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Archive for the ‘Anticancer Resistance’ Category


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

A mutated gene called RAS gives rise to a signalling protein Ral which is involved in tumour growth in the bladder. Many researchers tried and failed to target and stop this wayward gene. Signalling proteins such as Ral usually shift between active and inactive states.

 

So, researchers next tried to stop Ral to get into active state. In inacvtive state Ral exposes a pocket which gets closed when active. After five years, the researchers found a small molecule dubbed BQU57 that can wedge itself into the pocket to prevent Ral from closing and becoming active. Now, BQU57 has been licensed for further development.

 

Researchers have a growing genetic data on bladder cancer, some of which threaten to overturn the supposed causes of bladder cancer. Genetics has also allowed bladder cancer to be reclassified from two categories into five distinct subtypes, each with different characteristics and weak spots. All these advances bode well for drug development and for improved diagnosis and prognosis.

 

Among the groups studying the genetics of bladder cancer are two large international teams: Uromol (named for urology and molecular biology), which is based at Aarhus University Hospital in Denmark, and The Cancer Genome Atlas (TCGA), based at institutions in Texas and Boston. Each team tackled a different type of cancer, based on the traditional classification of whether or not a tumour has grown into the muscle wall of the bladder. Uromol worked on the more common, earlier form, non-muscle-invasive bladder cancer, whereas TCGA is looking at muscle-invasive bladder cancer, which has a lower survival rate.

 

The Uromol team sought to identify people whose non-invasive tumours might return after treatment, becoming invasive or even metastatic. Bladder cancer has a high risk of recurrence, so people whose non-invasive cancer has been treated need to be monitored for many years, undergoing cystoscopy every few months. They looked for predictive genetic footprints in the transcriptome of the cancer, which contains all of a cell’s RNA and can tell researchers which genes are turned on or off.

 

They found three subgroups with distinct basal and luminal features, as proposed by other groups, each with different clinical outcomes in early-stage bladder cancer. These features sort bladder cancer into genetic categories that can help predict whether the cancer will return. The researchers also identified mutations that are linked to tumour progression. Mutations in the so-called APOBEC genes, which code for enzymes that modify RNA or DNA molecules. This effect could lead to cancer and cause it to be aggressive.

 

The second major research group, TCGA, led by the National Cancer Institute and the National Human Genome Research Institute, that involves thousands of researchers across USA. The project has already mapped genomic changes in 33 cancer types, including breast, skin and lung cancers. The TCGA researchers, who study muscle-invasive bladder cancer, have looked at tumours that were already identified as fast-growing and invasive.

 

The work by Uromol, TCGA and other labs has provided a clearer view of the genetic landscape of early- and late-stage bladder cancer. There are five subtypes for the muscle-invasive form: luminal, luminal–papillary, luminal–infiltrated, basal–squamous, and neuronal, each of which is genetically distinct and might require different therapeutic approaches.

 

Bladder cancer has the third-highest mutation rate of any cancer, behind only lung cancer and melanoma. The TCGA team has confirmed Uromol research showing that most bladder-cancer mutations occur in the APOBEC genes. It is not yet clear why APOBEC mutations are so common in bladder cancer, but studies of the mutations have yielded one startling implication. The APOBEC enzyme causes mutations early during the development of bladder cancer, and independent of cigarette smoke or other known exposures.

 

The TCGA researchers found a subset of bladder-cancer patients, those with the greatest number of APOBEC mutations, had an extremely high five-year survival rate of about 75%. Other patients with fewer APOBEC mutations fared less well which is pretty surprising.

 

This detailed knowledge of bladder-cancer genetics may help to pinpoint the specific vulnerabilities of cancer cells in different people. Over the past decade, Broad Institute researchers have identified more than 760 genes that cancer needs to grow and survive. Their genetic map might take another ten years to finish, but it will list every genetic vulnerability that can be exploited. The goal of cancer precision medicine is to take the patient’s tumour and decode the genetics, so the clinician can make a decision based on that information.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/29117162

 

https://www.ncbi.nlm.nih.gov/pubmed/27321955

 

https://www.ncbi.nlm.nih.gov/pubmed/28583312

 

https://www.ncbi.nlm.nih.gov/pubmed/24476821

 

https://www.ncbi.nlm.nih.gov/pubmed/28988769

 

https://www.ncbi.nlm.nih.gov/pubmed/28753430

 

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Insights into the Metabolome

Curator: Larry H. Bernstein, MD, FCAP

FCAP

 

Updated 6/3/2016

 

Tapping the Metabolome

Genes, Transcripts, Proteins—All Have Come into Their “-Ome”     GEN May 15, 2016 (Vol. 36, No. 10)

http://www.genengnews.com/gen-articles/tapping-the-metabolome/5770/

 

 

The retina is responsible for capturing images from the visual field. Retinitis pigmentosa, which refers to a group of inherited diseases that cause retinal degeneration, causes a gradual decline in vision because retinal photoreceptor cells (rods and cones) die. Images on the left are courtesy of the National Eye Institute, NIH; image on the right is courtesy of Robert Fariss, Ph.D., and Ann Milam, Ph.D., National Eye Institute, NIH.

Metabolomics, the comprehensive evaluation of the products of cellular processes, can provide new findings and insight in a vast array of diseases and dysfunctions. Though promising, metabolomics lacks the standing of genomics or proteomics. It is, in a manner of speaking, the new kid on the “omics” block.

Even though metabolomics is still an emerging discipline, at least some quarters are giving it a warm welcome. For example, metabolomics is being advanced by the Common Fund, an initiate of the National Institutes of Health (NIH). The Common Fund has established six national metabolomics cores. In addition, individual agencies within NIH, such as the National Institute of Environmental Health Sciences (NIEHS), are releasing solicitations focused on growing more detailed metabolomics programs.

Whether metabolomic studies are undertaken with or without public support, they share certain characteristics and challenges. Untargeted or broad-spectrum studies are used for hypotheses generation, whereas targeted studies probe specific compounds or pathways. Reproducibility is a major challenge in the field; many studies cannot be reproduced in larger cohorts. Carefully defined guidance and standard operating procedures for sample collection and processing are needed.

While these challenges are being addressed, researchers are patiently amassing metabolomic insights in several areas, such as retinal diseases, neurodegenerative diseases, and autoimmune diseases. In addition, metabolomic sleuths are availing themselves of a growing selection of investigative tools.

A Metabolomic Eye on Retinal Degeneration

The retina has one of the highest metabolic activities of any tissue in the body and is composed of multiple cell types. This fact suggests that metabolomics might be helpful in understanding retinal degeneration. At least, that’s what occurred to Ellen Weiss, Ph.D., a professor of cell biology and physiology at the University of North Carolina School of Medicine at Chapel Hill. To explore this possibility, Dr. Weiss began collaborating with Susan Sumner, Ph.D., director of systems and translational sciences at RTI International.

Retinal degeneration is often studied through the use of genetic-mouse models that mimic the disease in humans. In the model used by Dr. Weiss, cells with a disease-causing mutation are the major light-sensing cells that degenerate during the disease. Individuals with the same or a similar genetic mutation will initially lose dim-light vision then, ultimately, bright-light vision and color vision.

Wild-type and mutant phenotypes, as well as dark- and light-raised animals, were compared, since retinal degeneration is exacerbated by light in this genetic model. Retinas were collected as early as day 18, prior to symptomatic disease, and analyzed. Although data analysis is ongoing, distinct differences have emerged between the phenotypes as well as between dark- and light-raised animals.

“There is a clear increase in oxidative stress in both light-raised groups but to a larger extent in the mutant phenotype,” reports Dr. Weiss. “There are global changes in metabolites that suggest mitochondrial dysfunction, and dramatic changes in lipid profiles. Now we need to understand how these metabolites are involved in this eye disease and the relevance of these perturbations.”

For example, the glial cells in the retina that upregulate a number of proteins in response to stress to attempt to save the retina are as likely as the light-receptive neurons to undergo metabolic changes.

“One of the challenges in metabolomics studies is assigning the signals that represent the metabolites or compounds in the samples,” notes Dr. Sumner. “Signals may be ‘unknown unknowns,’ compounds that have never been identified before, or ‘known unknowns,’ compounds that are known but that have not yet been assigned in the biological matrix.”

Internal and external libraries, such as the Human Metabolome Dictionary, are used to match signals. Whether or not a match exists, fragmentation patterns are used to characterize the metabolite, and when possible a standard is obtained to confirm identity. To assist with this process, the NIH Common Fund supports Metabolite Standard Synthesis Cores (MSSCs). RTI International holds an MSSC contract in addition to being a NIH-designated metabolomics core.

Mitochondrial Dysfunction in Alzheimer’s Disease     

Alzheimer’s disease (AD) is difficult to diagnose early due to its asymptomatic phase; accurate diagnosis occurs only in postmortem brain tissue. To evaluate familial AD, a rare inherited form of the disease, the laboratory of Eugenia Trushina, Ph.D., associate professor of neurology and associate professor of pharmacology at the Mayo Clinic, uses mouse models to study the disease’s early molecular mechanisms.

Synaptic loss underlies cognitive dysfunction. The length of neurons dictates that mitochondria move within the cell to provide energy at the site of the synapses. An initial finding was that very early on mitochondrial trafficking was affected reducing energy supply to synapses and distant parts of the cell.

During energy production, the major mitochondrial metabolite is ATP, but the organelle also produces many other metabolites, molecules that are implicated in many pathways. One can assume that changes in energy utilization, production, and delivery are associated with some disturbance.

“Our goal,” explains Dr. Trushina, “was to get a proof of concept that we could detect in the blood of AD patients early changes of mitochondria dysfunction or other changes that could be informative of the disease over time.”

A Mayo Clinic aging study involves a cohort of patients, from healthy to those with mild cognitive impairment (MCI) through AD. Patients undergo an annual battery of tests including cognitive function along with blood and cerebrospinal fluid sampling. Metabolic signatures in plasma and cerebrospinal fluid of normal versus various disease stages were compared, and affected mitochondrial and lipid pathways identified in MCI patients that progressed to AD.

“Last year we published on a new compound that goes through the blood/brain barrier, gets into mitochondria, and very specifically, partially inhibits mitochondrial complex I activity, making the cell resistant to oxidative damage,” details Dr. Trushina. “The compound was able to either prevent or slow the disease in the animal familial models.

“Treatment not only reduced levels of amyloid plaques and phosphorylated tau, it also restored mitochondrial transport in neurons. Now we have additional compounds undergoing investigation for safety in humans, and target selectivity and engagement.”

“Mitochondria play a huge role in every aspect of our lives,” Dr. Trushina continues. “The discovery seems counterintuitive, but if mitochondria function is at the heart of AD, it may provide insight into the major sporadic form of the disease.”

Distinguishing Types of Asthma

In children, asthma generally manifests as allergy-induced asthma, or allergic asthma. And allergic asthma has commonalities with allergic dermatitis/eczema, food allergies, and allergic rhinitis. In adults, asthma is more heterogeneous, and distinct and varied subpopulations emerge. Some have nonallergic asthma; some have adult-onset asthma; and some have obesity-, occupational-, or exercise-induced asthma.

Adult asthmatics may have markers of TH2 high verus TH2 low asthma (T helper 2 cell cytokines) and they may respond to various triggers—environmental antigens, occupational antigens, irritants such as perfumes and chlorine, and seasonal allergens. Exercise, too, can trigger asthma.

One measure that can phenotype asthmatics is nitric oxide, an exhaled breath biomarker. Nitric oxide is a smooth muscle relaxant, vasodilator, and bronchodilator that can have anti-inflammatory properties. There is a wide range of values in asthmatics, and a number of values are needed to understand the trend in a particular patient. L-arginine is the amino acid that produces nitric oxide when converted to L-citrulline, a nonessential amino acid.

According to Nicholas Kenyon, M.D., a pulmonary and critical care specialist who is co-director of the University of California, Davis Asthma Network (UCAN), some metabolomic studies suggest that there is a state of L-arginine depletion during asthma attacks or in severe asthma suggesting a lack of substrate to produce nitric oxide. Dr. Kenyon is conducting clinical work on L-arginine supplementation in a double-blind cross-over  intervention trial of L-arginine versus placebo. The 50-subject study in severe asthmatics should be concluded in early 2017.

Many new biologic therapies are coming to market to treat asthma; it will be challenging to determine which advanced therapy to provide to which patient. Therapeutics mostly target severe asthma populations and are for patients with evidence of higher numbers of eosinophils in the blood and lung, which include anti-IL-5 and (soon) anti-IL-13, among others.

Tools Development 

Waters is developing metabolomics applications that use multivariate statistical methods to highlight compounds of interest. Typically these applications combine separation procedures, accomplished by means of liquid chromatography or gas chromatography (LC or GC), with detection methods that rely on mass spectrometry (MS). To support the identification, quantification, and analysis of LC-MS data, the company provides bioinformatics software. For example, Progenesis QI software can interrogate publicly available databases and process information about isotopic patterns, retention times, and collision cross-sections.

Mass spectrometry (MS) is the gold standard in metabolomics and lipidomics. But there is a limit to what accurate mass and resolution can achieve. For example, neither isobaric nor isomeric species are resolvable solely by MS. New orthogonal analytical tools will allow more confident identifications.

To improve metabolomics separations before MS detection, a post-ionization separation tool, like ion mobility, which is currently used to support traditional UPLC-MS and MS imaging metabolomics protocols, becomes useful. The collision-cross section (CCS), which measures the shape of molecules, can be derived, and it can be used as an additional identification coordinate.

Other new chromatographic tools are under development, such as microflow devices and UltraPerformance Convergence Chromatography (UPC2), which uses liquid CO2 as its mobile phase, to enable new ways of separating chiral metabolites. Both UPC2 and microflow technologies have decreased solvent consumption and waste disposal while maintaining UPLC-quality performance in terms of chromatographic resolution, robustness, and reproducibility.

Informatics tools are also improving. In the latest versions of Waters’ Progenesis software, typical metabolomics identification problems are resolved by allowing interrogation of publicly available databases and scoring according to accurate mass, isotopic pattern, retention time, CCS, and either theoretical or experimental fragments.

MS imaging techniques, such as MALDI and DESI, provide spatial information about the metabolite composition in tissues. These approaches can be used to support and confirm traditional analyses without sample extraction, and they allow image generation without the use of antibodies, similar to immunohistochemistry.

“Ion-mobility tools will soon be implemented for routine use, and the use of extended CCS databases will help with metabolite identification,” comments Giuseppe Astarita Ph.D., principal scientist, Waters. “More applications of ambient ionization MS will emerge, and they will allow direct-sampling analyses at atmospheric pressure with little or no sample preparation, generating real-time molecular fingerprints that can be used to discriminate among phenotypes.”

Microflow Technology   

Microflow technology offers sensitivity and robustness. For example, at the Proteomics and Metabolomics Facility, Colorado State University, peptide analysis was typically performed using nanoflow chromatography; however, nanoflow chromatography is slow and technically challenging. Moving to microflow offered significant improvements in robustness and ease-of-use and resulted in improved chromatography without sacrificing sensitivity.

Conversely, small molecule applications were typically performed with analytical-scale chromatography. While this flow regime is extremely robust and fast, it can sometimes be limited in sensitivity. Moving to microflow offered significant improvements in sensitivity, 5- to 10-fold depending on the compound, without sacrificing robustness.

But broad-scale microflow adoption is hampered by a lack of available column chemistries and legacy HPLC or UPLC infrastructure that is not conducive to low-flow operation.

“We utilize microflow technology on all of our tandem quadrupole instruments for targeted quantitative assays,” says Jessica Prenni, Ph.D., director, Proteomics and Metabolomics Facility, Colorado State University. “All of our peptide quantitation is exclusively performed with microflow technology, and many of our small molecule assays. Application examples include endocannabinoids, bile acids and plant phytohormone panels.”

Compound annotation and comparability and transparency in data processing and reporting is a challenge in metabolomics research. Multiple groups are actively working on developing new tools and strategies; common best practices need to be adopted.

The continued growth of open-source spectral databases and new tools for spectral prediction from compound databases will dramatically impact the ability for metabolomics to result in novel discoveries. The move to a systems-level understanding through the combination of various omics data also will have a huge influence and be enabled by the continued development of open-source and user-friendly pathway-analysis tools.

 Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging

http://www.genengnews.com/gen-articles/paving-the-road-for-clinical-biomarkers/5757/

http://www.genengnews.com/Media/images/Article/thumb_ArcherDX_AnalyticalSensitivity2362411344.jpg

Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.

In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.

In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.

At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.

Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.

Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.

Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.

Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.

“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”

In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.

Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.

“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”

Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.

“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”

This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.

Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).

Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response.  …..

 

Researchers Uncover How ‘Silent’ Genetic Changes Drive Cancer

Fri, 06/03/2016 – 8:41amby Rockeller University

http://www.dddmag.com/news/2016/06/researchers-uncover-how-silent-genetic-changes-drive-cancer

“Traditionally, it has been hard to use standard methods to quantify the amount of tRNA in the cell,” says Tavazoie. The lead authors of the article, Hani Goodarzi, formerly a postdoc in the lab and now a new assistant professor at UCSF, and research assistant Hoang Nguyen, devised and applied a new method that utilizes state-of-the-art genomic sequencing technology to measure the amount of tRNAs in different cell types.

The team chose to compare breast tissue from healthy individuals with tumor samples taken from breast cancer patients–including both primary tumors that had not spread from the breast to other body sites, and highly aggressive, metastatic tumors.

They found that the levels of two specific tRNAs were significantly higher in metastatic cells and metastatic tumors than in primary tumors that did not metastasize or healthy samples. “There are four different ways to encode for the protein building block arginine,” explains Tavazoie. “Yet only one of those–the tRNA that recognizes the codon CGG–was associated with increased metastasis.”

The tRNA that recognizes the codon GAA and encodes for a building block known as glutamic acid was also elevated in metastatic samples.

The team hypothesized that the elevated levels of these tRNAs may in fact drive metastasis. Working in mouse models of primary, non-metastatic tumors, the researchers increased the production of the tRNAs, and found that these cells became much more invasive and metastatic.

They also did the inverse experiment, with the anticipated results: reducing the levels of these tRNAs in metastatic cells decreased the incidence of metastases in the animals.

How do two tRNAs drive metastasis? The researchers teamed up with members of the Rockefeller University proteomics facility to see how protein expression changes in cells with elevated levels of these two tRNAs.

“We found global increases in many dozens of genes,” says Tavazoie, “so we analyzed their sequences and found that the majority of them had significantly increased numbers of these two specific codons.”

According to the researchers, two genes stood out among the list. Known as EXOSC2 and GRIPAP1, these genes were strongly and directly induced by elevated levels of the specific glutamic acid tRNA.

“When we mutated the GAA codons to GAG– a “silent” mutation because they both spell out the protein building block glutamic acid–we found that increasing the amount of tRNA no longer increased protein levels,” explains Tavazoie. These proteins were found to drive breast cancer metastasis.

The work challenges previous assumptions about how tRNAs function and suggests that tRNAs can modulate gene expression, according to the researchers. Tavazoie points out that “it is remarkable that within a single cell type, synonymous changes in genetic sequence can dramatically affect the levels of specific proteins, their transcripts, and the way a cell behaves.”

 

Testing Blood Metabolites Could Help Tailor Cancer Treatment

6/03/2016 1 Comment by Institute of Cancer Research
http://www.dddmag.com/news/2016/06/testing-blood-metabolites-could-help-tailor-cancer-treatment

Scientists have found that measuring how cancer treatment affects the levels of metabolites – the building blocks of fats and proteins – can be used to assess whether the drug is hitting its intended target.

This new way of monitoring cancer therapy could speed up the development of new targeted drugs – which exploit specific genetic weaknesses in cancer cells – and help in tailoring treatment for patients.

Scientists at The Institute of Cancer Research, London, measured the levels of 180 blood markers in 41 patients with advanced cancers in a phase I clinical trial conducted with The Royal Marsden NHS Foundation Trust.

They found that investigating the mix of metabolic markers could accurately assess how cancers were responding to the targeted drug pictilisib.

Their study was funded by the Wellcome Trust, Cancer Research UK and the pharmaceutical company Roche, and is published in the journal Molecular Cancer Therapeutics.

Pictilisib is designed to specifically target a molecular pathway in cancer cells, called PI3 kinase, which has key a role in cell metabolism and is defective in a range of cancer types.

As cancers with PI3K defects grow, they can cause a decrease in the levels of metabolites in the bloodstream.

The new study is the first to show that blood metabolites are testable indicators of whether or not a new cancer treatment is hitting the correct target, both in preclinical mouse models and also in a trial of patients.

Using a sensitive technique called mass spectrometry, scientists at The Institute of Cancer Research (ICR) initially analysed the metabolite levels in the blood of mice with cancers that had defects in the PI3K pathway.

They found that the blood levels of 26 different metabolites, which were low prior to therapy, had risen considerably following treatment with pictilisib. Their findings indicated that the drug was hitting its target, and reversing the effects of the cancer on mouse metabolites.

Similarly, in humans the ICR researchers found that almost all of the metabolites – 22 out of the initial 26 – once again rose in response to pictilisib treatment, as seen in the mice.

Blood levels of the metabolites began to increase after a single dose of pictilisib, and were seen to drop again when treatment was stopped, suggesting that the effect was directly related to the drug treatment.

Metabolites vary naturally depending on the time of day or how much food a patient has eaten. But the researchers were able to provide the first strong evidence that despite this variation metabolites can be used to test if a drug is working, and could help guide decisions about treatment.

 

New Metabolic Pathway Reveals Aspirin-Like Compound’s Anti-Cancer Properties

http://www.genengnews.com/gen-news-highlights/new-metabolic-pathway-reveals-aspirin-like-compound-s-anti-cancer-properties/81252777/

Researchers at the Gladstone Institutes say they have found a new pathway by which salicylic acid, a key compound in the nonsteroidal anti-inflammatory drugs aspirin and diflunisal, stops inflammation and cancer.

In a study (“Salicylate, Diflunisal and Their Metabolites Inhibit CBP/p300 and Exhibit Anticancer Activity”) published in eLife, the investigators discovered that both salicylic acid and diflunisal suppress two key proteins that help control gene expression throughout the body. These sister proteins, p300 and CREB-binding protein (CBP), are epigenetic regulators that control the levels of proteins that cause inflammation or are involved in cell growth.

By inhibiting p300 and CBP, salicylic acid and diflunisal block the activation of these proteins and prevent cellular damage caused by inflammation. This study provides the first concrete demonstration that both p300 and CBP can be targeted by drugs and may have important clinical implications, according to Eric Verdin, M.D., associate director of the Gladstone Institute of Virology and Immunology .

“Salicylic acid is one of the oldest drugs on the planet, dating back to the Egyptians and the Greeks, but we’re still discovering new things about it,” he said. “Uncovering this pathway of inflammation that salicylic acid acts upon opens up a host of new clinical possibilities for these drugs.”

Earlier research conducted in the laboratory of co-author Stephen D. Nimer, M.D., director of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, and a collaborator of Verdin’s, established a link between p300 and the leukemia-promoting protein AML1-ETO. In the current study, scientists at Gladstone and Sylvester worked together to test whether suppressing p300 with diflunisal would suppress leukemia growth in mice. As predicted, diflunisal stopped cancer progression and shrunk the tumors in the mouse model of leukemia. ……

 

Novel Protein Agent Targets Cancer and Host of Other Diseases

http://www.genengnews.com/gen-news-highlights/new-protein-agent-targets-cancer-and-host-of-other-diseases/81252780/

Researchers at Georgia State University have designed a new protein compound that can effectively target the cell surface receptor integrin v3, mutations in which have been linked to a number of diseases. Initial results using this new molecule show its potential as a therapeutic treatment for an array of illnesses, including cancer.

The novel protein molecule targets integrin v3 at a novel site that has not been targeted by other scientists. The researchers found that the molecule induces apoptosis, or programmed cell death, of cells that express integrin v3. This integrin has been a focus for drug development because abnormal expression of v3 is linked to the development and progression of various diseases.

“This integrin pair, v3, is not expressed in high levels in normal tissue,” explained senior study author Zhi-Ren Liu, Ph.D., professor in the department of biology at Georgia State. “In most cases, it’s associated with a number of different pathological conditions. Therefore, it constitutes a very good target for multiple disease treatment.”

“Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, which binds to integrin αvβ3 outside the classical ligand-binding site,” the authors wrote. “We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3.”

The findings from this study were published recently in Nature Communications in an article entitled “Rational Design of a Protein That Binds Integrin αvβ3 Outside the Ligand Binding Site.”   …..

“We took a unique angle,” Dr. Lui noted. “We designed a protein that binds to a different site. Once the protein binds to the site, it directly triggers cell death. When we’re able to kill pathological cells, then we’re able to kill the disease.”

The investigators performed extensive cell and molecular testing that confirmed ProAgio interacts and binds well with integrin v3. Interestingly, they found that ProAgio induces apoptosis by recruiting caspase 8—an enzyme that plays an essential role in programmed cell death—to the cytoplasmic area of integrin v3. ProAgio was much more effective in inducing cell death than other agents tested.

 

Noncoding RNAs Not So Noncoding

Bits of the transcriptome once believed to function as RNA molecules are in fact translated into small proteins.

By Ruth Williams | June 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/46150/title/Noncoding-RNAs-Not-So-Noncoding

In 2002, a group of plant researchers studying legumes at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, discovered that a 679-nucleotide RNA believed to function in a noncoding capacity was in fact a protein-coding messenger RNA (mRNA).1 It had been classified as a long (or large) noncoding RNA (lncRNA) by virtue of being more than 200 nucleotides in length. The RNA, transcribed from a gene called early nodulin 40 (ENOD40), contained short open reading frames (ORFs)—putative protein-coding sequences bookended by start and stop codons—but the ORFs were so short that they had previously been overlooked. When the Cologne collaborators examined the RNA more closely, however, they found that two of the ORFs did indeed encode tiny peptides: one of 12 and one of 24 amino acids. Sampling the legumes confirmed that these micropeptides were made in the plant, where they interacted with a sucrose-synthesizing enzyme.

Five years later, another ORF-containing mRNA that had been posing as a lncRNA was discovered inDrosophila.2,3 After performing a screen of fly embryos to find lncRNAs, Yuji Kageyama, then of the National Institute for Basic Biology in Okazaki, Japan, suppressed each transcript’s expression. “Only one showed a clear phenotype,” says Kageyama, now at Kobe University. Because embryos missing this particular RNA lacked certain cuticle features, giving them the appearance of smooth rice grains, the researchers named the RNA “polished rice” (pri).

Turning his attention to how the RNA functioned, Kageyama thought he should first rule out the possibility that it encoded proteins. But he couldn’t. “We actually found it was a protein-coding gene,” he says. “It was an accident—we are RNA people!” The pri gene turned out to encode four tiny peptides—three of 11 amino acids and one of 32—that Kageyama and colleagues showed are important for activating a key developmental transcription factor.4

Since then, a handful of other lncRNAs have switched to the mRNA ranks after being found to harbor micropeptide-encoding short ORFs (sORFs)—those less than 300 nucleotides in length. And given the vast number of documented lncRNAs—most of which have no known function—the chance of finding others that contain micropeptide codes seems high.

Overlooked ORFs

From the late 1990s into the 21st century, as species after species had their genomes sequenced and deposited in databases, the search for novel genes and their associated mRNAs duly followed. With millions or even billions of nucleotides to sift through, researchers devised computational shortcuts to hunt for canonical gene and mRNA features, such as promoter regions, exon/intron splice sites, and, of course, ORFs.

ORFs can exist in practically any stretch of RNA sequence by chance, but many do not encode actual proteins. Because the chance that an ORF encodes a protein increases with its length, most ORF-finding algorithms had a size cut-off of 300 nucleotides—translating to 100 amino acids. This allowed researchers to “filter out garbage—that is, meaningless ORFs that exist randomly in RNAs,” says Eric Olsonof the University of Texas Southwestern Medical Center in Dallas.

Of course, by excluding all ORFs less than 300 nucleotides in length, such algorithms inevitably missed those encoding genuine small peptides. “I’m sure that the people who came up with [the cut-off] understood that this rule would have to miss anything that was shorter than 100 amino acids,” saysNicholas Ingolia of the University of California, Berkeley. “As people applied this rule more and more, they sort of lost track of that caveat.” Essentially, sORFs were thrown out with the computational trash and forgotten.

Aside from statistical practicality and human oversight, there were also technical reasons that contributed to sORFs and their encoded micropeptides being missed. Because of their small size, sORFs in model organisms such as mice, flies, and fish are less likely to be hit in random mutagenesis screens than larger ORFs, meaning their functions are less likely to be revealed. Also, many important proteins are identified based on their conservation across species, says Andrea Pauli of the Research Institute of Molecular Pathology in Vienna, but “the shorter [the ORF], the harder it gets to find and align this region to other genomes and to know that this is actually conserved.”

As for the proteins themselves, the standard practice of using electrophoresis to separate peptides by size often meant micropeptides would be lost, notes Doug Anderson, a postdoc in Olson’s lab. “A lot of times we run the smaller things off the bottom of our gels,” he says. Standard protein mass spectrometry was also problematic for identifying small peptides, says Gerben Menschaert of Ghent University in Belgium, because “there is a washout step in the protocol so that only larger proteins are retained.”

But as researchers take a deeper dive into the function of the thousands of lncRNAs believed to exist in genomes, they continue to uncover surprise micropeptides. In February 2014, for example, Pauli, then a postdoc in Alex Schier’s lab at Harvard University, discovered a hidden code in a zebrafish lncRNA. She had been hunting for lncRNAs involved in zebrafish development because “we hadn’t really anticipated that there would be any coding regions out there that had not been discovered—at least not something that is essential,” she says. But one lncRNA she identified actually encoded a 58-amino-acid micropeptide, which she called Toddler, that functioned as a signaling protein necessary for cell movements that shape the early embryo.5

Then, last year, Anderson and his colleagues reported another. Since joining Olson’s lab in 2010, Anderson had been searching for lncRNAs expressed in the heart and skeletal muscles of mouse embryos. He discovered a number of candidates, but one stood out for its high level of sequence conservation—suggesting to Anderson that it might have an important function. He was right, the RNA was important, but for a reason that neither Anderson nor Olson had considered: it was in fact an mRNA encoding a 46-amino-acid-long micropeptide.6

“When we zeroed in on the conserved region [of the gene], Doug found that it began with an ATG [start] codon and it terminated with a stop codon,” Olson says. “That’s when he looked at whether it might encode a peptide and found that indeed it did.” The researchers dubbed the peptide myoregulin, and found that it functioned as a critical calcium pump regulator for muscle relaxation.

With more and more overlooked peptides now being revealed, the big question is how many are left to be discovered. “Were there going to be dozens of [micropeptides]? Were there going to be hundreds, like there are hundreds of microRNAs?” says Ingolia. “We just didn’t know.”

see more at  http://www.the-scientist.com/?articles.view/articleNo/46150/title/Noncoding-RNAs-Not-So-Noncoding

Research at Micro- and Nanoscales

From whole cells to genes, closer examination continues to surprise.

By Mary Beth Aberlin | June 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/46129/title/Research-at-Micro–and-Nanoscales

Little things mean a lot. To any biologist, this time-worn maxim is old news. But it’s worth revisiting. As several articles in this issue of The Scientist illustrate, how researchers define and examine the “little things” does mean a lot.

Consider this month’s cover story, “Noncoding RNAs Not So Noncoding,” by TS correspondent Ruth Williams. Combing the human genome for open reading frames (ORFs), sequences bracketed by start and stop codons, yielded a protein-coding count somewhere in the neighborhood of 24,000. That left a lot of the genome relegated to the category of junk—or, later, to the tens of thousands of mostly mysterious long noncoding RNAs (lncRNAs). But because they had only been looking for ORFs that were 300 nucleotides or longer (i.e., coding for proteins at least 100 amino acids long), genome probers missed so-called short ORFs (sORFs), which encode small peptides. “Their diminutive size may have caused these peptides to be overlooked, their sORFs to be buried in statistical noise, and their RNAs to be miscategorized, but it does not prevent them from serving important, often essential functions, as the micropeptides characterized to date demonstrate,” writes Williams.

How little things work definitely informs another field of life science research: synthetic biology. As the functions of genes and gene networks are sussed out, bioengineers are using the information to design small, synthetic gene circuits that enable them to better understand natural networks. In “Synthetic Biology Comes into Its Own,” Richard Muscat summarizes the strides made by synthetic biologists over the last 15 years and offers an optimistic view of how such networks may be put to use in the future. And to prove him right, just as we go to press, a collaborative group led by one of syn bio’s founding fathers, MIT’s James Collins, has devised a paper-based test for Zika virus exposure that relies on a freeze-dried synthetic gene circuit that changes color upon detection of RNAs in the viral genome. The results are ready in a matter of hours, not the days or weeks current testing takes, and the test can distinguish Zika from dengue virus. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” commented one researcher about the test.

Moving around little things is the name of the game when it comes to delivering a package of drugs to a specific target or to operating on minuscule individual cells. Mini-scale delivery of biocompatible drug payloads often needs some kind of boost to overcome fluid forces or size restrictions that interfere with fine-scale manipulation. To that end, ingenious solutions that motorize delivery by harnessing osmotic changes, magnets, ultrasound, and even bacterial flagella are reviewed in “Making Micromotors Biocompatible.”

….  http://www.the-scientist.com/?articles.view/articleNo/46129/title/Research-at-Micro–and-Nanoscales

Cilengitide: The First Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesis and Clinical Evaluation

Anticancer Agents Med Chem. 2010 Dec; 10(10): 753–768.
doi:  10.2174/187152010794728639

Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany).

This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.

Integrins are heterodimeric receptors that are important for cell-cell and cell-extracellular matrix (ECM) interactions and are composed of one α and one β-subunit [1, 2]. These cell adhesion molecules act as transmembrane linkers between their extracellular ligands and the cytoskeleton, and modulate various signaling pathways essential in the biological functions of most cells. Integrins play a crucial role in processes such as cell migration, differentiation, and survival during embryogenesis, angiogenesis, wound healing, immune and non-immune defense mechanisms, hemostasis and oncogenic transformation [1]. The fact that many integrins are also linked with pathological conditions has converted them into very promising therapeutic targets [3]. In particular, integrins αvβ3, αvβ5 and α5β1 are involved in angiogenesis and metastasis of solid tumors, being excellent candidates for cancer therapy [47].

There are a number of different integrin subtypes which recognize and bind to the tripeptide sequence RGD (arginine, glycine, aspartic acid), which represents the most prominent recognition motif involved in cell adhesion. For example, the pro-angiogenic αvβ3 integrin binds various RGD-containing proteins, including fibronectin (Fn), fibrinogen (Fg), vitronectin (Vn) and osteopontin [8]. It is therefore not surprising that this integrin has been targeted for cancer therapy and that RGD-containing peptides and peptidomimetics have been designed and synthesized aiming to selectively inhibit this receptor [9, 10].

One classical strategy used in drug design is based on the knowledge about the structure of the receptor-binding pocket, preferably in complex with the natural ligand. However, this strategy, the so-called “rational structure-based design”, could not be applied in the field of integrin ligands since the first structures of integrin’s extracellular head groups were not described until 2001 for αvβ3 [11] (one year later, in 2002 the structure of this integrin in complex with Cilengitide was also reported [12]) and 2004 for αIIbβ3 [13]. Therefore, initial efforts in this field focused on a “ligand-oriented design”, which concentrated on optimizing RGD peptides by means of different chemical approaches in order to establish structure-activity relationships and identify suitable ligands.

We focused our interest in finding ligands for αvβ3 and based our approach on three chemical strategies pioneered in our group: 1) Reduction of the conformational space by cyclization; 2) Spatial screening of cyclic peptides; and 3)N-Methyl scan.

The combination of these strategies lead to the discovery of the cyclic peptidec(RGDf(NMe)V) in 1995. This peptide showed subnanomolar antagonistic activity for the αvβ3 receptor, nanomolar affinities for the closely related integrins αvβ5 and α5β1, and high selectivity towards the platelet receptor αIIbβ3. The peptide was patented together with Merck in 1997 (patent application submitted in 15.9.1995, opened in 20.3.1997) [14] and first presented with Merck’s agreement at the European Peptide Symposium in Edinburgh (September 1996) [15]. The synthesis and activity of this molecule was finally published in 1999 [16]. This peptide is now developed by Merck-Serono, (Darmstadt, Germany) under the name “Cilengitide” and has recently entered Phase III clinical trials for treating glioblastoma [17].  …..

The discovery 30 years ago of the RGD motif in Fn was a major breakthrough in science. This tripeptide sequence was also identified in other ECM proteins and was soon described as the most prominent recognition motif involved in cell adhesion. Extensive research in this direction allowed the description of a number of bidirectional proteins, the integrins, which were able to recognize and bind to the RGD sequence. Integrins are key players in the biological function of most cells and therefore the inhibition of RGD-mediated integrin-ECM interactions became an attractive target for the scientific community.

However, the lack of selectivity of linear RGD peptides represented a major pitfall which precluded any clinical application of RGD-based inhibitors. The control of the molecule’s conformation by cyclization and further spatial screening overcame these limitations, showing that it is possible to obtain privileged bioactive structures, which enhance the biological activity of linear peptides and significantly improve their receptor selectivity. Steric control imposed in RGD peptides together with their biological evaluation and extensive structural studies yielded the cyclic peptide c(RGDfV), the first small selective anti-angiogenic molecule described. N-Methylation of this cyclic peptide yielded the much potentc(RGDf(NMe)V), nowadays known as Cilengitide.

The fact that brain tumors, which are highly angiogenic, are more susceptible to the treatment with integrin antagonists, and the positive synergy observed for Cilengitide in combination with radio-chemotherapy in preclinical studies, encouraged subsequent clinical trials. Cilengitide is currently in phase III for GBM patients and in phase II for other types of cancers, with to date a promising therapeutic outcome. In addition, the absence of significant toxicity and excellent tolerance of this drug allows its combination with classical therapies such as RT or cytotoxic agents. The controlled phase III study CENTRIC was launched in 2008, with primary outcome measures due on September 2012. The results of this and other clinical studies are expected with great hope and interest.

Integrin Targeted Therapeutics

Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Integrins are heterodimeric cell surface receptors found in nearly all metazoan cell types, composed of non-covalently linked α and β subunits. In mammals, eighteen α-subunits and eight β-subunits have been identified to date 1. From this pool, 24 distinct heterodimer combinations have been observed in vivo that confer cell-to-cell and cell-to-ligand specificity relevant to the host cell and the environment in which it functions 2. Integrin-mediated interactions with the extracellular matrix (ECM) are required for the attachment, cytoskeletal organization, mechanosensing, migration, proliferation, differentiation and survival of cells in the context of a multitude of biological processes including fertilization, implantation and embryonic development, immune response, bone resorption and platelet aggregation. Integrins also function in pathological processes such as inflammation, wound healing, angiogenesis, and tumor metastasis. In addition, integrin binding has been identified as a means of viral entry into cells 3. ….

Combination of cilengitide and radiation therapy and temozolomide. The addition of cilengitide to radiotherapy and temozolomide based treatment regimens has shown promising preliminary results in ongoing Phase II trials in both newly diagnosed and progressive glioblastoma multiforme 139140. In addition to the Phase II objectives sought, these trials are significant in that they represent progress that has made in determining tumor drug uptake and in identifying a subset of patients that may benefit from treatment. In a Phase II trial enrolling 52 patients with newly diagnosed glioblastoma multiforme receiving 500 mg cilengitide twice weekly during radiotherapy and in combination with temozolomide for 6 monthly cycles following radiotherapy, 69% achieved 6 months progression free survival compared to 54 % of patients receiving radiotherapy followed by temozolomide alone. The one-year overall survival was 67 and 62 % of patients for the cilengitide combination group and the radiotherapy and temozolomide group, respectively. Non-hematological grade 3-4 toxcities were limited, and included symptoms of fatigue, asthenia, anorexia, elevated liver function tests, deep vein thrombosis and pulmonary embolism in across a total of 5.7% of the patients. Grade 3-4 hematological malignancies were more common and included lymphopenia (53.8%), thrombocytopenia (13.4%) and neutropenia (9.6%). This trial is significant in the fact that is has provided the first evidence correlating a molecular biomarker with response to treatment. Decreased methylguanine methyltransferase (MGMT) expression was associated with favorable outcome. Patients harboring increased MGMT promoter methylation appeared to benefit more from combined treatment with cilengitide than did patients lacking promoter methylation. The significance of the MGMT promoter methylation in predicting response is likely due to inclusion of temozolomide in the treatment combination.

A similar Phase II study evaluating safety and differences in overall survival among newly diagnosed glioblastoma multiforme patients receiving radiation therapy combined with temozolomide and varying doses of cilengitide is nearing completion. Preliminary reports specify that initial safety run-in studies in 18 patients receiving doses 500, 1000 and 2000 mg cilengitide found no dose limiting toxicities. Subsequently 94 patients were randomized to receive standard therapy plus 500 or 2000 mg cilengitide. Median survival time in both cohorts was 18.9 months. At 12 months the overall survival was 79.5 % (89/112 patients).

In the last two decades great progress has been made in the discovery and development of integrin targeted therapeutics. Years of intense research into integrin function has provided an understanding of the potential applications for the treatment of disease. Advances in structural characterization of integrin-ligand interactions has proved beneficial in the design and development of potent, selective inhibitors for a number of integrins involved in platelet aggregation, inflammatory responses, angiongenesis, neovascularization and tumor growth.

The αIIbβ3 integrin antagonists were the first inhibitors to make their way into clinical use and have proven to be effective and safe drugs, contributing to the reduction of mortality and morbidity associated with acute coronary syndromes. Interestingly, the prolonged administration of small molecules targeting this integrin for long-term prevention of thrombosis related complications have not been successful, for reasons that are not yet fully understood. This suggests that modulating the intensity, duration and temporal aspects of integrin function may be more effective than simply shutting off integrin signaling in some instances. Further research into the dynamics of platelet activation and thrombosis formation may elucidate the mechanisms by which integrin activation is modulated.

The introduction of α4 targeted therapies held great promise for the treatment of inflammatory diseases. The development of Natalizumab greatly improved the quality of life for multiple sclerosis patients and those suffering with Crohn’s Disease compared to previous treatments, but the role in asthma related inflammation could not be validated. Unfortunately for MS and Crohn’s patients, immune surveillance in the central nervous system was also compromised as a direct effect α4β7 antagonism, with potentially lethal effects. Thus Natalizumab and related α4β7 targeting drugs are now limited to patients refractory to standard therapies. The design and development of α4β1 antagonists for the treatment of Crohn’s Disease may offer benefit with decreased risks. The involvement of these integrins in fetal development also raises concerns for widespread clinical use.

Integrin antagonists that target angiogenesis are progressing through clinical trials. Cilengitide has shown promising results for the treatment of glioblastomas and recurrent gliomas, cancers with notoriously low survival and cure rates. The greatest challenge facing the development of anti-angiogenic integrin targeted therapies is the overall lack of biomarkers by which to measure treatment efficacy.

 

Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach

Biochem J. 2009 Nov 11; 424(2): 179–189. doi:  10.1042/BJ20090992
Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by α5β1 is poorly understood. Here we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of RGD and synergy sites in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish α5 subunit with the corresponding regions of human α5 we show that blades 1-4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller (D3-A3 loop) contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human α5β1 supports an important function for D3-A3 loop residues Trp-157 and Ala-158 in the binding of antagonists. These results will aid the development of reagents that block α5β1 functions in vivo.
Structural Basis of Integrin Regulation and Signaling
Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cellpathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 Å couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
The immune system relies heavily on integrins for (a) adhesion during leukocyte trafficking from the bloodstream, migration within tissues, immune synapse formation, and phagocytosis; and (b) signaling during costimulation and cell polarization. Integrins are so named because they integrate the extracellular and intracellular environments by binding to ligands outside the cell and cytoskeletal components and signaling molecules inside the cell. Integrins are noncovalently associated heterodimeric cell surface adhesion molecules. In vertebrates, 18 α subunits and 8 β subunits form 24 known αβ pairs (Figure 1). This diversity in subunit composition contributes to diversity in ligand recognition, binding to cytoskeletal components and coupling to downstream signaling pathways. Immune cells express at least 10 members of the integrin family belonging to the β2, β7, and β1 subfamilies (Table 1). The β2 and β7 integrins are exclusively expressed on leukocytes, whereas the β1 integrins are expressed on a wide variety of cells throughout the body. Distribution and ligand-binding properties of the integrins on leukocytes are summarized in Table 1. For reviews, see References 1 and 2. Mutations that block expression of the β2 integrin subfamily lead to leukocyte adhesion deficiency, a disease associated with severe immunodeficiency (3).
As adhesion molecules, integrins are unique in that their adhesiveness can be dynamically regulated through a process termed inside-out signaling or priming. Thus, stimuli received by cell surface receptors for chemokines, cytokines, and foreign antigens initiate intracellular signals that impinge on integrin cytoplasmic domains and alter adhesiveness for extracellular ligands. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction (outside-in signaling). These dynamic properties of integrins are central to their proper function in the immune system. Indeed, mutations or small molecules that stabilize either the inactive state or the active adhesive state—and thereby block the adhesive dynamics of leukocyte integrins—inhibit leukocyte migration and normal immune responses.

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Chemotherapy Benefit in Early Breast Cancer Patients

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

Agendia’s MammaPrint® First and Only Genomic Assay to Receive Level 1A Clinical Utility Evidence for Chemotherapy Benefit in Early Breast Cancer Patients

http://www.b3cnewswire.com/201604191373/agendias-mammaprintr-first-and-only-genomic-assay-to-receive-level-1a-clinical-utility-evidence-for-chemotherapy-benefit-in-early-breast-cancer-patients.

  • Clinical high-risk patients with a low-risk MammaPrint® result, including 48 percent node-positive, had five-year distant metastasis-free survival rate in excess of 94 percent, whether randomized to receive adjuvant chemotherapy or not
  • MammaPrint could change clinical practice by substantially de-escalating the use of adjuvant chemotherapy and sparing many patients an aggressive treatment they will not benefit from
  • Forty-six percent overall reduction in chemotherapy prescription among clinically high-risk patients

April 19, 2016 / B3C newswire / Agendia, Inc., together with the European Organisation for Research and Treatment of Cancer (EORTC) and Breast International Group (BIG), announced results from the initial analysis of the primary objective of the Microarray In Node-negative (and 1 to 3 positive lymph node) Disease may Avoid ChemoTherapy (MINDACT) study at the American Association for Cancer Research Annual Meeting 2016 in New Orleans, LA.

Using the company’s MammaPrint® assay, patients with early-stage breast cancer who were considered at high risk for disease recurrence based on clinical and biological criteria had a distant metastasis-free survival at five years in excess of 94 percent.The MammaPrint test—the first and only genomic assay with FDA 510(k) clearance for use in risk assessment for women of all ages with early stage breast cancer—identified a large group of patients for whom five-year distant metastasis–free survival was equally good whether or not they received adjuvant chemotherapy (chemotherapy given post-surgery).

“The MINDACT trial design is the optimal way to prove clinical utility of a genomic assay,” said Prof. Laura van ’t Veer, CRO at Agendia, Leader, Breast Oncology Program, and Director, Applied Genomics at UCSF Helen Diller Family Comprehensive Cancer Center. “It gives the level 1A clinical evidence (prospective, randomized and controlled) that empowers physicians to clearly and confidently know when chemotherapy is part of optimal early-stage breast cancer therapy.  In this trial, MammaPrint (70-gene assay) was compared to the standard of care physicians use today, to decide what is the best treatment option for an early-stage breast cancer patient.”

The MINDACT trial is the first prospective randomized controlled clinical trial of a breast cancer recurrence genomic assay with level 1A clinical evidence and the first prospective translational research study of this magnitude in breast cancer to report the results of its primary objective.

Among the 3,356 patients enrolled in the MINDACT trial, who were categorized as having a high risk of breast cancer recurrence based on common clinical and pathological criteria (C-high), the MammaPrint assay reduced the chemotherapy treatment prescription by 46 percent.Using the 70-gene assay, MammaPrint, 48 percent of lymph-node positive breast cancer patients considered clinically high-risk (Clinical-high) and genomic low-risk (MammaPrint-low) had an excellent distant metastasis-free survival at five years in excess of 94 percent.

“Traditionally, physicians have relied on clinical-pathological factors such as age, tumor size, tumor grade, lymph node involvement, and hormone receptor status to make breast cancer treatment decisions,” said Massimo Cristofanilli, MD, Associate Director of Translational Research and Precision Medicine at the Robert H. Lurie Comprehensive Cancer Center, Northwestern University in Chicago. “These findings provide level 1A clinical utility evidence by demonstrating that the detection of low-risk of distant recurrence reported by the MammaPrint test can be safely used in the management of thousands of women by identifying those who can be spared from a toxic and unnecessary treatment.”

MINDACT is a randomized phase III trial that investigates the clinical utility of MammaPrint, when compared (or – “used in conjunction with”) to the standard clinical pathological criteria, for the selection of patients unlikely to benefit from adjuvant chemotherapy. From 2007 to 2011, 6,693 women who had undergone surgery for early-stage breast cancer enrolled in the trial (111 centers in nine countries). Participants were categorized as low or high risk for tumor recurrence in two ways: first, through analysis of tumor tissue using MammaPrint at a central location in Amsterdam; and second, using Adjuvant! Online, a tool that calculates risk of breast cancer recurrence based on common clinical and biological criteria.

Patients characterized in both clinical and genomic assessments as “low- risk” are spared chemotherapy, while patients characterized as “high- risk” are advised chemotherapy. Those with conflicting results are randomized to use either clinical or genomic risk (MammaPrint) evaluation to decide on chemotherapy treatment.

The MINDACT trial is managed and sponsored by the EORTC as part of an extensive and complex partnership in collaboration with Agendia and BIG, and many other academic and commercial partners, as well as patient advocates.

“These MINDACT trial results are a testament that the science of the MammaPrint test is the most robust in the genomic breast recurrence assay market.  Agendia will continue to collaborate with pharmaceutical companies, leading cancer centers and academic groups on additional clinical research and in the pursuit of bringing more effective, individualized treatments within reach of cancer patients,” said Mark Straley, Chief Executive Officer at Agendia. “We value the partnership with the EORTC and BIG and it’s a great honor to share this critical milestone.”

Breast cancer is the most frequently diagnosed cancer in women worldwide(1). In 2012, there were nearly 1.7 million new breast cancer cases among women worldwide, accounting for 25 percent of all new cancer cases in women(2).

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Colon cancer and organoids

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

 

Guts and Glory

An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.

By Anna Azvolinsky    http://www.the-scientist.com/?articles.view/articleNo/45580/title/Guts-and-Glory

Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”

“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”

Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”

Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”

Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.

Clevers Creates

Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”

Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.

Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”

Clevers Concentrates

On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.

On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.

Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”

Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.

Clevers Catapults

COURTESY OF HANS CLEVERS AND JEROEN HUIJBEN, NYMUS

3DCrypt building and colon cancer.

Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.

Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”

On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.

Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”

Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”

Greatest Hits

  • Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
  • Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
  • First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
  • Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
  • Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
 Regenerative Medicine Comes of Age   
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
  • Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.

    An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.

    A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.

    Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.

    Many damage-repair strategies are at an  early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.

    Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.

  • Building Stem Cell Factories

    Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.

    “Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”

    Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.

    For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.

    • “You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.

    • Keeping an Eye on Aging Eyes

      The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.

      The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.

      These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.

      A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.

      The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.

      One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.

      “These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”

      Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.

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Pull at Cancer’s Levers

Curator: Larry H. Bernstein, MD, FCAP

 

Driving Cancer Immunotherapy 

The Stakes in Immuno-Oncology Are Too High for Researchers to Pull at Cancer’s Levers Blindly. Researchers Need a System.

  • Within the past decade or so, a revolutionary idea has emerged in the minds of scientists, physicians, and medical experts. Instead of using man-made chemicals to treat cancer, let us instead unleash the power of our own bodies upon the malignancy.

    This idea is the inspiration behind cancer immunotherapy, which is, according to most experts, a therapeutic approach that involves training the immune system to fight off cancer. In the words of one expert, cancer immunotherapy means “taking the immune system’s inherent properties and turbo charging those to fight cancer.”

    Cancer immunotherapy technologies are being developed to accomplish
    several tasks:

    • Enhance the molecular targeting of cancer cells
    • Report the rate of killing by specific immune agents
    • Direct immune cells toward tumor destruction.

    Since its inception, the field has evolved, and it continues to do so. It began with in vivo investigations of tumor growth and development, and it progressed through laboratory investigations of cellular morphology and survival curves. And now it is adopting pathway analysis to guide therapeutic development and improve patient care.

    To begin to understand cancer immunotherapy, one must understand how the immune system targets tumor cells. One of the prominent adaptive components of the immune system is the T cell, which responds to perceived threats through the massive increase in clonal T cells targeted in some way toward the diseased cell or pathogen.

  • The T-Cell Repertoire

    Adaptive Biotechnologies’ immunoSEQ Assay, a high-throughput research platform for immune system profiling, is designed to generate sequencer-ready libraries using highly optimized primer sets in a multiplex PCR format that targets T- and B-cell receptor genes. This image depicts how the assay’s two-step PCR process can be used to quantify the clonal diversity of immune cells.

    Immunologists call this process VJD rearrangement. It happens during T-lymphocyte development and affects three gene regions, the variable (V), the diversity (D), and the joining (J) regions. This rearrangement of the genetic code allow for the structural diversity in T-cell receptors responsible for antigenic specificity including antigenic targets on tumor cells. In the case of cancer, specificity is complicated because the tumor is actually part of the body itself, one of the reasons cancers naturally evade detection.

    The specificity problem would always hinder attempts to goad the immune system into attacking cancer, scientists realized, unless technologies emerged that could efficiently track the clonal diversity of T cells inside patients. Existing technologies, such as spectratyping, were inadequate.  In 2007, when Dr. Robins and his collaborators began developing the technology, only 10,000 T-cell receptor sequences had been reported in all the literature using older methodologies.

    “The immunology field of the time had no connection with high-throughput sequencing,” notes Dr. Robins, recalling his days as a computational biologist for the Fred Hutchinson Cancer Research Center. “It became clear that instead of using this old technology to look at T-cell receptors, we could just directly sequence them—if we could amplify them correctly.”

    With its first experiment, Dr. Robins’ team ended up with six million T-cell receptor sequences. “Our approach,” Dr. Robins modestly suggests, “kind of changed the scale of what we were able to do.” The team went on to develop advanced multiplex sequencing technology, doing work that essentially started the field of immune sequencing. “Previously,” maintains Dr. Robins, “no one had ever been able to quantitatively do a multiplex PCR.”

    Adaptive Biotechnologies’ product, the ImmunoSEQ® assay, uses several hundred primer pairs to quantify the clonal diversity of T cells. Using this technology, researchers and clinicians can focus on T-cell clones that are expanded specifically in or near a tumor or that are circulating in the blood stream.

    “You obviously can’t get a serial sample of the tumor,” explains Dr. Robins, “but you can get serial samples of blood,” allowing for immune cell repertoire tracking during the progression of a disease. The technology is already being used to assess leukemias in the clinic, directly tracking the leukemia itself based on the massive clonal expansion of a single cancerous B or T cell.

    Eventually, Dr. Robins’ team hopes to monitor serial changes in T cell clones before, during, and after therapeutic intervention. The team has even developed a tumor infiltrating lymphocyte (TIL) assay to examine clones that are attracted to tumors.

     

Circulating Tumor Cells

“Years ago, they were just interested in what was happening in the tumor,” says Daniel Adams, senior research scientist at Creatv MicroTech. “Now people have realized that the immune system is reacting to the tumor.”

Scientists such as Adams have been tracking tumor cells and tumor-modified stromal cells, as well as components of the non-adaptive immune system, directly within the bloodstream to examine changes that occur over time.

“We can’t go back in to re-biopsy the patient every year, or every time there is a recurrence,” says Adams, “It’s just not feasible.”

That is why Creatv MicroTech, with locations in Maryland and New Jersey, has developed the CellSieve, a mechanical cell filter. The CellSieve, which improves on older technology through better polymers and engineering, isolates circulating tumor cells (CTCs) and stromal cells in order to capture them for further clinical analysis.

Isolation, culture and expansion of cells isolated on CellSieve™. (A) MCF-7 cells spiked into vacutainers, isolated by filtration and cultured on CellSieve for 2-3 weeks. A 3 dimensional cluster attributed to this cell line is seen on the filter. (green=anti-cytokeratin, blue=DAPI) (B) PANC-1 cells spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. PANC-1 is seen growing as a monolayer on the filter. (C) SKBR3 cells are spiked into blood, filtered by CellSieve. The CTCs are identified by presence of anti-cytokeratin and anti-EpCAM, and absence of anti-CD45. After CTCs are counted, cells are subtyped by HER2 FISH. (D) SKBR3 cells are spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. Expanded colonies were directly analyzed as a whole colony and as individual cells, molecularly by HER2/CR17 FISH probes. (E) Circulating stromal cell, e.g. a 70 µm giant cancer associated macrophage can be identified for clinical use, myeloid marker in red. (F) A cell of interest can be identified and restained with immunotherapeutic biomarkers, e.g. PD-L1 (green) and PD-1 (purple). (G) After filtration, cells were identified with histopathological stains (e.g. H&E) for cytological analysis. (H) After H&E, external cell structures were analyzed by SEM. [Creatv MicroTech].

 

“As a patient goes through therapy, the patient’s resistance builds, and the cancer recurs in different subpopulations,” states Adams. “And after a few years, the original tumor mass is no longer applicable to what is growing in the patient farther down the road.”

Although CTCs are exceedingly rare in the bloodstream, with just one or two in every 5 to 10 mL of blood, and although these cells have a very low viability, the surviving CTCs have a high prognostic value.

“We looked at 30 to 40 breast cancer patients over two years,” reports Adams. “And we showed that if you have a dividing CTC, you have a 90% chance of dying in two years and a 100% chance of dying within two and half years.”

Furthermore, the immune system response can be tracked, says Adams, by examining stromal cells, which can also be collected with the CellSieve filtration device. That is, these cells can be collected serially. Much recent evidence supports the conclusion that stromal cells in the tumor environment co-evolve with the tumor, suggesting that stromal marker changes reflect tumor changes.

“There is this plethora of stromal cells and tumor cells out there in the circulation for you to look at,” declares Adams. “Once the cells are isolated, you can subject them to pathological approaches, biomarker approaches, or molecular approaches—or all of the above.”

A MicroTech Creatv study published in the Royal Society of Chemistry showed the efficacy of following up CTC isolation with techniques such as fluorescence in situ hybridization (FISH), histopathological analysis, and cell culture.

Cancer-Killing Assays

Diverse mechanisms are at play in cancer biology. Our understanding of these mechanisms contributes to a couple of virtuous cycles. It strengthens and is strengthened by diagnostic approaches, such as immune- and tumor-cell monitoring. The same could be said of therapeutic approaches. Cancer biology will inform and be informed by cancer immunotherapies such as adoptive cell transfer. To maintain the virtuous cycle, however, it will be necessary to conduct in vitro testing.

“There is no doubt that immunotherapy is going to play a major role in the treatment of cancer,” says Brandon Lamarche, Ph.D., technical communicator and scientist at ACEA Biosciences. “Regardless of what the route is, what is going to have to happen in terms of the research area is that you need an effective cell-killing assay.”

ACEA Biosciences, a San Diego-based company, has developed a microtiter plate that is coated with gold electrodes across 75% of the well bottoms. When the microtiter plates are placed in the company’s xCELLigence plate reader, the electrodes enable the detection of changes in cell morphology and viability through electrical impedance.

“The instrument provides a weak electric potential to the electrodes on the plate, so you get electrons flowing between these electrodes,” explains Dr. Lamarche. Researchers can then apply reagents or non-adherent immune cell suspensions to adherent cancer cells and examine the effect.

Dr. Lamarche asserts that the xCELLigence system overcomes problems that bedevil competing cell-killing assays. These problems include leaky and radioactive labels, such as chromium 51, and assays that can only provide users with an endpoint for cell killing. “With xCELLigence,” he insists, “you’re getting the full spectrum of what’s happening, and there’s all kinds of subtleties in the cell-killing curves that are very informative in terms of the biology.”

ACEA would like to see the xCELLigence system become the new standard in cell-killing assays from standard research to clinical testing on patient tumors. Dr. Lamarche envisions a day when patient tumor cells are quickly screened with therapeutic scenarios to determine the most efficacious killing option. “xCELLigence technology,” he suggests, “enables you to quickly sample a broad spectrum of conditions with a very simple workflow.”

Bioinformatics of Immuno-Oncology

From monitoring to treatment modalities, the field of cancer immunotherapy is aided by bioinformatics-minded data-mining experts, such as the analysts at Thompson-Reuters who are compiling data archives and applying advanced analytics to find new targets. “Essentially,” says Richard Harrison Ph.D., the company’s chief scientific officer for the life science division, “for every stage within pharmaceutical drug development, we have a database associated with that.”

The analysts at Thompson-Reuters curate and compile databases such as MedaCore and Cortellis, which they provide to their clients to help them with their research and clinical studies. “We can take customer data, and using our tools and our pathway maps, we can help them understand what their data is telling them,” explains Dr. Harrison.

Matt Wampole, Ph.D., a solutions scientist at Thompson-Reuters, spends his days reaching out and working with customers to help them understand and better use the company’s products. “Bench researchers,” he points out, “don’t necessarily know what is upstream of whatever expression change might be leading to a particular change in regulation.” Dr. Wampole indicates that he is part of a “solution team” that aids clients in determining important signaling cascades, regulators, and so on.

“We have a group of individuals who are very ‘skilling’ experts in the field,” Dr. Wampole continues, “including experts in the areas such as biostatistics, data curation, and data analytics. These experts help clients identify models, stratify patients, understand mechanisms, and look into disease mechanisms.”

Dr. Harrison sums up the Thompson-Reuters approach as follows: “We look for master regulators that can serve as both targets and biomarkers.” By examining the gene signatures from both the patient and from curated datasets, in the case of cancer immunotherapy, they hope to segregate patients according to what drugs will work best for them.

  • “We are working with a number of pharmaceutical companies to put our approach into practice for clinical trials,” informs Dr. Harrison. The approach has already been applied in several studies, including one that used data analysis of cell lines to help predict drug response in patients. Another study helped stratify glioblastoma patients.

  • Tumor-Targeted Delivery Platform

    PsiOxus Therapeutics, which is focused on immune therapeutics in oncology, has developed a patented platform for tumor-targeted delivery based on its oncolytic vaccine, Enadenotucirev (EnAd), which can be delivered systemically via intravenous administration.

    According to company officials, EnAd’s anti-cancer scope can be expanded by adding new genes, thereby enabling the creation of a broad range of unique immuno-oncology therapeutics. In a recent study conducted at the University of Oxford, researchers led by Philip G. Jakeman, Ph.D., sought to improve the models for evaluating cancer therapeutics by introducing ex vivo methodologies for research into colorectal cancer.

    The ex vivo approach utilized was able to exploit a major advantage by preserving the three-dimensional architecture of the tumor and its associated compartments, including immune cells. The study, which was presented at the International Summit on Oncolytic Viral Therapeutics in Quebec, showed the tissue slice model can provide a novel means to assessing an oncolytic vaccine in a system that more accurately recapitulates human tumors, provide a more stringent test for oncolytic viruses, such as EnAd, and allow study of the human immune cells within the tumor 3D context.

    By maintaining the components of the tumor immune microenvironment, this new methodology could become useful in analyzing anti-viral responses within tumors, or even in evaluating therapeutics that target immunosuppressive tumor micro-environments, noted the Oxford team.

     

 

Deciphering the Cancer Transcriptome

A Rogue’s Gallery of Malignant Outliers May Hide in Transcriptome Profiles That Emphasize Averages

http://www.genengnews.com/gen-articles/deciphering-the-cancer-transcriptome/5729/

 

The key link between genomic instability and cancer progression is transcriptome dynamics. The shifts in transcriptome dynamics that contribute to cancer evolution may come down to statistical outliers. [iStock/zmeel]

  • In recent years, scientists have adopted a gene-centric view of cancer, a tendency to see each malignant transformation as the consequence of alterations in a discrete number of genes or pathways. These alterations are, fortunately, absent from healthy cells, but they pervert malignant cells.

    The gene-centric view takes in molecular landscapes illuminated by genomic and transcriptomic technologies. For example, genomes can be cost-effectively sequenced within hours. Such capabilities have made it possible to interrogate associations between genotypes and phenotypes for increasing numbers of conditions, and to collect data from progressively larger patient groups.

    As genomic and transcriptomic technologies rise, they reveal much—but much remains hidden, too. Perhaps these technologies are less like the sun and more like the proverbial streetlight, the one that narrows our searches because we’re inclined to stay in the light, even though what we hope to find may lie in the shadows.

    “Each individual study that looks at the cancer transcriptome is impressive and tells a convincing story, but if we put several high-quality papers together, there are very few genes that overlap,” says Henry H. Heng, Ph.D., professor of molecular medicine, genetics, and pathology at Wayne State University. “This shows that something is wrong.”

  • Distinct Karyotypes

    One of the major observations in Dr. Heng’s lab is that the intra- and intertumor cellular heterogeneity results in nearly every cancer cell having a unique, distinct karyotype, that is, an important but often ignored genotype. “Biological systems need a lot of heterogeneity,” notes Dr. Heng. “People like to think that this is noise, but heterogeneity is a fundamental buffer system for biological function to be achievable. Moreover, it is the key agent for cellular adaptation.”

    To capture the degree of genomic heterogeneity at the genome level and its impact on cancer cell growth, Dr. Heng and colleagues performed serial dilutions to isolate single mouse ovarian surface epithelial cells that had undergone spontaneous transformation. Spectral karyotyping revealed that within a short timeframe each of these unstable cells exhibited a very distinct karyotype. In these unstable cells, cloning at the level of the karyotype was not possible.

    Stable cells exhibited a normal growth distribution, i.e., no subset of stable cells contributed disproportionately to the overall growth of the cell population. In contrast, unstable cell populations showed a non-normal growth distribution, with few cells contributing most to the cell population’s growth. For example, a single unstable colony contributed more than 70% to the cell population’s growth. This finding suggests that although average profiles can be used to describe non-transformed cells, they cannot be taken to represent the biology of malignant cells.

    “Most people who study the transcriptome want to get rid of the noise, but the noise is in fact the strategy that cancer uses to be successful,” explains Dr. Heng. “Each individual cancer cell is very weak but together the entity becomes very robust.”

    In a recent model that Dr. Heng and colleagues proposed, system inheritance visualizes chromosomes not merely as the vehicle for transmitting genetic information, but as the genetic network organizer that shapes the physical interactions between genes in the three-dimensional space. Based on this model, individual genes represent parts of the system. The same genes can be reorganized to form different systems, and chromosomal instability becomes more important than the contribution of individual genes and pathways to cancer biology.

    The vital link between genomic instability and cancer progression is transcriptome dynamics, and the shifts in those dynamics that contribute to cancer evolution may come down to statistical outliers.

    “Transcriptome studies rarely focus on single-cell analyses, which means important outliers are frequently ignored,” declares Dr. Heng. “This preoccupation with uninformative averages explains why we have learned so little despite having examined so many transcriptomes.”

  • Chimeras and Fusion Genes

    “Our focus is on chimeric RNA molecules,” says Laising Yen, Ph.D, assistant professor of pathology at Baylor College of Medicine. “This category of RNAs is very special because their sequences come from different genes.”

    In a study that was designed to capture chimeric RNAs in prostate cancer, Dr. Yen and his colleagues performed high-throughput sequencing of the transcriptomes from human prostate cancer samples. “We found far more chimeric RNAs, in terms of abundance, and a number of species that are not seen in normal tissue,” reports Dr. Yen. This approach identified over 2,300 different chimeric RNA species. Some of these chimeras were present in prostate cancer cell lines, but not in primary human prostate epithelium cells, which points toward their relevance in cancer.

    “Most of these chimeric RNAs do not have a genomic counterpart, which means that they could be produced by trans-splicing,” explains Dr. Yen. During trans-splicing, individual RNAs are generated and trans-spliced together as a single RNA, which provides a mechanism for generating a chimera.

    “The other possibility is that in cancer cells, where gene–gene boundaries are known to become broken, chimeras can be formed by cis-splicing from a very long transcript that encodes several neighboring genes located on the same chromosome,” informs Dr. Yen. Chimeric RNAs formed by either of these two mechanisms can potentially translate into fusion proteins, and these aberrant proteins may have oncogenic consequences.

    Another effort in Dr. Yen’s laboratory focuses on chromosomal aberrations in ovarian cancer. One of the hallmarks of ovarian cancer is the high degree of genomic rearrangement and the increased genomic instability.

    “When we looked at ovarian cancers, we did not find as many chimeric RNAs,” notes Dr. Yen. “But we found many fusion genes.” Gene fusions, similarly to chimeric RNAs, increase the diversity of the cellular proteome, which could be used selectively by cancer cells to increase their rates of proliferation, survival, and migration.

    A recent study in Dr. Yen’s lab identified BCAM-AKT2, a recurrent fusion gene that is specific and unique to high-grade serous ovarian cancer. BCAM-AKT2 is the only fusion gene in this malignancy that was proved to be translated into a fusion kinase in patients, which points toward its functional significance and potential therapeutic value.

    “Recurrent fusion genes, which are repeatedly found in many patients in precisely made forms, indicate that there is a reason that they are present,” concludes Dr. Yen. “This might have important therapeutic implications.”

  • Context-Specific Patterns

    “We contributed to a study of tumor gene expresssion that we are currently revisiting because so much more data has become available,” says Barbara Stranger, Ph.D., assistant professor, Institute for Genomics and Systems Biology, University of Chicago. “The data is being processed in homogenized analytic pipelines, and we can look at many more tumor types across the Cancer Genome Atlas than a few years ago.”

    Previously, Dr. Stranger and colleagues performed expression quantitative trait loci (eQTL) analyses to examine mRNA and miRNA expression in breast, colon, kidney, lung, and prostate cancer samples. This approach identified 149 known cancer risk loci, 42 of which were significantly associated with expression of at least one transcript.

    Causal alleles are being prioritized using a fine-mapping strategy that integrated the eQTL analysis with genome-wide DNAseI hypersensitivity profiles obtained from ENCODE data. These analyses are focusing on capturing differences across tumors and on performing comparisons with normal tissue, and one of the challenges is the lack of normal tissue from the same patients.

    “But still there is a lot of power in these analyses because they are based on large-scale genomic datasets. Also, these tumor datasets can be compared with large-scale normal tissue genomics datasets, such as the NIH’s Genotype-Tissue Expression (GTEx) project,” clarifies Dr. Stranger. “This helps us characterize differences between those tumors and normal tissue in terms of the genetics of gene regulation.”

    An ongoing effort in Dr. Stranger’s laboratory involves elucidating how the effect of genetic polymorphisms is shaped by context. Stimulated cellular states, cell-type differences, cellular senescence, and disease are some of the contexts that are known to impact genetic polymorphisms.

    “We have seen a lot of context specificity,” states Dr. Stranger. “Our observations suggest that a genetic polymorphism can have a specific effect in regulating a particular gene or transcript in one context, and another effect in another context.”

    Another example of cellular context is sex, and an active area of investigation in Dr. Stranger’s lab proposes to dissect the manner in which sex differences shape the regulatory effects of genetic polymorphisms.

    “Thinking about sex-specific differences is not very different from thinking about a different cellular environment,” notes Dr. Stranger.

    The expression of specific transcription factors can be determined by sex; consequently, a polymorphism that interacts with a transcription factor may have functional outcomes that can be seen in only one of the sexes.

    “There are gene-level and gene-splicing differences that we see in normal tissues between males and females, and we want to take the same approach and look at the cancer context to see whether the genetic regulation of gene expression and transcript splicing is different between individuals and whether it has a sex bias,” concludes Dr. Stranger. “Finally, we want to see how that differs in cancer relative to normal tissues.”

    Early Clinical Impact

An increasing number of clinicians are adding the cancer transcriptome to their precision medicine program. They have found that the transcriptome is important in identifying clinically impactful results. [iStock/DeoSum]

“Over the last two years,” says Andrew Kung, M.D., Ph.D., chief of the Division of Pediatric Hematology, Oncology, and Stem Cell Transplantation at Columbia University Medical Center, “we have included the cancer transcriptome as part of our precision medicine program.” Dr. Kung and colleagues developed a clinical genomics test that includes whole-exome sequencing of tumors and normal tissue and RNA-seq of the tumor.

“Our results show that the transcriptome is very important in identifying clinically impactful results,” asserts Dr. Kung. “The technology has really moved from a research tool to real clinical application.” In fact, the test has been approved by New York State for use in cancer patients.

The data from transcriptome profiling has enabled identification of translocations, verification of somatic alterations, and assessment of expression levels of cancer genes.  Dr. Kung and his colleagues are using genomic information for initial diagnosis and prognostic decisions, as well as the investigation of potentially actionable alterations and the monitoring of disease response.

To gain insight into gene-expression changes, transcriptome analysis usually compares two different types of tissues or cells. For example, analyses may attempt to identify differentially expressed genes in cancer cells and normal cells.

“In patients with cancer, we usually do not have access to the normal cell of origin, making it harder to identify the genes that are over- or under-expressed,” explains Dr. Kung. “Fortunately, the vast amounts of existing gene-expression data allow us to identify genes whose expression are most changed relative to models built on the expression data aggregated across large existing datasets.”

These genomic technologies were first used to augment the care of pediatric patients at Columbia. The technologies were so successful that they attracted philanthropic funding, which is being used to expand access to genomic testing to all children with high-risk cancer across New York City.

Other related articles published in this Open Access Online Scientific Journal include the following:

Immunotherapy in Combination, 2016 MassBio Annual Meeting  03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

Live Press Coverage: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/04/01/plenary-session-immunotherapy-in-combination-2016-massbio-annual-meeting-03312016-800-am-04012016-300-pm-royal-sonesta-hotel-cambridge-ma/

 

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AACR and Philly New Media Present a Town Hall Discussion on Precision Medicine

Cancer Precision Medicine: Big Ideas in Research, Treatment, and Prevention

A Town Hall Forum will discuss the latest findings with regard to precision medicine, its impact currently in cancer treatment, and future directions, discussed by some of the preeminent cancer researchers and oncologists in the country. This unprecedented event is being hosted by the American Association for Cancer Research (AACR) and Philadelphia Media Network – publisher of The Philadelphia Inquirer, Daily News, and Philly.com.

Given the following speakers, this event will have a large focus on use of cancer immunotherapy as well as new targets in the precision medicine arena.

Register today: Philly.com/CancerEvent – Use the promo code “AACR” for discounted $45 tickets.

When: Thursday, January 21, 2016 • Program: 2 pm • Networking reception: 5:30 pm.

Where:  The College of Physicians of Philadelphia • 19 South 22nd Street, Philadelphia, Pa.

The event will be held in Philadelphia at the College of Physicians of Philadelphia, home of the famous Mutter Museum.

Please follow the meeting coverage on @pharma_BI and using the following @ handle and # hastags of Twitter:

@AACR

@pharma_BI

@PhillyInquirer

#cbi16

#precisionmedicine

#endcancer

 

From Penn Medicine News Blog: Archives (please click on link below)

Penn’s Center for Personalized Diagnostics (CPD), which recently named Kojo S.J. Elenitoba-Johnson, MD, as its founding director, is diving deeper into cancer patients’ tumors with next generation DNA sequencing.

The genetic tests help refine diagnoses with greater precision than standard imaging tests and blood work by spotting known mutations that can inform the treatment plan. Since it launched in February 2013, the CPD has performed more than 4,000 advanced diagnostics, representing a wide range of cancers.  It’s also producing actionable findings: Of those tests, 75 percent found disease-associated mutations, revealing possible new treatment pathways.

This new CPD video helps breakdown how the process works, but a patient story can really help connect the dots. We’ve written about several people who benefited from the CPD, including one acute myeloid leukemia patient with an FLT3 mutation that made her a candidate for a targeted therapy, and another whose cholangiocarcinoma was successfully treated with a BRAF-targeted therapy after the mutation—typically associated with melanoma—was spotted.

ACC’s role as a national leader in personalized cancer care was also reinforced with the opening of the Center for Rare Cancers and Personalized Therapy in 2015.

Directed by Marcia Brose, MD, PhD, this virtual center enrolls patients into clinical trials based on genetic markers rather than tumor origin.  Patients with the same mutation, BRAF for instance, but different cancers, are part of the same clinical study investigating a targeted therapy.  A story, set to air on TV news affiliates across the country in the upcoming weeks, will feature a patient with a rare salivary tumor who ran out of treatment options, until a HRAS mutation identified through the CPD put him back on track, after switching to the drug tipifarnib. The patient responded well, and a recent scan revealed that his disease has stabilized.

“Philadelphia is a hotbed for healthcare innovation and groundbreaking scientific research—which becomes even more apparent as the ACC continues to move the needle in the precision medicine world,”Abramson Cancer Center (ACC) director Chi Van Dang, MD, PhD, said.  “Quickly evolving diagnostics and genetic tests, cancer vaccines, and powerful personalized therapies that use the body’s own immune system to fight off cancer: These are just a few of the medical advances being utilized today that are giving patients the greatest chance.”

For Media Inquiries see the following AACR contact information:

Julia Gunther
Assistant Director, Media and Public Relations
215-446-6896
Cell: 267-250-5441
Fax: 215-861-5937
julia.gunther@aacr.org
Gunther promotes the AACR’s meetings, journals, and initiatives to the media and the public.

Lauren Walens
Senior Manager, Media and Public Relations
215-446-7163
Fax: 267-765-1050
lauren.walens@aacr.org
Walens promotes the AACR’s meetings, journals, and initiatives to the media and the public. She also manages the AACR’s blog, Cancer Research Catalyst.

Lauren Riley
Senior Coordinator, Media and Public Relations
215-446-7155
Fax: 215-446-7291
lauren.riley@aacr.org
Riley is responsible for media relations promotion and support, conference newsroom logistics, writing and proofreading, website and news release copy, as well as office support for the Communications and Public Relations Department staff.

 

 

 

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Transformer Proteins against Cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Revision  1/13/20165

GEN News Highlights:  “Transformer” Proteins with Role in Cancer

http://www.genengnews.com/gen-news-highlights/transformer-proteins-with-role-in-cancer/81252133/

Investigators at the Scripps Research Institute (TSRI) and St. Jude Children’s Research Hospital say they have found how a protein involved in cancer twists and morphs into different structures.

“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said Ashok Deniz, Ph.D., associate professor at TSRI.

The study (“Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding”), published in Angewandte Chemie, focuses  on the nucleophosmin (NPM1) protein, which has many functions and, when mutated, has been shown to interfere with cells’ normal tumor suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.

Previous research led by study collaborators Richard Kriwacki, Ph.D., and Diana Mitrea, Ph.D., at St. Jude had demonstrated that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.

 

Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell. To shed light on how this transformation occurred, Dr. Deniz and his colleagues used a combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET), and circular dichroism, which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.

The researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. Such phosphorylation prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.

When NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.

 

Priya Banerjee, Ph.D., an American Heart Association-supported postdoctoral research associate at TSRI and the first author of the study, compared these complicated transitions to the morphing of a “Transformers” toy, where a robot can become a car and then a jet. “Phosphorylation and partner-binding are like different cellular switches driving these changes,” said Dr. Banerjee.

According to Dr. Banerjee, the new study also reveals many intermediate states between monomer and pentamer structures and that these states can be manipulated or “tuned” by changing conditions such as salt levels, phosphorylation, and partner binding, which may explain how cells regulate the protein’s multiple functions. The researchers said future studies could shed more light on the biological functions of these different structures and how they might be used in future cancer therapies.

The team added that combining the three techniques used in this study, plus a novel protein-labeling technique for single-molecule fluorescence, could be a useful strategy for studying other unstructured, “intrinsically disordered proteins” (IDPs), which are involved in a host of cellular functions, as well as neurodegenerative disease, heart disease, infectious disease, type 2 diabetes and other conditions.

 

Single ‘Transformer’ Proteins

http://www.technologynetworks.com/Proteomics/news.aspx?ID=186996

 

“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said TSRI Associate Professor Ashok Deniz, senior author of the new study along with Richard Kriwacki, faculty member at St. Jude.

The study  focuses on a protein called nucleophosmin (NPM1). This protein has many functions and, when mutated, has been shown to interfere with cells’ normal tumor-suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.

Previous research led by study collaborators Kriwacki and Diana Mitrea at St. Jude had shown that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.

Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell.

To shed light on how this transformation occurred, Deniz and his colleagues used an innovative combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET) and circular dichroism—which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.

Remarkably, the researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. This modification, called phosphorylation, prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.

Interestingly, when NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.

Priya Banerjee, an American Heart Association-supported postdoctoral research associate at TSRI and the first author of the study, compared these complicated transitions to the morphing of a “Transformers” toy, where a robot can become a car and then a jet. “Phosphorylation and partner-binding are like different cellular switches driving these changes,” said Banerjee.

Banerjee said the new study also reveals many intermediate states between monomer and pentamer structures—and that these states can be manipulated or “tuned” by changing conditions such as salt levels, phosphorylation and partner binding, which may explain how cells regulate the protein’s multiple functions. The researchers said future studies could shed more light on the biological functions of these different structures and how they might be used in future cancer therapies.

The researchers added that combining the three techniques used in this study, plus a novel protein-labeling technique for single-molecule fluorescence, could be a useful strategy for studying other unstructured, “intrinsically disordered proteins” (IDPS). IDPS are involved in a host of cellular functions, as well as neurodegenerative disease, heart disease, infectious disease, type 2 diabetes and other conditions.

TSRI and St. Jude Scientists Study Physics of Single ‘Transformer’ Proteins with Role in Cancer

http://www.scripps.edu/news/press/2015/20151221deniz.html

December 21, 2015 – A new study led by scientists at The Scripps Research Institute (TSRI) and St. Jude Children’s Research Hospital shows how a protein involved in cancer twists and morphs into different structures.

“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said TSRI Associate Professor Ashok Deniz, senior author of the new study along with Richard Kriwacki, faculty member at St. Jude.

The study, published recently in the journal Angewandte Chemie, focuses on a protein called nucleophosmin (NPM1). This protein has many functions and, when mutated, has been shown to interfere with cells’ normal tumor suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.

Previous research led by study collaborators Kriwacki and Diana Mitrea at St. Jude had shown that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.

Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell.

To shed light on how this transformation occurred, Deniz and his colleagues used an innovative combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET) and circular dichroism—which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.

Remarkably, the researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. This modification, called phosphorylation, prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.

Interestingly, when NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.

 

Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding  

Priya R. Banerjee, Diana M. Mitrea, Richard W. Kriwacki, Ashok, A. Deniz

     http://dx.doi.org:/10.1002/anie.201507728

As for many intrinsically disordered proteins, order–disorder transitions in the N-terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm-N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single-molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm-N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway-specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm-N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm-N order–disorder transition enabled a complex interplay of phosphorylation and partner-binding steps to modulate its folding landscape.

 

Conditionally and Transiently Disordered Proteins: Awakening Cryptic Disorder To Regulate Protein Function

Related articles:

Discovery of Small Molecules that Inhibit the Disordered Protein, p27(Kip1).

Iconaru LI, Ban D, Bharatham K, Ramanathan A, Zhang W, Shelat AA, Zuo J, Kriwacki RW.

Sci Rep. 2015 Oct 28;5:15686. doi: 10.1038/srep15686.

Select item 264270603.

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

Pettersson M, Quant M, Min J, Iconaru L, Kriwacki RW, Waddell MB, Guy RK, Luthman K, Grøtli M.

PLoS One. 2015 Oct 1;10(10):e0137867. doi: 10.1371/journal.pone.0137867. eCollection 2015.

Select item 264113004.

Discoveries and controversies in BCL-2 protein-mediated apoptosis.

Zheng JH, Viacava Follis A, Kriwacki RW, Moldoveanu T.

FEBS J. 2015 Sep 28. doi: 10.1111/febs.13527. [Epub ahead of print] Review.

PMID:
26411300
Select item 262360135.

Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis.

Follis AV, Llambi F, Merritt P, Chipuk JE, Green DR, Kriwacki RW.

Mol Cell. 2015 Aug 20;59(4):677-84. doi: 10.1016/j.molcel.2015.06.029. Epub 2015 Jul 30.

PMID:
26236013

 

 

A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

Xuan CaoTomonori KanekoJenny S. LiAn-Dong Liu, et al.

Nature Communications 2015; 6(7721)     http://dx.doi.org:/10.1038/ncomms8721

Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylation-mediated molecular switch comprising deleted in liver cancer 1 (DLC1), tensin-3 (TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3–DLC1 and PTEN–PI3K complexes into the TNS3–PI3K and PTEN–DLC1 complexes. Subsequently, the TNS3–PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN–DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration.

 

Protein Phosphorylation is an Important Tool…  Chapter 13.  in Protein Phosphorylation in Human Health 

Roberta Fraschini, Erica Raspelli and Corinne Cassani 

http://cdn.intechopen.com/pdfs-wm/38809.pdf

Protein phosphorylation is a reversible posttranslational modification that can modulate protein role in several physiological processes in almost every possible way. These include modification of its intrinsic biological activity, subcellular location, half-life and binding with other proteins. Protein phosphorylation is particularly important for the regulation of key proteins involved in the control of cell cycle progression.

Protein phosphorylation is the covalent binding of a phosphate group to some critical residues of the polypeptide. The phosphorylation state of a protein is given by a balance between the activity of protein kinases and protein phosphatases. Eukaryotic protein kinases transfer phosphate groups (PO43-) from ATP to an hydroxyl group of the lateral chain of specific serine, threonine or tyrosine residues on peptide substrates. In simple eukaryotic cells, like yeasts, Ser/Thr kinases are more common, while more complex eukaryotic cells, like human cells, have many Tyr kinases. Protein kinases recognize their substrates specifically and their active site consists of an activation loop and a catalytic loop between which substrates bind. Protein kinases differ from each other in the structure of their catalitic domain. A second class of enzymes, protein phosphatases, is responsible for the reverse reaction in which phosphate groups are removed from a protein. Phosphatases gain specificity by binding protein cofactors which facilitate binding to specific phosphoproteins. The active phosphatase often consists of a complex of the phosphatase catalytic subunit and a regulatory subunit.

The phosphorylation of specific residues induces structural changes that regulate protein functions by modulating protein folding, substrate affinity, stability and activity. For example, phosphorylation can cause switch-like changes in protein function, which can also lead to major modifications in the catalytic function of enzymes, including kinases and phosphatases. In addition, protein phosphorylation often leads to rearrangement in the structure of the protein that can induce changes in interacting partners or subcellular localization.

Phosphorylation acts as a molecular switch for many regulatory events in signalling pathways that drive cell division, proliferation, differentiation and apoptosis. In order to ensure an appropriate balance of protein phosphorylation, the cell can compartmentalize both protein kinases and phosphatases. Another kind of regulation can be achieved by the spatial distribution of kinases and phosphatases, that creates a gradient of phosphorylated substrates across different subcellular compartments. This spatial separation can also control the activity of other proteins or enzymes and the occurrence of other posttranslational modifications.

Molecular Biology of the Cell. 4th edition.   Protein Function.   http://www.ncbi.nlm.nih.gov/books/NBK26911/

We have seen that each type of protein consists of a precise sequence of amino acids that allows it to fold up into a particular three-dimensional shape, or conformation. But proteins are not rigid lumps of material. They can have precisely engineered moving parts whose mechanical actions are coupled to chemical events. It is this coupling of chemistry and movement that gives proteins the extraordinary capabilities that underlie the dynamic processes in living cells.

In this section, we explain how proteins bind to other selected molecules and how their activity depends on such binding. We show that the ability to bind to other molecules enables proteins to act as catalysts, signal receptors, switches, motors, or tiny pumps. The examples we discuss in this chapter by no means exhaust the vast functional repertoire of proteins. However, the specialized functions of many of the proteins you will encounter elsewhere in this book are based on similar principles.

All Proteins Bind to Other Molecules

The biological properties of a protein molecule depend on its physical interaction with other molecules. Thus, antibodies attach to viruses or bacteria to mark them for destruction, the enzyme hexokinase binds glucose and ATP so as to catalyze a reaction between them, actin molecules bind to each other to assemble into actin filaments, and so on. Indeed, all proteins stick, or bind, to other molecules. In some cases, this binding is very tight; in others, it is weak and short-lived. But the binding always shows great specificity, in the sense that each protein molecule can usually bind just one or a few molecules out of the many thousands of different types it encounters. The substance that is bound by the protein—no matter whether it is an ion, a small molecule, or a macromolecule— is referred to as a ligand for that protein (from the Latin word ligare, meaning “to bind”).

The ability of a protein to bind selectively and with high affinity to a ligand depends on the formation of a set of weak, noncovalent bonds—hydrogen bonds, ionic bonds, and van der Waals attractions—plus favorable hydrophobic interactions (see Panel 2-3, pp. 114–115). Because each individual bond is weak, an effective binding interaction requires that many weak bonds be formed simultaneously. This is possible only if the surface contours of the ligandmolecule fit very closely to the protein, matching it like a hand in a glove (Figure 3-37).

Figure 3-37. The selective binding of a protein to another molecule.

Figure 3-37

The selective binding of a protein to another molecule. Many weak bonds are needed to enable a protein to bind tightly to a second molecule, which is called aligand for the protein. A ligand must therefore fit precisely into a protein’s binding (more…)

The region of a protein that associates with a ligand, known as the ligand’s binding site, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids. These amino acids can belong to different portions of the polypeptide chain that are brought together when the protein folds (Figure 3-38). Separate regions of the protein surface generally provide binding sites for different ligands, allowing the protein’s activity to be regulated, as we shall see later. And other parts of the protein can serve as a handle to place the protein in a particular location in the cell—an example is the SH2 domain discussed previously, which is often used to move a protein containing it to sites in theplasma membrane in response to particular signals.

Figure 3-38. The binding site of a protein.

Figure 3-38

The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can make noncovalent bonds only (more…)

Although the atoms buried in the interior of the protein have no direct contact with the ligand, they provide an essential scaffold that gives the surface its contours and chemical properties. Even small changes to the amino acids in the interior of a protein molecule can change its three-dimensional shape enough to destroy a binding site on the surface.

The Details of a Protein’s Conformation Determine Its Chemistry

Proteins have impressive chemical capabilities because the neighboring chemical groups on their surface often interact in ways that enhance the chemical reactivity of amino acid side chains. These interactions fall into two main categories.

First, neighboring parts of the polypeptide chain may interact in a way that restricts the access of water molecules to aligand binding site. Because water molecules tend to form hydrogen bonds, they can compete with ligands for sites on the protein surface. The tightness of hydrogen bonds (and ionic interactions) between proteins and their ligands is therefore greatly increased if water molecules are excluded. Initially, it is hard to imagine a mechanism that would exclude a molecule as small as water from a protein surface without affecting the access of the ligand itself. Because of the strong tendency of water molecules to form water–water hydrogen bonds, however, water molecules exist in a large hydrogen-bonded network (see Panel 2-2, pp. 112–113). In effect, a ligand binding site can be kept dry because it is energetically unfavorable for individual water molecules to break away from this network, as they must do to reach into a crevice on a protein’s surface.

Second, the clustering of neighboring polar amino acid side chains can alter their reactivity. If a number of negatively charged side chains are forced together against their mutual repulsion by the way the protein folds, for example, the affinity of the site for a positively charged ion is greatly increased. In addition, when amino acid side chains interact with one another through hydrogen bonds, normally unreactive side groups (such as the –CH2OH on the serine shown inFigure 3-39) can become reactive, enabling them to enter into reactions that make or break selected covalent bonds.

Figure 3-39. An unusually reactive amino acid at the active site of an enzyme.

Figure 3-39

An unusually reactive amino acid at the active site of an enzyme. This example is the “catalytic triad” found in chymotrypsin, elastase, and other serine proteases (see Figure 3-14). The aspartic acid side chain (Asp 102) induces the histidine (more…)

The surface of each protein molecule therefore has a unique chemical reactivity that depends not only on which amino acid side chains are exposed, but also on their exact orientation relative to one another. For this reason, even two slightly different conformations of the same protein molecule may differ greatly in their chemistry.

Sequence Comparisons Between Protein Family Members Highlight Crucial Ligand Binding Sites

As we have described previously, many of the domains in proteins can be grouped into families that show clear evidence of their evolution from a common ancestor, and genome sequences reveal large numbers of proteins that contain one or more common domains. The three-dimensional structures of the members of the same domain family are remarkably similar. For example, even when the amino acid sequence identity falls to 25%, the backbone atoms in a domain have been found to follow a common protein fold within 0.2 nanometers (2 Å).

These facts allow a method called “evolutionary tracing” to be used to identify those sites in a protein domain that are the most crucial to the domain’s function. For this purpose, those amino acids that are unchanged, or nearly unchanged, in all of the known protein family members are mapped onto a structural model of the three-dimensional structure of one family member. When this is done, the most invariant positions often form one or more clusters on the protein surface, as illustrated in Figure 3-40A for the SH2 domain described previously (see Panel 3-2, pp. 138–139). These clusters generally correspond to ligand binding sites.

Figure 3-40. The evolutionary trace method applied to the SH2 domain.

Figure 3-40

The evolutionary trace method applied to the SH2 domain. (A) Front and back views of a space-filling model of the SH2 domain, with evolutionarily conserved amino acids on the protein surface colored yellow, and those more toward the protein interior colored (more…)

The SH2 domain is a module that functions in protein–protein interactions. It binds the protein containing it to a second protein that contains a phosphorylated tyrosine side chain in a specific amino acid sequence context, as shown in Figure 3-40B. The amino acids located at the binding site for the phosphorylated polypeptide have been the slowest to change during the long evolutionary process that produced the large SH2 family of peptide recognition domains. Becausemutation is a random process, this result is attributed to the preferential elimination during evolution of all organisms whose SH2 domains became altered in a way that inactivated the SH2-binding site, thereby destroying the function of the SH2 domain.

In this era of extensive genome sequencing, many new protein families have been discovered whose functions are unknown. By identifying the critical binding sites on a three-dimensional structure determined for one family member, the above method of evolutionary tracing is being used to help determine the functions of such proteins.

Proteins Bind to Other Proteins Through Several Types of Interfaces

Proteins can bind to other proteins in at least three ways. In many cases, a portion of the surface of one protein contacts an extended loop of polypeptide chain (a “string”) on a second protein (Figure 3-41A). Such a surface–string interaction, for example, allows the SH2 domain to recognize a phosphorylated polypeptide as a loop on a second protein, as just described, and it also enables a protein kinase to recognize the proteins that it will phosphorylate (see below).

Figure 3-41. Three ways in which two proteins can bind to each other.

Figure 3-41

Three ways in which two proteins can bind to each other. Only the interacting parts of the two proteins are shown. (A) A rigid surface on one protein can bind to an extended loop of polypeptide chain (a “string”) on a second protein. (B)(more…)

A second type of protein–protein interface is formed when two α helices, one from each protein, pair together to form acoiled-coil (Figure 3-41B). This type of protein interface is found in several families of gene regulatory proteins, as discussed in Chapter 7.

The most common way for proteins to interact, however, is by the precise matching of one rigid surface with that of another (Figure 3-41C). Such interactions can be very tight, since a large number of weak bonds can form between two surfaces that match well. For the same reason, such surface–surface interactions can be extremely specific, enabling aprotein to select just one partner from the many thousands of different proteins found in a cell.

 

Molecular Motors

Perhaps the most fascinating proteins that associate with the cytoskeleton are the molecular motors called motor proteins. These remarkable proteins bind to a polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move steadily along it. Dozens of different motor proteins coexist in every eucaryotic cell. They differ in the type of filament they bind to (either actin or microtubules), the direction in which they move along the filament, and the “cargo” they carry. Many motor proteins carry membrane-enclosed organelles—such as mitochondria, Golgi stacks, or secretory vesicles—to their appropriate locations in the cell. Other motor proteins cause cytoskeletal filaments to slide against each other, generating the force that drives such phenomena as muscle contraction, ciliary beating, and cell division.

Cytoskeletal motor proteins that move unidirectionally along an oriented polymer track are reminiscent of some other proteins and protein complexes discussed elsewhere in this book, such as DNA and RNA polymerases, helicases, and ribosomes. All of these have the ability to use chemical energy to propel themselves along a linear track, with the direction of sliding dependent on the structural polarity of the track. All of them generate motion by coupling nucleosidetriphosphate hydrolysis to a large-scale conformational change in a protein, as explained in Chapter 3.

The cytoskeletal motor proteins associate with their filament tracks through a “head” region, or motor domain, that binds and hydrolyzes ATP. Coordinated with their cycle of nucleotide hydrolysis and conformational change, the proteins cycle between states in which they are bound strongly to their filament tracks and states in which they are unbound. Through a mechanochemical cycle of filament binding, conformational change, filament release, conformational relaxation, and filament rebinding, the motor protein and its associated cargo move one step at a time along the filament (typically a distance of a few nanometers). The identity of the track and the direction of movement along it are determined by the motor domain (head), while the identity of the cargo (and therefore the biological function of the individual motor protein) is determined by the tail of the motor protein.

In this section, we begin by describing the three groups of cytoskeletal motor proteins. We then describe how they work to transport membrane-enclosed organelles or to change the shape of structures built from cytoskeletal filaments. We end by describing their action in muscle contraction and in powering the whiplike motion of structures formed from microtubules.

 

Actin-based Motor Proteins Are Members of the Myosin Superfamily

The first motor protein identified was skeletal muscle myosin, which is responsible for generating the force for muscle contraction. This myosin, called myosin II (see below) is an elongated protein that is formed from two heavy chains and two copies of each of two light chains. Each of the heavy chains has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long amino acid sequence that forms an extended coiled-coil that mediates heavy chain dimerization (Figure 16-51). The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other myosin molecules. These tail-tail interactions result in the formation of large bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends of the thick filament (Figure 16-52).

Figure 16-51. Myosin II.

Figure 16-51

Myosin II. (A) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long (green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization (more…)

Figure 16-52. The myosin II bipolar thick filament.

Figure 16-52

The myosin II bipolar thick filament. (A) Electron micrograph of a myosin II thick filament isolated from frog muscle. Note the central bare zone, which is free of head domains. (B) Schematic diagram, not drawn to scale. The myosin II molecules aggregate (more…)

Each myosin head binds and hydrolyses ATP, using the energy of ATP hydrolysis to walk toward the plus end of anactin filament. The opposing orientation of the heads in the thick filament makes the filament efficient at sliding pairs of oppositely oriented actin filaments past each other. In skeletal muscle, in which carefully arranged actin filaments are aligned in “thin filament” arrays surrounding the myosin thick filaments, the ATP-driven sliding of actin filaments results in muscle contraction (discussed later). Cardiac and smooth muscle contain myosins that are similarly arranged, although they are encoded by different genes.

When a muscle myosin is digested by chymotrypsin and papain, the head domain is released as an intact fragment (called S1). The S1 fragment alone can generate filament sliding in vitro, proving that the motor activity is contained completely within the head (Figure 16-53).

Figure 16-53. Direct evidence for the motor activity of the myosin head.

Figure 16-53

Direct evidence for the motor activity of the myosin head. In this experiment, purified S1 myosin heads were attached to a glass slide, and then actin filaments labeled with fluorescent phalloidin were added and allowed to bind to the myosin heads. (A) (more…)

It was initially thought that myosin was present only in muscle, but in the 1970’s, researchers found that a similar two-headed myosin protein was also present in nonmuscle cells, including protozoan cells. At about the same time, other researchers found a myosin in the freshwater amoeba Acanthamoeba castellanii that was unconventional in having a motor domain similar to the head of muscle myosin but a completely different tail. This molecule seemed to function as a monomer and was named myosin I (for one-headed); the conventional myosin was renamed myosin II (for two-headed).

Subsequently, many other myosin types were discovered. The heavy chains generally start with a recognizable myosin motor domain at the N-terminus, and then diverge widely with a variety of C-terminal tail domains (Figure 16-54). The new types of myosins include a number of one-headed and two-headed varieties that are approximately equally related to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). The myosin tails (and the tails of motor proteins generally) have apparently diversified during evolution to permit the proteins to dimerize with other subunits and to interact with different cargoes.

Figure 16-54. Myosin superfamily tree.

Figure 16-54

Myosin superfamily tree. (A) A family tree for a few of the many known members of the myosin superfamily. The length of the lines separating individual family members indicates the amount of difference in the amino acid sequence of the motor domain. Groups (more…)

Some myosins (such as VIII and XI) have been found only in plants, and some have been found only in vertebrates (IX). Most, however, are found in all eucaryotes, suggesting that myosins arose early in eucaryotic evolution. The yeastSaccharomyces cerevisiae contains five myosins: two myosin Is, one myosin II, and two myosin Vs. One can speculate that these three types of myosins are necessary for a eucaryotic cell to survive and that other myosins perform more specialized functions in multicellular organisms. The nematode C. elegans, for example, has at least 15 myosin genes, representing at least seven structural classes; the human genome includes about 40 myosin genes.

All of the myosins except one move toward the plus end of an actin filament, although they do so at different speeds. The exception is myosin VI, which moves toward the minus end.

The exact functions for most of the myosins remain to be determined. Myosin II is always associated with contractile activity in muscle and nonmuscle cells. It is also generally required for cytokinesis, the pinching apart of a dividing cell into two daughters (discussed in Chapter 18), as well as for the forward translocation of the body of a cell during cell migration. The myosin I proteins contain a second actinbinding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization and the protrusion of actin-rich structures at the cell surface. Myosin V is involved in vesicle and organelle transport. Myosin VII is found in the inner ear in vertebrates, and certain mutations in the gene coding for myosin VII cause deafness in mice and humans.

 

There Are Two Types of Microtubule Motor Proteins: Kinesins and Dyneins

Kinesin is a motor protein that moves along microtubules. It was first identified in the giant axon of the squid, where it carries membrane-enclosed organelles away from the neuronal cell body toward the axon terminal by walking toward the plus end of microtubules. Kinesin is similar structurally to myosin II in having two heavy chains and two light chains per active motor, two globular head motor domains, and an elongated coiled-coil responsible for heavy chain dimerization. Like myosin, kinesin is a member of a large protein superfamily, for which the motor domain is the only common element (Figure 16-55). The yeastSaccharomyces cerevisiae has six distinct kinesins. The nematode C. elegans has 16 kinesins, and humans have about 40.

Figure 16-55. Kinesin and kinesin-related proteins.

Figure 16-55

Kinesin and kinesin-related proteins. (A) Structures of four kinesin superfamily members. As in the myosin superfamily, only the motor domains are conserved. Conventional kinesin has the motor domain at the N-terminus of the heavy chain. The middle domain (more…)

There are at least ten families of kinesin-related proteins, or KRPs, in the kinesin superfamily. Most of them have the motor domain at the N-terminus of the heavy chain and walk toward the plus end of the microtubule. A particularly interesting family has the motor domain at the C-terminus and walks in the opposite direction, toward the minus end of the microtubule. Some KRP heavy chains lack a coiled-coil sequence and seem to function as monomers, analogous to myosin I. Some others are homodimers, and yet others are heterodimers. At least one KRP (BimC) can self-associate through the tail domain, forming a bipolar motor that slides oppositely oriented microtubules past one another, much as a myosin II thick filament does for actin filaments. Most kinesins carry a binding site in the tail for either a membrane-enclosed organelle or another microtubule. Many of the kinesin superfamily members have specific roles in mitotic and meiotic spindle formation and chromosome separation during cell division.

The dyneins are a family of minus-end-directed microtubule motors, but they are unrelated to the kinesin superfamily. They are composed of two or three heavy chains (that include the motor domain) and a large and variable number of associated light chains. The dynein family has two major branches (Figure 16-56). The most ancient branch contains thecytoplasmic dyneins, which are typically heavy-chain homodimers, with two large motor domains as heads. Cytoplasmic dyneins are probably found in all eucaryotic cells, and they are important for vesicle trafficking, as well as for localization of the Golgi apparatus near the center of the cell. Axonemal dyneins, the other large branch, include heterodimers and heterotrimers, with two or three motor-domain heads, respectively. They are highly specialized for the rapid and efficient sliding movements of microtubules that drive the beating of cilia and flagella (discussed later). A third, minor, branch shares greater sequence similarity with cytoplasmic than with axonemal dyneins but seems to be involved in the beating of cilia.

Figure 16-56. Dyneins.

Figure 16-56

Dyneins. Freeze-etch electron micrographs of a molecule of cytoplasmic dynein and a molecule of ciliary (axonemal) dynein. Like myosin II and kinesin, cytoplasmic dynein is a two-headed molecule. The ciliary dynein shown has three heads. Note that the (more…)

Dyneins are the largest of the known molecular motors, and they are also among the fastest: axonemal dyneins can move microtubules in a test tube at the remarkable rate of 14 μm/sec. In comparison, the fastest kinesins can move their microtubules at about 2–3 μm/sec.

 

The Structural Similarity of Myosin and Kinesin Indicates a Common Evolutionary Origin

The motor domain of myosins is substantially larger than that of kinesins, about 850 amino acids compared with about 350. The two classes of motor proteins track along different filaments and have different kinetic properties, and they have no identifiable amino acid sequence similarities. However, determination of the three-dimensional structure of the motor domains of myosin and kinesin has revealed that these two motor domains are built around nearly identical cores (Figure 16-57). The central force-generating element that the two types of motor proteins have in common includes the site of ATP binding and the machinery necessary to translate ATP hydrolysis into an allosteric conformational change. The differences in domain size and in the choice of track can be attributed to large loops extending outward from this central core. These loops include the actin-binding and microtubule-binding sites, respectively.

Figure 16-57. X-ray crystal structures of myosin and kinesin heads.

Figure 16-57

X-ray crystal structures of myosin and kinesin heads. The central nucleotide-binding domains of myosin and kinesin (shaded in yellow) are structurally very similar. The very different sizes and functions of the two motors are due to major differences (more…)

An important clue to how the central core is involved in force generation has come from the observation that the motor core also bears some structural resemblance to the nucleotidebinding site of the small GTPases of the Ras superfamily. As discussed in Chapter 3 (see Figure 3-74), these proteins exhibit distinct conformations in their GTP-bound (active) and GDP-bound (inactive) forms: mobile “switch” loops in the nucleotide-binding site are in close contact with the γ-phosphate in the GTP-bound state, but these loops swing out when the hydrolyzed γ-phosphate is released. Although the details of the movement are different for the two motor proteins, and ATP rather than GTP is hydrolyzed, the relatively small structural change in the active site—the presence or absence of a terminal phosphate—is similarly amplified to cause a rotation of a different part of the protein. In kinesin and myosin, a switch loop interacts extensively with those regions of the protein involved in microtubule and actin binding, respectively, allowing the structural transitions caused by the ATP hydrolysis cycle to be relayed to the polymer-binding interface. The relay of structural changes between the polymer-binding site and the nucleotide hydrolysis site seems to work in both directions, since theATPase activity of motor proteins is strongly activated by binding to their filament tracks.

Motor Proteins Generate Force by Coupling ATP Hydrolysis to Conformational Changes

Although the cytoskeletal motor proteins and GTP-binding proteins both use structural changes in their nucleoside-triphosphate-binding sites to produce cyclic interactions with a partner protein, the motor proteins have a further requirement: each cycle of binding and release must propel them forward in a single direction along a filament to a newbinding site on the filament. For such unidirectional motion, a motor protein must use the energy derived from ATP binding and hydrolysis to force a large movement in part of the protein molecule. For myosin, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm (see Figure 16-57), which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a piston-like helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix like a long lever, causing the far end of the helix to move by about 5.0 nm. These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement (Figure 16-58). In the myosin VI subfamily of myosins, which move backward (toward the minus end of the actin filament), the converter domain probably lies in a different orientation, so that the same piston-like movement of the small helix causes the lever arm to rotate in the opposite direction.

Figure 16-58. The cycle of structural changes used by myosin to walk along an actin filament.

Figure 16-58

The cycle of structural changes used by myosin to walk along an actin filament. (Based on I. Rayment et al., Science 261:50–58, 1993. © AAAS.)

In kinesin, instead of the rocking of a lever arm, the small movements of switch loops at the nucleotidebinding siteregulate the docking and undocking of the motor head domain to a long linker region that connects this motor head at one end to the coiled-coil dimerization domain at the other end. When the front (leading) kinesin head is bound to amicrotubule before the power stroke, its linker region is relatively unstructured. On the binding of ATP to this bound head, its linker region docks along the side of the head, which throws the second head forward to a position where it will be able to bind a new attachment site on the protofilament, 8 nm closer to the microtubule plus end than the binding site for the first head. The nucleotide hydrolysis cycles in the two heads are closely coordinated, so that this cycle of linker docking and undocking can allow the two-headed motor to move in a hand-over-hand (or head-over-head) stepwise manner (Figure 16-59A).

Figure 16-59. Comparison of the mechanochemical cycles of kinesin and myosin II.

Figure 16-59

Comparison of the mechanochemical cycles of kinesin and myosin II. The shading in the two circles representing the hydrolysis cycle indicates the proportion of the cycle spent in attached and detached states for each motor protein. (A) Summary of the (more…)

The coiled-coildomain seems both to coordinate the mechanochemical cycles of the two heads (motor domains) of thekinesin dimer and to determine its directionality of movement. Recall that whereas most members of the kinesin superfamily, with their motor domains at the N-terminus, move toward the plus end of the microtubule, a few superfamily members have their motor domains at the C-terminus and move toward the minus end. Since the motor domains of these two types of kinesins are essentially identical, how can they move in opposite directions? The answer seems to lie in the way in which the heads are connected. In high-resolution images of forward-walking and backward-walking members of the kinesin superfamily bound to microtubules, the heads that are attached to the microtubule are essentially indistinguishable, but the second, unattached heads are oriented very differently. This difference in tilt apparently biases the next binding site for the second head, and thereby determines the directionality of motor movement (Figure 16-60).

Figure 16-60. Orientation of forward- and backward-walking kinesin superfamily proteins bound to microtubules.

Figure 16-60

Orientation of forward- and backward-walking kinesin superfamily proteins bound to microtubules. These images were generated by fitting the structures of the free motor-protein dimers (determined by x-ray crystallography) onto a lower resolution image (more…)

Although both myosin and kinesin undergo analogous mechanochemical cycles, the exact nature of the coupling between the mechanical and chemical cycles is different in the two cases (see Figure 16-60). For example, myosin without any nucleotide is tightly bound to its actin track, in a so-called “rigor” state, and it is released from this track by the association of ATP. In contrast, kinesin forms a rigor-like tight association with a microtubule when ATP is bound to the kinesin, and it is hydrolysis of ATP that promotes release of the motor from its track.

Thus, cytoskeletal motor proteins work in a manner highly analogous to GTP-binding proteins, except that in motor proteins the small protein conformational changes (a few tenths of a nanometer) associated with nucleotide hydrolysis are amplified by special protein domains—the lever arm in the case of myosin and the linker in the case of kinesin—to generate large-scale (several nanometers) conformational changes that move the motor proteins stepwise along their filament tracks. The analogy between the GTPases and the cytoskeletal motor proteins has recently been extended by the observation that one of the GTP-binding proteins—the bacterial elongation factorG—translates the chemical energy of GTP hydrolysis into directional movement of the mRNAmolecule on the ribosome.

Motor Protein Kinetics Are Adapted to Cell Functions

The motor proteins in the myosin and kinesin superfamilies exhibit a remarkable diversity of motile properties, well beyond their choice of different polymer tracks. Most strikingly, a single dimer of conventional kinesin moves in a highly processive fashion, traveling for hundreds of ATPase cycles along a microtubule without dissociating. Skeletal muscle myosin II, in contrast, cannot move processively and makes just one or a few steps along an actin filament before letting go. These differences are critical for the motors’ various biological roles. A small number of kinesin molecules must be able to transport a mitochondrion all the way down a nerve cellaxon, and therefore require a high level of processivity. Skeletal muscle myosin, in contrast, never operates as a single molecule but rather as part of a huge array of myosin II molecules. Here processivity would actually inhibit biological function, since efficient muscle contraction requires that each myosin head perform its power stroke and then quickly get out of the way, to avoid interfering with the actions of the other heads attached to the same actin filament.

There are two reasons for the high degree of processivity of kinesin movement. The first is that the mechanochemical cycles of the two motor heads in a kinesin dimer are coordinated with each other, so that one kinesin head does not let go until the other is poised to bind. This coordination allows the motor protein to operate in a hand-over-hand fashion, never allowing the organelle cargo to diffuse away from the microtubule track. There is no apparent coordination between the myosin heads in a myosin II dimer. The second reason for the high processivity of kinesin movement is that kinesin spends a relatively large fraction of its ATPase cycle tightly bound to the microtubule. For both kinesin and myosin, the conformational change that produces the force-generating working stroke must occur while the motor protein is tightly bound to its polymer, and the recovery stroke in preparation for the next step must occur while the motor is unbound. But as we have seen in Figure 16-59, myosin spends only about 5% of its ATPase cycle in the tightly bound state and is unbound the rest of the time.

What myosin loses in processivity it gains in speed; in an array in which many motor heads are interacting with the sameactin filament, a set of linked myosins can move their filament a total distance equivalent to 20 steps during a single cycle time, while kinesins can move only two. Thus, myosins can typically drive filament sliding much more rapidly than kinesins, even though they hydrolyze ATP at comparable rates and take molecular steps of comparable length.

Within each motor protein class, movement speeds vary widely, from about 0.2 to 60 μm/sec for myosins, and from about 0.02 to 2 μm/sec for kinesins. These differences arise from a fine-tuning of the mechanochemical cycle. The number of steps that an individual motor molecule can take in a given time, and thereby the velocity, can be increased by either increasing the motor protein’s intrinsic ATPase rate or decreasing the proportion of cycle time spent bound to the filament track. Moreover, the size of each step can be changed by either changing the length of the lever arm (for example, the lever arm of myosin V is about three times longer than the lever arm of myosin II) or the angle through which the helix swings (Figure 16-61). Each of these parameters varies slightly among different members of the myosin and kinesin families, corresponding to slightly different protein sequences and structures. It is assumed that the behavior of each motor protein, whose function is determined by the identity of the cargo attached through its tail-domain, has been fine-tuned during evolution for speed and processivity according to the specific needs of the cell.

Figure 16-61. The effect of lever arm length on the step size for a motor protein.

Figure 16-61

The effect of lever arm length on the step size for a motor protein. The lever arm of myosin II is much shorter than the lever arm of myosin V. The power stroke in the head swings their lever arms through the same angle, so myosin V is able to take a (more…)

Motor Proteins Mediate the Intracellular Transport of Membrane-enclosed Organelles

A major function of cytoskeletal motors in interphase cells is the transport and positioning of membrane-enclosed organelles. Kinesin was originally identified as the protein responsible for fast axonal transport, the rapid movement of mitochondria, secretory vesicle precursors, and various synapse components down the microtubule highways of theaxon to the distant nerve terminals. Although organelles in most cells need not cover such long distances, their polarized transport is equally necessary. A typical microtubule array in an interphase cell is oriented with the minus ends near the center of the cell at the centrosome, and the plus ends extending to the cell periphery. Thus, centripetal movements of organelles toward the cell center require the action of minus-end-directed motor proteins such as cytoplasmic dynein, whereas centrifugal movements toward the periphery require plus-end-directed motors such as kinesins.

The role of microtubules and microtubule motors in the behavior of intracellular membranes is best exemplified by the part they play in organizing the endoplasmic reticulum (ER) and the Golgi apparatus. The network of ER membranetubules aligns with microtubules and extends almost to the edge of the cell, whereas the Golgi apparatus is located near the centrosome. When cells are treated with a drug that depolymerizes microtubules, such as colchicine or nocodazole, the ER collapses to the center of the cell, while the Golgi apparatus fragments and disperses throughout the cytoplasm(Figure 16-62). In vitro, kinesins can tether ER-derived membranes to preformed microtubule tracks, and walk toward the microtubule plus ends, dragging the ER membranes out into tubular protrusions and forming a membranous web very much like the ER in cells. Likewise, the outward movement of ER tubules toward the cell periphery is associated with microtubule growth in living cells. Conversely, dyneins are required for positioning the Golgi apparatus near the cell center, moving Golgi vesicles along microtubule tracks toward minus ends at the centrosome.

Figure 16-62. Effect of depolymerizing microtubules on the Golgi apparatus.

Figure 16-62

Effect of depolymerizing microtubules on the Golgi apparatus. (A) In this endothelial cell, the microtubules are labeled in red, and the Golgi apparatus is labeled in green (using an antibody against a Golgi protein). As long as the system of microtubules (more…)

The different tails and their associated light chains on specific motor proteins allow the motors to attach to their appropriate organelle cargo. For example, there is evidence for membrane-associated motor receptors, sorted to specific membrane-enclosed compartments, that interact directly or indirectly with the tails of the appropriate kinesin family members. One of these receptors seems to be the amyloid precursor protein, APP, which binds directly to a light chainon the tail of kinesin-I and is proposed to be a transmembrane motor proteinreceptormolecule in nerve-cell axons. It is the abnormal processing of this protein that gives rise to Alzheimer’s disease, as discussed in Chapter 15.

For dynein, attachment to membranes is known to be mediated by a large macromolecular assembly. Cytoplasmic dynein is itself a huge proteincomplex, and it requires association with a second large protein complex called dynactinto translocate organelles effectively. The dynactin complex includes a short actinlike filament that is made of the actin-related protein Arp1 (distinct from Arp2 and Arp3, the components of the ARP complex involved in the nucleation of conventional actin filaments). Membranes of the Golgi apparatus are coated with the proteins ankyrin and spectrin, which have been proposed to associate with the Arp1 filament in the dynactin complex to form a planar cytoskeletal array reminiscent of the erythrocyte membranecytoskeleton (see Figure 10-31). The spectrin array probably gives structural stability to the Golgi membrane, and—via the Arp1 filament—it may mediate the regulatable attachment of dynein to the organelle (Figure 16-63).

Figure 16-63. A model for the attachment of dynein to a membrane-enclosed organelle.

Figure 16-63

A model for the attachment of dynein to a membrane-enclosed organelle. Dynein requires the presence of a large number of accessory proteins to associate with membrane-enclosed organelles. Dynactin is a large complex (red)that includes components that (more…)

Motor proteins also have a significant role in organelle transport along actin filaments. The first myosin shown to mediate organelle motility was myosin V, a two-headed myosin with a large step size (see Figure 16-61). In mice, mutations in the myosin V gene result in a “dilute” phenotype, in which fur color looks faded. In mice (and humans),membrane-enclosed pigment granules, called melanosomes, are synthesized in cells called melanocytes beneath the skin surface. These melanosomes move out to the ends of dendritic processes in the melanocytes, from where they are delivered to the overlying keratinocytes that form the skin and fur. Myosin V is associated with the surface of melanosomes, and it is able to mediate their actin-based movement in a test tube (Figure 16-64). In dilute mutant mice, the melanosomes are not delivered to the keratinocytes efficiently, and pigmentation is defective. Other myosins, including myosin I, are associated with endosomes and a variety of other organelles.

Figure 16-64. Myosin V on melanosomes.

Figure 16-64

Myosin V on melanosomes. (A) Phase-contrast image of a portion of a melanocyte isolated from a mouse. The black spots are melanosomes, which are membrane-enclosed organelles filled with the skin pigment melanin. (B) The same cell labeled with a fluorescent (more…)

Motor Protein Function Can Be Regulated

The cell can regulate the activity of motor proteins, allowing it to change either the positioning of its membrane-enclosed organelles or its whole-cell movements. One of the most dramatic examples is provided by fish melanocytes. These giant cells, which are responsible for rapid changes in skin coloration in several species of fish, contain large pigment granules that can alter their location in response to neuronal or hormonal stimulation (Figure 16-65). These pigment granules aggregate or disperse by moving along an extensive network of microtubules. The minus ends of these microtubules are nucleated by the centrosome and are located in the center of the cell, while the plus ends are distributed around the cell periphery. The tracking of individual pigment granules reveals that the inward movement is rapid and smooth, while the outward movement is jerky, with frequent backward steps (Figure 16-66). Both dynein and kinesinare associated with the pigment granules. The jerky outward movements apparently result from a tug-of-war between the two motor proteins, with the stronger kinesin winning out overall. When the kinesin light chains become phosphorylated after a hormonal stimulation that signals skin color change, kinesin is inactivated, leaving dynein free to drag the pigment granules rapidly toward the cell center, changing the fish’s color. In a similar way, the movement of other membrane organelles coated with particular motor proteins is controlled by a complex balance of competing signals that regulate both motor protein attachment and activity.

Figure 16-65. Regulated melanosome movements in fish pigment cells.

Figure 16-65

Regulated melanosome movements in fish pigment cells. These giant cells, which are responsible for changes in skin coloration in several species of fish, contain large pigment granules, or melanosomes (brown). The melanosomes can change their location (more…)

Figure 16-66. Bidirectional movement of a melanosome on a microtubule.

Figure 16-66

Bidirectional movement of a melanosome on a microtubule. An isolated melanosome (yellow) moves along a microtubule on a glass slide, from the plus end toward the minus end. Halfway through the video sequence, it abruptly switches direction and moves from (more…)

Myosin activity can also be regulated by phosphorylation. In nonmuscle cells, myosin II can be phosphorylated on a variety of sites on both heavy and light chains, affecting both motor activity and thick filament assembly. The myosin II can exist in two different conformational states in such cells, an extended state that is capable of forming bipolar filaments, and a bent state in which the tail domain apparently interacts with the motor head. Phosphorylation of the regulatory light chain by the calcium-dependent myosin light-chain kinase (MLCK) causes the myosin II to preferentially assume the extended state, which promotes its assembly into a bipolar filament and leads to cell contraction (Figure 16-67). Myosin light-chain phosphorylation is an indirect target of activated Rho, the small GTPasediscussed previously whose activation causes a reorganization of the actincytoskeleton into contractile stress fibers. The MLCK is also activated during mitosis, causing myosin II to assemble into the contractile ring that is responsible for dividing the mitotic cell into two. Regulation of other members of the myosin superfamily is not as well understood, but control of these myosins is also likely to involve site-specific phosphorylations.

Figure 16-67. Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments.

Figure 16-67

Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments. (A) The controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called regulatory light chain, shown (more…)

Summary

Motor proteins use the energy of ATP hydrolysis to move along microtubules or actin filaments. They mediate the sliding of filaments relative to one another and the transport of membrane-enclosed organelles along filament tracks. All known motor proteins that move on actin filaments are members of the myosin superfamily. The motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family. The myosin and kinesin superfamilies are diverse, with about 40 genes encoding each type of protein in humans. The only structural element shared among all members of each superfamily is the motor “head” domain. These heads can be attached to a wide variety of “tails,” which attach to different types of cargo and enable the various family members to perform different functions in the cell. Although myosin and kinesin walk along different tracks and use different mechanisms to produce force and movement by ATP hydrolysis, they share a common structural core, suggesting that they are derived from a common ancestor.

Two types of specialized motility structures in eucaryotic cells consist of highly ordered arrays of motor proteins that move on stabilized filament tracks. The myosin-actin system of the sarcomere powers the contraction of various types of muscle, including skeletal, smooth, and cardiac muscle. The dyneinmicrotubule system of the axoneme powers the beating of cilia and the undulations of flagella.

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Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
Bookshelf ID: NBK26888

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