the long non-coding RNA CDKN2B-AS1 could potentially contribute to HT development. Such a study has now been done. The findings suggest that SNPs rs10757274, rs2383207, rs10757278, and rs1333049, particularly those within the CDKN2B-AS1 gene, and related haplotypes may confer increased susceptibility to HT development. (unpublished)

Case in point. Lipoprotein Lipase and Atherosclerosis

Lipoprotein lipase (LPL) plays a pivotal role in lipids and metabolism of lipoprotein. Dysfunctions of LPL have been found to be associated with dyslipidemia, obesity and insulin resistance.Dyslipidemia, obesity and insulin resistance are risk factor of atherosclerosis. Japanese investigators have  hypothesized that elevating LPL activity would cause protection of atherosclerosis. (unpublished).

Case in  point. Holocaust survivors pass on stress.

Descendants of Holocaust Survivors Have Altered Stress Hormones

Parents’ traumatic experience may hamper their offspring’s ability to bounce back from trauma
By Tori Rodriguez | Feb 12, 2015
Scientifc American
 

A person’s experience as a child or teenager can have a profound impact on their future children’s lives, new work is showing. Rachel Yehuda, a researcher in the growing field of epigenetics and the intergenerational effects of trauma, and her colleagues have long studied mass trauma survivors and their offspring. Their latest results reveal that descendants of people who survived the Holocaust have different stress hormone profiles than their peers, perhaps predisposing them to anxiety disorders.

Yehuda’s team at the Icahn School of Medicine at Mount Sinai and the James J. Peters Veterans Affairs Medical Center in Bronx, N.Y., and others had previously established that survivors of the Holocaust have altered levels of circulating stress hormones compared with other Jewish adults of the same age. Survivors have lower levels of cortisol, a hormone that helps the body return to normal after trauma; those who suffered post-traumatic stress disorder (PTSD) have even lower levels.

Case in point. Genome engineering with CRISPR-Cas9

The new frontier of genome engineering with CRISPR-Cas9

GENOME EDITING

Jennifer A. Doudna* and Emmanuelle Charpentier*
Science Nov 2014; 346(6213) 1258096:1077 – 1087.
http://dx.doi.org:/10.1126/science.1258096

BACKGROUND: Technologies for making and manipulating DNA have enabled advances in biology ever since the discovery of the DNA double helix. But introducing site-specific modifications in the genomes of cells and organisms remained elusive. Early approaches relied on the principle of site-specific recognition of DNA sequences by oligonucleotides, small molecules, or self-splicing introns. More recently, the site-directed zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) using the principles of DNAprotein recognition were developed. However, difficulties of protein design, synthesis, and validation remained a barrier to

SUMMARY

The field of biology is now experiencing a transformative phase with the advent of facile genome engineering in animals and plants using RNA-programmable CRISPR-Cas9. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems, which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR associated protein Cas9 is an endonuclease that uses a guide sequence within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA. The dual tracrRNA:crRNA was engineered as a single guide RNA (sgRNA) that retains two critical features: a sequence at the 5  side that determines the DNA target site by Watson-Crick base-pairing and a duplex RNA structure at the 3 side that binds to Cas9. This finding created a simple two-component system in which changes in the guide sequence of the sgRNA program Cas9 to target any DNA sequence of interest. The simplicity of CRISPR-Cas9 programming, together with a unique DNA cleaving mechanism, the capacity for multiplexed target recognition, and the existence of many natural type II CRISPR-Cas system variants, has enabled remarkable developments using this cost-effective and easy-to-use technology to precisely and efficiently target, edit, modify, regulate, and mark genomic loci of a wide array of cells and organisms.

Figure (not shown)

The Cas9 enzyme (blue) generates breaks in double-stranded DNA by using its two catalytic centers (blades) to cleave each strand of a DNA target site (gold) next to a PAM sequence (red) and matching the 20-nucleotide sequence (orange) of the single guide RNA (sgRNA). The sgRNA includes a dual-RNA sequence derived from CRISPR RNA (light green) and a separate transcript (tracrRNA, dark green) that binds and stabilizes the Cas9 protein. Cas9-sgRNA–mediated DNA cleavage produces a blunt double-stranded break that triggers repair enzymes to disrupt or replace DNA sequences at or near the cleavage site. Catalytically inactive forms of Cas9 can also be used for programmable regulation of transcription and visualization of genomic loci.

This Review illustrates the power of the technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders. CRISPR-Cas9 is having a major impact on functional genomics conducted in experimental systems. Its application in genome-wide studies will enable large-scale screening for drug targets and other phenotypes and will facilitate the generation of engineered animal models that will benefit pharmacological studies and the understanding of human diseases. CRISPR-Cas9 applications in plants and fungi also promise to change the pace and course of agricultural research. Future research directions to improve the technology will include engineering or identifying smaller Cas9 variants with distinct specificity that may be more amenable to delivery in human cells. Understanding the homology-directed repair mechanisms that follow Cas9-mediated DNA cleavage will enhance insertion of new or corrected sequences into genomes. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy.

Case in point.

ZFN, TALEN and CRISPR/Cas-based methods for genome engineering

Thomas Gaj1,2,3, Charles A. Gersbach4,5, and Carlos F. Barbas III1,2,3 1The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA 3Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA 4Department of Biomedical Engineering, Duke University, Durham, NC, USA 5Institutes for Genome Sciences and Policy, Duke University, Durham, NC, USA

Trends Biotechnol . 2013 July ; 31(7): 397–405. http://dx.doi.org:/10.1016/j.tibtech.2013.04.004

Abstract Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of CRISPR/Cas-based RNA-guided DNA endonucleases.

Keywords zinc-finger; TALE; CRISPR; nuclease; genome engineering

Classical and contemporary approaches for establishing gene function With the development of new and affordable methods for whole-genome sequencing, and the design and implementation of large-scale genome annotation projects, scientists’ are poised to deliver upon the promises of the Genomic Revolution to transform basic science and personalized medicine. The resulting wealth of information presents researchers with a new primary challenge of converting this enormous amount of data into functionally and clinically relevant knowledge. Central to this problem is the need for efficient and reliable methods that enable investigators to determine how genotype influences phenotype. Targeted gene inactivation via homologous recombination is a powerful method capable of providing conclusive information for evaluating gene function.

Several factors impede the use of these methods:

  • the low efficiency at which engineered constructs are correctly inserted into the chromosomal target site,
  • the need for time-consuming and labor-insensitive selection/screening strategies, and
  • the potential for adverse mutagenic effects.

Targeted gene knockdown by RNA interference (RNAi) has provided researchers with a rapid, inexpensive and high-throughput alternative to homologous recombination. However, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects, and provides only temporary inhibition of gene function. These restrictions impede researchers’ ability to directly link phenotype to genotype and limit the practical application of RNAi technology.

In the past decade, a new approach has emerged that enables investigators to directly manipulate virtually any gene in a diverse range of cell types and organisms. This core technology – commonly referred to as “genome editing” – is based on the use of engineered nucleases composed of sequence-specific DNA-binding domains fused to a non-specific DNA cleavage module. These chimeric nucleases enable efficient and precise genetic modifications by inducing targeted DNA double-strand breaks (DSBs) that stimulate the cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR).

Case in point.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference (RNAi).

RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function, but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects. Other recent approaches to targeted genome modification – zinc-finger nucleases (ZFNs), and transcription-activator like effector nucleases (TALENs) – enable researchers to generate permanent mutations by introducing double stranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli, but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus.

Three types of CRISPR mechanisms have been identified, of which type II has been the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (not shown).

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence.

Investment in CRISPR technology

CRISPR Therapeutics is a biopharmaceutical company created to translate CRISPR-Cas9, a breakthrough genome-editing technology, into transformative medicines for serious human diseases. We are uniquely positioned to translate CRISPR-Cas9 technology into human therapeutics, thanks to its multi-disciplinary team of world-renowned academics, clinicians and drug developers.

CRISPR Therapeutics’ vision is to cure serious human diseases at the molecular level using CRISPR-Cas9. The company is headquartered in Basel, Switzerland and has operations in London, UK and Cambridge, Massachusetts.

The biopharmaceutical company that is focused on translating CRISPR-Cas9 gene-editing technology into transformative medicines for serious human diseases, congratulates its scientific founder, Dr. Emmanuelle Charpentier, for being named to TIME Magazine’s TIME 100 Most Influential People in the World alongside fellow CRISPR-Cas9 discoverer, Dr. Jennifer Doudna. In addition, Dr. Emmanuelle was awarded the Louis Jeantet Prize for Medicine, considered the most prestigious European award for researchers in the life sciences, for her discovery of the CRISPR-Cas9 gene editing tool. She will receive the award in a ceremony in Geneva, Switzerland, on April 22, 2015.

Dr. Charpentier has received numerous additional awards for her research, including in 2014 the Alexander von Humboldt Professorship, the Dr Paul Janssen Award, the Grand-Prix Jean-Pierre Lecocq (French Academy of Sciences), the Göran Gustafsson Prize (Royal Swedish Academy of Sciences) and in 2015 the Breakthrough Prize in Life Sciences. She was also selected as one of the American Foreign Policy magazine’s 100 Leading Global Thinkers for 2014.

Cambridge-based Editas Medicine announced a $120 million Series B round led by Bill Gates’s chief advisor for science and technology, Boris Nikolic. The list of financiers teaming with Nikolic reads like a rolodex of so-called crossover investors. Nikolic, who joined Editas’ board, made the investment through what’s been called “bng0,” a new U.S.-based investment company backed by “large family offices with a global presence and long-term investment horizon” and formed specifically to invest in Editas. CEO Katrine Bosley confirmed that Gates is one of the individuals investing in Editas alongside Nikolic. Editas has become the first of the group not only to attract crossover backers, but to begin discussing the diseases that its targeting.

Caribou Biosciences, one of the biotech startups working to advance a much-watched new technology for precise gene editing, has raised an $11 million Series A round from venture capital firms and Swiss drug giant Novartis.

The money will help Berkeley, CA-based Caribou speed up its efforts to adapt a versatile genome editing technique co-discovered by one of its founders, UC Berkeley professor Jennifer Doudna, for a range of uses, including drug research and development, and industrial technology.

Doudna and her collaborator, Emmanuelle Charpentier of the Helmholtz Center for Infection Research in Braunschweig, Germany, and Umeå University in Sweden, figured out how to transform a bacterial defense against viral infection into a tool to edit out abnormal sections of genes, such as those that cause hereditary diseases.

Caribou’s gene editing platform is based on two elements of that bacterial molecular machinery: a guiding mechanism called CRISPR (clustered, regularly interspaced palindromic repeats), and an enzyme called Cas9, or CRISPR-associated protein 9, molecular scissors that cut a segment of DNA. Caribou was founded in 2011 to commercialize the work from Doudna’s lab.

 

 

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Posted in Academic Publishing, Amino acids, Anaerobic Glycolysis, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Ca2+ triggered activation, Cachexia, Calcium, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Competition in regenerative stem cell science, Curation, Curation methodology, Cytoskeleton, Dehydrogenase, Diabetes Mellitus, Discovery process, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Explanatory, Fatty acids, Folate and B12, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Genomic Endocrinology, Genomic Expression, Glycobiology: Biopharmaceutical Production, Health Economics and Outcomes Research, Hexokinase, Historical relevance, Inosine nucleotides, Interviews with Scientific Leaders, Iron deficiency, Kinase, Lipid metabolism, Lipids, Liver & Digestive Diseases Research, Loss of function gene, Metabolism, Metabolomics, Methylation, Mg++, Molecular Genetics & Pharmaceutical, mtDNA, Mutant Gene Expression, Myelodysplasia, Myelofibrosis, Myocardial Ischemia, Myocardial Metabolism, Na-K-ATPase, Neurodegenerative Diseases, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrigenomics, Nutrition, Nutrition and Phytochemistry, Nutrition Disorders, Nutritional Supplements: Atherogenesis, Open Access Journals, Oxidative phosphorylation, Pentose monophosphate shunt, Phosphatase, Phosphorylase, Phosphorylation, PKC, Plant extract, Proteins, Proteomics, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, Pyruvate Kinase, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, RNA Biology, S-nitrosylation, Signaling, Signaling & Cell Circuits, Skeletal muscle regeneration, Small Molecules in Development of Therapeutic Drugs, Theoretic convergence, Transcriptomics, Translational Science, Ubiquitinylation, Warburg effect, tagged , , , , , , , , , , , , , , , , , on August 15, 2015| Leave a Comment »

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

Article ID #180: Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle. Published on 8/15/2015

WordCloud Image Produced by Adam Tubman

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

http://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Severe IBS Symptoms: Clinical Trial results on delivery of peppermint oil to the small intestine through a system (IBgard®)

Posted in Cell Biology, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Drug Delivery Platform Technology, Metabolism on July 8, 2015| Leave a Comment »

Severe IBS Symptoms: Clinical Trial results on delivery of peppermint oil to the small intestine through a system (IBgard®)

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Written by Freelance medical writer Walter Alexander

 

IBgard® Delivery of Mint Oil Effective in Patients with Severe IBS Symptoms
WASHINGTON DC, May 17, 2015—In IBSREST™ (Irritable Bowel Syndrome Reduction Evaluation and Safety Trial), sustained delivery of peppermint oil to the small intestine through a system (IBgard®) that minimizes release in the stomach and colon, resulted in significant reductions in severe or unbearable irritable bowel syndrome (IBS) symptoms over four weeks as compared with placebo. Other formulations of mint oil tend to remain in the stomach or pass all the way to the colon, potentially causing cause heartburn, nausea and anal burning.

About 25 percent of IBS patients, said Brooks D. Cash, MD, professor of medicine at University of South Alabama, Mobile, AB, at his poster at the Digestive Disease Week 2015 annual meeting, describe their symptoms as severe. Dr. Cash noted that derangements in gut immunity, microbiota, sensation, motility, secretion, and digestion have all been proposed as possible etiologies of IBS. L-menthol, the main constituent of peppermint oil, has anti-spasmodic, anti-carminative, topical analgesic, anti-infective and 5-HT3 receptor antagonism properties. IBSREST™ evaluated the efficacy and tolerability of IBgard, an ultra-purified peppermint oil, in a population enriched with severe/unbearable symptoms.High symptom severity reflects higher intensity and frequency of individual symptoms, leading in IBS patients to lower quality of life, work disruptions, and frequent physician visits (more than one per month). Dr. Cash noted also that among treatments for the three IBS subtypes (M:mixed/alternating; D: diarrhea; C:constipation), approved products are lacking for IBS-M and options for IBS-D are limited. “Our study was selective for patients with severe symptoms because that is where the unmet need is,” Dr. Cash said in an interview. He pointed out further that in studies of other treatments, more severe patients tend to respond less well. The targeted delivery of mint oil in solid microspheres to the small intestine was expected to address the unmet need, according to Dr. Cash.

The randomized, placebo-controlled trial included 72 patients (mean age ~41 years) who met Rome III criteria for IBS-D or IBS-M, had average daily IBS-related abdominal pain of ≥4 on a 0-10 scale, and a Total IBS Symptom Score (TISS) of ≥2 on a 0-4 scale. The TISS scale encompasses 8 symptoms (abdominal pain or discomfort, bloating or distention, pain at evacuation, urgency, constipation, diarrhea, mucus or gas and sense of incomplete evacuation). After a 3-week period for symptom severity assessment and prohibited medication washout, subjects were randomly allocated to receive IBgard 180 mg TID or identical placebo for 4 weeks.

After 28 days, the reduction from baseline in number of severe and unbearable symptoms (average of frequency and intensity ≥3) was -66 percent for IBgardas compared with -42 percent with placebo (P=0.0212). The reduction in patient-reported severe or unbearable abdominal pain intensity at 28 days was -79.4 percent for IBgard and-40.5 percent for placebo (P=0.0009). Trends in percent reduction in severe or unbearable individual intensity scores were favorable for IBgard across all 8 severe/unbearable parameters (from 71 percent to 90 percent), more than at 24 hour assessment (30 percent to 47 percent).“Severe symptom patients responded as well as those with less severe symptom patients. That’s very reassuring,” Dr. Cash said.

IBgard was well tolerated and safe. No patients withdrew from the study on account of treatment emergent adverse events.

Dr. Cash concluded, “Over 4 weeks, IBgard was effective at improving the composite IBS symptom score, and the individual IBS symptom components, including severe or unbearable abdominal pain intensity at 4 weeks.”

 

SOURCE

From: Gail Thornton <gailsthornton@yahoo.com>

Reply-To: Gail Thornton <gailsthornton@yahoo.com>

Date: Wednesday, July 8, 2015 at 11:23 AM

To: Aviva Lev-Ari <avivalev-ari@alum.berkeley.edu>

Subject: Article for Pharmaceutical Business Intelligence on IBgard

 

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Nonhematologic Cancer Stem Cells [11.2.3]

Posted in Anticancer Resistance, Biochemical pathways, Biological Networks, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Genomics, Curation, Disease Biology, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Metabolism, Metabolomics, Methylation, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Personal Health Applications: Tech Innovations serves HealhCare, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Population Health Management, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , , , , , , , , on May 22, 2015| Leave a Comment »

Nonhematologic Cancer Stem Cells [11.2.3]

Writer and Curator: Larry H. Bernstein, MD, FCAP 

Nonhematologic Stem Cells

11.2.3.1 C8orf4 negatively regulates self-renewal of liver cancer stem cells via suppression of NOTCH2 signalling

Pingping Zhu, Yanying Wang, Ying Du, Lei He, Guanling Huang, et al.
Nature Communications May 2015; 6(7122). http://dx.doi.org:/10.1038/ncomms8122

Liver cancer stem cells (CSCs) harbor self-renewal and differentiation properties, accounting for chemotherapy resistance and recurrence. However, the molecular mechanisms to sustain liver CSCs remain largely unknown. In this study, based on analysis of several hepatocellular carcinoma (HCC) transcriptome datasets and our experimental data, we find that C8orf4 is weakly expressed in HCC tumors and liver CSCs. C8orf4 attenuates the self-renewal capacity of liver CSCs and tumor propagation. We show that NOTCH2 is activated in liver CSCs. C8orf4 is located in the cytoplasm of HCC tumor cells and associates with the NOTCH2 intracellular domain, which impedes the nuclear translocation of N2ICD. C8orf4 deletion causes the nuclear translocation of N2ICD that triggers the NOTCH2 signaling, which sustains the stemness of liver CSCs. Finally, NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Altogether, C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Like stem cells, CSCs are characterized by self-renewal and differentiation simultaneously9. Not surprisingly, CSCs share core regulatory genes and developmental pathways with normal tissue stem cells. Accumulating evidence shows that NOTCH, Hedgehog and Wnt signaling pathways are implicated in the regulation of CSC self-renewal4. NOTCH signaling modulates many aspects of metazoan development and tissue stemness10, 11. NOTCH receptors contain four members (NOTCH1–4) in mammals, which are activated by engagement with various ligands. The aberrant NOTCH signaling was first reported to be involved in the tumorigenesis of human T-cell leukaemia12, 13. Recently, a number of studies have reported that the NOTCH signaling pathway is implicated in regulating self-renewal of breast stem cells and mammary CSCs14, 15. However, how the NOTCH signaling regulates the liver CSC self-renewal remains largely unknown.

C8orf4, also called thyroid cancer 1 (TC1), was originally cloned from a papillary thyroid carcinoma and its surrounding normal thyroid tissue16. C8orf4 is ubiquitously expressed across a wide range of vertebrates with the sequence conservation across species. A number of studies have reported that C8orf4 is highly expressed in several tumors and implicated in tumorigenesis17, 18, 19. In addition, C8orf4 augments Wnt/β-catenin signaling in some cancer cells20, 21, suggesting it may be involved in the regulation of self-renewal of CSCs. However, the biological function of C8orf4 in the modulation of liver CSC self-renewal is still unknown. Here we show that C8orf4 is weakly expressed in HCC and liver CSCs. NOTCH2 signaling is highly activated in HCC tumors and liver CSCs. C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

C8orf4 is weakly expressed in HCC tissues and liver CSCs

To search for driver genes in the oncogenesis of HCC, we performed genome-wide analyses using several online-available HCC transcriptome datasets by R language and Bioconductor approaches. After analysing gene expression profiles of HCC tumor and peri-tumor tissues, we identified >360 differentially expressed genes from both Park’s cohort (GSE36376; ref. 22) and Wang’s cohort (GSE14520; refs 23, 24). Of these changed genes, we focused on C8orf4, which was weakly expressed in HCC tumors derived from both Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) (Fig. 1a). Lower expression of C8orf4 was further confirmed in HCC samples by quantitative reverse transcription–PCR (qRT–PCR) and immunoblotting (Fig. 1b,c). In this study, HCC patient samples we used included all subtypes of HCC. In addition, these observations were further validated by immunohistochemical (IHC) staining (Fig. 1d). These data indicate that C8orf4 is weakly expressed in HCC tumor tissues.

C8orf4 is weakly expressed in HCC tumours and liver CSCs

C8orf4 is weakly expressed in HCC tumours and liver CSCs

Figure 1. C8orf4 is weakly expressed in HCC tumours and liver CSCs

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f1.jpg

(a)C8orf4 is weakly expressed in HCC patients. Using R language and Bioconductor methods, we analyzed C8orf4 expression in HCC tumor and peri-tumor tissues provided by Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) datasets. (b,c) C8orf4 expression levels were verified in HCC patient samples by quantitative RT–PCR (qRT–PCR) (b) and immunoblotting (c). β-actin served as a loading control. 18S: 18S rRNA. (d) HCC samples were assayed by immunohistochemical staining. Scale bar—left: 50 μm; right: 20 μm. (e) C8orf4 is weakly expressed in CD13+CD133+ cells sorted from Huh7 cells and primary HCC samples. C8orf4 messenger RNA (mRNA) was measured by qRT–PCR. Six HCC samples got similar results. (f) C8orf4 is much more weakly expressed in oncospheres than non-sphere tumor cells. Non-sphere: Huh7 or HCC primary cells that failed to form spheres. (g) HCC sample tissues were co-stained with anti-C8orf4 and anti-CD13 or anti-CD133 antibodies, then counterstained with DAPI for confocal microscopy. White arrows indicate CD13+ or CD133+ cells. Scale bars: 20 μm. For a,b, data are shown as box and whisker plot. Boxes represent interquartile range (IQR); upper and lower edge corresponds to the 75th and 25th percentiles, respectively. Horizontal lines within boxes represent median levels of gene intensity. Whiskers below and above boxes extend to the 5th and 95th percentiles, respectively. For e and f, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. P, peri-tumor; T, tumor.

 

Notably, C8orf4 was also weakly expressed in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) by analysis of its expression profiles derived from online datasets (GSE14897; ref. 25 and GSE25417; ref. 26) (Supplementary Fig. 1a,b). C8orf4 was also lowly expressed in normal liver stem cells (Supplementary Fig. 1c,d), suggesting that C8orf4 may be involved in the regulation of self-renewal of liver stem cells. Thus, we propose that C8orf4 might play a role in the maintenance of liver CSCs. Since CD13 and CD133 were widely used as liver CSC surface markers, we sorted CD13+CD133+ cells from Huh7 and Hep3B HCC cell lines as well as HCC samples, serving as liver CSCs. We observed that C8orf4 was weakly expressed in liver CSCs enriched from both HCC cell lines and patient samples (Fig. 1e). Six HCC samples were analyzed for these experiments. Similar results were obtained in CD13+CD133+ cells from Hep3B cells. Furthermore, we performed sphere formation experiments using Huh7 cells and HCC primary sample cells, and detected expression levels of C8orf4. We observed that C8orf4 was dramatically reduced in the oncospheres generated by both HCC cell lines and patient samples (Fig. 1f). In addition, we noticed that C8orf4 expression was negatively correlated with liver CSC markers such as CD13 and CD133 in HCC samples (Fig. 1g), suggesting lower expression of C8orf4 in liver CSCs. Moreover, C8orf4 was mainly located in the cytoplasm of tumour cells. Altogether, C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs.

C8orf4 negatively regulates self-renewal of liver CSCs

We then wanted to look at whether C8orf4 plays a critical role in the self-renewal maintenance of liver CSCs. C8orf4 was knocked out in Huh7 cells through a CRISPR/Cas9 system (Fig. 2a). TwoC8orf4-knockout (KO) cell strains were established and C8orf4 was completely deleted in these two strains. C8orf4 deletion dramatically enhanced oncosphere formation (Fig. 2b). We co-stained SOX9, a widely used progenitor marker, and Ki67, a well-known proliferation marker, in C8orf4 KO sphere cells. We found that SOX9 was strongly stained in C8orf4 KO sphere cells (Supplementary Fig. 2a). In contrast, Ki67 staining was not significantly altered in C8orf4 KO sphere cells versus WT sphere cells. We also digested sphere cells and examined the SOX9 and Ki67 expression by flow cytometry. Similar results were achieved (Supplementary Fig. 2b). Importantly, through serial passage of CSC sphere cells, similar observations were obtained in the fourth generation oncosphere assay (Supplementary Fig. 2c,d). These data suggest that C8orf4 is involved in the regulation of liver CSC self-renewal.

(not shown)

Figure 2: C8orf4 knockout enhances self-renewal of liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f2.jpg

  • C8orf4-deficient Huh7 cells were established using a CRISPR/Cas9 system. T7 endonuclease I cleavage confirmed the efficiency of sgRNA (left panel, white arrowheads), and C8orf4-knockout efficiency was confirmed by western blot (right panel). Two knockout cell lines were used.  C8KO#1:C8orf4KO#1;  C8KO#2: C8orf4KO#2. (b) C8orf4-deficient cells enhanced sphere formation activity. Calculated ratios are shown in the right panel. (c) C8orf4-deficient or WT Huh7 cells (1 × 106) were injected into BALB/c nude mice. Tumor sizes were observed every 5 days. (d) C8orf4 deficiency enhances tumor-initiating capacity. Diluted cell numbers of Huh7 cells were implanted into BALB/c nude mice for tumor initiation. Percentages of tumor-formation mice were calculated (left panel), and frequency of tumor-initiating cells was calculated using extreme limiting dilution analysis (right panel). Error bars represent the 95% confidence intervals of the estimation. (e) Expression levels of CD13 andCD133 were analyzed in C8orf4-knockout Huh7 cells. (f) C8orf4 was silenced in HCC primary cells and C8orf4 depletion enhanced sphere formation activity. Calculated ratios are shown at the right panel. Three HCC specimens obtained similar results. (g) C8orf4-overexpressing Huh7 cells were established (left panel). C8orf4-overexpressing Huh7 cells and control Huh7 cells were cultured for sphere formation. (h,i) Xenograft tumor growth (h) and frequency of tumor-initiating cells (i) for C8orf4-overexpressing Huh7 cells were analyzed as c,d. (j) C8orf4 overexpression reduces expression of CD133 and CD13 in Huh7 cells. (k) C8orf4 was transfected in HCC primary cells and cultured for sphere formation. Three HCC patient samples obtained similar results. Scale bars: b,f,g,k, 500 μm. Student’s t-test was used for statistical analysis,    *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data represent at least three independent experiments. oeC8orf4, overexpression of C8orf4; oeVec, overexpression vector.

In addition, C8orf4-deficient Huh7 cells overtly increased xenograft tumour growth (Fig. 2c). We then performed sphere formation and digested oncospheres formed by C8orf4-deficient or WT cells into single-cell suspension, then subcutaneously implanted 1 × 104, 1 × 103, 1 × 102 and 10 cells into BALB/c nude mice. Tumour formation was examined for tumour-initiating capacity at the third month. C8orf4 deficiency remarkably enhanced tumour-initiating capacity and liver CSC ratios (Fig. 2d). In addition, C8orf4 deletion significantly enhanced expression levels of the liver CSC markers such as CD13 and CD133 (Fig. 2e). We also silenced C8orf4 in HCC primary cells using a lentivirus infection system and established C8orf4-silenced cells. Two pairs of short hairpin RNA (shRNA) sequences obtained similar knockdown efficiency. C8orf4 knockdown remarkably promoted sphere formation and xenograft tumour growth (Fig. 2f and Supplementary Fig. 2e). These data indicate that C8orf4 deletion potentiates the self-renewal of liver CSCs.
We next overexpressed C8orf4 in Huh7 cells and HCC primary cells using lentivirus infection. We observed that C8orf4 overexpression in Huh7 cells remarkably reduced sphere formation and xenograft tumour growth (Fig. 2g,h). In addition, C8orf4 overexpression remarkably reduced tumour-initiating capacity and expression of liver CSC markers (Fig. 2i,j). Similar results were observed by C8orf4 overexpression in HCC primary cells (Fig. 2k). We tested three HCC samples with similar results. Overall, C8orf4 negatively regulates the maintenance of liver CSC self-renewal and tumour propagation.

C8orf4 suppresses NOTCH2 signaling in liver CSCs

To further determine the underlying mechanism of C8orf4 in the regulation of liver CSCs, we analyzed three major self-renewal signaling pathways, including Wnt/β-catenin, Hedgehog and NOTCH pathways, in C8orf4-deleted Huh7 cells and HCC primary cells. We found that only NOTCH target genes were remarkably upregulated in C8orf4-deficient cells (Fig. 3a), whereasC8orf4 deficiency did not significantly affect the Wnt/β-catenin or the Hedgehog pathway. Given that the NOTCH family receptors have four members, we wanted to determine which NOTCH member was involved in the C8orf4-mediated suppression of liver CSC stemness. We noticed that only NOTCH2 was highly expressed in both Huh7 cells and HCC samples (Fig. 3b). In addition, this result was also confirmed by analysis of NOTCH expression levels derived from Wang’s cohort (GSE14520) and Petel’s cohort (E-TABM-36; ref. 27) (Fig. 3c). Moreover, we analysed expression profiles of C8orf4 and NOTCH target genes using Park’s cohort (GSE36376) and Wurmbach’s cohort (GSE6764; ref. 28). These cohort datasets provided several Notch signaling and its target genes. HEY1, NRARP and HES6 genes were highly expressed in HCC tumour tissues (GSE6764; ref. 28), which were further confirmed in HCC samples by real-time PCR (Supplementary Fig. 3a,b). Furthermore, HEY1, NRARP and HES6 genes have been reported to be relatively specific NOTCH target genes. We then examined these three genes as the NOTCH2 target genes throughout this study. We found that the C8orf4 expression level was negatively correlated with the expression levels of HEY1 and HES6, suggesting that C8orf4 inhibited NOTCH signaling in HCC patients (Fig. 3d). Finally these results were further confirmed in HCC samples by qRT-PCR (Fig. 3e). To further explore the activation status of NOTCH2 signaling in liver CSCs, we examined the expression levels of NOTCH downstream target genes in oncospheres and CD13+CD133+ cells derived from both Huh7 cells and HCC cells. We observed that NOTCH target genes were highly expressed in liver CSCs (Fig. 3f,g). These observations were verified by immunoblotting (Fig. 3h). In addition, the expression levels of NRARP, HES6 and HEY1 were positively related to the expression levels of EpCAM and CD133 derived from Zhang’s cohort (GSE25097; ref. 29) and Wang’s cohort (GSE14520; Supplementary Fig. 3c,d). These data suggest that the NOTCH2 signaling plays a critical role in the maintenance of self-renewal of liver CSCs.

(not shown)

Figure 3: C8orf4 suppresses NOTCH2 signaling in liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f3.jpg

(a) C8orf4 deficiency or depletion activates NOTCH signaling. The indicated major stemness signalling pathways were analysed in C8orf4-knockout Huh7 cells (left panel) and C8orf4-silenced primary cells of HCC samples (right panel). (b) Four receptor members of NOTCH family were examined in both Huh7 cells (left panel) and 29 pairs of HCC samples (right panel). (c) NOTCH receptors were analyzed from Wang’s cohort (left panel) and Petel’s cohort (right panel) datasets. (d) HEY1 and HES6 were highly expressed in C8orf4low samples by analysis of Park’s cohort (upper panel) and Wurmbach’s cohort (lower panel). (e) Expression levels of HEY1 and HES6 along with C8orf4 were analysed in HCC samples by qRT–PCR. (f,g) Expression levels of NRARP, HEY1 and HES6 in spheres generated by Huh7 cells and HCC primary cells (f) and in CD13+CD133+ cells sorted from Huh7 cells and HCC primary cells (g). Non-sphere: Huh7 cells or HCC cells that failed to form spheres. (h) HEY1, HES6 and NRARP expression in sphere and non-sphere cells was detected by immunoblotting. β-actin was used as a loading control. For c,d, data are shown as box and whisker plot. Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For a,b,f,g, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

C8orf4 interacts with NOTCH2 that is critical for liver CSCs

On ligand–receptor binding, the NOTCH receptor experiences a proteolytic cleavage by metalloprotease and γ-secretase, releasing a NOTCH extracellular domain (NECD) and a NOTCH intracellular domain (NICD), respectively30. Then the active NICD undergoes nuclear translocation and activates the expression of NOTCH downstream target genes31.Then we constructed the NOTCH2 extracellular domain (N2ECD) and intracellular domain (N2ICD) and examined the interaction with C8orf4 via a yeast two-hybrid approach. Interestingly, we found that C8orf4 interacted with N2ICD, but not N2ECD (Fig. 4a). The interaction was validated by co-immunoprecipitation (Fig. 4b). Through domain mapping, the ankyrin repeat domain of NOTCH2 was essential and sufficient for its association with C8orf4 (Fig. 4c). Taken together, C8orf4 interacts with the N2ICD domain of NOTCH2.

Figure 4: C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs.

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f4.jpg

(a) C8orf4 interacts with N2ICD. Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD) fused C8orf4 and Gal4-activating domain (AD) fused N2ICD. p53 and large T antigen were used as a positive control. (b) Recombinant Flag-N2ICD and GFP–C8orf4 were incubated for co-immunoprecipitation. (c) The ankyrin repeat AR domain is essential and sufficient for the interaction of C8orf4 with N2ICD. Various N2ICD truncation constructs were co-transfected with GFP–C8orf4 for domain mapping. NLS: nuclear location signal. (d) NOTCH2 was knocked down in Huh7 cells and detected by qRT–PCR and immunoblotting. (e) NOTCH2-silenced Huh7 cells were cultured for sphere formation assays. Two pairs of shRNAs against NOTCH2 obtained similar results. (f,g) Xenograft tumor growth (f) and frequency of tumor-initiating cells (g) for NOTCH2-silenced Huh7 cells were analyzed. (h) NOTCH2 was silenced in HCC primary cells and NOTCH2 depletion declined sphere formation activity. Three HCC specimens obtained similar results. (i) Sphere formation capacity was examined in differently treated HCC primary cells. (j) HCC primary cells were treated with indicated lentivirus and implanted into BALB/c nude mice for xenograft tumor growth assays. Scale bars: e,h,i, 500 μm, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation; NS, not significant.

To further verify the role of NOTCH2 in the maintenance of liver CSC self-renewal, we knocked down NOTCH2 in Huh7 cells and established stably depleted cell lines by two pairs of NOTCH2 shRNAs (Fig. 4d). NOTCH2 knockdown dramatically reduced sphere formation (Fig. 4e), as well as attenuated xenograft tumor growth and tumor-initiating capacity (Fig. 4f,g). Similar observations were achieved in NOTCH2-depleted HCC primary cells (Fig. 4h). In addition, we found that simultaneous knockdown of NOTCH2 and overexpression of C8orf4 failed to reduce sphere formation capacity compared with individual knockdown of NOTCH2 (Fig. 4i), suggesting that NOTCH2 and C8orf4 affected sphere formation through the same pathway. Meanwhile, C8orf4 knockdown failed to rescue the sphere formation ability of NOTCH2-depleted HCC primary cells (Fig. 4i). Similar observations were obtained in Huh7 cells (Supplementary Fig. 4). Finally, NOTCH2 depletion in C8orf4-silenced Huh7 cells or HCC primary cells also abrogated the C8orf4 depletion-mediated enhancement of xenograft tumor growth (Fig. 4j), suggesting that C8orf4 acted as upstream of NOTCH2 signaling. These data suggest that C8orf4 suppresses the liver CSC stemness through inhibiting the NOTCH2 signaling pathway.

C8orf4 blocks nuclear translocation of N2ICD

As shown in Fig. 1g, C8orf4 was mainly localized in the cytoplasm in tumor cells of HCC samples. To confirm these observations, we stained C8orf4 in several HCC cell lines and noticed that C8orf4 also resided in the cytoplasm of Huh7 cells and Hep3B cells (Fig. 5a and Supplementary Fig. 5a). These results were further validated by cellular fractionation (Fig. 5b). Importantly, C8orf4 KO led to nuclear translocation of N2ICD (Fig. 5c). In addition, we also examined the intracellular location of N2ICD in Huh7 spheres. We found that C8orf4 deletion caused complete nuclear translocation of N2ICD in oncosphere cells (Fig. 5d,e), while N2ICD was mainly located in the cytoplasm of WT oncosphere cells. However, we found that C8orf4 KO did not affect subcellular localization of β-catenin (Supplementary Fig. 5b,c). Through luciferase assays, C8orf4 transfection did not significantly influence promoter transcription activity of Wnt target genes such as TCF1, LEF and SOX4 (Supplementary Fig. 5d). These data indicate that C8orf4 resides in the cytoplasm of HCC cells and inhibits nuclear translocation of N2ICD.

C8orf4 deletion causes the nuclear translocation of N2ICD

C8orf4 deletion causes the nuclear translocation of N2ICD

Figure 5: C8orf4 deletion causes the nuclear translocation of N2ICD.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f5.jpg

(a) C8orf4 resides in the cytoplasm of Huh7 cells. Huh7 cells were permeabilized and stained with anti-C8orf4 antibody, then counterstained with PI for confocal microscopy. (b) Cellular fractionation was performed and detected by immunoblotting. (c,d) C8orf4 knockout causes the nuclear translocation of N2ICD. C8orf4-deficient Huh7 cells (c) and sphere cells (d) were permeabilized and stained with anti-C8orf4 and anti-N2ICD antibodies, then counterstained with DAPI followed by confocal microscopy. (e) Cellular fractionation was performed in C8orf4-deficient sphere and WT sphere cells followed by immunoblotting. (f) C8orf4-deficient Huh7 cells were implanted into BALB/c nude mice. Xenograft tumors were analyzed by immunohistochemical staining. Red arrowheads denote nuclear translocation of N2ICD. (g) C8orf4-overexpressing Huh7 cells were permeabilized for immunofluorescence staining. (h) Cellular fractionation was performed in C8orf4-overexpressing Huh7 cells for immunoblotting. (i,j) C8orf4 was overexpressed in N2ICD-overexpressing Huh7 cells followed by immunofluorescence staining (i) and immunoblotting (j). (k) NOTCH target genes were measured in cells treated as in i by real-time PCR. Scale bars: a,c,d,g,i, 10 μm; f, 40 μm. Student’s t-test was used for statistical analysis, **P<0.01;***P<0.001, data are shown as mean±s.d.. Data represent at least three independent experiments.

To further determine whether C8orf4 inhibits the NOTCH2 signaling in the propagation of xenograft tumors, we examined the distribution of N2ICD and NOTCH2 target gene activation inC8orf4-deficient xenograft tumor tissues. We found that C8orf4-deficient tumors displayed much more nuclear translocation of N2ICD compared with WT tumors (Fig. 5f). Expectedly, C8orf4-deficient tumors showed elevated expression levels of NOTCH2 target genes such as HEY1, HES6 and NRARP (Supplementary Fig. 5e). Furthermore, C8orf4 overexpression blocked the nuclear translocation of N2ICD (Fig. 5g,h). Consequently, C8orf4-overexpressing tumors showed much less N2ICD nuclear translocation and reduced expression levels of NOTCH2 target genes compared with control tumors (Supplementary Fig. 5f,g). Of note, C8orf4 overexpression in N2ICD-overexpressing Huh7 cells still blocked nuclear translocation of N2ICD (Fig. 5i,j). Consequently, C8orf4 overexpression abolished the activation of Notch2 signaling (Fig. 5k). These results suggest that C8orf4 deletion causes the nuclear translocation of N2ICD leading to activation of NOTCH2 signaling.

NOTCH2 signalling is required for the stemness of liver CSCs

To further verify the role of NRARP and HEY1 in the maintenance of liver CSC self-renewal, we knocked down these two genes in Huh7 cells and established stably depleted cell lines by two pairs of shRNAs. As expected, NRARP knockdown dramatically reduced sphere formation (Fig. 6a,b). NRARP knockdown also attenuated tumor-initiating capacity and liver CSC ratios (Fig. 6c). Similar results were achieved in NRARP-silenced HCC primary cells (Fig. 6d,e). Similarly, HEY1 silencing remarkably reduced sphere formation derived from Huh7 and HCC primary cells (Fig. 6f–i), as well as declined xenograft tumor growth and tumor-initiating capacity (Supplementary Fig. 6a,b). In sum, NOTCH2 signaling is required for the maintenance of liver CSC self-renewal.

(not shown)

Figure 6: Depletion of NRARP and HEY1 impairs stemness of liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f6.jpg

(a,b) NRARP-silenced Huh7 cells were established (a) and showed reduced sphere formation capacity (b). Two pairs of shRNAs against NRARP obtained similar results. (c) NRARP-silenced Huh7 cells decline tumour-initiating capacity (left panel) and reduce liver CSC frequency (right panel). Error bars represent the 95% confidence intervals of the estimation. (d,e) NRARP was knocked down in HCC primary cells (d) and sphere formation was detected (e). Three HCC samples were tested with similar results. (f,g) HEY1-silenced Huh7 cells were established (f) and sphere formation was assayed (g). Two pairs of shRNAs against HEY1 obtained similar results. (h,i) HEY1 was knocked down in HCC primary cells (h) and HEY1 depletion impaired sphere formation capacity (i). Three HCC samples were tested with similar results. Scale bars: b,e,g,i, 500 μm. For a,b,d–i, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01;  ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

NOTCH2 signaling is correlated with HCC severity

As shown above, the NOTCH2 signaling was highly activated in liver CSCs and involved in the regulation of liver CSC stemness. We further examined the relationship of NOTCH2 signaling with the progression of HCC. First, we analyzed NOTCH2 activation levels in HCC tumor tissues and peri-tumor tissues derived from Park’s cohort (GSE36376). We observed that HEY1, HES6 and NRARP were highly expressed in the tumor tissues of HCC patients (Fig. 7a). Consistently, high expression levels of HEY1, HES6 and NRARP in HCC tumors were validated by Zhang’s cohort (GSE25097) (Fig. 7b). Importantly, high expression of these three genes was confirmed in HCC samples through quantitative RT–PCR (Fig. 7c), as well as immunoblotting (Fig. 7d). To confirm a causative link between low C8orf4 expression level and nuclear N2ICD, we examined 93 HCC samples (31 peri-tumor, 37 early stage of HCC patients and 25 advanced stage of HCC patients) with immunohistochemistry staining. We observed that nuclear staining of N2ICD appeared in ~10% tumor cells in the majority of early HCC patients we tested (Fig. 7e,f). In advanced HCC patients, nuclear staining of N2ICD in tumor cells increased to ~30% in almost all the advanced HCC patients we examined. Consequently, HEY1 staining existed in ~10% tumor cells with scattered distribution and increased to 30% tumor cells in the advanced HCC patients (Fig. 7e). Consistently, low expression of C8orf4 was well correlated with activation of NOTCH2 signaling (Fig. 7e,f).

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

Figure 7: NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f7.jpg

(a,b) NOTCH target genes were highly expressed in HCC tumour tissues derived from Park’s cohort (a) and Zhang’s cohort (b). (c) High expression levels of NOTCH target genes in HCC tumor tissues were verified by qRT–PCR. (d) HEY1 expression in HCC tumor tissues was detected by western blot. (e) IHC staining for N2ICD, C8orf4 and HEY1. These images represent 93 HCC samples. Scale bars, 50 μm. (f) IHC images were calculated using Image-Pro Plus 6. (g) Expression levels of NOTCH target genes were elevated in HCC tumors and advanced HCC patients derived from Wang’s cohort. (h) HEY1 expression level was positively correlated with prognosis prediction of HCC patients analyzed by Petel’s cohort and Wang’s cohort. HCC samples were divided into two groups according to HEY1 expression levels followed by Kaplan–Meier survival analysis. For a–c, data are shown as box and whisker plot, Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For f,g, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001; data are shown as mean ± standard deviation. Experiments were repeated at least three times. aHCC, advanced HCC; CL, cirrhosis liver; eHCC, early HCC; IL, inflammatory liver; NL, normal liver; NS, not significant.

Serial passages of colonies or sphere formation in vitro, as well as transplantation of tumor cells, are frequently used to assess the long-term self-renewal capacities of CSCs32. We used HCC primary cells for serial passage growth in vitro and tested the expression levels of C8orf4, HEY1 and SOX9. We found that C8orf4 expression was gradually reduced over serial passages in oncosphere cells (Supplementary Fig. 7a). Consequently, the expression of NOTCH2 targets such as HEY1 and SOX9 was gradually increased in oncosphere cells during serial passages (Supplementary Fig. 7b). In addition, N2ICD nuclear translocation appeared in oncosphere cells with high expression of HEY1 plus low expression of C8orf4 (termed as C8orf4−/N2ICDnuc/HEY1+cells) (Supplementary Fig. 7c). These data suggest that the C8orf4−/N2ICDnuc/HEY1+ fraction cells represent a subset of liver CSCs.

Through analyzing Wang’s cohort (GSE54238), we noticed that the NOTCH2 activation levels were positively correlated with the development and progression of HCC (Fig. 7g). By contrast, the NOTCH2 pathway was not activated in inflammation liver, cirrhosis liver and normal liver (Fig. 7f). Consistently, similar observations were achieved by analysis of Zhang’s cohort (GSE25097) (Supplementary Fig. 7d). In addition, the NOTCH2 activation levels were consistent with clinicopathological stages of HCC patients derived from Wang’s cohort (GSE14520) (Supplementary Fig. 7e). Finally, HCC patients with higher expression of HEY1 displayed worse prognosis derived from Petel’s cohort (E-TABM-36) and Wang’s cohort (GSE14520) (Fig. 7h). These two cohorts (E-TABM-36 and GSE14520) have survival information of HCC patients. Taken together, the NOTCH2 activation levels in tumor tissues are consistent with clinical severity and prognosis of HCC patients.

Discussion

CSC have been identified in many solid tumors, including breast, lung, brain, liver, colon, prostate and bladder cancers4, 6, 33. CSCs have similar characteristics associated with normal tissue stem cells, including self-renewal, differentiation and the ability to form new tumors. CSCs may be responsible for cancer relapse and metastasis due to their invasive and drug-resistant capacities34. Thus, targeting CSCs may become a promising therapeutic strategy to deadly malignancies35, 36. However, it remains largely unknown about hepatic CSC biology. In this study, we used CD13 and CD133 to enrich CD13+CD133+
subpopulation cells as liver CSCs. Based on analysis of several online-available HCC transcriptome datasets, we found that C8orf4 is weakly expressed in HCC tumors as well as in CD13+CD133+ liver CSCs. NOTCH2 signaling is required for the maintenance of liver CSC self-renewal. C8orf4 resides in the cytoplasm of tumor cells and interacts with N2ICD, blocking the nuclear translocation of N2ICD. Lower expression of C8orf4 causes nuclear translocation of N2ICD that activates NOTCH2 signaling in liver CSCs. NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Therefore, C8orf4 negatively regulates self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Elucidating signaling pathways that maintains self-renewal of liver CSCs is pivotal for the understanding of hepatic CSC biology and the development of novel therapies against HCC. Several signaling pathways, such as Wnt/β-catenin, transforming growth factor-beta, AKT and STAT3 pathways, have been defined to be implicated in the regulation of liver CSCs37. Not surprisingly, some liver CSC subsets and normal tissue stem cells may share core regulatory genes and common signaling pathways. The NOTCH signaling pathway plays an important role in development via cell-fate determination, proliferation and cell survival38, 39. The NOTCH family receptors contain four members in mammals (NOTCH1–4), which are activated by binding to their corresponding ligands. A large body of evidence provides that NOTCH signaling is implicated in carcinogenesis40. However, the role of NOTCH signaling in liver cancer is controversial. A previous study reported that NOTCH1 signaling suppresses tumor growth of HCC41. Recently, several reports showed that NOTCH signaling enhances liver tumor initiation42, 43, 44. Importantly, a recent study showed that various NOTCH receptors have differential functions in the development of liver cancer45. Here we demonstrate that NOTCH2 signaling is activated in HCC tumor tissues and liver CSCs, which is required for the maintenance of liver CSC self-renewal.

C8orf4, also known as TC1, was originally cloned from a papillary thyroid cancer16, 46. The copy number variations of C8orf4 are associated with acute myeloid leukemia and other hematological malignancies19, 47. C8orf4 has been reported to be implicated in various cancers. C8orf4 was highly expressed in thyroid cancer, gastric cancer and breast cancer16, 20, 46. C8orf4 has been reported to enhance Wnt/β-catenin signaling in cancer cells that is associated with poor prognosis20, 21. However, C8orf4 is downregulated in colon cancer48. In this study, we show that C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs. Our observations were confirmed by two HCC cohort datasets. Importantly, C8orf4 negatively regulates the NOTCH2 signaling to suppress the self-renewal of liver CSCs. Therefore, C8orf4 may exert distinct functions in the regulation of various malignancies.

NOTCH receptors consist of noncovalently bound extracellular and transmembrane domains. Once binding with membrane-bound Delta or Jagged ligands, the NOTCH receptors undergoes a proteolytic step by metalloprotease and γ-secretase, generating NECD and NICD fragments11, 31. The NICD, a soluble fragment, is released in the cytoplasm on proteolysis. Then the NICD translocates to the nucleus and binds to the transcription initiation complex, leading to activation of NOTCH-associated target genes49. However, it is largely unclear how the NICD is regulated during NOTCH signaling activation. Here we show that N2ICD binds to C8orf4 in the cytoplasm of liver non-CSC tumor cells, which impedes the nuclear translocation of N2ICD. By contrast, in liver CSCs, lower expression of C8orf4 causes the nuclear translocation of N2ICD, leading to activation of NOTCH signaling.

CSCs or tumour-initiating cells, behave like tissue stem cells in that they are capable of self-renewal and of giving rise to hierarchical organization of heterogeneous cancer cells4. Thus, CSCs harbour the stem cell properties of self-renewal and differentiation. Actually, the CSC model cannot account for tumorigenesis in all tumours. CSCs could undergo genetic evolution, and the non-CSCs might switch to CSC-like cells4. These results highlight the dynamic nature of CSCs, suggesting that the clonal evolution and CSC models can act in concert for tumorigenesis. Furthermore, low C8orf4 expression in tumor cells results in overall Notch2 activation, which then may have more of a progenitor signature and be more aggressive. These cells would likely have a growth advantage in non-adherent conditions and express many of the stemness markers. The dynamic nature of CSCs or persistent NOTCH2 activation may contribute to the high number of C8orf4−/N2ICDnuc/HEY1+ cells in advanced HCC tumors and correlation in the patient cohort.

A recent study showed that NOTCH2 and its ligand Jag1 are highly expressed in human HCC tumors, suggesting activation of NOTCH2 signaling in HCC45. In addition, inhibiting NOTCH2 or Jag1 dramatically reduces tumor burden and growth. However, suppression of NOTCH3 has no effect on tumor growth. Dill et al.43 reported that Notch2 is an oncogene in HCC. Notch2-driven HCC are poorly differentiated with a high expression level of the progenitor marker Sox9, indicating a critical role of Notch2 signaling in liver CSCs. Here we found that NOTCH2 and its target genes such as NRARP, HEY1 and HES6 are highly expressed in HCC samples. In addition, depletion of NRARP and HEY1 impairs the stemness maintenance of liver CSCs and tumor propagation. Moreover, the expression levels of NRARP, HEY1 and HES6 in tumors are positively correlated with clinical severity and prognosis of HCC patients. Finally, the NOTCH2 activation status is positively related to the clinicopathological stages of HCC patients. Altogether, C8orf4 and NOTCH2 signaling can be detected for the diagnosis and prognosis prediction of HCC patients, as well as used as targets for eradicating liver CSCs for future therapy.

11.2.3.2 Quantifying the Landscape for Development and Cancer from a Core Cancer Stem Cell Circuit

The authors developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. The landscape exhibits four basins of attraction, representing cancer stem cell, stem cell, cancer and normal cell states. They also uncovered certain key genes and regulations responsible for determining the switching between different states. [Cancer Res]

Chunhe Li and Jin Wang
Cancer Res May 13, 2015; 75(10).
http://dx.doi.org:/10.1158/0008-5472.CAN-15-0079

Cancer presents a serious threat to human health. The understanding of the cell fate determination during development and tumor genesis remains challenging in current cancer biology. It was suggested that cancer stem cell (CSC) may arise from normal stem cells, or be transformed from normal differentiated cells. This gives hints on the connection between cancer and development. However, the molecular mechanisms of these cell type transitions and the CSC formation remain elusive. We quantified landscape, dominant paths and switching rates between cell types from a core gene regulatory network for cancer and development. Stem cell, CSC, cancer, and normal cell types emerge as basins of attraction on associated landscape. The dominant paths quantify the transition processes among CSC, stem cell, normal cell and cancer cell attractors. Transition actions of the dominant paths are shown to be closely related to switching rates between cell types, but not always to the barriers in between, due to the presence of the curl flux. During the process of P53 gene activation, landscape topography changes gradually from a CSC attractor to a normal cell attractor. This confirms the roles of P53 of preventing the formation of CSC, through suppressing self-renewal and inducing differentiation. By global sensitivity analysis according to landscape topography and action, we identified key regulations determining cell type switchings and suggested testable predictions. From landscape view, the emergence of the CSCs and the associated switching to other cell types are the results of underlying interactions among cancer and developmental marker genes. This indicates that the cancer and development are intimately connected. This landscape and flux theoretical framework provides a quantitative way to understand the underlying mechanisms of CSC formation and interplay between cancer and development. Major Findings: We developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. Landscape exhibits four basins of attraction, representing CSC, stem cell, cancer and normal cell states. We quantified the kinetic rate and paths between different attractor states. We uncovered certain key genes and regulations responsible for determining the switching between different states.

11.2.3.3 IMP3 Promotes Stem-Like Properties in Triple-Negative Breast Cancer by Regulating SLUG

Scientists observed that insulin-like growth factor-2 mRNA binding protein 3 (IMP3) expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and demonstrated that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells. [Oncogene]

S Samanta, H Sun, H L Goel, B Pursell, C Chang, A Khan, et al.
Oncogene , (18 May 2015) |
http://dx.doi.org:/10.1038/onc.2015.164

IMP3 (insulin-like growth factor-2 mRNA binding protein 3) is an oncofetal protein whose expression is prognostic for poor outcome in several cancers. Although IMP3 is expressed preferentially in triple-negative breast cancer (TNBC), its function is poorly understood. We observed that IMP3 expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and we demonstrate that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells (CSCs). The mechanism by which IMP3 contributes to this phenotype involves its ability to induce the stem cell factor SOX2. IMP3 does not interact with SOX2 mRNA significantly or regulate SOX2 expression directly. We discovered that IMP3 binds avidly to SNAI2 (SLUG) mRNA and regulates its expression by binding to the 5′ UTR. This finding is significant because SLUG has been implicated in breast CSCs and TNBC. Moreover, we show that SOX2 is a transcriptional target of SLUG. These data establish a novel mechanism of breast tumor initiation involving IMP3 and they provide a rationale for its association with aggressive disease and poor outcome.

11.2.3.4 Type II Transglutaminase Stimulates Epidermal Cancer Stem Cell Epithelial-Mesenchymal Transition

Researchers investigated the role of type II transglutaminase (TG2) in regulating epithelial mesenchymal transition (EMT) in epidermal cancer stem cells. They showed that TG2 knockdown or treatment with TG2 inhibitor, resulted in a reduced EMT marker expression, and reduced cell migration and invasion. [Oncotarget]

ML Fisher, G Adhikary, W Xu, C Kerr, JW Keillor, RL Ecker
Oncotarget May 08, 2015;

Type II transglutaminase (TG2) is a multifunctional protein that has recently been implicated as having a role in ECS cell survival. In the present study we investigate the role of TG2 in regulating epithelial mesenchymal transition (EMT) in ECS cells. Our studies show that TG2 knockdown or treatment with TG2 inhibitor, results in a reduced EMT marker expression, and reduced cell migration and invasion. TG2 has several activities, but the most prominent are its transamidase and GTP binding activity. Analysis of a series of TG2 mutants reveals that TG2 GTP binding activity, but not the transamidase activity, is required for expression of EMT markers (Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α), and increased ECS cell invasion and migration. This coupled with reduced expression of E-cadherin. Additional studies indicate that NFϰB signaling, which has been implicated as mediating TG2 impact on EMT in breast cancer cells, is not involved in TG2 regulation of EMT in skin cancer. These studies suggest that TG2 is required for maintenance of ECS cell EMT, invasion and migration, and suggests that inhibiting TG2 GTP binding/G-protein related activity may reduce skin cancer tumor survival.

Epidermal squamous cell carcinoma (SCC) is among the most common cancers and the frequency is increasing at a rapid rate [1,2]. SCC is treated by surgical excision, but the rate of recurrence approaches 10% and the recurrent tumors are aggressive and difficult to treat [2]. We propose that human epidermal cancer stem (ECS) cells survive at the site of tumor excision, that these cells give rise to tumor regrowth, and that therapies targeted to kill ECS cells constitute a viable anti-cancer strategy. An important goal in this context is identifying and inhibiting activity of key proteins that are essential for ECS cell survival. Working towards this goal, we have developed systems for propagation of human ECS cells [3]. These cells display properties of cancer stem cells including self-renew and high level expression of stem cell marker proteins [3].

In the present study we demonstrate that ECS cells express proteins characteristic of cells undergoing EMT (epithelial-mesenchymal transition). EMT is a morphogenetic process whereby epithelial cells lose epithelial properties and assume mesenchymal characteristics [4]. The epithelial cells lose cell-cell contact and polarity, and assume a mesenchymal migratory phenotype. There are three types of EMT. This first is an embryonic process, during gastrulation, when the epithelial sheet gives rise to the mesoderm [5]. The second is a growth factor and cytokine-stimulated EMT that occurs at sites of tissue injury to facilitate wound repair [6]. The third is associated with epithelial cancer cell acquisition of a mesenchymal migratory/invasive phenotype. This process mimics normal EMT, but is not as well controlled and coordinated [4, 7, 8]. A number of transcription factors (ZEB1, ZEB2, snail, slug, and twist) that are expressed during EMT suppress expression of epithelial makers, including E-cadherin, desmoplakin and claudins [4]. Snail proteins also activate expression of vimentin, fibronectin and metalloproteinases [4]. Snail factors are not present in normal epithelial cells, but are present in the tumor cells and are prognostic factors for poor survival [4].

An important goal is identifying factors that provide overarching control of EMT in cancer stem cells. In this context, several recent papers implicate type II transglutaminase (TG2) as a regulator of EMT [9–12]. TG2, the best studied transglutaminase, was isolated in 1957 from guinea pig liver extract as an enzyme involved in the covalent crosslinks proteins via formation of isopeptide bonds [13]. However, subsequent studies reveal that TG2 also serves as a scaffolding protein, regulates cell adhesion, and modulates signal transduction as a GTP binding protein that participates in G protein signaling [14]. TG2 is markedly overexpressed in cancer cells, is involved in cancer development [15–18], and has been implicated in maintaining and enhancing EMT in breast and ovarian cancer [10, 12, 19, 20]. The G protein function may have an important role in these processes [10, 21–23].

In the present manuscript we study the role of TG2 in regulating EMT in human ECS cells. Our studies show that TG2 is highly enriched in ECS cells. We further show that these cells express EMT markers and that TG2 is required to maintain EMT protein expression. TG2 knockdown, or treatment with TG2 inhibitor, reduces EMT marker expression and ECS cell survival, invasion and migration. TG2 GTP binding activity is absolutely required for maintenance of EMT protein expression and EMT-related responses. However, in contrast to breast cancer [9, 10], we show that TG2 regulation of EMT is not mediated via NFκB signaling.

TG2 is required for expression of EMT markers

EMT is a property of tumor stem cells that confers an ability to migrate and invade surrounding tissue [24–26]. We first examined whether ECS cells express EMT markers. Non-stem cancer cells and ECS cells, derived from the SCC-13 cancer cell line, were analyzed for expression of EMT markers. Fig. 1A shows that a host of EMT transcriptional regulators, including Twist, Snail and Slug, are increased in ECS cells (spheroid) as compared to non-stem cancer cells (monolayer). This is associated with increased levels of vimentin, fibronectin and N-cadherin, which are mesenchymal proteins, and reduced expression of E-cadherin, an epithelial marker. HIF-1α, an additional marker frequently associated with EMT, is also elevated. We next examined whether TG2 is required to maintain EMT marker expression. SCC-13 cell-derived ECS cells were grown in the presence of control- or TG2-siRNA, to reduce TG2, and the impact on EMT marker level was measured. Fig. 1B shows that loss of TG2 is associated with reduced expression of Twist, Snail, vimentin and HIF-1α. To further assess the role of TG2, we utilized SCC13-Control-shRNA and SCC13-TG2-shRNA2 cell lines. These lines were produced by infection of SCC-13 cells with lentiviruses encoding control- or TG2-specific shRNA. Fig. 1C shows that SCC13-TG2-shRNA2 cells express markedly reduced levels of TG2 and that this is associated with reduced expression of EMT associated transcription factors and target proteins, and increased expression of E-cadherin. To confirm this, we grew SCC13-Control-shRNA and SCC13-TG2-shRNA2 cells as monolayer cultures for immunostain detection of EMT markers. As shown in Fig. 2A, TG2 levels are reduced in TG2-shRNA expressing cells, and this is associated with the anticipated changes in epithelial and mesenchymal marker expression.

Tumor cells that express EMT markers display enhanced migration and invasion ability [24–26]. We therefore examined the impact of TG2 reduction on these responses. To measure invasion, control-shRNA and TG2-shRNA cells were monitored for ability to move through matrigel. Fig. 2B shows that loss of TG2 reduces movement through matrigel by 50%. We further show that this is associated with a reduction in cell migration using a monolayer culture wound closure assay. The control cells close the wound completely within 14 h, while TG2 knockdown reduces closure rate (Fig. 2C).

TG2 inhibitor reduces EMT marker expression and EMT functional responses

NC9 is a recently developed TG2-specific inhibitor [27, 28]. We therefore asked whether pharmacologic inhibition of TG2 suppresses EMT. SCC-13 cells were treated with 0 or 20 μM NC9. Fig. 3A shows that NC9 treatment reduces EMT transcription factor (Twist, Snail, Slug) and EMT marker (vimentin, fibronectin, N-cadherin, HIF-1α) levels. Consistent with these changes, the level of the epithelial marker, E-cadherin, is elevated. Fig. 3B and 3C show that pharmacologic inhibition of TG2 activity also reduces EMT biological response. Invasion (Fig. 3B) and cell migration (Fig. 3C) are also reduced.

Identification of TG2 functional domain required for EMT

We next performed studies to identify the functional domains and activities required for TG2 regulation of EMT. TG2 is a multifunctional enzyme that serves as a scaffolding protein, as a transamidase, as a kinase, and as a GTP binding protein [21]. The two best studied functions are the transamidase and GTP binding/G-protein related activities [21]. Transamidase activity is observed in the presence of elevated intracellular calcium, while GTP binding-related signaling is favored by low calcium conditions (reviewed in [21]). To identify the TG2 activity required for EMT, we measured the ability of wild-type and mutant TG2 to restore EMT in SCC13-TG2-shRNA2 cells, which have reduced TG2 expression (Fig. 4A). SCC13-TG2-shRNA2 cells display reduced expression of EMT markers including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α, and increased expression of the epithelial maker, E-cadherin, as compared to SCC13-Control-shRNA cells. Expression of wild-type TG2, or the TG2-C277S or TG2-W241A mutants, restores marker expression in SCC13-TG2-shRNA2 cells (Fig. 4A). TG2-C277S and TG2-W241A lack transamidase activity [10,29–31]. In contrast, TG2-R580A, which lacks G-protein activity [29–31], and TG2-Y516F, which retains only partial G-protein activity [30], do not efficiently restore marker expression. These findings suggest that the TG2 GTP binding function is required for EMT.

We next assayed the ability of the TG2 mutants to restore EMT functional responses-invasion and migration. Fig. 4B, 4C shows that wild-type TG2, TG2-C277S and TG2-W241A restore the ability of SCC13-TG2-shRNA2 cells to invade matrigel, but TG2-R580A and Y516F are less active. Fig. 4D shows a similar finding for cell migration, in that the TG2-R580A and Y517F mutant are only partially able to restore SCC13-TG2-shRNA2 cell migration. These findings suggest that TG2 GTP binding/G-protein related activity is required for EMT-related migration and invasion by skin cancer cells.

Role of TG2 in regulating EMT in A431 cells

The number of available epidermis-derived squamous cell carcinoma cell lines is limited, and so we compared our findings with A431 cells. A431 cells are squamous cell carcinoma cells established from human vulvar skin. A431 cells were grown as monolayer (non-stem cancer cells) and spheroids (ECS cells) and after 10 d the cells were harvested and assayed for expression of TG2 and EMT makers. Fig. 5A shows that TG2 levels are elevated in ECS cells and that this is associated with increased levels of mesenchymal markers, including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α. In contrast, E-cadherin levels are reduced. We next examined the impact of TG2 knockdown on EMT marker expression. Fig. 5B shows that mesenchymal markers are globally reduced and E-cadherin level is increased. As a biological endpoint of EMT, we examine the impact of TG2 knockdown on spheroid formation and found that TG2 loss leads to reduced spheroid formation (Fig. 5C). We next examined the impact of NC9 treatment on EMT and found a reduction in EMT markers expression associated with an increase in epithelial (E-cadherin) marker level (Fig. 5D). This loss of EMT marker expression is associated with reduced matrigel invasion (Fig. 5E), reduced spheroid formation (Fig. 5F) and reduced cell migration (Fig. 5G).

Role of NFκB

Previous studies in breast [18, 32–36], ovarian cancer [12, 37, 38], and epidermoid carcinoma [11] indicate that NFκB signaling mediates TG2 impact on EMT. We therefore assessed the role of NFκB in skin cancer cells. As shown in Fig. 6A, the increase in TG2 level observed in ECS cells (spheroids) is associated with reduced NFκB level. In addition, NFκB level is increased in TG2 knockdown cells (Fig. 6B). Thus, increased NFκB is not associated with increased TG2. We next assessed the impact of NFκB knockdown on TG2 control of EMT marker expression. Fig. 6C shows that TG2 is required for increased expression of EMT markers (HIF-1α, snail, twist, N-cadherin, vimentin and fibronectin) and reduced expression of the E-cadherin epithelial marker; however, knockdown of NFκB expression does not interfere with TG2 regulation of these endpoints. We next examined the effect of TG2 knockdown on NFκB and IκBα localization. The fluorescence images in Fig. 6D suggest that TG2 knockdown with TG2-siRNA does not alter the intracellular localization of NFκB or IκBα. This is confirmed by subcellular fractionation assay (Fig. 6E) which compares NFκB level in SCC13-TG2-Control and SCC13-TG2-shRNA2 (TG2 knockdown) cells. We also monitored NFκB subcellular distribution following treatment with NC9, the TG2 inhibitor. Fig. 6F shows that cytoplasmic/nuclear distribution of NFκB is not altered by NC9. Finally, we monitored the impact of TG2 expression on NFκB binding to a canonical NFκB-response element. Increased NFκB binding to the response element is a direct measure of NFκB activity [10]. Fig. 6G shows that overall binding is reduced in nuclear (N) extract prepared from ECS cells (spheroids) as compared to non-stem cancer cells (monolayer), and that NFkB binding, as indicated by gel supershift assay, is also slightly reduced in ECS cell extracts. These findings indicate that NFkB binding is slightly reduced in ECS cells, which are TG2-enriched (Fig. 1A).

We next monitored the role of NFκB on biological endpoints of EMT. Fig. 7A and 7B show that TG2 knockdown reduces migration through matrigel, but NFκB knockdown has no impact. Likewise, TG2 knockdown reduces wound closure, but NFκB knockdown does not. These findings suggest that NFκB does not mediate the pro-EMT actions of TG2 in epidermal squamous cell carcinoma.

The metastatic cascade, from primary tumor to metastasis, is a complex process involving multiple pathways and signaling cascades [39–41]. Cells that complete the metastatic cascade migrate away from the primary tumor through the blood to a distant site and there form a secondary tumor. Identifying the mechanisms that allow cells to survive this journey and form secondary tumors is an important goal. The processes involved in epithelial-mesenchymal transition (EMT) are important cancer therapy targets, as EMT is associated with enhanced cancer cell migration and stem cell self-renewal. EMT regulators, including Snail, Twist, Slug, are increased in expression in EMT and control expression of genes associated with the EMT phenotype [42].

TG2 is required for EMT

We have characterized a population of ECS cells derived from epidermal squamous cell carcinoma [3]. The present studies show that these cells, which display enhanced migration and invasion, possess elevated levels of TG2. Moreover, these cells are enriched in expression of transcription factors associated with EMT (Snail, Slug, and Twist, HIF-1α) as well as mesenchymal structural proteins including vimentin, fibronectin and N-cadherin. Consistent with a shift to mesenchymal phenotype, E-cadherin, an epithelial marker, is reduced in level. Additional studies show that TG2 knockdown results in a marked reduction in EMT marker expression and that this is associated with reduced ability of the cells to migrate to close a scratch wound and reduced movement in matrigel invasion assays. We also examined the impact of treatment with a TG2 inhibitor. NC9 is an irreversible active site inhibitor of TG2, that locks the enzyme in an open conformation [28, 43–45]. NC9 treatment of ECS cells results in decreased levels of Snail, Slug and Twist. These transcription factors suppress E-cadherin expression [46] and their decline in level is associated with increased levels of E-cadherin. NC9 inhibition of TG2 also reduces expression of vimentin, fibronectin and N-cadherin, and these changes are associated with reduced cell migration and reduced invasion through matrigel.

(Figures are not shown)

We also examined the role of TG2 in A431 squamous cell carcinoma cells derived from the vulva epithelium. TG2 is elevated in A431-derived ECS cells, as are EMT markers, and knockdown of TG2, with TG2-siRNA, reduces EMT marker expression and spheroid formation. Studies with NC9 indicate that NC9 inhibits A431 spheroid formation, EMT, migration and invasion. These studies indicate that TG2 is also required for EMT and migration and invasion in A431 cells. Based on these findings we conclude that TG2 is essential for EMT, migration and invasion, and is likely to contribute to metastasis in squamous cell carcinoma.

TG2 GTP binding activity is required for EMT

TG2 is a multifunctional enzyme that can act as a transamidase, GTP binding protein, protein disulfide isomerase, protein kinase, protein scaffold, and DNA hydrolase [21, 29, 44, 47]. The two most studied functions are the transamidase and GTP binding functions [29, 44, 47]. To identify the TG2 activity responsible for induction of EMT, we studied the ability of TG2 mutants to restore EMT in SCC13-TG2-shRNA2 cells, which express low levels of TG2 and do not express elevated levels of EMT markers or display EMT-related biological responses. These studies show that wild-type TG2 restores EMT marker expression and the ability of the cells to migrate on plastic and invade matrigel. TG2 mutants that retain GTP binding activity (TG2-C277S and TG2-W241A) also restore EMT. In contrast, TG2-R580A, which lacks GTP binding function, does not restore EMT. This evidence suggests that the GTP binding function is essential for TG2 induction of the EMT phenotype in ECS cells. Recent reports suggest that the TG2 is important for maintenance of stem cell survival in breast [9, 10, 17] and ovarian [12, 38, 48] cancer cells. Moreover, our findings are in agreement of those of Mehta and colleagues who reported that the TG2 GTP binding function, but not the crosslinking function, is required for TG2 induction of EMT in breast cancer cells [10].

TG2, NFκB signaling and EMT

To gain further insight into the mechanism of TG2 mediated EMT, we examined the role of NFκB. NFκB has been implicated as mediating EMT in breast, ovarian, and pancreatic cancer; however, NFκB may have a unique role in epidermal squamous cell carcinoma. In keratinocytes, NFκB has been implicated in keratinocyte dysplasia and hyperproliferation [49]. However, inhibition of NFκB function has also been shown to predispose murine epidermis to cancer [50]. Here we show that TG2 levels are elevated and NFκB levels are reduced in ECS cells as compared to non-stem cancer cells, and that TG2 knockdown is associated with increased NFκB level. In addition, TG2 knockdown, or inhibition of TG2 by treatment with NC9, does not altered the nuclear/cytoplasmic distribution of NFκB. We further show that elevated levels of TG2 in spheroid culture results in a slight reduction in NFκB binding to the NFκB response element, as measured by gel mobility supershift assay. These molecular assays strongly suggest that NFκB does not mediate the action of TG2 in epidermal cancer stem cells. Moreover, knockdown of NFκB-p65 in TG2 positive cells does not result in a reduction in Snail, Slug and Twist, or mesenchymal marker proteins expression, and concurrent knockdown of TG2 and NFκB does not reduce EMT marker protein levels beyond that of TG2 knockdown alone. These findings suggest that NFκB is not an intermediary in TG2-stimulated EMT in ECS cells. This is in contrast to the required role of NFκB in mediating TG2 induction of cell survival and EMT in breast cancer cells [18, 32, 33] and ovarian cancer [12, 37, 38] and epidermoid carcinoma [11].

11.2.3.5 CD24+ Ovarian Cancer Cells are Enriched for Cancer Initiating Cells and Dependent on JAK2 Signaling for Growth and Metastasis

Investigators showed that CD24+ and CD133+ cells have increased tumorsphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24– cells, had significantly greater tumor initiation and tumor growth capacity. [Mol Cancer Ther]

D Burgos-Ojeda, R Wu, K McLean, Yu-Chih Chen, M Talpaz, et al.
Molec Cancer Ther May 12, 2015; 14(5)
http://dx.doi.org:/10.1158/1535-7163.MCT-14-0607

Ovarian cancer is known to be composed of distinct populations of cancer cells, some of which demonstrate increased capacity for cancer initiation and/or metastasis. The study of human cancer cell populations is difficult due to long requirements for tumor growth, inter-patient variability and the need for tumor growth in immune-deficient mice. We therefore characterized the cancer initiation capacity of distinct cancer cell populations in a transgenic murine model of ovarian cancer. In this model, conditional deletion of Apc, Pten, and Trp53 in the ovarian surface epithelium (OSE) results in the generation of high grade metastatic ovarian carcinomas. Cell lines derived from these murine tumors express numerous putative stem cell markers including CD24, CD44, CD90, CD117, CD133 and ALDH. We show that CD24+ and CD133+ cells have increased tumor sphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24- cells, had significantly greater tumor initiation and tumor growth capacity. No preferential tumor initiating or growth capacity was observed for CD44+, CD90+, CD117+, or ALDH+ versus their negative counterparts. We have found that CD24+ cells, compared to CD24- cells, have increased phosphorylation of STAT3 and increased expression of STAT3 target Nanog and c-myc. JAK2 inhibition of STAT3 phosphorylation preferentially induced cytotoxicity in CD24+ cells. In vivo JAK2 inhibitor therapy dramatically reduced tumor metastases, and prolonged overall survival. These findings indicate that CD24+ cells play a role in tumor migration and metastasis and support JAK2 as a therapeutic target in ovarian cancer.

11.2.3.6 EpCAM-Antibody-Labeled Noncytotoxic Polymer Vesicles for Cancer Stem Cells-Targeted Delivery of Anticancer Drug and siRNA

Researchers designed and synthesized a novel anti-epithelial cell adhesion molecule (EpCAM)-monoclonal-antibody-labeled cancer stem cells (CSCs)-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA. [Biomacromolecules]

Jing Chen , Qiuming Liu , Jiangang Xiao , and Jianzhong Du
Biomacromolecules May 19, 2015. (just published)
http://dx.doi.org:/10.1021/acs.biomac.5b00551

Cancer stem cells (CSCs) have the capability to initiate tumor, to sustain tumor growth, to maintain the heterogeneity of tumor, and are closely linked to the failure of chemotherapy due to their self-renewal and multilineage differentiation capability with an innate resistance to cytotoxic agents. Herein, we designed and synthesized a novel anti-EpCAM (epithelial cell adhesion molecule)-monoclonal-antibody-labeled CSCs-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA (to overcome CSCs drug resistance by silencing the expression of oncogenes). This vesicle shows high delivery efficacy of both anticancer drug doxorubicin hydrochloride (DOX∙HCl) and siRNA to the CSCs because it is labeled by the monoclonal antibodies to the CSCs-surface-specific marker. Compared to non-CSCs-targeting vesicles, the DOX∙HCl or siRNA loaded CSCs-targeting vesicles exhibited much better CSCs killing and tumor growth inhibition capabilities with lower toxicity to normal cells (IC50,DOX decreased by 80%), demonstrating promising potential applications in nanomedicine.

11.2.3.7 Survival of Skin Cancer Stem Cells Requires the Ezh2 Polycomb Group Protein

Investigators showed that Ezh2 is required for epidermal cancer stem (ECS) cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. They also showed that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduced Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. [Carcinogenesis]

G Adhikary, D Grun, S Balasubramanian, C Kerr, J Huang and RL Eckert
Carcinogenesis (2015)
http://dx.doi.org:/10.1093/carcin/bgv064

Polycomb group (PcG) proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation, and that treatment with Ezh2 inhibitors may be a strategy for reducing epidermal cancer stem cell survival and suppressing tumor formation.

11.2.3.8 Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer

R Thakur, R Trivedi, N Rastogi, M Singh & DP Mishra
Scientific Reports May 14, 2015; 5(10194)
http://dx.doi.org:/10.1038/srep10194

Cancer stem cells (CSCs) are responsible for aggressive tumor growth, metastasis and therapy resistance. In this study, we evaluated the effects of Shikonin (Shk) on breast cancer and found its anti-CSC potential. Shk treatment decreased the expression of various epithelial to mesenchymal transition (EMT) and CSC associated markers. Kinase profiling array and western blot analysis indicated that Shk inhibits STAT3, FAK and Src activation. Inhibition of these signaling proteins using standard inhibitors revealed that STAT3 inhibition affected CSCs properties more significantly than FAK or Src inhibition. We observed a significant decrease in cell migration upon FAK and Src inhibition and decrease in invasion upon inhibition of STAT3, FAK and Src. Combined inhibition of STAT3 with Src or FAK reduced the mammosphere formation, migration and invasion more significantly than the individual inhibitions. These observations indicated that the anti-breast cancer properties of Shk are due to its potential to inhibit multiple signaling proteins. Shk also reduced the activation and expression of STAT3, FAK and Src in vivo and reduced tumorigenicity, growth and metastasis of 4T1 cells. Collectively, this study underscores the translational relevance of using a single inhibitor (Shk) for compromising multiple tumor-associated signaling pathways to check cancer metastasis and stem cell load.

Breast cancer is the most common endocrine cancer and the second leading cause of cancer-related deaths in women. In spite of the diverse therapeutic regimens available for breast cancer treatment, development of chemo-resistance and disease relapse is constantly on the rise. The most common cause of disease relapse and chemo-resistance is attributed to the presence of stem cell like cells (or CSCs) in tumor tissues1, 2. CSCs represent a small population within the tumor mass, capable of inducing independent tumors in vivo and are hard to eradicate2. Multiple signaling pathways including Receptor Tyrosine Kinase (RTKs), Wnt/β-catenin, TGF-β, STAT3, Integrin/FAK, Notch and Hedgehog signaling pathway helps in maintaining the stem cell programs in normal as well as in cancer cells3, 4, 5, 6. These pathways also support the epithelial-mesenchymal transition (EMT) and expression of various drug transporters in cancer cells. Cells undergoing EMT are known to acquire stem cell and chemo-resistant traits7. Thus, the induction of EMT programs, drug resistance and stem cell like properties are interlinked7. Commonly used anti-cancer drugs eradicate most of the tumor cells, but CSCs due to their robust survival mechanisms remain viable and lead to disease relapse8. Studies carried out on patient derived tumor samples and in vivo mouse models have demonstrated that the CSCs metastasize very efficiently than non-CSCs9, 10, 11. Therefore, drugs capable of compromising CSCs proliferation and self-renewal are urgently required as the inhibition of CSC will induce the inhibition of tumor growth, chemo-resistance, metastasis and metastatic colonization in breast cancer.

Shikonin, a natural dietary component is a potent anti-cancer compound12, 13. Previous studies have shown that Shk inhibits the cancer cell growth, migration, invasion and tumorigenic potential12. Shk has good bioavailability, less toxicity and favorable pharmacokinetic and pharmacodynamic profiles in vivo12. In a recent report, it was shown that the prolonged exposure of Shk to cancer cells does not cause chemo-resistance13.Other studies have shown that it inhibits the expression of various key inflammatory cytokines and associated signaling pathways12, 14. It decreases the expression of TNFα, IL12, IL6, IL1β, IL2, IFNγ, inhibits ERK1/2 and JNK signaling and reduces the expression of NFκB and STAT3 transcription factors14, 15. It inhibits proteasome and also modulates the cancer cell metabolism by inhibiting tumor specific pyurvate kinase-M214,15, 16. Skh causes cell cycle arrest and induces necroptosis in various cancer types14. Shk also inhibits the expression of MMP9, integrin β1 and decreases invasive potential of cancer cells14, 17. Collectively, Shk modulates various signaling pathways and elicits anti-cancer responses in a variety of cancer types.

In breast cancer, Shk has been reported to induce the cell death and inhibit cell migration, but the mechanisms responsible for its effect are not well studied18, 19. Signaling pathways modulated by Shk in cancerous and non-cancerous models have previously been shown important for breast cancer growth, metastasis and tumorigenicity20. Therefore in the current study, we investigated the effect of Shk on various hallmark associated properties of breast cancer cells, including migration, invasion, clonogenicity, cancer stem cell load and in vivo tumor growth and metastasis.

Shk inhibits cancer hallmarks in breast cancer cell lines and primary cells

We first examined the effect of Shk on various cancer hallmark capabilities (proliferation, invasion, migration, colony and mammosphere forming potential) in breast cancer cells. MTT assay was used to find out effect of Shk on viability of breast cancer cells. Semi-confluent cultures were exposed to various concentrations of Shk for 24 h. Shk showed specific anti-breast cancer activity with IC50 values ranging from 1.38 μM to 8.3 μM in MDA-MB 231, MDA-MB 468, BT-20, MCF7, T47D, SK-BR-3 and 4T1 cells (Fig. 1A). Whereas the IC50 values in non-cancerous HEK-293 and human PBMCs were significantly higher indicating that it is relatively safe for normal cells (Fig. S1A). Shk was found to induce necroptotic cell death consistent with previous reports (Fig. S1B). Treatment of breast cancer cells for 24 h with 1.25 μM, 2.5 μM and 5.0 μM of Shk significantly reduced their colony forming potential (Fig. 1B). To check the effect of Shk on the heterogeneous cancer cell population, we tested it on patient derived primary breast cancer cells. Shk reduced the viability and colony forming potential of primary breast cancer cells in dose dependent manner (Fig. 1C,D). Further we checked its effects on migration and invasion of breast cancer cells. Shk (2.5 μM) significantly inhibited the migration of MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cells (Fig. 1E). It also inhibited the cell invasion in dose dependent manner (Fig. 1F and S1C, S1D, S1E,S1F). We further examined its effect on mammosphere formation. MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cell mammosphere cultures were grown in presence or absence of 1.25 μM, 2.5 μM and 5.0 μM Shk for 24 h. After 8 days of culture, a dose dependent decrease in the mammosphere forming potential of these cells was observed (Figs. 1G,H). Collectively, these results indicated that Shk effectively inhibits the various hallmarks associated with aggressive breast cancer.

(not shown)

Figure 1: Shk inhibits multiple cancer hallmarks

Shk reduces cancer stem cell load in breast cancer

As Shk exhibited strong anti-mammosphere forming potential; therefore it was further examined for its anti-cancer stem cell (CSC) properties. Cancer stem cell loads in breast cancer cells were assessed using Aldefluor assay which measures ALDH1 expression. MDA-MB 231 cells with the highest number of ALDH1+ cells were selected for further studies (Fig. S2A). We also checked the correlation between ALDH1 expression and mammosphere formation. Sorted ALDH1+ cells were subjected to mammosphere cultures. ALDH1+ cells formed highest number of mammospheres compared to ALDH1-/low and parent cell population, indicating that ALDH1+ cells are enriched in CSCs (Fig. S2B). Shk reduced the Aldefluor positive cells in MDA-MB 231 cells after 24 h of treatment (Fig. 2A,B). Next, we examined the effect of Shk on the expression of stem cell (Sox2, Oct3/4, Nanog, AldhA1 and c-Myc) and EMT (Snail, Slug, ZEB1, Twist, β-Catenin) markers, associated with the sustenance of breast CSCs. Shk (2.5 μM) treatment for 24 h reduced the expression of these markers (Fig. 2C and S2D). Shk also reduced protein expression of these markers in dose dependent manner (Fig. 2D,E and S2C).

(not shown)

Figure 2: Shk decreases stem cell load in breast cancer cells and enriched CD44+,CD24−/low breast cancer stem cells.

To further confirm anti-CSC properties of Shk, we checked the effect of shikonin on the load of CD44+ CD24− breast CSCs in MCF7 cells grown on matrigel. Shikonin reduced CD44+ CD24− cell load in dose dependent manner after 24 h of treatment (Fig S2E). We also tested its effects on the enriched CSC population. CD44+ CD24− cells were enriched from MCF7 cells using MagCellect CD24− CD44+ Breast CSC Isolation Kit (Fig. S2F). Enriched CSCs formed highest number of mammosphere in comparison to parent MCF7 cell population or negatively selected CD24+ cells (Fig. S2G). Enriched CSCs were treated with indicated doses of Shk (0.625 μM, 1.25 μM and 2.5 μM) for 24 h and were either analyzed for ALDH1 positivity or subjected to colony or mammosphere formation. 2.5 μM dose of Shk reduced ALDH1+ cells by 50% and inhibited colony and mammosphere formation (Fig. S2H, 2F, 2G and 2H). Shk also reduced the mRNA expression of CSC markers in CD44+ CD24− cells and patient derived primary cancer cells (Fig. 2I,J). These results collectively indicated that Shk inhibits CSC load and associated programs in breast cancer.

Shk is a potent inhibitor of STAT3 and poorly inhibits FAK and Src

To identify the molecular mechanism responsible for anti-cancer properties of Shk, we used a human phospho-kinase antibody array to study a subset of phosphorylation events in MDA-MB 231 cells after 6h of treatment with 2.5 μM Shk. Amongst the 46 phospho-antibodies spotted on the array, the relative extent of phosphorylation of three proteins decreased to about ≳ 2 fold (STAT3, 3.3 fold; FAK, 2.5 fold and Src, 1.8 fold) upon Shk treatment (Fig. 3A,B). These proteins (STAT3, FAK and Src) are known to regulate CSC proliferation and self renewal21, 22, 23. Therefore, we focused on these proteins and the result of kinase-array was confirmed by western blotting. Shk effectively inhibits STAT3 at early time point (1 h) while activation of FAK and Src decreased on or after 3 h (Fig. 3C) confirming Shk as a potent inhibitor of STAT3. Shk also reduced the protein expression of STAT3, FAK and Src at 24 h (Fig. 3C).

(not shown)

Figure 3. Shk inhibits STAT3, FAK and Src signaling pathways.

We also observed that Shk does not inhibit JAK2 at initial time-points (Fig. 3C). This raised a possibility that Shk either regulates STAT3 independent of JAK2 or it binds directly to STAT3. To check the first probability, we activated STAT3 by treating the cells with IL6 (100 ng ml−1) for 1 h followed by treatment with Shk (2.5 μM) for 1 h. Both immunofluorescence and western-blotting results showed that Shk inhibited activated STAT3 without inhibiting JAK2 (Fig. S3A, S3B) confirming that Shk inhibits JAK2 mediated activation of STAT3 possibly by binding directly to STAT3. For further confirmation, we performed an in silico molecular docking analysis to examine binding of Shk with the STAT3 SH2 domain. In a major conformational cluster, Shk occupied Lys-707, Lys-709 and Phe-710 binding sites in the STAT3 SH2 domain similar to the STAT3 standard inhibitor S3I-201 (Fig. S3C and S3D). The binding energy of Shk to STAT3 was −4.20 kcal mol−1. Collectively, these results showed that Shk potently inhibits STAT3 activation and also attenuates FAK and Src activation.

STAT3, Src and FAK are differentially expressed and activated in breast CSCs (BCSCs)

STAT3 and FAK are known to play an important role in proliferation and self-renewal of CSCs in various cancer types including breast cancer21, 22, 24. Src also support CSC phenotype in some cancer types, but there are limited reports of its involvement in breast cancer25. Therefore, we checked the expression and activation of STAT3, FAK and Src in CSCs and non-CSCs. Here we used two methods to enrich the CSCs and non-CSCs. In the first method, the MDA-MB 231 cells were subjected to mammosphere formation for 96 h. After 96 h, mammosphere and non-mammosphere forming cells were clearly visible (Fig. 4A). These mammosphere and non-mammosphere forming cells were separated by using a 70 micron cell strainer. Mammospheres were subjected to two subculture cycles to enrich CSCs. With each passage, the viable single cells (non-mammosphere forming cells) and mammospheres were collected in RIPA lysis buffer and western blotting was done (Fig. 4B). We found that the activation and expression of the STAT3, FAK and Src is higher in enriched mammosphere cultures (Fig. 4C). In the second method, CD44+ CD24− cells were isolated from MCF7 cultures using MagCellect Breast CSC Isolation Kit. STAT3, FAK and Src activation and their mRNA and protein expression were assessed in enriched CSCs and were compared to parent MCF7 cell population. STAT3, FAK and Src all were differentially activated in CSCs (Fig. 4E). High mRNA as well as protein expressions of all the three genes was also observed in CSCs (Fig. 4D,E). Collectively, these results indicate that STAT3, FAK and Src are over expressed and activated in BCSCs.

Figure 4: STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

  • Representative picture indicating mammosphere and single suspended cells. (B) Schematic outline of mammosphere enrichment. (C) Protein expression and activation of STAT3, FAK and Src was determined in single suspended cells (non-mammosphere forming cells) and mammospheres by western blot. The full size blots corresponding to the cropped blot images are given in  S10. (D) Gene expression of STAT3, FAK and Src was determined in MCF7 parent population and CD44+ CD24−/low MCF7 cells using PCR. The full agarose gel images corresponding to the cropped images are given in Fig. S10. (E) Protein expression and activation of STAT3, FAK and Src was in CD44+ 24− cells and parent population.

STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f4.jpg

STAT3 is important for mammosphere formation and CSC programs in breast cancer

As our results indicated that the expression and activation of STAT3, FAK and Src is high in BCSCs and Shk is capable of inhibiting these signaling proteins; therefore to find out functional relevance of each protein and associated effects on their pharmacological inhibition by Shk, we used specific inhibitors against these three. Effect of these inhibitors was first tested on the mammosphere forming potential of MDA-MB 231, MDA-MB 468 and MCF7 cells. A drastic reduction in the mammosphere formation was observed upon STAT3 inhibition. FAK and Src inhibition also reduced the primary and secondary mammosphere formation but STAT3 inhibition showed most potent effect (Fig. 5A and S4). Further, we also checked the effect of these inhibitors on the expression of various CSC and EMT related markers in MDA-MB 231 cells. STAT3 inhibition decreased the expression of most of the CSC and EMT markers (Fig. 5B). These two findings indicated that STAT3 inhibition is more effective in reducing mammosphere forming potential and weakens major CSC programs and the anti-CSC potential of Shk is possibly due to its strong STAT3 inhibitory effect.
(not shown)

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

Figure 5: STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer.

http://www.nature.com/srep/2015/150514/srep10194/carousel/srep10194-f5.jpg

(A) Bar graph represents number of mammospheres formed from 2500 cells in presence and absence of indicated treatments. MDA-MB 231, MDA-MB 468 and MCF7 24 h mammosphere cultures were treated with Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). After 24 h, treatments were removed and cells were allowed to grow in fresh mammosphere culture media for 8 days. (B) Expression of various stem cell and EMT related transcription factors and markers were detected using western blotting in MDA-MB 231 cells with or without indicated treatments. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) MDA-MB 231, MDA-MB 468 and MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. Bar graph represents average of three independent experiments. (D) MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. After 24 h, cells were treated with DMSO (untreated) or Shk (treated) as indicated in the bar graph. Data are shown as the mean ±SD. (*) p < 0.05 and (**) p < 0.01.

To further check the involvement of these pathways in CSCs, we cultured MDA-MB 231, MDA-MB 468 and MCF7 cells in the presence of either IL6 (100ng ml−1), EGF (25 ng ml−1) or Fibronectin (1 μg ml−1) coated surface for two population doublings. Cells were then subjected to mammosphere formation. In IL6 pre-treated cultures, there was a sharp rise in mammosphere formation, indicating that the STAT3 activation shifts CSC and non-CSC dynamics towards CSCs (Fig. 5C). IL6 is previously known to induce the conversion of non-CSC to CSC via STAT3 activation26. In MCF7 cells, mammosphere forming potential after IL6 pre-treatment increased nearly by three fold. Therefore, we further checked the effectiveness of Shk on mammosphere forming potential in pre-treated MCF7 cells. It was found that Shk inhibits mammosphere formation most effectively in IL6 pre-treated cultures (Fig. 5D). However, in EGF and Fibronectin pre-treated cultures, Shk was relatively less effective. This was possibly due to its weak FAK and Src inhibitory potential. Collectively, these results illustrated that STAT3 activation is significantly correlated with the mammosphere forming potential of breast cancer cells and its inhibition by a standard inhibitor or Shk potently reduce the mammosphere formation.

Shk inhibit CSCs load by disrupting the STAT3-Oct3/4 axis

In breast cancer, STAT3 mediated expression of Oct3/4 is a major regulator of CSC self-renewal26, 27. As we observed that both Shk and STAT3 inhibitors decreased the Oct3/4 expression (Figs. 2C and 5B), we further checked the effect of STAT3 activation on ALDH1+ CSCs and Oct3/4 expression. On IL6 pre-treatment, number of ALDH1+ cells increased in all three (MDA-MB 231, MDA-MB 468 and MCF7) cancer cells (Fig. 6A). MCF7 cells showed highest increase. Therefore, to check the effect of STAT3 inhibition on CSC load, we incubated IL6 pre-treated MCF7 cells with Shk and STAT3 inhibitor for 24 h and analyzed for ALDH1 positivity. It was observed that both Shk and STAT3 inhibitor reduced the IL6 induced ALDH1 positivity from 10% to < 2% (Fig. 6B). These results suggested that Shk induced inhibition of STAT3 and decrease in BCSC load is interlinked. We further checked the effect of STAT3 activation status on Oct3/4 expression in MDA-MB 231, MDA-MB 468 and MCF7 cells. We observed that expression of Oct3/4 increases with the increase in STAT3 activation (Fig. 6C–E).

(not shown)

Figure 6: STAT3 activation status and its effect on cancer stem cell load

STAT3 transcriptional activity is important in maintaining CSC programs28, 29. Therefore, we also examined the effect of Shk on STAT3 promoter activity. STAT3 reporter assay was performed in presence of IL6 and Shk; it was found that Shk reduced the promoter activity of STAT3 in a dose dependent manner (Fig. S5). Collectively, these results showed that Shk mediated STAT3 inhibition are responsible for decrease in CSC load and Oct3/4 associated stem cell programs.

Shk inhibits mammosphere formation, migration and invasion through inhibition of STAT3, FAK and Src in breast cancer cells

As the earlier results (Fig. 1) showed that Shk inhibits cell migration and invasion in breast cancer cells, we further examined the effect of STAT3, FAK and Src inhibitors on cell migration and invasion in MDA-MB 231 cells. It was found that STAT3 inhibitor poorly inhibits cell migration while both Src and FAK inhibitors were effective in reducing cell migration (Fig. 7A). All the three inhibitors decreased the cell invasion and MMP9 expression significantly (Fig. 7B and S6). It was also observed that effect of all these inhibitors, except STAT3 inhibitor on mammosphere formation and FAK inhibitor on cell migration, were not comparable to that of Shk. Shk inhibited all these properties more effectively than individual inhibition of STAT3, FAK and Src. This made us to assume that the ability of Shk to inhibit multiple signaling molecules simultaneously is the reason behind its potent anti-cancer effect. To check this notion, we combined STAT3, FAK and Src inhibitors with each other and examined the effect of combinations on invasion, migration and mammosphere forming potential in MDA-MB 231 cells. We observed further decrease in cell migration and invasion on combining STAT3 and FAK, STAT3 and Src, or FAK and Src (Figs. 7A,B). Combination of FAK and Src was not very effective in inhibiting mammosphere formation in MDA-MB 231 cells and CD44+ CD24− MCF7 CSCs. However, their combination with STAT3 decreased the mammosphere forming potential equivalent to that of Shk (Fig. 7C,D). We also compared the mammosphere forming potential of Shk with Salinomycin (another anti-CSC agent) and found that at 2.5 μM dose of Shk was almost two times more potent than Salinomycin (Fig. S7). Collectively, these results indicated that Shk inhibits multiple signaling proteins (STAT3, FAK and Src) to compromise various aggressive breast cancer hallmarks.

Figure 7: Combination of FAK, Src and STAT3 inhibitors is more potent than individual inhibition against various cancer hallmarks.

combination-of-fak-src-and-stat3-inhibitors-is-more-potent-than-individual-inhibition-against-various-cancer-hallmarks

combination-of-fak-src-and-stat3-inhibitors-is-more-potent-than-individual-inhibition-against-various-cancer-hallmarks

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f7.jpg

  • Cell migration and (B) cell invasion potential of MDA-MB 231 cells was assessed in the presence of Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). Various combinations of these inhibitors were also used STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM + AZM 475271; 10 μM) and FAK+Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cell migration and cell invasion was assessed through scratch cell migration assay and transwell invasion after 24 h of treatments. (C,D) Mammosphere forming potential of MDA-MB 231 cells and CD44+ CD24−/low enriched MCF7 cells was assessed in presence of similar combination of STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM+ AZM 475271; 10 μM) and FAK + Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cells were subjected to mammosphere cultures for 24 h and treated with the indicated inhibitors for next 24 h, followed by media change and growth of mammospheres were monitored for next 8 days. Data are shown as the mean ±SD. (**) p < 0.01.

Shk inhibits breast cancer growth, metastasis and decreases tumorigenicity

To explore whether Shk may have therapeutic potential for breast cancer treatment in vivo, we tested Shk against 4T1-induced breast cancer syngenic mouse model. 4T1 cells (mouse breast cancer cells) are capable of growing fast and metastasize efficiently in vivo30. Prior to the in vivo experiments, we checked the effect of Shk on ALDH1 positivity and on activation of STAT3, FAK and Src in 4T1 cells in vitro. Shk effectively decreased the ALDH1+ cells and inhibited STAT3, FAK and Src in 4T1 cells in vitro (Fig. S8A and S8B). For in vivo tumor generation, 1 × 106 cells were injected subcutaneously in the fourth nipple mammary fat pad of BALB/c mice. When the average size of tumors reached around 50 mm3, mice were divided into three groups, vehicle and two Shk treated groups each received either 2.5 mg Kg−1 or 5.0 mg Kg−1 Shk. Shk was administered via the intraperitoneal injection on every alternate day. It significantly suppressed the tumor growth in 4T1 induced syngenic mouse model (Fig. 8A). The average reduction in 4T1 tumor growth was 49.78% and 89.73% in 2.5 mg Kg−1 and 5.0 mg Kg−1 groups respectively compared with the vehicle treated group (Fig. 8A). No considerable change in body weight of the treated group animals was observed (Fig. S9A). We further examined the effect of Shk on the tumor initiating potential of breast cancer cells. 4T1 induced tumors were excised from the control and treatment groups on the second day after 4th dose of Shk was administered. Tumors were dissociated; cells were allowed to adhere and then re-injected into new animals for secondary tumor formation. Growth of secondary tumors was monitored till day 15 post-reinjection. Shk treated groups showed a marked decrease in secondary tumor formation (Fig. 8D). We also observed a drastic reduction in the number of metastatic nodules in the lungs of treatment group animals (Fig. 8F). The reduction in the metastatic load was not proportional to the decrease in tumor sizes; however within the treatment group, some animals with small tumors were carrying higher number of metastatic nodules. As FAK is an important mediator of cancer metastasis and metastatic colonization, we further examined the effects of Shk on metastatic colonization. For this, 1 × 105 4T1 cells were injected to BALB/c mice through tail vein. Animals were divided into three groups, as indicated above. Shk and vehicle were administered through intraperitoneal injections at alternate days starting from the 2nd day post tail vein injections till 33rd day. The average reduction in total number of metastatic nodules was 88.6% – 90.5% in Shk treated mice compared to vehicle control (Fig. 8F). An inset picture (Fig. 8A lower panel) represents lung morphology of vehicle control and treated groups. We further examined the activation and expression status of STAT3, FAK and Src between vehicle control and treated group tumors. There were low expression and activation of STAT3, FAK and Src in treated tumors as compared to the vehicle control (Fig. 8B,C). Similar trend was observed in ALDH1 expressions (Fig. 8B). Further, the mice tumor sections were subjected to immunohistochemistry, immunofluorescence and hematoxylin and eosin (H&E) staining to study histology and expression of key proteins being examined in this study. Fig. 8G shows representative images of H&E staining, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), STAT3 and Oct3/4 immunostaining. PCNA expression was low while TUNEL positive cells were high in tumor tissues of Shk treated groups. STAT3 and Oct3/4 expression was low in Shk treated groups. These results collectively demonstrated that Shk modulates the expression and activation of STAT3, FAK and Src in vivo and is effective in suppressing tumorigenic potential and metastasis in syngenic mouse model.

Figure 8: Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo.

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f8.jpg

  • Shk inhibited 4T1 tumor growth. Bar graph represents the average tumor volumes in vehicle control and Shk treated tumor bearing mice (n = 6). (*) p < 0.05 and (**) p < 0.01. Inset picture of upper panel represents tumor sizes and lower pane represents lung morphology in vehicle control and Shk treatment groups. (B) Western blot examination of indicated proteins for their expression and activation in vehicle control and treated tumor groups. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) Gene expression of stem cell and EMT markers in tumor tissues excised from the vehicle control and Shk treated groups (n = 3). (D) Number of secondary tumors formed after injecting indicated cell dilutions from Vehicle treated and Shk treated 4T1 tumors. (E) Number of lung nodules formed in mice injected with 4T1 mouse mammary tumor cells in the mammary fat pad and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks (n = 6). (F) Number of lung nodules in mice injected with 4T1 mouse mammary tumor cells through tail vein and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks. (n = 8) (G) Representative panel of the histological H&E staining, immunofluorescence staining for the STAT3, Oct3/4, cell proliferation marker PCNA and DNA damage indicator-TUNEL staining of tumor sections from vehicle and treatment groups.

Recent studies have shown that aggressiveness, therapy resistance and disease relapse in breast cancer is attributed to a small population of CSCs involved in continuous self-renewal and differentiation through signaling pathways similar to that of the normal stem cells31. Therapeutic targeting of CSCs therefore, has profound clinical implications for cancer treatment31. Recent studies indicated that therapies / agents targeting both differentiated cancer cells and CSCs may possibly have significant therapeutic advantages32. Therefore, it is imperative to look for novel therapeutic agents with lesser side effects urgently for effective targeting of CSCs. In search of novel, nontoxic anti-CSC agents, attention has been focused on natural agents in recent times33,34. In this study, we have used a natural napthoquinone compound, Shk with established antitumorigenic, favorable pharmacokinetic and toxicity profiles and report for the first time its potent anti-CSC properties. Shk significantly inhibits breast cancer cell proliferation in vitro, ex vivoand in vivo. It decreases the cell migration and invasion of breast cancer cells in vivo, as well as inhibits tumorigenicity, metastasis and metastatic colonization in a syngenic mouse model of breast cancer in vivo. These finding suggest a strong potential of Shk in breast cancer therapy.

We assessed the effect of Shk on the CSC load in breast cancer cells through various functional assays (tumorsphere in vitro and syngenic mouse model of breast cancer in vivo) and quantification of specific stem cell markers. In breast cancer, CD44+ CD24− cells and ALDH1+ cells are considered to be BCSCs21, 25. Shk significantly decreased the mammosphere formation (Fig. 1H, S1G and 2H), ALDH1+ cell and CD44+ CD24− cell loads in vitro (Fig. 2B, S2E and S2H). It also reduced the expression of CSC markers (Oct3/4, Sox2, Nanog, c-Myc and Aldh1) in vivo andin vitro (Fig. 2C,D, S2C and S2D). These genes are known to regulate stem cell programs and in cancer, they are established promoters and regulators of CSC phenotype35, 36, 37, 38, 39, 40. Decrease in the expression of these genes on Shk treatment indicates its potential to suppress CSC programs. Tumor initiating potential (tumorigenicity) is the bona fide measure of CSCs. Reduction in the tumorigenic potential of cells isolated form Shk treated tumors indicates in vivoanti-CSC effects of Shk.

We further demonstrated that Shk is a potent inhibitor of STAT3 and it also inhibits FAK and Src (Fig. 3A–C). Its STAT3 inhibitory property was found to be responsible for its anti-CSC effects (Figs. 6B and 7B). STAT3 and FAK inhibitors are previously known to compromise CSC growth41,42. Here, we found that pharmacological inhibition of STAT3 was more effective in compromising CSC load than FAK and Src inhibitions (Fig. 5A). STAT3 activation through IL6 increases mammosphere formation more significantly than Src and FAK activation through EGF and Fibronectin (Fig. 5C). This indicates that IL6-STAT3 axis is a key regulator of BCSC dynamics.

11.2.3.9 Ovatodiolide Sensitizes Aggressive Breast Cancer Cells to Doxorubicin Anticancer Activity, Eliminates Their Cancer Stem Cell-Like Phenotype, and Reduces Doxorubicin-Associated Toxicity

Investigators evaluated the usability of ovatodiolide (Ova) in sensitizing triple negative breast cancer (TNBC) cells to doxorubicin (Doxo), cytotoxicity, so as to reduce Doxo effective dose and consequently its adverse effects. Ova-sensitized TNBC cells also lost their cancer stem cell-like phenotype evidenced by significant dissolution and necrosis of formed mammospheres, as well as their terminal differentiation. [Cancer Lett]

11.2.3.10 Glabridin Inhibits Cancer Stem Cell-Like Properties of Human Breast Cancer Cells: An Epigenetic Regulation of miR-148a/SMAd2 Signaling

The authors report that glabridin (GLA) attenuated the cancer stem cell (CSC)-like properties through microRNA-148a (miR-148a)/transforming growth factor beta-SMAD2 signal pathway in vitro and in vivo. In MDA-MB-231 and Hs-578T breast cancer cell lines, GLA enhanced the expression of miR-148a through DNA demethylation. [Mol Carcinog]

11.2.3.11 Ginsenoside Rh2 Inhibits Cancer Stem-Like Cells in Skin Squamous Cell Carcinoma

The effects of ginsenoside Rh2 (GRh2) on Lgr5-positive cancer stem cells (CSCs) were determined by flow cytometry and by tumor sphere formation. Scientists found that GRh2 dose-dependently reduced skin squamous cell carcinoma viability, possibly through reduced the number of Lgr5-positive CSCs. [Cell Physiol Biochem]

Liu S. Chen M. Li P. Wu Y. Chang C. Qiu Y. Cao L. Liu Z. Jia C.
Cell Physiol Biochem 2015;36:499-508
http://dx.doi.org:/10.1159/000430115

Background/Aims: Treatments targeting cancer stem cells (CSCs) are most effective cancer therapy, whereas determination of CSCs is challenging. We have recently reported that Lgr5-positive cells are cancer stem cells (CSCs) in human skin squamous cell carcinoma (SCC). Ginsenoside Rh2 (GRh2) has been shown to significantly inhibit growth of some types of cancers, whereas its effects on the SCC have not been examined. Methods: Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by fow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for Atg7 and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Results: We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability. Conclusion: Taken together, our data suggest that GRh2 inhibited SCC growth, possibly through reduced the number of Lgr5-positive CSCs. This may be conducted through an interaction Carcinoma account for more than 80% of all types of cancer worldwide, and squamous cell carcinoma (SCC) is the most frequent carcinoma. Skin SCC causes a lot of mortality yearly, which requires a better understanding of the molecular carcinogesis of skin SCC for developing efficient therapy [1,2]. Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng, and has proven therapeutic effects on inflammation [3] and a number of cancers [4,5,6,7,8,9,10,11,12,13,14], whereas its effects on the skin SCC have not been examined.

Cancer stem cells (CSCs) are cancer cells with great similarity to normal stem cells, e.g., the ability to give rise to various cell types in a particular cancer [15,16]. CSCs are highly tumorigenic, compared to other non-CSCs. CSCs appear to persist in tumors as a distinct population and CSCs are believed to be responsible for cancer relapse and metastasis after primary tumor resection [15,16,17,18]. Recently, the appreciation of the critical roles of CSCs in cancer therapy have been continuously increasing, although the identification of CSCs in a particular cancer is still challenging.

To date, different cell surface proteins have been used to isolate CSCs from a variety of cancers by flow cytometry. Among these markers for identification of CSCs, the most popular ones are prominin-1 (CD133), side population (SP) and increased activity of aldehyde dehydrogenase (ALDH). CD133 is originally detected in hematopoietic stem cells, endothelial progenitor cells and neuronal and glial stem cells. Later on, CD133 has been shown to be expressed in the CSCs from some tumors [19,20,21,22,23], but with exceptions [24]. SP is a sub-population of cells that efflux chemotherapy drugs, which accounts for the resistance of cancer to chemotherapy. Hoechst (HO) has been experimentally used for isolation of SP cells, while the enrichment of CSCs by SP appears to be limited [25]. Increased activity of ALDH, a detoxifying enzyme responsible for the oxidation of intracellular aldehydes [26,27], has also been used to identify CSCs, using aldefluor assay [28,29]. However, ALDH has also been detected in other cell types, which creates doubts on the purity of CSCs using ALDH method [30,31]. Moreover, all these methods appear to be lack of cancer specificity.

The Wnt target gene Lgr5 has been recently identified as a stem cell marker of the intestinal epithelium, and of the hair follicle [32,33]. Recently, we reported that Lgr5 may be a potential CSC marker for skin SCC [34]. We detected extremely high Lgr5 levels in the resected skin SCC specimen from the patients. In vitro, Lgr5-positive SCC cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Elimination of Lgr5-positive SCC cells completely inhibited cancer cell growth in vitro.

Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by flow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for autophagy-related protein 7 (Atg7) and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Atg7 was identified based on homology to Pichia pastoris GSA7 and Saccharomyces cerevisiae APG7. In the yeast, the protein appears to be required for fusion of peroxisomal and vacuolar membranes. The protein shows homology to the ATP-binding and catalytic sites of the E1 ubiquitin activating enzymes. Atg7 is a mediator of autophagosomal biogenesis, and is a putative regulator of autophagic function [35,36,37,38]. We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability.

Transduction of SCC cells with GFP under Lgr5 promoter

We have recently shown that Lgr5 is CSC marker for skin SCC [34]. In order to examine the role of GRh2 on SCC cells, as well as a possible effect on CSCs, we transduced human skin SCC cells A431 [34] with a lentivirus carrying GFP reporter under Lgr5 promoter (Fig. 1A). The Lgr5-positive cells were green fluorescent in culture (Fig. 1B), and could be analyzed or isolated by flow cytometry, based on GFP (Fig. 1C).

(not shown)

Fig. 1. Transduction of SCC cells with GFP under Lgr5 promoter. (A) The structure of lentivirus carrying GFP reporter under Lgr5 promoter. (B) The pLgr5-GFP-transduced A431 cells in culture. Lgr5-positive cells were green fluorescent. Nuclear staining was done by DAPI. (C) Representative flow chart for analyzing pLgr5-GFP-transduced A431 cells by flow cytometry based on GFP. Gated cells were Lgr5-positive cells. Scar bar is 20µm.

GRh2 dose-dependently inhibits SCC cell growth

Then, we examined the effect of GRh2 on the viability of SCC cells. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. We found that from 0.01mg/ml to 1mg/ml, GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (Fig. 2A), or a MTT assay (Fig. 2B). Next, we questioned whether GRh2 may have a specific effect on CSCs in SCC cells. Thus, we analyzed GFP+ cells, which represent Lgr5-positive CSCs in pLgr5-GFP-transduced A431 cells after GRh2 treatment. We found that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (Fig. 2C), and by quantification (Fig. 2D). We also examined the capability of the GRh2-treated cells in the formation of tumor sphere. We found that GRh2 dose-dependently deceased the formation of tumor sphere-like structure, by quantification (Fig. 2E), and by representative images (Fig. 2F). Together, these data suggest that GRh2 dose-dependently inhibited SCC cell growth, possibly through inhibition of CSCs.

Fig. 2. GRh2 dose-dependently inhibits SCC cell growth. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. (A-B) GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (A), or a MTT assay (B). (C-D) GFP+ cells after GRh2 treatment were analyzed by flow cytometry, showing that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (C), and by quantification (D). The capability of the GRh2-treated cells to form tumor sphere-like structures was examined, shown by quantification (E), and by representative images (F). *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f02.JPG

GRh2 treatment decreases β-catenin and increases autophagy in SCC cells

We analyzed the molecular mechanisms underlying the cancer inhibitory effects of GRh2 on SCC cells. We thus examined the growth-regulatory proteins in SCC. From a variety of proteins, we found that GRh2 treatment dose-dependently decreases β-catenin, and dose-dependently upregulated autophagy-related proteins Beclin, Atg7 and increased the ratio of LC3 II to LC3 I, by quantification (Fig. 3A), and by representative Western blots (Fig.3B). Since β-catenin signaling is a strong cell-growth stimulator and autophagy can usually lead to stop of cell-growth and cell death, we feel that the alteration in these pathways may be responsible for the GRh2-mediated suppression of SCC growth.

(not shown)

Figure 3. GRh2 treatment decreases β-catenin and increases autophagy in SCC cells.

http://www.karger.com/Article/ShowPic/430115?image=000430115_f03.JPG

Inhibition of autophagy abolishes the effects of GRh2 on β-catenin

In order to find out the relationship between β-catenin and autophagy in this model, we inhibited autophagy using a shRNA for Atg7, and examined its effect on the changes of β-catenin by GRh2. First, the inhibition of Atg7 in A431 cells by shAtg7 was confirmed by RT-qPCR (Fig. 4A), and by Western blot (Fig. 4B). Inhibition of Atg7 resulted in abolishment of the effects of GRh2 on other autophagy-associated proteins (Fig. 4B), and resulted in abolishment of the inhibitory effect of GRh2 on β-catenin (Fig. 4B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 4C). Together, inhibition of autophagy abolishes the effects of GRh2 on β-catenin. Thus, the regulation of GRh2 on β-catenin needs autophagy-associated proteins.

Fig. 4. Inhibition of autophagy abolishes the effects of GRh2 on β-catenin.

A431 cells were transfected with shRNA for Atg7, or scrambled sequence (scr) as a control. (A) RT-qPCR for Atg7. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f04.JPG

Overexpression of β-catenin abolishes the effects of GRh2 on autophagy

Next, we inhibited the effects of GRh2 on β-catenin by overexpression of β-catenin in A431 cells. First, the overexpression of β-catenin in A431 cells was confirmed by RT-qPCR (Fig. 5A), and by Western blot (Fig. 5B). Overexpression of β-catenin resulted in abolishment of the effects of GRh2 on autophagy-associated proteins (Fig. 5B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 5C). Together, inhibition of β-catenin signaling abolishes the effects of GRh2 on autophagy. Thus, the regulation of GRh2 on autophagy needs β-catenin signaling. This model is thus summarized in a schematic (Fig. 6), suggesting that GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.

http://www.karger.com/Article/ShowPic/430115?image=000430115_f05.JPG

Fig. 5. Overexpression of β-catenin abolishes the effects of GRh2 on autophagy. A431 cells were transfected with β-catenin, or scrambled sequence (scr) as a control. (A) RT-qPCR for β-catenin. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f06.JPG

Fig. 6. Schematic of the model. GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.

Understanding the cancer molecular biology of skin SCC and identification of an effective treatment are both critical for improving the current therapy [1]. Lgr5 has been recently identified as a novel stem cell marker of the intestinal epithelium and the hair follicle, in which Lgr5 is expressed in actively cycling cells [32,33]. Moreover, we recently showed that Lgr5-positive are CSCs in skin SCC [34]. Thus, specific targeting Lgr5-positive cells may be a promising therapy for skin SCC.

In the current study, we analyzed the effects of GRh2 on the viability of SCC. Importantly, we not only found that GRh2 dose-dependently decreases SCC cell viability, but also dose-dependently decreased the number of Lgr5-positive CSCs in SCC cells. These data suggest that the CSCs in SCC may be more susceptible for the GRh2 treatment, and the decreases in CSCs may result in the decreased viability in total SCC cells. This point was supported by following mechanism studies. Activated β-catenin signaling by WNT/GSK3β prevents degradation of β-catenin and induces its nuclear translocation [39]. Nuclear β-catenin thus activates c-myc, cyclinD1 and c-jun to promote cell proliferation, and activates Bcl-2 to inhibit apoptosis [39]. High β-catenin levels thus are a signature of CSCs. Therefore, it is not surprising that CSCs are more affected than other cells when GRh2 targets β-catenin signaling.

In addition, GRh2 appears to target autophagy. Although altered metabolism may be beneficial to the cancer cells, it can create an increased demand for nutrients to support cell growth and proliferation, which creates metabolic stress and subsequently induces autophagy, a catabolic process leading to degradation of cellular components through the lysosomal system [40]. Cancer cells use autophagy as a survival strategy to provide essential biomolecules that are required for cell viability under metabolic stress [40]. However, autophagy not only results in a staring in cell growth, but also may result in cell death [40]. Increases in autophagy may substantially decrease cancer cell growth. Thus, GRh2 has its inhibitory effect on skin SCC cells through a combined effect on cell proliferation (by decreasing β-catenin) and autophagy [40].

Interestingly, our data suggest an interaction between β-catenin and autophagy. This finding is consistent with previous reports showing that autophagy negatively modulates Wnt/β-catenin signaling by promoting Dvl instability [41,42], and with other studies showing that β-catenin regulates autophagy [38,43,44].

Of note, we have checked other SCC lines and essentially got same results. Together with our previous reports showing that Lgr5-positive cells are CSCs in skin SCC [34], these findings thus highlight a future engagement of Lgr5-directed GRh2 therapy, which could be performed in a sufficiently frequent manner, to substantially improve the current treatment for skin SCC.

Normal vs Cancer Thyroid Stem Cells: The Road to Transformation
The authors discuss new insights into thyroid stem cells as a potential source of cancer formation in light of the available information on the oncogenic role of genetic modifications that occur during thyroid cancer development. Understanding the fine mechanisms that regulate tumor transformation may provide new ground for clinical intervention in terms of prevention, diagnosis and therapy. [Oncogene] Abstract
Cancer Stem Cells: A Potential Target for Cancer Therapy
The identification of cancer stem cells (CSCs) and a better understanding of the complex characteristics of CSCs will provide invaluable diagnostic, therapeutic and prognostic targets for clinical application. The authors introduce the dysregulated properties of CSCs in cancers and discuss the possible challenges in targeting CSCs for cancer treatment. [Cell Mol Life Sci] Abstract
Targeting Cancer Stem Cells Using Immunologic Approaches
Wicha, M; Chang, A; Yingxin, X; Xiaolian, Z; Ning, N; Liu, Shuang, Q, L; Pan, Q		 Stem Cells 2015-04-15 4.15 | Apr 22
Targeting Notch, Hedgehog, and Wnt Pathways in Cancer Stem Cells: Clinical Update
Ivy, P; Takebe, N		 Nat Rev Clin Oncol 2015-04-07 4.14 | Apr 15
Hypoxia-Inducible Factors in Cancer Stem Cells and Inflammation
Liu, Y; Peng, G		 Trends Pharmacol Sci 2015-04-06 4.14 | Apr 15
NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions
Tang, D; Chao, HP; Wang, J; Yang, Tao; Jeter, C		 Stem Cells 2015-03-26 4.12 | Apr 1

Two Genes Control Breast Cancer Stem Cell Proliferation and Tumor Properties

May 22, 2015 by
https://beyondthedish.wordpress.com/2015/05/22/two-genes-control-breast-cancer-stem-cell-proliferation-and-tumor-properties/
When mothers breastfeed their babies, they depend upon a unique interaction of genes and hormones to produce milk and deliver it to their hungry little tyke. Unfortunately, this same cocktail of genes and hormones can also lead to breast cancer, especially if the mother has her first pregnancy after age 30.

 A medical research group at the Medical College of Georgia at Georgia Regents University has established that a gene called DNMT1 plays a central role in the maintenance of the breast (mammary gland) stem cells that enable normal rapid growth of the breasts during pregnancy. This same gene, however, can also maintain those cancer stem cells that enable breast cancer. According to their work, the DNMT1 gene is highly expressed in the most common types of breast cancer.

Also, another gene called ISL1, which encodes a protein that puts the brakes on the growth of breast stem cells, is nearly silent in the breasts during pregnancy and in breast cancer stem cells.

Dr. Muthusamy Thangaraju, a biochemist at the Medical College of Georgia, who is the corresponding author of this study, which was published in the journal Nature Communications, said, “DNMT1 directly regulates ISL1. If the DNMT1 expression is high, this ISL1 gene is low.” Thangaraju and his team first observed the connection between DNMT1 and ISL1 when they knocked out the DNMT1 gene mice and noted an increase in the expression of ISL1. These results inspired them to examine the relationship of these two genes in human breast cancer cells.

Thangaraju and his co-workers discovered that ISL1 is silent in most human breast cancers. Furthermore, they demonstrated that restoring higher levels of ISL1 to human breast cancer cells dramatically reduced cancer stem cell populations, the growth of these cells, and their ability to spread throughout the tissue; all of which are hallmarks of cancer.

Conversely, Thangaraju and his team knocked out the DNMT1 gene in a breast-cancer mouse model, the breast will not develop as well. However, according to Thangaraju, this same deletion will also prevent the formation of about 80 percent of breast tumors. In fact, DNMT1 also down-graded super-aggressive, triple-negative breast cancers, which are negative for the estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-).

The findings from this work also point toward new therapeutic targets for breast cancer and new strategies to diagnose early breast cancer. For example, a blood test for ISL1 might provide a marker for the presence of early breast cancer. Additionally, the anti-seizure medication valproic acid is presently being used in combination with chemotherapy to treat breast cancer, and this drug increases the expression of ISL1. This might explain why valproic acid works for these patients, according to Thangaraju. Workers in Thangaraju’s laboratory are already screening other small molecules that might work as well or better than valproic acid.

Mammary stem cells maintain the breasts during puberty as well as pregnancy, which are both periods of dynamic breast cell growth. During pregnancy, breasts may generate 300 times more cells as they prepare for milk production. Unfortunately, these increased levels of cell growth might also include the production of tumor cells, and the mutations that lead to breast cancer increase in frequency with age. If the developing fetus dies before she comes to term, immature breast cells that were destined to become mature mammary gland cells can more easily become cancer, according to Rajneesh Pathania, a GRU graduate student who is the first author of this study.

DNMT1 is essential for maintaining a variety of stem cell types, such as hematopoietic stem cells, which produce all types of blood cells. However, the role of DNMT1 in the regulation of breast-specific stem cells that make mammary gland tissue and may enable breast cancer has not been studied to this point.

For reasons that are unclear, there is an increased risk of breast cancer if the first pregnancy occurs after age 30 or if mothers lose their baby during pregnancy or have an abortion. Women who never have children also are at increased risk, but multiple term pregnancies further decrease the risk, according to data compiled and analyzed by the American Cancer Society.

Theories to explain these phenomena include the coupling of the hormone-induced maturation of breast cells that occurs during pregnancy with an increase in the potential to produce breast cancer stem cells. Most breast cancers thrive on estrogen and progesterone, which are both highly expressed during pregnancy and help fuel stem cell growth. During pregnancy, stem cells also divide extensively and as their population increases, DNMT1 levels also increase.

In five different types of human breast cancer, researchers found high levels of DNMT1 and ISL1 turned off. Even in a laboratory dish, when they reestablished normal expression levels of ISL1, human breast cancer cells and stem cell activity were much reduced, Thangaraju said.

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Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Posted in Biomarkers & Medical Diagnostics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Prevention: Research & Programs, Cancer Screening, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Curation, Cytoskeleton, Diagnostic Immunology, Diagnostics and Lab Tests, Disease Biology, Drug Toxicity, Genomic Expression, Monoclonal Immunotherapy, tagged , , , , , , , on May 12, 2015| Leave a Comment »

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Writer and Curator: Larry H. Bernstein, MD, FCAP

This contribution has the following structure:

9.1.1 Viruses in carcinogenesis

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal Antibodies in Cancer Therapy

9.1.4 Aptamers

9.1.5 Tumor Suppressors

9.1 Vaccines

9.1.1  Viruses in carcinogenesis

  • HPV-associated cervical cancer
  • HPV-associated head and neck cancer: a virus-related cancer epidemic

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

HIV Infection and Cancer Risk

HIV and cancer of the cervix

Anal cancer: an HIV-associated cancer

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Types of Cancer: AIDS/HIV related malignancies

Cytokines in cancer pathogenesis and cancer therapy

Dendritic Cells as Therapeutic Vaccines against Cancer

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal antibodies

Monoclonal antibodies in cancer therapy

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

9.1 Vaccines

9.1.1  Viruses in carcinogenesis

HPV-associated cervical cancer

http://www.cancer.net/navigating-cancer-care/prevention-and-healthy-living/hpv-and-cancer

Human papillomavirus (HPV) is a virus that is usually passed on during direct skin-to-skin contact, most commonly sex. In fact, HPV is the most common sexually transmitted disease in the United States. Most men and women are not aware they have an HPV infection because they do not develop any symptoms or health problems. Certain HPV types can cause precancerous lesions (areas of abnormal tissue) or cancer.

More than 40 of the viruses are called “genital type” HPVs. These viruses are spread from person to person when their genitals come into contact, usually during vaginal or anal sex. They can also be passed on through oral sex.

Genital HPV types can infect the genital area of women, including the vulva (outer portion of the vagina), the vagina, and the cervix (the lower, narrow part of a woman’s uterus), as well as the genital area of men, including the penis. In both men and women, genital HPV can infect the anus and some areas of the head and neck.

Nearly all cervical cancers are caused by HPV infection. Strong scientific evidence shows that a lasting HPV infection is required for cervical cancer to begin developing. Whether a woman who is infected with HPV will develop cervical cancer depends on a number of factors, including the type of HPV infection she has. Of the cervical cancers related to HPV, about 70% are caused by two strains, HPV-16 or HPV-18. In women who have HPV, smoking may increase the risk of cervical cancer.

Warts and precancerous lesions can be removed through cryotherapy (freezing); loop electrosurgical excision procedure (LEEP), which uses electric current to remove abnormal tissue; or surgery.

Receiving an HPV vaccine reduces your risk of infection. The U.S. Food and Drug Administration (FDA) approved two vaccines that help prevent HPV infection: Gardasil and Cervarix. It is important to note that the vaccines cannot cure an existing HPV infection.

Purpose of the vaccines. The goal of HPV vaccination is to prevent a lasting HPV infection after a person is exposed to the virus. Gardasil, introduced in 2006, helps prevent infection from the two HPVs known to cause most cervical cancers and precancerous lesions in the cervix. The vaccine also prevents against the two low-risk HPVs known to cause 90% of genital warts. Gardasil is approved for the prevention of cervical, vaginal, and vulvar cancers in girls and women ages nine to 26. It is also approved to prevent anal cancer in women and men and genital warts in men and boys in the same age range. Cervarix, introduced in 2009, is approved for the prevention of cervical cancer in girls and women ages 10 to 25.

Effectiveness and safety of the vaccines. Data show the HPV vaccinations are safe and highly effective in preventing a lasting infection of the HPV types they target. Because it takes many years before a precancerous lesion develops into an invasive cancer, it will likely take several more years before there is evidence that the number of cancer cases in vaccinated individuals has been reduced.

HPV-associated head and neck cancer: a virus-related cancer epidemic
Shanthi Marur, Gypsyamber D’Souza, William H Westra, Arlene A Forastiere
Lancet Oncol 2010; 11: 781–89
http://dx.doi.org:/10.1016/S1470-2045(10)70017-6

A rise in incidence of oropharyngeal squamous cell cancer—specifically of the lingual and palatine tonsils—in white men younger than age 50 years who have no history of alcohol or tobacco use has been recorded over the past decade. This malignant disease is associated with human papillomavirus (HPV) 16 infection. The biology of HPV-positive oropharyngeal cancer is distinct with P53 degradation, retinoblastoma RB pathway inactivation, and P16 upregulation. By contrast, tobacco-related oropharyngeal cancer is characterized by TP53 mutation and downregulation of CDKN2A (encoding P16). The best method to detect virus in tumor is controversial, and both in-situ hybridization and PCR are commonly used; P16 immunohistochemistry could serve as a potential surrogate marker. HPV-positive oropharyngeal cancer seems to be more responsive to chemotherapy and radiation than HPV-negative disease. HPV 16 is a prognostic marker for enhanced overall and disease-free survival, but its use as a predictive marker has not yet been proven. Many questions about the natural history of oral HPV infection remain under investigation. For example, why does the increase in HPV-related oropharyngeal cancer dominate in men? What is the potential of HPV vaccines for primary prevention? Could an accurate method to detect HPV in tumor be developed? Which treatment strategies reduce toxic effects without compromising survival? Our aim with this review is to highlight current understanding of the epidemiology, biology, detection, and management of HPV-related oropharyngeal head and neck squamous cell carcinoma, and to describe unresolved issues.

Cancers of the head and neck arise from mucosa lining the oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract, and nasophaynx. By far the most common histological type is squamous cell carcinoma, and grade can vary from well-differentiated keratinizing to undifferentiated non-keratinizing. An increase in incidence of oropharyngeal squamous cell carcinoma—specifically in the tonsil and tongue base—has been seen in the USA, most notably in individuals aged 40–55 years. Patients with oropharyngeal cancer are mainly white men. Unlike most tobacco-related head and neck tumors, patients with oropharyngeal carcinoma usually do not have a history of tobacco or alcohol use. Instead, their tumors are positive for oncogenic forms of the human papillomavirus (HPV), particularly 16 type. About 60% of oropharyngeal squamous cell cancers in the USA are positive for HPV 16. HPV-associated head and neck squamous cell carcinoma seems to be a distinct clinical entity, and this malignant disease has a better prognosis than HPV-negative tumors, due in part to increased sensitivity of cancers to chemotherapy and radiation therapy. Although HPV is now recognized as a causative agent for a subset of oropharyngeal squamous cell carcinomas, the biology and natural history of oropharyngeal HPV infection and the best clinical management of patients with HPV-related head and neck squamous cell tumors is not well understood.

Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52 100 oropharyngeal cancers) and 301 408 deaths.1 Although HPV has been long known to be an important cause of anogenital cancer, only in recent times has it been recognized as a cause of a subset of head and neck squamous cell carcinomas.2 More than 100 different types of HPV exist,3 and at least 15 types are thought to have oncogenic potential.4 However, most (>90%) HPV-associated head and neck squamous cell cancers are caused by one virus type, HPV 16, the same type that leads to HPV-associated anogenital cancers. The proportion of head and neck squamous cell carcinomas caused by HPV varies widely (figure 1),5–16 largely because of the burden of tobacco-associated disease in this population of tumors. Tobacco, alcohol, poor oral hygiene, and genetics remain important risk factors for head and neck tumors overall, but HPV is now recognized as one of the primary causes of oropharyngeal squamous cell cancers. In the USA, about 40–80% of oropharyngeal cancers are caused by HPV, whereas in Europe the proportion varies from around 90% in Sweden to less than 20% in communities with the highest rates of tobacco use (figure 1).

The incidence of head and neck cancers overall in the USA has fallen in recent years, consistent with the decrease in tobacco use in this region. By contrast, incidence of HPV-associated oropharyngeal cancer seems to be rising, highlighting the increasing importance of this causal association.17–19 In a US study in which data of the Surveillance, Epidemiology, and End Results (SEER) program were used, incidence of oropharyngeal tumors (which are most likely to be HPV-associated) rose by 1·3% for base of tongue cancers and by 0·6% for tonsillar cancers every year between 1973 and 2004. By contrast, incidence of oral cavity cancers (not associated with HPV) declined by 1·9% every year during the same period.17 The age-adjusted incidence of tonsillar cancer increased 3·5-fold in women and 2·6-fold in men between 1970 and 2002.24 Augmented incidence of HPV-associated oropharyngeal cancers represents an emerging viral epidemic of cancer.

Why is increased incidence of HPV-associated oropharyngeal cancer most pronounced in young individuals? This effect could be attributable to changes in sexual norms (i.e., more oral sex partners or oral sex at an earlier age in recent than past generations) combined with fewer tobacco-associated cancers in young cohorts, making the outcomes of HPV-positive cancers more visible. Can the higher rates of HPV-associated oropharyngeal cancers in men compared with women be accounted for solely by differences in sexual behavior, or are biological differences in viral clearance present that could contribute to the higher burden of these cancers in men? HPV prevalence in cervical rather than penile tissue might boost the chances of HPV infection when performing oral sex on a woman, contributing to the higher rate of HPV-associated oropharyngeal cancer in men.

Tobacco use has fallen in past decades, and the corresponding rise in proportion of head and neck cancers that are oropharyngeal in origin has been striking, both in the USA and internationally. SEER data suggest that about 18% of all head and neck carcinomas in the USA were located in the oropharynx in 1973, compared with 31% of such squamous cell tumors in 2004.19 Similarly, in Sweden, the proportion of oropharyngeal cancers caused by HPV has steadily increased, from 23% in the 1970s to 57% in the 1990s, and as high as 93% in 2007.13,25 These data indicate that HPV is now the primary cause of tonsillar malignant disease in North America and Europe.

Findings of initial studies suggest that oral HPV frequency increases with age. Prevalent oral HPV infection is detected in 3–5% of adolescents26–28 and 5–10% of adults.14,29 We do not yet know whether the natural history of oral HPV or risk factors for persistent HPV infection in the oropharynx differ from those known for anogenital HPV infection (table 1). Data suggest oral HPV prevalence is amplified with number of sexual partners and is more typical in men, in HIV-infected individuals, and in current tobacco users.26–28,30,31

In view of the importance of tobacco use in head and neck squamous cell carcinoma, most cases of this malignant disease seen in non-smokers are unsurprisingly HPV-related. However, oral HPV infection is common in smokers and non-smokers and is an important cause of oropharyngeal cancer in both groups. For example, in case series, only 13–16% of individuals with HPV-positive head and neck squamous cell cancer did not smoke or drink alcohol.32,33 Although a higher proportion of individuals with HPV-positive compared with HPV-negative tumors are non-smokers or neither smoke nor drink alcohol, many with HPV-positive disease have a history of alcohol and tobacco use. In fact, 10–30% of HPV-positive head and neck squamous cell carcinomas were recorded in heavy tobacco and alcohol users.32,33 This finding underscores that HPV-associated malignant disease not only arises in people who do not smoke or drink alcohol but also occurs in people with the traditional risk factors of tobacco and alcohol use.

HPV detection may ultimately serve a more comprehensive role than mere prognostication. Detection of HPV is emerging as a valid biomarker for discerning the presence and progress of disease encompassing all aspects of patients’ care, from early cancer detection,41 to more accurate tumor staging (e.g., localization of tumor origin),42,43 to selection of patients most likely to benefit from specific treatments,44 to post-treatment tumor surveillance.45,46 Consequently, there is a pressing need for a method of HPV detection that is highly accurate, reproducible from one diagnostic laboratory to the next, and practical for universal application in the clinical arena. Despite growing calls for routine HPV testing of all oropharyngeal carcinomas, the best method for HPV detection is not established. Various techniques are currently in use, ranging from consensus and type-specific PCR methods, real-time PCR assays to quantify viral load, type-specific DNA in-situ hybridization, detection of serum antibodies directed against HPV epitopes, and immunohistochemical detection of surrogate biomarkers (e.g., P16 protein). Although PCR-based detection of HPV E6 oncogene expression in frozen tissue samples is generally regarded as the gold standard for establishing the presence of HPV, selection of assays for clinical use will ultimately be influenced by concerns relating to sensitivity, specificity, reproducibility, cost, and feasibility. Development of non-fluorescent chromogens has enabled visualization of DNA hybridization by conventional light microscope; furthermore, adaptation of in-situ hybridization to formalin-fixed and paraffin-embedded tissues has made this technique compatible with standard tissue-processing procedures and amenable to retrospective analysis of archival tissue blocks. Most PCR-based methods, on the other hand, need a high level of technical skill and are best used with fresh-frozen samples.

Limitations of any one detection assay can be offset by algorithms that combine the strengths of complementary assays.50 A highly feasible strategy incorporates P16 immunohistochemistry and HPV in-situ hybridization. In view of sensitivity that approaches 100%, P16 immunostaining is a good first-line assay for elimination of HPV-negative cases from any additional analysis. Since specificity is almost 100%, a finding positive for HPV 16 on in-situ hybridization reduces the number of false-positive cases by P16 staining alone. A P16-positive, HPV 16-negative result singles out a subset of tumors that qualifies for rigorous analysis for other (i.e., non-HPV 16) oncogenic virus types.

HPV in-situ hybridisation and P16 immunostaining as a practical diagnostic approach to discernment of HPV status can be applied readily to cytological preparations, including fi ne-needle aspirates from patients with cervical lymph-node metastases.41,52 Further expansion of HPV testing to blood and other body fl uids would advance the role of HPV as a clinically relevant biomarker, but these specimens would need other detection platforms. PCR-based detection of HPV DNA in blood (53) and saliva (54) of patients after treatment of their HPV-positive cancers suggests a future role in tumour surveillance. Detection of serum antibodies to HPV-related epitopes can predict the HPV status of head and neck cancers, and this method has been advocated as a way to project clinical outcomes and guide treatment without the constraints of tissue acquisition.53,55

The increasing prevalence of oropharyngeal cancer in young populations and substantially amplified survival rates with current treatment approaches stands in contrast to survival achieved in older individuals with comorbid disorders associated with tobacco and alcohol history. Several characteristics of patients with head and neck cancer have been linked with favorable prognosis, including non-smoker, minimum exposure to alcohol, good performance status, and no comorbid disorders, all of which are related to HPV-positive tumor status. Findings of retrospective analyses suggest that individuals with HPV-positive oropharyngeal cancer have higher response rates to chemotherapy and radiation and increased survival62–65 compared with those with HPV-negative tumors. Augmented sensitivity to chemotherapy and radiotherapy has been attributed to absence of exposure to tobacco and presence of functional unmutated TP53.63,64,66 Increased survival of patients with HPV-positive cancer is also possibly attributable in part to absence of field cancerization related to tobacco and alcohol exposure.67

Survival outcomes for individuals with HPV 16-positive and P16-positive oropharyngeal tumors were similar. Failure data indicated significantly diminished rates of locoregional failure and second primary tumour in patients with HPV-positive oropharyngeal cancer compared with those with HPV-negative tumors; distant metastases did not differ between the two groups. When survival was assessed after adjustment for tobacco exposure, in individuals who smoked, those with HPV-positive oropharyngeal tumors and fewer than 20 pack-years had 2-year overall survival of 95%, compared with 80% in those with HPV-positive cancers and 20 pack-years or more, and 63% in HPV-negative cancers and 20 pack-years or more. By comparison with people with HPV-positive oropharyngeal tumors who smoked and had fewer than 20 pack-years, the hazard of death was raised for those with HPV-negative tumors and 20 pack-years or more (hazard ratio 4·33) and those with HPV-positive cancers and 20 pack-years or more (1·79). These data indicate clearly that tobacco exposure alters the biology of HPV-positive oropharyngeal tumors and is an important prognostic factor.

An association between HPV-positive, P16-positive oropharyngeal tumors and survival outcomes was reported in another retrospective analysis of a large phase 3 trial of chemoradiation, which included more than 800 patients enrolled from international sites.72 This substudy analysis looked at 195 available tumor samples in patients with an oropharyngeal primary cancer, of which 28% were HPV-positive and 58% were P16-positive. Individuals with HPV-positive cancers had 2-year overall survival of 94% and 2-year failure-free survival of 86% compared with 77% (p=0·007) and 75% (p=0·035), respectively, in those with HPV-negative tumors. When co-expression of HPV and P16 was correlated with survival outcomes, individuals with HPV-positive, P16-positive tumors had 2-year overall survival of 95% compared with 88% in those with HPV-negative, P16-positive cancers and 71% (p=0·003) in those with HPV-negative, P16-negative tumors. Similar results were noted for 2-year failure-free survival (89%, 86%, and 69%, respectively; p=0·002) and time to locoregional failure (93%, 95%, and 84%, respectively; p=0·051). By multivariable analysis, HPV 16 and P16 were identified as independent prognostic factors.

ECOG proposes induction chemotherapy with a triple drug regimen to reduce tumor burden to subclinical disease (clinical complete response at primary site) followed by lower dose radiation (total dose 54 Gy) and concurrent cetuximab. Overall survival and progression-free survival outcomes will be assessed and compared with results of the 2008 ECOG study.70 The main aim of this planned study is to assess potential for a lower dose of radiation to control disease and to investigate toxic effects and quality-of-life variables.

In summary, tumor HPV status is a prognostic factor for overall survival and progression-free survival and might also be a predictive marker of response to treatment. The method of in-situ hybridization provides a feasible approach for implementation in most diagnostic pathology laboratories, and immunohistochemical staining for P16 could be useful as a surrogate marker for HPV status. Seemingly, locoregional recurrence—but not the rate of distant disease—is diminished in patients with HPV-positive tumors. Smoking and tobacco exposure might modify survival and recurrence of HPV-positive tumors and should be considered in future trials for risk stratification of patients with HPV-positive malignant disease.

HCV and cancer

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide
Joseph F. Perz, Armstrong GL, Farrington LA,  Hutin YJF, Bell BP
J Hepatol 2006; 45:529-538
http://dx.doi.org:/10.1016/j.jhep.2006.05.013

End-stage liver disease accounts for one in forty deaths worldwide. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) are well-recognized risk factors for cirrhosis and liver cancer, but estimates of their contributions to worldwide disease burden have been lacking. Methods: The prevalence of serologic markers of HBV and HCV infections among patients diagnosed with cirrhosis or hepatocellular carcinoma (HCC) was obtained from representative samples of published reports. Attributable fractions of cirrhosis and HCC due to these infections were estimated for 11 WHO-based regions. Results: Globally, 57% of cirrhosis was attributable to either HBV (30%) or HCV (27%) and 78% of HCC was attributable to HBV (53%) or HCV (25%). Regionally, these infections usually accounted for >50% of HCC and cirrhosis. Applied to 2002 worldwide mortality estimates, these fractions represent 929,000 deaths due to chronic HBV and HCV infections, including 446,000 cirrhosis deaths (HBV: n = 235,000; HCV: n = 211,000) and 483,000 liver cancer deaths (HBV: n = 328,000; HCV: n = 155,000). Conclusions: HBV and HCV infections account for the majority of cirrhosis and primary liver cancer throughout most of the world, highlighting the need for programs to prevent new infections and provide medical management and treatment for those already infected.

Among primary liver cancers occurring worldwide, hepatocellular carcinoma (HCC) represents the major histologic type and likely accounts for 70% to 85% of cases [2]. Cirrhosis precedes most cases of HCC, and may exert a promotional effect via hepatocyte regeneration [3,4]. Compared with other causes of cirrhosis, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is associated with a higher risk of developing HCC [3,5]. Alcohol abuse represents a leading cause of cirrhosis and is also a major contributor. dietary aflatoxin exposure in parts of Africa and Asia has been associated with primary liver cancer, especially in hosts with chronic HBV infection [8].

An understanding of the relative contribution of various etiologies to disease burden is important for setting public health priorities and guiding prevention programs [10,11]. The World Health Organization’s Global Burden of Disease (GBD) 2000 project aims to quantify the burden of premature morbidity and mortality from over 130 major causes [1,12]. Liver cancer and cirrhosis are included in the analysis, but with the exception of alcohol, the etiologies underlying these diseases have not been well accounted for [1,11,13]. In particular, HBV and HCV infections have been poorly characterized in previous WHO estimates since these were based primarily on the acute effects of infection and omitted the associated burdens of chronic liver disease [10,11].

The attributable fraction represents the proportion of disease occurrence that potentially would be prevented by eliminating a given risk factor. For cirrhosis, a systematic analysis of attributable fractions has been lacking altogether. For HCC, previous estimates of the attributable fractions due to HBV and HCV are available but are not comprehensive and do not correspond to the regional designations and related conventions of the current GBD project [14].

The prevalence of HBV and HCV infection among cirrhosis and HCC patients varied considerably within and between regions (Tables 2 and 3). These variations tended to reflect known patterns of HBV and HCV infection endemicity [99,100]. For example, in countries where HCV infection has long been endemic, such as Japan and Egypt, there were high prevalences of HCV infection among cirrhosis and HCC patients. The same held true for China and most of the African nations in our sample regarding HBV infection. Areas such as these, where HBV infection predominated, appeared to have a younger population of HCC cases, which is thought to reflect the preponderance of infections acquired early in life (e.g., perinatal HBV transmission) [8]. Patterns of HBV and HCV co-infection were also notable.

When we applied the HBV- and HCV-attributable fractions we derived to 2002 worldwide mortality estimates [1], we found that approximately 929,000 deaths from cirrhosis (n = 446,000) and primary liver cancer (n = 483,000) were likely due to chronic viral hepatitis infections. HBV infection accounted for 563,000 deaths (235,000 from cirrhosis and 328,000 from liver cancer) and HCV infection accounted for 366,000 deaths (211,000 from cirrhosis and 155,000 from liver cancer).

We showed that chronic viral hepatitis infections likely account for the majority of both cirrhosis and HCC globally and in nearly all regions of the world. One of the strengths of our analysis was that it employed simple and transparent methods. Our estimates of attributable fractions were derived from reviews of published studies reporting the prevalence of HBV and HCV infections in patients with cirrhosis or HCC in all regions of the world. Alternate approaches rely on estimates of the prevalence of risk factors and corresponding relative risks in the source populations. However, errors associated with extrapolating exposure or hazard from one population to another are a major source of uncertainty in efforts to characterize international health risks [12]. Given the lack of representative data regarding HBV and HCV infection prevalences worldwide along with uncertainties in deriving region specific risk estimates, we believe ours is the preferred approach.

Our findings help illustrate the great need for programs aimed at preventing HBV or HCV transmission. In 1992, WHO recommended that all countries include hepatitis B vaccine in their routine infant immunization programs. As of 2003, WHO/UNICEF estimated 42% hepatitis B vaccination coverage among the global birth cohort [106]. Therefore, implementation of this strategy, which represents the most effective way of preventing chronic HBV infection and related end stage liver disease, is far from complete [107,108]. Other key primary prevention strategies include screening blood donors and maintaining infection control practices to prevent the transmission of healthcare-related HBV and HCV infections [105,109,110]. In countries where these activities have not been fully implemented, they should be given a high priority. In most developed countries, injection drug use and high-risk sexual behaviors represent the major risk factors for HCV infection and HBV infection, respectively, indicating the importance of related prevention efforts (e.g., reducing the numbers of new initiates to injection drug use).

The role of programs to identify, counsel, and provide medical management for the many persons already infected with HBV or HCV requires careful consideration [105,110]. Counseling that includes advice regarding avoidance of alcohol and education regarding modes of transmission can help reduce the risks for developing chronic disease or spreading infection to susceptible persons. The widespread application of therapeutic interventions also has the potential to accelerate the declines in end-stage liver disease that will eventually follow from hepatitis B vaccination and other primary prevention efforts [104,107]. Recent advances have occurred in the therapeutic management of chronic hepatitis B and chronic hepatitis C, but treatments are long and involve substantial costs and side effects [111–113]. Countries will need to consider the potential benefits of treatment while insuring that scarce healthcare resources are allocated in a manner that does not undermine primary prevention efforts [114].

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

Huang J1, Magnusson M, Törner A, Ye W, Duberg AS.
Br J Cancer. 2013 Nov 26; 109(11):2917-23.
http://dx.doi.org:/10.1038/bjc.2013.689

A few studies indicated that hepatitis C and hepatitis B virus (HCV/HBV) might be associated with pancreatic cancer risk. The aim of this nationwide cohort study was to examine this possible association. Methods: Hepatitis C virus-
and hepatitis B virus-infected individuals were identified from the national surveillance database from 1990 to 2006, and followed to the end of 2008. The pancreatic cancer risk in the study population was compared with the general population by calculation of Standardized Incidence Ratios (SIRs), and with a matched reference population using a Cox proportional hazards regression model to calculate hazard ratios (HRs). Results: In total 340 819 person-years in the HCV cohort and 102 295 in the HBV cohort were accumulated, with 34 and 5 pancreatic cancers identified, respectively. The SIRHCV was 2.1 (95% confidence interval, CI: 1.4, 2.9) and the SIRHBV was 1.4 (0.5, 3.3). In the Cox model analysis, the HR for HCV infection was 1.9 (95% CI: 1.3, 2.7), diminishing to 1.6 (1.04, 2.4) after adjustment for potential confounders.
Conclusion: Our results indicated that HCV infection might be associated with an increased risk of pancreatic cancer but further studies are needed to verify such association. The results in the HBV cohort indicated an excess risk, however, without statistical significance due to lack of power.

Pancreatic cancer is one of the most rapidly fatal malignancies with a 5-year survival rate below 5%. The long-term survival is poor also for early diagnosed patients treated with resection surgery (Jemal et al, 2010). In Europe, it was estimated in a prediction model that in the year 2012 there would be 75 000–80 000 deaths from pancreatic cancer, which is the fourth most common cause of cancer-associated death for both men and women (Malvezzi et al, 2012). The incidence of pancreatic cancer is higher in the Nordic countries and Central Europe than in other parts of the world (Bosetti et al, 2012).

Tobacco smoking is a well-established risk factor for pancreatic cancer (Iodice et al, 2008), and a similar magnitude of excess risk as smoking was found among the users of Scandinavian snus (moist snuff) (Boffettaet al, 2005; Luo et al, 2007). Besides, accumulating evidence consistently shows that old age, male sex, diabetes mellitus, hereditary pancreatitis, chronic pancreatitis and family history are positively associated with this carcinoma (Pandol et al, 2012). Albeit the biological mechanism is unclear, recent epidemiological studies indicated that some infections, such as exposure to Helicobacter pylori (Trikudanathan et al, 2011), poor oral health (Michaud et al, 2007), hepatitis C virus (HCV) (Hassan et al, 2008; El-Serag et al, 2009) or hepatitis B virus (HBV) (Hassan et al, 2008; Iloeje et al, 2010; Wang et al, 2012a, 2012b) might be associated with pancreatic cancer risk.

Globally, ∼170 million people are chronically infected with HCV (World Health Organization, 1997) and an estimated 350 million with HBV (Custer et al, 2004). The prevalence rates of HCV and HBV infection vary widely in the world, and Sweden is a low endemic country with an estimated 0.5% of the population infected with HCV (Duberg et al, 2008a) and even lower rate for HBV infection. Both chronic HCV and HBV infections are main causes of hepatocellular carcinoma (HCC). Previous findings demonstrated that HBV may replicate within the pancreas (Shimoda et al, 1981; Yoshimura et al, 1981) and that HCV could be associated with pancreatitis (Alvares-Da-Silva et al, 2000; Torbenson et al, 2007). Some studies support that HCV and HBV may have a role in the development of pancreatic cancer, but the evidence is far from conclusive (Hassan et al, 2008; El-Serag et al, 2009; Iloeje et al, 2010; Wang et al, 2012a, 2012b), and more studies are needed. Towards this end, we utilised Swedish population-based nationwide registers, with documentation of all diagnosed HCV- and HBV-infected individuals in Sweden, to explore the association of HCV or HBV infection and the risk of pancreatic cancer.

Baseline characteristics of the HCV and HBV cohorts are presented in Table 1. In the HCV and chronic HBV cohorts the mean follow-up time were 9.1 and 9.4 years, with a total of 360 154 and 107 986 person-years at risk, respectively. There was a clear male dominance in the HCV cohort, and median age at entry into the HCV or HBV cohorts (notification date) was 38 and 31 years, respectively. A marked difference between cohorts was observed regarding the aspect of country of origin; HCV-infected individuals were more likely from Nordic countries, but persons with chronic HBV infection were often immigrants from non-Nordic countries.

Hepatitis C virus cohort

In the HCV cohort, there were 34 pancreatic cancer cases observed during 340 819 person-years of follow-up (first 6 months of follow-up excluded), whereas 16.5 were expected, yielding a statistically significant increased risk of pancreatic cancer (SIR: 2.1; 95% CI: 1.4, 2.9). The SIR did not alter substantially across sex or estimated duration of HCV infection (Table 2). The majority of cases were among the patients who were born before 1960.

From the Cox regression model, an ∼90% excessive risk for pancreatic cancer (HR 1.9; 95% CI: 1.3, 2.7) was observed after adjustment for age, sex and county of residence, which is similar to the result from the SIR analysis. This excess risk diminished somewhat but remained statistically significant after further adjustment for potential confounders (HR 1.6; 95% CI: 1.04, 2.4). The results did not vary markedly when stratified by sex (Table 3). In the additional analyses, excluding all individuals ever hospitalized with acute and/or chronic pancreatitis, the results did not alter notably (data not shown).

In the HCV cohort, the Standardized Incidence Ratio (SIR) for lung cancer was 2.3 (95% CI: 1.9, 2.7) and the Hazard Ratio (HR) for lung cancer was 2.2 (95% CI: 1.8, 2.7), decreasing to 1.6 (95% CI: 1.3, 2.1) after adjustment for the potential confounders used in the pancreatic cancer analyses.

Chronic HBV cohort

A total of five pancreatic cancer cases were found during 102 295 person-years of follow-up (first 6 months excluded), whereas 3.5 were expected. Compared with the age- and sex-matched Swedish general population, a 40% excess risk of pancreatic cancer was found in the chronic HBV cohort (SIR: 1.4; 95% CI: 0.5, 3.3), but without statistical significance. Because of the small number of pancreatic cancer cases, there was not enough power for additional stratified analyses (Table 4).

The Cox regression model revealed similar results as the SIR analysis. The point estimates were somewhat higher (HR=2.0 from the model adjusted for only matching factors and HR=1.8 from the fully adjusted model), but still statistically non-significant (Table 5). The SIR for lung cancer in the chronic HBV infection cohort was 1.7 (95% CI: 1.1, 2.5).

This population-based large cohort study revealed a doubled risk of pancreatic cancer among HCV-infected patients compared with the Swedish general population. The excess risk was persistent across strata by sex or duration of infection. Although further adjustment for potential confounders, i.e., chronic obstructive pulmonary disease (related to smoking), diabetes mellitus, chronic pancreatitis and alcohol-related disease, resulted in an attenuated relative risk, this finding still supports the hypothesis that HCV infection might be associated with an increased risk of pancreatic cancer. Besides, the result indicated a moderate excessive risk of pancreatic cancer among HBV-infected patients according to different statistical approaches, but the size of the study cohort and the observed number of cancers were too small to draw a sound conclusion. Pancreatic cancer is more common in older age groups, and the small number of pancreatic cancers among the HBV cohort was probably an effect of the relatively young cohort, concordant with the epidemiology of chronic hepatitis B in Sweden.

The strengths of this register-based study include population-based cohort design, relatively large sample size, independently collected data on documentation of HCV/HBV notifications and pancreatic cancer occurrence and high completeness of follow-up.

The parallel (laboratory and clinician) notification system of HCV/HBV infections in Sweden has a high coverage of those with a diagnosed infection; it is estimated that about 75–80% of HCV infections are diagnosed, but there still remain unknown infections, not yet diagnosed or documented. In addition, a small portion of the reported patients could have a resolved infection, spontaneously or by treatment, this could (probably insignificant) lower the risk in the HCV and HBV cohort.

The number of unidentified HCV/HBV-coinfected individuals is probably low in the studied cohorts. However, in the HCV cohort there could be some patients who were never diagnosed with hepatitis B but have serologic markers of a past HBV infection. In these patients we cannot exclude the possibility of occult hepatitis B.

The biological mechanism of the association between HCV and pancreatic cancer is unclear. However, virtually, the pancreas and liver share the common blood vessels and ducts, and prior evidence demonstrated that the pancreas is a remote location for hepatitis virus inhabitation and replication (Hassan et al, 2008). HCV infection is associated with type 2 diabetes, which is both a risk factor and might be a consequence of pancreatic cancer (Mehta et al, 2000; Sangiorgio et al, 2000). Besides, previous studies reported that subclinical/acute pancreatitis (Katakura et al, 2005) and hyperlipasemia (Yoffe et al, 2003) may be extrahepatic manifestations of HCV infection. In addition, pancreatic involvement was observed among patients who suffered from chronic hepatitis infection, resulting in mild pancreatic damage accompanied with increased serum levels of pancreatic enzyme (Taranto et al, 1989; Katakura et al, 2005). Immune response may lead to chronic inflammation in the targeted organs after long time persistent infection with HCV. Therefore, hepatitis C virus conceivably serves as a biological agent that may indirectly have a role in inflammation-associated pancreatic carcinogenesis. Although still unclear to what extent chronic inflammation contributes to pancreatic cancer development, it is postulated that HCV can induce inflammatory microenvironment with high concentration of growth factors and cytokines. This may exert effects by accumulating alterations in driver genes and promoting cancer cell growth and proliferation.

HIV AIDS and Cancer

http://www.cancer.gov/cancertopics/causes-prevention/risk/infectious-agents/hiv-fact-sheet

Key Points

  • People infected with human immunodeficiency virus (HIV) have a higher risk of some types of cancer than uninfected people.
  • A weakened immune system caused by infection with HIV, infection with other viruses, and traditional risk factors such as smoking all contribute to this higher cancer risk.
  • Highly active antiretroviral therapy and lifestyle changes may reduce the risk of some types of cancer in people infected with HIV.
  • The National Cancer Institute (NCI) conducts and supports a number of research programs aimed at understanding, preventing, and treating HIV infection, acquired immunodeficiency syndrome-related cancers, and cancer-associated viral diseases.
  1. Do people infected with human immunodeficiency virus (HIV) have an increased risk of cancer?

Yes. People infected with HIV have a substantially higher risk of some types of cancer compared with uninfected people of the same age (1). Three of these cancers are known as “acquired immunodeficiency syndrome (AIDS)-defining cancers” or “AIDS-defining malignancies”: Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. A diagnosis of any one of these cancers marks the point at which HIV infection has progressed to AIDS.

People infected with HIV are several thousand times more likely than uninfected people to be diagnosed with Kaposi sarcoma, at least 70 times more likely to be diagnosed with non-Hodgkin lymphoma, and, among women, at least 5 times more likely to be diagnosed with cervical cancer (1).

In addition, people infected with HIV are at higher risk of several other types of cancer (1). These other malignancies include anal, liver, and lung cancer, and Hodgkin lymphoma.

People infected with HIV are at least 25 times more likely to be diagnosed with anal cancer than uninfected people, 5 times as likely to be diagnosed with liver cancer, 3 times as likely to be diagnosed with lung cancer, and at least 10 times more likely to be diagnosed with Hodgkin lymphoma (1).

People infected with HIV do not have increased risks of breast, colorectal, prostate, or many other common types of cancer (1). Screening for these cancers in HIV-infected people should follow current guidelines for the general population

HIV and cancer of the cervix

Z.M. Chirenje
bestpracticeobgyn April 2005; 19(2):269–276
http://dx.doi.org/10.1016/j.bpobgyn.2004.10.002

Cancer of the cervix is the second most common cause of cancer-related death in women worldwide, and in some low resource countries accounts for the highest cancer mortality in women. The highest burden of the HIV/AIDS epidemic is currently in sub-Saharan Africa, where more than half of the people infected are women who have no access to cervical cancer screening. The association between HIV and invasive cervical cancer is complex, with several studies now clearly demonstrating an increased risk of pre-invasive cervical lesions among HIV-infected women. However, there have not been significantly higher incidence rates of invasive cervical cancer associated with the HIV epidemic. The highest numbers of HIV-infected women are in poorly-resourced countries, where the natural progression of HIV disease in the absence of highly active antiretroviral treatment sometimes results in deaths from opportunistic infections before the onset of invasive cervical cancer. This chapter will discuss the association of HIV and cervical intraepithelial neoplasia, the treatment of pre-invasive lesions, and invasive cervical cancer in HIV-infected women. The role of screening and the impact of antiretroviral treatment on the progression of pre-invasive and invasive cancer will also be discussed.

Anal cancer: an HIV-associated cancer

Klencke BJ, Palefsky JM
Hematology/oncology Clinics of North America [2003, 17(3):859-872]
http://dx.doi.org:/1016/S0889-8588(03)00039-X

Although not yet included in the Centers for Disease Control definition of AIDS, anal cancer clearly occurs more commonly in HIV-infected patients. An effective screening program for those groups who are at highest risk might be expected to impact rates of anal cancer just as significantly as did cervical Pap screening programs for the incidence of cervical cancer. Despite a relatively low rate of progression from AIN to invasive cancer, the scope of the problem is enormous based on the prevalence of anal HPV infection and the size of the HIV-infected, at-risk population. Thus, the potential benefits of screening, detection, and the development of more effective therapy also are enormous. Currently, therapeutic HPV vaccines for AIN represent an exciting avenue of research in HPV-related anogenital disease. Invasive anal cancer and HSIL (which is believed to be the precursor lesion) are expected to become increasingly important health problems for both HIV-infected men and women as their life expectancy lengthens. Although HAART may have improved the ability of many to tolerate CMT, it appears that toxicity of this therapy continues to be a problem for a proportion of HIV-infected subjects. The acute side effects present specific challenges to the clinician and patient, have an immediate impact on the patient’s plan of care and dose intensity of the treatment, and ultimately may impact the outcome of the planned treatment. Late toxicity may influence the long-term quality of life. Small patient numbers, variable radiation therapy doses, limited information about viral load, and a potential confounding effect of higher CD4+ levels make it difficult to draw any conclusions about the effect of HAART on anal cancer outcome. Large, prospective studies will be required before solid conclusions about the impact of various factors on anal cancer prognosis and outcome can be drawn.

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Hirokazu Tamamura, and Nobutaka Fujii
Exp Opin on Ther Targets Dec 2005; 9(6): 1267-1282 http://dx.doi.org:/10.1517/14728222.9.6.1267

CXCR4 is the receptor of the chemokine CXCL12, which is involved in progression and metastasis of several types of cancer cells, HIV infection and rheumatoid arthritis. The authors developed selective CXCR4 antagonists, T22 and T140, initially as anti-HIV agents, which inhibit T cell line-tropic (X4-) HIV-1 infection through their specific binding to CXCR4. Recently, T140 analogues have also been shown to inhibit CXCL12-induced migration of breast cancer cells, leukaemia T cells, pancreatic cancer cells, small cell lung cancer cells, chronic lymphocytic leukaemia B cells, pre-B acute lymphoblastic leukaemia cells and so on in vitro. Biostable T140 analogues significantly suppressed pulmonary metastasis of breast cancer cells and melanoma cells in mice. Furthermore, these compounds significantly suppressed the delayed-type hypersensitivity response induced by sheep red blood cells and collagen-induced arthritis, which represent in vivo mouse models of arthritis. Thus, T140 analogues proved to be attractive lead compounds for chemotherapy of these problematic diseases. This article reviews recent research on T140 analogues, referring to several other CXCR4 antagonists.

Types of Cancer: AIDS/HIV related malignancies

http://cancer.northwestern.edu/cancertypes/cancer_type.cfm?category=1

People with HIV/AIDS are at high risk for developing certain cancers, such as Kaposi’s sarcoma, non-Hodgkin lymphoma, and cervical cancer. For people with HIV, these three cancers are often called “AIDS-defining conditions,” meaning that if a person with HIV has one of these cancers it can signify the development of AIDS. The connection between HIV/AIDS and certain cancers is not completely understood, but the link likely depends on a weakened immune system. Most types of cancer begin when normal cells begin to change and grow uncontrollably, forming a mass called a tumor. A tumor can be benign (noncancerous) or malignant (cancerous, meaning it can spread to other parts of the body). The types of cancer most common for people with HIV/AIDS are described in more detail below.

Kaposi’s sarcoma

Kaposi’s sarcoma is a type of skin cancer, which has traditionally occurred in older men of Jewish or Mediterranean descent, young men in Africa, or people who have received organ transplantation. Today, Kaposi’s sarcoma is found most often in homosexual men with HIV/AIDS and related to an infection with the human herpesvirus 8 (HHV-8). Kaposi’s sarcoma in people with HIV is often called epidemic Kaposi’s sarcoma. HIV/AIDS-related Kaposi’s sarcoma causes lesions to arise in multiple sites in the body, including the skin, lymph nodes, and organs such as the liver, spleen, lungs, and digestive tract.

Non-Hodgkin lymphoma

HIV/AIDS-related NHL is the second most common cancer associated with HIV/AIDS, after Kaposi’s sarcoma. There are many different subtypes of NHL. The most common subtypes of NHL in people with HIV/AIDS are primary central nervous system lymphoma (affecting the brain and spinal fluid), found in 20% of all NHL cases in people with HIV/AIDS, primary effusion lymphoma (causing fluid to accumulate around the lungs or in the abdomen), or intermediate and high-grade lymphoma. More than 80% of lymphomas in people with HIV/AIDS are high-grade B-cell lymphoma, while 10% to 15% of lymphomas among people with cancer who do not have HIV/AIDS are of this type. It is estimated that between 4% and 10% of people with HIV/AIDS develop NHL.

Other types of cancer

Other, less common types of cancer that may develop in people with HIV/AIDS are Hodgkin’s lymphoma, angiosarcoma (a type of cancer that begins in the lining of the blood vessels), anal cancer, liver cancer, mouth cancer, throat cancer, lung cancer, testicular cancer, colorectal cancer, and multiple types of skin cancer including basal cell carcinoma, squamous cell carcinoma, and melanoma.

Treatment options for the most common treatments for HIV/AIDS-related cancers are listed by the specific type of cancer. Treatment options and recommendations depend on several factors, including the type and stage of cancer, possible side effects, and the patient’s preferences and overall health.

Palliative care can help a person at any stage of illness. People often receive treatment for the cancer and treatment to ease side effects at the same time. In fact, patients who receive both often have less severe symptoms, better quality of life, and report they are more satisfied with treatment.

Palliative treatments vary widely and often include medication, nutritional changes, relaxation techniques, and other therapies. You may also receive palliative treatments similar to those meant to eliminate the cancer, such as chemotherapy, surgery, and radiation therapy.

It is extremely important that all patients with HIV/AIDS and an associated cancer receive treatment with highly active antiretroviral treatment (HAART) both during the cancer treatments and afterwards. HAART can effectively control the virus in most patients. Better control of the HIV infection decreases the side effects of many of the treatments, may decrease the chance of a recurrence, and can improve a patient’s chance of recovery from the cancer.

The treatment of HIV/AIDS-related Kaposi sarcoma usually cannot cure the cancer, but it can help relieve pain or other symptoms. This can be followed by palliative care for Kaposi sarcoma. Antiviral treatment for HIV/AIDS helps reduce a person’s chance of getting Kaposi sarcoma and can reduce the severity of Kaposi sarcoma. HAART helps treat the tumor and reduce the symptoms associated with Kaposi sarcoma for people with HIV/AIDS. It is usually used before other treatments, such as chemotherapy.

Curettage and electrodesiccation. In this procedure, the cancer is removed with a curette, a sharp, spoon-shaped instrument. The area can then be treated with electrodesiccation, which uses an electric current to control bleeding and kill any remaining cancer cells. Many patients have a flat, pale scar from this procedure.

Cryosurgery. Cryosurgery, also called cryotherapy or cryoablation, uses liquid nitrogen to freeze and kill cells. The skin will later blister and shed off. This procedure will sometimes leave a pale scar. More than one freezing may be needed.

In photodynamic therapy, a light-sensitive substance is injected into the lesion that stays longer in cancer cells than in normal cells. A laser is directed at the lesion to destroy the cancer cells.

Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells. A doctor who specializes in giving radiation therapy to treat cancer is called a radiation oncologist. The most common type of radiation treatment is called external-beam radiation therapy, which is radiation given from a machine outside the body. When radiation therapy is given using implants, it is called internal radiation therapy or brachytherapy. External-beam radiation therapy may be given as a palliative treatment. A radiation therapy regimen (schedule) usually consists of a specific number of treatments given over a set period of time.

Side effects from radiation therapy may include fatigue, mild skin reactions, upset stomach, and loose bowel movements. Most side effects go away soon after treatment is finished. Learn more about radiation therapy.

Chemotherapy may help control advanced disease, although curing HIV/AIDS-related Kaposi sarcoma with chemotherapy is extremely rare. Usually, for HIV/AIDS-related Kaposi sarcoma, chemotherapy is used to help relieve symptoms and to lengthen a patient’s life. Common drugs for Kaposi sarcoma include: liposomal doxorubicin (Doxil), paclitaxel (Taxol, LEP-ETU, Abraxane), and vinorelbine (Navelbine, Alocrest).

The side effects of chemotherapy depend on the individual and the dose used, but they can include fatigue, risk of infection, nausea and vomiting, hair loss, loss of appetite, and diarrhea. These side effects usually go away once treatment is finished.

HIV/AIDS-related Kaposi sarcoma may receive alpha-interferon (Roferon-A, Intron A, Alferon), which appears to work by changing the surface proteins of cancer cells and by slowing their growth. Immunotherapy is generally used for people who are in the good-risk category in the immune system (I) factor of the TIS staging system (see Stages). The most common side effects of alpha-interferon are low levels of white blood cells and flu-like symptoms.

The main treatments for HIV/AIDS-related non-Hodgkin lymphoma are chemotherapy, targeted therapy, and radiation therapy.

Treatments for women with the precancerous condition called CIN (see   Overview) are generally not as effective for women with HIV/AIDS because of a weakened immune system. Often, the standard treatment for HIV/AIDS can lower the symptoms of CIN.

Women with invasive cervical cancer and HIV/AIDS that is well-controlled with medication, generally receive the same treatments as women who do not have HIV/AIDS. Common treatment options include surgery, radiation therapy, and chemotherapy.

Cytokines in cancer pathogenesis and cancer therapy

Glenn Dranoff
Nature Reviews Cancer Jan 2004; 4(11-22) http://dx.doi.org:/10.1038/nrc1252

The mixture of cytokines that is produced in the tumor microenvironment has an important role in cancer pathogenesis. Cytokines that are released in response to infection, inflammation and immunity can function to inhibit tumor development and progression. Alternatively, cancer cells can respond to host-derived cytokines that promote growth, attenuate apoptosis and facilitate invasion and metastasis. A more detailed understanding of cytokine–tumor-cell interactions provides new opportunities for improving cancer immunotherapy.

Dendritic Cells as Therapeutic Vaccines against Cancer
Jacques Banchereau and A. Karolina Palucka
Nature Reviews Immunology APR 2005; 5:296-306
http://cnc.cj.uc.pt/BEB/private/pdfs/2007-2008/Immunology/E/Rev_paper_E4.pdf

Mouse studies have shown that the immune system can reject tumours, and the identification of tumor antigens that can be recognized by human T cells has facilitated the development of immunotherapy protocols. Vaccines against cancer aim to induce tumor-specific effector T cells that can reduce the tumor mass, as well as tumor-specific memory T cells that can control tumor relapse. Owing to their capacity to regulate T-cell immunity, dendritic cells are increasingly used as adjuvants for vaccination, and the immunogenicity of antigens delivered by dendritic cells has now been shown in patients with cancer. A better understanding of how dendritic cells regulate immune responses will allow us to better exploit these cells to induce effective anti-tumor immunity.

Vaccines against infectious agents are one success of immunology and have spared countless individuals from diseases such as polio, measles, hepatitis B and tetanus8 . However, progress in the development of vaccines against infectious agents has been largely empirical and not always successful, as many infectious diseases still evade the immune system, particularly chronic infections such as tuberculosis, malaria and HIV infection. Further progress will be made through rational design based on our increased understanding of how the immune system works and how the induction of protective immunity is regulated. The same principle applies to vaccines against cancer, particularly as cancer is a chronic disease, and when it becomes clinically visible, tumor cells and their products have already been interacting with and affecting host cells for a considerable time to ensure the survival of the tumor. Ex vivo-generated, antigen-loaded DCs have now been used as vaccines to improve immunity9 . Numerous studies in mice have shown that DCs loaded with tumor antigens can induce therapeutic and protective antitumor immunity10. The immunogenicity of antigens delivered by DCs has been shown in patients with cancer9 or chronic HIV infection11, thereby providing proof of principle that using DCs as vaccines can work. Despite this, the efficacy of therapeutic vaccination against cancer has recently been questioned12 because of the undeniably limited rate of objective tumor regressions that has been observed in clinical studies so far. However, the question is not whether DC vaccines work but how to orient further studies to refine the immunological and clinical parameters of vaccination with DCs to improve its efficacy.

Vaccines against cancer Early studies in mice showed that the immune system can recognize and reject tumours13 and that immunodeficient mice (lacking interferon-γ (IFN-γ) and recombination-activating gene 2) have an increased incidence of cancer14 (BOX 1). In humans, the incidence of some cancers is increased in immunodeficient patients15 and is increased with age, owing to Immunosenescence16. These observations support the scientific rationale for immunotherapy for cancer. The term immunotherapy refers to any approach that seeks to mobilize or manipulate the immune system of a patient for therapeutic benefit17. In this regard, there are numerous strategies for improving the resistance of a patient to cancer. These include non-specific activation of the immune system with microbial components or cytokines, antigen-specific adoptive immunotherapy with antibodies and/or T cells, and antigen-specific active immunotherapy (that is, vaccination). The main limitation of using antibodies is that target proteins need to be expressed at the cell surface. By contrast, targets for T cells are usually peptides derived from intracellular proteins, which are presented at the cell surface in complexes with MHC molecules18. The identification of defined tumor antigens in humans19,20 prompted the development of adoptive T-cell therapy. Yet, the most attractive strategy is vaccination, which is expected to induce both therapeutic T-cell immunity (in the form of tumor-specific effector T cells) and protective T-cell immunity (in the form of tumor-specific memory T cells that can control tumor relapse)21–23. Numerous approaches for the therapeutic vaccination of individuals who have cancer have been developed, including the use of the following: autologous and allogeneic tumor cells (which are often modified to express various cytokines), peptides, proteins and DNA vaccines9,23–26. The observed results are variable; however, in many cases, a tumour-specific immune response has been induced, and tumor regressions, albeit limited, have occurred. These approaches rely on random encounter of the vaccine with host DCs. A lack of encounter of the vaccine antigen with DCs might result in the absence of an immune response. Alternatively, an inappropriate encounter — for example, with unactivated DCs or with the ‘wrong’ subset of DCs — might lead to silencing of the immune response27. Both of these situations could explain some of the shortcomings of current cancer vaccines. Furthermore, we do not know how tumor antigens need to be delivered to DCs in vivo to elicit an appropriate immune response.

Immature and mature dendritic cells have different functions. A | Immature dendritic cells (DCs) induce tolerance. Tissue DCs constantly sample their environment, capture antigens and migrate in small numbers to draining lymph nodes. In the absence of inflammation, the DCs remain in an immature state, and antigens are presented to T cells in the lymph node without costimulation, leading to either the deletion of T cells or the generation of inducible regulatory T cells. B | Mature DCs induce immunity. Tissue inflammation induces the maturation of DCs and the migration of large numbers of mature DCs to draining lymph nodes. The mature DCs express peptide–MHC complexes at the cell surface, as well as appropriate co-stimulatory molecules. This allows the priming of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTLs), the activation of B cells and the initiation of an adaptive immune response. To control the immune response, CD4+CD25+ regulatory T (TReg)-cell populations are also expanded. [ADCC, antibody-dependent  cell-mediated cytotoxicity; NK, natural killer; TCR, T-cell receptor].

Box 1 |

Mice

  • The immune system can reject tumors
  • Immune-mediated rejection of chemically induced tumours13
  • Increased cancer incidence in immunodeficient mice14

Humans

  • Increased cancer incidence in immunodeficient patients15
  • Increased cancer incidence with age (immunosenescence)16
  • Cancer regression in patients with paraneoplastic neurological disorders that are mediated by onconeuronal antibodies and specific CD8+ T cells136

Dendritic cells DC subsets. There are thought to be two main pathways of differentiation into DCs2,31 (FIG. 2). The myeloid pathway generates two subsets: Langerhans cells, which are found in stratified epithelia such as the skin; and interstitial DCs, which are found in all other tissues32. The lymphoid pathway generates plasmacytoid DCs (pDCs), which secrete large amounts of type I IFNs (IFN-α and IFN-β) after viral infection33,34. DCs and their precursors show remarkable functional plasticity. For example, pDCs form one of the first barriers to the expansion of intruding viruses, thereby functioning, through the release of type I IFNs, as part of the innate immune response. Subsequently, these cells differentiate into DCs that can present antigens to T cells, thereby functioning as members of the adaptive immune system35,36. Monocytes can differentiate into either macrophages, which function as scavengers, or DCs that induce specific immune responses37,38. Different cytokines skew the in vitro differentiation of monocytes into DCs with different phenotypes and functions (FIG. 3). So, after activation (for example, by granulocyte/ macrophage colony-stimulating factor, GM-CSF), monocytes that encounter interleukin-4 (IL-4) become DCs known as IL-4-DCs29,30,39. By contrast, after encounter with IFN-α, tumour-necrosis factor (TNF) or IL-15, activated monocytes differentiate into IFN-α-DCs40–43, TNF-DCs44 or IL-15-DCs45, respectively. Whether, in vivo, all interstitial DCs are derived from monocytes remains to be established, but myeloid DCs that are isolated from human peripheral blood also give rise to different DC types after exposure to different cytokines. Each of these DC subsets has both common and unique biological functions, which are determined by a unique combination of cell-surface molecule expression and cytokine secretion. For example, whereas IL-4-DCs are a homologous population of immature cells that is devoid of Langerhans cells, TNFDCs are heterogeneous and include both CD1a+ Langerhans cells and CD14+ interstitial DCs44.In vitro experiments showed that Langerhans cells and interstitial DCs that were generated from cultures of CD34+ hematopoietic progenitors differ in their capacity to activate lymphocytes: interstitial DCs induce the differentiation of naive B cells into immunoglobulin-secreting plasma cells4,32, whereas Langerhans cells seem to be particularly efficient activators of cytotoxic CD8+ T cells. They also differ in their cytokine-secretion pattern (only interstitial DCs produce IL-10) and their enzymatic activity4,32, which might be fundamental for the selection of peptides that are presented to T cells. Indeed, different enzymes are likely to degrade a given antigen into different sets of peptides, as has recently been shown for the HIV protein Nef 46. This then leads to different sets of peptide–MHC complexes being presented and thereby to distinct repertoires of antigen-specific T cells. So, these unique DCs are likely to yield unique immune effectors, thereby allowing the broad immune response that is required to combat permanently evolving microorganisms and tumors.

Distinct DC subsets induce distinct types of immune response. DCs have a crucial role in determining the type of response that is induced. There is evidence that either polarized DCs or distinct DC subsets might provide T cells with different signals that determine the class of immune response31. So, in mice, splenic CD8α+ DCs prime naive CD4+ T cells to produce TH1 cytokines in a process that involves IL-12, whereas splenic CD8α– DCs prime naive CD4+ T cells to produce TH2 cytokines47,48. Furthermore, this polarization into different T-cell subsets also depends on the signal received by a DC, as shown by the induction of IL-12 production and polarization towards TH1 cells when DCs are activated with Escherichia coli lipopolysaccharide (LPS), but the absence of IL-12 production and polarization towards TH2 cells when the same type of DC is exposed to LPS from Porphyromonas gingivalis 49. In humans, CD40 ligand (CD40L)-activated monocyte-derived DCs prime TH1-cell responses through an IL-12-dependent mechanism, whereas pDCs activated with IL-3 and CD40L have been shown to secrete negligible amounts of IL-12 and to prime TH2-cell responses50. So, both the type of DC subset and the activation signals to which DCs are exposed are important for polarization of T cells.

Mouse proof-of-principle in vivo studies

  • Ex vivo-generated, antigen-loaded dendritic cells (DCs) induce antigen-specific T-cell immunity137
  • Ex vivo gene-loaded DCs can induce humoral immunity138
  • Ex vivo-generated, antigen-loaded DCs induce tumor-specific immunity139,140
  • Ex vivo-generated DCs are superior to other types of vaccine141
  • Ex vivo-generated immature DCs induce tolerance142
  • Combination therapy with ex vivo-generated DCs improves vaccine efficacy112,113

This is an important parameter in vaccination against cancer, as type 1 immunity (including IFN-γ secretion) is desirable, whereas type 2 immunity (including IL-4 or IL-10 secretion) is considered deleterious. DCs and immune tolerance. DCs can induce and maintain immune tolerance27, both central and peripheral.

Central Tolerance depends on mature thymic DCs, which are essential for the deletion of newly generated T cells that have a receptor that recognizes self-components51. However, central tolerance might not be effective for all antigens. Furthermore, many self-antigens might not have access to the thymus, and others are only expressed later in life. So, there is a requirement for Peripheral Tolerance, which occurs in lymphoid organs and is mediated by immature DCs (FIG. 1a). Immature DCs present tissue antigens to T cells in the absence of appropriate co-stimulation, leading to T-cell Anergy or deletion27 or to the development of IL-10-secreting Inducible Regulatory T Cells52,53. The research groups of Nussenzweig and Steinman54 have elegantly shown that fusion proteins targeted to immature DCs lead to the induction of antigen-specific tolerance. By contrast, concomitant activation of these DCs with CD40- specific antibody results in a potent immune response, because the DCs are induced to express a large number of co-stimulatory molecules55. However, mature DCs might also contribute to peripheral tolerance by promoting the clonal expansion of naturally occurring CD4+CD25+ REGULATORY T (TReg) CELLS56, as discussed later. Therefore, the biology of DCs offers several targets for the control of cellular immunity. The parameters that need to be considered include DC-related factors, host-related factors and combining DC vaccines with other therapies.

Subsets of human dendritic cells. (Fig not shown). The population of dendritic cells (DCs) in the peripheral blood, which can be mobilized by treatment with FLT3L (fms-related tyrosine kinase 3 ligand), contains both CD11c+ myeloid DCs and CD11c– plasmacytoid DCs. So far, most studies of DCs have been carried out with DCs generated by culturing monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4); this simple procedure yields a homogenous population of DCs that resemble interstitial DCs, and the population is devoid of Langerhans cells. These DCs are immature and require exogenous factors for maturation. DCs can also be generated by culturing CD34+ haematopoietic progenitor cells (HPCs) or peripheral-blood monocytes with GM-CSF and tumour-necrosis factor (TNF). In this way, two DC subsets can be obtained: Langerhans cells, which might have improved efficacy for eliciting cytotoxic T lymphocytes; and interstitial DCs, which resemble monocyte-derived DCs. Adding IL-4 to CD34+ HPC cultures in the presence of GM-CSF and TNF inhibits the differentiation of Langerhans cells. [Green boxes indicate cell types that can be induced by culture with GM-CSF and TNF. Yellow boxes indicate cell types that can be induced by culture or mobilization with FLT3L].

Plasticity of monocyte-derived dendritic cells. (Fig not shown). Activated monocytes can differentiate into different types of dendritic cell (DC) after encounter with different cytokines. These distinct DCs will influence the differentiation of lymphocytes into immune effectors with different functions, leading to varied immune responses. For example, interleukin-15-DCs (IL-15- DCs) are remarkably more efficient at priming and maturation of rare antigen-specific cytotoxic T lymphocytes (CTLs) than are IL-4-DCs. Thymic stromal lymphopoietin-DCs (TSLP-DCs) induce CD4+ T cells to differentiate into pro-inflammatory T helper 2 (TH2) cells, which secrete large amounts of IL-13 and tumor-necrosis factor (TNF)143, whereas interferon-α-DCs (IFN-α-DCs) induce CD4+ T cells to differentiate into TH1 cells, which secrete IFN-γ and IL-10. The properties and function of TNF-DCs remain to be determined. [FLT3L, fms-related tyrosine kinase 3 ligand; GM-CSF, granulocyte/macrophage colony-stimulating factor].

Antigen loading. Loading MHC class I and class II molecules at the cell surface of DCs with peptides derived from defined antigens is the most commonly used strategy for DC-based vaccination22,87. Although this technique is important for proof-of-principle studies, the use of peptides has limitations: the restriction of a peptide to a given HLA type; the limited number of well-characterized Tumor-Associated Antigens; the relatively rapid turnover of exogenous peptide– MHC complexes, resulting in comparatively low antigen presentation by the time that the DC arrives in the draining lymph node after injection; and the induction of a restricted repertoire of T-cell clones, thereby limiting the ability of the immune system to control tumor-antigen variation. Yet another level of complexity is brought about by the use of MODIFIED HETEROCLITIC PEPTIDES. Some synthetic peptides, even those derived from immune-dominant antigens, do not bind MHC class I molecules with high affinity, possibly explaining their limited immunogenicity in vivo88. Therefore, the generation of peptide analogues with increased affinity for MHC class I molecules (known as heteroclitic peptides) could be used to improve peptide immunogenicity89,90. However, recent elegant studies in patients with malignant melanoma show that T cells elicited in vivo by vaccination with heteroclitic MART1 (melanoma antigen recognized by autologous T cells) or glycoprotein 100 (gp100) peptide show poor recognition of the endogenous melanoma-derived peptide and less efficient tumor-cell lysis compared with T cells specific for the native peptide91.

Immunoregulatory mechanisms

Naturally occurring CD4+CD25+ regulatory T cells

Cell-mediated suppression independent of interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β);
clonal expansion is regulated by mature dendritic cells (DCs)

Inducible regulatory T cells

Cytokine-mediated suppression through IL-10 and/or TGF-β; induction and clonal expansion is regulated by immature DCs

Natural killer T cells

Cytokine-mediated suppression through IL-13

Vaccine-induced B cells?

Cytokine-mediated regulation through IL-4, IL-6 and IL-10; competition with DCs for antigen uptake

Tumor-specific interferon-γ-secreting T cells?

Immunoediting and selection of escape variants (not discussed in main text)

Immune correlates of efficacy of dendritic-cell-based vaccines

  • Induction of broad tumour-specific T-cell immunity: T cells specific for several tumour antigens
  • Induction of effector T cells: T cells with immediate capacity to recognize tumour antigens and secrete cytokines such as tumour-necrosis factor and interferon-γ
  • Induction of memory T cells: T cells that secrete interleukin-2 and proliferate on re-exposure to tumour antigen
  • Induction of T cells that kill tumour cells
  • Decreased number of T cells with regulatory function

DCs are an attractive target for therapeutic manipulation of the immune system to increase otherwise insufficient immune responses to tumour antigens. However, the complexity of the DC system requires rational manipulation of DCs to achieve protective or therapeutic immunity. So, further research is needed to analyse the immune responses induced in patients by distinct ex vivo-generated DC subsets that are activated through different pathways. The ultimate ex vivo-generated DC vaccine will be heterogeneous and composed of several subsets, each of which will target a specific immune effector. These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs, which is the next step in the development of DC-based vaccination. Indeed, distinct DC subsets express unique cell-surface molecules, such as different lectins131: Langerhans cells express langerin, which is crucial for the formation of Birbeck granules132,133; interstitial DCs express DCSIGN (dendritic-cell-specific intercellular-adhesionmolecule-3-grabbing non-integrin), which is involved in interactions with T cells and in DC migration but is also used by pathogens (such as HIV) to hijack the immune system; and pDCs express yet another lectin, BDCA2 (blood DC antigen 2)134,135. Such differential expression of cell-surface molecules might allow specific in vivo targeting of DC subsets for induction of the desired type of immune response.

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

Elke Jäger, Yao-Tseng Chen, Jan W. Drijfhout, Julia Karbach, et al.
J Exp Med. 1998 Jan 19; 187(2): 265–270.
A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2−, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

There is growing evidence for humoral and cellular immune recognition of cancer by the autologous human host (1–6). Based on CTL-dependent lysis of cultured melanoma cell lines, several categories of autoimmunogenic tumor antigens have been characterized, including differentiation antigens of specific cell lineages (7–9), individual antigens caused by point mutations (10, 11), and tumor antigens, such as MAGE, which are expressed in a variable proportion of different tumor types, but are silent in most normal tissues except the testis (12). CTL responses against melanoma antigens induced by peptide vaccines in vivo have been associated with a favorable development of advanced melanoma in some patients (6, 13). As immunoselection of antigen-negative tumor cell variants has been observed during peptide vaccination (14), the molecular characterization of additional CTL-defined tumor antigens is needed to develop polyvalent vaccines with broader immunotherapeutic effects.

Sahin et al. have recently introduced a powerful new methodology for identifying human tumor antigens eliciting humoral immune response (5). The method has been called SEREX, for serological expression cloning of recombinant cDNA libraries of human tumors. Novel and previously defined tumor antigens have been identified by the SEREX method, including MAGE-1 and tyrosinase, both originally identified by cloning the epitopes recognized by CTLs. Thus, antibody screening of cDNA libraries prepared from human tumors can be used to identify antigens eliciting a cellular immune response, including CTLs, circumventing the need for established cultured autologous cell lines and stable CTL lines.

We have recently identified a novel human tumor antigen by SEREX analysis of a human esophageal cancer (15). The antigen, NY-ESO-1, belongs to a growing number of human tumor antigens we have called “cancer–testis” antigens that include MAGE, GAGE, BAGE (1), and SSX2 (HOM-MEL-40) (5, 16). These antigens have the following characteristics: (a) they are expressed in a variable portion of a wide range of cancers, (b) their normal tissue expression is generally restricted to the testis, and (c) they are generally coded for by genes on the X chromosome. In a recent survey of sera from normal individuals and cancer patients, antibodies against NY-ESO-1 were found in ∼10% of patients with melanoma, ovarian cancer, and other cancers, but not in normal individuals (Stockert, E., manuscript in preparation). One patient with a high NY-ESO-1 antibody response was found to have specific CTL reactivity against cultured autologous melanoma cells. In the present study, we report that NY-ESO-1 encodes the CTL target in this patient and identify the NY-ESO-1 peptides that are recognized.

High-titer Antibody Reactivity against NY-ESO-1.

Melanoma patient NW38 presented with extensive metastases to inguinal lymph nodes having large areas of necrosis. Reverse transcriptase PCR of tumor RNA showed that this tumor expressed NY-ESO-1. Based on the hypothesis that exposure of the immune system to large amounts of intracellular tumor proteins released from the necrotic tumor might elicit a strong humoral immune response, the serum of patient NW38 was tested for specific reactivity against recombinant NY-ESO-1 protein. Fig. ​Fig.11 shows the reactivity of NW38 serum with the recombinant NY-ESO-1 protein, with a lysate of NY-ESO-1–transfected COS-7 cells, and with a lysate of the autologous NY-ESO-1 messenger RNA–positive tumor cell line NW-MEL-38. A 22-kD protein species was identified in both cell lysates, and comigrated with the purified recombinant NY-ESO-1 protein. The identity of this protein species as NY-ESO-1 was further confirmed by using an anti–NY-ESO-1 mouse monoclonal antibody. Reactivity against recombinant NY-ESO-1 protein was still detectable at a serum dilution of 1:100,000. No reactivity was detected against a lysate of untransfected COS-7 cells.
The correlation between NY-ESO-1 expression and NW38-IVS-1 reactivity suggested NY-ESO-1 as the antigenic target. To prove this, COS-7 cells were transfected with NY-ESO-1 cDNA and different MHC class I molecules and used as targets for NW38-IVS-1. Reactivity was measured in a standard TNF-α release assay. TNF release was found after stimulation of NW38-IVS-1 with COS-7 cells cotransfected with HLA-A2− and NY-ESO-1 cDNA. No reactivity was detected after stimulation with cotransfectants of pcDNA3.1(−)-NY-ESO-1− and pcDNA1Amp-HLA-A1 cDNA, COS-7 cells transfected with pcDNA3.1(−), or untransfected COS-7 cells (Fig. ​(Fig.3).3).

Peptide-specific CTLs.

26 different peptides encoded by NY-ESO-1 with theoretical binding motifs to the HLA-A2.1 molecule were tested for specific recognition by NW38-IVS-1. The target cells were peptide-pulsed T2 cells. Of these 26 peptides, three were recognized by NW38-IVS-1 as determined by a standard51Cr–release assay (Table ​(Table1).1). The peptide sequences SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT are located between positions 155 and 167 of the NY-ESO-1 protein (15), and show overlapping sequences. The 11-mer SLLMWITQCFL (2 in Table ​Table1)1) and the 9-mer SLLMWITQC (12 in Table ​Table1)1) consist of identical amino acids at positions 1–9.

To provide additional confirmation of the peptide specificity, the 26 synthetic peptides were individually incubated with HLA-A2–transfected COS-7 cells and tested in the TNF release assay. Consistent with the results of 51Cr–release assay, specific TNF-α release was detected in tests with peptides SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. NY-ESO-1/HLA-A2 transfectants were used as a positive control in these assays (Fig. ​(Fig.4).4).

The search for tumor antigens that induce specific immune responses in cancer patients is the ongoing challenge in tumor immunology. Evidence for a specific humoral response to human cancer came from serological analysis of cell surface reactivity of sera from cancer patients for autologous cancer cells, an approach called autologous typing (4). However, with only a few exceptions, this approach did not allow for the structural definition of the antigenic target. An autologous typing system also provided the first evidence for the development of CTLs with specificity for human melanoma cells (3, 17, 21–24). Using specific antitumor CTLs as probes, a number of CTL targets have been cloned on the basis of MHC class I–restricted recognition (1, 6). However, this approach involves cultured cancer cell lines and stable CTL lines from the same patient, two requirements that cannot easily be met with many tumor types. With the demonstration that genes coding for CTL-recognized tumor antigens elicit humoral immunity and can be cloned by SEREX methodology, a technically less demanding approach defining immunogenic tumor antigens is now available, one that extends the range of analysis to tumor types that are not easily adaptable to in vitro growth and are not sensitive targets for CTLs. A number of novel tumor antigens have been defined by SEREX, including two new members of the cancer–testis antigenic family, SSX2 (HOM-MEL-40) (5, 16), and NY-ESO-1 (15).

In this study, we identified a melanoma patient, NW38, with high-titered antibody against NY-ESO-1. This patient had a large and highly necrotic tumor, and the sustained release of intracellular antigens that are usually inaccessible to the immune system may account for the high NY-ESO-1 titer. The establishment of an autologous cell line that typed NY-ESO-1 positive provided target cells for assessing CTL reactivity in this patient. A CTL line was established from this patient that lysed the autologous melanoma cell line in an HLA-A2–restricted fashion. Using target cells transfected with NY-ESO-1 and HLA-A2, the specificity of CTL reactivity was found to be coded by NY-ESO-1. Computer analysis of the NY-ESO-1 sequence identified 26 peptides with HLA-A2–binding motifs. Screening of these peptides presented by T2 cells identified three sequences that were confirmed to be specifically recognized by NW38-IVS-1. This is the first conclusive demonstration of simultaneous antibody and CTL responses against a cancer–testis antigen in a single patient.

The strategy used in this study to generate and analyze CTL reactivity to a SEREX-defined antigen can be used as a model for investigating cellular immune responses to the growing list of other SEREX antigens. Identification of clones in SEREX requires high-titered IgG antibody, and the development of such antibodies requires the help of CD4+ T cells. In this sense, SEREX can be thought of as a method to define the CD4+ T cell repertoire to human tumor antigens. Also, the presence of both NY-ESO-1 antibody and CTLs in patient NW38 suggests that screening for an antibody response may be a simple and effective way to identify patients with concomitant CTL reactivity, and this possibility is now being tested in other patients with NY-ESO-1 antibody. In the absence of autologous tumor cell lines, CD8+ T cells can be stimulated with autologous antigen-presenting cells that have been transfected with the coding gene or fed purified protein antigens. A similar strategy can be used to identify peptide targets for CD4+ T cells.

A major objective in defining immunogenic human tumor targets is to explore their use in the development of cancer vaccines, and a number of clinical trials with various vaccine constructs are currently underway. Although tumor regression is the desired goal of a therapeutic vaccine, this end point cannot be expected to be an effective way to develop maximally immunogenic tumor vaccines. For this purpose, reliable immunological assays are needed to monitor the specificity and strength of specific immune reactions generated by the vaccine. With the exception of vaccines aimed at inducing a humoral immune response such as GM2 ganglioside vaccines, most vaccine trials are designed to stimulate cellular immunity, particularly the development of CTLs and CD4+ T cells. These have been difficult to detect in vaccine trials with MAGE peptides (25), and difficult to interpret in trials with vaccines containing melanocyte differentiation antigens, since CTLs against these antigens can be generated in vitro from nonvaccinated melanoma patients as well as normal individuals (26, 27). However, de novo induction and increase of preexisting CTL reactivity have been detected after vaccination with melanocyte differentiation antigens and observed to be associated with cancer regressions in a limited number of patients (13). The demonstration of a simultaneous antibody and CTL response to NY-ESO-1 in the same patient suggests that serological methods may be useful in monitoring vaccine trials with NY-ESO-1 and other tumor antigens eliciting a humoral immune response.

9.1.3 Monoclonal Antibodies in Cancer Therapy

R K Oldham
JCO September 1983; 1(9): 582-590
http://jco.ascopubs.org/content/1/9/582.short


The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal antibodies, the possibility of specifically targeted therapy is being considered. Early trials of monoclonal antibody in experimental animals and humans have indicated its ability to traffic to specific tumor sites and to localize on or around the tumor cells displaying antigens to which the antibody is directed. This evidence of specific targeting, along with preliminary evidence of therapeutic efficacy for monoclonal antibodies and immunoconjugates with drugs, toxins, and isotopes is encouraging. The current status of clinical trials with monoclonal antibodies is reviewed and an example of the experimental approach for the development of immunoconjugates in animal models is presented.

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

Robert K. Oldham, Robert O. Dillman
JCO Apr 10, 2008: 26(11): 1774-1777
http://dx.doi.org:/10.1200/JCO.2007.15.7438

In 1983, it was apparent that a major problem with current modalities of cancer treatment was the lack of specificity for the cancer cell.1 It was predicted that a major advancement in treatment of cancer would be the development of a class of agents that would have a greater degree of specificity for the tumor cell. Based on many animal studies and the treatment of fewer than 100 patients, it was evident in 1983 that monoclonal antibodies would be that major advance.

The first patient treated in the United States with monoclonal antibody therapy was a patient with non-Hodgkin’s lymphoma.2 Nadler et al2 described the treatment using a murine monoclonal antibody designated AB 89. Although treatment was not successful in inducing a significant clinical response, it did represent the first proof of principle in humans that a monoclonal antibody could induce transient decreases in the number of circulating tumor cells, induce circulating dead cells, and form complexes with circulating antigen, all with minimal toxicity to the patient. Antibody could be detected on the surface of circulating lymphoma cells, and free antigen in the serum decreased with each infusion of antibody. After two courses of milligram doses of AB 89, a final and third course with 1.5 g of antibody was administered during a 6-hour period. A marked reduction in circulating antigen was noted, but these studies suggested to the authors that the quantity of circulating antigen was too great to effectively deliver AB 89 to the patient’s tumor cells in a therapeutically effective manner.2

In the Journal of Clinical Oncology review article cited earlier,1 evidence was reviewed from animal tumor models that clearly demonstrated both specificity and therapeutic efficacy with little serious toxicity. Whereas passive serotherapy of human cancer had shown little success,3 it was apparent in the earlier review that monoclonal antibodies could be used in the treatment of leukemia and lymphoma.4,5 In 1983, a review of the literature revealed approximately 10 published studies and one in-press article of therapeutic trials of monoclonal antibody therapy in humans. All of these studies used murine monoclonal antibodies and were phase I/II studies. Most were in leukemia or lymphoma, but the earliest solid tumor studies were also underway in melanoma6 and GI cancer.1

By 1983, the pioneers in monoclonal antibody research believed that a new era of cancer therapy had begun, and for the first time, true specific and targeted therapy was underway using hybridoma technology to produce monoclonal antibodies with exquisite specificity. It was also apparent, based on animal model studies, that monoclonal antibodies could be a vehicle to bring immunoconjugate therapy to the clinic by conjugating monoclonal antibodies to drugs, toxins, and radioisotopes using the specificity of the monoclonal antibody to carry enhanced killing capacity directly to the tumor cells. Thus, the era of monoclonal antibody therapy, as well as immunoconjugate therapy, had begun.

Although there was much excitement (and skepticism) about this new treatment modality (the use of a form of biologic therapy with great specificity in patients with advanced cancer) there were also problems and limitations. As presented in Table 1, there were clinical toxicities with murine monoclonal antibodies, most of which were secondary to the interaction with the target antigen.7 However, the major limitation was their immunogenicity. Murine proteins are highly immunogenic, and it was soon found that only a few infusions of these foreign proteins could be given to patients with cancer because of the development of human antimouse antibody.8 Another problem quickly became apparent, in that some of the antigens on cancer cell surfaces modulated off the surface and into the circulation when antibody attached. Modulation could also cause internalization of the complex. It was recognized that this could represent a therapeutic advantage by using the antibody as carrier to internalize the toxic component of an immunoconjugate, potentially making it more therapeutically active.

In 1983, few specific antigens found only in cancer cells had been identified, and there was much debate about the specificity of these antigens. Many of the antigens to which monoclonal antibodies were made were embryonic antigens or shared antigens found on cancer cells and some normal cells. Therefore, although the specificity of the antibody was exquisite for the antigen, the specificity for the antibody or immunoconjugate for cancer was not absolute. One fairly clear exception occurred early in the 1980s when Levy et al9 developed monoclonal antibodies to the idiotype of B-lymphoma cells. The first patient given this anti-idiotypic antibody had a complete response to therapy, and his lymphoma went into a sustained remission that lasted for years. As a direct result of these early studies with anti-idiotypic antibodies, there is now a series of idiotype vaccines that are in phase III trials in patients with low-grade follicular lymphomas.10 These anti-idiotype vaccines will likely be the first truly custom-tailored, personalized anticancer vaccines to be approved for therapeutic use.

The major limitation of murine monoclonal antibody therapy was the immunogenicity of the mouse protein; a variety of investigators postulated that for monoclonal antibody therapy to be truly successful, human or humanized antibodies would be necessary. It was also known 25 years ago that the half-life of murine antibodies in the circulation was brief, and because of human antimouse antibody, became briefer with each infusion of murine monoclonal antibody. Previous studies of human immunoglobulin in clinical trials had demonstrated a much longer half-life for human immunoglobulin, which predicted that once human or humanized antibodies were available, the therapeutic efficacy of monoclonal antibodies and their immunoconjugates might be considerably enhanced.1
How has the field of monoclonal antibody and immunoconjugate therapy fared since the predictions of the early 1980s? Twenty-five years later, considerable progress has been made in this field.11,12 The US Food and Drug Administration has approved 21 monoclonal antibody products, with six of these biologic drugs approved specifically for cancer (Table 2). It was a landmark date in November 1997 when rituximab became the first monoclonal antibody approved specifically for cancer therapy.13 In addition to these six unconjugated monoclonal antibody therapies, one drug immunoconjugate, gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst, Madison, NJ), has been approved. This humanized monoclonal antibody to CD33 is approved for use in acute myelogenous leukemia and uses the antibody conjugated to calicheamicin, a potent enediyene antibiotic originally isolated from aMicromonospora echoinospora.14 Two radioisotope-antibody conjugates, ytrrium-90 ibritumomab tiuxetan (Zevalin; Cell Therapeutics Inc, Seattle, WA) and iodine-131 tositumomab (Bexxar; GlaxoSmithKline, Middlesex, United Kingdom) have been approved.15 The murine form of these antibodies was retained in order to expedite clearance from the circulation. Both radiolabeled antibodies target the CD20 antigen on lymphoma cells.

Unlike the immunoconjugates, which are currently infrequently used, each of the six unconjugated antibodies approved for cancer therapy is currently frequently used in the treatment of humans with cancer. The use of techniques to humanize or chimarize monoclonal antibodies to decrease their murine components has been an important advance in the field. These molecules have a long half-life in the blood stream, and can interact with human complement or effector cells of the patient’s immune system. They behave in a manner similar to naturally occurring immunoglobulin and work along the lines of our normal antibody-based immune response as effective agents in treating patients with cancer.16

Rituximab has become the largest-selling biologic drug in clinical oncology, and is active in a variety of human lymphomas and chronic lymphocytic leukemia.17,18 This is a chimeric monoclonal antibody targeting the CD20 antigen found on both normal B cells and on most low-grade and some higher grade B-cell lymphomas. It is effective as a single agent in induction and maintenance therapy. It is primarily used, however, in combination with standard chemotherapies in the treatment of patients with non-Hodgkin’s B-cell lymphomas and chronic lymphocytic leukemia.19-22

A second monoclonal antibody that has proven highly effective in the clinic is trastuzumab, a humanized antibody that reacts with the second part of the human epidermal growth factor receptor 2.23 Like rituximab, it is effective as a single agent in induction and maintenance therapy, but is used primarily in conjunction with chemotherapy for patients with human epidermal growth factor receptor 2/neu–positive breast cancer.24,25

Alemtuzumab is a humanized monoclonal antibody targeting the CD52 antigen found on B lymphocytes and is used primarily for chronic lymphocytic leukemia.26 Like the two previously cited monoclonal antibody therapies, alemtuzumab is effective as induction and maintenance therapy. Alemtuzumab is also reactive with T lymphocytes, and unlike the other two antibodies, it is typically not combined with chemotherapy because of the increased risk of infection.(26)

Another humanized monoclonal antibody, bevacizumab, has been applied more broadly in human solid tumors because it targets vascular endothelial growth factor, which is the ligand for a receptor found on blood vessels.(27) Because this receptor is on endothelial cells, bevacizumab seems to be effective by reducing the blood supply to tumor nodules, thereby slowing or interrupting growth. Initially approved for advanced colorectal cancer,(28) it is now used in a variety of human solid tumors including cancers of the lung, kidney, and breast.(29-31)

The last two antibodies approved for clinical use were cetuximab (a chimeric antibody), and panitumumab (a completely human antibody). Both target the epidermal growth factor receptors found on a variety of human tumors.(32,33) Cetuximab was originally approved for use in combination with chemotherapy in metastatic colorectal cancer.(34) It also enhances chemotherapy and radiation therapy of squamous cell cancers of the head and neck.(35) Panitumumab was approved based on its single-agent activity in refractory colorectal cancer and is being combined with chemotherapy as well.

At the end of 2007, 25 years of clinical studies have resulted in the approval of six unconjugated, humanized, or chimeric monoclonal antibodies for cancer therapy along with one drug immunoconjugate and two radioisotope immunoconjugates. Although few in number, these monoclonal antibodies are changing the face of cancer therapy, bringing us closer to more specific and more effective biologic therapy of cancer as opposed to nonspecific cytotoxic chemicals.

Modern recombinant techniques have made it possible to rapidly produce both chimeric antibodies and humanized antibodies, and totally human antibodies are also being produced. Identification of surface receptors that are integral to proliferation and apoptosis has provided more targets for monoclonal antibodies beyond those originally identified by the murine immune system. In 2008, there are more than 100 monoclonal antibody–based biologic drugs in hundreds of clinical trials. Many of these are in phase II and phase III and will be coming before the US Food and Drug Administration for approval in the next few months and years. At long last, immunoconjugates are proving efficacious with acceptable toxicity and will extend our diagnostic (36) and therapeutic armamentarium (37) from mainly unconjugated monoclonal antibodies to a broad array of highly active and specific immunoconjugates.

On this silver anniversary for our 1983 review, “Monoclonal Antibodies in Cancer Therapy, ” we can confidently predict that progress toward more specific and less toxic therapy for human cancer is in our near future. The developments during the past 25 years in both biologic drugs and targeted small molecules place us on the verge of more cures with less toxicity for our patients with cancer.

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Dan Peer1,7, Jeffrey M. Karp2,3,7, Seungpyo Hong, et al. 
Nature Nanotechnology 2, 751 – 760 (2007)
http://dx.doi.org:/10.1038/nnano.2007.387

Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells. This review examines some of the approved formulations and discusses the challenges in translating basic research to the clinic. We detail the arsenal of nanocarriers and molecules available for selective tumor targeting, and emphasize the challenges in cancer treatment.

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer
Vaishali Bagalkot, L Zhang, E Levy-Nissenbaum, S Jon, PW Kantoff, et al.
Nano Letters 2007; 7(10):3065-3070
http://dx.doi.org:/10.1021/nl071546n

We report a novel quantum dot (QD)−aptamer(Apt)−doxorubicin (Dox) conjugate [QD−Apt(Dox)] as a targeted cancer imaging, therapy, and

sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD−Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD−Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor−acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor−quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Semiconductor nanocrystals known as quantum dots (QDs)

have been increasingly utilized as biological imaging and labeling probes because of their unique optical properties, including broad absorption with narrow photoluminescence spectra, high quantum yield, low photobleaching, and resistance to chemical degradation. In some cases, these unique properties have conferred advantages over traditional fluorophores such as organic dyes.1-4 The surface modification of QDs with antibodies, aptamers, peptides, or small

molecules that bind to antigens present on the target cells or tissues has resulted in the development of sensitive and specific targeted imaging and diagnostic modalities for in vitro and in vivo applications.5-7 More recently, QDs have been engineered to carry distinct classes of therapeutic agents for simultaneous imaging and therapeutic applications.8,9 While these combined imaging therapy nanoparticles represent an exciting advance in the field of nanomedicine, it would be ideal to engineer “smart” multifunctional nanoparticles that are capable of performing these tasks while sensing the delivery of drugs in a simple and easily detectable manner. One way to achieve this goal is to develop multifunctional nanoparticles capable of sensing the release of the therapeutic modality by a change in the fluorescence of the imaging modality.

Figure 1. (a) Schematic illustration of QD-Apt(Dox) Bi-FRET system. In the first step, the CdSe/ZnS core-shell QD are surface functionalized with the A10 PSMA aptamer. The intercalation of Dox within the A10 PSMA aptamer on the surface of QDs results in the formation of the QD-Apt(Dox) and quenching of both QD and Dox fluorescence through a Bi-FRET mechanism: the fluorescence of the QD is quenched by Dox while simultaneously the fluorescence of Dox is quenched by intercalation within the A10 PSMA aptamer resulting in the “OFF” state. (b)

Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis. The release of Dox from the QD-Apt(Dox) conjugates induces the recovery of fluorescence from both QD and Dox (“ON” state), thereby sensing the intracellular delivery of Dox and enabling the synchronous fluorescent localization and killing of cancer cells.

Figure 3. Fluorescence spectra. (a) QD-Apt conjugate (1 µM) with increasing molar ratio of Dox (from top to bottom: 0, 0.1, 0.3, 0.6, 1, 1.5, 2.1, 2.8, 3.5, 4.5, 5.5, 7, and 8) at an excitation of 350 nm. (b) Dox (10 µM) with increasing molar ratio of QD-Apt conjugate (from top to bottom: 0.02, 0.04, 0.07, 0.09, 0.12, 0.14, and 0.16) at an excitation of 480 nm.

In conclusion, herein we report to our knowledge the first example of a multifunctional nanoparticle that can detect cancer cells at a single cell level while intracellularly releasing a cytotoxic dose of a therapeutic agent in a reportable manner. We demonstrate the specificity and sensitivity of this cancer imaging, therapy and sensing nanoparticle conjugate system in vitro by using PCa cell lines. By functionalizing the surface of fluorescent QD with the A10 PSMA aptamer, and intercalating Dox into the double-stranded CG sequence of the A10 PSMA aptamer, we developed a targeted QD-Apt(Dox) conjugate with reversible Bi-FRET properties. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of Dox on these conjugates. The presence of additional Dox may enhance the selfquenching effect of QD-Apt(Dox) conjugates thereby improving their imaging sensitivity, while the higher dose of Dox may enhance the therapeutic efficacy of the conjugates. Furthermore, through the use of other disease-specific aptamers or other targeting molecules, similar multifunctional nanoparticles may potentially be developed for additional important medical applications

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

Hongguang Sun1, Xun Zhu2, Patrick Y Lu3, Roberto R Rosato, et al.
Molecular Therapy Nucleic Acids(2014) 3, e182;
http://dx.doi.org:/10.1038/mtna.2014.32

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

The terms “aptamer” and “SELEX” were introduced by two independent groups in 1990.1,2 The term “aptamer” refers to small nucleic acid ligands that exhibit specific therapeutic functions and an unambiguous binding affinity for their targets. Conversely, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology is the method used for aptamer development. Although using small molecule nucleic acids as therapeutics has been explored for decades, development of SELEX and aptamer technology revolutionized this field.

The most important property of an aptamer, from the Latin aptus (to fit), is its high target selectivity. These short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into specific three-dimensional (3D) structures with dissociation constants usually in the pico- to nano-molar range.3 Moreover, in contrast to other nucleic acid molecular probes, aptamers interact with and bind to their targets through structural recognition (Figure 1), a process similar to that of an antigen-antibody reaction. Thus, aptamers are also referred to as “chemical antibodies.”

Figure 1.

Schematic diagram of aptamer binding to its target.

Full figure (43K)

Due to their small size and oligonucleotide properties, aptamers offer several advantages over protein antibodies in both their extensive clinical applicability and a less challenging industrial synthesis process. Specifically, (i) aptamers can penetrate tissues faster and more efficiently due to their significantly lower molecular weight (8–25 kDa aptamers versus ~150 kDa of antibodies). Therefore, aptamers penetrate tissues barriers and reach their target sites in vivo more efficiently than the larger-sized protein antibodies. (ii) Aptamers are virtually nonimmunogenic in vivo. In principal, as aptamers are oligonucleotides they should not be recognized by the immune system. In practice, a recent clinical study showed that aptamers did not stimulate an immune response in vivo,4,5 as compared to protein antibodies that are highly immunogenic, especially following repeat injections. (iii) Aptamers are thermally stable. Based on the intrinsic property of oligonucleotides, even after a 95 °C denaturation, aptamers can refold into their correct 3D conformations once cooled to room temperature. In comparison, protein-based antibodies permanently lose their activity at high temperatures. More importantly, a well-established synthesis protocol and chemical modification technology lead to (iv) rapid, large-scale aptamer synthesis and modification capacity that includes a variety of functional moieties; (v) low structural variation during chemical synthesis; and (vi) have lower production costs. Moreover, aptamers specifically recognize a wide range of targets, such as ions, drugs, toxins, peptides, proteins, viruses, bacteria, cells, and even tissues.6,7,8,9,10,11,12 In the clinic, aptamer-based therapeutics are gaining momentum. For example, Macugen, a modified RNA aptamer, specifically targets vascular endothelial growth factor. It has been approved by the US Food and Drug Administration (FDA)13 for the treatment of wet age-related macular degeneration and is under evaluation for other conditions.14 In the cancer setting, AS1411 targets nucleolin, a protein over-expressed in a variety of tumors. It is currently being evaluated as a potential treatment option in solid tumors and acute myeloid leukemia.15 An updated list of therapeutic aptamers undergoing clinical trials is included in ref. 16 and Table 1. Taken together, these clinical studies highlight many possible uses that aptamers may have in a variety of biomedical fields, including therapeutics.17
Table 1 – A list of therapeutic aptamers undergoing clinical trials.

Since aptamer technology was first introduced, the RNA-based sequence library has been widely used for SELEX. Based on the existing evidence, it is believed that the presence of a 2′-OH group and non-Watson-Crick base pairing allows RNA aptamer oligonucleotides to fold into more diverse 3D structures than ssDNA molecules. Consequently, using the more flexible RNA sequences simplifies the development of high-affinity and -specificity aptamers. Despite their advantages, RNA sequences are very sensitive to nucleases present in biological environments and can be rapidly degraded.18 To increase nuclease resistance of RNA-based aptamers, several chemical modifications have been investigated. Evidence shows that 2′-OH group and phosphodiester linkages of RNA sequences are the sites of nuclease hydrolysis. Subsequently, substitutions of the 2′-OH functional group by 2′-fluoro, 2′-amino, or 2′-O-methoxy motifs, and/or changes to the phosphodiester backbone with boranophosphate or phosphorothioate are the most common modifications aimed at increasing nuclease resistance.19 More recently, Wu et al. developed a novel chemical modification method to increase siRNA stability, in which phosphorodithioate and 2′-O-Methyl were simultaneously substituted in the same nucleotide.20
This modification method significantly enhanced siRNA stability and represents a potential new direction for utilization of RNA-based therapies in complex biological systems. Other effective modifications recently reported utilize the locked nucleic acid technology16,21 or generate “mirror” RNA sequence structures, termed spiegelmers.22 These modifications result in structural changes to the RNA sequences, which cannot be digested by nucleases.

In addition to RNA aptamers, ssDNA-based aptamers have also been developed. Due to their lack of 2′-OH groups, DNA molecules are naturally resistant to 2′-endonucleases and are stable in biological environments. Recently, our group developed a biostable DNA-based aptamer specific for CD30, a protein biomarker that is over-expressed in Hodgkin and anaplastic large cell lymphomas. Functional analysis demonstrated that this ssDNA-based aptamer exhibited high CD30 binding affinity as low as 2 nmol/l and was stable in human serum for up to 8 hours. Conversely, an RNA-based CD30 aptamer was digested within 10 minutes under similar conditions.23
In summary, unique chemical features and biological functions have made aptamers a very attractive tool in biomedical research over the past two decades. Currently, there are over 4,000 published articles referenced in the PubMed database that include the term “aptamer.” Research areas that include aptamer technology cover bioassays, drug development, cell detection, tissue staining, in vitro and in vivo imaging, nanotechnology, and targeted therapy. As chemical antibodies, aptamers represent an excellent alternative to replace or supplement protein antibodies, which have been extensively used in the clinic.

Aptamers Specifically Targeting Cell Surface Biomarkers

Using SELEX technology to develop aptamers for cell surface biomarkers

SELEX, the methodology used to develop aptamers specific for a target of interest, is based on a repetitive amplification and enrichment process. The SELEX process follows several steps: first, a random ssDNA oligonucleotide library is chemically synthesized to contain between 1014–1015 unique random sequences flanked by conserved primer binding sites. This step utilizes the following universal scheme: 5′-sense primer sequence-(random sequence)-antisense primer sequence-3′, where the primer sequence ranges from 18 to 22 bases and the random sequence contains 20–40 nucleic acids. The general procedure consists of labeling the 5′-sense primer with a fluorochrome reporter for monitoring aptamer selection, while the 3′-antisense primer is labeled with an affinity molecule, such as biotin, that is used to separate single-stranded oligonucleotides generated in each amplification round. This random ssDNA library can be used directly to select an initial pool of DNA aptamers. Conversely, generation of RNA aptamers requires two extra steps. Specifically, a pool of random ssDNA oligonucleotides is generated, T7 RNA polymerase promoter sequence is added to the 5′-sense primer, and the DNA is then used as a template for T7 RNA polymerase-based transcription in the 5′ to 3′ direction. During the second SELEX step, the oligonucleotide library is heated and rapidly cooled to promote the formation of 3D structures. The library is then mixed with the target of interest for specific binding enrichment. In the third step, the unbound sequences are discarded through the use of membranes, columns, magnetic beads, and capillary electrophoresis.6,24,25 In the fourth step, the enriched sequences are amplified in vitro by either PCR (DNA aptamers) or RT-PCR (RNA aptamers) to generate a new sequence library for the next round of SELEX. The amplified sequence library may go through further negative-target selection, which eliminates the nonspecific sequences generated by binding of nontarget moieties. Lastly, aptamer selection goes through 4–20 rounds of amplification and enrichment. The exact number of required amplification and selection steps depends on the aptamer target being a purified protein or a living cell, and on the evolution of the aptamer sequence library, as that established by gel electrophoresis, flow cytometry (for target binding), classical cloning or sequencing methods, or by high throughput Next-Generation Sequencing (NGS). In recent years, the traditional SELEX method had also been modified to include the capillary electrophoresis (CE) SELEX, toggle selection, photo-SELEX, bead-based selection, X-Aptamers, and Slow Off-rate Modified Aptamers (SOMAmers) in order to maximize affinity and specificity, to improve the speed of selection and success rate, and to provide additional properties to the selected aptamers.26,27,28,29,30,31

Similar to protein antibody development, purified recombinant proteins or peptides expressed in prokaryotic or eukaryotic systems can be used as targets for aptamers selected by the SELEX method. However, because of the posttranslational modifications, especially in the case of highly glycosylated proteins, purified proteins or peptides often cannot fold into the correct 3D structure that is formed under physiologic conditions.32 Consequently, the newly synthesized aptamers may not be able to selectively recognize and interact with their corresponding targets, which would result in failure of the biomedical application. As this is a common problem, it is very important to choose biomarkers in their native conformation for aptamers selection. Taking this issue into an account, a modified SELEX technology that uses whole living cells, Cell-based SELEX (or Cell-SELEX), was recently established.33 To develop cell-specific aptamers, the Cell-SELEX method uses whole living cells that express surface biomarkers of interest. However, the presence of many different cell surface molecules in addition to the target biomarker(s) results in the synthesis of many unrelated/unwanted aptamers. Therefore, in addition to all the SELEX steps described above, Cell-SELEX technology also utilizes control cells that do not express the target biomarker(s) during the counter-selection step.33

Well-characterized biomarkers that are endogenously expressed at high levels, such as the ErbB superfamily, MUC1, EpCAM, and CD30, offer the best potential for cell-based aptamer development. Subsequently, cell lines that have high endogenous expression of cell-specific or cancer type-specific biomarker(s) are commonly used for Cell-SELEX. However, if such cell lines are unavailable, a biomarker of interest could be over-expressed in a particular cell line via gene transfection and the parental cells used for counter-selection. Using this approach, aptamers targeting the cancer stem cell (CSC) biomarker CD133 have been recently developed.34 In this study, CD133 cDNA was transfected into HEK293T cells that were then used for aptamer enrichment, with the parental HEK293T cells serving as a negative control. Similarly, an aptamer specific for the human receptor tyrosine kinase was recently developed.35

Figure 2.

Schematic diagram of our hybrid-SELEX method for selection of CD30-specific ssDNA aptamer. In our experiment, the hybrid-SELEX process is divided into (a) the cell-based SELEX selection and (b) CD30 protein-based SELEX enrichment. First, CD30-expressing lymphoma cells are used for positive selection and CD30-negative Jurkat cells are used in negative counter-selection. After 20 rounds of selection, the enriched aptamer pool is incubated with CD30 protein immobilized on magnetic beads for five additional rounds of enrichment. SELEX, Systematic Evolution of Ligands by EXponential enrichment.

Full figure and legend (183K)

Aptamers specific for cell surface biomarkers

Cell surface biomarkers are functionally important molecules involved in many biological processes, such as signal transduction, cell adhesion and migration, cell–cell interactions, and communication between the intra- and extra-cellular environments. An abnormal expression of cell surface biomarkers is often related to tumorigenesis.50 Clinically, it is estimated that about 60% of cancer-targeting drugs, including therapeutic antibodies and small molecule inhibitors, target cell surface biomarkers,51 making them attractive for disease treatment. In the last decade, many aptamers targeting cell surface biomarkers have been developed through the advancement of both the protein- and/or cell-based SELEX technologies (see Table 2 for detailed list). These aptamers have been extensively studied for diagnosis and/or treatment of hematological malignancies,7,23,49 lung,52,53,54 liver,55 breast,56,57 ovarian,58 brain,59,60colorectal,61 and pancreatic cancers,46 as well as for identification and characterization of CSCs.34,62

Aptamer-Mediated Targeted Therapies

Traditional cancer treatment approaches, such as chemotherapy, radiotherapy, photodynamic therapy, and photothermal therapy can cause serious side effects in patients due to their associated nonspecific toxicity. To minimize these side effects, a concept of personalized, targeted therapy has been gaining momentum. One of the main clinical approaches for targeted cancer therapy employs antibody-based drugs. Although antibody-mediated therapy is highly specific and results in fewer side effects, potential immunogenicity and high cost of production may limit its clinical applications. To overcome these obstacles, oligonucleotide aptamer-based targeted therapeutics and specific drug delivery systems have recently been explored. These studies revealed numerous advantages offered by the aptamer technology over protein-based antibody therapies, with some of these described in the section below.
Aptamer-drug conjugates

Aptamer-drug conjugation (ApDC) is a very simple yet effective model of noncovalently or covalently conjugating aptamer sequences directly with therapeutic agents (Figure 3). For example, aptamer-conjugated Doxorubicin (Dox), a chemotherapeutic agent extensively used in the treatment of various cancers, has recently been shown to have enhanced therapeutic efficacy over Dox alone. Mechanistically, Dox cytotoxicity is caused by its intercalation into the nucleic acid structure at the preferred paired CG or GC sites with subsequent inhibition of cancer cell proliferation. Taking advantage of its propensity for intercalation, Dox can be noncovalently conjugated to oligonucleotide aptamers containing CG/GC sequences through a simple incubation step. A recent report by Subramanian et al. describes the effectiveness of aptamer-Dox conjugates in the treatment of retinoblastoma.63 In their study, a 2′-fluoro modified RNA aptamer EpDT3 (specific for EpCAM, a CSC marker), was noncovalently conjugated with Dox. After binding to EpCAM molecules expressed at the cancer cell surface, the EpDT3-Dox conjugates were preferentially internalized by the cancer and not by the healthy cells, greatly enhancing therapeutic efficacy and reducing treatment-associated side effects. Several other studies also utilized aptamer-Dox conjugates for cancer therapy, such as HER2 aptamer-Dox conjugates targeting breast cancer,64 MUC1 aptamer-Dox conjugates targeting lung cancer,65 and PSMA aptamer-Dox conjugates targeting prostate cancer.66 Despite their obvious advantages, several concerns related to the use of aptamer-Dox conjugate have been raised. These include (i) instability of the aptamer-drug conjugate due to the reversible nature of noncovalent conjugation process; (ii) short circulating half-life of aptamer-drug conjugates in vivo due to their low molecular weight; and (iii) poor drug payload capacity due to a very simple structure of aptamers. These three disadvantages and technological approaches to improve them are described in greater detail below.

Figure 3.

Schematic diagram of noncovalent or covalent aptamer-drug conjugation.

Full figure (40K)

To enhance the stability of drug loading, Dox can be covalently conjugated to aptamer sequences via a functional linker moiety. For example, the DNA aptamer sgc8 possesses a strong affinity for PTK7 kinase that is abundantly expressed on the surface of CCRF-CEM T-cell acute lymphoblastic leukemia cells. To enhance its stability, this aptamer was covalently conjugated with Dox through an acid-labile linker.67 Once the sgc8 aptamer-Dox conjugate was preferentially bound and internalized by the target cells, the acid-labile linker was easily cleaved in the acidic lysosomal environment, releasing Dox and effectively killing target cells.67 On the other side of the spectrum, covalent conjugation is the most commonly used method of aptamer-drug conjugation, especially for agents that cannot intercalate into the nucleic acid structure or whose intercalation would disrupt aptamer structure.68 Evidence suggests that these covalently conjugated aptamer-drug compounds are significantly more stable than the corresponding noncovalently conjugated intercalations.69

Conjugation of aptamers with high molecular weight polymers, such as polyethylene glycol (PEG), has been examined in order to increase aptamer molecular weight. Specifically, PEG has been widely used in drug modifications, including synthesis of Macugen aptamers. This modification, resulting in PEGylated aptamers, not only increased the aptamer molecular weight and prolonged its circulating half-life, but also enhanced its stability and decreased its toxic accumulation in nontarget tissues.70,71

Finally, in order to increase aptamer-drug payload capacity, an innovative model named aptamer-tethered DNA nanotrains (aptNTrs) was recently introduced by Zhu et al. to deliver Dox to cancer cells.72 In this study, structure of the sgc8 aptamer that targets PTK7 was modified by adding a DNA trigger probe on the 5′-end. Consequently, the modified aptamer acted as a locomotive for targeting, while two hairpin monomers containing Dox intercalation sites acted as boxcars to deliver the drug. After self-assembly, the newly synthesized sgc8 aptamer-NTrs displayed high drug payload capacity, with the drug/sgc8 aptamer-NTr molar ratio of 50:1. Importantly, sgc8 aptamer-NTrs-Dox conjugates were preferentially internalized by the target cells, thereby inhibiting tumor cell growth in vitro and in vivo.72

Another strategy for increasing the aptamer payload capacity involves the construction of polyvalent aptamers. Polyvalent aptamers exhibit an increased target affinity and are more rapidly internalized by their target cells. To demonstrate this, Boyacioglu et al. developed a new DNA aptamer they termed SZTI01 against PSMA.69 First, a dimeric aptamer complex (DAC) was created for specific delivery of Dox to PSMA-expressing cancer cells. Then, the SZTI01aptamer was modified on the 3′-terminus with either a dA16 or dT16 single-stranded tail that contained CpG sites for loading Dox, and the two monomers were annealed in a 1:1 ratio to form the DAC structure. The results of the study showed that DACs have a high Dox payload capacity with the Dox/DAC molar ratio of about 4:1, and the DACs-Dox conjugates were stable under physiological conditions for up to 8 hours.69 In another study, a DNA aptamer targeting MUC1 was truncated and an aptamer containing three repeats of the active targeting region, termed L3, was synthesized. Although the Dox payload capacity was not specifically modified in the L3 aptamer, the L3-Dox conjugates showed a stronger affinity to target cells and lower cytotoxicity to off-target cells than the parental MUC1 aptamer.73 Finally, polyvalent aptamers can also be constructed through the rolling circle amplification (RCA) technology. Using the RCA method and the sgc8 aptamer sequence as a circular template, a polyvalent sgc8 aptamer, termed Poly-Aptamer-Drug, was synthesized.74 It was determined that the Dox payload capacity of the polyvalent sgc8 aptamer increased tenfold, as compared to the monovalent sgc8 aptamer. Moreover, because of their 40-fold greater binding affinity, the Poly-Aptamer-Drug conjugates were more effective than their monovalent counterparts in targeting and killing leukemia cells.74

Although Dox presents itself as a very attractive chemotherapeutic agent for use in aptamer conjugation, other drugs, such as Gemcitabine (Gem) and photosensitizers, can also be targeted to cancer cells through the aptamer technology. Gem is an FDA-approved deoxycytidine analog (dFdC) used for anticancer therapy. To deliver Gem specifically to pancreatic cancer cells, Ray et al. developed a novel aptamer-Gem polymer model. In this model, a single-stranded RNA polymer contained Gem that was enzymatically synthesized through a mutant T7 RNA polymerase-mediated transcription reaction and fused with a nuclease-resistant 2′-fluoro-modified RNA aptamer (E07) that selectively binds to EGFR on pancreatic cancer cells. The E07 aptamer structure was modified by introducing a 24-nucleotide sequence at the 3′ end and using it as an adaptor for Gem polymer binding. Following an annealing step, the Gem polymer complementary bound with the E07 aptamer and preferentially targeted the EGFR-expressing pancreatic cancer cells, inhibiting cell proliferation.75

Compared with the traditional chemotherapeutic agents, controlled conditional prodrug photosensitizers have also been extensively used for aptamer-mediated drug delivery. In this therapeutic approach, termed photodynamic therapy, or photodynamic therapy, photosensitizers are activated by light irradiation and induce production of intracellular reactive oxygen species, resulting in cytotoxicity. A study by Ferreira et al. describes the development of a DNA aptamer specific for MUC1 and covalently conjugated at the 5′ end with the photosensitizer chlorin e6.76 Upon light irradiation, MUC1-expressing epithelial cancer cells were preferentially killed with cytotoxicity about 500-fold higher than that of the control cells. Similar studies have reported using a necleolin aptamer (AS1411)-TMPyP4 for targeting breast cancer77 and the EGFR aptamer (R13)-TF70 for treatment of lung cancer.78

Finally, approaches to extend the scope of aptamer application have also been developed. Similar to bi-specific antibodies, bi-specific or even tri-specific aptamers can be constructed. A bi-specific aptamer for targeting different cells was recently described by Zhu et al. In their study, specific DNA aptamers sgc8 and sgd5a were conjugated through a dsDNA linker. Compared to each mono-aptamer, this bi-specific aptamer (named SD) could recognize its target cell simultaneously with equal specificity and affinity, while Dox intercalation into the dsDNA induced target cell cytotoxicity.79 In the same study, a Y-shape dsDNA linker was used to construct a tri-specific aptamer that also recognized its target cells with high specificity and affinity.79 Clinically, Min et al. proposed using a bi-specific aptamer for prostate cancer therapy. It is well established that prostate tumors may contain both PSMA-positive and -negative cell types. Thus, this study utilized two aptamers, a 2′-fluoro modified RNA aptamer targeting PSMA-expressing cells and a DUP-1 peptide aptamer specific to PSMA-negative cells, conjugated through streptavidin. Moreover, intercalating Dox into the PSMA aptamer of this bi-specific aptamer model could serve as a tool to target all prostate cancer cell types.80

Aptamer-nanoparticle therapeutics

Nanoparticles (NPs) are attractive vehicles to increase both the half-life and the drug payload capacity of aptamer-mediated drug delivery. In addition to their common features, such as biocompatibility for clinical applications, large surface for enhanced aptamer and drug loading, and uniform size and shape for excellent biodistribution, NPs have other individual physical and chemical properties defined by their materials. For example, copolymers and liposomes are biodegradable, while metal materials offer exceptional photothermal and magnetic performance.

Conclusion

Antibody-based targeted therapeutics provide high target specificity and affinity. However, their potential for immunogenicity is of a great concern, as is their high production cost, both of which have limited their clinical applicability. As discussed in this review, when compared to protein antibodies, oligonucleotide aptamers offer many advantages, including simple chemical synthesis, virtual nonimmunogenicity, smaller size, faster tissue penetration, ease of modification with different functional moieties, low cost of production, and high biological stability. Therefore, aptamers have become a promising new class of molecular ligands that could replace or supplement protein antibodies. In summary, aptamer technology has a strong market value and may be applied in various biomedical fields, including in vitro cancer cell detection, in vivo tumor imaging, and targeted cancer therapy (Figure 7).

Figure 7.

Summary of various aptamer applications.

Full figure (58K)

Although aptamer technology has a great potential in the biomedical field, several technical challenges remain and must be addressed. These include: (i) how can aptamers be rapidly adapted for specific targets by decreasing false-positive/-negative selection? Primarily dependent on the natural properties of targets of interest, such as proteins versus cells or tissues, the process of aptamer selection is usually time-consuming, and the success rate is sometimes low. To improve the speed and success rate, novel methods for aptamer selection have been recently described. They include bead-based selection, that can select aptamers as rapidly as a single round of selection,27,28 and the SOMAmer, which improves the aptamer production success rate from less than 30% to over 50%.29,30 More recently, a study by Cho et al. devised a Quantitative Parallel Aptamer Selection System (QPASS) method, which integrates microfluidic selection, NGS, and in situ-synthesized aptamer arrays. This approach allows for the simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel.116 In addition to QPASS, evolving modifications to the Cell-SELEX approach are beginning to address difficulties with successful removal of the influence stemming from the presence of dead cells, slow enrichment aptamers recognizing targets of interest, and contamination with unwanted aptamer sequences. As described above, utilization of the above-mentioned FACS-mediated SELEX44,45 and hybrid-SELEX23 offers novel approaches that address these technical challenges.

(ii) How can we select cancer-relevant targets for aptamer development and clinical applications? Tumorigenesis is a dynamic process that includes multiple constantly changing factors. Therefore, a one-size-fits-all cancer-specific biomarker is unlikely to ever be identified. Yet, it has been established that certain biomarkers present in healthy tissues are highly expressed in cancer cells. Moreover, certain biomarkers are associated with particular cancer cell types making them to be considered as useful targets for development of targeted cancer therapy. However, while use of cancer cells to identify biomarkers and to develop therapeutic agents is a reasonable approach, cultured cells, especially immortalized cell lines, greatly differ from tumor tissues in vivo. To overcome these limitations and to select more reliable cancer-relevant biomarkers for aptamer development, several innovative SELEX methods have been recently described. Of particular interest are the tissue-based SELEX117 and the in vivo-SELEX,118 which offer target selection under more relevant pathologic conditions. This cell/tissue-specific biomarker selection can also be utilized for development of noncancer related therapies, as shown for aptamers targeting the adipose tissue in obesity119 and for aptamers designed to penetrate the blood-brain barrier in order to combat brain diseases.120 Hence, we believe that the careful selection of cancer-associated biomarkers and cell/tissue type-specific biomarkers will expand the scopes of aptamer applicability and improve the feasibility of clinical applications.

(iii) What methods could improve aptamer biostability in vivo? Unmodified RNA-based aptamers are very susceptible to the nuclease-mediated degradation in vivo. Although many chemical modifications aimed at increasing biostability of the RNA aptamers have been developed, including 2′-modifications, 3′-modifications, phosphodiester backbone modifications,19,20 and utilizations of novel nucleic acids (locked nucleic acid and Spiegelmers),16,21,22 their effectiveness is still limited. When it was first described, PEGylation was a very attractive strategy for prolonging aptamer circulation half-life and enhancing their biostability. However, a recent report showed that the in vivo use of PEGylated aptamers induced production of anti-PEG antibodies,121 emphasizing the need for the development of alternative approaches.

(iv) How can aptamer technology be modified to achieve a more effective drug delivery? Many drug delivery systems described in this review are tested in vitroor in animal models. Yet, as with any compound that is translated from the bench to the bedside, aptamer-drug conjugates may behave differently in a human patient than they do in laboratory animals. Therefore, aptamer-drug conjugation remains an important challenge that must be considered. Specifically, various coupling approaches lead to different pharmacokinetics, biodistribution, and tolerability in vivo, which in turn greatly affect treatment effectiveness. In the same vein, we must consider the effectiveness of aptamer-mediated target gene therapy. Gene therapy, including siRNA and miRNA aimed at silencing specific genes, is considered the next generation therapeutic approach. However, silencing a single pathogenic gene may not be a viable therapeutic option because tumorigenesis is a process regulated by multiple genes and signaling pathways. Therefore, combining targeted therapeutics with gene therapy may represent the most effective strategy. Such combinational therapy approaches can greatly improve the therapeutic efficacy while reducing the required dosages of both drugs and small molecule RNAs,122 and, more importantly, may offer new alternatives to combat chemotherapy-resistant cancers.110

(v) The last important point to consider is whether aptamer-mediated biotherapies can become effective, FDA-approved medications. Following Macugen approval by the FDA, many aptamer-mediated biotherapies have been evaluated in clinical trials. Of particular interest is AS1411, an antitumor aptamer that has completed several Phase I clinical trials.15 Trial results are promising and offer useful insights into further modifications that could be applied to therapeutic aptamer development.

Taken together, although some technical challenges remain to be addressed, oligonucleotide aptamers have become an attractive and promising tool for targeted cancer therapy. As more clinical data are accumulated, we and others will be better equipped to optimize aptamer formulations, leading to the expansion of aptamer use in the clinic.

9.1.5 Tumor Suppressors

Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility
Prerna Malaney, Ravi R Pathak, Bin Xue, VN Uversky & Vrushank Davé
Scientific Reports 20 June 2013; 3(2035)
http://dx.doi.org:/10.1038/srep02035

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTEN’s protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

The concept of “Intrinsic Disorder” in proteins has rapidly gained attention as the preponderance and functional roles of IDPs are increasingly being identified in eukaryotic proteomes1, 2. Structured proteins adopt energetically stable three-dimensional conformations with minimum free energy. In contrast, IDPs, due to their unique amino acid sequence arrangements, cannot adopt energetically favorable conformations and, thus, lack stable tertiary structure in vitro3. This structural plasticity allows IDPs to operate within numerous functional pathways, conferring multiple regulatory functions4, 5, 6. Indeed, mutations in and dysregulation of IDPs are associated with many diseases including cancer1, 6, 7, signifying that IDPs play vital roles in functional pathways. Evidence suggests that ~80% of proteins participating in processes driving cancer contain IDRs6. For example, tumor suppressor p53 as an IDP, functions via its C-terminal IDR, which simultaneously exists in different conformations, each of which function differently1. Since PTEN is the second most frequently mutated tumor suppressor with versatile functions8, we hypothesized that PTEN may contain IDR(s) that can be exploited for therapeutic targeting in cancers and diseases associated with pathogenic PI3K/Akt/mTOR (Phosphoinositide 3-Kinase/Akt/ mammalian Target of Rapamycin) signaling9, 10, 11.

PTEN (phosphatase and tensin homolog), a 403 amino acid dual protein/lipid phosphatase converts phosphatidylinositol(3,4,5)-triphosphate (PIP3) to phosphatidylinositol(4,5)-bisphosphate (PIP2), thereby regulating the PI3K/Akt/mTOR pathway involved in oncogenic signaling, cell proliferation, survival and apoptosis12. PTEN, as a protein phosphatase, autodephosphorylates itself13. Deficiency or dysregulation of PTEN drives endometrial, prostate, brain and lung cancers, and causes neurological defects14, 15. PTEN is activated after membrane association16, providing conformational accessibility to the catalytic phosphatase domain (PD) that converts PIP3 to PIP216(Figure 1a). Because PTEN reduces PIP3 levels and inhibits pathogenic PI3K signaling, therapeutically targeting PTEN to the membrane to enhance its activity is of significance in treating several pathologies including cancer.

Figure 1: PTEN: A newly identified IDP.

PTEN - A newly identified IDP. srep02035-f1

PTEN – A newly identified IDP. srep02035-f1

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f1.jpg

(a) Diagrammatic representation of PTEN structure. PTEN, a 403 amino acid protein, comprises of PBM: PIP2 Binding Module (AA 1–13; in green), a phosphatase Domain (AA 14–185; in pink), C2 Domain (AA 190–350; in blue), C-terminal region or Tail (AA 351–400; in orange) and a PDZ binding domain (AA 401–403; in dark blue). The PDZ-binding motif is considered as a part of the C-terminal region. *Figure not to scale. (b) Crystal structure of PTEN. Only the phosphatase (in pink) and C2 domain (in blue) are amenable to crystallization. The first seven residues and the last 50 residues represent unstructured/loosely-folded regions that are yet to be crystallized. These regions represent the N- and C-termini of PTEN, respectively. (Source: RCSB Protein Data Bank). (c) Disorder analysis of PTEN. PONDR-VLXT and PONDR-FIT prediction tools were used to determine the disorder score of PTEN. Any value above 0.5 indicates intrinsic disorder. There are several disordered stretches within the PTEN protein, however, the most prominent of these disordered regions is a 50 amino-acid stretch located at the C-terminus of the PTEN protein. (d) IDPs are enriched in polar (R, Q, S, T, E, K, D, H) and structure breaking (G, P) amino acids and are depleted in hydrophobic (I, L, V, M, A), aromatic (Y, W, F) and cysteine (C) and asparagine (N) residues. The amino acid sequence of PTEN highlights these classes of residues with their relative distribution. (e) Composition profiling for full-length PTEN (in green), its ordered domain (in yellow) and its IDR (in red). The tool used is Composition Profiler (Vacic et al, 2007). As shown in the graph, the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E), structure breaking residues (specifically P) and is depleted in all hydrophobic residues, cysteine and all aromatic residues. (f) Histogram representing the percentage of hydrophobic, polar, aromatic, structure breaking, cysteine and asparagines residues in ordered vs. disordered regions. The disordered region has an amino acid composition in line with the definition of IDPs.

PTEN crystal structure revealed that the PD and membrane-binding C2 domains are ordered (Figure 1b); however, the structures of the N-terminus, the CBR3 loop and the 50 amino-acid C-tail remain undetermined17. The C-tail is of particular significance due to its ability to regulate PTEN membrane association, activity, function, stability18, 19, 20, 21. Herein, we identify PTEN as an IDP with its C-tail being intrinsically disordered. The PTEN C-tail IDR is heavily phosphorylated by a number of kinases and regulates the majority of PTEN functions, including a large number of PPIs that forms the PTEN primary and secondary interactomes, comprising critical functional protein hubs, most of which are related to cancer. Our analysis provides a mechanistic insight into the functioning of the PTEN C-tail IDR at the systems level, including inter- and intra-molecular interactions that will aid in designing drugs to enhance the lipid phosphatase activity of PTEN for the pharmacotherapy of cancers and pathological conditions driven by hyperactive PI3K-signaling.

PTEN is an IDP

Utilizing two disorder prediction software programs, PONDR-VLXT and PONDR-FIT22, 23, we have identified PTEN as a bona fide IDP. PTEN has a highly disordered, functionally versatile, C-tail encompassing amino acids 351–403 (Figure 1a and 1c). A PDZ-binding motif (amino acids 401–403) is part of the disordered region. Thus, the PTEN C-tail IDR facilitates interactions with a vast repertoire of PDZ domain-containing proteins (Figs. 1a and 2d). The unique amino acid composition of IDRs dictates their structural plasticity3, 23, 24. IDRs are enriched in polar and structure-breaking amino acid residues, depleted in hydrophobic and aromatic residues and, rarely, contain Cys and Asn residues1, 23, 24. The ordered region of PTEN (AA 1–350) has 25% hydrophobic, 43% polar, 9% structure breaking, 13% aromatic and 9% Cys and Asn residues. In contrast, the PTEN C-tail (AA 351–403) is enriched in polar (66%) and structure breaking (11%) residues and is depleted in hydrophobic (11%), aromatic (6%) and Cys and Asn residues (6%), indicating an ideal profile for the IDR (Figs. 1d and 1f ). Further, compositional analysis of PTEN using the Composition Profiler24 reveals that the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E) and structure breaking residues (specifically P) but is depleted in all aromatic and hydrophobic residues in addition to cysteine. (Figure 1e), again exhibiting universal characteristics of IDPs. Taken together, we establish the PTEN C-tail as a functional IDR and classify PTEN as a new IDP.

Figure 2: The functional relevance of the PTEN IDR.

The functional relevance of the PTEN IDR. srep02035-f2

The functional relevance of the PTEN IDR. srep02035-f2

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f2.jpg

(a) The number of mutations observed in PTEN over its 403 amino-acid stretch is plotted. Fewer mutations are observed in the tail region (in red) possibly indicating the deleterious nature of mutations in the functionally critical C-terminal region. [Source: Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC), Human Gene Mutation Database (HGMD)]. (b) Number of mutations in every successive 50 amino-acid stretch of the PTEN protein. The last 50 amino-acid stretch, representing the tail region has at least one-eighth the number of mutations seen in any other 50 amino-acid stretch along PTEN, pointing to its critical function in cell homeostasis. (c) Correlation of mutations with the amino acid composition of PTEN. The ratio of mutations in specific residues in the disordered vs. ordered region are represented in this graph. The residues considered here are those used to define IDRs: hydrophobic, polar, aromatic, structure-breaking, cysteine and asparagine residues. Compared to the other classes of residues, mutations in aromatic residues are much higher in the disordered region when compared to the ordered region. (d) The PTEN primary interactome. Forty proteins interact with known regions of PTEN. There are approximately 340 more proteins that interact with PTEN at sites that are yet to be determined (see Supplementary Table S2). Proteins shown in pink interact with the phosphatase domain, those in blue interact with the C2 domain and those in orange interact with the disordered tail. (Visualization tool: Cytoscape). (e) The PTEN C-tail has a higher propensity for PPIs. Of the 40 mapped proteins, 60% interact with the disordered indicating a strong correlation between degree of disorder and the number of protein interactions. (f) Most proteins within the PTEN interactome are highly disordered. Approximately 80% of PTEN-interacting proteins within the primary interactome are disordered, as indicated in red. The proteins within the interactome that are ordered are indicated in blue.

Low mutability of PTEN IDR suggests critical biological functions

Mutations in PTEN are associated with several types of cancers14. To correlate PTEN mutations to its structure, we analyzed all human PTEN mutations deposited in the COSMIC Database (http://www.sanger.ac.uk/genetics/CGP/cosmic/). The disordered PTEN C-tail IDR shows unusually low mutability (~8-fold less) compared to any other 50 amino-acid stretch of PTEN (Figure 2a and 2b). To confirm our finding of the low mutability of the C-tail region, we also analyzed all human PTEN mutations deposited in the Human Gene Mutation Database (HGMD,http://www.hgmd.cf.ac.uk/ac/index.php)25 (Figure 2a), cBioPortal for Cancer Genomics26, 27(Supplementary Figure S1) and the Roche Cancer Genome Database28 (Supplementary Figure S1) which was consistent with the COSMIC database mutational data. It is likely that evolutionary pressure maintains a survival advantage and ipso facto abrogates progeny with mutations in highly functional protein sequences29, 30, 31. Thus, the functionally versatile PTEN C-tail IDR cannot afford mutations, hence showing least number of mutations. It is equally likely that mutations in individual residues within the IDR are well tolerated, as the evolutionary pressure may have shifted to maintaining global biophysical properties and structural malleability of the IDR to safeguard the critical protein function29. In either case, on a global scale, the versatile structural pliability of the PTEN IDR dictates functional diversity and biological activities29. Thus, the slightest functional perturbation in the PTEN IDR due to mutations, either within the IDR or in domains interacting with it, could disrupt cellular homeostasis as seen in cancers and neurodegenerative disorders associated with PTEN mutations. This is supported by our data indicating that PTEN, as an IDP when mutated, causes several cancers14.

Moreover, the PTEN C-tail IDR exhibits preferential mutations in aromatic residues compared to the ordered region (Figure 2c). The ratio of mutations in aromatic residues in the disordered to ordered region is much higher than any other class of residues (structure breaking, hydrophobic, polar, Cys and Asn), likely attributed to the structure-imparting property of aromatic residue32. Specifically, aromatic residues within IDRs engage in stacking interactions, enhancing nucleation between distinct residues at functional protein-protein interaction interfaces32. Thus loss of this critical structural and functional property imparted by aromatic residues is associated with a disease phenotype. In summary, the disordered PTEN C-tail IDR has functionally evolved to contain a combination of peptides that cannot tolerate mutations.

Disorderliness in PTEN primary interactome drives functional networks

Protein-Protein Interactions (PPIs) typically occur between conserved, structurally rigid regions of two or more proteins, particularly ordered proteins that display energetically favorable, highly-folded conformations. Intriguingly, IDPs lack tertiary structure, yet engage in PPIs, albeit with lower affinities but high specificity1. The lack of structure within IDPs enhances their biophysical landscape, conferring them with the ability to attain structural complementarities required for PPIs. Since IDPs do not conform to a stable structure, they are less compact, providing a larger physical interface and energetic adaptability to interact with multiple proteins1, 7. Thus, conditional folding within IDPs is effectively utilized for interaction with a multitude of binding partners, enabling them to shuttle between several signaling cascades as efficient “cogs”, mediating and regulating PPIs4,7, 33, 34, 35, 36. Indeed, we discovered that PTEN, being an IDP, interacted with more than 400 proteins (Supplementary Table S1) when a combination of online software, literature search and database mining tools were used. Proteins with known PTEN interaction domains were classified as “mapped” (Figure 2d and Supplementary Table S1), whereas those with uncharacterized/predicted interactions were designated as “unmapped” proteins (Supplementary Table S1). Derivation of PTEN primary interactome from the mapped proteins using Cytoscape (http://www.cytoscape.org/) indicated that PTEN disorderliness is efficiently used for interaction with 40 proteins, most existing in distinct functional pathways (Figure 2d, 2e and Supplementary Table S2).

Interestingly, within the PTEN primary interactome, 60% of interactions occurred within the disordered C-tail region. Furthermore, disorder analysis on the primary interactome revealed that 33 proteins (>82%) were IDPs, of which two-thirds interacted with the C-tail IDR (Figure 2e, 2f andSupplementary Table S3), indicating a high propensity for disorder-disorder (D-D)-type interactions.

In order to study evolutionary conservation of the PTEN C-tail and its interactions across species, several sequence alignments were performed (Figure 3a). Sequence alignment of the entire PTEN protein from different animal species shows a good conservation of the catalytic phosphatase domain between vertebrates and invertebrates with 100% sequence conservation for the dual specificity phosphatase catalytic motif HCKAGKGR8 (Supplementary Figure S2). The C-tail shows good conservation in the vertebrate species, likely indicating the recent emergence of the function of PTEN C-tail region in regulating PTEN activity and enriching its PPI potential, translating to its versatile functions. In order to examine the conservation across species for the PTEN C-tail interacting proteins, a literature search was conducted to identify experimentally verified domains/motifs involved in interaction with the C-tail. The domains involved in these interactions with the C-tail for 13 proteins with relevant literature sources for these interactions are part of Supplementary Figure S3. Subsequent sequence alignments for these thirteen proteins (Supplementary Figure S3) shows good sequence homology for the domains/motifs involved in interaction with the PTEN C-tail. These findings support the concept that the PTEN C-tail has evolved in vertebrates to incorporate features that allow it to interact with these proteins.

Figure 3: Sequence conservation in PTEN and its interacting partners reflects functionality.

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f3.jpg

(a) Sequence alignment of the PTEN protein for vertebrate and invertebrate animals. Green color indicates sequence similarity while red indicates sequence dissimilar amino acid residues. All comparisons are made with respect to the human PTEN protein. (b) Network analysis for PTEN was performed to assess its potential as a network hub. The network shows multiple secondary interactions within the 40 mapped proteins, indicating their role in multiple signaling cascades mediated via PTEN. The proteins SMAD2/3, AR, PCAF, ANAPC7, B-arrestin 1 and p53 appear to be critical within these signaling cascades and also happen to be intrinsically disordered (Supplementary Table S3), reinforcing the concept of preferential interactions between disordered proteins. (Analysis Tool: Metacore by GeneGo).

Further, to assess whether PTEN acts as a functional hub protein and regulates pathways through its protein-binding partners, we performed functional network analysis using the Analyze Network option from MetaCore (GeneGo Inc, Thomson Reuters, 2011) (Figure 3b). The PTEN primary interactome was used as input with PTEN as the central node. We identified multiple interactions not only between PTEN (node) and SMAD2/3, AR, PCAF, ANAPC3, ANAPC4, Caveolin, β-arrestin 1 and p53 (edges), but also amongst the edge proteins themselves (Figure 3b). Interestingly, all the edge proteins are themselves highly disordered (Supplementary Table S3). Further supporting this finding, our functional enrichment revealed that 13 proteins (one-third) of the PTEN primary interactome were cancer-related and highly disordered (Figure 4a, Supplementary Table S3 and S4).

Figure 4: Derivation and disorder analysis of the PTEN cancer interactome.

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f4.jpg

  • Derivation of the PTEN Cancer Interactome. Functional enrichment of the PTEN primary interactome identified 13 cancer-related proteins which are also intrinsically disordered. Subsequently, the PTEN secondary interactome was derived from the primary PTEN interacting proteins. A subset of the secondary interactome was designated as the PTEN Cancer Interactome and it represents the proteins that interact with the 13 cancer-related proteins of the primary interactome. (b) PTEN Cancer Interactome. PTEN is the primary node that interacts with the 13 cancer-related proteins representing the partial primary interactome. Proteins that interact with each of the 13 cancer-related proteins comprise the secondary interactome. Disordered proteins are represented in red while ordered proteins are shown in blue. Cancer-related proteins in the PTEN primary interactome were identified using IPA (Ingenuity® Systems, ingenuity.com). (c) We identified 40 proteins that are part of the PTEN primary interactome of which 13 are highly disordered (IDP) and identified as potential cancer network hubs based on functional network analysis. We further identify 299 IDPS from the secondary PTEN interactome. A filter for cancer-related proteins revealed that approximately two-thirds of the IDPs that form the secondary interactome (193 out of 299) are involved in oncogenesis, suggesting a high degree of functional enrichment. (Functional network analysis was performed using IPA (Ingenuity® Systems,www.ingenuity.com).Full size image (805 KB)

Pliant PTEN secondary interactome relays function of the primary network

The disorderliness of the PTEN primary interactome prompted us to investigate the possibility that PTEN radiates its function via a malleable network of IDPs that extends beyond the primary interactome. Therefore, we derived the PTEN secondary interactome (Supplementary Table S5) and ascertained the interaction of 13 cancer-related proteins identified in the primary interactome (Figure 4a). The entire PTEN secondary interactome consisted of 299 IDPs, of which 193 IDPs (two-thirds) were associated with the 13 cancer-related proteins, generating a “PTEN-Cancer Interactome” (Figure 4, Supplementary Table S5 and S6). Thus, two-third of the IDPs within the PTEN secondary interactome associates with one-third of the cancer related IDPs within the PTEN primary interactome, indicating that cancer-related functions are driven by IDPs in the PTEN interactome and that the flexibility of IDP-IDP interactions modulates diverse functions; dysregulation of which causes cancers.

Functional network analysis of the 193 cancer-related IDPs identified 31 proteins that shared multiple nodes (Figure 5a and Supplementary Table S6). We overlaid this network with the cancer-related IDPs of the primary interactome to predict functionally critical protein hubs (indicated in yellow circles in Figure 5a and b). Our analysis revealed 16 proteins as highly populated hubs, most enriched in disordered regions, again demonstrating that a high degree of structural and functional association between the hubs required IDP-IDP interactions (Figure 5b). The involvement of these hubs in multiple, critical oncogenic signaling pathways make them attractive drug targets in the field of clinical oncology. Our bioinformatic analysis resonates well with observed biological phenomena as seen in the case of MDM2 protein, which is a major PPI hub regulating p53. Interaction of the human androgen receptor (AR) protein and MDM2 influences prostate cell growth and apoptosis37. Mdm2-Daxx interaction activates p53 following DNA damage38, and Daxx binds and inhibits AR function39. Conversely, the breast cancer susceptibility gene 1 (BRCA1) interacts directly with AR and enhances AR target genes, such as p21(WAF1/CIP1), that may result in the increase of androgen-induced cell death in prostate cancer cells40. Further, BRCA1 complexes with Smad3 and is inactivated, leading to early-onset familial breast and ovarian cancer41. Within the same network, MDM2 inhibits the transcriptional activity of SMAD proteins including SMAD342, thereby, emerging as a major player in prostrate, breast and ovarian cancer. Loss of PTEN, on the other hand, results in resistance to apoptosis by activating the MDM2-mediated antiapoptotic mechanism. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome (Figure 5b). Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis (Figure 5c). These findings support the prevailing concept of preferential interaction between disordered regions of two distinct proteins; with PTEN being the common disordered interacting hub, giving functional centrality to PTEN in many critical cellular pathways.

Figure 5: Predicting functionally relevant network hubs in the PTEN cancer interactome.

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

http://www.nature.com/srep/2013/130620/srep02035/images/srep02035-f5.jpg

(a) Methodology to identify functional hubs within the PTEN Cancer Interactome. The PTEN Cancer Interactome contains 193 IDPs that are potential hubs. Over-represented IDPs (or IDPs with multiple occurrences) in the PTEN Cancer Interactome would have a greater propensity to function as hubs. Upon sorting for over-represented IDPs the list of 193 proteins is brought down to 31 proteins. In order to assess the possibility of these 31 proteins as functional hubs a network analysis is warranted. (b) We identified 31 potential hubs based on multiple associations from within the 193 cancer-associated IDPs of the PTEN secondary interactome. Regulatory networks derived from these 31 proteins were overlaid with a similar network from the 13 cancer-related proteins. Based on the number of associations within the network, we identify 16 potential functional hubs in the PTEN cancer interactome (indicated in yellow). Regulatory interactions were generated using the Transcriptome Browser tool (Lopez et al, 2008). (c) Functional network analysis of the 16 predicted hubs. In order to assess the functional association of the 16 predicted hubs with PTEN – a network analysis with PTEN as a central node was done. The analysis identifies MDM2 protein, a major regulator of p53, as one of the major PPI hubs in the PTEN cancer interactome. A number of other critical cancer-related proteins, such as AR, SMAD2/3 and PDGFRB that are part of the PTEN primary interactome, feature prominently in the PTEN cancer interactome. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome. Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis. (Functional network analysis was performed using IPA (Ingenuity® Systems, www.ingenuity.com).

To further validate our methodology in using intrinsic disorder and cancer as filters to identify key signaling hubs, we compared our data sets with a previously published cancer signaling data set. We derived 7 common hubs (Supplementary Table S7), which were extended using the expansive human signaling network described previously43, 44, 45, 46 to obtain the PTEN associated cancer interactome (Figure 6a). An extensive disease associated network analysis using IPA validated our predictions as all the seven predicted hubs had an extensive cross-talk across multiple cancer disease types (Figure 6b).

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

Biochemical features modulating PTEN PPIs. srep02035-f7

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f6.jpg

(a) A PTEN linked cancer network was derived using seven of the 16 predicted cancer hubs that were common with the human cancer associated gene set. The associated partners of the seven hubs were extracted from the human signaling network (Cui et al, 2007, Awan et al, 2007, Li et al, 2012 and Newman et al, 2013). Red color denotes the potential cancer hubs and blue color are their associated partners. Topological analysis identifies p53 as the most significant network hub in the PTEN linked cancer network (Supplementary Table S7). (b) Disease associated network of PTEN cancer hubs. A functional network was constructed with the seven topologically relevant hubs identified previously using the Core Analysis function from the IPA suite to derive the primary network (denoted as MP). A disease network was constructed using the Path Designer option and disease associated biological functions were overlaid on the primary network. Fx denotes the different functions associated with the members of the networks.

Modulation of PTEN PPIs by linear binding motifs

Recent evidence has shown that IDPs mediate PPIs via short linear amino acid sequences (~20 residues) called Molecular Recognition Elements (MoREs) or Molecular Recognition Features (MoRFs)35, 47. MoRFs undergo disorder-to-order transitions upon binding and adopt thermodynamically stable well-defined structures47, increasing the propensity of IDPs to interact with a vast repertoire of proteins. MoRFs also display molecular recognition elements that capture the binding partner proteins with high specificity. These partner-dependent conformational differences are critical to imparting versatile binding properties to IDRs35.

Since the PTEN IDR engages in multiple PPIs, we tested the possibility for the existence of MoRFs. The MORFP red algorithm48 revealed that PTEN contains major MoRF sites at amino acids 273–279 (part of the disordered CBR3 loop of the C2 domain), amino acids 339–347 (in close vicinity of the disordered C-tail) and amino acids 395–403 (part of the disordered C-tail) (Figure 7a and Supplementary Figure S4). The primary restriction of MoRFs to the PTEN C-tail IDR or adjacent regions indicates that these MoRFs directly participate in modulating PPI functions (Figure 7a). However, mutational analysis within MoRFs is required to establish their active role in functional PPIs.

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

Biochemical features modulating PTEN PPIs. srep02035-f7

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f7.jpg

(a) MoRFs in the PTEN C-tail IDR. MoRFpred (Disfani et al, 2012), a computational tool, was used to identify MoRF regions within the PTEN protein (Supplementary Figure S4). The MoRFs in the vicinity of the C-tail IDR are highlighted in red. Interestingly, all of the major MoRFs (with a length greater than 5 residues) are observed in the vicinity of disordered regions (either part of the disordered CBR3 loop of the C2 domain or the C-tail IDR) indicating a positive correlation between intrinsic disorder and PPIs. (b) ELMs in PTEN C-tail IDR. Eukaryotic Linear Motifs (or ELMs) are 3–11 amino acid long sequences that mediate PPIs. IDRs are particularly enriched in ELMs (Dinkel et al, 2012). The linear motifs occurring in the disordered segment of PTEN (tail + PDZ domain) have been highlighted. The motifs with a high conservation score (>0.75) are indicated in red. Interestingly, all of the motifs with a high conservation score are restricted to the C-tail IDR. (c) Phosphorylation sites in the C-tail IDR. Phosphorylation of PTEN, particularly on serine and threonine residues in the disordered region, regulates the function and stability of PTEN. Phosphorylation occurs at Ser 362, Thr 366, Ser 370, Ser 380, Thr 382, Thr 383, Ser 385 by various enzymes such as Casein Kinase II, Glycogen synthase kinase 3-B and Polo-like kinase 3. Each of these phosphorylation events helps regulate the availability and stability of the PTEN molecule within the cell.

Protein-protein interactions are also facilitated by very short motifs (3–10 amino acids) called Short Linear Motifs (SLiMs) or Eukaryotic Linear Motifs (ELMs)49, 50. Because of their short sequences, ELMs arise/disappear by simple point mutations, providing the evolutionary plasticity that the ordered protein domains lack. Thus, ELMs easily adapt to novel interactions in signaling pathways, where rapid assembly/disassembly of multi-protein complexes is a prerequisite. The frequent occurrence of ELMs in a typical proteome indicates their critical cellular functions. Consistent with this notion, a higher density of ELMs are observed in hub proteins and IDPs50. Since ELMs have short sequences, they interact with low-affinity, however, they engage in highly cooperative binding in protein complexes, triggering productive signaling50. Therefore, at increased intracellular local concentrations they competitively bind to mutually overlapping physiological targets of each other as seen with PDZ, SH2 and PTB interaction domains found in cancer-associated proteins and in IDRs49, 50. As PTEN contains a PDZ-binding motif within the IDR (Figure 1a and c), we probed for the existence and features of ELMs in PTEN using The Eukaryotic Linear Motif Resource (http://elm.eu.org). We identified 34 different classes of ELMs in PTEN that mediate PPIs (Supplementary Figure S5). Interestingly, the four ELMs that are most conserved (conservation score>0.75) occurred within the PTEN C-tail IDR, indicating its high level of functional/biological significance (Figure 7b). ELM functions are further modulated by post-translational modifications, mainly by phosphorylation50. Indeed, the PTEN IDR possesses nine phosphorylation sites51, 52(Figure 7c).

PTEN phosphorylation modulates intramolecular association and PPI function

Post-translational Modifications (PTMs) in IDPs facilitate PPIs5. Modifying enzymes readily dock on structurally flexible IDRs, making them a hot spot for PTMs4, 7, 53, 54. Consistent with this notion, regulatory cancer-associated proteins have twice as much disorder and undergo more frequent phosphorylation/dephosphorylation than other cellular proteins as predicted by DISPHOS (a DISorder-enhanced PHOSphorylation prediction software)54, implicating a tight interconnection between protein phosphorylation and disorder. Consistent with the function of PTM in IDRs, clustering of Ser and Thr phosphorylation sites (Figure 7c) in the C-tail IDR regulates PTEN stability, membrane association and activity19, 20. Phosphorylation in the PEST [proline (P), glutamic acid (E), serine (S) and threonine (T)] domain within the C-tail IDR (amino acids 352 to 399) inhibits degradation of PTEN51. Casein kinase II (CK II), Glycogen synthase kinase 3-beta (GSK3-β) and PLK3 (Polo-like kinase 3) phosphorylate Ser and Thr residues within the IDR, each providing a distinct function51 (Figure 7c). The microtubule-associated serine/threonine (MAST), serine/threonine kinase 11(STK11) or LKB1 and casein kinase I (CKI) kinases have also been implicated in PTEN phosphorylation. STK11/LKB1 modifies T383, while CKI modifies T366, S370 and S38552. Indeed, our DISPHOS prediction for C-tail IDRs supports these experimental observations (Supplementary Figure S6).

Substrate-kinase interactions are typically of the disordered-ordered (D-O) type and are stabilized by hydrogen bonding (Figure 7c), a hallmark of IDRs54. Indeed, computational analysis revealed that large ordered regions comprising the catalytic domains of CKII, GSK3B, PLK3, Rak, and Src kinases interact with the C-tail IDR (Supplementary Table S8), indicating that PTEN engages in D-O type intermolecular interactions with the modifying kinases.

At the intramolecular level, phosphorylation at C-tail residues triggers a conformational change in PTEN, inhibiting its membrane association and, therefore, its lipid phosphatase activity18, 19, 21, 55. The phosphorylated C-tail IDR folds onto the PD and C2 domains giving rise to the “closed-closed” conformation of PTEN (Figure 8a) that is incapable of interaction with the membrane18, 20. The “closed- closed” form of PTEN is enzymatically inactive and cannot convert PIP3 to PIP2. The identification of the exact resides involved in this intramolecular interaction remains an active area of research18, 20, 56.

Figure 8: Targeting PTEN C-tail IDR.
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Most PTEN functions emanate from the C-tail IDR, including aberrant PPIs that hyper-activate oncogenic pathways. (a) Phosphorylation mediates an intramolecular interaction in the PTEN molecule. Phosphorylation causes a conformational change in PTEN converting it to the enzymatically inactive “closed closed ” form wherein the flexible tail folds onto residues in the C2 and phosphatase domain, thereby making it incapable of interacting with the membrane. Dephosphorylation (by an unknown phosphatase or via auto-dephosphorylation) converts PTEN to the “open-closed” form. Electrostatic interactions, mediated by the PBM, further convert PTEN to the “open-open” form wherein it binds to the membrane and acts as a lipid phosphatase converting PIP3 to PIP2, thereby, abrogating signaling via the PI3K/Akt/mTOR pathways. Subsequent to membrane binding, several E3 ubiquitin ligases polyubiquitinate PTEN marking it for proteasomal degradation. Phosphorylation, by inducing the intramolecular interaction, masks the ubiquitination sites thereby increasing the half-life of the PTEN protein within the cell. Therefore, phosphorylation negatively regulates PTEN function but positively regulates its stability. (b) PTEN IDR engages in PPIs of the disorder:order type (D-O type). As revealed in the present study, this occurs via the use of a MoRF or SLiM region. Therefore, designing a peptidomimetic drug molecule that competes with the PTEN MoRF/SLiM binding to the ordered protein will abrogate PTEN binding, therefore PTEN function. PTEN IDR is highly accessible to multiple kinases that phosphorylate and modulate PTEN function, mainly its inhibition via intra-molecular interactions. PTEN inhibition hyper-activates the PI3K/AKT/mTOR pathway, which increase the oncogenic potential of the cell and drives cancer growth. Therefore, targeting the PTEN C-tail IDR with small molecules that bind and sterically hinder PTEN phosphorylation and/or intra-molecular interactions will be an ideally adjunctive therapy to multiple inhibitor therapy targeting of the PI3/AKT/mTOR pathway.

It was recently shown that the phosphorylation events of PTEN occur in two independent cascades of ordered events, with the S380–S385 cluster being modified prior to the S361–S70 cluster52. Even within the two clusters, the phosphorylation events follow a specific pattern with a distributive kinetic mechanism. Not surprisingly, distributive kinetics is energetically favorable on protein domains that are highly disordered with multiple ensembles of flexible structures52. Thus the dynamic nature of these phosphorylation events is contingent to the inherent flexibility in the PTEN structure driven by intrinsically disordered C-tail crucial for PTEN stability and localization within the cell (Figure 8a).

Targeting intrinsic disorder in PTEN and its interactome

Drug targeting to critical protein regions can mitigate aberrant cellular processes driving oncogenesis57. However, despite numerous clinical trials with molecularly targeted therapies, failure rates for cancer treatments remain high. Conventional therapies targeting pathway-specific kinases suffer from “off-target effects” and often fail due to the emergence of compensatory and alternative pathways58. As a novel approach, facile drug targeting to IDRs within critical signaling hub proteins is highly plausible59, 60, 61. Moreover, as IDRs undergo extensive PTMs53 and engage in PPIs4, 34, 36, the multitude of resulting protein interactions (normal and aberrant) can be targeted concomitantly with a cocktail of distinct inhibitors, which dampens oncogenic signaling60.

Indeed, targeting PPIs is a more selective treatment strategy over conventional enzyme inhibitors60. However, disruption of multiple ordered interfaces within PPIs by small molecule inhibitors remains challenging62. The advantage of targeting IDPs engaged in PPIs is that, unlike ordered proteins, they engage in PPIs via MoRFs or ELMs, which are small peptide regions that bind with low affinity and thus are susceptible to disruption by small molecule inhibitors59. Consistent with this notion, small molecules disrupted highly disordered complexes of p53-Mdm2 and c-Myc-Max interactions by inducing order upon binding60, 63. Likewise, targeting the PTEN C-tail IDR may reduce its intra- and inter-molecular interactions and limit accessibility to enzymes mediating PTMs (Figure 8b), providing a means to increase PTEN activity. Our analysis shows that since the C-tail IDR is rich in conserved MoRFs/SLiMS, targeting these regions will prove to be a rational therapeutic modality for a large number of cancers that show compromised PTEN activity or hyperactivation of the oncogenic PI3K/AKT/mTOR pathway9, 10, 11. Since reductions in the levels and activity of PTEN are sufficient to drive oncogenesis11, 14, 15, increasing PTEN activity is an ideal therapy for cancers associated with hyperactive PI3K-signaling.

Discussion

Recent studies on genome- and proteome-wide molecular alterations in diseases indicate that pathological conditions are caused by perturbations in complex, highly interconnected biological networks64. Thus, current reductionist approach of studying structure-function relationship in diseases has limited our abilities to discover effective targeted therapeutics. In an attempt to overcome these limitations, in the current study, we have undertaken a novel approach to drug discovery that exploits systems and network biology at the structural, topological and functional level. Using PTEN, a tumor suppressor, we have applied computational and systems biology approaches and integrated extensive data-mining and biochemical properties of IDP interactions to reach a finer understanding of PTEN function. These results have identified PTEN C-tail IDR and several hub proteins in PTEN-driven molecular network implicated in human diseases as therapeutic targets, enhancing the repertoire of clinically relevant biological targets for pharmacotherapy.

Our derivation and analysis of PTEN primary and secondary interactome indicates that altered levels or interactions of IDPs perturb myriad cellular signaling pathways, leading to pathological conditions including cancer. IDPs have the propensity to aggregate and cause cellular toxicity65. Therefore, PTEN as an IDP has evolved a mechanism, wherein, the level of active PTEN, its cellular localization and PTEN-PPIs are regulated via phosphorylation of the C-tail IDR. Furthermore, evolutionarily conserved ELMs and MoRFs that we have identified within the C-tail IDR may play a critical role in orchestrating the formation and function of the PTEN interactome.

Increase in complexity of PPIs is either directed by the number and type of proteins or by increasing the number of interactions required to execute cellular functions66. To delineate how PTEN executes myriad functions, we first derived the PTEN primary interactome. We found 40 proteins to directly interact on the PTEN molecule, out of which 25 were associated with the C-tail IDR, consistent with the concept that disorderliness within PTEN executes its myriad functions. To enhance our understanding of PTEN functions in the context of multiple distinct pathways at the systems-level, we delineated functional networks operating within the primary interactome. Our findings showed a high degree of cross-talk between edges, implying that shared regulatory modules, comprised of multiple signaling cascades, operate via PTEN-mediated interaction networks. When these networks are altered, diseases ensue with extreme functional penalties. We also found that the edge proteins were themselves highly disordered indicating that disorderliness within the PTEN primary interactome confers functional versatility. Supporting this notion, 13 proteins that were functionally classified as cancer-related were also highly disordered forming a pliable “PTEN-Cancer Interactome”. Thus, PTEN lesions influence the flexibility of IDP-IDP interactions modulating diverse functions, likely causing cancer.

Owing to the inherent ability of PPIs to be flexible while being complex, specific cellular functions are readily fine-tuned as per the biological demands. Emerging evidence suggests that certain features on the IDRs are recognized as a way of conferring plasticity to protein interaction networks. Consistent with this concept, our data suggest that PTEN, a hub protein containing an IDR, likely utilizes MoRFs and ELMs, gets differentially modified via PTMs, acquiring complementary structures to engage and modulate PPI activity by facilitating adaptive binding to multiple protein partners in many cellular pathways. Thus, our present work provide a novel entrée in targeting intrinsic disorder in PTEN and its interactome to dampen the aberrant PI3K-signaling that drives many cancers. First, imparting order to the PTEN structure may help dampen multiple oncogenic signaling pathways mediated via the 16 hub proteins identified in the present study, by limiting their affinity for PPIs. Second, targeting intrinsic disorder in PTEN and its interactome can become an adjunctive or alternative approach to the use of various kinase inhibitors, which are toxic and have many off-target effects when used to mitigate the aberrant hyperactivation of PI3K/AKT/mTOR oncogenic signaling pathway. Taken together, the present findings provide a novel entrée to design strategies for drug discovery and may become a logical intervention in the pharmacotherapy of cancer and other PTEN-associated disease treatment modalities.

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Realtime Clinical Expert Support

Posted in BioIT: BioInformatics, Biomarkers & Medical Diagnostics, Bone marrow derived cells, Chemical Biology and its relations to Metabolic Disease, tagged , , , , on May 10, 2015| Leave a Comment »

Medical Informatics View

Chapter 1 Statement of Inferential    Second Opinion

Realtime Clinical Expert Support

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options.

 

Keywords: Entropy, Maximum Likelihood Function, separatory clustering, peripheral smear, automated hemogram, Anomaly, classification by anomaly, multivariable and multisyndromic, automated second opinion

Abbreviations: Akaike Information Criterion, AIC;  Bayes Information Criterion, BIC, Systemic Inflammatory Response Syndrome, SIRS.

 

Background: The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Intent: We are proposing an innovation that supercedes the main design elements of a DASHBOARD and utilizes the conjoined syndromic features of the disparate data elements.  So the important determinant of the success of this endeavor is that it facilitates both the workflow and the decision-making process with a reduction of medical error. Continuing work is in progress in extending the capabilities with model datasets, and sufficient data because the extraction of data from disparate sources will, in the long run, further improve this process.  For instance, the finding of  both ST depression on EKG coincident with an elevated cardiac biomarker (troponin), particularly in the absence of substantially reduced renal function. The conversion of hematology based data into useful clinical information requires the establishment of problem-solving constructs based on the measured data.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

Progressive modification of the measured features of the hemogram has delineated characteristics expressed as measurements of size, density, and concentration, resulting in many characteristic features of classification. In the diagnosis of hematological disorders proliferation of marrow precursors, the domination of a cell line, and features of suppression of hematopoiesis provide a two dimensional model.  Other dimensions are created by considering the maturity of the circulating cells.  The application of rules-based, automated problem solving should provide a valid approach to the classification and interpretation of the data used to determine a knowledge-based clinical opinion. The exponential growth of knowledge since the mapping of the human genome enabled by parallel advances in applied mathematics that have not been a part of traditional clinical problem solving.  As the complexity of statistical models has increased the dependencies have become less clear to the individual.  Contemporary statistical modeling has a primary goal of finding an underlying structure in studied data sets.  The development of an evidence-based inference engine that can substantially interpret the data at hand and convert it in real time to a “knowledge-based opinion” could improve clinical decision-making by incorporating multiple complex clinical features as well as duration of onset into the model.

An example of a difficult area for clinical problem solving is found in the diagnosis of SIRS and associated sepsis.  SIRS (and associated sepsis) is a costly diagnosis in hospitalized patients.   Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard.  The early diagnosis of SIRS/sepsis is made by the application of defined criteria (temperature, heart rate, respiratory rate and WBC count) by the clinician.   The application of those clinical criteria, however, defines the condition after it has developed and has not provided a reliable method for the early diagnosis of SIRS.  The early diagnosis of SIRS may possibly be enhanced by the measurement of proteomic biomarkers, including transthyretin, C-reactive protein and procalcitonin.  Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift).  The use of such markers, obtained by automated systems in conjunction with innovative statistical modeling, provides a promising approach to enhance workflow and decision making.   Such a system utilizes the conjoined syndromic features of disparate data elements with an anticipated reduction of medical error.  This study is only an extension of our approach to repairing a longstanding problem in the construction of the many-sided electronic medical record (EMR).  In a classic study carried out at Bell Laboratories, Didner found that information technologies reflect the view of the creators, not the users, and Front-to-Back Design (R Didner) is needed.

Costs would be reduced, and accuracy improved, if the clinical data could be captured directly at the point it is generated, in a form suitable for transmission to insurers, or machine transformable into other formats.  Such data capture, could also be used to improve the form and structure of how this information is viewed by physicians, and form a basis of a more comprehensive database linking clinical protocols to outcomes, that could improve the knowledge of this relationship, hence clinical outcomes.

 

 

How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed,  expressed by McGowan and Winstead-Fry (J J McGowan and P Winstead-Fry. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October; 87(4): 462–470.)   PMCID: PMC226622    Copyright notice

 

 

 

 

They introduce Problem Knowledge Couplers as a clinical decision support software tool that  recognizes that functionality must be predicated upon combining unique patient information, but obtained through relevant structured question sets, with the appropriate knowledge found in the world’s peer-reviewed medical literature.  The premise of this is stated by LL WEED in “Idols of the Mind” (Dec 13, 2006): “ a root cause of a major defect in the health care system is that, while we falsely admire and extol the intellectual powers of highly educated physicians, we do not search for the external aids their minds require”.  HIT use has been focused on information retrieval, leaving the unaided mind burdened with information processing.

 

 

The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.  In order to optimize the interface for physician, the system would have a “front-to-back” design, with the call up for any patient ideally consisting of a dashboard design that presents the crucial information that the physician would likely act on in an easily accessible manner.  The key point is that each item used has to be closely related to a corresponding criterion needed for a decision.  Currently, improved design is heading in that direction.  In removing this limitation the output requirements have to be defined before the database is designed to produce the required output.  The ability to see any other information, or to see a sequential visualization of the patient’s course would be steps to home in on other views.  In addition, the amount of relevant information, even when presented well, is a cognitive challenge unless it is presented in a disease- or organ-system structure.  So the interaction between the user and the electronic medical record has a significant effect on practitioner time, ability to minimize errors of interpretation, facilitate treatment, and manage costs.  The reality is that clinicians are challenged by the need to view a large amount of data, with only a few resources available to know which of these values are relevant, or the need for action on a result, or its urgency. The challenge then becomes how fundamental measurement theory can lead to the creation at the point of care of more meaningful actionable presentations of results.  WP Fisher refers to the creation of a context in which computational resources for meeting the challenges will be incorporated into the electronic medical record.  The one which he chooses is a probabilistic conjoint (Rasch) measurement model, which uses scale-free standard measures and meets data quality standards. He illustrates this by fitting a set of data provided by Bernstein (19)(27 items for the diagnosis of acute myocardial infarction (AMI) to a Rasch multiple rating scale model testing the hypothesis that items work together to delineate a unidimensional measurement continuum. The results indicated that highly improbable observations could be discarded, data volume could be reduced based on internal, and increased ability of the care provider to interpret the data.

 

Classified data a separate issue from automation

 Feature Extraction. This further breakdown in the modern era is determined by genetically characteristic gene sequences that are transcribed into what we measure.  Eugene Rypka contributed greatly to clarifying the extraction of features in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Even though there may be a large number of measured values, the variety is reduced by this compression, even though there is risk of loss of information.  Yet the real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information”, and Akaike found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka.  Of course, this was made far less complicated by the genetic complement that defines its function, which made  more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:

 

The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Extension of conditions and presentation to the electronic medical record (EMR)

We have published on the application of an automated inference engine to the Systemic Inflammatory Response (SIRS), a serious infection, or emerging sepsis.  We can report on this without going over previous ground.  Of considerable interest is the morbidity and mortality of sepsis, and the hospital costs from a late diagnosis.  If missed early, it could be problematic, and it could be seen as a hospital complication when it is not. Improving on previous work, we have the opportunity to look at the contribution of a fluorescence labeled flow cytometric measurement of the immature granulocytes (IG), which is now widely used, but has not been adequately evaluated from the perspective of diagnostic usage.  We have done considerable work on protein-energy malnutrition (PEM), to which the automated interpretation is currently in review.  Of course, the

cholesterol, lymphocyte count, serum albumin provide the weight of evidence with the primary diagnosis (emphysema, chronic renal disease, eating disorder), and serum transthyretin would be low and remain low for a week in critical care.  This could be a modifier with age in providing discriminatory power.

 

Chapter  3           References

 

The Cost Burden of Disease: U.S. and Michigan. CHRT Brief. January 2010. @www.chrt.org

The National Hospital Bill: The Most Expensive Conditions by Payer, 2006. HCUP Brief #59.

 

Rudolph RA, Bernstein LH, Babb J: Information-Induction for the diagnosis of

myocardial infarction. Clin Chem 1988;34:2031-2038.

Bernstein LH (Chairman). Prealbumin in Nutritional Care Consensus Group.

Measurement of visceral protein status in assessing protein and energy malnutrition: standard of care. Nutrition 1995; 11:169-171.

Bernstein LH, Qamar A, McPherson C, Zarich S, Rudolph R. Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory. Yale J Biol Med 1999; 72: 5-13.

 

Kaplan L.A.; Chapman J.F.; Bock J.L.; Santa Maria E.; Clejan S.; Huddleston D.J.; Reed R.G.; Bernstein L.H.; Gillen-Goldstein J. Prediction of Respiratory Distress Syndrome using the Abbott FLM-II amniotic fluid assay. The National Academy of Clinical Biochemistry (NACB) Fetal Lung Maturity Assessment Project.  Clin Chim Acta 2002; 326(8): 61-68.

 

Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.

 

Bernstein L, Bradley K, Zarich SA. GOLDmineR: Improving models for classifying patients with chest pain. Yale J Biol Med 2002; 75, pp. 183-198.

Ronald Raphael Coifman and Mladen Victor Wickerhauser. Adapted Waveform Analysis as a Tool for Modeling, Feature Extraction, and Denoising. Optical Engineering, 33(7):2170–2174, July 1994.

 

R. Coifman and N. Saito. Constructions of local orthonormal bases for classification and regression. C. R. Acad. Sci. Paris, 319 Série I:191-196, 1994.

 

Chapter 4           Clinical Expert System

Realtime Clinical Expert Support and validation System

We have developed a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .

 

The patient was treated for SIRS and the blood tests were repeated during the following week. The full combined record of our system’s assessment of the patient, as derived from the further Hematology tests, is illustrated below. The yellow line shows the diagnosis that corresponds to the first blood test (as also shown in the image above). The red line shows the next diagnosis that was performed a week later.

 

 

 

 

 

 

 

 

As we can see the following treatment, the SIRS risk as a major concern was eliminated and the system provides a positive feedback for the treatment of the physician.

 

Method for data organization and classification via characterization metrics.

Our database organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighbor hood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity. We offer a method that addresses the complexity and enables rather than disables the practitioner.  The method identifies outliers and combines data according to commonality of features.

 

 

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Diabetes is caused by Leaky Calcium Channels in Pancreatic Beta Cells – research @Columbia University Medical Center: The Role of RyR2 in Regulation of Insulin Release and Glucose Homeostasis

Posted in Ca2+ triggered activation, Calcium, Calcium Signaling, Cell Biology, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Clinical Diagnostics, Diabetes Mellitus, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Endocrine Diseases on April 14, 2015| Leave a Comment »

Diabetes is caused by Leaky Calcium Channels in Pancreatic Beta Cells – research @Columbia University Medical Center: The Role of RyR2 in Regulation of Insulin Release and Glucose Homeostasis

Reporter: Aviva Lev-Ari, PhD, RN

Cellular Defect Linked to Diabetes

Leaky calcium channels in pancreatic beta cells can lead to high blood sugar

VIEW VIDEO

http://newsroom.cumc.columbia.edu/blog/2015/04/07/cellular-defect-linked-diabetes/?elq=c55ba8ff64104a0b8e2c82d78749fe88&elqCampaignId=9&elqaid=12507&elqat=1&elqTrackId=aefc67f3855b40fe8b0a4461f3b0ca74

“Pancreatic beta cells were found to have leaky RyR2s, which were disrupting the function of mitochondria that provide cells with energy required for insulin release. The dysfunction was consistent with mitochondrial alterations that have been described in pancreatic beta cells from patients with type 2 diabetes,” said Dr. Santulli.

See article

http://newsroom.cumc.columbia.edu/blog/2015/04/07/cellular-defect-linked-diabetes/?elq=c55ba8ff64104a0b8e2c82d78749fe88&elqCampaignId=9&elqaid=12507&elqat=1&elqTrackId=aefc67f3855b40fe8b0a4461f3b0ca74

 

pancreatic beta cells

Electron microscope image of a pancreatic beta cell, showing malformed mitochondria resulting from calcium leakage; the purple circle represents an insulin granule. (Credit: Dr. Gaetano Santulli)

 

About:

 

The paper is titled, “Calcium release channel RyR2 regulates insulin release and glucose homeostasis.”

The other contributors are: Gennaro Pagano (Imperial College, London, UK, University of Molise, Campobasso, Italy, and Federico II University, Naples, Italy), Celestino Sardu (Leiden University Medical Center, Leiden, Netherlands, Second University of Naples, Naples, Italy, and Catholic University of the Sacred Heart, John Paul II Foundation for Research and Treatment, Campobasso, Italy), Wenjun Xie (CUMC), Steven Reiken (CUMC), Salvatore Luca D’Ascia (Department of Cardiology and Arrhythmology, Clinical Institute Città Studi Hospital, Milan, Italy), Michele Cannone (Giuseppe Tatarella Hospital, Cerignola, Foggia, Italy), Nicola Marziliano (Niguarda Ca’ Granda Hospital, Milan, Italy, and University Hospital of Parma, Parma, Italy), Bruno Trimarco (Federico II University), Theresa A. Guise (Indiana University School of Medicine, Indianapolis, Indiana), and Alain Lacampagne (14U1046 INSERM, UMR 9214, CNRS, CHRU Montpellier, Montpellier, France)

The study was funded by grants from the American Heart Association (13POST16810041), the Schaefer Foundation, the Phillip Foundation, and the National Institutes of Health (R01HL061503, R01HL102040, and R01AR060037).

Dr. Marks is a consultant and board member of ARMGO Pharma, Inc., which is targeting RyR channels for therapeutic purposes. The other authors declare no financial or other conflicts of interest.

Columbia University Medical Center provides international leadership in basic, preclinical, and clinical research; medical and health sciences education; and patient care. The medical center trains future leaders and includes the dedicated work of many physicians, scientists, public health professionals, dentists, and nurses at the College of Physicians and Surgeons, the Mailman School of Public Health, the College of Dental Medicine, the School of Nursing, the biomedical departments of the Graduate School of Arts and Sciences, and allied research centers and institutions. Columbia University Medical Center is home to the largest medical research enterprise in New York City and State and one of the largest faculty medical practices in the Northeast. For more information, visit cumc.columbia.edu or columbiadoctors.org.

 

Other related articles on the role of Calcium in Health and in Disease were published in this Open Access Online Scientific Journal, include the following: 

 

Identification of Biomarkers that are Related to the Actin Cytoskeleton – Part I

Larry H Bernstein, MD, FCAP

 

Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility – Part II

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

 

Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease – Part III

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN

 

The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets – Part IV

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

 

Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter – Part V

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

 

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD – Part VI

Aviva Lev-Ari, PhD, RN

 

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses – Part VII

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism – Part VIII

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor – Part IX

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission – Part X

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Sensors and Signaling in Oxidative Stress – Part XI

Larry H. Bernstein, MD, FCAP

 

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD)) – Part XII

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

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