Feeds:
Posts
Comments

Posts Tagged ‘oncotherapy’

Novel Oncologic Approach by Drug Trapping

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Tim Sandle, PhD just reported on this interesting and novel method of chemotherapy for cancer.

Fighting cancer by trapping drugs in tumors

By Tim Sandle     Jan 27, 2016 inScience

Read more: http://www.digitaljournal.com/science/fighting-cancer-by-trapping-drugs-in-tumors/article/455971#ixzz3yvjl23Mn

A research group have developed a novel means to fight cancer. Because anti-cancer drugs only work for a limited time, the new method succeeds in trapping the drug inside the tumor so it works for longer.

Cancer drugs vary in their mechanism of activity. Some are intended to attack the tumor from the outside whereas others are designed to attack the tumor from within. An example of the latter is the drug cilengitide which affects blood veseels and it is intended to cut-off the nutrient supply to cancerous cells. It is with medications designed to work from the inside that the new research as focused on.

 The problem is with drugs designed to work from the inside is they have a short life. The drugs are often absorbed into cancer cells and become ineffective. For this reason, researchers wanted to find a means to lock the drug into the tumor for longer.

The process of doing so involves creating pockets or ‘depots’, of microscopic sizes, to enable anti-cancer drugs to remain trapped inside tumors. To do this they developed nanocarriers, to wrap the anti-cancer drug into. The nanocarrier is covered with even smaller nanoscpasules composed of hyaluronic acid gel. The capsules contain an enzyme, and they are injected into the blood stream.

On reaching the tumor site, the capsules dissolve as a consequence of an enzyme located on the tumor surface. The carriers are then deposited inside the tumor. The depot is larger enough to prevent absorption by the cancer cell. The nanocarrier is designed to breakdown slowly and to produce a slow-release of the anti-cancer drug within.

The feat of biomedical engineering was tested out on mice. Here it was found the anti-cancer medication was 10 times more effective against tumors, increasing the shrinkage rate, when compared to the same drugs administered conventionally.

The study was designed as a “proof of concept.” Having established this, further studies will be set up to test out the effectiveness of the method.

The research was conducted at two centers: North Carolina State University and the University of North Carolina at Chapel Hill. The research findings are published in the journal NanoLetters, in a paper titled “Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellular Drug-Delivery Depots.”

Read more: http://www.digitaljournal.com/science/fighting-cancer-by-trapping-drugs-in-tumors/article/455971#ixzz3yvkJodV2

SJ Williams, PhD

I wonder if they get the same effect as with the earlier attempts at producing either prodrugs linked to antibodies (like the ADEPT method) which did work except for some severe dose-limiting toxicities. The other phenomenon to consider is ‘bystander effect’ and if this approach produces such an effect or not. Irregardless increased distribution of drug is extremely important and would be nice to see in further studies if dose could be minimized.

Read Full Post »

Perspectives on Anti-metastatic Effects in Cancer Research 2015

Curator: Larry H. Bernstein, MD, FCAP

 

Combining Kinetic Ligand Binding and 3D Tumor Invasion Technologies to Assess Drug Residence Time and Anti-metastatic Effects of CXCR4 Inhibitors

Application Note 3D Cell Culture, ADME/Tox, Cell Imaging, Cell-Based Assays
BioTek Instruments, Inc. P.O. Box 998, Highland Park, Winooski, Vermont 05404-0998
Brad Larson and Leonie Rieger, BioTek Instruments, Inc., Winooski, VT
Nicolas Pierre, Cisbio US, Inc., Bedford, MA
Hilary Sherman, Corning Incorporated, Life Sciences, Kennebunk, ME

http://vertassets.blob.core.windows.net/download/ba9da411/ba9da411-a56c-42d3-a1a0-8c128224947f/cisbio_residence_time_app_note_final.pdf

Metastasis, the spread of cancer cells from the original tumor to secondary locations within the body, is linked to approximately 90% of cancer deaths1 . The expression of chemokine receptors, such as CXCR4 and CCR7, is tightly correlated with the metastatic properties of breast cancer cells. In vivo, neutralizing the interaction of CXCR4 and its known ligand, SDF1-α (CXCL12), significantly impaired the metastasis of breast cancer cells and cell migration2 . Traditionally, the discovery of novel agents has been guided by the affinity of the ligand for the receptor under equilibrium conditions, largely ignoring the kinetic aspects of the ligandreceptor interaction. However, awareness of the importance of binding kinetics has started to increase due to accumulating evidence3, 4, 5, 6 suggesting that the in vivo effectiveness of ligands may be attributed to the time a particular ligand binds to its receptor (drug-target residence time).

Similarly, appropriate in vitro cell models have also been lacking to accurately assess the ability of novel therapies to inhibit tumor invasion. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells7 . Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format.

Here we examine the drug-target residence time of various CXCR4 inhibitors using a direct, homogeneous ligand binding assay and CXCR4 expressing cell line in a kinetic format. This inhibitor panel was further tested in a 3D tumor invasion assay to determine whether there is a correlation between the molecule’s CXCR4 residence time and inhibition of the phenotypic effect of tumor invasion. MDA-MB-231 breast adenocarcinoma cells, known to be invasive, and metastasize to lung from primary mammary fat pad tumors8 , were included, in addition to primary human dermal fibroblasts. Cellular analysis algorithms provided accurate quantification of changes to the original tumoroid structure, as well as invadopodia development. The combination presents an accurate, yet easy-to-use method to assess target-based and phenotypic effects of new, potential anti-metastatic drugs.

……

Cytation™ 5 Cell Imaging Multi-Mode Reader Cytation 5 is a modular multi-mode microplate reader that combines automated digital microscopy and microplate detection. Cytation 5 includes filter- and monochromator-based microplate reading; the microscopy module provides high resolution microscopy in fluorescence, brightfield, color brightfield and phase contrast. With special emphasis on live-cell assays, Cytation 5 features temperature control to 65 °C, CO2 / O2 gas control and dual injectors for kinetic assays. Shaking and Gen5 software are also standard. The instrument was used to image spheroids, as well as individual cell invasion through the Matrigel matrix.

Tag-lite® Receptor Ligand Binding Assay

Figure 1. Tag-lite® Receptor Ligand Binding Assay Procedure. The Tag-lite CXCR4 assay relies on a fully functional SNAP-tag fused CXCR4 receptor and fluorescently labeled ligand SDF1-α. Being homogeneous, the binding assay allows for binding events to be precisely recorded in time. The assay can be used to derive the kinetic binding parameters of unlabeled compounds by application of the Motulsky and Mahan equations.

……

Results and Discussion

Drug-Target Residence Time

Determination Association Kinetics of SDF1-α-d2 Labeled Ligand

The final Drug-Target Residence Time value takes into account the observed on and off rates of the unlabeled inhibitors as well as the labeled SDF1-α-d2 ligand, and is computed by incorporation of the Motulsky and Mahan equation9 . The first step to calculate the final value was to perform an associative binding experiment using a concentration range of 0-100 nM of the d2 acceptor fluor labeled ligand. Binding was monitored kinetically over a period of 40 minutes.

Figure 2. Association binding graph of SDF1-α-d2. Observed associative binding curves calculated from HTRF ratios of wells containing SDF1-α-d2 ligand concentrations ranging from 0-100 nM. Non-specific binding values subtracted from total ratios to determine observed specific binding.

Binding increases over time until it plateaus after several minutes (Figure 2). The plateau in an association experiment depends on the concentration of labeled SDF1-α used. Higher plateaus will be obtained with higher concentrations. Fitting of the curves with Graph Pad Prism yields the observed association rate values for all concentrations tested or kobs.

The Kd value of the labeled ligand was also determined by plotting the HTRF ratios generated after a binding equilibrium was reached with the different concentrations of ligand tested.

Figure 3. SDF1-α-d2 saturation binding curve. HTRF ratios generated upon the achievement of binding equilibrium of tested [SDF1-α-d2].

In a saturation binding experiment, increasing concentrations of labeled SDF1-α result in increased binding. Saturation is obtained when no further binding can be recorded. The ligand concentration that binds to half the receptor sites at equilibrium or Kd was 29 nM.

An assessment of whether the labeled SDF1-α ligand follows the Law of Mass action can also be carried out. If the system does follow the Law of Mass action then kobs increases linearly with increasing concentrations of SDF1-α.

Due to the linear shape of the curve, and an R2 value >0.9, Law of Mass Action was proven for the labeled SDF1-α ligand. This allowed for the use of Graph Pad Prism software to derive association and dissociation rate constants from the linear regression line. The rate constant values experimentally found or mathematically derived are summarized in Table 1. kon,SDF1-α-d2 and koff ,SDF1-α-d2 were 0.001 nM-1.s-1 and 0.04 s-1, respectively

Table   SDF1-α-d2 Kinetic Binding Characterization

Association Kinetics of SDF1-α-d2 Labeled Ligand In the theory developed by Motulsky and Mahan, an unlabeled competitor is co-incubated with a labeled ligand during a kinetic association experiment. Here, a single concentration of the SDF1-α-d2 ligand, 25 nM, was co-incubated with multiple concentrations of the unlabeled SDF1-α competitors in the presence of the CXCR4 expressing cells. Kinetic binding of the labeled ligand was then monitored over time.

Figure 5. Kinetics of Competitive Binding. Plot of specific binding HTRF ratios over time for the SDF1-α-d2 ligand when in the presence of 100, 10, or 1 nM concentrations of (A.) AMD 3100, (B.) AMD 3465, or (C.) IT1t.

From the curve fitting of the observed SDF1-α-d2 kinetic binding, and incorporation of the Law of Mass Action linear regression line, k(off) (Min-1) values were then calculated. Final residence time (R) values could then be determined using the following formula:

R = 1/k(off)

Therefore, molecules having a lower k(off) rate reside at the target receptor for longer periods of time.

Table 2. SDF1-α Competitor Dissociation Rate and Residence Time Values.

From the shape of the curves in Figure 5, and a comparison of the residence time values generated for the labeled ligand and unlabeled competitors (Table 2), qualitative and quantitative assumptions regarding the various competitors can then be made. First, if the competitor dissociates faster from its target than the ligand (smaller R value), such as is seen with AMD 3100 (Figure 5A), the specific binding of the ligand will slowly and monotonically approach its equilibrium in time. However, when the competitor dissociates slower (larger R value), the association curve of the ligand consists of two phases, starting with a typical “overshoot” and then a decline until a new equilibrium is reached. Competitors whose residence times are greater than that of the SDF1-α-d2 ligand, such as AMD 3465 and IT1t (Figure 5B and C), may then exhibit a stronger inhibitory response when used in the confirmatory phenotypic 3D tumor invasion assay.

Interruption of Invasion via SDF1-α Ligand Binding Inhibition As stated previously, interruption of the interaction between CXCR4 and its known ligand, SDF1-α, impairs metastasis of breast cancer and cell migration2 . Therefore, a phenotypic assessment of the CXCR4 inhibitor panel was then performed to determine whether changes in the level of tumor migration could be detected, and more importantly, if compounds exhibiting longer residence times compared to SDF1-α-d2 exhibited a higher inhibitory effect on migration through the 3D matrix. MDA-MB-231 breast adenocarcinoma cells, co-cultured with human dermal fibroblasts, were used as the in vitro tumor model. This breast cancer cell line has been previously shown to express the CXCR4 receptor10.

Figure 6. Image-based Monitoring of MDA-MB-231/Fibroblast Tumor Invasion. Overlaid brightfield and fluorescent images captured using a 4x objective, after a 0 and 5 day incubation period with AMD 3465, IT1t, and CTCE 9908. Imaging channel representation: Brightfield – Total cells and invadopodia; GFP – MDA-MB-231 cells; RFP – Fibroblasts.

Figure 7. Quantification of Invasive Tumor Area. 4x overlaid images captured following 5 day (A.) 100 and (B.) 0 μM IT1t incubation with tumoroids. Object masks automatically drawn by Gen5 using the following criteria: Threshold: 5000 RFU; Min. Object Size: 400 μm; Max. Object Size: 1500 μm; Image Smoothing Strength: 0; Background Flattening Size: Auto.

Cellular analysis is performed with the Cytation 5 using the brightfield signal to quantify the extent of invasion. Minimum and maximum object sizes, as well as brightfield threshold values are set such that a precise object mask is automatically drawn around each tumoroid in its entirety (Figure 7A and B). The same criteria are used for all images evaluated during the experiment. This allows for a quantitative comparison of the area covered within each object mask to be completed.

Figure 8. Tumor Invasion Inhibition Determination. Graphs of individual tumoroid areas on day 0, and subsequent to five day invasion period in the presence of inhibitor concentrations.

The 4x images displayed (Figure 6), as well as the graphs in Figure 8, demonstrating total tumoroid area coverage before and after the incubation period illustrate the ability of CXCR4 inhibitors to interrupt tumor invasion consistent with the previously determined residence time. AMD 3465 and IT1t, which exhibit a residence time longer than SDF1-α-d2, effectively minimize tumor invasion in a dose dependent manner. The decrease in MDAMB-231 GFP and fibroblast RFP expression exhibited after a 5 day 100 μM IT1t incubation, also seen after a 7 day AMD 3465 incubation of the same concentration (data not shown), may also indicate the chronic cytotoxic effects that elevated dosing of these compounds can have on both cancer and stromal cells. All other compounds show little to no effect on the ability of the tumoroid to migrate through the 3D matrix. While AMD 3465 and ITt1 display the same sub-nanomolar potency, AMD3465 prevails as a CXCR4 inhibitor due to its greater residence time.

Conclusions The Tag-lite CXCR4 ligand binding assay provides a simple, yet robust cell-based approach to determine kinetic binding of known receptor ligands, as well as competitive binding of test molecules. The simultaneous dual emission capture and injection capabilities of the Synergy Neo allow accurate calculations of kinetic association and dissociation rates to be made when used in conjunction with the Tag-lite® assay. Corning Spheroid Microplates then provide an easy-to-use, consistent method to perform spheroid aggregation and confirmatory 3D tumor invasion assays. Imaging of spheroid formation, as well as invading structures can be performed by the Cytation™ 5 using brightfield or fluorescent channels to easily track tumoroid invasion. The flexible cellular analysis capacity of the Gen5™ Data Analysis Software also allows for accurate assessment of 3D tumor invasion during the entire incubation period. The combination of assay chemistry, cell model, kinetic microplate and image-based monitoring, in addition to cellular analysis provide an ideal method to better understand the target-based and phenotypic effects of potential inhibitors of tumor invasion and metastasis.

References

  1. Saxe, Charles. ‘Unlocking The Mysteries Of Metastasis’. ExpertVoices 2013. http://www.cancer.org/ cancer/news/expertvoices/post/2013/01/23/unlockingthe-mysteries-of-metastasis.aspx. Accessed 16 Mar. 2015.
  2. Müller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M., McClanahan, T., Mruphy, E., Yuan, W., Wagner, S., Barrera, J., Mohar, A., Verástegui, E., Zlotnik, A. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001, 410, 50-56.
  3. Swinney, D. Biochemical mechanisms of drug action: what does it take for success? Nat Rev Drug Discov. 2004, 3, 801-808.
  4. Copeland, R., Pompliano, D., Meek, T. Drugtarget residence time and its implications for lead optimization. Nat Rev Drug Discov. 2006,5, 730-739.
  5. Tummino, P., Copeland, R. Residence time of receptor-ligand complexes and its effect on biological function. Biochemistry. 2008, 47, 5481-5492.
  6. Zhang, R., Monsma, F. The importance of drug-target residence time. Curr Opin Drug Discov Devel. 2009, 12, 488-496.
  7. Mao, Y., Keller, E., Garfield, D., Shen, K., Wang, J. Stromal cells in tumor microenvironment and breast cancer. Cancer Metast Rev. 2013, 32, 303-315.
  8. Kamath, L., Meydani, A., Foss, F., Kuliopulos, A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res. 2001, 61, 5933-5940.
  9. Motulsky, H., Mahan, L. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol. 1984, 25, 1-9.
  10. Sun, Y., Mao, X, Fan, C, Liu, C., Guo, A., Guan, S., Jin, Q., Li, B., Yao, F., Jin, F. CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis. Tumor Biol. 2014, 35, 7765-7773.

 

 

Inspired by Nature

Researchers are borrowing designs from the natural world to advance biomedicine.

By Daniel Cossins | August 1, 2015
http://mobile.the-scientist.com/article/43625/inspired-by-nature

When biomedical engineer Jeff Karp has questions, he looks to animals for answers. In 2009, Karp gathered his team at the Brigham and Women’s Hospital in Boston to brainstorm novel ways to capture circulating tumor cells (CTCs) in the bloodstream. They mulled over the latest microfluidic devices. Then the conversation turned to the New England Aquarium, and to jellyfish.

Scientists have tried to grab cancer cells from blood ever since they discovered that tumors shed malignant cells that migrate throughout the vasculature—a process known as metastasis. “If you pluck out these cells, you have a direct indicator of what the cancer looks like,” says Karp. “Then you can screen drugs to get those that will have the greatest impact.” Doctors might also be able to detect such cells during the earliest stages of metastatic cancer, when it’s more readily treatable.

CANCER-CELL CAPTURE DEVICE: Jellyfish’s long, sticky tentacles grab prey and other food particles from water. Researchers have copied this design by coating the channels of a microfluidic chip with long, tentacle-like strands of DNA that bind a protein on the surface of leukemia cells. The device can process 10 times more blood than existing chips in the same amount of time.
See full infographic: JPG SANDCASTLE WORM: PHEBE LI FOR THE SCIENTIST. DIAGRAM: KIMBERLY BATTISTA

The problem is, CTCs make up a tiny fraction of cells in the bloodstream of a person with cancer, meaning an effective diagnostic must process relatively large volumes of blood. However, an existing test, which uses magnetic particles to isolate CTCs, processes just 7.5 milliliters of blood, only a fraction of one percent of the 5 liters of blood in an adult human. Dialysis-like microfluidic devices promise to handle larger volumes and improve efficiency, but the best current prototypes still feature extremely narrow microchannels to ensure CTCs pass within reach of CTC-binding antibodies along the perimeter. “Channel height is extremely low in a lot of the proposed devices, meaning you can barely flow any blood through,” says Karp. (See “Capturing Cancer Cells on the Move,” The Scientist, April 2014.)

Karp wanted to change that. “We asked ourselves, ‘What creatures can capture things at a distance?’” he recalls. One of his graduate students suggested jellyfish, whose long, sticky tentacles grab prey and other food particles from water. Within a year, Karp and his colleagues had designed a microfluidic chip on which 800-micron-wide microchannels are lined with long, tentacle-like strands of DNA that bind a protein on the surface of leukemia cells as they pass through the channels. (See illustration below.) In 2012, Karp showed that the jellyfish-inspired device could process 10 times more blood than existing chips in the same amount of time and trap an average of 50 percent of circulating leukemia cells.1 Karp estimates that a device the size of the standard microscope slide could collect hundreds or thousands of tumor cells in minutes. Encouraged by such results, Karp’s team is now improving the platform, designing chips that can catch any CTC of interest.

The jellyfish is far from the only intriguing organism to have served as a blueprint for scientists in the field of bioinspired medicine. Researchers have taken cues from the adhesive chemistry perfected by mussels and marine worms to create tissue glues that stick in wet and turbulent conditions; from red blood cell membranes to help drug-carrying nanoparticles avoid immune attack; and from the slippery slides that help carnivorous pitcher plants catch prey to produce novel antibacterial surfaces. (See “Bioinspired Antibacterial Surfaces.”) Nature, it seems, provides a compendium of biomedical solutions.

“Nature has used the power of evolution by natural selection to develop the most efficient ways to solve all kinds of problems,” says Donald Ingber, founding director of the Wyss Institute for Biologically Inspired Engineering in Boston. “We’ve uncovered so much about how nature works, builds, controls, and manufactures from the nanoscale up. Now we’re starting to leverage those biological principles.”

Sticking points

Looking to nature is not a new concept, and bioinspiration is just one of several approaches bioengineers employ to devise new medical treatments and devices. But in the last few years, the approach has come to the fore with several promising new products, even if most of them remain a few years away from human trials. “Almost every research institute now has a center for biomimicry or biologically inspired engineering,” says Ingber. “It’s just reaching that tipping point where it’s going to begin to have an impact.”

TISSUE GLUE: The sandcastle worm (Phragmatopoma californica) builds reef-like shelters by gluing together grains of sand with two separate secretions: one containing negatively charged polyphosphate proteins and the other positively charged polyamine proteins. Researchers mimicked this idea with synthetic polyelectrolytes to create an injectible fluid that can patch fetal membrane ruptures in an in vitro model.
See full infographic: JPG SANDCASTLE WORM: PHEBE LI FOR THE SCIENTIST. DIAGRAM: KIMBERLY BATTISTA

Medical adhesion is one area where bioinspiration promises to make an impression. Stitches and staples are still the standard for suturing wounds and closing up surgical incisions, but these technologies can damage tissue, leave gaps for bacteria to infiltrate, and increase the risk of inflammation. For years, surgeons have been in need of new medical adhesives that can bond tissue strongly inside the body without provoking inflammation.

Heeding the call, bioengineers have again turned to the sea. Phillip Messersmith of the University of California, Berkeley, for example, is focused on the protein-filled secretions marine mussels use to fasten themselves to wave-battered rocks. The proteins in these liquid secretions are rich in an amino acid called dihydroxyphenylalanine (DOPA), which features reactive catechol chains. These catechol chains bond tightly with each other in a mussel’s own secretions but also bond with metal atoms present on the surface of rocks. Using this strategy as a blueprint, Messersmith and colleagues chemically synthesized a variant of DOPA to crosslink biocompatible polymers.

Their glue has successfully fastened transplanted insulin-producing islet cells to the outer surface of the liver and nearby tissues in mice.2 The technique could potentially provide an alternative to standard methods of islet transplantation in which islets are infused into the liver vasculature, where they trigger an inflammatory response that quickly kills off about half of the transplanted cells—and impairs the surviving cells’ ability to produce therapeutic insulin. The researchers are also testing the bioinspired adhesive’s ability to repair ruptured fetal membranes, which can lead to premature birth and other serious complications. (See “Mimicking Mussels,” The Scientist, April 2013.)

 

Cancer Invasion and Metastasis: Molecular and Cellular Perspective

Tracey A. Martin, Lin Ye, Andrew J. Sanders, Jane Lane, and Wen G. Jiang*.

* Metastasis and Angiogenesis Research Group, Institute of Cancer and Genetics, Cardiff University School of Medicine, Department of Surgery, University Hospital of Wales, Cardiff, UK.

Metastatic Cancer: Clinical and Biological Perspectives edited by Rahul Jandial.

Read this chapter in the Madame Curie Bioscience Database here.

Metastasis is the leading reason for the resultant mortality of patients with cancer. The past few decades have witnessed remarkable progress in understanding the molecular and cellular basis of this lethal process in cancer. The current article summarizes some of the key progress in this area and discusses the role of cell junctions, cell adhesions, epithelial-mesenchymal transition, angio and lymphangiogenesis and organ specific metastasis.

Of primary importance in the prognosis of cancer patients is the sequence of events leading to the development of tumor cell invasion and metastasis. The course of tumor metastasis entails a series of stages that lead to the formation of secondary tumors in distant organs and is, largely, responsible for the mortality and morbidity of cancer.

Once tumor cells acquire the ability to penetrate the surrounding tissues, the process of invasion is instigated as these motile cells pass through the basement membrane and extracellular matrix, progressing to intravasation as they penetrate the lymphatic or vascular circulation. The metastatic cells then journey through the circulatory system invading the vascular basement membrane and extracellular matrix in the process of extravasation. Ultimately, these cells will attach at a new location and proliferate to produce the secondary tumor. Concentrating research efforts on identifying and understanding the mechanisms concerned in tumor cell invasion may lead to limiting tumor progression and, as a result, to a reduction in mortality for many cancer patients. In the following, we have summarized some of the recent progress in the area of cell adhesion, epithelial to mesenchymal transition, angiogenesis, lymphangiogenesis and organ specific metastasis in cancer.

Go to:

Cancer Invasion and Metastasis: The Role of Cell Adhesion Molecules

Cancer metastasis is the spread of cancer cells to tissues and organs beyond where the tumor originated and the formation of new tumors (secondary and tertiary foci) is the single event that results in the death of most patients with cancer. At the time of cancer diagnosis, at least half of the patients already present clinically detectable metastatic disease.1 A higher number of patients will also have micrometastases that would be beyond conventional detection techniques. Thus, metastasis is the most life threatening event in patients with cancer. The process is composed of a number of sequential events which must be completed in order for the tumor cell to successfully metastasize, the so called metastatic cascade. This process contributes to the complexity of cancer as a multiplex disease. During the metastatic cascade, changes in cell-cell and cell-matrix adhesion are of paramount importance.2

The metastatic cascade can be broadly separated into three main processes: invasion, intravasation and extravasation. The loss of cell-cell adhesion capacity allows malignant tumor cells to dissociate from the primary tumor mass and changes in cell-matrix interaction enable the cells to invade the surrounding stroma; the process of invasion. This involves the secretion of substances to degrade the basement membrane and extracellular matrix and also the expression/ suppression of proteins involved in the control of motility and migration. The tumor must also initialize angiogenesis, without which the tumor would fail to develop, as local diffusion for transport of nutrients to and removal of waste products from the tumor site would suffice for tumors up to 2 mm in diameter.3 The blood vessel within the tumor’s vicinity can then provide a route for the detached cells to enter the circulatory system and metastasize to distant sites; the process of intravasation.4,5 Interaction between the tumor cell and the surrounding stroma is extremely important in the development of tumor angiogenesis.6 Once the tumor cell has arrived at a likely point of intravasation, it interacts with the endothelial cells by undergoing biochemical interactions (mediated by carbohydratecarbohydrate locking reactions, which occur weakly but quickly) develops adhesion to the endothelial cells to form stronger bonds, and thus penetrates the endothelium and the basement membrane; the process of extravasation. The new tumor can then proliferate at this secondary focus.

The metastatic cascade is therefore dependent on the loss of adhesion between cells, which results in the dissociation of the cell from the primary tumor, and subsequently the ability of the cell to attain a motile phenotype via changes in cell to matrix interaction.

Cellular Junctions

Epithelial cells are characterized by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. The cell to cell adhesion complex runs from the apical to the basal membranes and is composed of Tight Junctions (TJ), Adherens Junctions (AJ), Gap Junctions (GJ), Desmosomes and integrins (Fig. 1).

Figure 1.

Schematics showing the arrangement of cell-cell junctions and cell-matrix interactions.

Tight Junctions (TJ)

The permeability of epithelial and endothelial cells is governed by the TJ and they are located at the apical membrane of the cell,79 (Fig. 1). The TJ is a region where the plasma membrane of adjacent cells forms a series of contacts that appear to completely occlude the extracellular space thus creating an intercellular barrier and intramembrane diffusion fence.10 In epithelial cells the TJ functions in an adhesive manner and can prevent cell dissociation.11 TJ in endothelial cells function as a barrier through which molecules and inflammatory cells can pass. Interaction with and penetration of the vascular endothelium by dissociated cancer cells is an important step in the formation of cancer metastases. TJ are the first barrier that cancer cells must overcome in order to metastasize. We have previously demonstrated that TJ of vascular endothelium in vivo function as a barrier between blood and tissues against metastatic cancer cells.12 Early studies demonstrated a correlation between the reduction of TJ and tumor differentiation and experimental evidence has emerged to place TJ in the frontline as the structure that cancer cells must overcome in order to metastasize.1215Although a considerable body of work exists on TJ and their role in a number of diseases, following the early work of Martinez-Paloma16 and others,17,18 it is only in recent years that there has been an upsurge in studies investigating their possible role in tumorigenesis and metastasis.

There have now been numerous studies on colorectal cancer,1921 pancreatic cancers2224 and an increasing number of studies performed on breast cancer.2527 Changes in both tumor and endothelial cells are necessary for successful growth and spread of cancer cells and these changes are somewhat similar. A change in cancer cells by upregulation or downregulation of relevant TJ proteins results in loss of cellcell association, cell contact inhibition, leading to uncontrolled growth, loss of adhesion to and degradation of the basement. These must be a concurrent loss of cellcell association in the endothelium and modulation of TJ proteins involved in facilitating the passage of the cancer cells through this barrier.

HGF/SF (hepatocyte growth factor), a cytokine secreted by stromal cells and key to the development and progression of cancer, particularly during metastasis has been shown to be capable of modulating expression and function of TJ molecules in human breast cancer cell lines.28 HGF decreased trans-epithelial resistance and increased paracellular permeability of human breast cancer cell lines, MDA-MB-231 and MCF-7. Q-PCR showed that HGF modulated the levels of several TJ molecule (occludin, claudin-1 and -5, JAM-1 and -2) mRNA transcripts in MDA-MB-231 and MCF-7 cells. Such data shows that HGF disrupts TJ function in human breast cancer cells by effecting changes in the expression of TJ molecules at both the mRNA and protein levels and that regulation of TJ could be of fundamental importance in the prevention of metastasis of breast cancer cells. Regulation of vascular permeability is one of the most important functions of endothelial cells, and endothelial cells from different organ sites show different degrees of permeability.29 Tumor blood vessels are more permeable on macro-molecular diffusion than normal tissue vessels. However, the cause and mechanism of hyperpermeability of human vessels had not been clear. Tumor cells release a number of factors that can assist their transmigration through the endothelium after treating endothelial cells with conditioned media from a highly invasive and metastatic melanoma cell line,29 with TJ being irreversibly damaged (as assessed using TER-trans-epithelial resistance). In fact, HGF has been shown to decrease TER and increase PCP (paracellular permeability) in human endothelial cells.8

An increasing number of studies have shown that numerous TJ components are directly or indirectly involved in cancer progression including ZO-1, ZO-2, claudin-7, claudin-1 and occludin.25 When human tissues and breast cancer cell lines were amplified for functional regions of occludin, tumor tissues showed truncated and/or variant signals. There was also considerable variation in the expression of occludin in the 10 human breast cancer cell lines investigated. Western blotting demonstrated that variants in the MDA-MB-231 and MCF-7 human breast cancer cell lines did not fit the expected occludin signals for changes in phosphorylation status. Immunostaining showed similarly disparate levels of expression. Ribozyme knockdown resulted in increased invasion, reduced adhesion and significantly reduced TJ functions. Q-RT-PCR analysis of 124 tumor and 33 background human breast tissues showed occludin to be significantly decreased in patients with metastatic disease. Immunohistochemical staining showed a decreased expression of occludin in the tumor sections. This study demonstrated for the first time that occludin is differentially expressed in human breast tumor tissues and cell lines. This loss of or aberrant expression has clear repercussions as to the importance of occludin in maintaining TJ integrity in breast tissues,25 (Fig. 2). Highly differentiated adenocarcinomas with well developed TJ provide an important insight into the usefulness of TJ molecules and are possible prognostic indicators and future targets for therapy. In breast cancer, ZO-1 has been demonstrated to be decreased in poorly differentiated tumors and correlated with increasing Grade and TNM (tumor-nodal) status.30 There are a respectable number of reports describing the dysregulation of transmembrane proteins in human cancers and in cell lines. This dysregulation can be the result of both upregulation and downregulation of expression, epigenetic changes and changes in activation and location of the proteins.

Adherens Junctions (AJ)

AJ are cellcell microdomains that provide adherent strength and localize to the basal side of the TJ31 (Fig. 1). The integral membrane proteins of the AJ are of the cadherin family, with E-cadherin being most abundant in epithelia and VE-cadherin in endothelia (Fig. 1). Nectins are also found in AJ of epithelia. In polarized epithelia of vertebrates, the AJ is part of the tripartite junctional complex localized at the juxtaluminal region, which comprises the TJ, AJ, and desmosome aligned in this order from the apical end of the junction.32 In this type of epithelia, the AJ is specifically termed the zonula adherens or adhesion belt, as it completely encloses the cells along with the F-actin lining, called the circumferential actin belt.33 The AJs in other cell types assume different morphologies with the AJ in fibroblastic cells being spotty and discontinuous34 while those in neurons are organized into tiny puncta as a constituent of the synaptic junctions.35 A major function of AJs is to maintain the physical association between cells, as disruption of them causes loosening of cellcell contacts, leading to disorganization of tissue architecture.33

Classical or type I cadherins mediate adhesion at the adherens, cellcell or cellmatrix adhesive junctions that are linked to microfilaments. Type I classical cadherins are composed of five tandem extracellular cadherin domains (EC1-EC5), a single segment transmembrane domain and a distinct, highly conserved cytoplasmic tail that specifically binds catenins.36 In addition to cadherin homophilic binding, it has been reported that cadherin is also capable of heterophilic interactions with numerous extracellular and intracellular proteins. The key to their adhesive activity is the interaction between the catenin-binding sequence and submembrane plaque proteins β-catenin or plakoglobin (γ-catenin), which form the link to the actin cytoskeleton. α-catenin binds to a short region close to the N terminus of β-catenin forming a stable bond between the complex and the actin cytoskeleton.36 In addition to α-, β-, and γ-catenin, a fourth catenin-like protein capable of binding cadherin, p120ctn, has emerged as a key regulator of cadherin function.37 p120ctn was originally identified as a substrate for receptor tyrosine kinases and like the other catenin molecules, binds directly to the cytoplasmic domain of cadherin.37

Nectins are transmembrane proteins that are found in both TJ and AJ. In AJ, during the process of early cellcell contacts, nectins first accumulate at the contacts, and then cadherins follow them, suggesting that the former may guide the latter in their junctional localization. Nectin interaction serves for recruiting cadherins to heterotypic cellcell borders, which are otherwise distributed throughout cellcell borders.33 Thus, nectins recruit cadherins to the synaptic contacts formed between two distinct domains of hippocampal neurons, i.e., axons and dendrites, which express nectin-1 and nectin-3, respectively.38 Thus, nectins show important cooperation with classic cadherins in generating heterotypic cellcell contacts.33

Evidence has long accumulated to point toward a pivitol role for E-cadherin and the catenin complex in the control of cancer cell dissociation and spread. Tumor invasion and metastasis, both hallmarks of tumor malignancy, frequently coincide with the loss of E-cadherin-mediated cell-cell adhesion. Expression of E-cadherin, the most abundant adhesion molecule in adherens junctions of epithelia, is downregulated in most, if not all, epithelial cancers.39 Several studies have shown that reconstitution of a functional E-cadherin adhesion complex suppresses the invasive phenotype of many different tumor cell types.4042 In the context of cancer, E-cadherin has been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial-mesenchymal transition (EMT) in epithelial tumor progression.

Recent studies in triple-negative breast cancer (TNBC), which is characterized by negativity for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2), have shown there is a high risk breast cancer that lacks specific targets for treatment selection. Chemotherapy is, therefore, the primary systemic modality used in the treatment of this disease, but reliable parameters to predict the chemosensitivity of TNBC have not been clinically available.43 Patients with E-cadherin-negative and Ki67-positive expression showed significantly worse overall survival time than those with either E-cadherin-positive or Ki67-negative expression. Multivariate analysis showed that the combination of E-cadherin-negative and Ki67-positive expression was strongly predictive of poor overall survival in TNBC patients receiving adjuvant chemotherapy. The authors demonstrated that adjuvant therapy is beneficial for Stage II TNBC patients and that the combination of E-cadherin and Ki67 status might be a useful prognostic marker indicating the need for adjuvant chemotherapy in Stage II TNBC patients.43

E-cadherin inactivation with loss of cell adhesion is the hallmark of lesions of the lobular phenotype and E-cadherin is typically absent, as seen by immunohistochemistry in both lobular carcinoma in situ and invasive lobular lesions, suggesting it occurs early in the neoplastic process. In invasive lobular lesions, the cadherin-catenin complex was examined; complete complex dissociation was defined as negative membranous E-cadherin, α- and β-catenin expression.44 E-cadherin was found to be absent in all lesions and positive in all normal tissues. Membranous a and β-catenin expressions decreased with the transition from lobular lesions to invasive lesions, while TWIST expression increased. Gene expression paralleled IHC-staining patterns with a stepwise downregulation of E-cadherin, α and β-catenins from normal to lobular to invasive lesions, and increasing expression of TWIST from normal to lobular to invasive lesions. The decreasing membranous catenin expression in tandem with increasing levels of TWIST across the spectrum of lobular lesions suggests that cadherin-catenin complex dissociation is a progressive process in human breast cancer.44

Desmosomes

In cell-cell junctions, desmosomes form adherent points in the form of a continuum of cells within tissues by linkage of their integral membrane proteins (desmocollin and desmoglein) via desmoplakins (plakophilin and plakoglobin) to intermediate filaments31,45 (Fig. 1). Desmosomes are crucial for tissue integrity by their very strong adherence that resists calcium-depletion in developed tissue, but can be regulated by protein kinase C when dynamic remodelling of cellcell adhesion is required.45 Desmosomes not only provide mechanical stability but also facilitate cellcell communication through signal transmission.46 The desmosome is divided into three parallel identifiable zones, arranged symmetrically on the cytoplasmic faces of the plasma membranes of bordering cells and separated by the extracellular domain, which in mature desmosomes is bisected by a dense midline. Each desmosomal plaque consists of a thick outer dense plaque and a translucent inner dense plaque. The five major desmosomal components are the desmosomal cadherins, represented by desmogleins (14) and desmocollins (13), the armadillo family members, plakoglobin and the plakophilins (13), and the plakin linker protein desmoplakin, which anchors the intermediate keratin filaments.46

Recent studies using mouse genetic approaches have uncovered a role for desmosomes in tumor suppression, demonstrating that desmosome downregulation occurs before that of adherens junctions to drive tumor development and early invasion, suggesting a two-step model of adhesion dysfunction in cancer progression.47 Studies have shown that an increased expression of desmosome proteins, such as Desmoglein 2 and 3 and PKP3, can be observed in certain cancers of the skin, head and neck, prostate and lung compared with normal tissue, and that this overexpression is associated with enhanced tumor progression.46,4850

Reduced expression of Desmocollin 2 has been reported in colorectal carcinomas, suggesting that it may play a role in the development and/or progression of colorectal cancer. Kolegraff et al.51 reported that the loss of Desmocollin-2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin-dependent transcription and proliferation following Desmocollin-2 knockdown and attenuated the in vivo growth of Desmocollin-2 -deficient cells. This provides evidence that loss of Desmocollin-2 contributes to the growth of colorectal cancer cells and highlights a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling.51

Oral squamous cell carcinomas and pre-malignant dysplasia can be suβ-classified according to their in vitro replicative lifespan, where the immortal dysplasia and carcinoma subsets have p16(ink4a) and p53 dysfunction, telomerase deregulation and genetic instability and the mortal subset do not. It has been demonstrated that desmosomal proteins exhibit a distinct expression pattern in oral mucosa when compared with epidermis in vivo. Microarray data from a large panel of lines shows that the transcript levels of Desmoglein 2 and Desmocollin2/3 are reduced in immortal dysplasia and carcinoma cells.52 Interestingly, Desmoglein 2 was upregulated. Reduction of Desmoglein 3 and upregulation of Desmoglein 2 were found in two independent microarray data sets. Significantly, we demonstrated that reduction of Desmoglein 3 and upregulation of Desmoglein 2 was reversible in vitro by using RNAi-mediated knockdown of Desmoglein 2 in carcinoma cells. The remaining desmosomal proteins were largely disrupted or internalized and associated with retraction of keratin intermediate filaments in oral squamous cell carcinomas lines. These findings suggest dysfunction and loss of desmosomal components are common events in the immortal class of oral squamous cell carcinomas and that these events may precede overt malignancy.52

There are numerous links between the desmosome and the adherens junction. A decrease in the levels of the desmosomal plaque protein, plakophilin3, leads to a decrease in desmosome size and cell-cell adhesion. Gosavi et al.53investigated whether plakophilin3 is required for desmosome formation. Plakophilin3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared with vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that plakophilin3, plakoglobin and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either plakoglobin or E-cadherin led to a decrease in the levels of plakophilin3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that plakoglobin and E-cadherin recruit plakophilin 3 to the cell border to initiate desmosome formation.53

Gap Junctions (GJ)

GJ are unique cell-to-cell channels that allow diffusion of small metabolites, second messengers, ions and other molecules between neighboring cells31 (Fig. 1). GJ communication is essential for electrical transduction, signaling and nutrition. The channels can be open or closed, a highly dynamic process regulated at multiple levels, with the integral membrane proteins forming these channels in vertebrates being the connexins of which over 20 family members have now been identified in humans; connexin43 the most abundantly expressed connexin.31 ZO-1 acts as a scaffold in GJ and recruits signaling proteins. Connexins are also known to interact with Occludin and also form complexes with CAR and β-catenin.54

For decades, cancer was associated with GJ defects. However, more recently it appeared that connexins can be re-expressed and participate in cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer are known to be connexin deficient, Lamiche et al.55 investigated whether their bone-targeted metastatic behavior could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates in the differentiation of osteoblastic cells. It appeared that Cx43 behaved differently in those cell lines and induced different phenotypes. In LNCaP, connexin43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those connexin43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of connexin43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of connexin43 may govern the ability of prostate cancer cells to metastasize in bones.55

In colorectal tumors, loss of connexin43 expression is correlated with significantly shorter relapse-free and overall survival. Connexin43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis and was found to colocalize with β-catenin and reduce Wnt signaling.56 This study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. Connexin43 was downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. Such data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.56

Integrins and Selectins

There is accumulating evidence for the role of integrins and selectins in cancer progression of various cancer types, including colon and lung carcinomas and melanomas.57 While selectin-mediated tumor cells arrest and adhesion contribute to metastasis, integrin-mediated interaction from both tumor cells and the surrounding environment further contribute to cancer progression.

Integrins

Integrins are large and complex transmembrane glycoproteins that consist of two distinct chains, α and β-subunits, which form a non-covalent heterodimer and combine to form 24 unique canonical α/β receptors.57 Integrins mediate cell adhesion and directly bind components of the extracellular matrix, such as fibronectin, vitronectin, laminin, or collagen and provide anchorage for cell motility and invasion. Integrins mediate bidirectional signaling where intracellular signals induce alterations in the conformation.57 Integrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. As many human tumors originate from epithelial cells, integrins expressed on epithelial cells are generally also present in tumor cells and therefore, integrins have become linked with patient survival and metastatic status. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Van den Hoogen et al.58 examined the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/ progenitor phenotype in human prostate cancer. Stable knockdown of αv integrin expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/ progenitor cell characteristics (α2 integrin, CD44, and ALDH(hi)) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. This indicates that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity.58,59

Lu et al.59 investigated the expression of osteopontin and integrin αv (ITGAV, main receptor of the osteopontin) in laryngeal and hypopharyngeal squamous cell carcinoma and any correlation of the expression quantity with tumor biological behavior. The expression quantity of osteopontin and integrin αv in primary and metastatic carcinomas is significantly higher than in normal tissues. The expression of osteopontin and integrin αv in the well-differentiated group was significantly lower than in moderately and poorly differentiated groups; the expression quantity of osteopontin and integrin αv in groups with lymph node metastasis was significantly higher than in groups without lymph node metastasis. The authors conclude that the expression of osteopontin and integrin αv significantly influenced the differentiation and metastasis of the laryngeal and hypopharyngeal squamous cell carcinoma. Overexpression of both proteins may have contributed to invasion and metastasis of the laryngeal and hypopharyngeal squamous cell carcinoma, and therefore, they both may have value as a target for chemotherapy in laryngeal and hypopharyngeal squamous cell carcinoma treatment.59

Selectins

The selectins: E-selectin, P-selectin, and L-selectin are adhesion molecules that are crucial for binding of circulating leukocytes to vascular endothelium during the inflammatory response to injury or infection. Accumulated evidence indicates that selectins regulate adhesion of circulating cancer cells to the walls of blood vessels.60 Selectin ligands are transmembrane glycoproteins expressed on leukocytes and cancer cells that promote bond formations with selectins to mediate inflammatory processes and selectins and their ligands also participate in signal transduction to regulate diverse cellular functions.60

Haematogenous metastasis of small cell lung cancer is still a poorly understood process and represents the life threatening event in this malignancy.61 In particular, the rate-limiting step within the metastatic cascade is not yet clearly defined although, many findings indicate that extravasation of circulating tumor cells is crucially important as most tumor cells within the circulation undergo apoptosis. If extravasation of small cell lung cancer tumor cells mimics leukocyte-endothelial interactions, small cell lung cancer cells should adhere to E- and P-selectins expressed on the luminal surface of activated endothelium. The adhesion to E- and P-selectin under physiological shear stress with regard to adhesive events, rolling behavior and rolling velocity was determined in the human small cell lung cancer cell lines SW2, H69, H82, OH1 and OH3. OH1 SCLC cells adhered best to recombinant human (rh) E-selectin FC-chimeras and human lung endothelial cells (HPMEC), H82 small cell lung cancer cells adhered best to activated human umbilical vein endothelial cells (HUVEC) under physiological shear stress. As OH1 cells had also produced by far the highest number of spontaneous lung metastases when xenografted into pfp/rag2 mice in previous experiments the findings implicate that adhesion of small cell lung cancer cells to E-selectin is of paramount importance in small cell lung cancer metastasis formation.61

Cell-Matrix Interactions

Controlled interaction between the cells and the extracellular matrix is essential for many processes, including normal development, migration and proliferation.31 Interaction between the cell and the matrix can occur through a number of routes; cell adhesion molecules (CAM) including integrins, selectins, cadherins, the Ig superfamily, CD44 and focal adhesions.

Integrins

Integrin-mediated adhesions to the extracellular matrix are among the first adhesion junctions where bidirectional signaling occurs.31 At the extracellular side integrins bind directly to the extracellular matrix which includes collagen, fibronectin and laminins etc. Cytoplasmic partners include talins, paxillin, focal adhesion kinase and linkage to α-actinin and actin-stress fibers. These focal adhesion complexes control a variety of signaling pathways regulated by the interplay with the extracellular partners. Substantial cross-talk between the diverse cellcell and cellextracellular matrix junctions has been found, and the architecture of the epithelial monolayer is highly regulated by their concerted actions.31

Cell Adhesion Molecules (CAM)

Cell adhesion molecules (CAM) facilitate cellular processes such as cell proliferation, migration, and differentiation and are essential during development and for maintaining the integrity of tissue architecture in adults.62 CAMs include cadherins, integrins, selectins, and the immunoglobulin superfamily (IgSF). In normal tissue, CAM expression is tightly regulated. However, aberrant expression of CAMs disrupts normal cell-cell and cell-matrix interactions and can facilitate tumor formation and metastasis. A number of IgSF members have been identified as biomarkers for cancer progression and have also been associated with metastatic progression in a range of huma tumors.62

CD44

CD44 is a multifunctional cell surface adhesion molecule that is involved in cell-cell and cell-matrix interaction and has been implicated in tumor cell invasion and metastasis. In humans, the CD44 family is encoded by a single gene located on chromosome 11p13 and comprises at least 20 exons. Exons 15, 1618 and 20, are spliced together to form a CD44 transcript that has become known as the standard isoform (CD44s). At least ten exons can be alternatively spliced and inserted into the standard isoform at an insertion site between exons 5 and 16 to give rise to variant isoforms of CD44. Thus, exons 615 are variant exons and are typically identified as v1v10.63 CD44 is the principal ligand for hyaluronic acid (HA), a major component of the extracellular matrix. However CD44 can also bind to other ECM components including collagen, fibronectin, laminin and non-ECM component such as osteopontin and serglycin. CD44 is expressed on a variety of cells and tissues including T- lymphocytes, B-cells, monocytes, granulocytes, erythrocytes, many epithelial cell types; Keratinocytes, chondrocytes, mesothelial and some endothelial cells. It is also expressed in many cancer cell types and their metastases in particular; high molecular weight forms of CD44 show restricted expression in tumors and may correlate with tumor development and metastasis and have potential diagnostic and prognostic value in some cancers. Additionally, it has been shown in experimental models that CD44 can inhibit tumor growth and metastatic spread. Further investigation is still needed but CD44 may yet prove to be a potential target for cancer therapy.63

The importance of non-coding RNA transcripts in regulating microRNA (miRNA) functions, especially the 3′ untranslated region (UTR), has been revealed in recent years. Genes encoding the extracellular matrix normally produce large mRNA transcripts including the 3UTR. How these large transcripts affect miRNA functions and how miRNAs modulate the extracellular matrix protein expression are largely unknown. Jeyapalan and Yang64 demonstrated that the overexpression of the CD44 3UTR results in enhanced cell motility, invasion and cell adhesion in human breast carcinoma cell line MDA-MB-231. They also found that expression of the CD44 3UTR enhances metastasis in vivo. Computational analysis indicated that miRNAs that interact with the CD44 3UTR also have binding sites in other matrix encoding mRNA 3UTRs, including collagen type 1α1 (Col1α1) repressed by miR-328 and fibronectin type 1 (FN1) repressed by miR-5123p, miR-491 and miR-671. Protein analysis demonstrated that expression of CD44, Col1a1, and FN1 were synergistically upregulated in vitro and in vivo upon transfection of the CD44 3UTR. The non-coding 3UTR of CD44 interacts with multiple miRNAs that target extracellular matrix properties and thus can be used to antagonize miRNA activities.64

CD44 is also a causal factor for tumor invasion, metastasis and acquisition of resistance to apoptosis. CD44 knockdown using inducible short hairpin RNA (shRNA) significantly reduces cell growth and invasion. Short hairpin RNA against CD44 and pGFP-V-RS-vector was used for knockdown of CD44 expression in SW620 colon cancer cells. Short hairpin RNA against CD44 reduced the expression of CD44. Cell proliferation, migration and invasion were markedly inhibited and apoptosis was increased in shRNA CD44-transfected cells. Knockdown of CD44 decreased the phosphorylation of PDK1, Akt and GSK3β, and β-catenin levels. Decreased phosphorylated Akt led to an increase in phosphorylated FoxO1 and induced cell cycle arrest in the G0-G1 phase and a decrease in the S phase. The levels of Bcl-2 and Bcl-xL expression were downregulated, while the levels of BAX expression and cleaved caspase-3, -8 and -9 were increased. CD44 knockdown by way of shRNA inhibited cell proliferation and induced cell apoptosis which suggests that it could be used as a therapeutic intervention with the anti-survival/pro-apoptotic machinery in human colon cancer.65

Focal Adhesions

Focal adhesion kinase (FAK), a crucial mediator of integrin and growth factor signaling, is a novel and promising target in cancer therapy. FAK resides within focal adhesions which are contact points between extracellular matrix (ECM) and cytoskeleton, and increased expression of the kinase has been linked with cancer cell migration, proliferation and survival.66 Migration is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement.67 Signaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase interacts with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. Wang et al.67 showed that the autophosphorylation of Tyr(397) in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains. This suggests that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cortactin N-terminus associates with F-actin, while its C-terminus interacts with focal adhesions. The tyrosine phosphorylation of cortactin by the FAK-Src complex modulates its interaction with FAK and increases its turnover at focal adhesions to promote cell motility.67

Clinical Considerations

A number of cell adhesion molecules have now become classed as clinical indicators and there is a clear trend toward using them for prognosis or diagnosis. The number of studies identifying these molecules as biomarkers are legion and cannot be thoroughly reviewed here. Some timely examples are as follows: The TJ transmembrane protein claudin-7 has achieved status as a prognostic indicator in invasive ductal carcinoma of the breast68 and is a candidate expression marker for distinguishing chromophobe renal cell carcinoma from other renal tumor subtypes, including the morphologically similar oncocytoma.69 Moreover, decreased claudin-7 correlated with high tumor grade in prostate cancer70 and is able to regulate the expression of prostate specific antigen.71 When considering potential targets for therapy, claudin-1 has been found to act as a cancer invasion/metastasis suppressor in addition to its use as a prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.72 E-Cadherin and vimentin have now been described predictive markers of outcome among patients with non-small cell lung cancer treated with erlotinib.73

Go to:

Epithelial-Mesenchymal Transition

Cell Motility

A major factor shaping the metastatic character of cancer cells lies in their motility. Cell motility and migration is crucial to normal development and is a major component of organogenesis, inflammation and wound healing. However, changes in the signaling pathways directing its regulation can lead to the pathological processes of tumor cell invasion and metastasis.

The development and progression of cell motility is orchestrated by a sequence of specific biophysical, interdependent processes involving cytoskeletal modifications, changes in cell-substrate adhesive properties and alterations in the extracellular matrix. Reacting to a stimulus, a cell will commence polarization and extend protrusions in the direction of migration74 which originates with extension of the leading edge by protrusion of lamellipodia and/or filopodia, driven by actin polymerisation and filament elongation, with frequently associated membrane ruffling,75 which extends the cell body to then produce new, distal adhesion sites. Following protrusion, adhesion is instigated between the cell and substratum at the leading edge accomplished largely by integrin and non-integrin receptors binding to specific extracellular matrix protein domains.74,76 Subsequently, actomyosin-mediated contraction of the cell occurs with resultant forward motion of the cell body, initiated by contractile forces being generated at or near the leading edge, coupled with detachment of the trailing edge from the substratum. In addition, the migrating cell secretes the proteases required to break down the extracellular matrix proteins thus providing a pathway for the advancing cell.

Several molecules have been identified as having important roles to play in the signaling processes leading to cell motility/migration, with the associated loss of epithelial characteristics and gain of a migratory and mesenchymal phenotype. Thus, the acquisition of a mesenchymal-like cell phenotype provides one of the major characteristics of metastatic progression of most carcinomas.

Mechanisms of EMT

There is growing acknowledgment that the detachment and escape of cells from the primary tumor mimics the developmental process known as epithelialmesenchymal transition (EMT) (Fig. 3), a dynamic process permitting polarized epithelial cells to go through multiple biochemical and morphological changes enabling them to assume a mesenchymal phenotype with enhanced migratory and invasive capabilities.7780

Figure 3.

Schematic description of EMT/MET showing effectors of these processes; dissociation/ association of cell to cell adhesions together with characteristic markers of either epithelial or mesenchymal cells.

Initiation of the process of EMT entails the loss of cell-cell adhesions; activation of transcription factors; alterations in expression of specific cell-surface proteins; reorganization and expression of cytoskeletal proteins; and production of ECM degrading enzymes. Consequently, the course of EMT involves a shift in the characteristic morphology and gene expression pattern of epithelial cells resulting in the acquisition of a characteristic mesenchymal, migratory phenotype.81,82

EMT Progression

Epithelial cells present a highly polarized morphology, intimately linked by cell-cell junctions in the form of TJ, AJ, desmosomes and GJ. Loss of these intercellular connections provides a critical step during EMT allowing for physical detachment of cancer cells from the primary tumor. Thus, EMT is characterized by the combined loss of epithelial cell junction proteins, including E-cadherin, α-catenin, claudins, occludin and ZO-1, an increased expression of mesenchymal markers, such as N-cadherin, vimentin and fibronectin, as well as reorganization of the cytoskeleton, which collectively results in the loss of apical-basal cell polarity and the attainment of a spindle-shaped morphology.77,83

Loss of expression of the cellcell adhesion molecule E-cadherin is a characteristic trait of EMT in development and in the progression of epithelial tumors to invasive, metastatic cancers. The loss of E-cadherin is generally seen to coincide with a gain of expression of the mesenchymal cadherin, N-cadherin in many cancer types; this ‘cadherin switch’ is thought to be necessary for tumor cells to gain invasive properties and is also a characteristic of EMT.39

It is evident from recent studies that EMT-inducing signals are, in part, initiated by growth factors, including hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor β (TGFβ). These induce downstream activation of a number of EMT-inducing transcription factors including Snail, Slug, Twist and zinc finger E-box binding homeobox 1 (ZEB1).81,8486

EMT Biomarkers

A number of biomarkers have been found to be useful indicators for EMT (Table 1.).

Table 1.

Biomarkers of EMT .

E-Cadherin

It is essential that weakening of cell-cell adhesion occurs to allow cells to become motile and metastasise and a modification in the adhesive properties of cells is a necessary element of the metastatic process. Cell adhesion molecules (CAMs) regulate cell-cell and cell-matrix adhesion and are implicated in almost all stages of metastasis, therefore alterations in normal levels of CAMs such as E-cadherin will be significant in tumor progression. E-cadherin is a member of a family of Ca2+ dependent CAMs made up of intracellular, extracellular and transmembrane domains. These domains play vital roles in cellular recognition during morphogenesis and development and are responsible for cell-cell adhesion87 thus holding a central role in the maintenance of tissue integrity. E-cadherin and its adhesion complex play an essential function in the adhesion of breast cancer cells, being involved in the control of tumor progression and metastasis. Members of the complex, such as β-catenin, act as regulators of cell adhesion, and also of cell signaling and transcription regulation.88 Studies exploring the expression of E-cadherin and α-catenin in tumor tissues have shown that loss of both molecules is linked to an increased invasiveness of tumor cells.89 Evidence for this comes from in vitro and in vivo studies which demonstrate that E-cadherin expression is inversely correlated with the motile and invasive behavior of tumor cells and also with metastasis in cancer patients.90 Further studies have revealed that the relocalization of β-catenin to the nucleus correlates with the acquisition of the mesenchymal phenotype,91,92 and is associated with the loss of E-cadherin. This reduction of cell surface E-cadherin causes the cells to be receptive to initiation of EMT.93 Numerous reports have indicated that E-cadherin plays a role in meningiomas, tumors of the central nervous system; with upregulation and nuclear localization of β-catenin in 60% of anaplastic memingiomas.94

Transcription Factors in EMT

Important transcription factors shown to be significant in EMT, as they affect the regulation of E-cadherin expression, are Slug and Snail (SNAI1),95 Zeb-185 and Twist.96,97 Importantly, Snail has been identified as having a significant role in the differentiation of epithelial cells into mesenchymal cells during embryonic development98,99 with Slug and Snail effecting the downregulation of E-cadherin expression by binding directly to two proximal E2-boxes of the E-cadherin promoter.84,100 It has been shown that Snail and E-cadherin expression are inversely correlated in squamous cell carcinoma101 and cancer of the breast.102 Snail also represses expression of genes encoding tight junction components, such as claudins and occludins.103

The basic helix-loop-helix protein Twist is also a key transcription factor in EMT and is known to trigger EMT mechanisms possibly by the regulation of the E-cadherin to N-cadherin switch. It is not known if E-cadherin expression can be repressed directly by Twist however, forced N-cadherin expression exerts a dominant effect over E-cadherin in breast cancer cells.104,105 Similarly, expression of N-cadherin in normal epithelial cells results in downregulation of E-cadherin expression.104 Work on glioblastoma (GBM) by Mikheeva et al.106 has shown that TWIST1 promotes GBM invasion through instigation of mesenchymal molecular and cellular changes. This study showed, however, that this effect was not reliant on a cadherin switch as a reduction in levels of E-cadherin and consequent increase in N-cadherin did not occur with TWIST1 overexpression.

Nevertheless many of the genes regulated by TWIST1 in GBM cell lines mirror those which it regulates in cancer metastasis which suggests some overlap with that of TWIST1-mediated EMT in carcinomas.106 In work on medulloblastoma, evidence for a significant role for EMT has been seen with intermittent hypoxic conditions in the tumor microenvironment.107 Hypoxia is recognized as a factor involved in overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) with overexpression promoting uPAR-mediated survival signaling in various cancers.108 Likewise, hypoxia/overexpression of uPAR in cancer cells promotes EMT and thus invasiveness and metastasis. The study by Gupta also showed that when medulloblastoma cells are exposed to intermittent hypoxia this initiates various molecular and phenotypic changes consistent with EMT, as the cell signaling molecules vimentin, N-cadherin, Snail are overexpressed in these medulloblastoma cells with a reduction in the epithelial markers ZO-1 and E-cadherin.

EMT-Related Factors

Bone Morphogenetic Protein (BMP7)

Numerous signaling pathways have been implicated in the initiation of EMT, in particular, TGF-β1 has been identified as a potent initiator of EMT in renal tubular epithelial cells,109 and also in cancer cells, stimulating cell invasion and metastasis.110 However, it has been reported that a member of the TGF-β superfamily, bone morphogenetic protein 7 (BMP-7) reverses TGF-β induced EMT by induction of E-cadherin.111 Indeed, BMP-7 has been shown to regulate epithelial homeostasis in the human mammary gland by preserving the epithelial phenotype.79 Similarly, a decrease in BMP-7 expression in human breast cancer leads to the acquisition of a bone metastatic phenotype,79 with loss of BMP-7 being associated with a more invasive and motile mesenchymal phenotype, in PC-3 prostate cancer cells.112Furthermore, systemic administration of recombinant BMP-7 to mice with severe renal fibrosis has resulted in reversal of EMT with repair of damaged epithelial structures111 as BMP-7 acts to reverse TGF-β1 induced EMT by upregulating E-cadherin in renal cells. Linked with this, BMP member growth and differentiation factor 9 (GDF-9) has been shown to promote the invasiveness of PC-3 cells together with an induction in the expression of genes including SNAI1, RhoC, ROCK-1 and N-cadherin, while reducing levels of E-cadherin. Thus in PC-3 cells, GDF-9 signaling via ALK-5, promotes cell invasiveness via a complex signaling network working collectively to trigger EMT, thus aiding in the aggressiveness and progression of prostate cancer cells.113

Matrix Metalloproteinases (MMPs)

The matrix metalloproteinases (MMPs) are an important component of cell invasion capable of degrading a range of extracellular matrix proteins allowing cancer cells to migrate and invade. In epithelial ovarian cancer TGFβ and EGF act as inducers of MMP2 production and enhance cell motility,114 while in breast cancer there is an upregulation of MMP9.115

In oral squamous cell carcinoma Snail and Slug are seen to act as regulators of TGFβ triggered EMT, with Snail upregulating MMP2 and MMP9 initiating EMT; while Slug and Snail maintain longer term EMT by stimulating MMP9 expression.116 The MMPs not only function in membrane/ matrix degradation but are also involved in cell adhesion. Treatment of MCF-7 cells with MMP7 results in E-cadherin cleavage producing an 80kDa fraction which is detectable in the serum and urine of cancer patients and has been proposed as a biomarker.117 Similarly, MMP9 appears to cleave the TJ molecule Occludin (personal communication).

Epithelial Protein Lost in Neoplasm (EPLIN)

The cytoskeletal protein EPLIN has been identified as a key molecule linking the cadherin-catenin complex to F-actin and stabilizing the Zona Adherens in MDCK and DLD-1 cells.118 It is an actin cross linking protein that bundles actin in the cells and stabilizes the cytoskeletal filaments. By doing so, EPLIN protein inhibits cell motility, and has been found to be downregulated in a number of oral, breast and prostate cancer cell lines. Forced expression of EPLIN in the EPLIN-α negative breast cancer cell line, MDA MB-231 has been shown to reduce migration and invasion in these cells so reducing their aggressiveness.119 Similarly, overexpression of EPLIN in the PC-3 cell line results in a reduction in both in vivo and in vitro growth potential together with a reduction in cell invasiveness and ability to adhere to extracellular matrix.120

Thus, EPLIN could be seen to be acting as a tumor suppressor. Recently, biochemical and functional evidence has exposed EPLIN as a negative regulator of EMT and invasiveness in prostate cancer cells. Evidence has emerged to show that a downregulation of EPLIN significantly disrupts epithelial structures, initiates actin cytoskeleton remodelling via the EPLIN link between actin filaments and β-catenin, affects explicit gene expression profiles and triggers a pro-EMT program.121

A great deal of energy has been focused, over the last four decades, on the elucidation of the molecular mechanisms governing EMT/MET since the concepts were first defined by Hay (1968).122 Evidence has emerged that the process of EMT can be classified into three different subtypes; type 1 associated with implantation, embryo formation, and organ development; type 2 EMT associated with wound healing, tissue regeneration, and organ fibrosis and type 3 EMT which arises in neoplastic cells in relation to tumor growth and cancer progression, occurring in cells that have gone through epigenetic changes in genes that support the instigation of localized tumors. Many investigators have found that applying the principles of carcinoma EMT to their studies has aided in the understanding of tumor cell invasion in various cancer types and pinpointed many of the genes specifically associated with EMT in relation to tumor growth and metastasis. Continued studies will hopefully provide significantly more information concerning the molecular mechanisms that drive EMT, in relation to the effects of EMT on the progression of carcinomas and will possibly offer new approaches and targets to prevent the most fatal characteristic of tumorigenesis-metastasis.

Go to:

Angiogenesis and Lymphangiogenesis in Cancer Metastasis

Introduction to Angiogenesis and Lymphangiogenesis

The growth of new blood or lymphatic vessels from pre-existing vessels (the process of angiogenesis or lymphangiogenesis) is essential in physiological events such as reproduction, development, wound-healing and immunity. However, imbalance or manipulation of these essential processes is seen in a number of disease states and these processes are frequently involved in cancer progression and metastasis.123,124

Angiogenic and Lymphangiogenic Cascade

The angiogenic process is made up of a complex multi-step cascade, which is tightly regulated through the balance of a number of pro- and anti- angiogenic factors. Tumor cells frequently tip this balance in favor of blood vessel production through the secretion of pro-angiogenic factors as summarized in Fig. 4. The production of angiogenic factors from a source tissue or tumor bind to and activate endothelial cells of a neighboring blood vessel. Following activation, the endothelial cells begin to produce enzymes that break down the basement membrane of the blood vessel creating tiny pores. Endothelial cells then proliferate and migrate through these pores, toward the angiogenic source, a mechanism that involves a variety of adhesion molecules to aid movement of the new blood vessel toward the source and also the production of various enzymes, such as matrix metalloproteinases, at the sprouting tip, to facilitate this movement through the extrα-cellular matrix. Endothelial cells of the new vessel then undergo a tubule formation phase, where these cells roll to form a tube like structure before establishment of a blood vessel loop between the source and the existing vessel. Finally, structural stabilization of this loop is obtained through recruitment of additional cell types, such as smooth muscle cells, providing support to the vessel and allowing blood flow to the angiogenic source.125

Figure 4.

Summary of key steps involved in the angiogenic cascade

While the vasculature system and lymphatic system are structurally different, the process of lymphangiogenesis shares similarities with the angiogenesis process. New lymphatic vessel growth can be stimulated by a variety of factors such as members of the vascular endothelial growth factor (VEGF) family (e.g., VEGF-C and VEGF-D), which induce sprouting of new vessels and proliferation of lymphatic endothelial cells (LEC),126,127 a process which, similar to angiogenesis, is utilized by metastasising tumor cells. Key angiogenic and lymphangiogenic factors are summaried inTable 2.

Table 2.

Key angiogenic and lymphangiogenic factors .

Therapeutic Potential of Angiogenesis and Lymphangiogenesis in Targeting Cancer Metastasis

While lymphangiogenesis and angiogenesis are essential in numerous physiological processes they are also commonly involved in disease states, in particular the progression of cancer and metastasis.

Angiogenesis and Anti-Angiogenesis Strategies in Cancer

The importance of angiogenesis in advanced tumor development has been known for many years. Without their own vasculature, tumors are unable to grow beyond a size of approximately 23 mm and are limited by their reliance on simple diffusion to obtain required resources.3,128 To overcome this, cancer cells often secrete certain factors to encourage new blood vessel growth to the tumor (tumor angiogenesis). These new blood vessels provide the required resources for advanced and rapid development of the tumor and also provide direct links with the vascular system to the tumor, facilitating metastatic invasion into this system and dissemination around the body.

There are a number of factors that have been demonstrated to enhance angiogenesis such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and, given the importance of tumor angiogenesis in facilitating advanced tumor growth and metastatic spread, research into effective targeting of tumor angiogenesis has been a key area of interest in the scientific community, employing various strategies to disrupt or block new blood vessel growth to the developing tumor.

VEGF is perhaps one of the best known and established angiogenesis regulators to date and given its major role in angiogenesis, it has been subjected to vast scientific study. The VEGF family itself consists of several members, which signal through a number of VEGF receptors, however, the main angiogenesis regulator in normal physiology and cancer appears to be VEGF (also known as VEGF-A) and the VEGF receptor-2 (VEGFR-2 or FLK).129,130 Early research established the importance of VEGF in regulating endothelial proliferation and survival and its ability to promote angiogenesis using in vitro models.131 Given its vital role in tumor angiogenesis, specific targeting of VEGF signaling has been one of the key avenues in developing anti-angiogenic therapies. One such strategy has employed the development and use of a VEGF neutralising antibody termed Bevacizumab (also known as Avastin). This therapy has been approved for use in a variety of cancer types, such as non-squamous non-small-cell lung cancer and colorectal cancer.130 Scientific research into the benefits of Bevacizumab is ongoing, with studies examining and demonstrating the potential of Bevacizumab in additional cancer types such as epithelial ovarian cancer, where previous trials have yielded promising results.132

HGF represents another potential target for the treatment of cancer progression and angiogenesis. The role of HGF in contributing to cancer progression has been well demonstrated within the literature. This is largely due to the ability of HGF to promote pro-metastatic traits such as motogenesis, morphogenesis, mitogenesis and angiogenesis.133 HGF has the capacity to enhance angiogenesis both directly and in-directly, either through its motogenic or morphogenic effects on endothelial cells or through its capacity to enhance other pro-angiogenic factors such as VEGF and its receptor.133,134 Several earlier studies conducted in our labs have highlighted the potential anti-angiogenic application for targeting HGF. HGF treatment in vivo was found to enhance the expression of tumor endothelial markers (TEMs) in tumors obtained from the inoculation of PC-3 prostate cancer cells into CD1 athymic nude mice. However, the addition of NK4, a HGF antagonist, to the treatment was able to prevent the elevation of these TEMs in the tumors.135 Similarly, in a breast cancer in vivo model HGF treatment was found to enhance vessel formation in tumors arising from MDA-MB-231 inoculation into CD1 athymic nude mice using immunohistochemical staining (IHC) analysis of resulting tumor tissues. In keeping with its role, addition of NK4 again prevented the enhanced angiogenesis seen in HGF treatment groups.136 In both studies HGF treatment caused enhanced tumor development, whereas co-treatment could suppress these increases in tumor growth.135,136

Given its involvement in the processes of angiogenesis and tumor progression, inhibitors to the cMET tyrosine kinase receptor of HGF have been developed as treatment regimes. Strategies such as Foretinib, an oral multikinase inhibitor targeting a variety of proteins including cMET and the VEGF receptor have been developed and are being assessed for their efficacy.137

Lymphangiogenesis and Anti-Lymphangiogenesis in Cancer

The area of lymphangiogenesis and the potential of anti-lymphangiogenic therapies in the treatment of cancer has been somewhat over-shadowed by research into anti-angiogenic strategies and the relative lack of pro-lymphangiogenic markers. However, the last 15 -20 years has seen the identification of lymphangiogenic markers and markers of lymphatic endothelial cells, such as lymphatic vessel endothelial hyaluronan receptor-1 LYVE-1138 and vascular endothelial growth factor receptor-3 (VEFGR-3).139 Studies such as these have aided in the progression of this field of research and demonstrated its importance in cancer metastasis.

Lymphatic metastases are common, with a number of cancers first metastasising to regional lymph nodes. The determination of lymph node involvement is an important factor in determining the aggressive nature of a particular cancer, with lymphatic metastasis commonly being associated with a poorer patient outlook.140 Scientific research, examining the role of VEGF-C and D in mouse models has demonstrated the potential of these factors to enhance tumor lymphatics and promote metastatic spread of tumor cells.141,142 In keeping with this, a number of recent studies have reported the association of lymphatic factors such as VEGF-C and D and the VEGFR3 receptor with lymph node metastasis and patient survival.143145 Taken together, these studies highlight the importance of tumor lymphangiogenesis in cancer spread and survival and demonstrate the potential for anti-lymphatic therapies, targeting factors such as VEGF-C, -D or the VEGFR3 receptor, to limit cancer spread and enhance survival rates.

In summary, anti-angiogenesis and anti-lympangiogenesis therapies hold great potential in combating the ongoing problem of cancer metastasis and the poor survival rates associated with cancer spread. Research and development of drugs in this area have so far begun to yield positive results with therapies such as Bevacizumab being implemented in the treatment of several cancer types. However, resistance to these anti-angiogenic strategies are possible and thus further research into new and multi target inhibitors of angiogenesis and lymphangiogenesis is essential in the ongoing fight against cancer spread.

Go to:

Organ Specific Metastasis

Cancer metastases are responsible for the majority of cancer-related deaths. From a primary tumor to a distant site and eventually developing a secondary tumor, cancerous cells need to proceed along a series of interrelated and sequential steps, including invasion through extracellular matrix, intravasation, survival in the circulation, extravasation into a distant site, and progressive growth at that site. The metastatic procedure is an inefficient process whereby the vast majority of circulating tumor cells are not able to progressively grow at distant sites. A latent period may exist between infiltration of cancer cells at a distant site and colonization leading progressively to the growth of a secondary tumor. Such a period can be as long as a couple of years seen in some metastases of breast cancer after initial management, and it can also be as short as a few months in lung cancer which may develop a metastasis rapidly within a few months of diagnosis. The cellular origin, intrinsic properties of the tumor, tissue affinities and circulation patterns determine not only the sites of tumor spread, but also the temporal course and severity of metastasis to vital organs. In addition to the above aspects of metastases, certain metastatic cells exhibit tissue tropism, preferring to grow in certain organs (Table 3). In breast cancer, for example, metastasis affects the bone and the lung, and less frequently the liver, brain, and adrenal medulla. Although the genetic and epigenetic basis of these metastatic properties is yet to be fully established, acquisition of the ability to complete each step involved in metastasis is thought to be driven by the accumulation of genetic mutations and epigenetic events that may result in a cells acquisition of metastatic traits during the process of developing a secondary tumor.

Table 3.

Common metastatic sites of certain solid tumors .

The organs mostly assaulted by metastases are lung, liver, brain and bone146 (Fig. 5). The lungs are the commonest site of metastases for many primary tumors. However, there is a great difference in propensity between the malignancies. It is just as high as 90% in melanomas at autopsy. The lungs serve as first filter for tumor cells spreading through blood circulation in malignancies whose venous drainage flows directly into the lungs. The tumors of testis, melanoma, osteosarcoma, and head and neck tumors have the highest incidence of pulmonary metastases.146 The liver is one of the most common sites for metastatic disease, accounting for 25% of all metastases to solid organs.147 In the United States and Europe, secondary liver neoplasms are far more common than primary hepatic neoplasms. In the adult oncology patient, most are metastatic carcinomas, of which adenocarcinomas are the predominant subtype, followed by squamous cell carcinomas and neuroendocrine carcinomas. Other tumor types that metastasize to the liver include melanomas, lymphomas, and rarely sarcomas. The most frequent metastasis to the brain occurs in patients with lung, breast, melanoma, renal, and colorectal tumors.148 In 2700 cases from the Memorial Sloan-Kettering Cancer Center in New York, the distribution of primary cancers was as follows: 48% lung, 15% breast, 9% melanoma, 1% lymphoma (mainly non-Hodgkin), 3% GI (3% colon and 2% pancreatic), 11% genitourinary (21% kidney, 46% testes, 5% cervix, 5% ovary), 10% osteosarcoma, 5% neuroblastoma, and 6% head and neck tumor . Once metastasis to the brain is diagnosed, the median survival of untreated patients is 12 mo. Bone metastases are most commonly seen in prostate, breast and lung cancer, which are leading malignancies in female and/or male having the highest incidence and mortality rates.149151 Bone metastasis usually leads to severe morbidities, which always persist until the death of patients, including bone pain, hypercalcemia, pathological fracture, spinal cord compression and consequent paralysis. In the following part, we generally reviewed the process and molecular mechanisms of organ specific metastases with a focus on bone metastasis.

Figure 5.

Organ specific metastases from primary tumors.

Metastatic Course, Routes and Steps

At an early stage, cancerous cells are confined to the primary site within the boundary of certain surrounding tissues. As the disease progresses, some cancer cells, as the result of genetic/ epigenetic predisposition, environmental interaction/stimulation, and indeed the combination of these elements, become more aggressive and begin to breach the surrounding structure. These cells would either directly invade the surrounding tissue, or disseminate via lymphatic and hematogenous routes. Direct invasion may result in the spreading of cancer cells to surrounding tissues and neighboring organs. For example, the local invasion of prostate cancer, can affect the erectile nerves, seminal vesicles, bladder and rectum nearby the prostate. The lymphatic and vascular routes differ from cancer to cancer according to their primary sites, however, frequently result in the systemic spread of cancer cells to distant organs, including bones, lung, and liver. For example, the primary lymphatic drainage of the prostate is via the internal iliac, perivesical, external iliac, obturator, and presacral nodes. The secondary lymphatic drainage includes the inguinal, common iliac and parα-aortic nodes. These nodes are therefore prime locations when one searches for the involved positive lymph nodes. Since the end of last century, a new technique, sentinel lymph node dissection has been developed and introduced in the detection, staging and management of lymph node involvement in cancer. The detection of a positive sentinel node indicates the need for a wide dissection of lymph nodes during surgery.

Both lymphatic and hematogenous dissemination frequently occur, even during early stages of the disease, and are seen in a vast majority of the patients who have an advanced cancer. To determine if systemic spread ‘occurred’ or not is a highly controversial topic, a conclusion of which is dependent on a wide variety of factors, from the type of samples to test, location and timing of sampling, techniques to detect cancer cells, to the interpretation of the presence of cancer cells or a cancer cell in a sample. Nonetheless, brain, bone, lung and liver are the most leading hematogenous sites from certain solid tumors.152155

The process of metastasis is complex and arduous, which incorporates multiple cells, factors and stages. During the development and progression of primary tumors, certain clones of tumor cells will have the required genotypic and phenotypic characteristics to enable themselves to interact with the local microenvironment. For example, tumor cells release VEGF to initiate angiogenesis, thus enhancing the blood supply to the tumor. The stromal cells are rich sources of protein factors that directly act on cancer cells thus driving the growth of tumors and dissemination of cancer cells. On the other hand, some of the stromal cell derived factors will directly induce angiogenesis thus supporting the growth and spread of an aggressive tumor. A good example of these stromα-derived protein factors is hepatocyte growth factor (HGF), a cytokine secreted by the stroma cells, which has been implicated in the angiogenesis and the dissemination of tumor cells.133 The disruption of intercellular adhesion in the tumor causes some tumor cells to detach from the tumor mass (detachment), followed by these cells invading through the extracellular matrix, a process so-called invasion which incorporates the motility, migration of tumor cells and breakdown of extracellular matrix. Some tumor cells will penetrate the blood vessels, thus entering the circulation (intravasation). From this point, these tumor cells move away from the primary site and circulate in the blood circulation where, they would encounter resistance by the immune system and the mechanical stresses of blood flow. Some tumor cells will eventually survive and adopt a process to leave the blood circulation, known as extravasation, in which cells adhere and penetrate the blood vessel again (a virtual reversal of the intravasation process). Once the tumor cells escape from the circulation, they will have to survive and finally develop a secondary tumor at the other site, in this case in bone. This complex process also needs the integration of multiple factors and events, such as invasion of tumor, angiogenesis and the interaction between tumor cells and the local microenvironment at a distant site/organ.

Metastasis Regulators

The interrelated and sequential multi-steps of metastasis require certain transformations of cancer cells at each step, from primary site to metastatic site. Numerous genes and molecules have been implicated into this dynamic and adaptable evolution of metastatic cancer cells, including suppressors and promoters of metastasis which may be altered genetically or epigenetically in accordance with the requirements at each step. Initiating factors for tumor progression and metastasis are critical and essential, particularly for dissociation and invasion which allow cancer cells to leave primary sites. The genes that determine these activities have been defined as metastasis initiation genes.156,157 These genes could promote cell motility, epithelial mesenchymal transition (EMT), extracellular matrix degradation, angiogenesis or evasion of the immune system. For example, EMT is mediated by developmental programmes that are under the control of aberrantly regulated transcription factors, such as Twist1, Snai1 and Snai2 (also known as Slug). Other determinants of invasion are components and modulators of certain pathways which include hepatocyte growth factor (HGF), VEGF and ERK pathways. Metastatic growth is also initiated by the suppression of non-coding RNAs, such as miR-126 and miR-335 in breast and gastric carcinomas.158,159 Some of the initiating factors that allow transformed cells to invade the surrounding tissue and attract a supportive stroma facilitate the dissemination of cancer cells and probably continue to do so after cancer cells infiltrate distant tissues. This is why some prognosis signatures of a malignancy can also be utilized as a signature to predict metastases.153

Metastasis suppressor genes are defined by their ability to inhibit metastasis at any step of the metastatic cascade. These metastasis suppressor genes inhibit metastasis of cancer cells, in vivo, without blocking tumorigenicity. To date, some metastasis suppressor genes have been identified, such as nonmetastatic gene 23 (NM23), Kangai 1 (KAI1), KISS1, mitogen-activated protein kinase 4 (MKK4), breast cancer metastasis suppressor 1 (BRMS1), Rho GDP dissociation inhibitor 2 (RhoGDI2), cofactor required for Sp1 transcriptional activation subunit 3 (CRSP3) and Vitamin D3 upregulated protein 1 (VDUP1). Deregulation of these metastasis suppressor genes has been indicated in certain solid tumors.160162

‘Seeds’ and ‘Soil’ Crosstalk between Cancer Cells and the Microenvironment during Bone Metastasis

Bone metastasis has been characterized as either osteolytic or osteoblastic. This classification actually represents two extremes of a continuum in which dysregulation of the normal bone remodelling process occurs. Patients can have both osteolytic and osteoblastic metastases or mixed lesions containing both elements. Most metastatic bone tumors from breast cancer have predominantly osteolytic lesions. In contrast, the metastatic lesions from prostate cancer are predominantly osteoblastic. During osteoblastic bone metastases, the balance between bone resorption and bone formation is tipped in favor of the latter. Patients suffer severe bone pain and the poor quality of bone produced in osteoblastic bone metastases frequently leads to bone fractures. Models to investigate osteoblastic metastases are rather rare, compared with models of osteolytic metastasis. Mechanisms, by which a metastatic lesion becomes osteoblastic or osteolytic remain unclear. However, a number of factors produced by cancer cells, such as platelet-derived growth factor (PDGF), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), VEGF, Wingless and NT-1 (WNT1), parathyroid hormone related protein (PTHrP), urokinase-type plasminogen activator (uPA), prostate specific antigen (PSA), endothelin-1 (ET-1) and BMPs, have been implicated in osteoblastic lesions.

The question of why the bone is the most preferred metastatic site of some solid tumors (breast, prostate and lung cancer) has aroused intense interest. One would first contemplate the anatomical characteristics of the organs at primary sites. The blood supply to the organs may provide a shortcut for the hematogenous dissemination of tumor cells from primary tumor to certain bones. For example, a rich venous plexus surrounds the prostate and connects to the venous drainage of the spine: this collection of veins (Batson’s plexus) is potentially one of the reasons why the lumbosacral spinal metastases are common in advanced prostate cancer.163 However, the anatomical explanation is not able to explain why the other axial skeleton, skull and ribs may also be involved in the bone metastasis from prostate cancer.

The ‘seed and soil’ theory proposed by Paget may provide some clue from a different standpoint.164 Osteotropic ‘seeds’ (tumor cells) may be developed during the progression of prostate cancer. These tumor cells may have acquired specific genetic phenotype, or activation of specific cytokine and proteases. These features direct the metastasis to bone. For example, elevated expression of BMPs and TGF-β in prostate cancer cells have been implicated in bone metastasis.165168 The “seeds” may also attach to the bone endothelium more effectively than to the endothelia of other organs.169 It has been suggested that the protease-activated receptor (PAR1, thrombin receptor) and integrin αVβ3 which are highly expressed in primary prostate cancer cell lines and metastatic prostate cancer cells derived from bone metastasis, may contribute to the bone metastases through facilitating the attachment of tumor cells to blood vessel walls and the process of extravasation.170173 The vascular endothelial growth factor (VEGF) secreted by the tumor cells may also contribute to the bone metastasis due to both the promotion of angiogenesis and the activation of osteoblasts.174176

On the other hand, bone also provides a fertile “soil” for the “seeds”. The bone matrix synthesized by osteoblasts has a particular abundance of cytokines and non-collagen proteins, which may attract prostate cancer cells and allow them to survive and proliferate in the bone matrix. For example, BMPs and TGF-β enriched in bone matrix can facilitate the development of bone metatstasis. Osteonectin, osteopontin, osteocalcin, and bone sialoprotein can also modulate the properties of prostate cancer cells and facilitate the spreading and growth, including promoting their migration, invasion and proliferation.177182 Bone turnover, as a characteristic of the adult bone, occurs most often in the bones rich in trabecular bone, such as the vertebrae, proximal femur, calcaneous, and ultradistal radius. During the bone turnover, cytokines and NCPs released or synthesized through bone resorption and bone formation thus generate a fertile ‘soil’. This may supplement the explanation of the favorite locations in bone metastases.

During the development of bone metastasis from prostate cancer, the interactions among tumor cells, bone cells and bone matrix constitute a “vicious cycle” of osteoblast/ osteoclast-mediated bone metastasis. For example, during the osteoblastic bone metastases of prostate cancer, cancer cells produce osteogenic factors such as ET-1, BMPs and PDGF, to activate osteoblasts. The osteoblasts differentiated from their progenitor cells deposit new matrix for bone formation. However, this unmineralised new matrix provides a more fertile soil to tumor cells, which is enriched with growth factors and NCPs. These factors help prostate cancer cells survive and proliferate in the bone microenvironment. The prostate cancer cells then further activate osteoblasts. In addition to this vicious cycle, at certain stages, both tumor-derived factors and osteoblasts expressing RANKL can activate osteoclasts, leading to some level of bone resorption, and subsequently generate bigger space for dominant osteoblastic lesion. The cytokines and NCPs released from bone matrix during bone resorption can also enhance this “vicious cycle” through facilitating proliferation of both prostate cancer cells and osteoblasts.

Go to:

Conclusion

Metastasis, the leading cause of mortality in patients with cancer, is receiving increasing attention in both scientfic and clinical research. Yet the mechanisms remain poorly understood and methods in combatting metastasis remain limited. It is however pleasing to observe some of the major progresses in this vital area of cancer research. With the increasing knowledge in gene expression, cellular behavior, biological events in the spread paths of cancer cells, there are now new prospects of taking some of the observations into the diagnosis, prognosis and treatment in the metastatic disease. For example, new knowledge on barrier function and paracelluar permeability may allow one to devise new direction in controlling the trepassing cancer cells and their entry into the destination tissues and organs. New biomarkers in areas such as epithelial to mesenchymal transtion offer new opportunities in predictive methods of metastatic potential of a primary tumor and new target for therapy. Angiogenesis has already been a fruitful area in new therapies and the organ specific spread of a solid tumor may allow new method of detection and a new way of targeting metastatic tumor cells. Although enormous challenges remain, it is anticipated that these lines of research will steadily find their into clinical practice.

Go to:

Acknowledgment

The authors wish to thank Cancer Research Wales, the Albert Hung Foundation, the Breast Cancer Hope Foundation, and the Welsh Assembly Government for supporting their work.

Go to:

References

1.

DeVita VT Jr., Young RC, Canellos GP. Combination versus single agent chemotherapy: a review of the basis for selection of drug treatment of cancer. Cancer. 1975;35:98–110. http://dx.doi.org/10.1002/1097-0142(197501)35:1<98::AID-CNCR2820350115>3.0.CO;2-B . [PubMed]

2.

Martin TA, Jiang WG. Loss of tight junction barrier function and its role in cancer metastasis. Biochim Biophys Acta. 2009;1788:872–91. http://dx.doi.org/10.1016/j.bbamem.2008.11.005 . [PubMed]

3.

Brooks PC. Cell adhesion molecules in angiogenesis. Cancer Metastasis Rev. 1996;15:187–94.http://dx.doi.org/10.1007/BF00437471 . [PubMed]

4.

Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992;267:10931–4. [PubMed]

5.

Folkman J. Fighting cancer by attacking its blood supply. Sci Am. 1996;275:150–4.http://dx.doi.org/10.1038/scientificamerican0996-150 . [PubMed]

6.

Ono M, Torisu H, Fukushi J, Nishie A, Kuwano M. Biological implications of macrophage infiltration in human tumor angiogenesis. Cancer Chemother Pharmacol. 1999;43(Suppl):S69–71.http://dx.doi.org/10.1007/s002800051101 . [PubMed]

7.

Jiang WG, Bryce RP, Horrobin DF, Mansel RE. Regulation of tight junction permeability and occludin expression by polyunsaturated fatty acids. Biochem Biophys Res Commun. 1998;244:414–20.http://dx.doi.org/10.1006/bbrc.1998.8288 . [PubMed]

8.

Jiang WG, Martin TA, Matsumoto K, Nakamura T, Mansel RE. Hepatocyte growth factor/scatter factor decreases the expression of occludin and transendothelial resistance (TER) and increases paracellular permeability in human vascular endothelial cells. J Cell Physiol. 1999;181:319–29. http://dx.doi.org/10.1002/(SICI)1097-4652(199911)181:2<319::AID-JCP14>3.0.CO;2-S . [PubMed]

9.

Tsukita S, Furuse M. Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol. 1999;9:268–73. http://dx.doi.org/10.1016/S0962-8924(99)01578-0 . [PubMed]

10.

Wong V, Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol. 1997;136:399–409. http://dx.doi.org/10.1083/jcb.136.2.399 . [PMC free article] [PubMed]

11.

Hollande F, Blanc EM, Bali JP, Whitehead RH, Pelegrin A, Baldwin GS, et al. HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line. Am J Physiol Gastrointest Liver Physiol. 2001;280:G910–21.[PubMed]

12.

Martin TA, Mansel RE, Jiang WG. Antagonistic effect of NK4 on HGF/SF induced changes in the transendothelial resistance (TER) and paracellular permeability of human vascular endothelial cells. J Cell Physiol.2002;192:268–75. http://dx.doi.org/10.1002/jcp.10133 . [PubMed]

13.

Ren J, Hamada J, Takeichi N, Fujikawa S, Kobayashi H. Ultrastructural differences in junctional intercellular communication between highly and weakly metastatic clones derived from rat mammary carcinoma. Cancer Res.1990;50:358–62. [PubMed]

14.

Satoh H, Zhong Y, Isomura H, Saitoh M, Enomoto K, Sawada N, et al. Localization of 7H6 tight junction-associated antigen along the cell border of vascular endothelial cells correlates with paracellular barrier function against ions, large molecules, and cancer cells. Exp Cell Res. 1996;222:269–74.http://dx.doi.org/10.1006/excr.1996.0034 . [PubMed]

15.

Hoevel T, Macek R, Mundigl O, Swisshelm K, Kubbies M. Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells. J Cell Physiol. 2002;191:60–8.http://dx.doi.org/10.1002/jcp.10076 . [PubMed]

16.

Martinez-Paloma A. Ultrastructural modifications of intercellular junctions in some epithelial tumors Lab Invest1970. 22 605 14. [PubMed]

17.

Inoue T, Shimono M, Yamamura T, Saito I, Watanabe O, Kawahara H. Acinic cell carcinoma arising in the glossopalatine glands: a report of two cases with electron microscopic observations. Oral Surg Oral Med Oral Pathol. 1984;57:398–407. http://dx.doi.org/10.1016/0030-4220(84)90159-2 . [PubMed]

18.

Mullin JM, O’Brien TG. Effects of tumor promoters on LLC-PK1 renal epithelial tight junctions and transepithelial fluxes. Am J Physiol. 1986;251:C597–602. [PubMed]

19.

Bornholdt J, Friis S, Godiksen S, Poulsen SS, Santoni-Rugiu E, Bisgaard HC, et al. The level of claudin-7 is reduced as an early event in colorectal carcinogenesis. BMC Cancer. 2011;11:65. http://dx.doi.org/10.1186/1471-2407-11-65 . [PMC free article] [PubMed]

20.

Nakagawa S, Miyoshi N, Ishii H, Mimori K, Tanaka F, Sekimoto M, et al. Expression of CLDN1 in colorectal cancer: a novel marker for prognosis. Int J Oncol. 2011;39:791–6. [PubMed]

21.

Wang X, Tully O, Ngo B, Zitin M, Mullin JM. Epithelial tight junctional changes in colorectal cancer tissues.ScientificWorldJournal. 2011;11:826–41. http://dx.doi.org/10.1100/ tsw.2011.86 . [PubMed]

22.

Takai E, Tan X, Tamori Y, Hirota M, Egami H, Ogawa M. Correlation of translocation of tight junction protein Zonula occludens-1 and activation of epidermal growth factor receptor in the regulation of invasion of pancreatic cancer cells. Int J Oncol. 2005;27:645–51. [PubMed]

23.

Zhou L, Tan X, Wang W, Wang B, Dai X, Liu J. Analysis of invasion-metastasis in pancreatic cancer: Correlation between the expression and arrangement of tight junction protein-2 and cell dissociation in pancreatic cancer cells.Mol Med Report. 2010;3:149–53. [PubMed]

24.

Kojima T, Takasawa A, Kyuno D, Ito T, Yamaguchi H, Hirata K, et al. Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.Exp Cell Res. 2011;317:2288–98. http://dx.doi.org/10.1016/j. yexcr.2011.06.020 . [PubMed]

25.

Martin TA, Mansel RE, Jiang WG. Loss of occludin leads to the progression of human breast cancer. Int J Mol Med. 2010;26:723–34. http://dx.doi.org/10.3892/ijmm_00000519 . [PubMed]

26.

Myal Y, Leygue E, Blanchard AA. Claudin 1 in breast tumorigenesis: revelation of a possible novel “claudin high” subset of breast cancers. J Biomed Biotechnol. 2010;2010:956897. http://dx.doi.org/10.1155/2010/956897 . [PMC free article] [PubMed]

27.

Wu Q, Liu Y, Ren Y, Xu X, Yu L, Li Y, et al. Tight junction protein, claudin-6, downregulates the malignant phenotype of breast carcinoma. Eur J Cancer Prev. 2010;19:186–94.http://dx.doi.org/10.1097/CEJ.0b013e328337210e . [PubMed]

28.

Martin TA, Watkins G, Mansel RE, Jiang WG. Hepatocyte growth factor disrupts tight junctions in human breast cancer cells. Cell Biol Int. 2004a;28:361–71. http://dx.doi. org/10.1016/j.cellbi.2004.03.003 . [PubMed]

29.

Utoguchi N, Mizuguchi H, Dantakean A, Makimoto H, Wakai Y, Tsutsumi Y, et al. Effect of tumour cell-conditioned medium on endothelial macromolecular permeability and its correlation with collagen. Br J Cancer.1996;73:24–8. http://dx.doi.org/10.1038/bjc.1996.5 . [PMC free article] [PubMed]

30.

Martin TA, Watkins G, Mansel RE, Jiang WG. Loss of tight junction plaque molecules in breast cancer tissues is associated with a poor prognosis in patients with breast cancer. Eur J Cancer. 2004b;40:271725.http://dx.doi.org/10.1016/j.ejca.2004.08.008 . [PubMed]

31.

Giepmans BN, van Ijzendoorn SC. Epithelial cell-cell junctions and plasma membrane domains. Biochim Biophys Acta. 2009;1788:820–31. http://dx.doi.org/10.1016/j.bbamem.2008.07.015 . [PubMed]

32.

Farquhar MG, Palade GE. Junctional complexes in various epithelia. J Cell Biol. 1963;17:375–412.http://dx.doi.org/10.1083/jcb.17.2.375 . [PMC free article] [PubMed]

33.

Meng W, Takeichi M. Adherens junction: molecular architecture and regulation. Cold Spring Harb Perspect Biol.2009;1:a002899. http://dx.doi.org/10.1101/cshperspect.a002899 . [PMC free article] [PubMed]

34.

Yonemura S, Itoh M, Nagafuchi A, Tsukita S. Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J Cell Sci.1995;108:127–42. [PubMed]

35.

Uchida N, Honjo Y, Johnson KR, Wheelock MJ, Takeichi M. The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J Cell Biol. 1996;135:767–79.http://dx.doi.org/10.1083/jcb.135.3.767 . [PMC free article] [PubMed]

36.

Gooding JM, Yap KL, Ikura M. The cadherin-catenin complex as a focal point of cell adhesion and signalling: new insights from three-dimensional structures. Bioessays. 2004;26:497–511. http://dx.doi.org/10.1002/bies.20033 . [PubMed]

37.

Reynolds AB, Daniel J, McCrea PD, Wheelock MJ, Wu J, Zhang Z. Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol Cell Biol. 1994;14:8333–42. [PMC free article] [PubMed]

38.

Togashi H, Miyoshi J, Honda T, Sakisaka T, Takai Y, Takeichi M. Interneurite affinity is regulated by heterophilic nectin interactions in concert with the cadherin machinery. J Cell Biol. 2006;174:141–51.http://dx.doi.org/10.1083/jcb.200601089 . [PMC free article] [PubMed]

39.

Cavallaro U, Christofori G. Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer.2004;4:118–32. http://dx.doi.org/10.1038/nrc1276 . [PubMed]

40.

Vleminckx K, Vakaet L Jr., Mareel M, Fiers W, van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell. 1991;66:107–19. http://dx.doi.org/10.1016/0092-8674(91)90143-M . [PubMed]

41.

Luo J, Lubaroff DM, Hendrix MJ. Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection. Cancer Res. 1999;59:3552–6. [PubMed]

42.

Hsu MY, Meier FE, Nesbit M, Hsu JY, Van Belle P, Elder DE, et al. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors. Am J Pathol. 2000;156:1515–25. http://dx.doi.org/10.1016/ S0002-9440(10)65023-7 . [PMC free article] [PubMed]

43.

Kashiwagi S, Yashiro M, Takashima T, Aomatsu N, Ikeda K, Ogawa Y, et al. Advantages of adjuvant chemotherapy for patients with triple-negative breast cancer at Stage II: usefulness of prognostic markers E-cadherin and Ki67. Breast Cancer Res. 2011;13:R122. http://dx.doi.org/10.1186/ bcr3068 . [PMC free article] [PubMed]

44.

Morrogh M, Andrade VP, Giri D, Sakr RA, Paik W, Qin LX, et al. Cadherin-catenin complex dissociation in lobular neoplasia of the breast. Breast Cancer Res Treat. 2012;132:641–52. http://dx.doi.org/10.1007/s10549-011-1860-0 . [PMC free article] [PubMed]

45.

Garrod D, Chidgey M. Desmosome structure, composition and function. Biochim Biophys Acta. 2008;1778:572–87. http://dx.doi.org/10.1016/j.bbamem.2007.07.014 . [PubMed]

46.

Brooke MA, Nitoiu D, Kelsell DP. Cell-cell connectivity: desmosomes and disease. J Pathol. 2012;226:158–71.http://dx.doi.org/10.1002/path.3027 . [PubMed]

47.

Dusek RL, Attardi LD. Desmosomes: new perpetrators in tumour suppression. Nat Rev Cancer. 2011;11:317–23.http://dx.doi.org/10.1038/nrc3051 . [PMC free article] [PubMed]

48.

Furukawa C, Daigo Y, Ishikawa N, Kato T, Ito T, Tsuchiya E, et al. Plakophilin 3 oncogene as prognostic marker and therapeutic target for lung cancer. Cancer Res. 2005;65:7102–10. http://dx.doi.org/10.1158/0008-5472.CAN-04-1877 . [PubMed]

49.

Brennan D, Mahoney MG. Increased expression of Dsg2 in malignant skin carcinomas: A tissuemicroarray based study. Cell Adh Migr. 2009;3:148–54. http://dx.doi.org/10.4161/ cam.3.2.7539 . [PMC free article] [PubMed]

50.

Breuninger S, Reidenbach S, Sauer CG, Strbel P, Pfitzenmaier J, Trojan L, et al. Desmosomal plakophilins in the prostate and prostatic adenocarcinomas: implications for diagnosis and tumor progression. Am J Pathol.2010;176:2509–19. http://dx.doi.org/10.2353/ajpath.2010.090737 . [PMC free article] [PubMed]

51.

Kolegraff K, Nava P, Helms MN, Parkos CA, Nusrat A. Loss of desmocollin-2 confers a tumorigenic phenotype to colonic epithelial cells through activation of Akt/b-catenin signaling. Mol Biol Cell. 2011;22:1121–34.http://dx.doi.org/10.1091/mbc.E10-10-0845 . [PMC free article] [PubMed]

52.

Teh MT, Parkinson EK, Thurlow JK, Liu F, Fortune F, Wan H. A molecular study of desmosomes identifies a desmoglein isoform switch in head and neck squamous cell carcinoma. J Oral Pathol Med. 2011;40:67–76.http://dx.doi.org/10.1111/j.1600-0714.2010.00951.x . [PubMed]

53.

Gosavi P, Kundu ST, Khapare N, Sehgal L, Karkhanis MS, Dalal SN. E-cadherin and plakoglobin recruit plakophilin3 to the cell border to initiate desmosome assembly. Cell Mol Life Sci. 2011;68:1439–54.http://dx.doi.org/10.1007/s00018-010-0531-3 . [PubMed]

54.

Derangeon M, Spray DC, Bourmeyster N, Sarrouilhe D, Herv JC. Reciprocal influence of connexins and apical junction proteins on their expressions and functions. Biochim Biophys Acta. 2009;1788:76878.http://dx.doi.org/10.1016/j.bbamem.2008.10.023 . [PMC free article] [PubMed]

55.

Lamiche C, Clarhaut J, Strale PO, Crespin S, Pedretti N, Bernard FX, et al. The gap junction protein Cx43 is involved in the bone-targeted metastatic behaviour of human prostate cancer cells. Clin Exp Metastasis.2012;29:111–22. http://dx.doi.org/10.1007/s10585-011-9434-4 . [PubMed]

56.

Sirnes S, Bruun J, Kolberg M, Kjenseth A, Lind GE, Svindland A, et al. Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome. Int J Cancer. 2011. http://dx.doi.org/10.1002/ijc.26392 . [PubMed]

57.

Bendas G, Borsig L. Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins. Int J Cell Biol. 2012;2012:676731. http://dx.doi. org/10.1155/2012/676731 . [PMC free article] [PubMed]

58.

van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC, van der Pluijm G. Integrin αv expression is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Am J Pathol.2011;179:2559–68. http://dx.doi.org/10.1016/j. ajpath.2011.07.011 . [PMC free article] [PubMed]

59.

Lu JG, Li Y, Li L, Kan X. Overexpression of osteopontin and integrin αv in laryngeal and hypopharyngeal carcinomas associated with differentiation and metastasis. J Cancer Res Clin Oncol. 2011;137:1613–8.http://dx.doi.org/10.1007/s00432-011-1024-y . [PubMed]

60.

St Hill CA. Interactions between endothelial selectins and cancer cells regulate metastasis. [Elite Ed] Front Biosci.2012;17:3233–51. [PubMed]

61.

Richter U, Schrder C, Wicklein D, Lange T, Geleff S, Dippel V, et al. Adhesion of small cell lung cancer cells to E- and P-selectin under physiological flow conditions: implications for metastasis formation. Histochem Cell Biol.2011;135:499–512. http://dx.doi.org/10.1007/s00418-0110804-4 . [PubMed]

62.

Wai Wong C, Dye DE, Coombe DR. The role of immunoglobulin superfamily cell adhesion molecules in cancer metastasis. Int J Cell Biol. 2012;2012:340296. http://dx.doi. org/10.1155/2012/340296 . [PMC free article] [PubMed]

63.

Martin TA, Harrison G, Mansel RE, Jiang WG. The role of the CD44/ezrin complex in cancer metastasis. Crit Rev Oncol Hematol. 2003;46:165–86. http://dx.doi.org/10.1016/ S1040-8428(02)00172-5 . [PubMed]

64.

Jeyapalan Z., Yang B. B. The non-coding 3’UTR of CD44 induces metastasis by regulating extracellular matrix functions. J Cell Sci. 2012 [PubMed]

65.

Park YS, Huh JW, Lee JH, Kim HR. shRNA against CD44 inhibits cell proliferation, invasion and migration, and promotes apoptosis of colon carcinoma cells. Oncol Rep. 2012;27:339–46. [PubMed]

66.

Schwock J, Dhani N, Hedley DW. Targeting focal adhesion kinase signaling in tumor growth and metastasis.Expert Opin Ther Targets. 2010;14:77–94. http://dx.doi. org/10.1517/14728220903460340 . [PubMed]

67.

Wang W, Liu Y, Liao K. Tyrosine phosphorylation of cortactin by the FAK-Src complex at focal adhesions regulates cell motility. BMC Cell Biol. 2011;12:49. http://dx.doi.org/10.1186/14712121-12-49 . [PMC free article] [PubMed]

68.

Bernardi MA, Logullo AF, Pasini FS, Nonogaki S, Blumke C, Soares FA, et al. Prognostic significance of CD24 and claudin-7 immunoexpression in ductal invasive breast cancer. Oncol Rep. 2012;27:28–38. [PubMed]

69.

Hornsby CD, Cohen C, Amin MB, Picken MM, Lawson D, Yin-Goen Q, et al. Claudin-7 immunohistochemistry in renal tumors: a candidate marker for chromophobe renal cell carcinoma identified by gene expression profiling.Arch Pathol Lab Med. 2007;131:1541–6. [PubMed]

70.

Sheehan GM, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP Jr., Ross JS. Loss of claudins-1 and -7 and expression of claudins-3 and -4 correlate with prognostic variables in prostatic adenocarcinomas. Hum Pathol.2007;38:564–9. http://dx.doi.org/10.1016/j.humpath.2006.11.007 . [PubMed]

71.

Zheng JY, Yu D, Foroohar M, Ko E, Chan J, Kim N, et al. Regulation of the expression of the prostate-specific antigen by claudin-7. J Membr Biol. 2003;194:187–97. http://dx.doi. org/10.1007/s00232-003-2038-4 . [PubMed]

72.

Chao YC, Pan SH, Yang SC, Yu SL, Che TF, Lin CW, et al. Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma. Am J Respir Crit Care Med. 2009;179:12333.http://dx.doi.org/10.1164/rccm.200803-456OC . [PubMed]

73.

Richardson F, Young GD, Sennello R, Wolf J, Argast GM, Mercado P, et al. The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy. Anticancer Res. 2012;32:537–52. [PubMed]

74.

Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science. 2003;302:1704–9. http://dx.doi.org/10.1126/science.1092053 . [PubMed]

75.

Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992;70:401–10. http://dx.doi.org/10.1016/0092-8674(92)90164-8 . [PubMed]

76.

Faassen AE, Schrager JA, Klein DJ, Oegema TR, Couchman JR, McCarthy JB. A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. J Cell Biol. 1992;116:521–31. http://dx.doi.org/10.1083/ jcb.116.2.521 . [PMC free article] [PubMed]

77.

Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol. 2005;17:548–58. http://dx.doi.org/10.1016/j.ceb.2005.08.001 . [PubMed]

78.

Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol.2006;7:131–42. http://dx.doi.org/10.1038/nrm1835 . [PubMed]

79.

Buijs JT, Henriquez NV, van Overveld PG, van der Horst G, ten Dijke P, van der Pluijm G. TGF-beta and BMP7 interactions in tumour progression and bone metastasis. Clin Exp Metastasis. 2007;24:60917.http://dx.doi.org/10.1007/s10585-007-9118-2 . [PubMed]

80.

Guarino M. Epithelial-mesenchymal transition and tumour invasion. Int J Biochem Cell Biol. 2007;39:2153–60.http://dx.doi.org/10.1016/j.biocel.2007.07.011 . [PubMed]

81.

Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–54.http://dx.doi.org/10.1038/nrc822 . [PubMed]

82.

Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 2003;15:740–6. http://dx.doi.org/10.1016/j.ceb.2003.10.006 . [PubMed]

83.

Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, et al. Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res. 2008;68:937–45. http://dx.doi.org/10.1158/0008-5472.CAN-07-2148 . [PubMed]

84.

Cano A, Prez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000;2:76–83.http://dx.doi.org/10.1038/35000025 . [PubMed]

85.

Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer. 2007;7:415–28. http://dx.doi. org/10.1038/nrc2131 . [PubMed]

86.

Medici D, Hay ED, Olsen BR. Snail and Slug promote epithelial-mesenchymal transition through betacatenin-T-cell factor-4-dependent expression of transforming growth factor-beta3. Mol Biol Cell. 2008;19:4875–87.http://dx.doi.org/10.1091/mbc.E08-05-0506 . [PMC free article] [PubMed]

87.

Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251:1451–5.http://dx.doi.org/10.1126/science.2006419 . [PubMed]

88.

Jiang WG, Mansel RE. E-cadherin complex and its abnormalities in human breast cancer. Surg Oncol.2000;9:151–71. http://dx.doi.org/10.1016/S0960-7404(01)00010-X . [PubMed]

89.

Zschiesche W, Schnborn I, Behrens J, Herrenknecht K, Hartveit F, Lilleng P, et al. Expression of E-cadherin and catenins in invasive mammary carcinomas. Anticancer Res. 1997;17(1B):561–7. [PubMed]

90.

Jiang WG. E-cadherin and its associated protein catenins, cancer invasion and metastasis. Br J Surg. 1996;83:437–46. http://dx.doi.org/10.1002/bjs.1800830404 . [PubMed]

91.

Gottardi CJ, Wong E, Gumbiner BM. E-cadherin suppresses cellular transformation by inhibiting betacatenin signaling in an adhesion-independent manner. J Cell Biol. 2001;153:1049–60.http://dx.doi.org/10.1083/jcb.153.5.1049 . [PMC free article] [PubMed]

92.

Stockinger A, Eger A, Wolf J, Beug H, Foisner R. E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity. J Cell Biol. 2001;154:1185–96.http://dx.doi.org/10.1083/jcb.200104036 . [PMC free article] [PubMed]

93.

Kim K, Lu Z, Hay ED. Direct evidence for a role of beta-catenin/LEF-1 signaling pathway in induction of EMT.Cell Biol Int. 2002;26:463–76. http://dx.doi.org/10.1006/cbir.2002.0901 . [PubMed]

94.

Pecina-Slaus N, Cicvara-Pecina T, Kafka A. Epithelial-to-mesenchymal transition: possible role in meningiomas [Elite Ed] Front Biosci (Elite Ed) 2012;4:889–96. http://dx.doi. org/10.2741/427 . [PubMed]

95.

Martin TA, Goyal A, Watkins G, Jiang WG. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol. 2005;12:488–96.http://dx.doi.org/10.1245/ASO.2005.04.010 . [PubMed]

96.

Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, et al. Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol. 2002;161:1881–91.http://dx.doi.org/10.1016/S0002-9440(10)64464-1 . [PMC free article] [PubMed]

97.

Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117:927–39.http://dx.doi.org/10.1016/j.cell.2004.06.006 . [PubMed]

98.

Leptin M, Grunewald B. Cell shape changes during gastrulation in Drosophila. Development. 1990;110:73–84.[PubMed]

99.

Smith DE, Franco del Amo F, Gridley T. Isolation of Sna, a mouse gene homologous to the Drosophila genes snail and escargot: its expression pattern suggests multiple roles during postimplantation development.Development. 1992;116:1033–9. [PubMed]

100.

Batlle E, Sancho E, Franc C, Domnguez D, Monfar M, Baulida J, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000;2:84–9.http://dx.doi.org/10.1038/35000034 . [PubMed]

101.

Yokoyama K, Kamata N, Hayashi E, Hoteiya T, Ueda N, Fujimoto R, et al. Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro. Oral Oncol. 2001;37:6571.http://dx.doi.org/10.1016/S1368-8375(00)00059-2 . [PubMed]

102.

Blanco MJ, Moreno-Bueno G, Sarrio D, Locascio A, Cano A, Palacios J, et al. Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene. 2002;21:32416.http://dx.doi.org/10.1038/sj.onc.1205416 . [PubMed]

103.

Ikenouchi J, Matsuda M, Furuse M, Tsukita S. Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci. 2003;116:1959–67.http://dx.doi.org/10.1242/jcs.00389 . [PubMed]

104.

Islam S, Carey TE, Wolf GT, Wheelock MJ, Johnson KR. Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion. J Cell Biol.1996;135:1643–54. http://dx.doi.org/10.1083/jcb.135.6.1643 . [PMC free article] [PubMed]

105.

Nieman MT, Prudoff RS, Johnson KR, Wheelock MJ. N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J Cell Biol. 1999;147:631–44. http://dx.doi.org/10.1083/jcb.147.3.631 . [PMC free article] [PubMed]

106.

Mikheeva SA, Mikheev AM, Petit A, Beyer R, Oxford RG, Khorasani L, et al. TWIST1 promotes invasion through mesenchymal change in human glioblastoma. Mol Cancer. 2010;9:194. http://dx.doi.org/10.1186/1476-4598-9-194 . [PMC free article] [PubMed]

107.

Gupta R, Chetty C, Bhoopathi P, Lakka S, Mohanam S, Rao JS, et al. Downregulation of uPA/uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells. Int J Oncol. 2011;38:733–44. [PubMed]

108.

Lester RD, Jo M, Montel V, Takimoto S, Gonias SL. uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells. J Cell Biol. 2007;178:425–36. http://dx.doi.org/10.1083/ jcb.200701092 . [PMC free article] [PubMed]

109.

Strutz F, Zeisberg M, Ziyadeh FN, Yang CQ, Kalluri R, Mller GA, et al. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int. 2002;61:1714–28. http://dx.doi.org/10.1046/j.1523-1755.2002.00333.x . [PubMed]

110.

Akhurst RJ, Derynck R. TGF-beta signaling in cancer–a double-edged sword. Trends Cell Biol. 2001;11:S44–51. [PubMed]

111.

Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, et al. BMP-7 counteracts TGFbeta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med. 2003;9:964–8.http://dx.doi.org/10.1038/nm888 . [PubMed]

112.

Ye L, Lewis-Russell JM, Kynaston H, Jiang WG. Endogenous bone morphogenetic protein-7 controls the motility of prostate cancer cells through regulation of bone morphogenetic protein antagonists. J Urol.2007;178:1086–91. http://dx.doi.org/10.1016/j.juro.2007.05.003 . [PubMed]

113.

Bokobza SM, Ye L, Kynaston H, Jiang WG. Growth and differentiation factor 9 (GDF-9) induces epithelial-mesenchymal transition in prostate cancer cells. Mol Cell Biochem. 2011;349:33–40.http://dx.doi.org/10.1007/s11010-010-0657-5 . [PubMed]

114.

Xu Z, Jiang Y, Steed H, Davidge S, Fu Y. TGFβ and EGF synergistically induce a more invasive phenotype of epithelial ovarian cancer cells. Biochem Biophys Res Commun. 2010;401:376–81.http://dx.doi.org/10.1016/j.bbrc.2010.09.059 . [PubMed]

115.

Wang X, Lu H, Urvalek AM, Li T, Yu L, Lamar J, et al. KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene. 2011;30:1901–11.http://dx.doi.org/10.1038/onc.2010.563 . [PMC free article] [PubMed]

116.

Qiao B, Johnson NW, Gao J. Epithelial-mesenchymal transition in oral squamous cell carcinoma triggered by transforming growth factor-beta1 is Snail family-dependent and correlates with matrix metalloproteinase-2 and -9 expressions. Int J Oncol. 2010;37:663–8. [PubMed]

117.

Lynch CC, Vargo-Gogola T, Matrisian LM, Fingleton B. Cleavage of E-Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA. J Oncol.2010;2010:530745. http://dx.doi.org/10.1155/2010/530745 . [PMC free article] [PubMed]

118.

Abe K, Takeichi M. EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc Natl Acad Sci U S A. 2008;105:13–9. http://dx.doi.org/10.1073/pnas.0710504105 . [PMC free article] [PubMed]

119.

Jiang WG, Martin TA, Lewis-Russell JM, Douglas-Jones A, Ye L, Mansel RE. Eplin-alpha expression in human breast cancer, the impact on cellular migration and clinical outcome. Mol Cancer. 2008;7:71.http://dx.doi.org/10.1186/1476-4598-7-71 . [PMC free article] [PubMed]

120.

Sanders AJ, Martin TA, Ye L, Mason MD, Jiang WG. EPLIN is a negative regulator of prostate cancer growth and invasion. J Urol. 2011;186:295–301. http://dx.doi.org/10.1016/j.juro.2011.03.038 . [PubMed]

121.

Zhang S, Wang X, Osunkoya AO, Iqbal S, Wang Y, Chen Z, et al. EPLIN downregulation promotes epithelial-mesenchymal transition in prostate cancer cells and correlates with clinical lymph node metastasis. Oncogene.2011;30:4941–52. http://dx.doi.org/10.1038/onc.2011.199 . [PMC free article] [PubMed]

122.

Hay ED. Organization and fine structure of epithelium and mesenchyme in the developing chick embryo. In: Epithelial-mesenchymal interactions. In: Fleischmajer R, Billingham RE, editors. Baltimore: Williams & Wilkins; 1968. pp. 31–55.

123.

Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007;6:273–86.http://dx.doi.org/10.1038/nrd2115 . [PubMed]

124.

Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146:87387.http://dx.doi.org/10.1016/j.cell.2011.08.039 . [PubMed]

125.

Pandya NM, Dhalla NS, Santani DD. Angiogenesis–a new target for future therapy. Vascul Pharmacol.2006;44:265–74. http://dx.doi.org/10.1016/j.vph.2006.01.005 . [PubMed]

126.

Al-Rawi MA, Jiang WG. Lymphangiogenesis and cancer metastasis. Front Biosci. 2011;16:723–39.http://dx.doi.org/10.2741/3715 . [PubMed]

127.

Albrecht I, Christofori G. Molecular mechanisms of lymphangiogenesis in development and cancer. Int J Dev Biol. 2011;55:483–94. http://dx.doi.org/10.1387/ijdb.103226ia . [PubMed]

128.

Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–6.http://dx.doi.org/10.1056/NEJM197111182852108 . [PubMed]

129.

Klagsbrun M, D’Amore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev.1996;7:259–70. http://dx.doi.org/10.1016/S1359-6101(96)00027-5 . [PubMed]

130.

Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473:298–307. http://dx.doi.org/10.1038/nature10144 . [PMC free article] [PubMed]

131.

Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;18:425.http://dx.doi.org/10.1210/er.18.1.4 . [PubMed]

132.

Sato S, Itamochi H. Bevacizumab and ovarian cancer. Curr Opin Obstet Gynecol. 2012;24:8–13.http://dx.doi.org/10.1097/GCO.0b013e32834daeed . [PubMed]

133.

Jiang WG, Martin TA, Parr C, Davies G, Matsumoto K, Nakamura T. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit Rev Oncol Hematol. 2005;53:35–69.http://dx.doi.org/10.1016/j.critrevonc.2004.09.004 . [PubMed]

134.

Wojta J, Kaun C, Breuss JM, Koshelnick Y, Beckmann R, Hattey E, et al. Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells. Lab Invest. 1999;79:427–38.[PubMed]

135.

Davies G, Mason MD, Martin TA, Parr C, Watkins G, Lane J, et al. The HGF/SF antagonist NK4 reverses fibroblast- and HGF-induced prostate tumor growth and angiogenesis in vivo. Int J Cancer. 2003;106:348–54.http://dx.doi.org/10.1002/ijc.11220 . [PubMed]

136.

Martin TA, Parr C, Davies G, Watkins G, Lane J, Matsumoto K, et al. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis. 2003;24:1317–23. http://dx.doi.org/10.1093/carcin/bgg072 . [PubMed]

137.

Eder JP, Shapiro GI, Appleman LJ, Zhu AX, Miles D, Keer H, et al. A phase I study of foretinib, a multi-targeted inhibitor of c-Met and vascular endothelial growth factor receptor 2. Clin Cancer Res. 2010;16:3507–16.http://dx.doi.org/10.1158/1078-0432.CCR-10-0574 . [PubMed]

138.

Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol. 1999;144:789–801.http://dx.doi.org/10.1083/jcb.144.4.789 . [PMC free article] [PubMed]

139.

Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, Dumont D, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A. 1995;92:3566–70. http://dx.doi.org/10.1073/pnas.92.8.3566 . [PMC free article] [PubMed]

140.

McAllaster JD, Cohen MS. Role of the lymphatics in cancer metastasis and chemotherapy applications. Adv Drug Deliv Rev. 2011;63:867–75. http://dx.doi.org/10.1016/j.addr.2011.05.014 . [PubMed]

141.

Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med. 2001;7:192–8. http://dx.doi.org/10.1038/84643 . [PubMed]

142.

Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo R, et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med. 2001;7:186–91. http://dx.doi.org/10.1038/84635 . [PubMed]

143.

Feng Y, Wang W, Hu J, Ma J, Zhang Y, Zhang J. Expression of VEGF-C and VEGF-D as significant markers for assessment of lymphangiogenesis and lymph node metastasis in non-small cell lung cancer. Anat Rec (Hoboken) 2010;293:802–12. http://dx.doi.org/10.1002/ar.21096 . [PubMed]

144.

Kilvaer TK, Valkov A, Sorbye S, Smeland E, Bremnes RM, Busund LT, et al. Profiling of VEGFs and VEGFRs as prognostic factors in soft tissue sarcoma: VEGFR-3 is an independent predictor of poor prognosis.PLoS One. 2010;5:e15368. http://dx.doi.org/10.1371/journal. pone.0015368 . [PMC free article] [PubMed]

145.

Raica M, Cimpean AM, Ceausu R, Ribatti D. Lymphatic microvessel density, VEGF-C, and VEGFR-3 expression in different molecular types of breast cancer. Anticancer Res. 2011;31:1757–64. [PubMed]

146.

Debois JM. 2002. TxNxM1: The Anatomy and Clinics of Metastatic Cancer, Kluwer Academic Publisher.

147.

Abbruzzese JL, Abbruzzese MC, Lenzi R, Hess KR, Raber MN. Analysis of a diagnostic strategy for patients with suspected tumors of unknown origin. J Clin Oncol. 1995;13:2094–103. [PubMed]

148.

Schouten LJ, Rutten J, Huveneers HA, Twijnstra A. Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma. Cancer. 2002;94:2698–705.http://dx.doi.org/10.1002/cncr.10541 . [PubMed]

149.

Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer.2002;2:584–93. http://dx.doi.org/10.1038/nrc867 . [PubMed]

150.

Hess KR, Varadhachary GR, Taylor SH, Wei W, Raber MN, Lenzi R, et al. Metastatic patterns in adenocarcinoma. Cancer. 2006;106:1624–33. http://dx.doi.org/10.1002/cncr.21778 . [PubMed]

151.

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–49.http://dx.doi.org/10.3322/caac.20006 . [PubMed]

152.

Bubendorf L, Schpfer A, Wagner U, Sauter G, Moch H, Willi N, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol. 2000;31:578–83. http://dx.doi.org/10.1053/hp.2000.6698 . [PubMed]

153.

Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. 2005;115:44–55. [PMC free article] [PubMed]

154.

Schlter K, Gassmann P, Enns A, Korb T, Hemping-Bovenkerk A, Hlzen J, et al. Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential. Am J Pathol.2006;169:1064–73. http://dx.doi.org/10.2353/ ajpath.2006.050566 . [PMC free article] [PubMed]

155.

Nguyen DX, Bos PD, Massagu J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer.2009;9:274–84. http://dx.doi.org/10.1038/nrc2622 . [PubMed]

156.

Nguyen DX, Massagu J. Genetic determinants of cancer metastasis. Nat Rev Genet. 2007;8:341–52.http://dx.doi.org/10.1038/nrg2101 . [PubMed]

157.

Chiang AC, Massagu J. Molecular basis of metastasis. N Engl J Med. 2008;359:2814–23.http://dx.doi.org/10.1056/NEJMra0805239 . [PMC free article] [PubMed]

158.

Feng R, Chen X, Yu Y, Su L, Yu B, Li J, et al. miR-126 functions as a tumour suppressor in human gastric cancer. Cancer Lett. 2010;298:50–63. http://dx.doi.org/10.1016/j.canlet.2010.06.004 . [PubMed]

159.

Yang R, Dick M, Marme F, Schneeweiss A, Langheinz A, Hemminki K, et al. Genetic variants within miR-126 and miR-335 are not associated with breast cancer risk. Breast Cancer Res Treat. 2011;127:54954.http://dx.doi.org/10.1007/s10549-010-1244-x . [PubMed]

160.

Steeg PS. Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer. 2003;3:55–63.http://dx.doi.org/10.1038/nrc967 . [PubMed]

161.

Malik FA, Sanders AJ, Jones AD, Mansel RE, Jiang WG. Transcriptional and translational modulation of KAI1 expression in ductal carcinoma of the breast and the prognostic significance. Int J Mol Med. 2009;23:273–8.[PubMed]

162.

Malik FA, Sanders AJ, Kayani MA, Jiang WG. Effect of expressional alteration of KAI1 on breast cancer cell growth, adhesion, migration and invasion. Cancer Genomics Proteomics. 2009;6:205–13. [PubMed]

163.

Batson OV. The function of the vertebral veins and their role in the spread of metastases. 1940. Clin Orthop Relat Res. 1995:4–9. [PubMed]

164.

Paget S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 1989;8:98–101. [PubMed]

165.

Autzen P, Robson CN, Bjartell A, Malcolm AJ, Johnson MI, Neal DE, et al. Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies. Br J Cancer. 1998;78:1219–23.http://dx.doi.org/10.1038/bjc.1998.658 . [PMC free article] [PubMed]

166.

De Pinieux G, Flam T, Zerbib M, Taupin P, Bellahcne A, Waltregny D, et al. Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression in localized human prostatic adenocarcinoma as predictors of clinical outcome: a clinicopathological and immunohistochemical study of 43 cases. J Urol.2001;166:1924–30. http://dx.doi.org/10.1016/S00225347(05)65722-9 . [PubMed]

167.

Shariat SF, Shalev M, Menesses-Diaz A, Kim IY, Kattan MW, Wheeler TM, et al. Preoperative plasma levels of transforming growth factor beta(1) (TGF-beta(1)) strongly predict progression in patients undergoing radical prostatectomy. J Clin Oncol. 2001;19:2856–64. [PubMed]

168.

Masuda H, Fukabori Y, Nakano K, Takezawa Y, CSuzuki T, Yamanaka H. Increased expression of bone morphogenetic protein-7 in bone metastatic prostate cancer. Prostate. 2003;54:268–74.http://dx.doi.org/10.1002/pros.10193 . [PubMed]

169.

Lehr JE, Pienta KJ. Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line. J Natl Cancer Inst. 1998;90:118–23. http://dx.doi.org/10.1093/jnci/90.2.118 . [PubMed]

170.

Chay CH, Cooper CR, Gendernalik JD, Dhanasekaran SM, Chinnaiyan AM, Rubin MA, et al. A functional thrombin receptor (PAR1) is expressed on bone-derived prostate cancer cell lines. Urology. 2002;60:760–5.http://dx.doi.org/10.1016/S0090-4295(02)01969-6 . [PubMed]

171.

Cooper CR, Chay CH, Pienta KJ. The role of alpha(v)beta(3) in prostate cancer progression. Neoplasia.2002;4:191–4. http://dx.doi.org/10.1038/sj.neo.7900224 . [PMC free article] [PubMed]

172.

Cooper CR, Chay CH, Gendernalik JD, Lee HL, Bhatia J, Taichman RS, et al. Stromal factors involved in prostate carcinoma metastasis to bone. Cancer. 2003;97(Suppl):739–47. http://dx.doi.org/10.1002/cncr.11181 . [PubMed]

173.

Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4:123–31. http://dx.doi.org/10.2174/1389450033346830 . [PubMed]

174.

Krupski T, Harding MA, Herce ME, Gulding KM, Stoler MH, Theodorescu D. The role of vascular endothelial growth factor in the tissue specific in vivo growth of prostate cancer cells. Growth Factors. 2001;18:287–302.http://dx.doi.org/10.3109/08977190109029117 . [PubMed]

175.

Chen J, De S, Brainard J, Byzova TV. Metastatic properties of prostate cancer cells are controlled by VEGF. Cell Commun Adhes. 2004;11:1–11. http://dx.doi.org/10.1080/15419060490471739 . [PubMed]

176.

Dai J, Kitagawa Y, Zhang J, Yao Z, Mizokami A, Cheng S, et al. Vascular endothelial growth factor contributes to the prostate cancer-induced osteoblast differentiation mediated by bone morphogenetic protein. Cancer Res.2004;64:994–9. http://dx.doi.org/10.1158/0008-5472.CAN03-1382 . [PubMed]

177.

Jacob K, Webber M, Benayahu D, Kleinman HK. Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone. Cancer Res. 1999;59:4453–7. [PubMed]

178.

Rosol TJ. Pathogenesis of bone metastases: role of tumor-related proteins. J Bone Miner Res. 2000;15:844–50.http://dx.doi.org/10.1359/jbmr.2000.15.5.844 . [PubMed]

179.

Denhardt DT, Giachelli CM, Rittling SR. Role of osteopontin in cellular signaling and toxicant injury. Annu Rev Pharmacol Toxicol. 2001;41:723–49. http://dx.doi.org/10.1146/annurev. pharmtox.41.1.723 . [PubMed]

180.

Fizazi K, Yang J, Peleg S, Sikes CR, Kreimann EL, Daliani D, et al. Prostate cancer cells-osteoblast interaction shifts expression of growth/survival-related genes in prostate cancer and reduces expression of osteoprotegerin in osteoblasts. Clin Cancer Res. 2003;9:2587–97. [PubMed]

181.

Zhang JH, Tang J, Wang J, Ma W, Zheng W, Yoneda T, et al. Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model. Int J Oncol. 2003;23:1043–8. [PubMed]

182.

Khodavirdi AC, Song Z, Yang S, Zhong C, Wang S, Wu H, et al. Increased expression of osteopontin contributes to the progression of prostate cancer. Cancer Res. 2006;66:883–8. http://dx.doi.org/10.1158/0008-5472.CAN-05-2816 . [PubMed]

183.

Resnick MB, Konkin T, Routhier J, Sabo E, Pricolo VE. Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study. Mod Pathol. 2005;18:511–8.http://dx.doi.org/10.1038/modpathol.3800301 . [PubMed]

184.

Montgomery E, Mamelak AJ, Gibson M, Maitra A, Sheikh S, Amr SS, et al. Overexpression of claudin proteins in esophageal adenocarcinoma and its precursor lesions. Appl Immunohistochem Mol Morphol. 2006;14:24–30.http://dx.doi.org/10.1097/01.pai.0000151933.04800.1c . [PubMed]

185.

Sommers CL, Heckford SE, Skerker JM, Worland P, Torri JA, Thompson EW, et al. Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines.Cancer Res. 1992;52:5190–7. [PubMed]

186.

Zajchowski DA, Bartholdi MF, Gong Y, Webster L, Liu HL, Munishkin A, et al. Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells. Cancer Res. 2001;61:5168–78.[PubMed]

187.

Ponnusamy MP, Lakshmanan I, Jain M, Das S, Chakraborty S, Dey P, et al. MUC4 mucin-induced epithelial to mesenchymal transition: a novel mechanism for metastasis of human ovarian cancer cells. Oncogene.2010;29:5741–54. http://dx.doi.org/10.1038/onc.2010.309 . [PMC free article] [PubMed]

188.

Wang J, Chen L, Li Y, Guan XY. Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma. PLoS One. 2011;6:e24967.http://dx.doi.org/10.1371/journal.pone.0024967 . [PMC free article] [PubMed]

189.

Sarri D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J. Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res. 2008;68:989–97.http://dx.doi.org/10.1158/0008-5472.CAN-07-2017 . [PubMed]

190.

Davies BR, Barraclough R, Davies MP, Rudland PS. Production of the metastatic phenotype by DNA transfection in a rat mammary model. Cell Biol Int. 1993;17:871–9. http://dx.doi. org/10.1006/cbir.1993.1150 . [PubMed]

191.

Andersen K, Mori H, Fata J, Bascom J, Oyjord T, Mlandsmo GM, et al. The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis. Dev Biol. 2011;352:181–90.http://dx.doi.org/10.1016/j.ydbio.2010.12.033 . [PMC free article] [PubMed]

192.

Cavallaro U. N-cadherin as an invasion promoter: a novel target for antitumor therapy? Curr Opin Investig Drugs.2004;5:1274–8. [PubMed]

193.

Cme C, Magnino F, Bibeau F, De Santa Barbara P, Becker KF, Theillet C, et al. Snail and slug play distinct roles during breast carcinoma progression. Clin Cancer Res. 2006;12:5395–402. http://dx.doi.org/10.1158/1078-0432.CCR-06-0478 . [PubMed]

194.

Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L, Bruyneel E, et al. The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell. 2001;7:1267–78.http://dx.doi.org/10.1016/S1097-2765(01)00260-X . [PubMed]

195.

Blissett AR, Garbellini D, Calomeni EP, Mihai C, Elton TS, Agarwal G. Regulation of collagen fibrillogenesis by cell-surface expression of kinase dead DDR2. J Mol Biol. 2009;385:902–11.http://dx.doi.org/10.1016/j.jmb.2008.10.060 . [PMC free article] [PubMed]

196.

Pea C, Garca JM, Silva J, Garca V, Rodrguez R, Alonso I, et al. E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations. Hum Mol Genet. 2005;14:3361–70.http://dx.doi.org/10.1093/hmg/ddi366 . [PubMed]

197.

Spoelstra NS, Manning NG, Higashi Y, Darling D, Singh M, Shroyer KR, et al. The transcription factor ZEB1 is aberrantly expressed in aggressive uterine cancers. Cancer Res. 2006;66:3893–902.http://dx.doi.org/10.1158/0008-5472.CAN-05-2881 . [PubMed]

198.

Conn G, Soderman DD, Schaeffer MT, Wile M, Hatcher VB, Thomas KA. Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line. Proc Natl Acad Sci U S A. 1990;87:1323–7.http://dx.doi.org/10.1073/pnas.87.4.1323 . [PMC free article] [PubMed]

199.

Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem. 1998;273:13313–6.http://dx.doi.org/10.1074/jbc.273.21.13313 . [PubMed]

200.

Dimmeler S, Dernbach E, Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett. 2000;477:258–62.http://dx.doi.org/10.1016/S0014-5793(00)01657-4 . [PubMed]

201.

Khurana R, Simons M. Insights from angiogenesis trials using fibroblast growth factor for advanced arteriosclerotic disease. Trends Cardiovasc Med. 2003;13:116–22. http://dx.doi. org/10.1016/S1050-1738(02)00259-1 . [PubMed]

202.

DAmore PA, Smith SR. Growth factor effects on cells of the vascular wall: a survey. Growth Factors.1993;8:61–75. http://dx.doi.org/10.3109/08977199309029135 . [PubMed]

203.

Otrock ZK, Mahfouz RA, Makarem JA, Shamseddine AI. Understanding the biology of angiogenesis: review of the most important molecular mechanisms. Blood Cells Mol Dis. 2007;39:212–20.http://dx.doi.org/10.1016/j.bcmd.2007.04.001 . [PubMed]

204.

Falcone DJ, McCaffrey TA, Haimovitz-Friedman A, Garcia M. Transforming growth factor-beta 1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor. J Cell Physiol.1993;155:595–605. http://dx.doi.org/10.1002/jcp.1041550317 . [PubMed]

205.

Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–60. http://dx.doi.org/10.1038/nm0603-653 . [PubMed]

206.

Thurston G. Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis.Cell Tissue Res. 2003;314:61–8. http://dx.doi.org/10.1007/ s00441-003-0749-6 . [PubMed]

207.

Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, et al. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol. 1999;154:385–94. http://dx.doi. org/10.1016/S0002-9440(10)65285-6 . [PMC free article] [PubMed]

208.

Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD, et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 2002;21:1505–13.http://dx.doi.org/10.1093/emboj/21.7.1505 . [PMC free article] [PubMed]

209.

He Y, Karpanen T, Alitalo K. Role of lymphangiogenic factors in tumor metastasis. Biochim Biophys Acta.2004;1654:3–12. [PubMed]

Copyright © 2013 Landes Bioscience.

In this Page

 

 

Yirong Li 30.31

NYU Langone Medical Center

How do cancer cells survive in blood circulation?

I am wondering how cancer cells escape from immunosystem and survive during blood circulation. Is there some ways to isolate cancer cells during their blood circulation?

Constantine Kaniklidis · 88.51 · 7.14 · No Surrender Breast Cancer Foundation (NSBCF)

This excellent question is essentially about two related subprocesses that constitute the “early game” of the metastatic process, namely (1) intravasation which is the endothelial transmigration of tumor cells into blood vessels in the vasculature, and (2) hematogenous survival (tumor cell survival in the circulatory system), which together are called hematogenous dissemination. I will give, below, a reasonably brief sketch of these subprocesses here (distilled from a lengthier 40+ page review of the metastatic process and cascade I recently completed [Kaniklidis, C. The Early Game of Metastasis: Tumor Cell Intravasation and Hematogenous Survival. (pending)].

The Metastatic Process: Brief summary
The multi-step process of metastasis is a complex and coordinated choreography encompassing:
(1) local infiltration of tumor cells into the surrounding/adjacent tissue (tumor cell penetration through the ECM / the basement membrane),
(2) intravasation (endothelial transmigration of tumor cells into vessels),
(3) hematogenous survival and translocation, that is, the tumor cell survival in the circulatory system and its translocation through the bloodstream to microvessels of distant tissues
(4) extravasation (exit from bloodstream, and
(5) adaption to the foreign microenvironment of distant site tissue and subsequent colonization (cell proliferation and the formation of a macroscopic secondary tumor) in competent organs.

Subprocesses (2) and (3) together, that is, the combination of intravasation + hematogenous survival constitute collectively what is known as hematogenous dissemination.

Intravasation
Tumor cells intravasation, which is the endothelial transmigration of cancer cells into vessels, involves two different types of motility: tumor cells can intravasate the blood, or the lymph vasculature, although dissemination via the hematogenous route seems to represents the major mechanism for dispersal of metastatic carcinoma cells [1], and for both routes, the process is mechanistically via interaction of tumor cells with the vascular endothelium. Note however that although the primary main route of the metastatic spread has generally until recently been the blood / circulation system, mounting evidence suggests that the lymphatic system is also a key player in cancer cell dissemination. But as to the central matter of endothelial transmigration of tumor cells, there remains indeterminacy and continued debate on the question of active versus passive dissemination, that is, as to whether (1) tumor cells actively migrate through blood and lymph vasculature as a response to phenomenon like growth factor gradients, or (2) do so passively by “crawling” into the vasculature even in the absence an active cell migration machinery, leading to a neatly phrased article title from Lance Munn and colleagues, namely “Do cancer cells crawl into vessels, or are they pushed?” [2].

There are a number of molecular phenomena that facilitate endothelial transmigration, that is, the crossing by tumor cells of the pericyte and endothelial cell barriers that constitute the microvessel walls:

(1) Twist:
Jing Yang et al. have shown in a murine breast cancer model that the transcription factor, Twist, appears to allow the step of intravasation and hence functions as an EMT-inducing transcription factor and thus a key regulator of metastasis [3], both augmenting EMT (epithelial-to-mesenchymal) transitions and promoting the rate of hematogenous intravasation.

(2) Chemoattractive Gradients and the Role of EGF / CSF-1:
In addition, a second mechanism is at play, as documented in the breast cancer context, that involves what is called chemoattractive gradients, confirmed by direct visualization using multiphoton microscopy by researchers at the Albert Einstein College of Medicine [4,5]. These direct observations demonstrated how perivascular macrophages in mammary tumors are critically involved in intravasation and hematogenous survival, and that these perivascular macrophages synergistically induce tumor cell intravasation even in the absence of local angiogenesis. These perivascular macrophages are recruited by the tumor cells to the injured site (Condeelis), inducing intravasation into the blood system via chemoattractive gradients generated by these same perivascular macrophages, with crosstalk and collaboration between the tumor microenvironment and the tumor cells at the intravasation site is enabled thorough a positive-feedback loop constituted by the reciprocal secretion of epidermal growth factor (EGF) created by the macrophages and colony stimulating factor-1 (CSF-1) by the tumor cells, jointly augmenting chemotaxis and the intravasation process, with EGF promoting tumor cell migration into the hematogenous vasculature through interaction with the EGF receptor, and CSF-1 expressed on the tumor cells functioning as a potent chemoattractant for CSF-1 receptor positive macrophages [6,7].

(3) Transforming Growth Factor-beta (TGF-beta):
In mammary carcinoma, the cytokine TGF-beta (transforming growth factor-beta) enhances intravasation via increased penetration of microvessel walls, suggesting that transient TGF-beta signalling is critical for blood-born metastasis [8].

(4) VEGF and Neoangiogenesis:
Via VEGF and neoangiogenesis, tumor cells stimulate formation of new blood vessels within the local microenvironment, with the neovasculature created by tumor cells being prone to leakiness, and ultimately facilitate intravasation [9].

Hematogenous Survival: Survival in Vasculature
But after successful invasion of the hematogenous vasculature, tumor cells must survive a perilous microenvironment of challenging hurdles that include hemodynamic shear forces turbulence, surveillance from and attack by immune cells especially natural killer (NK) cells, and lack of substratum, and entrapment-by-size in early-encountered capillary beds, which occurs usually even in the first capillary bed encountered by the tumor cells consequent to the fact that the diameter of most tumor cells is too large for passage through small capillaries [10].

A main defense for hematogenous survival used by tumor cells is using platelets as a shield, by binding coagulation factors on the platelets, forming an embolus aggregate that protects the tumor cells from immune-cell-mediated lysis / destruction, as well as decreases the level and impact of the circulation system’s hemodynamic shear forces and turbulence, to enhance survival [11-14].

In addition, tumor cells are physically shielded from the stress of blood flow, shear forces and turbulence, as well as from lysis by NK cells by two related processes:

(1) activation of the coagulation cascade and
(2) formation of platelet-rich thrombi around tumor cells in the vasculature [15-18]. The process,

in essence, is that tumor cell tissue factor triggers thrombin formation that initiates both coagulation and platelet activation, which in turn enhance metastatic spread. And Fibrin can be bound by integrins on tumor cells and on activated platelets, triggering the formation of tumor-cell–fibrin–platelet aggregates. These large aggregates and emboli then have the strength and resiliency to survive hematogenous shear forces and turbulence [17,19-22]

And it appears that the normal anti-tumor reactivity of NK immune cells can be subverted by a platelet-derived coating (called MHC Class I) which disguises the tumor cell with a pseudo-normal phenotype, exempting it from immune response and attack. [23,24].

References
1. Gupta GP, Massagué J. Cancer metastasis: building a framework. Cell 2006 Nov 17; 127(4):679-95.
2. Bockhorn M, Jain RK, Munn LL. Active versus passive mechanisms in metastasis: do cancer cells crawl into vessels, or are they pushed? Lancet Oncol 2007; 8(5):444-8.
3. Yang J, Mani SA, Donaher JL, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004 Jun 25; 117(7):927-39.
4. Condeelis J, Segall JE. “Intravital imaging of cell movement in tumours,” Nat Rev Cancer. 2003 Dec;3(12):921-30;
5. Wyckoff JB, Wang Y, Lin EY, et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 2007 Mar 15; 67(6):2649-56.
6. Wyckoff J, Wang W, Lin EY, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 2004 Oct 1; 64(19):7022-9.
7. Goswami S, Sahai E, Wyckoff JB, et al. Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer Res. 2005 Jun 15;65(12):5278-83.
8. Giampieri S, Manning C, Hooper S, Jones L, Hill CS, Sahai E. Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol. 2009 Nov;11(11):1287-96.
9. Carmeliet P, Jain RK. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 2011; 10(6):417-27.
10. Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006; 12(8):895-904.
11. Palumbo JS. Mechanisms linking tumor cell-associated procoagulant function to tumor dissemination. Semin Thromb Hemost 2008; 34(2):154-60.
12. Im JH, Fu W, Wang H, et al. Coagulation facilitates tumor cell spreading in the pulmonary vasculature during early metastatic colony formation. Cancer Res 2004; 64(23): 8613–8619.
13. Palumbo JS, Talmage KE, Massari JV, et al. Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and -independent mechanisms. Blood 2007 110(1):133–141.
14. Khamis ZI, Sahab ZJ, Sang QX. Active roles of tumor stroma in breast cancer metastasis. Int J Breast Cancer 2012; 2012:574025.
15. Palumbo JS, Talmage KE, Massari JV, et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 2005; 105:178–85.
16. [Erpenbeck L, Schon MP. Deadly allies: the fatal interplay between platelets and metastasizing cancer cells. Blood 2010; 115:3427–36.
17. Gay LJ, Felding-Habermann B. Contribution of platelets to tumour metastasis. Nat Rev Cancer 2011; 11:123–34.
18. Degen JL , Palumbo JS . Hemostatic factors, innate immunity and malignancy. Thromb Res 2012; 129 Suppl 1:S1–5.
19. Liu Y, Jiang P, Capkova K, et al. Tissue factor activated coagulation cascade in the tumor microenvironment is critical for tumor progression and an effective target for therapy. Cancer Res 2011; 71:6492–502.]
20. Labelle M, Begum S, Hynes RO. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 2011; 20:576–90.
21. Camerer E, Qazi AA, Duong DN, Cornelissen I, Advincula R, Coughlin SR. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood 2004;104:397–401.
22. [Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. Cell 2011 Oct 14; 147(2):275-92.
23. Placke T, Oergel M, Schaller M, J, et al. Platelet-derived MHC Class I confers a pseudo-normal phenotype to cancer cells that subverts the anti-tumor reactivity of natural killer immune cells. Cancer Res 2012; 72:440–8.
24. Nieswandt B, Hafner M, Echtenacher B, Mannel DN. Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res 1999 59:1295–1300.

Constantine Kaniklidis, Director of Medical Research,
No Surrender Breast Cancer Foundation (NSBCF)
European Association for Cancer Research (EACR)

Christopher Daniel Duntsch · 79.15 · 110.86 · Hybrid Bioscience, Inc., Synthetic Investments, Inc., The University of Tennessee Health Science Center

This is simple to model. Epigenetic and genetic changes result in cells having a gain of function, developing the ability to migrate, to express extracellular MMPs and related proteins, to migrate from a primary focus and get into the lymphatic system and hemotopoietic system, to up regulate cell surface proteins that allow binding a distant sites, to survive immunosurveillance (rarely) by changing HLA/B expression and other immunoprotiens and / or cell surface antigens, or to express anti-immune proteins such as FAS Ligan, to express proteins to digest and create pathways into distant organs. Much research as of late suggest the most difficult thing for a metastatic cancer cell to do is to survive in a foreign tissue, because it is a hostile environment. Other research suggests cancer stem cells adapt by creating pseudo environments around said metastatic cells that create an environment similar to the primary foci. Regardless, most research demonstrates that for every cancer stell attempting to metastasize, a small fraction succeed.

Salwa Hassan Teama · 11.71 · 2.35 · Ain Shams University

Cancer is a prominent cause of death worldwide. When cancer disseminates from the primary lesion to other vital organs, it becomes a devastating disease. In fact, it is the metastatic process that leads to 90% of cancer-related deaths Metastatic tumors are spread over the entire human body and are more difficult to remove or treat than the primary tumor. In a patient with metastatic disease, circulating tumor cells (CTCs) can be found in venous blood. These circulating tumor cells are part of the metastatic cascade.

METASTATIC PROCESS
A complex multi-step event, this biological process requires tumor cells to break free of the primary solid tumor, penetrate into the blood or lymphatic circulation, and ultimately extravasate out of the circulation and into an organ or tissue distant from the primary lesion.
Cancer occurs after a cell is progressively genetically damaged and turns into a cell bearing a malignant phenotype. These cells are able to undergo uncontrolled abnormal mitosis, which leads to an increase of these cancerous cells at that location. In absence of regular control mechanisms a heterogeneous population of cells is created and these cancerous cells together form the primary tumor. A tumor is considered benign if it lacks the ability to invade other tissue. When cells acquire the ability to penetrate and infiltrate surrounding normal tissues, the cancer is considered malignant and has the potential to metastasize.
Before tumor cells can start to metastasize, they need to succeed in stimulating angiogenesis. In this way tumor cells gain direct access to the blood circulation. This leads to improved access to the nutrients and oxygen carried by the blood, but also an opportunity for the tumor cells to enter the blood stream. An alternative route for tumor cells to end up in the blood circulation is through the lymphatic system. Tumor cells circulating in the blood can reach in principle most sites of the body, but different kinds of cancer create metastasis at different sites. For example breast cancer generally creates metastases in liver, lung and bone while prostate cancer most often metastasizes in bone. This preference is driven by two processes. The first is mechanical of nature, a large amount of CTC arrests in the first capillary bed they encounter. The second is more biological, the CTCs will form a metastasis in tissue only if they are able to extravasate out of the blood stream and the local environment is suitable for them to grow. This preference has been noted for the first time by
Stephen Paget and is known as the seed and soil hypothesis. Tumor cells thus have a preference for a certain site, and this opens an interesting research field to identify the cell surface molecules on the tumor cells and the endothelial cells aligning the capillaries at the specific sites.
More:http://www.ifcc.org/media/209935/eJIFCC%20n%203_2012_07%20Van%20Dalum.pdf
http://www.aacc.org/publications/cln/2008/november/Pages/Series1108.aspx

What are CTC?
Epithelial tumors or carcinomas represent about 80% of all cancers. CTC originate from the epithelium and are not present in the circulating peripheral blood of individuals free of neoplastic disease. Derived from clones of the primary tumor, these cells can be detected before the primary tumor is identified and often persist even after the primary tumor has been removed.
More:http://www.aacc.org/publications/cln/2008/november/Pages/Series1108.aspx

Cancer cell heterogeneity
Heterogeneity among cancer cells (Pleiomorphism) was a common and predominant features of most common solid tumors.
Heterogeneity and Clonality
Cancer cells are genetically unstable and as the population expands the probability of mutation increases. This is in turn lead to possibilities that epigenetic mechanisms could also exert selective differential selective pressure on heterogeneous cancer cell population.
Wolman (1982, 1986) considered that genetic and chromosomal instability were the potential source of genetic heterogeneity among all the tumors and that variation in local environment selective pressure and differential survival may contribute to cellular heterogeneity within expanding tumors also heterogeneity itself might permit selection and increase the number of aberrant cells responsible for tumor progression and metastasis
More:http://link.springer.com/article/10.1023%2FA%3A1010614909387?LI=true#page-2

Developing tumors must acquire nutrients to ensure their rapid growth. Second, they must escape the attack from the host immune system.
The vast majority of tumor cells that enter the circulation are rapidly eliminated by factors such as blood turbulence,natural killer cells, and macrophages.
Nitric oxide secretion by activated macrophages and endothelial cells is a major cytotoxic mediator responsible for the destruction of tumor cells passing through capillary beds. In addition, activation of apoptosis also contributes to eliminate metastatic cells.
In contrast, fibrin deposits, platelet aggregation, and adhesion around the tumor emboli may protect circulating cells from mechanical trauma, facilitate their arrest in capillary beds, and protect tumor emboli from destruction by host immunity .
More: http://www.molmed.org/content/1999/5_99/5_99_Fournier.PDF

Recent studies suggest that these phenomena could be related and that tumor cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy. This specific metabolism helps tumor cells to avoid the immune attack from the host by blocking or avoiding the immune attack. By changing their metabolism, tumor cells produce or sequester a variety of amino acids, lipids and chemical compounds that directly alter immune function therefore promoting immune evasion. A second group of metabolism-related modification targets the major histocompatibility complex-I (MHC-I) and related molecules. Tumor MHC-I presents tumor-associated antigens (TAAs) to cytotoxic Tcells (CTLs) and hence, sensitizes cancer cells to the cytolytic actions of the anti-tumor adaptive immune response. Blocking tumor mitochondrial activity decreases expression of MHC-I molecules at the tumor cell surface. And peroxynitrite (PNT), produced by tumor-infiltrating myeloid cells, chemically modifies MHC-I avoiding TAA expression in the plasma membrane.
These evidences on the role of tumor cell metabolism on tumor immune escape open the possibility of combining drugs designed to control tumor cell metabolism with new procedures of anti-tumor immunotherapy.
From tumor cell metabolism to tumor immune escape
More: http://hal.archives-ouvertes.fr/docs/00/72/67/17/PDF/villalba_et_al-IJBCB.pdf

 

Andrew Sunters · 52.96 · 160.24 · Royal Veterinary College

An interesting question, and is at the heart of one of the “hallmarks of cancer” and the “enabling characteristics” of cancer in Hanahan and Weinbergs theory of the hallmarks of cancer, which is a good starting place. One of the hallmarks is the ability to locally invade tissue and form distant metastases. Whilst there is debate about how well these cells survive, it is obvious that some do survive. Two strategies are for the cells to coat them selves with something to impare immune recognition, and this has been shown to be platelets or other cell types, as Naresh and others point out. Another is related to mutations-in tumours such as colon tumours which commonly lacking mismatch repair genes frameshift mutations generate nonsensical proteins which when expressed on the MHC can attract the interest of the immune system. Loss of the MHC or the protein machinery which process antigen peptides for MHC presentation means that these mutant peptides are not seen by the immune system, and the cancer cells can avoid detection to some degree. I would urge you to read the paper and updates and discussion:

http://www.sciencedirect.com/science/article/pii/S0092867400816839

 

 

 

Primitive Human Leukemia Cells Grown in Lab

Rogue stem cells are at the root of all leukemias

http://www.technologynetworks.com/HTS/news.aspx?ID=185776

Chronic myeloid leukemia (CML), a family of cancers that affect blood and bone marrow, is treatable in many instances. But the therapies used to keep the cancer in check have no effect on the primitive stem cells, also known as leukemia stem cells, that cause the disease in the first place – leaving patients susceptible to a relapse if they go off their meds.

Now, using cells from a patient with CML, researchers at the University of Wisconsin-Madison have found the recipe to generate cells with properties of primitive human leukemia cells in the lab. The work establishes a potent model for studying CML stem cells and identifying new drugs that could potentially provide better treatment options for leukemia.

“Treatment doesn’t eliminate the stem cells that cause chronic myeloid leukemia,” explains Igor Slukvin, a UW-Madison professor of pathology and laboratory medicine and an expert on stem cells and human blood. “We know we can treat CML, but we can’t cure it. The stem cells persist.”

Slukvin and his collaborators report their work online. By genetically reprogramming the patient’s bone marrow cells, the Wisconsin group was able to turn back the developmental clock and make all-purpose induced pluripotent stem cells (iPSCs), which capture the underlying genetic alterations driving the leukemia. Directing the induced primitive CML cells to become early blood cells, researchers were able to generate cells that share many properties of leukemia stem cells, including increased long-term survival and proliferation as well as innate resistance to drugs.

The drugs now used to treat CML are known as tyrosine kinase inhibitors; they work by stopping the progression and proliferation of the cancer cells emanating from the bone marrow. Though these drugs are very effective, patients risk relapse if they stop taking the medication. Moreover, in some patients the leukemia cells develop resistance to the drug, making it less effective.

The ability to make leukemia stem cells in the lab using reprogrammed adult bone marrow cells from patients will give scientists a new way to explore the development and progression of the disease in a laboratory dish. Currently, only mouse models of leukemia stem cells are available in the lab.

The advent of a human cell model opens the door to exploring the differences in how the disease manifests itself within different people. “The induced cells capture the genetic abnormality of the individual patient,” says Slukvin, creating the potential for more personalized treatment of the disease.

In addition, the new cell model creates a path to capture the genetic variations of the disease as it manifests itself in individual patients. Because iPSCs arise from a single cell, the selection of individual cloned iPSC cells makes it possible to capture the diversity of genetic alterations within individual stem cells and study their effects.

Because the disease transitions through chronic, accelerated and acute phases in patients, the new model may also let scientists study the progression of leukemia in the lab.

“If we make iPSCs from stored patient samples collected at different stages of diseases,” notes Slukvin, “we can produce from these iPSCs primitive leukemia cells that capture different stages of leukemia progression.”

An important potential application is that the lab-created human stem cells, like other types of synthesized stem cells or their derivatives, can also be used to learn more about leukemia stem cell survival factors and develop high-throughput drug screens where chemical compounds can be tested for efficacy and safety as potential drug candidates. Slukvin and his team, in fact, used the new system to discover a novel primitive leukemia cell survival factor, a protein known as olfactomedin 4.

Using drugs or antibodies to target the protein, which helps the primitive CML cells survive, may open an avenue to clear the leukemia and potentially cure the disease, although much work remains to be done to achieve such an outcome.

Neoplastic blood cells become pluripotent

Igor I. Slukvin

In this issue of Blood, Ye and colleagues show that CD34+ cells obtained from patients with JAK2-V617F MPDs could be reprogrammed to iPSCs and be differentiated back into hematopoietic progenitors.1

Myeloproliferative disorders (MPDs) represent a group of clonal hematopoietic progenitor/stem cell disorders associated with excess production of cells of myeloid lineages, resulting in an increase in one or more mature peripheral blood elements. This group of myeloproliferative neoplasms includes polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic myeloid leukemia (CML), and other rarer disorders. Whereas CML was the first blood cancer known to be linked to a chromosomal translocation, the JAK2-V617F mutation associated with PV was discovered only several years ago.2 However, the mechanisms of transformation by JAK2-V617F mutation are not well understood, particularly why the same mutation causes different phenotypes including PV, ET, or PMF. It has been hypothesized that disease manifestation depends on the cell affected by the original mutation, the genetic background of the patient, or the level of JAK2-V617F activity. The work by Ye et al provides a novel approach to ask and answer important questions about MPD pathogenesis, by modeling development of myeloproliferative neoplasia in vitro using patient-specific induced pluripotent stem cells (iPSCs).1

In 2006, the Yamanaka group revealed that mouse skin fibroblasts could be reprogrammed to pluripotency via ectopic expression of 4 transcription factors.3 A year later, iPSCs were obtained from human fibroblasts.4,5 These discoveries opened opportunities to generate disease-specific iPSCs carrying a particular genetic trait at the cellular level. As proof of this concept, iPSCs have been generated from fibroblasts obtained from patients with several genetic diseases including the inherited bone marrow failure syndrome Fanconi anemia.6 However, a fibroblast-based approach would not work for acquired blood diseases such as MPDs or leukemia, because cytogenetic abnormalities defining such diseases are limited to bone marrow cells in most of the cases. Several months ago, Loh et al demonstrated that iPSCs could be generated by reprogramming mobilized peripheral blood CD34+cells.7 The work published in this issue of Blood by Ye et al is the first description of successful reprogramming of CD34+ cells from patients with acquired blood diseases.1 Using retroviral vectors encoding Oct4, Sox2, Klf4, and c-Myc genes, Ye et al generated iPSCs from CD34+ cells obtained from healthy controls and MPD patients carrying the JAK2-V617F mutation. While MPD-derived iPSCs retained the JAK2-V617F mutation, they had a normal karyotype, embryonic stem cell–like phenotype, and pluripotent differentiation potential. When control and diseased iPSCs were differentiated back into CD34+CD45+ hematopoietic progenitors, the progenitors derived from MPD-iPSCs recapitulated the features of somatic CD34+ cells from which the iPSCs were originally derived. Similar to somatic MPD CD34+ cells, iPSC-derived CD34+CD45+ cells demonstrated enhanced erythropoiesis and up-regulation of genes known to be increased in PV.

This study clearly demonstrates how iPSC technology could be used to model acquired blood diseases. This technology would be of particular value for the study of blood disorders such as myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, and others for which animal models are not available or difficult to create. In addition, iPSCs carrying leukemia-specific cytogenetic translocation could be used to analyze how cancer stem cells develop. Importantly, the iPSC-based approach would be helpful in addressing the role of genetic background in manifestation of neoplastic blood disorders. Because iPSCs are capable of indefinite self-renewal, diseased blood cells can be generated continuously in the laboratory, eliminating the need for a constant supply of hematopoietic progenitors from the patients. In particular, a continuous supply of genetically diverse diseased blood cells for drug screening and discovery could be created. Because multiple types of cells can be generated from iPSCs, interaction of diseased blood cells with endothelial or stromal cells could be modeled in vitro. However, several important issues related to iPSC models of blood diseases remain to be addressed. It is known that the hematopoietic differentiation potential of iPSC lines generated from the same starting material varies significantly.8 If several clones were generated from iPSCs, which clones should be selected to make an appropriate conclusion regarding differences in differentiation potential? What would be an appropriate control for diseased versus nondiseased iPSCs? For studies of acquired blood diseases, iPSC lines can be generated from hematopoietic cells and fibroblasts or bone marrow mesenchymal stem cells (see figure). In this way, iPSCs with the same genetic background, but different in terms of presence or absence of acquired mutations, will be available for comparative analysis. The majority of disease-specific iPSCs have been made using retroviral vectors. Although the impact of exogenous expression is unclear, the possibility remains that retroviral integration and background expression of pluripotency genes may affect the behavior of iPSC-derived hematopoietic progenitors. Recently developed new reprogramming methods allowing for the generation of transgene-free iPSCs will be helpful to overcome this limitation.

http://d3md5dngttnvbj.cloudfront.net/content/bloodjournal/114/27/5409/F1.medium.gif

The use of iPSCs in modeling for acquired blood disease. Bone marrow samples from patients with acquired blood diseases can be used to obtain mutation-free mesenchymal stem cells (MSCs) and CD34+ cells or other types of hematopoietic progenitors (HPs) carrying disease-associated mutation. Alternatively, diseased peripheral blood CD34+ cells and fibroblasts or other types of cells lacking mutation from the same patient can be used. By reprogramming cells with or without genetic abnormality from the same patient, iPSCs with the same genetic background but different in expression of mutation can be generated. Using an in vitro differentiation system, hematopoietic precursors at different stages of maturation and terminally differentiated cells can be obtained for studies of disease pathogenesis. Transplantation of de novo generated cells with neoplasia-specific mutation into immunocompromised mice can be used to address emergence of blood cancer stem cells. Drug screening and discovery is another obvious and immediate benefit of iPSC technology for development of new therapies for blood diseases.

 

REFERENCES

    1. Ye Z,
    2. Zhan H,
    3. Mali P,
    4. et al

. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood2009;114(27):5473-5480.

Abstract/FREE Full Text

    1. James C,
    2. Ugo V,
    3. Le Couedic JP,
    4. et al

. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434(7037):1144-1148.

CrossRefMedline

    1. Takahashi K,
    2. Yamanaka S

. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4):663-676.

CrossRefMedlineWeb of Science

    1. Yu J,
    2. Vodyanik MA,
    3. Smuga-Otto K,
    4. et al

. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318(5858):1917-1920.

Abstract/FREE Full Text

    1. Takahashi K,
    2. Tanabe K,
    3. Ohnuki M,
    4. et al

. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861-872.

CrossRefMedline

    1. Raya A,
    2. Rodriguez-Piza I,
    3. Guenechea G,
    4. et al

. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature2009;460(7251):53-59.

CrossRefMedlineWeb of Science

    1. Loh YH,
    2. Agarwal S,
    3. Park IH,
    4. et al

. Generation of induced pluripotent stem cells from human blood. Blood 2009;113(22):5476-5479.

Abstract/FREE Full Text

    1. Choi K,
    2. Yu J,
    3. Smuga-Otto K,
    4. et al

. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 2009;27(3):559-567.

CrossRefMedlineWeb of Science

 

 

 

Gene Test Finds Which Breast Cancer Patients Can Skip Chemo

9/28/2015   Marilynn Marchione, AP Chief Medical Writer

In this Sept. 5, 2013 file photo, chemotherapy is administered to a cancer patient via intravenous drip in Durham, N.C. In a study sponsored by the National Cancer Institute and results published online Monday, Sept. 28, 2015, by the New England Journal of Medicine, a gene-activity test that was used to gauge early-stage breast cancer patient’s risk accurately identified a group of women whose cancers are so likely to respond to hormone-blocking drugs that adding chemo would do little if any good while exposing them to side effects and other health risks. (Gerry Broome, Associated Press) Many women with early-stage breast cancer can skip chemotherapy without hurting their odds of beating the disease – good news from a major study that shows the value of a gene-activity test to gauge each patient’s risk.

The test accurately identified a group of women whose cancers are so likely to respond to hormone-blocking drugs that adding chemo would do little if any good while exposing them to side effects and other health risks. In the study, women who skipped chemo based on the test had less than a 1 percent chance of cancer recurring far away, such as the liver or lungs, within the next five years.

“You can’t do better than that,” said the study leader, Dr. Joseph Sparano of Montefiore Medical Center in New York.

An independent expert, Dr. Clifford Hudis of New York’s Memorial Sloan Kettering Cancer Center, agreed.

“There is really no chance that chemotherapy could make that number better,” he said. Using the gene test “lets us focus our chemotherapy more on the higher risk patients who do benefit” and spare others the ordeal.

The study was sponsored by the National Cancer Institute. Results were published online Monday by the New England Journal of Medicine and discussed at the European Cancer Congress in Vienna.

The study involved the most common type of breast cancer – early stage, without spread to lymph nodes; hormone-positive, meaning the tumor’s growth is fueled by estrogen or progesterone; and not the type that the drug Herceptin targets. Each year, more than 100,000 women in the United States alone are diagnosed with this.

The usual treatment is surgery followed by years of a hormone-blocking drug. But many women also are urged to have chemo, to help kill any stray cancer cells that may have spread beyond the breast and could seed a new cancer later. Doctors know that most of these women don’t need chemo but there are no great ways to tell who can safely skip it.

A California company, Genomic Health Inc., has sold a test called Oncotype DX since 2004 to help gauge this risk. The test measures the activity of genes that control cell growth, and others that indicate a likely response to hormone therapy treatment.

Past studies have looked at how women classified as low, intermediate or high risk by the test have fared. The new study is the first to assign women treatments based on their scores and track recurrence rates.

Of the 10,253 women in the study, 16 percent were classified as low risk, 67 percent as intermediate and 17 percent as high risk for recurrence by the test. The high-risk group was given chemotherapy and hormone-blocking drugs. Women in the middle group were randomly assigned to get hormone therapy alone or to add chemo. Results on these groups are not yet ready – the study is continuing.

But independent monitors recommended the results on the low-risk group be released, because it was clear that adding chemo would not improve their fate.

After five years, about 99 percent had not relapsed, and 98 percent were alive. About 94 percent were free of any invasive cancer, including new cancers at other sites or in the opposite breast.

“These patients who had low risk scores by Oncotype did extraordinarily well at five years,” said Dr. Hope Rugo, a breast cancer specialist at the University of California, San Francisco, with no role in the study. “There is no chance that for these patients, that chemotherapy would have any benefit.”

Dr. Karen Beckerman, a New York City obstetrician diagnosed with breast cancer in 2011, said she was advised to have chemo but feared complications. A doctor suggested the gene test and she scored very low for recurrence risk.

“I was convinced that there was no indication for chemotherapy. I was thrilled not to have to have it,” and has been fine since then, she said.

Mary Lou Smith, a breast cancer survivor and advocate who helped design the trial for ECOG, the Eastern Cooperative Oncology Group, which ran it, said she thought women “would be thrilled” to skip chemo.

“Patients love the idea of a test” to help reduce uncertainty about treatment, she said. “I’ve had chemotherapy. It’s not pretty.”

The test costs $4,175, which Medicare and many insurers cover. Others besides Oncotype DX also are on the market, and Hudis said he hopes the new study will encourage more, to compete on price and accuracy.

“The future is bright” for gene tests to more precisely guide treatment, he said.

Source: Associated Press

http://www.biosciencetechnology.com/news/2015/09/gene-test-finds-which-breast-cancer-patients-can-skip-chemo-0?

 

 

Sequencing Metastatic Cancers Could Lead to Improved Therapies

  • Unravelling the genetic sequences of cancer that has spread to the brain could offer unexpected targets for effective treatment, according to a study (“Genomic characterization of brain metastases and paired primary tumors reveals branched evolution and potential therapeutic targets”) published in Cancer Discovery.

Scientists say they found that the original, or primary, cancer in a patient’s body may have important differences at a genetic level from cancer that has spread to the patient’s brain. This insight could suggest new lines of treatment.

Priscilla Brastianos, M.D., a neurooncologist and director of the Brain Metastasis program at Massachusetts General Hospital, points out that “brain metastases are a devastating complication of cancer. Approximately eight to ten percent of cancer patients will develop brain metastases, and treatment options are limited. Even where treatment is successfully controlling cancer elsewhere in the body, brain metastases often grow rapidly.”

She and her colleagues studied tissue samples from 104 adults with cancer. In collaboration with researchers at the Broad Institute, they analyzed the genetics of biopsies taken from the primary tumor, brain metastases, and normal tissues in each adult. For 20 patients, they also had access to metastases elsewhere in the body.

The team discovered that, in every patient, the brain metastasis and primary tumor shared some of their genetics, but there were also key differences. In 56% of patients, genetic alterations that potentially could be targeted with drugs were found in the brain metastasis but not in the primary tumor.

“We found genetic alterations in brain metastases that could affect treatment decisions in more than half of the patients in our study,” notes Dr. Brastianos. “We could not detect these genetic alterations in the biopsy of the primary tumor. This means that when we rely on analysis of a primary tumor we may miss mutations in the brain metastases that we could potentially target and treat effectively with drugs.”

This study also found that if a patient had more than one brain metastasis, each was genetically similar. The researchers used their findings to map the evolution of a cancer through a patient’s body, and draw up a phylogenetic tree for each patient to demonstrate how the cancer had spread and where each metastasis had come from.

They concluded that brain metastases and the primary tumor share a common genetic ancestor. Once a cancer cell, or clone, has moved from the primary site to the brain, it continues to develop and amass genetic mutations. The genetic similarity of the brain metastases in individual patients suggests that each brain metastasis has developed from a single clone entering the brain.

The genetic changes in brain metastases are independent of any occurring at the same time in the primary tumor, and in metastases elsewhere in the body, the researchers said. Characterization of the genetics of a patient’s primary cancer can be used to optimize treatment decisions, so that drugs that target specific mutations in the cancer can be chosen. However, brain metastases are not routinely biopsied and analyzed.

“When brain metastasis tissue is available as part of clinical care, we are suggesting sequencing and analysis of that sample,” continues Dr. Brastianos. “It may offer more therapeutic opportunities for the patient. Genetic characterization of even a single brain metastasis may be superior to that of the primary tumor or a lymph node biopsy for selection of a targeted treatment.”

http://www.genengnews.com/gen-news-highlights/sequencing-metastatic-cancers-could-lead-to-improved-therapies/81251786/

 

Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells

Kejin Hu,1Junying Yu,1Kran Suknuntha,2Shulan Tian,3Karen Montgomery,4Kyung-Dal Choi,1Ron Stewart,3James A. Thomson,3 and Igor I. Slukvin corresponding author 1,2

Blood. 2011 Apr 7; 117(14): e109–e119.        http://dx.doi.org:/10.1182/blood-2010-07-298331

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using nonintegrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non–B- or non–T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods

The advent of reprogramming technology has opened up the possibility of obtaining patient-specific induced pluripotent stem cells (iPSCs) for the study of blood diseases and for potential therapeutic applications. Although skin fibroblasts initially were used to obtain human iPSCs,1,2 several studies demonstrated successful reprogramming of CD34+ cells from CB or mobilized peripheral blood.3,4 Recently, T cells and peripheral blood mononuclear cells have also been successfully reprogrammed to iPSCs.5–7 Because genetic abnormalities are limited to hematopoietic cells in many blood diseases, successful reprogramming of blood cells represents a major advance in establishing iPSC-based models for hematologic diseases. However, because the current reprogramming methods use virus-based delivery of reprogramming factors, permanent integration of transgene and/or vector sequences into the genome, residual transgene expression, low efficiency, and slow kinetics remain the major problems surrounding this technology. To overcome these problems, several approaches have been used, including transient transfection, RNA transfection, the “PiggyBac” system, protein transduction, the Cre-LoxP excision system, minicircle vectors, and episomal plasmids.8–13 Nevertheless, limitations related to low reprogramming efficiency and/or genomic integration and complexity of genetic manipulations are still not completely resolved, and the suitability of these newest techniques for blood reprogramming remains unknown.

We recently developed a method for obtaining human iPSCs free of vector and transgene sequences from human fibroblasts using nonintegrating episomal vectors.14 In the present study, we have demonstrated that this technology could be applied to efficiently reprogram mononuclear cells from human BM and CB to pluripotency with up to 100 times more reprogramming efficiency compared with fibroblasts. The iPSCs generated by this method were free of transgene and vector sequences and were able to differentiate back to the blood, albeit with lower efficiency compared with fibroblast-derived iPSCs. Using the same protocol, we also efficiently reprogrammed a BM sample from a patient with chronic myeloid leukemia (CML), and were able to obtain transgene-free iPSCs with unique, patient-specific complex chromosomal translocation, which would be impossible to generate using currently available genetic-engineering methods. The elimination of genomic integration and background transgene expression, some of which are oncogenes, is a critical step toward advancing iPSC technology for the modeling of blood diseases and therapeutic applications.

Generation of iPSCs from mononuclear cells

Frozen CB mononuclear cells were obtained from AllCells. BM mononuclear cells from normal donors and from a patient with CML in the chronic phase were purchased from AllCells. Total BM cells intended for final disposition were also obtained from the University of Wisconsin Hospital and Clinics. Whole BM was cultured overnight in expansion medium consisting of StemSpan SFEM (StemCell Technologies) supplemented with Ex-Cyte (0.2%; Celliance) and recombinant human IL-3 (10 ng/mL), IL-6 (100 ng/mL), SCF (100 ng/mL), and FMS-related tyrosine kinase-3 ligand (Flt3L;100 ng/mL; all from PeproTech). The next day, Histopaque (Sigma-Aldrich) separation was performed to obtain the mononuclear cells. For reprogramming, BM mononuclear cells were cultured in expansion medium for 2 days (Figure 1A). After removing the dead cells by spinning over a 20% Percoll gradient (Sigma-Aldrich), 1 × 105 to 3.7 × 106viable cells were transfected with combination 19 of reprogramming factors (9 μg of pEP4EO2SET2K and pEP4EO2SEN2K and 6 μg of pCEP4M2L)14 using the CD34+ Nucleofector kit (Lonza). After an additional 2 days of culturing in expansion medium and removing the dead cells by Percoll density centrifugation, cells were transferred onto MEFs and cultured in iPSC medium. Starting from day 10, MEF-conditioned medium was used, and this was changed every day. The individual iPSC colonies were picked up for expansion from days 17-21. CB mononuclear cells were reprogrammed using the same conditions with or without the addition of 1μM thiazovivin (Stemgent).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/figure/F1/

Figure 1

Efficient generation of transgene-free iPSCs from BM mononuclear cells. (A) Schematic diagram of reprogramming protocol. (B) Kinetics of morphologic changes after blood reprogramming. (C-D) Comparison of reprogramming efficiency between blood cells and …

High efficiency of reprogramming of mononuclear cells from human BM and CB

For the production of iPSCs, BM mononuclear cells were cultured in serum-free expansion medium supplemented with human SCF, IL-3, IL-6, and Flt3L for 2 days to expand hematopoietic progenitors, and transfected with episomal vectors (combination 19)14 by nucleofection. After an additional 2 days of culture in hematopoietic medium, floating cells were transferred onto MEF feeders (Figure 1A). Cells in coculture underwent a series of changes, including morphologic transformation from round to cuboidal shape, with eventual formation of ALP+ colonies with typical ESC morphology at approximately day 17-21 of culture (Figure 1B-C). By picking up 50 of 88 high-quality iPSC colonies, we were able to obtain 47 iPSC lines in a single reprogramming experiment, representing 352 iPSC lines per 106 transfected cells. This high reprogramming efficiency of blood cells was reproduced in another experiment (Figure 1D). In contrast, we obtained only a few iPSC lines by transfection of 106 fibroblasts with episomal plasmids expressing the same set of reprogramming factors.14 To confirm superior efficiency of BM-cell reprogramming, we performed side-by-side reprogramming experiments with BM mononuclear cells and neonatal fibroblasts and evaluated the number of ALP+ colonies after the first passage. As shown in Figure 1C, reprogrammed BM mononuclear cells generated a much higher number of ALP+ colonies compared with fibroblasts in 2 independent experiments. BM iPSCs expressed the typical ESC markers OCT4, SOX2, NANOG, LIN28, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and ALP as determined by RT-PCR and flow cytometry (Figure 1E,J). We also observed up-regulation of other ESC signature genes REX1 (ZFP42), GDF3, DNMT3B, andTDGF1, which were not present in our reprogramming cocktails (Figure 1F,J). As expected, BM iPSCs lost expression of the pan-hematopoietic markers CD45 and CD43 (data not shown) and genes typically found in the BM hematopoietic cells (Figure 2C). To characterize the molecular properties of BM iPSCs, we performed a global analysis of the gene expression of blood-derived iPSCs and compared them with 5 hESC lines and 3 iPSCs derived from fibroblasts using plasmid combination 19 (DF19 iPSC lines).14 In this analysis, we also included 2 iPSC lines derived from fibroblasts using the same set of reprogramming factors but using expression vectors with different transgene arrangements (combination 6, DF6 iPSC lines).14Global analysis of gene expression confirmed the similarity of BM iPSCs to 5 hESC and 5 fibroblast iPSC lines. As shown in Figure 2A, BM iPSCs clustered together with hESCs and fibroblast-derived iPSCs, but were distant from the parental BM cells. Similarly, analysis of scatter plots shows a much tighter correlation of reprogrammed BM cells with hESCs than with parental cells (Figure 2B). The pluripotency of iPSC-derived cell lines was confirmed using a teratoma-formation assay with demonstration of derivatives of all 3 germ layers (Figure 1G). Whereas we detected an abnormal karyotype in one BM iPSC line, the majority of them maintained the normal karyotype (Figure 1H).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710002.gif

Figure 2

Global analysis of gene expression in hESCs and iPSCs generated from BM, CB, and fibroblasts and their parental cells. (A) Pearson correlation analysis of global gene expression. (B) Scatter plots comparing the global gene-expression profiles of BM9 iPSC …

Although we used single-cell subcloning to isolate cells that had lost episomal plasmids in our previous reprogramming studies,14 our initial subcloning experiments with BM iPSCs demonstrated that all clones obtained at passage 15 were transgene-free (Figure 1I). Based on these experiments, we concluded that episomal plasmids were cured from BM iPSCs faster than we had previously thought. To analyze the kinetics of episomal plasmid loss, we extracted episomal DNA at different passage from 10 random BM iPSC lines. We found that episomal DNA was lost progressively, and was absent in some samples as early as passage 3. By passage 7, we did not detect any transgene in 7 of 10 lines checked with multiple pairs of primers (Figure 1K).

We applied a similar approach to the reprogramming of mononuclear cells of CB. Although the efficiency of reprogramming was much lower, we were able to obtain 6 CB iPSCs from approximately 3 × 106transfected CB mononuclear cells. By adding small-molecule thiazovivin21 to reprogramming cultures, we were able to increase the reprogramming efficiency of CB cells by more than 10 times (Figure 3B). We obtained a total of 22 CB iPSC lines from 2 reprogramming experiments. All CB iPSCs displayed the typical hESC phenotype and gene-expression profile (Figure 3A,G). Six selected CB iPSC lines showed pluripotency in the teratoma assay and were free of episomal vectors and genomic integration CB iPSCs (Figure 3E-F).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710003.gif

Figure 3

Reprogramming of CB mononuclear cells with nonintegrating constructs. (A) All 22 CB iPSC lines express hESC-specific surface markers as indicated, and express OCT4, NANOG, and SOX2. iPSC lines checked are: CB iPSC1 to CB iPSC6, CB iPSCT1 to CB iPSCT10, …

Hematopoietic differentiation potential of blood-derived iPSCs

To test hematopoietic differentiation potential of blood-derived iPSCs, we used iPSC cocultured with OP9.22As we showed previously, hematopoietic differentiation from hESCs proceeds through the formation of a population of CD34+ cells, which includes CD34+CD43+ hematopoietic progenitors, CD34+CD31+CD43−endothelial cells, and CD34+CD31−CD43− mesenchymal cells. The 3 major populations of CD43+hematopoietic cells include CD235a/CD41a+ erythro-megakaryocytic progenitors and lin−CD43+CD45−and CD45+ multipotent progenitors.18 Earlier, we found that fibroblast-derived iPSCs and hESCs follow a very similar pattern of hematopoietic differentiation, although significant variation in blood-forming potential was observed between different iPSC clones. In addition, we noted that the generation of 4 iPSC clones was sufficient to ensure that at least one clone showed good hematopoietic differentiation potential.23 Testing of 4 BM iPSC lines revealed a similar differentiation pattern of BM iPSCs (Figure 4A). However, opposite our expectations, all 4 BM iPSCs produced fewer CD43+ hematopoietic progenitors than H1 hESCs or transgene-free fibroblast-derived iPSCs obtained using a similar method. Screening 5 additional BM iPSCs and 6 CB iPSCs failed to reveal a clone with higher differentiation potential, indicating that our blood-derived iPSCs were somewhat resistant to differentiating back to the blood in coculture with OP9 (Figure 4B). Because recent studies have suggested that lymphoid cell–derived iPSCs differentiate into blood less efficiently than CD34+ cell–derived iPSCs,7 we evaluated the rearrangement of TCR and IGH genes in our cells to determine whether our iPSCs originated from lymphoid cells. As shown in Figure 5, all 9 tested iPSC lines lacked rearrangements of TCR and IGH, indicating that their origin was non–B- or non–T-lymphoid cells.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710004.gif

Figure 4

Hematopoietic differentiation potential of BM- and CB-derived iPSCs. (A) In coculture with OP9, blood-derived iPSCs generate a CD34+ population of cells with typical subsets including CD43+hematopoietic progenitors, CD31+CD43− endothelial cells, …

Figure 5

Analyses of TCR and IGH rearrangement in BM and CB iPSC lines. (A) PCR analyses of TCRB rearrangements. (B) PCR analyses of TCRG rearrangement. (C) PCR analyses of IGH rearrangements. FR indicates framework. (D) Specimen controls. M indicates the 50-bp…

Reprogramming of BM samples with CML

Reprogramming of neoplastic BM cells provides an opportunity to address the effect of oncogenes and patient-specific chromosomal abnormalities on the development of the leukemia phenotype in vitro. However the virus-based approach for reprogramming leukemic cells is highly undesirable because of genomic integration and background expression of reprogramming factors, some of which are oncogenes. Therefore, we applied episomal vectors to generate transgene-free iPSCs from a patient with CML in the chronic phase. We picked, expanded, and froze 50 CML iPSC lines from a single reprogramming. As with normal BM, we were able to generate multiple transgene-free CML iPSC lines with typical features of pluripotent stem cells. Two transgene-free CML iPSC lines were selected and characterized (Figure 6). RT-PCR analysis revealed that both CML iPSCs retained typical BCR-ABL fusion (Figure 6H). Moreover, the CML iPSCs were found to have a complex karyotype with a 4-way translocation between chromosomes 1, 9, 22, and 11 that was present in the patient BM (Figure 7). CML iPSC lines lacked rearrangement of TCR or IGH, indicating derivation from nonlymphoid cells (Figure 5). After hematopoietic differentiation, these cell lines generated CD43+ hematopoietic progenitors, which included typical subsets of CD235a/CD41a+ erythro-megakaryocytic and lin−CD34+CD43+CD45+/− multipotent progenitors (Figure 6E). In a colony-forming assay, these differentiated CML iPSCs formed all types of hematopoietic colonies, including granulocyte, erythrocyte, monocyte, megakaryocyte and giant granulocyte-macrophage colonies (Figure 6F).

Figure 6

Generation of iPSCs from BM samples from a patient in the chronic phase of CML. (A) Flow cytometric analysis of hESC-specific marker expression in CML iPSC15 and CML iPSC17. (B) Bright-field image demonstrating typical hESC morphology of CML iPSCs growing …

Figure 7

Karyograms of BM cells from a patient with CML and the 2 iPSCs derived from these cells. Top left panel shows spectral karyogram of CML iPSC15. SKY analysis demonstrates the 4-way translocation between chromosomes 1, 9, 11, and 22, shown here by classification-colored …

Current methods for blood reprogramming rely on use of genome-integrating viruses and require several rounds of viral infection. Our data show that iPSC lines free of any transgene or vector sequence could be obtained using EBV-based episomal vectors. The efficiency of reprogramming blood cells by this method was at least 100 times higher than that of fibroblasts and was similar or higher to reported reprogramming efficiency using virus-based methods. Although previous studies have demonstrated the generation of iPSCs from blood using CD34+ cells3,4 or T cells,5–7 these methods require the isolation of progenitors or mature blood cells before reprogramming. We demonstrated that successful reprogramming could be achieved using just 106-107 mononuclear cells from CB or BM without any additional purification steps. Moreover, iPSCs with rearranged TCR or IGH may be undesirable for potential therapeutic applications and modeling of lymphoid development, because prearranged antigen-receptor genes are expressed precociously in early hematopoietic progenitors, leading to abnormal hematopoietic and lymphoid development and predisposition for lymphomas.24 A selective reprogramming of nonlymphoid cells using our method makes it possible to obtain iPSCs lacking TCR and IGH rearrangements using nonseparated mononuclear cells. Reprogramming of blood cells with episomal vectors occurs more rapidly than fibroblasts and is associated with a loss of episomal DNA in the majority of iPSC lines after 7 passages, thus eliminating the requirement for extensive additional subcloning steps. Human BM and CB represent the most accessible sources of somatic cells, with extensive and diverse archived samples available. Successful reprogramming of frozen blood samples containing less than 107 mononuclear cells in the present study clearly demonstrates the applicability of the described method for the generation of transgene-free iPSCs without rearranged antigen-receptor genes from archived samples of normal and diseased blood cells for studies of hematopoietic development, blood disease pathogenesis, and drug screening, and potentially for therapeutic purposes.

397 Induced Pluripotent Stem Cell Model of Chronic Myeloid Leukemia Revealed Olfactomedin 4 As a Novel Survival Factor for Primitive Leukemia Cells

Program: Oral and Poster Abstracts
Type: Oral

Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Strategies to Circumvent Therapy Resistance

Kran Suknuntha, MD, PhD1*, Yuki Ishii, PhD2*, Kejin Hu, PhD3*, Mcintosch Brayan, PhD4*, David T. Yang, MD5,…, Jean YJ Wang, PhD2*, James Thomson, PhD, DVM6* and Igor Slukvin, MD, PhD

56th ASH Meeting 2014   https://ash.confex.com/ash/2014/webprogram/Paper70688.html

CML is a myeloproliferative disorder characterized by unregulated growth of predominantly myeloid cells, and their subsequent accumulation in the bone marrow and peripheral blood. CML originates in hematopoietic stem cells (HSCs) with t(9;22)(q34;q11.2) translocation, which causes the constitutively expression of the BCR-ABL kinase driving the expansion of leukemic progeny. Ex vivo cultures of CML-derived cell lines and primary CML cells, ectopic expression ofBCR-ABL in CD34+ cells and mouse models have provided important insights into CML pathogenesis and led to the development of targeted therapy for this neoplastic disease with BCR-ABL thyrosine kinase inhibitor (TKI), imatinib. Despite these achievements, in many cases CML remains incurable because of innate resistance of CML leukemia stem cells (LSCs) to TKI. Thus, a definitive cure for leukemia requires identifying novel therapeutic targets to eradicate LSCs. However, the rarity of LSCs within the pool of malignant cells remains a major limiting factor for their study in humans.  Recently we generated transgene-free iPSCs from the bone marrow mononuclear cells of a patient in the chronic phase of CML (CML15 iPSCs and CML17 iPSCs) and showed that these iPSCs capture the entire genome of neoplastic cells, including the unique 4-way translocation between chromosomes 1, 9, 22, and 11 that was present in the patient bone marrow (BM) (Hu et al., Blood 2011). By differentiating CML iPSCs back to the blood we were able to generate iCD34+primitive hematopoietic cells with typical LSC properties, including HSC phenotype (lin-CD34+CD45+CD90+CD117+CD45RA-RholowALDHhigh), adhesion defect, increased long-term survival and proliferation, and innate resistance to TKI imatinib. By analyzing transcriptome of CML and normal BM iCD34+ cells treated or non-treated with imatinib we discovered OLFM4 as top-ranking gene, which is selectively upregulated by imatinib in CML, but not normal BM iCD34+ cells. Using siRNA, we demonstrated that OLFM4 knockdown potentiate imatinib-induced apoptosis and suppression of CFCs in iCD34+ cells, thereby indicating that OLFM4 is involved in regulation of imatinib resistance and survival of de novo generated primitive CML cells. To find out whether findings obtained using iCD34+ cells can be translated to somatic cells, we evaluated the expression and functional role of OLFM4 in CD34+ cells obtained from parental bone marrow and bone marrow from the several other CML patients in the chronic phase. Using immunohistochemistry and RT-PCR we confirmed OLFM4 expression in lin-CD34+ and CD34- bone marrow cells from patients. Knockdown OLFM4 with siRNA in somatic CML lin-CD34+ potentiated imatininb-induced CFC suppression, abrogated LTC-ICs and engraftment of lin-CD34+ cells in NSGW41 mice,  thereby indicating that OLFM4 is critical for survival of CML LSCs.  In summary, we showed that reprogramming leukemia cells to pluripotency and then differentiating them back into blood cells can be used as a novel approach to produce an unlimited number of primitive hematopoietic cells with LSC properties and identify of novel LSC survival factors and drug targets. We validated this approach by demonstrating the successful application of the iPSC-based platform to discover OLFM4 as a novel LSC survival factor in patients in the chronic phase of CML.

 

Scientists Discover How Cancer Cells Escape Blood Vessels
12/16/2015 –  Anne Trafton, MIT News Office    http://www.biosciencetechnology.com/news/2015/12/scientists-discover-how-cancer-cells-escape-blood-vessels

A rounded cancer cell (top left) sends out nanotubes connecting with endothelial cells. Genetic material can be injected via these nanotubes, transforming the endothelial cells and making them more hospitable to additional cancer cells. (Image: Sengupta Lab)

Scientists at MIT and Massachusetts General Hospital have discovered how cancer cells latch onto blood vessels and invade tissues to form new tumors — a finding that could help them develop drugs that inhibit this process and prevent cancers from metastasizing.

Cancer cells circulating in the bloodstream can stick to blood vessel walls and construct tiny “bridges” through which they inject genetic material that transforms the endothelial cells lining the blood vessels, making them much more hospitable to additional cancer cells, according to the new study.

The researchers also found that they could greatly reduce metastasis in mice by inhibiting the formation of these nanobridges.

“Endothelial cells line every blood vessel and are the first cells in contact with any blood-borne element. They serve as the gateway into and out of tumors and have been the focus of intense research in vascular and cancer biology. These findings bring these two fields together to add greater insight into control of cancer and metastases,” said Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology, a member of MIT’s Institute for Medical Engineering and Science, and one of the leaders of the research team.

The lead author of the paper, which appears in the Dec. 16 issue of Nature Communications, is Yamicia Connor, a graduate student in the Harvard-MIT Division of Health Sciences and Technology (HST). The paper’s senior author is Shiladitya Sengupta, an assistant professor at HST and at Harvard Medical School.

Building bridges

Metastasis is a multistep process that allows cancer to spread from its original site and form new tumors elsewhere in the body. Certain cancers tend to metastasize to specific locations; for example, lung tumors tend to spread to the brain, and breast tumors to the liver and bone.

To metastasize, tumor cells must first become mobile so they can detach from the initial tumor. Then they break into nearby blood vessels so they can flow through the body, where they become circulating tumor cells (CTCs). These CTCs must then find a spot where they can latch onto the blood vessel walls and penetrate into adjacent tissue to form a new tumor.

Blood vessels are lined with endothelial cells, which are typically resistant to intruders.

“Normal endothelial cells should not enable a cancer cell to invade, but if a cancer cell can connect with an endothelial cell, and inject signals that enable this endothelial cell to be controlled and completely transformed, then it facilitates metastasis,” Sengupta said.

The researchers first spotted tiny bridges between cancer cells and endothelial cells while using electron microscopy to study the interactions between those cell types. They speculated that the cancer cells might be sending some kind of signal to the endothelial cells.

“Once we saw that these structures allowed for a ubiquitous transfer of a lot of different materials, microRNAs were an obvious interesting molecule because they’re able to very broadly control the genome of a cell in ways that we don’t really understand,” Connor said. “That became our focus.”

MicroRNA, discovered in the early 1990s, helps a cell to fine-tune its gene expression. These strands of RNA, about 22 base pairs long, can interfere with messenger RNA, preventing it from being translated into proteins.

In this case, the researchers found, the injected microRNA makes the endothelial cells “sticky.” That is, the cells begin to express proteins on their surfaces that attract other cells to adhere to them. This allows additional CTCs to bind to the same site and penetrate through the vessels into the adjacent tissue, forming a new tumor.

“It’s almost like the cancer cells are cooperating with each other to facilitate migration,” Sengupta said. “You just need maybe 1 percent of the endothelial cells to become sticky, and that’s good enough to facilitate metastasis.”

Non-metastatic cancer cells did not produce these invasive nanobridges when grown on endothelial cells.

Erkki Ruoslahti, a professor of cell, molecular, and developmental biology at the University of California at Santa Barbara, said that the discovery is an important advance in understanding tumor metastasis.

“I found it particularly interesting that the transfer of regulatory macromolecules from tumor cells to endothelial cells via intercellular nanotubes appears to be more effective (at least over relatively short distances) than exosome-mediated transfer, which has received a lot of attention lately,” said Ruoslahti, who was not part of the research team.

Shutting down metastasis

The nanobridges are made from the proteins actin and tubulin, which also form the cytoskeleton that gives cells their structure. The researchers found that they could inhibit the formation of these nanobridges, which are about 300 microns long, by giving low doses of drugs that interfere with actin.

When the researchers gave these drugs to mice with tumors that normally metastasize, the tumors did not spread.

Sengupta’s lab is now trying to figure out the mechanism of nanobridge formation in more detail, with an eye toward developing drugs that act more specifically to inhibit the process.

“If we can first understand how these structures are formed, then we can try to design targeted therapies to inhibit their formation, which could be a promising new area for developing drugs that specifically target metastasis,” Connor said.

Source: Massachusetts Institute of Technology

 

 

 

Back-to-the-Future with Tumor Cell-Based Avatars

Researchers Looking for Alternatives to Individual Avatars Have Found Reason to Be Hopeful in Tumor-Cell Based Predictive Models

Formidable barriers, including time and expense required to breed and maintain mice engrafted with human tumor tissue, impede the widespread use of mouse avatars.

  • Mice grafted with human tumors, known as patient-derived xenograft (PDX) mice, have migrated from cancer research labs to the clinic.  But as limitations to modeling patient individual tumors in mice emerge, some investigators are turning to cell-based models and applying new methodologies to support and grow cells in culture.

Conceived by Heinz-Herbert Fiebig and colleagues at the University of Freiburg in the early 1980s, it was hoped that PDX mice would more accurately reflect an individual patient’s tumor in a model system and predict tumor responses to drug therapies.  Dr. Fiebig is the founder and CEO of Oncotest, a company that specializes in preclinical pharmacological contract research.

Since their introduction, commercial labs, including Oncotest, the Jackson Laboratory, and Discovery Group plc Horizon (Horizon), have provided access to a wide range of PDX mice made from donated tumor tissue.  The tissue, cryopreserved for future use after biopsy, serves as the basis for offering drug-testing services to researchers and pharmaceutical companies. Oncotest, for example, says it provides drug-testing services to 16 of the 20 largest pharmaceutical companies, using a library of more than 350 PDX mouse models.

And beyond PDX mouse model libraries for pharma companies, companies now offer individualized avatar mice directly to patients developed using their own tumors.  Champions Oncology provides mouse avatars directly to patients, at a cost of $10,000 to $12,000.  Proponents of these mouse models say they can facilitate the identification of a personalized therapeutic regimen, may prove more useful than genomic analysis, and eliminate the cost and toxicity associated with nontargeted chemotherapeutics.

But formidable barriers impede the widespread use of mouse avatars, scientists say, including the time and expense required to breed and maintain mice engrafted with human tumor tissue.  Development of an individualized avatar takes anywhere from three to six months, more time than some critically ill patients can survive and, in about 30% of cases, Champions points out it hasn’t been able to grow the patient’s tumor in mice.

In a study published in Cancer in April 2014, Justin Stebbing, M.D., Ph.D., and colleagues at Imperial College, London, reported that they worked with Champions to develop avatars with the company’s TumorGraft system for 22 patients with advanced sarcoma. But nine patients died before the results were ready. “Within a couple of months after their surgery or biopsy, they get chemotherapy and they pass away,” says Champions CEO Ronnie Morris. “We build the avatar, but the patient can’t use it.”

In this study, the scientists said that of implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although several patients died before completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of the 16 (81%) remaining individuals. No patients died during the TumorGraft-predicted therapy.

On the other hand the authors noted that a primary advantage of Champions’ TumorGraft is “that it allows discrimination between the different standard-of-care therapies that may be available, as well as other potential treatments not normally indicated for that tumor.

“Our increased understanding of tumor heterogeneity, even within a single subtype, means that knowing how patients with the same tumor previously responded to a particular drug is no guarantee that the current patient will respond similarly. TumorGraft overcomes this problem by helping guide oncologists to those treatments that are most likely to provide a positive clinical outcome.”

  • Search for Alternatives

Given the obstacles to using individual avatars to guide patient therapy, researchers in several laboratories are currently looking for alternatives, turning in some cases to tumor-cell based predictive models in a back to the future approach utilizing up-to-date pharmacogenomics and novel cell culture technologies to improve the longstanding odds against success culture of tumor cells from biopsied material.

The team of Jeffrey Engelman, M.D., Ph.D., director of thoracic oncology and molecular therapeutics at Massachusetts General Hospital Cancer Center, has successfully established cell culture models from biopsy samples of lung cancer patients for functional pharmacologic studies. Dr. Engelman noted that while “Genetics has been extremely useful to guiding treatment, in many cases tumor genetics are ambiguous or do not reveal a mutation that informs a therapeutic strategy. These functional pharmacologic studies can identify effective therapeutic choices even when the genetics fail to do so.”

Dr. Engelman and colleagues described in Science a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome drug resistance. Their cell culture models were derived from patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected to genetic analyses and a pharmacological screen.

With the system they could identify multiple effective drug combinations, they said.  These included the combination of ALK and mitogen-activated protein kinases (MAPK) inhibitors active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation. A combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant-resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, the authors said their strategy could help direct therapeutic choices for individual patients.

  • Several Approaches

Noting the historical difficulty of coaxing tumor cells obtained from tumor biopsies to grow in culture, Dr. Engelman told GEN that his team typically tries three or four different approaches to optimize the growth of cells from a single biopsy, including 3D culture, organoids, and feeder layers to support the best cancer cell growth.  “We want to get the biopsy to the high-throughput screening phase as quickly as possible and get the results to inform patient therapy as quickly as possible,” he said.

While the application described in their publication involved lung cancer, he notes that his lab is trying the approach on breast cancer, colorectal tumors, and melanoma.  “What’s interesting for us is that there are cancers for which no work has ever been done before,” he noted.

To date, the investigators are “not applying the cell culture technology to the clinic, but are inching closer to doing so,” Dr. Engleman said. “We are confident in the results we get from the screen and believe the data is quite valuable, but we want to make sure there is clinical outcome with therapeutics prior to having a patient enroll in a clinical trial or embark on a specific therapy.”

Dr. Engelman also believes that the technology can be commercialized, but that he is “focused on making it work.” These initial studies demonstrated success in developing NSCLC models NSCLC models in 50% of collected specimens. However, the team believes that success rates could be further improved by using biopsies acquired for specifically for cell line generation.

The authors noted that with their pharmacologic platform, they discovered several previously undescribed combinations in EGFR mutant and ALK-positive lung cancers that were validated in follow-up studies and in vivo.  They speculate that a similar approach could be explored in the future as a diagnostic test to identify therapeutic strategies for individual patients (under the auspices of an IRB-approved protocol).

In their study, they screened the cells after they became fully established cell lines, often requiring two to six months, a time frame that would make this approach less than ideal as a routine diagnostic test. But they say, their results of the program provides the  groundwork for performing screens on viable cells obtained within weeks of a biopsy using newer technologies that would permit screening of the cancer cells while still in the presence of the stroma present in the biopsy.

In a proof of concept study in Nature Methods, investigators working at MGH, Harvard Medical School, the Karolinska Institute, and other institutions showed that circulating tumor cells (CTCs) can be captured in viable form and used to establish cell cultures, potentially bypassing the need for a biopsy as a source of tumor cells to culture.

The investigators captured the CTCs using microchip technology (the Cluster-Chip) developed to capture CTC clusters independently of tumor-specific markers from unprocessed blood.  The device isolates the CTC clusters through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and, the investigators said,  even two-cell clusters can be efficiently captured.

Maheswaran et al., in Cancer Research, used the device to show that the culture of CTCs in the blood of patients with breast cancer enabled them to study patterns of drug susceptibility linked to the genetic context that is unique to an individual tumor.

The investigators established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets.

The authors noted that with optimization of CTC culture conditions, this strategy could help identify the best therapies for individual cancer patients over the course of their disease.

These and other investigators believe, that cell-based methods, once optimized, could bypass the need for whole animal cancer avatars, providing another resource to help inform the choice of therapies likely to be effective in a given patient.

http://www.genengnews.com/insight-and-intelligence/back-to-the-future-with-tumor-cell-based-avatars/77900518

 

 

 

 

 

 

Linking Phenotypes and Modes of Action Through High-Content Screen Fingerprints

The Use of High-Content Screening as a Powerful Technique for Monitoring Phenotypic Responses

Felix Reisen, Amelie Sauty de Chalon, Martin Pfeifer, Xian Zhang, Daniela Gabriel, Paul Selzer

Fig. 2. Phenotypes of snuclei are colored purple, the cytoplasm redix tool compounds targeting different cellular compartments. In all figures nuclei are colored purple, the cytoplasm red.

  • In today’s drug discovery campaigns we observe a clear trend toward more complex assay environments. While target-based high-throughput screening (HTS) still plays an important role, phenotypic screening techniques are gaining importance. Phenotypic screening assays are believed to be more closely linked to a given disease state than target-based approaches where the molecular hypothesis might not be relevant for disease pathogenesis.

One approach to phenotypic drug discovery is high-content screening (HCS), an HTS technique based on automated microscopy. HCS allows for highly multiplexed assay readouts that can be used to simultaneously assay several modes of action or toxicity. Additionally, HCS enables screening in a controlled and disease-relevant environment by even using patient-derived cell cultures.

While there are many advantages to phenotypic screening, additional knowledge about the targets being modulated to bring about the desired phenotype can be highly beneficial, for example, in lead optimization, by helping interpretation of structure activity relationships. In addition, knowledge of the target can also help to identify related targets that may bring about challenges in designing selective lead molecules.

Various techniques have been developed to support target identification for compounds active in phenotypic assays. These include approaches such as affinity chromatography, biochemical fractionation, radioactive ligand binding assays, drug affinity responsive target stability. Alternative approaches are based on in vivo chemical genomic assays developed in yeast Saccharomyces cerevisiae or in silico approaches using historic knowledge about compound target associations. In silico methods predict possible targets for a compound by comparing the similarity of the compound’s profile (using chemical similarity, gene expression profile, or HCS experiments) to those of previously characterized compounds with known target.

For the rest of the story, click here.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. GEN presents here one article “Linking Phenotypes and Modes of Action Through High-Content Screen Fingerprints.” Authors of the paper are Felix Reisen, Amelie Sauty de Chalon, Martin Pfeifer, Xian Zhang, Daniela Gabriel, and Paul Selzer.

http://www.genengnews.com/insight-and-intelligence/linking-phenotypes-and-modes-of-action-through-high-content-screen-fingerprints/77900527/

 

 

Immuno-Oncology Landscape Expands

New Techniques Enable Closer Look into Genetic & Cellular Alterations in Tumor Microenvironment

  • For years, researchers and physicians have suspected, and have worked to demonstrate, how the immune system affects susceptibility to, defense against, and progression of certain cancers. It is now understood that the immune system has the ability to influence the fate of developing cancers by not only functioning as a tumor promoter that facilitates cellular transformation, promotes tumor growth, and sculpts tumor cell immunogenicity, but also as an extrinsic tumor suppressor that either destroys developing tumors or restrains their expansion.

In the last few decades, drugs, biologicals, and vaccines targeting certain attributes of the immune system, known as immunotherapeutics, have become available, and emerging clinical data suggest that cancer immunotherapy is likely to become a key part of the clinical management of cancer for years to come.

Although immunotherapies represent a major step forward in cancer care, providing in some cases unprecedented response rates, there is still much work to do to discover new druggable targets, find biomarkers to predict response, as well as gain deeper understanding of why some cancer types are incredibly responsive to immunotherapeutic treatments while others are not.

  • How Immunotherapies Work

Figure 1.  Inhibitory costimulatory checkpoints are a natural immune mechanism for self-tolerance and minimization of collateral tissue damage. Inhibitory checkpoint receptors such as PD-1, LAG-3, TIM-3, and CTLA-4 are expressed by T cells, and their ligands are expressed by macrophage and dendritic cells. Tumor cells can express multiple inhibitory ligands to repress T-cell function and thereby evade clearance by the immune system.

  • A deeper understanding of cancer as a disease requires the acknowledgement of its inherent heterogeneity. As with the cancer cells within a tumor, the immunological microenvironments in which they grow are similarly heterogeneous. Emerging and well-established scientific tools and techniques for the analysis of cancer cells, immune cells and their microenvironment can be combined to yield new insights into the nature of tumorigenesis, immune system recruitment, and treatment optimization.In general, immunotherapies direct an individual’s immune system to fight cancer by either stimulating it to attack cancer cells or by introducing manufactured immune system components to augment immune function. Immunotherapy treatments work in different ways. Some boost the body’s immune system in a very general way. Others help train the immune system to attack cancer cells specifically.
  • On an immuno-oncological level, the genetic and cellular alterations that define a cancer cell provide the immune system with the means to be recruited to the tumor and generate T-cell responses to recognize and eradicate those cells. Elimination of cancer by T cells is only one step in the cancer immunity cycle. T-cell activation is controlled by both stimulatory and inhibitory checkpoints. Tumors use the expression of inhibitory ligands as a mechanism of suppressing cytotoxic T-cell response and inducing an immunosuppressive environment.
  • Identification of specific cancer T-cell inhibitory signals, such as PD-L1, has prompted the development of a new class of cancer immunotherapy that specifically hinders immune effector inhibition, reinvigorating and potentially expanding preexisting anticancer immune responses (Figure 1).
  • The presence of environment-altering immunosuppressive innate myeloid lineages in the tumor microenvironment may further explain the limited activity observed with previous immune-based therapies and why these therapies may be more effective in combination with agents that target other steps of the cycle.
  • Understanding the Tumor and Its Microenvironment

In addition, the presence and quantity of various immune cell types in the tumor microenvironment may have prognostic value. Many scientists believe that a deepening appreciation of oncology genomics and the quantity and type of antigens expressed by the tumor cells, when coupled with an analysis of the patient’s immune system, will greatly progress the field and unlock the next generation of immunotherapies.

Flow cytometry and immunohistochemistry are established tools for the labeling and analysis of immunological and oncology cellular components. New techniques are likewise becoming more widely used that enable simultaneous detection of proteins and nucleic acids at single-cell resolution.

New Cellular Analysis Tools

  • eBioscience, a business unit of Affymetrix, has recently expanded commercialization of two such novel assays that provide exciting new technologies in the armament of cellular analysis techniques for immuno-oncology research. The first is PrimeFlow™ RNA Assay, which is the only commercially available assay for the simultaneous detection of RNA and protein expression within millions of cells at single-cell resolution using a standard flow cytometer. The assay is compatible with cell surface and intracellular antibody staining, using traditional fluorochromes for multiparameter cellular analysis.
  • With this technology an immune-oncology researcher could explore gene expression heterogeneity among different rare tumor-infiltrating immune cell subsets with single-cell resolution and without laborious cell sorts, as well as compare kinetics of both RNA and protein in the same cell.

http://www.genengnews.com/Media/images/Article/thumb_eBioscience_Fig21361229223.jpg

Figure 2. The PrimeFlow RNA Assay workflow contains several steps: antibody staining, fixation and permeabilization including intracellular staining if desired, followed by target hybridization with a target-specific probe set containing 20 to 40 oligonucleotide pairs. Next, branched DNA signal amplification is achieved through a series of sequential hybridization steps consisting of pre-amplifiers, amplifiers, and labeled probes, followed by detection by flow cytometric analysis. This results in excellent specificity, low background, and a high signal-to-noise ratio. For simplicity, two RNA targets are shown in the schematic above (red and green), and only 3 of the 20 to 40 oligonucleotide target probe pairs per target RNA are shown.

http://www.genengnews.com/gen-articles/immuno-oncology-landscape-expands/5577/

  • S. Shalapour et al. recently published a study in the journal Nature (April 29, 2015) applying these techniques to mouse models of castrate-resistant prostate cancer demonstrating that the presence of a very specific and rare (0.04–3% of total) B cell population in the tumor microenvironment correlates to a immunotherapeutic response allowing a CTL-dependent eradication of oxaliplatin-treated tumors.
  • ViewRNA® In Situ Hybridization (ISH) Cell and Tissue Assays comprise the second new technique from eBioscience. Similar to the PrimeFlow RNA assay, but compatible with microscopy, these assays enable the visualization of single-copy RNA transcripts within adherent and suspended single cells or single cells in tissue sections, and in the case of ViewRNA ISH Tissue Assays, the spatial separation of tumor subclones by phenotypic RNA expression. Similarly, this technique can be used to visualize and quantitate cellular and molecular attributes of tumor-infiltrating immune cells to elucidate biomarkers of resistance and response. Leveraging these novel cell analysis approaches, immuno-oncology researchers can analyze cellular diversity in the tumor microenvironment as well as the diversity of immune cell responses at a single-cell level.
  • Breakthrough responses to new immunotherapies are stimulating a renewed interest in basic immune biology. With our quest to develop strategies to harness the human immune response against cancer to achieve durable responses and/or complete eradication of cancer in patients safely, we must explore multiple approaches simultaneously. Which immune checkpoints can be manipulated? Are there dual therapies that can be applied to improve responses? Are there biomarkers inherent to the immune system in general, the specific tumor and the tumor microenvironment that can be used to stratify responders?
  • Multiple approaches to cancer therapy exist, and few are as complicated as immune-based therapy. That being said, few therapies in recent history have demonstrated such extraordinary and durable responses for the patients who do respond. As such, many believe that this will be an intensifying area of research and clinical focus for years to come.

 

 

New Research for Prostate Cancer Therapies

Dr. Glenn Bubley has been treating patients with prostate cancer for more than 25 years.

“When a patient’s diagnosis is latter-stage prostate cancer, the standard treatment is androgen deprivation therapy [ADT],” says Bubley, Director of the Genitourinary Cancer Program in the Cancer Center at Beth Israel Deaconess Medical Center. “ADT works by lowering testosterone production and thereby depriving prostate tumors of the ‘fuel’ that helps them grow.”

But, he adds, although this hormone therapy is almost always effective, all tumors eventually grow resistant to ADT — and cancer recurs. Over the past two years, Bubley has been part of a BIDMC scientific team that has been testing a targeted treatment alternative for late-stage prostate cancer using a unique type of study known as a “Co-Clinical Trial.”

This new approach to clinical research — in which specially-created mouse models with genetic mutations are matched with tumor tissue from human cancer patients in order to test new therapies — was developed by BIDMC Cancer Center Director Pier Paolo Pandolfi, MD, PhD.

“Targeted therapies are designed to attack cancers by pinpointing the genes and genetic mutations that underlie diseases,” says Pandolfi (right). “The problem is that cancer cells are genetically complex, sometimes containing hundreds of genetic mutations. We needed to develop a way to cut down on all this ‘genetic noise’ to get at the root of the disease. The Co-Clinical Trial enables us to streamline and expedite the process in order to more quickly test a variety of new cancer drugs.”

Here’s how it works: In the Co-Clinical Trial, human participants are matched with animal models that have been genetically engineered to carry different combinations of just a few major human prostate cancer genes.

“When the animals develop tumors — just as the human patients did — they will receive the same therapies as the patients receive,” says Bubley (right). But, he adds, because each animal has only a few mutations, the researchers will be able to quickly assess which treatments are effective and which are not — and will be able to go back and adjust treatment accordingly for the human patients.

A particular advantage to this approach, say Bubley and Pandolfi, will be the ability to test combinations of different drugs to treat prostate cancer and overcome ADT resistance.

“Going forward, we think that combinations of targeted and conventional therapies may prove to be effective, particularly for drug-resistant disease,” says Bubley. “And the only realistic way to be able to quickly test numerous different drug combinations will be through the Co-Clinical Trial process.”

http://www.bidmc.org/YourHealth/BIDMCInteractive/BIDMC-Bulletin/Archives/Nov15/Leading-Edge.aspx#sthash.vUwp5TAi.dpuf

 

 

 

 

Nanocarriers May Carry New Hope for Brain Cancer Therapy

Fri, 11/20/2015 – DOE/Lawrence Berkeley National Laboratory

http://www.dddmag.com/news/2015/11/nanocarriers-may-carry-new-hope-brain-cancer-therapy

 

At only 20 nanometers in size and featuring a unique hierarchical structure, 3HM nanocarriers meet all the size and stability requirements for effectively delivering therapeutic drugs to brain cancer tumors. Credit: Ting Xu, Berkeley Lab

 

Glioblastoma multiforme, a cancer of the brain also known as “octopus tumors” because of the manner in which the cancer cells extend their tendrils into surrounding tissue, is virtually inoperable, resistant to therapies, and always fatal, usually within 15 months of onset. Each year, glioblastoma multiforme (GBM) kills approximately 15,000 people in the United States. One of the major obstacles to treatment is the blood brain barrier, the network of blood vessels that allows essential nutrients to enter the brain but blocks the passage of other substances. What is desperately needed is a means of effectively transporting therapeutic drugs through this barrier. A nanoscience expert at Lawrence Berkeley National Laboratory (Berkeley Lab) may have the solution.

 

Ting Xu, a polymer scientist with Berkeley Lab’s Materials Sciences Division who specializes in self-assembling bio/nano hybrid materials, has developed a new family of nanocarriers formed from the self-assembly of amphiphilic peptides and polymers. Called “3HM” for coiled-coil 3-helix micelles, these new nanocarriers meet all the size and stability requirements for effectively delivering a therapeutic drug to GBM tumors. Amphiphiles are chemical compounds that feature both hydrophilic (water-loving) and lipophilic (fat-loving) properties. Micelles are spherical aggregates of amphiphiles.

 

In a recent collaboration between Xu, Katherine Ferrara at the University of California (UC) Davis, and John Forsayeth and Krystof Bankiewicz of UC San Francisco, 3HM nanocarriers were tested on GBM tumors in rats. Using the radioactive form of copper (copper-64) in combination with positron emission tomography (PET) and magnetic resonance imaging (MRI), the collaboration demonstrated that 3HM can cross the blood brain barrier and accumulate inside GBM tumors at nearly double the concentration rate of current FDA-approved nanocarriers.

 

“Our 3HM nanocarriers show very good attributes for the treatment of brain cancers in terms of long circulation, deep tumor penetration and low accumulation in off-target organs such as the liver and spleen,” says Xu, who also holds a joint appointment with the UC Berkeley’s Departments of Materials Sciences and Engineering, and Chemistry. “The fact that 3HM is able to cross the blood brain barrier of GBM-bearing rats and selectively accumulate within tumor tissue, opens the possibility of treating GBM via intravenous drug administration rather than invasive measures. While there is still a lot to learn about why 3HM is able to do what it does, so far all the results have been very positive.”

 

Glial cells provide physical and chemical support for neurons. Approximately 90-percent of all the cells in the brain are glial cells which, unlike neurons, undergo a cycle of birth, differentiation, and mitosis. Undergoing this cycle makes glial cells vulnerable to becoming cancerous. When they do, as the name “multiforme” suggests, they can take on different shapes, which often makes detection difficult until the tumors are dangerously large. The multiple shapes of a cancerous glial cell also make it difficult to identify and locate all of the cell’s tendrils. Removal or destruction of the main tumor mass while leaving these tendrils intact is ineffective therapy: like the mythical Hydra, the tendrils will sprout new tumors.

 

Although there are FDA approved therapeutic drugs for the treatment of GBM, these treatments have had little impact on patient survival rate because the blood brain barrier has limited the accumulation of therapeutics within the brain. Typically, GBM therapeutics are ferried across the blood brain barrier in special liposomes that are approximately 110 nanometers in size. The 3HM nanocarriers developed by Xu and her group are only about 20 nanometers in size. Their smaller size and unique hierarchical structure afforded the 3HM nanocarriers much greater access to rat GBM tumors than 110-nanometer liposomes in the tests carried out by Xu and her colleagues.

 

“3HM is a product of basic research at the interface of materials science and biology,” Xu says. “When I first started at Berkeley, I explored hybrid nanomaterials based on proteins, peptides and polymers as a new family of biomaterials. During the process of understanding the hierarchical assembly of amphiphilic peptide-polymer conjugates, my group and I noticed some unusual behavior of these micelles, especially their unusual kinetic stability in the 20 nanometer size range. We looked into critical needs for nanocarriers with these attributes and identified the treatment of GBM cancer as a potential application.”

 

Copper-64 was used to label both 3HM and liposome nanocarriers for systematic PET and MRI studies to find out how a nanocarrier’s size might affect the pharmacokinetics and biodistribution in rats with GBM tumors. The results not only confirmed the effectiveness of 3HM as GBM delivery vessels, they also suggest that PET and MRI imaging of nanoparticle distribution and tumor kinetics can be used to improve the future design of nanoparticles for GBM treatment.

 

“I thought our 3HM hybrid materials could bring new therapeutic opportunities for GBM but I did not expect it to happen so quickly,” says Xu, who has been awarded a patent for the 3HM technology.

 

 

 

 

 

Read Full Post »

P13K delta-gamma anticancer agent

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RP 6350, Rhizen Pharmaceuticals S.A. and Novartis tieup for Rhizen’s inhaled dual Pl3K-delta gamma inhibitor

by DR ANTHONY MELVIN CRASTO Ph.D

 

(A)           and                         (Al)                  and                (A2)

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (Compound A1 is RP 6350).

 

str1

 

RP 6350, RP6350, RP-6350

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

mw 415

Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation  In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PATENTS……..WO 11/055215 ,  WO 12/151525.

  • Antineoplastics; Small molecules
  • Mechanism of Action Phosphatidylinositol 3 kinase delta inhibitors; Phosphatidylinositol 3 kinase gamma inhibitors
  • Phase I Haematological malignancies
  • Preclinical Multiple myeloma

 

Swaroop K. V. S. Vakkalanka,
COMPANY Rhizen Pharmaceuticals Sa

https://clinicaltrials.gov/ct2/show/NCT02017613

 

PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

Company Rhizen Pharmaceuticals S.A.
Description Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Molecular Target Phosphoinositide 3-kinase (PI3K) delta ; Phosphoinositide 3-kinase (PI3K) gamma
Mechanism of Action Phosphoinositide 3-kinase (PI3K) delta inhibitor; Phosphoinositide 3-kinase (PI3K) gamma inhibitor
Therapeutic Modality Small molecule

 

Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
 Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
 1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

POSTER SESSIONS
Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
  • Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
  • RP6530, a novel, small molecule PI3K delta/gamma
  • Activity and selectivity of RP6530 for PI3K delta and gamma isoforms

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.

Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.

Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.

Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Disclosures:Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership.Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

 

PI3K Dual Inhibitor (RP-6530)


Therapeutic Area Respiratory , Oncology – Liquid Tumors , Rheumatology Molecule Type Small Molecule
Indication Peripheral T-cell lymphoma (PTCL) , Non-Hodgkins Lymphoma , Asthma , Chronic Obstructive Pulmonary Disease (COPD) , Rheumatoid Arthritis
Development Phase Phase I Rt. of Administration Oral

Description

Rhizen is developing dual PI3K gamma/delta inhibitors for liquid tumors and inflammatory conditions.

Situation Overview

Dual Pl3K inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases manifested by a reduction in neutrophilia and TNF in response to LPS. Scientific evidence for PI3-kinase involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches, which makes it a potential target for treatment of respiratory disease. Resistance to conventional therapies such as corticosteroids in several patients has been attributed to an up-regulation of the PI3K pathway; thus, disruption of PI3K signaling provides a novel strategy aimed at counteracting the immuno-inflammatory response. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ? and ? isoforms would be expected to attenuate the progression of immune response encountered in most variations of airway inflammation and arthritis.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.

 

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.png

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.pngtps:/

Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.

 

Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.

PATENT
WO2014195888, DUAL SELECTIVE PI3 DELTA AND GAMMA KINASE INHIBITORS

This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above

Figure imgf000094_0001

15 14 10 12 12a

REFERENCES
April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
  • 01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
  • 18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
  • 18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014

 

WO2011055215A2 Nov 3, 2010 May 12, 2011 Incozen Therapeutics Pvt. Ltd. Novel kinase modulators
WO2012151525A1 May 4, 2012 Nov 8, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO2013164801A1 May 3, 2013 Nov 7, 2013 Rhizen Pharmaceuticals Sa Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
US20110118257 May 19, 2011 Rhizen Pharmaceuticals Sa Novel kinase modulators
US20120289496 May 4, 2012 Nov 15, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO 2011055215

 

 

Read Full Post »

Gene Amplification and Activation of the Hedgehog Pathway

Curator: Larry H Bernstein, MD, FCAP

 

Hedgehog signaling pathway: an overview

​​Proteins of the Hedgehog (Hh) family are powerful signaling molecules that act as morphogens during development in both vertebrates and invertebrates.

Hh was first discovered in a genetic screen performed on cuticle embryo, that aimed to understand the body segmentation of Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). In this screen, mutant embryos for Hh developed as prickly little balls similar to a hedgehog (so the name of the protein).

The core components of the Hh pathway were initially identified in Drosophila​ and are conserved in vertebrates, where the pathway has maintained the same mechanisms of action through species (although with some exceptions). Most interesting, deregulation of the Hh pathway leads to developmental defects and cancer.

Hh signaling cascade in Drosophila

Hh maturation, release and movement

Hh is first synthesized as a precursor. It undergoes autoproteolytic cleavage where a cholesterol molecule (Porter et al., 1996), and a palmitic acid molecule (Ingham and McMahon, 2001) are added to the final product. The primary role of these modifications is to direct the mature signal to interact with a set of cellular components that are responsible of the Hh secretion, movement and reception. In particular, cholesterol is involved in Hh trafficking and movement (Gallet et al., 2003), whereas palmitoylation in Hh signaling (Chamoun et al., 2001; Liu et al., 2007).

Once Hh is modified, it is ready to be secreted from the cells (Burke et al., 1999). After secretion, Hh interacts with the extracellular matrix and has to find a way to move through it to reach the receiving cells, forming a concentration gradient.

Several models have been proposed to explain how Hh can move far from its source, such as its movement inside a special structures called lipoprotein particles (Bolanos-Garcia and Miguel, 2003; Olofsson et al., 1999) and through its interaction with heparan sulphate proteoglycans (HSPGs) (Jia et al., 2003; Nakato et al., 1995).

At the plasma membrane

Hh signal transduction is initiated at the plasma membrane where Hh interacts with its 12 transmembrane protein receptor Patched (Ptc) (Ingham and McMahon,2001). The interaction between Hh and Ptc is facilitated by the Ihog/Cdo family of coreceptors (Zhang et al., 2010). The binding between Ptc and Hh has two main important roles:

  1. Limiting the spreading of Hh: the binding between Hh and Ptc results in their internalization, targeting Hh to lysosomes for degradation (Gallet and Therond, 2005).
  2. Increase of Smoothened (Smo) expression and activation: (Chen and Struhl, 1996; Denef et al., 2000; Lum et al., 2003; Taipale et al., 2002) this gives rise to a cascade of signal transmission that function to regulate the transcription factor Cubitus interruputs (Ci) (Alexandre et al., 1996; Méthot and Basler, 1999).

Once Hh binds Ptc, the seven-pass transmembrane protein Smo undergoes several phosphorylation events (Hh dose-dependent) (Fan et al., 2012). Smo phosphorylation occurs at its cytoplasmic tail (C-tail) which contains several phosphorylation sites of PKA, CK1, GSK3 (Zhang et al., 2004). The main consequences of Smo phosphorylation are:

  1. Promoting Smo cell surface expression by inhibiting ubiquitation-mediated endocytosis and degradation (Fan et al., 2012).
  2. Controlling Smo conformation, which occurs on the C-tail itself of the Smo dimer that lead to an INACTIVE (C-tails far from each other in the absense of Hh) or ACTIVE (C-tails opening and approach in the presence of Hh). This conformation change is exclusively due to the phosphorylation events (Zhao et al., 2007).

Within the cytoplasm

The activation or inhibition of the Hh pathway is regulated by a multi-protein complex (Hh signaling complex, HSC) downstream of Smo. The components of the HSC complex are:

  • The transcription factor Ci
  • The serine/threonine kinase Fused (Fu)
  • ​The kinesin-like molecule Costal 2 (Cos2), which also binds to PKA, CK1 and GSK3, all implicated in the Hh signaling pathway (Aza-Blanc et al., 1997).
  • Suppressor of fused (Sufu)

The HSC complex is associated with microtubules in the absense of Hh (Robbins et al., 1997; Sisson et al., 1997; Stegman et al., 2000). In the presence of Hh, the complex dissociates from the microtubule and the Cos-Fu-Ci complex interacts with the C-tail of Smo (Hooper, 2003; Ingham et al., 1991; Lum et al., 2003; Ogden et al., 2003; Ruel et al., 2003) whereas the Sufu-Ci complex remains cytoplasmic.

Both Cos-Fu-Ci and Sufu-Ci complexes regulate the status of the transcription factor Ci. Ci is a 155 kDa protein (Ci-FL, full length) that contains a zinc finger domain responsible for its DNA binding (Slusarski et al., 1995). Ci is converted to an ACTIVE FORM (Ci-A, 155 kDa) responsible for target gene activation in the presence of Hh, or to a REPRESSOR FORM (Ci-R, 75 kDa), that still bind DNA but inhibit the pathway in the absence of Hh.

Control of the active/inactive form of Ci is mediated by phosphorylation events that are mainly under the control of Cos2. In the absense of Hh, Cos2-Fu-Ci and Sufu-Ci complexes promote Ci-R formation preventing its activation (Robbins et al., 1997; Sisson et al., 1997; Wang et al., 2000; Wang and Holmgren, 2000; Wang and Jiang, 2004; Zhang et al., 2004). In the presence of Hh, the Cos2-Fu-Ci complex interacts with the C-tail of Smo domains, which is regulated by Cos2 phosphorylation (Liu et al., 2007; Nybakken et al., 2002; Ranieri et al., 2012; Ranieri et al., 2014; Ruel et al., 2007), promoting Ci-A formation and consequent pathway activation.

Figure 1. Drosophila Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs, such as Dally and Dally-like (Dlp). In the absense of Hh, Ptc inhibits Smo allowing Ci to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Ci to a partial proteolytic cleavage (mediated by Slimb/β​TRCP) to generate the repressor form (Ci-R). Binding of Hh to its receptor Ptc and co-receptor Ihog releases Ptc inhibition on Smo, which undergoes phosphorylation mainly by PKA and CK1. Consequently, Smo accumulates at the cell surface recruiting the Cos2-Fu-Ci complex. Here, according to the amount of Hh received by the cell, phosphorylation events on Cos2 and Fu regulate the activation of Ci and therefore of the pathway itself.

Hh signaling orthologues in vertebrates

In mammals, there are three paralogous Hh genes: Sonic hedgehog (Shh, the most broadly expressed and best studied Hh molecule), Indian hedgehog (Ihh, primarily involved in bone differentiation) and Desert hedgehog (Dhh, involved in gonad differentiation).

The main difference between Hh signaling in Drosophila and vertebrates is the requirement for the vertebrate intraflaggular transport (IFT), which consists of large multisubunits complexes that are responsible for the bidirectional transport of proteins between the base and the tip cilia (Huangfu et al., 2003).

Both Ptc and Smo can localize to primary cilia in a mutually exclusive way, where the binding of Shh to Ptc allows Smo to move into the cilium, promoting pathway activation through the Gli transcription factors (Rohatgi et al., 2007).

Main similarities and differences between Drosophila and vertebrate Hh signaling are:

  • The Smo structure is highly conserved between Drosophila and vertebrates. Interestingly, the phospho-sites on the Smo C-tail and their dimerization mechanism is conserved as well, though the kinases involved are slightly different (Chen et al., 2011).
  • There are three Ci homologues known as Gli1, Gli2 and Gli3. Gli1 and Gli2 are transcriptional activators, whereas Gli3 functions as a transcriptional repressor (Ding et al., 1998; Matise et al., 1998; Park et al., 2000; Tempé et al., 2006).
  • Unlike Drosophila Sufu, vertebrate Sufu has a central and very important role in the Shh pathway (Svä​rd et al., 2006). However, the two proteins share high sequence homology (Merchant et al., 2004; Stone et al., 1999).
  • The Cos2 homologues, kif7 and kif27, have conserved their negative role within the pathway by controlling Gli’s function and abundance (Cheung et al., 2009; Tay et al., 2005; Wilson et al., 2009).
  • Mammalian Fu can associate to kif27 and being involved in ciliogenesis, while a compensatory Fu kinase, associated with kif7, is necessary for Hh signaling (Wilson et al., 2009).

These suggest an evolutionary conservation in the Shh intracellular cascade, though further studies are necessary to better understand the molecular functions of the protein involved.

 

 

http://a.static-abcam.com/CmsMedia/Media/mammal.png

Figure 2. Mammal Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs (such as GPC3, GPC4 and GPC6). In the absence of Hh, Ptc inhibits Smo allowing Gli to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Gli to a partial proteolytic cleavage (mediated by β​-TRCP) to generate the repressor form (Gli-R). In the presence of Hh, binding of Hh to its receptor Ptc and co-receptor Cdo releases Ptc inhibition on Smo, which undergoes phosphorylation by mainly CK1 and GRK2. Consequently, Smo accumulates at the cell surface (within the cilia). Sufu is the major negative regulator of the pathway (kif7 is a minor one). In the presence of Hh, Sufu destabilization and degradation allows the release of its repression on Gli, with consequent pathway activation.

References

  • Alexandre C, Jacinto A and Ingham PW (1996). Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev 10, 2003–2013.
  • Aza-Blanc P1, Ramírez-Weber FA, Laget MP, Schwartz C and Kornberg TB (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell, 89, 1043–1053.
  • Bolanos-Garcia VM and Miguel RN (2003). On the structure and function of apolipoproteins: more than a family of lipid-binding proteins. Prog Biophys Mol Biol, 83, 47–68.
  • Burke R, Nellen D, Bellotto M, Hafen E, Senti KA, Dickson BJ and Basler K (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell, 99, 803–815.
  • Chamoun Z, Mann RK, Nellen D, von Kessler DP, Bellotto M, Beachy PA and Basler K (2001). Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science, 293, 2080–2084.
  • Chen Y and Struhl G (1996). Dual roles for patched in sequestering and transducing Hedgehog. Cell, 1, 553–563.
  • Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J and Jiang J (2011). Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol, 9, e1001083.

 

 

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

Carminia Maria Della Corte1Claudio Bellevicine2Giovanni Vicidomini3Donata Vitagliano1Umberto Malapelle2Marina Accardo4Alessio Fabozzi1Alfonso Fiorelli3Morena Fasano1Federica Papaccio1Erika Martinelli1Teresa Troiani1Giancarlo Troncone2Mario Santini3Roberto Bianco5Fortunato Ciardiello1, and Floriana Morgillo1,*

Clin Cancer Res October 15, 201521; 4686  http://dx.doi.org:/ 10.1158/1078-0432.CCR-14-3319  http://clincancerres.aacrjournals.org/content/21/20/4686.full

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.

Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines.

Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.

In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.

Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

This article is featured in Highlights of This Issue, p. 4497

Translational Relevance

The amplification of SMO in non–small cell lung cancer (NSCLC) resistant to EGFR-TKIs opens new possibilities of treatment for those patients who failed first-line EGFR-targeted therapies. The synergistic interaction of the Hedgehog (Hh) and MET pathways further support the rationale for a combined therapy with specific inhibitors. In addition, Hh pathway activation is essential for the acquisition of mesenchymal properties and, as such, for the aggressiveness of the disease. Also, in EGFR wild-type NSCLC models, inhibition of Hh, along with inhibition of EGF receptor (EGFR), can revert the resistance to anti-EGFR targeted drugs. In addition, inhibition of the Hh pathway sensitizes EGFR wild-type NSCLC to standard chemotherapy. These data encourage further evaluation of Hh inhibitors as novel therapeutic agents to overcome tyrosine kinase inhibitor (TKI) resistance and to revert epithelial-to-mesenchymal transition (EMT) in NSCLC.

 

Read :

  1. CCR 20th Anniversary Commentary: Triple-Negative Breast Cancer in 2015–Still in the Ballpark
  2. Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions
  3. Molecular Pathways: At the Crossroads of Cancer Epigenetics and Immunotherapy
  4. Differential Expression of Immune-Regulatory Genes Associated with PD-L1 Display in Melanoma: Implications for PD-1 Pathway Blockade
  5. The Allelic Context of the C797S Mutation Acquired upon Treatment with Third-Generation EGFR Inhibitors Impacts Sensitivity to Subsequent Treatment Strategies

 

Tyrosine kinase inhibitors (TKI) against the EGF receptor (EGFR) represent the first example of molecularly targeted agents developed in the treatment of non–small cell lung cancer (NSCLC) and are, currently, useful treatments after failure of first-line chemotherapy and, more importantly, for the first-line treatment of patients whose tumors have EGFR-activating gene mutations (1). However, after an initial response, all patients experience disease progression as a result of resistance occurrence. Recognized mechanisms of acquired resistance to anti-EGFR-TKIs in EGFR-mutated NSCLC are METgene amplification or the acquisition of secondary mutations such as the substitution of a threonine with a methionine (T790M) in exon 20 of the EGFR gene itself (2). However, these molecular changes are able to identify only a portion of patients with cancer defined as “non-responders” to EGFR-targeted agents. A number of molecular abnormalities in cancer cells may partly contribute to resistance to anti-EGFR agents (2, 3). Our group and others have shown that epithelial-to-mesenchymal transition (EMT) is a critical event in the metastatic switch and is generally associated with resistance to molecularly targeted agents in NSCLC models (4, 5). EMT is a process characterized by loss of polarity and dramatic remodeling of cell cytoskeleton through loss of epithelial cell junction proteins, such as E-cadherin, and gain of mesenchymal markers, such as vimentin (6). The clinical relevance of EMT and drug insensitivity comes from studies showing an association between epithelial markers and sensitivity to erlotinib in NSCLC cell lines, suggesting that EMT-type cells are resistant to erlotinib (7). In particular, recent data suggest that cancer cells with EMT phenotype demonstrate stem cell–like features and strategies reverting EMT could enhance the therapeutic efficacy of EGFR inhibitors (4, 5).

The Hedgehog (Hh) signaling cascade has emerged as an important mediator of cancer development and metastatic progression. The Hh signaling pathway is composed of the ligands sonic, Indian, and desert hedgehog (Shh, Ihh, Dhh, respectively) and the cell surface molecules Patched (PTCH) and Smoothened (SMO). In the absence of Hh ligands, PTCH causes suppression of SMO; however, upon ligand binding to PTCH, SMO protein leads to activation of the transcription factor GLI1, which in turn translocates into the nucleus, leading to the expression of Hh induced genes (8). The Hh signaling pathway is normally active in human embryogenesis and in tissue repair, as well as in cancer stem cell renewal and survival. This pathway is critical for lung development and its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated in bronchial epithelial cells exposed to cigarette smoke extraction. In particular, the activation of this pathway happens at an early stage of carcinogenesis when cells acquire the ability to growth in soft agar and as tumors when xenografted in immunocompromised mice. Treatment with Hh inhibitors at this stage can cause complete regression of tumors (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Reactivation of the Hh pathway with induction of EMT has been implicated in the carcinogenesis of several cancer types (15). Inhibition of the Hh pathway can reverse EMT and is associated with enhanced tumor sensitivity to cytotoxic agents (16). Recently, upregulation of the Hh pathway has been demonstrated in the NSCLC cell line A549, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype with increased cell motility and invasion (17).

The aim of the present work was to study the role of the Hh signaling pathway as mechanism of resistance to EGFR-TKIs in different models of NSCLC.

 

Methods ….

 

Results

Activation of Hh signaling pathway in NSCLC cell lines with resistance to EGFR-TKIs

We established an in vitro model of acquired resistance to the EGFR-TKI gefitinib using the EGFR exon 19 deletion mutant (delE746-A750) HCC827 human NSCLC cell line by continuous culturing these cells in the presence of increasing doses of gefitinib. HCC827 cells, which were initially sensitive to gefitinib treatment (in vitro IC50 ∼ 80 nmol/L), became resistant (HCC827-GR cells) after 12 months of continuous treatment with IC50 > 20 μmol/L. This cell line was also cross-resistant to erlotinib and to the irreversible EGFR kinase inhibitor BIBW2992 (afatinib; data not shown). Sequencing of the EGFR gene in gefitinib-resistant HCC827-GR cells showed the absence of EGFRT790M mutation (data not shown). After the establishment of HCC827-GR cells, we characterized their resistant phenotype by protein expression analysis. While the activation of EGFR resulted efficiently inhibited by gefitinib treatment both in HCC827 and in HCC827-GR cells, phosphorylation of AKT and MAPK proteins persisted in HCC827-GR cells despite the inhibition of the upstream EGFR (Fig. 1A).

Figure 1.

Figure 1.

Activation of Hh signaling pathway in NSCLC cell lines resistant to EGFR-TKIs. A, Western blot analysis of EGFR and of downstream signaling pathways in parental EGFR-mutated human lung adenocarcinoma HCC827 cells and in their gefitinib-resistant derivative (HCC827-GR). β-Actin was included as a loading control. B, Western blot analysis of Hh pathway, MET, and selected epithelial- and mesenchymal-related proteins in a panel of EGFR-TKI–sensitive (HCC827, H322, and Calu-3) and -resistant (HCC827-GR, H1299, Calu-3 ER, H460) NSCLC cell lines. β -Actin was included as a loading control. C, FISH analysis of gain in MET andSMO gene copy number in HCC827 and HCC827-GR. D, top, GLI-driven luciferase expression in HCC827 and HCC827-GR cells before and after depletion of GLI1 in both cell lines; bottom, evidence of GLI1 mRNA downregulation by siRNA. β-Actin was included as a loading control. E, MTT cell proliferation assays in HCC827-GR and PC9 cancer cell transfected with an empty vector or SMO expression plasmid with the indicated concentrations of gefitinib for 3 days. Bottom, Western blotting for evaluation of SMO after transfection.

HCC827-GR cells exhibited a mesenchymal phenotype with increased ability to invade, to migrate, and to grow in an anchorage-independent manner (Fig. 2A–C). Therefore, we next examined whether HCC827-GR cell line exhibits molecular changes known to occur during the EMT. Indeed, we found expression of vimentin and SLUG proteins and loss of E-cadherin protein expression in gefitinib-resistant cells as compared with gefitinib-sensitive cells (Fig. 1B). Although activation of the AXL kinase and NF-κB (20–22) have been described as known mechanisms of EGFR-TKI resistance, the analysis of their activation status resulted not significantly different among our cell lines. However, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

Figure 2.

http://clincancerres.aacrjournals.org/content/21/20/4686/F2.medium.gif

Figure 2.

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines. A, invasion assay. B, migration assay, C, anchorage-independent colony formation in soft agar. D, cell proliferation measured with the MTT assay in parental human lung adenocarcinoma HCC827 cells and in HCC827-GR derivative. The results are the average ± SD of 3 independent experiments, each done in triplicate.

Recently, expression of Shh and activation of the Hh pathway have been correlated to the TGFβ-induced EMT in A549 lung cancer cells (17). To investigate the expression profile of Hh signaling components in this in vitro model of acquired resistance to anti-EGFR–TKIs, we performed Western blot analysis for Shh, GLI1, 2, 3, SMO, and PTCH in HCC827-GR cells. While Shh levels did not differ between HCC827 and HCC827-GR cells, a significantly increased expression of SMO and GLI1 was found in HCC827-GR cells as compared with parental cells (Fig. 1B). No differences in the levels of GLI2 and 3 were observed (data not shown). Of interest, also PTCH protein levels resulted increased in HCC827-GR cells. This is of relevance, as PTCH is a target gene of GLI1 transcriptional activity and increased PTCH levels indicate activation of Hh signaling. We further analyzed expression and activation of MET, as a known mechanism of acquired resistance to anti-EGFR drugs in NSCLC. Indeed, MET phosphorylation resulted strongly activated in HCC827-GR cells (Fig. 1B). Analysis of the MET ligand levels, HGF, by ELISA assay, did not evidence any significant difference in conditioned media of our cells (data not shown). As previous studies have demonstrated MET gene amplification in NSCLC cell lines with acquired resistance to gefitinib (23), we evaluated MET gene copy number by FISH analysis and D-PCR in HCC827 and in HCC827-GR cell lines. The mean MET gene copy number was similar between gefitinib-sensitive and gefitinib-resistant HCC827 cell line (Fig. 1C).

Of interest, while we were working to these experiments, data on SMO gene amplification in EGFR-mutated NSCLC patients with acquired resistance to anti-EGFR targeted drugs were reported on rebiopsies performed at progression, revealing SMO amplification in 2 of 16 patients (12.5%; ref. 24). For this reason, we evaluated by FISH SMO gene copy number in HCC827-GR cells, in which the mean SMO gene copy number was 4-fold higher than that of parental HCC827 cells, indicating SMO gene amplification (Fig. 1C).

We further analyzed the expression and the activation of these molecules on a larger panel of EGFR-WT NSCLC cell lines, including NSCLC cells sensitive to EGFRTKIs, such as H322 and Calu-3 cells, NSCLC cell lines with intrinsic resistance to EGFR-TKIs, such as H1299 and H460 cells and Calu-3 ER (erlotinib-resistant) cells, which represents an in vitromodel of acquired resistance to erlotinib obtained from Calu-3 cells (refs. 4, 18; Supplementary Table S1). As shown in Fig. 1B, similarly to HCC827-GR cells, the Hh signaling pathway resulted in activation of these NSCLC models of intrinsic or acquired resistance to EGFR-TKI.

To further investigate the presence of specific mutations in the Hh pathway components, we sequenced DNA from our panel of NSCLC cell lines by Ion Torrent NGS; results indicated the absence of specific mutations in Hh-related genes (data not shown).

Because GLI1 is a transcription factor, we tested the functional significance of increased expression of this gene in the EGFR-sensitive and -resistant cell lines, using a GLI1-responsive promoter within a luciferase reporter expression vector (Fig. 1D). Analysis of luciferase activity of HCC827-GR cells revealed a 6- to 7-fold increase in GLI-responsive promoter activity as compared with HCC827 cells (P < 0.001), suggesting that transcriptional activity of GLI1 is significantly higher in gefitinib-resistant HCC827-GR cells. Furthermore, depletion of GLI1 protein expression by transfection with a GLI1-specific siRNA expression vector led to approximately 65% decrease in GLI1-driven promoter activity in HCC827-GR (P < 0.01; Fig. 1D). To determine whether SMO expression may promote resistance to gefitinib, 2 cell lines harboring the mutated EGFR gene, HCC827 and PC9 cells, and the sensitive EGFR-WT cell line Calu-3, were transiently transfected with an SMO expression plasmid. When treated with gefitinib, transfected cells exhibited a partial loss of sensitivity to the EGFR inhibition (Fig. 1E).

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines

As previously mentioned, HCC827-GR cells acquired expression of vimentin and SLUG and loss of E-cadherin when compared with gefitinib-sensitive HCC827 cancer cells along with an increased ability to invade, migrate, and form colonies in semisolid medium (Fig. 2A–C). We next evaluated whether the Hh pathway activation was necessary for gefitinib acquired resistance by genetically or by pharmacologically inhibiting Hh components in the HCC827-GR cell line. Knockdown of GLI1 by a GLI1siRNA approach had a very little effect on HCC827-GR cells. However, when gefitinib treatment (1 μmol/L) was performed in HCC827-GR cells after GLI1 blockade, invasion, migration, and colony-forming capabilities were significantly inhibited (Fig. 2A–C). Next, we evaluated the effects of 2 small-molecule inhibitors of SMO, such as LDE225 and vismodegib. Treatment with LDE225 (1 μmol/L;Fig. 2A–D) or with vismodegib (1 μmol/L; data not shown) alone did not significantly affect the viability and the invasion and migration abilities of HCC827-GR cells. Combined treatment with gefitinib and LDE225 (1 μmol/L) or vismodegib (1 μmol/L) caused inhibition of these parameters in HCC827-GR cells (Fig. 2A–C).

Taken together, these data show that Hh activation is required for acquisition of gefitinib resistance in HCC827-GR cells.

As overexpression and activation of MET was found in HCC827-GR cells, we evaluated whether inhibition of MET phosphorylation by PHA-665752 could restore gefitinib sensitivity in this model. Although abrogation of MET signaling in combination with the inhibition of EGFR signaling marginally affected gefitinib sensitivity of HCC827-GR cells, surprisingly, inhibition of MET synergistically enhanced the effects of Hh inhibition in HCC827-GR cells (Fig. 2A–D) in terms of invasion, migration, colony-forming, and proliferation abilities, indicating a significant synergism between these 2 signaling pathways. The triple inhibition of EGFR, SMO, and MET did not result in any additional antiproliferative effects (data not shown).

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKI in EGFR-dependent NSCLC cell lines

To study the role of Hh pathway in the regulation of key signaling mediators downstream of the EGFR and to explore the interaction between Hh and MET pathways, we further characterized the effects of Hh inhibition alone and in combination with EGFR or MET inhibitor on the intracellular signaling by Western blotting. As illustrated in Fig. 3A, treatment of HCC827-GR cells with the SMO inhibitor LDE225, gefitinib or with the MET inhibitor PHA-665772, for 72 hours, did not affect total MAPK and AKT protein levels and activation. A marked decrease of the activated form of both proteins was observed only when LDE225 was combined with PHA-665772, at greater level than inhibition of EGFR and MET, suggesting that the Hh pathway cooperates with MET to the activation of both MAPK and AKT signaling pathways. In addition, vimentin expression, induced during the acquisition of gefitinib resistance, was significantly decreased after Hh inhibition, suggesting that the Hh pathway represents a key mediator of EMT in this model. The combination of MET and Hh inhibitors strongly induced cleavage of the 113-kDa PARP to the 89-kDa fragment, indicating an enhanced programmed cell death.

Figure 3.

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKIs in HCC827-GR cells. A, Western blot analysis of Hh, MET, and EGFR activation and their downstream pathways activation following treatment with the indicated concentration LDE225 and PHA-556752 on HCC827-GR NSCLC cell line. β-Actin was included as a loading control. B, co-immunoprecipitation for the interaction between MET and SMO. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti- SMO (top) or anti-MET (bottom). The immunoprecipitates were subjected to Western blot analysis (WB) with indicated antibodies. Control immunoprecipitation was done using control mouse preimmune serum (PS). C, GLI-driven luciferase expression in HCC827-GR cells during treatment with gefitinib, LDE225, PHA-556752, or their combinations. D, co-immunoprecipitation for the interaction between SUFU and GLI1. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti-GLI1 (top) or anti-SUFU (bottom) antibodies. The immunoprecipitates were subjected to Western blot analysis with indicated antibodies. Control immunoprecipitation was done using control mouse PS.

Of interest, the inhibition of SMO by LDE225 also reduced the activated, phosphorylated form of MET (Fig. 3A), revealing an interaction between SMO and MET receptors. To address this issue, we hypothesized a direct interplay between both receptors. SMO immunoprecipitates from HCC827-GR cells showed greater MET binding than that from the parental HCC827 cells (Fig. 3B). As MET has been demonstrated to interact with HER3 to mediate resistance to EGFR inhibitors (25), we explored the expression of HER3 in SMO immunoprecipitates. Protein expression analysis did not show any association with HER3; similar results were obtained with EGFR protein expression analysis in the immunoprecipitates (data not shown).

The increased SMO/MET heterodimerization observed in HCC827-GR cells was partially reduced by the inhibition of SMO or MET with LDE225 or PHA-665752, respectively, and to a greater extent with the combined treatment (Fig. 3B). These results support the hypothesis that Hh and MET pathways interplay at level of their receptors.

To study whether the cooperation between these 2 pathways appears also at a downstream level, and considering that, as shown in Fig. 3A, MET inhibition partially reduces the levels of GLI1 and PTCH proteins, we analyzed luciferase expression of GLI1 reporter vector in HCC827-GR cells after treatment with LDE225, PHA-665752, or both. As shown in Fig. 3C, transcriptional activity of GLI1 resulted strongly decreased by the combined treatment. In particular, treatment with single-agent LDE225 did not abrogate the transcriptional activity of GLI1 suggesting a GLI1 noncanonical activation. In addition, single-agent PHA-665752 reduced GLI1-dependent signal, suggesting a role for MET in GLI1 regulation. To better investigate these findings, we hypothesized that MET can regulate GLI1 activity through its nuclear translocation. We, therefore, analyzed the binding ability of SUFU, a known cytoplasmic negative regulator of GLI1, following treatment of HCC827-GR cells with LDE225 and/or PHA-665752. Indeed, interaction between SUFU and GLI1 was markedly decreased in HCC827-GR cells as compared with HCC827 cells (Fig. 3D), which further confirmed the role of the activation of Hh pathway in this gefitinib-resistant NSCLC model. Furthermore, while combined treatment with LDE225 and PHA-665752 strongly increased the binding between GLI1 and SUFU, suggesting an inhibitory effect on GLI1 activity, also treatment with the MET inhibitor PHA-665752 alone favored the interaction of GLI1 with SUFU (Fig. 3D), indicating a role of MET on the activation of GLI1. This phenomenon could be a consequence of the decreased interplay between SMO and MET receptors or the effect of a direct regulation of GLI1 by MET.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts

We finally investigated the in vivo antitumor activity of Hh inhibition by LDE225, alone and in combination with gefitinib or with the MET inhibitor in nude mice bearing HCC827-GR cells. Treatment with gefitinib, as single agent, did not cause any change in tumor size as compared with control untreated mice, confirming that the in vitro model of gefitinib resistance is valid also in vivo. Treatment with LDE225 or with PHA-665752 as single agents caused a decrease in tumor size even stronger than that observed in vitro, suggesting a major role of these drugs on tumor microenvironment. However, combined treatments, such as LDE225 plus gefitinib or LDE225 plus PHA-665752, significantly suppressed HCC827-GR tumor growth with a major activity of LDE225 plus PHA-665752 combination. Indeed at 21 days from the starting of treatment, the mean tumor volumes in mice bearing HCC827-GR tumor xenografts and treated with LDE225 plus gefitinib or with LDE225 plus PHA-665752 were 24% and 2%, respectively, as compared with control untreated mice (Fig. 4A). Figure 4B shows changes in tumor size from baseline in the 6 groups of treatment. A total of eight mice for each treatment group were considered. Combined treatment of LDE225 plus gefitinib caused objective responses in 5 of 8 mice (62.5%). Of interest, the most active treatment combination was LDE225 plus PHA-665752 with complete responses in 8 of 8 mice (100%).

Figure 4.

http://clincancerres.aacrjournals.org/content/21/20/4686/F4.medium.gif

Figure 4.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts. A, athymic nude mice were injected subcutaneously into the dorsal flank with 107 HCC827-GR cancer cells. After 7 to 10 days (average tumor size, 75 mm3), mice were treated as indicated in Materials and Methods for 3 weeks. HCC827-GR xenografted mice received only vehicle (control group), gefitinib (100 mg/kg daily orally by gavage), LDE225 (20 mg/kg intraperitoneally three times a week), PHA-665752 (25 mg/kg intraperitoneally twice a week), or their combination. Data represent the average (±SD). The Student t test was used to compare tumor sizes among different treatment groups at day 21 following the start of treatment. B, waterfall plot representing the change in tumor size from baseline in the 6 groups of treatment. A total of 8 mice for each treatment group were evaluated. C, effects of combined LDE225 and PHA-665752 on expression of MET, PTCH, and vimentin. Tissues were stained with hematoxylin and eosin (H&E). Representative section from each condition.

We then studied the effects of gefitinib, LDE225, PHA-665752, and their combinations on the expression of PTCH, MET, and vimentin in tumor xenografts biopsies from mice of each group of treatment (Fig. 4C and Supplementary Table S2). We measured PTCH expression, as it represents a direct marker of Hh activation. While vimentin staining was particularly intense in control and gefitinib-treated tumors, treatment with LDE225 alone and in combination with PHA-665752 significantly reduced the intensity of the staining further confirming the role of Hh inhibition on the reversal of mesenchymal phenotype. Of interest, MET immunostaining resulted in a consistent nuclear positivity: this particular localization has been described as a marker of poor outcome and tendency to a mesenchymal phenotype (26). Although the combination of LDE225 and gefitinib resulted in a significant reduction of tumor growth with a concomitant reduction in staining intensity of vimentin, the combination of LDE225 and PHA-665752 was the most effective treatment, with 8 of 8 (100%) mice having a complete response in their tumors. In fact, histologic evaluations of these tumors found only fibrosis and no viable cancer cells. According to Western blot analysis of protein extracts harvested from the HCC827-GR xenograft tumors, the levels of phospho-EGFR, phospho-MET, and GLI1 resulted in a decrease after treatment with the respective inhibitor. Interestingly, the combined treatment with LDE225 and PHA-665752 resulted in a stronger inhibition of phospho-MAPK and phospho-AKT (Supplementary Fig. S1).

Role of the Hh pathway in mediating resistance to EGFR inhibitors in EGFR-WT NSCLC

As shown in Fig. 1B, although H1299, H460, and Calu-3 ER lacked SMO amplification (data not shown), these cells displayed Hh pathway activation. We further conducted luciferase expression analysis that showed a 8- to 9-fold increase in GLI1-dependent promoter activity in these lines as compared with EGFR inhibitor–sensitive H322 and Calu-3 cells, suggesting that transcriptional activity of GLI1 is higher in EGFR-TKI–resistant EGFR-WT NSCLC lines (Supplementary Fig. S2A). Similar to HCC827-GR cells, these cells showed also activation of MET. However, as reported in previous studies (4), MET inhibition alone or in combination with EGFR inhibition or with SMO inhibition resulted ineffective in inhibiting cancer cell proliferation and survival (data not shown).

We therefore tested the effects of Hh inhibition, by silencing GLI1 or by using LDE225, alone and/or in combination with erlotinib. Although knockdown of GLI1 or treatment with LDE225 (1 μmol/L) did not significantly affect NSCLC cell viability, combined treatment with erlotinib restored sensitivity to erlotinib (Supplementary Fig. S2B).

In addition, H1299, Calu-3 ER, and H460 cells exhibited significantly higher invasive and migratory abilities than H322 and Calu-3 cells and inhibition of Hh pathway significantly reduced these abilities. Collectively, these results suggest that Hh pathway activation mediates the acquisition of mesenchymal properties in EGFR-WT lung adenocarcinoma cells with erlotinib resistance (Supplementary Fig. S2B–S2D).

We next evaluated the effects of LDE225 alone and/or in combination with erlotinib on the activation of downstream pathways. Erlotinib treatment result was unable to decrease the phosphorylation levels of AKT and MAPK in H1299 and Calu-3 ER cells (Fig. 5A). However, when LDE225 was combined with erlotinib, a strong inhibition of AKT and MAPK activation was observed in these EGFR inhibitor–resistant cells (Fig. 5A). Furthermore, flow cytometric analysis revealed that combined treatment with both erlotinib and LDE225 significantly enhanced the apoptotic cell percentage to 65% and 70% (P < 0.001) in H1299 and Calu-3 ER cells, respectively (Fig. 5B), confirmed by the induction of PARP cleavage after the combined treatment (Fig. 5A). These findings suggest that Hh pathway drives proliferation and survival signals in NSCLC cells in which EGFR is blocked by erlotinib, and only the inhibition of both pathways can induce strong antiproliferative and proapoptotic effects. The in vitro synergism between EGFR and SMO was confirmed alsoin vivo. Combination of erlotinib and LDE225 significantly suppressed growth of Calu-3 ER xenografted tumors in nude mice (Supplementary Fig. S1F).

Figure 5.

Activation of Hh signaling pathway mediates resistance to EGFR-TKI in EGFR-WT NSCLC cell lines. A, Western blot analysis of EGFR and its downstream pathways activation, including PARP cleaved form, following treatment with the indicated concentration LDE225 and erlotinib on Calu-3, Calu-3 ER, and H1299 NSCLC cell line. β-Actin was included as a loading control. B, apoptosis was evaluated as described in Supplementary Materials and Methods with annexin V staining in Calu-3, Calu-3-GR, and H1299 cancer cells, which were treated with the indicated concentration LDE225 and erlotinib. Columns, mean of 3 identical wells of a single representative experiment; bars, top 95% confidence interval; ***, P < 0.001 for comparisons between cells treated with drug combination and cells treated with single agent.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy

To extend our preclinical observations, we further investigated the effects of Hh pathway inhibition on sensitivity of EGFR-WT NSCLC cells to standard chemotherapy used in this setting and mostly represented by cisplatin.

To investigate the role of the Hh pathway in mediating resistance also to chemotherapy, we evaluated the efficacy of cisplatin and Hh inhibition treatment alone or in combination on the colony-forming ability in semisolid medium of H1299 and H460 cell lines (Fig. 6). Treatment with cisplatin alone resulted in a dose-dependent inhibition of colony formation with an IC50 value of 13 and 11 μmol/L for H1299 and H460 cells, respectively. However, when combined with LDE225, the treatment resulted in a significant synergistic antiproliferative effect in both NSCLC cell lines (Fig. 6). Together, these results indicate that treatment of EGFR-WT NSCLC cells with Hh inhibitors could improve sensitivity of NSCLCs to standard chemotherapy.

Figure 6.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy. Anchorage-independent colony formation in soft agar in human lung adenocarcinoma H1299 and H460. The results are the average ± SD of 3 independent experiments, each done in triplicate. For defining the effect of the combined drug treatments, any potentiation was estimated by multiplying the percentage of cells remaining by each individual agent. The synergistic index was calculated as previously described (19). In the following equations, A and B are the effects of each individual agent and AB is the effect of the combination. Subadditivity was defined as %AB/(%A%B) < 0.9; additivity was defined as %AB/(%A%B) = 0.9–1.0; and supra-additivity was defined as %AB/(%A%B) > 1.0.

Discussion

Resistance to currently available anticancer drugs represents a major clinical challenge for the treatment of patients with advanced NSCLC. Our previous works (4, 18) reported that whereas EGFR-TKI–sensitive NSCLC cell lines express the well-established epithelial markers, cancer cell lines with intrinsic or acquired resistance to anti-EGFR drugs express mesenchymal characteristics, including the expression of vimentin and a fibroblastic scattered morphology. This transition plays a critical role in tumor invasion, metastatic dissemination, and the acquisition of resistance to therapies such as EGFR inhibitors. Among the various molecular pathways, the Hh signaling cascade has emerged as an important mediator of cancer development and progression (8). The Hh signaling pathway is active in human embryogenesis and tissue repair in cancer stem cell renewal and survival and is critical for lung development. Its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated by cigarette smoke extraction exposure in bronchial epithelial cells (12). In particular, the activation of this pathway correlated with the ability to growth in soft agar and in mice as xenograft and treatment with Hh inhibitors showed regression of tumors at this stage (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Recently, alterations of the SMO gene (mutation, amplification, mRNA overexpression) were found in 12.2% of tumors of The Cancer Genome Atlas (TCGA) lung adenocarcinomas by whole-exome sequencing (27). The incidence of SMO mutations was 2.6% and SMO gene amplifications were found in 5% of cases. SMO mutations and amplification strongly correlated with SHH gene dysregulation (P < 0.0001). In a small case report series, 3 patients with NSCLC with Hh pathway activation had been treated with the SMO inhibitor LDE225 with a significant reduction in tumor burden, suggesting that Hh pathway alterations occur in NSCLC and could be an actionable and valuable therapeutic target (27). Recently, upregulation of Shh, both at the mRNA and at the protein levels, was demonstrated in the A549 NSCLC cell line, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype (17, 28, 29) and mediated increased cell motility, invasion, and tumor cell aggressiveness (30, 31).

In the present study, SMO gene amplification has been identified for the first time as a novel mechanism of acquired resistance to EGFR-TKI in EGFR-mutant HCC827-GR NSCLC cells. These data are in agreement with the results of a cohort of patients with EGFR-mutant NSCLC that were treated with EGFR-TKIs (24). Giannikopoulus and colleagues have demonstrated the presence of SMO gene amplification in tumor biopsies taken at occurrence of resistance to EGFR-TKIs in 2 of 16 patients (24). In both cases, theMET gene was also amplified. In this respect, although the MET gene was not amplified in HCC827-GR cells, we found a significant functional and structural interaction between MET and Hh pathways in these cells. In fact, the combined inhibition of both SMO and MET exerted a significant antiproliferative and proapoptotic effect in this model, demonstrated by tumor regressions with complete response in 100% of HCC827-GR tumors xenografted in nude mice.

Several MET inhibitors have been evaluated in phase II/III clinical studies in patients with NSCLC, with controversial results. Most probably, blocking MET receptor alone is not enough to revert the resistant phenotype, as it is implicated in several intracellular interactions, and the best way to overcome resistance to anti-EGFR-TKIs is a combined approach, with Hh pathway inhibitors.

In the context of EMT, Zhang and colleagues demonstrated that AXL activation drives resistance in erlotinib-resistant subclones derived from HCC827, independently from MET activation in the same subclone, and that its inhibition is sufficient to restore erlotinib sensitivity by inhibiting downstream signal MAPK, AKT, and NF-κB (21). In addition, Bivona and colleagues described in 3 HCC827 erlotinib-resistant subclones increased RELA phosphorylation, a marker of NF-κB activation, in the absence of MET upregulation, and demonstrated that NF-κB inhibition enhanced erlotinib sensitivity, independently from AKT or MAPK inhibition (22). Differently, we detected Hh and MET hyperactivation in our resistance model HCC827-GR without a clear increase in AXL and NF-κB activation.

Although the level of activation of AXL and NF-κB did not result in contribution to resistance in our model, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

In a preclinical model, the evolution of resistance can depend strictly from the selective activation of specific pathways, whereas different mechanisms can occur simultaneously in patients with NSCLC, due to tumor heterogeneity. Thus, all data regarding EFGFR-TKIs resistance have to be considered equally valid.

We further extended the evaluation of the Hh pathway to NSCLC cell lines harboring the wild-type EGFR gene and demonstrated that Hh is selectively activated in NSCLC cells with intrinsic or acquired resistance to EGFR inhibition and occurred in the context of EMT.

To further validate these data, we blocked SMO or downregulated GLI1 RNA expression in NSCLC cells that had undergone EMT, and this resulted in resensitization of NSCLC cells to erlotinib and loss of vimentin expression, indicating an mesenchymal-to-epithelial transition promoted by the combined inhibition of EGFR and Hh. Inhibition of the Hh pathway alone was not sufficient to reverse drug resistance but required concomitant EGFR inhibition to block AKT and MAPK activation and to restore apoptosis, indicating that the prosurvival PI3K/AKT pathway and the mitogenic RAS/RAF/MEK/MAPK pathways likely represent the level of interaction of EGFR and Hh signals.

In EGFR-WT NSCLC models, the role of MET amplification/activation is less clear, and in our experience, its inhibition did not increase the antitumor activity of SMO inhibitors.

In addition, Hh inhibition contributed to increase the response to cisplatin treatment which is the standard chemotherapeutic option used in EGFR-WT NSCLC patients and in EGFR-mutated patients after progression on first-line EGFR-TKI, thus representing a valid contribution to achieve a better disease control in those patients without oncogenic activation or after progression on molecularly targeted agents.

Collectively, the results of the present study provide experimental evidence that activation of the Hh pathway, through SMO amplification, is a potential novel mechanism of acquired resistance in EGFR-mutated NSCLC patients that occurs concomitantly with MET activation, and the combined inhibition of these 2 pathways exerts a significant antitumor activity. In light of these results, screening of SMO alteration is strongly recommended in EGFR-mutated NSCLC patients with acquired resistance to EGFR-TKIs at first progression.

 

Hedgehog: functions and mechanisms

Markku Varjosalo and Jussi Taipale1

Genes & Dev. 2008. 22:2454-2472    Copyright © 2008, Cold Spring Harbor Laboratory Press  http://dx.doi.org:/10.1101/gad.1693608

The Hedgehog (Hh) family of proteins control cell growth, survival, and fate, and pattern almost every aspect of the vertebrate body plan. The use of a single morphogen for such a wide variety of functions is possible because cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time cells are exposed to Hh. The Hh gradient is shaped by several proteins that are specifically required for Hh processing, secretion, and transport through tissues. The mechanism of cellular response, in turn, incorporates multiple feedback loops that fine-tune the level of signal sensed by the responding cells. Germline mutations that subtly affect Hh pathway activity are associated with developmental disorders, whereas somatic mutations activating the pathway have been linked to multiple forms of human cancer. This review focuses broadly on our current understanding of Hh signaling, from mechanisms of action to cellular and developmental functions. In addition, we review the role of Hh in the pathogenesis of human disease and the possibilities for therapeutic intervention.

 

The origin of the name Hedgehog derives from the short and “spiked” phenotype of the cuticle of the Hh mutant Drosophila larvae. Mutations in the Hh gene were identified by Nusslein-Volhard and Wieschaus (1980) in their large-scale screen for mutations that impair or change the development of the fruit fly larval body plan. Drosophila Hh DNA was cloned in the early 1990s (Lee et al. 1992; Mohler and Vani 1992; Tabata et al. 1992; Tashiro et al. 1993). In addition to Drosophila,Hh genes have also been found in a range of other invertebrates including Hirudo medicinalis (leech) and Diadema antillarum (sea urchin) (Chang et al. 1994;Shimeld 1999; Inoue et al. 2002). It is important to note that the model organismCaenorhabditis elegans (roundworm) has no Hh ortholog, even though it has several proteins homologous to the Hh receptor Ptc (Kuwabara et al. 2000).

Hh orthologs from vertebrates—including Mus musculus (mouse), Danio rerio(zebrafish), and Gallus gallus (chicken)—were cloned in 1993 (Echelard et al. 1993;Krauss et al. 1993; Riddle et al. 1993; Chang et al. 1994). Cloning of the firstRattus rattus (rat) and human Hh genes were reported shortly thereafter, in 1994 and 1995, respectively (Roelink et al. 1994; Marigo et al. 1995). The vertebrate genome duplication (Wada and Makabe 2006) has resulted in expansion of the Hhgenes, which can be categorized into three subgroups: the Desert Hedgehog(Dhh), Indian Hedgehog (Ihh), and Sonic Hedgehog (Shh) groups (Echelard et al. 1993). The Shh and Ihh subgroups are more closely related to each other than to the Dhh subgroup, which in turn is closest to Drosophila Hh. Avians and mammals have one Hh gene in each of the three subgroups, but due to another whole-genome duplication (Jaillon et al. 2004) and further rearrangements, zebrafish has three extra Hh homologs, one in the Shh subgroup: tiggywinkle hedgehog (Twhh) (Ekker et al. 1995), and two others in the Ihh group; echidna hedgehog (Ehh) (Currie and Ingham 1996); and qiqihar hedgehog (Qhh) (Fig. 1A; Ingham and McMahon 2001).

Figure 1.

Figure 1.

(A) Phylogram illustrating the evolution of the Hh proteins. The different Hh proteins were aligned using Prankster (Loytynoja and Goldman 2005). Hh subgroups are indicated by a color code, as follows: Dhh (blue), Shh (green), and Ihh (red). (B) The central conserved components of the Hh signaling pathway and their role in forward signaling. Positively and negatively acting pathway components are in green and red, respectively. Note that most interactions between components are inhibitory. The conserved kinases involved in regulation of Ci/GLI processing from activator forms (Ci/GLI-A) to repressor forms (Ci/GLI-R) are casein kinases (CKs) 1α and 1ε, glycogen synthase kinase-3β (GSK3β), and protein kinase A (PKA). (C) The four negative (red) and two positive (green) transcriptional feedback loops of the Hh pathway. Ci/GLI-positive feedback to itself is mediated by GLI1 in mammals. HIP and FoxA2 are only found in vertebrates, and Engrailed (En) has been characterized as a regulator of Hh only in Drosophila. Both Drosophilaand mammalian names of the components are given separated by a slash.

Components of the Hh signal transduction pathway have been identified primarily using Drosophila genetics (for example, see Lee et al. 1992; Alcedo et al. 1996;van den Heuvel and Ingham 1996; Burke et al. 1999; Chamoun et al. 2001; Jacob and Lum 2007b). Mechanisms by which the Hh signal is transduced has been further characterized using Drosophila and mouse cell culture models (Fig. 1B,C; e.g., see Kinto et al. 1997; C.H. Chen et al. 1999; Chuang and McMahon 1999;Taipale et al. 2000; Lum et al. 2003a; Nybakken et al. 2005; Varjosalo et al. 2006). In both vertebrates and invertebrates, binding of Hh to its receptor Patched (Ptc) activates a signaling cascade that ultimately drives the activation of a zinc-finger transcription factor (Ci in Drosophila, GLI1–3 in mammals), leading to the expression of specific target genes (Huangfu and Anderson 2006; Jacob and Lum 2007a; Varjosalo and Taipale 2007).

Although many of the key components are conserved in vertebrates, the mammalian Hh signaling pathway is incompletely understood and harbors some differences and additional pathway components (see below). It was long thought that the main difference between Drosophila and mammalian Hh signaling was that mammals had multiple orthologs of many pathway components, including Hh, Ptc, and Ci. However, the roles of mammalian orthologs of two critical components of the Drosophila pathway, the protein kinase Fused (Fu) and the atypical kinesin Costal2 (Cos2), appear not to be conserved (Chen et al. 2005; Merchant et al. 2005; Svard et al. 2006; Varjosalo et al. 2006). This suggests that the mechanisms of Hh signal transduction from the receptor to the Ci/GLI transcription factors have evolved differentially after separation of the vertebrate and invertebrate lineages approximately 1 billion years ago (Hedges 2002; Varjosalo and Taipale 2007).

Developmental functions and expression of mammalian Hh proteins

The Hh proteins act as morphogens controlling multiple different developmental processes (Fig. 2). All mammalian Hh proteins are thought to have similar physiological effects—the differences in their roles in development result from diverse pattern of expression (McMahon et al. 2003; Sagai et al. 2005).

Figure 2.

Shh controls mouse development from an embryo to an adult. (Top) The embryo cartoons show aspects of expression of the Hh target gene patched (blue) during mouse embryonic development. (Bottom) Bars show approximate embryonic stages when Shh, Ihh, and/or Dhh (color code in bottom left) control developmental processes in the indicated tissues or cell types. The approximate embryonic stage is indicated by dpc and Theiler stage (TS) (Theiler 1989). References: the role of Hh in early embryogenesis prior to TS 15 (Chiang et al. 1996; Zhang et al. 2001; Astorga and Carlsson 2007); limb development (Ahn and Joyner 2004); cranial neural crest (Jeong et al. 2004); cardiac septation (Goddeeris et al. 2008); gastrointestinal system (Madison et al. 2005); bladder (Haraguchi et al. 2007); lung (White et al. 2007); prostate (Berman et al. 2004); pancreas (Hebrok et al. 2000); testis development (Yao et al. 2002); retina (Sigulinsky et al. 2008); kidney (Hu et al. 2006); hair (St-Jacques et al. 1998; Jeong et al. 2004); taste buds (Miura et al. 2001); ear (Riccomagno et al. 2002); ovary (Wijgerde et al. 2005; Pangas 2007); tooth (Cobourne et al. 2001, 2004); bone growth (St-Jacques et al. 1999); cerebellum growth (Hatton et al. 2006; Sillitoe and Joyner 2007).

Dhh expression is largely restricted to gonads, including sertoli cells of testis and granulosa cells of ovaries (Bitgood et al. 1996; Yao et al. 2002; Wijgerde et al. 2005). Consistent with its expression in a very narrow tissue range, Dhh-deficient mice do not show notable phenotypes is most tissues and are viable. However, males are infertile due to complete absence of mature sperm (Bitgood et al. 1996).

Ihh is specifically expressed in a limited number of tissues, including primitive endoderm (Dyer et al. 2001), gut (van den Brink 2007), and prehypertrophic chondrocytes in the growth plates of bones (Vortkamp et al. 1996; St-Jacques et al. 1999). Approximately 50% of Ihh−/− embryos die during early embryogenesis due to poor development of yolk-sac vasculature. Surviving embryos display cortical bone defects as well as aberrant chondrocyte development in the long bones (St-Jacques et al. 1999; Colnot et al. 2005). Homozygous hypomorphic mutations of IHH in humans cause acrocapitofemoral dysplasia, a congenital condition characterized by bone defects and short stature (Hellemans et al. 2003).

Shh is the most broadly expressed mammalian Hh signaling molecule. During early vertebrate embryogenesis, Shh expressed in midline tissues such as the node, notochord, and floor plate controls patterning of the left–right and dorso-ventral axes of the embryo (Sampath et al. 1997; Pagan-Westphal and Tabin 1998;Schilling et al. 1999; Watanabe and Nakamura 2000; Meyer and Roelink 2003). Shh expressed in the zone of polarizing activity (ZPA) of the limb bud is also critically involved in patterning of the distal elements of the limbs (Riddle et al. 1993;Chang et al. 1994; Johnson et al. 1994; Marti et al. 1995). Later in development, during organogenesis, Shh is expressed in and affects development of most epithelial tissues (Fig. 2).

Deletion of Shh leads to cyclopia, and defects in ventral neural tube, somite, and foregut patterning. Later defects include, but are not limited to, severe distal limb malformation, absence of vertebrae and most of the ribs, and failure of lung branching (Chiang et al. 1996; Litingtung et al. 1998; Pepicelli et al. 1998).

The different Hh ligands often act in the same tissues during development, and can function partially redundantly (Fig. 2). For example, Shh and Ihh act together in early embryonic development, and their combined loss phenocopies the loss of the Hh receptor component Smoothened (Smo), leading to early embryonic lethality due to defects in heart morphogenesis and extraembryonic vasculogenesis (Zhang et al. 2001; Astorga and Carlsson 2007).

Regulatory elements affecting mammalian Hh expression

Of the mammalian Hh genes, only the mechanisms controlling Shh expression have been studied in detail. The expression pattern of Shh is the result of the combined action of multiple enhancer-elements, which act independently to control Shh transcription in different tissues and expression domains. Both local-acting and very distal elements have been identified (Fig. 3).

Figure 3.

Regulation of mammalian Shh gene expression. (Top) Enhancer-elements driving expression of the mouse Shh gene in different neural domains (left) and in posterior margin of the embryonic limb buds (right). Approximate expression domains of the elements are indicated by blue color. Black lines perpendicular to the neural tube indicate zona limitans intrathalamica (ZLI) and midbrain–hindbrain junction. (Bottom) Known genes in the ∼1 Mb genomic region upstream of the human Shh gene (University of California at Santa Cruz genome browser, assembly 36). Note that only one transcriptional start site of another gene appears to be between the most distal conserved Shh enhancer (MFCS1) and the Shh gene itself.

Two independent enhancers—Shh floor plate enhancer 1 (SFPE1) and SFPE2, located at −8 kb and in intron 2, respectively—act to direct reporter expression exclusively to the floor plate of the hindbrain and spinal cord (Epstein et al. 1999). A third element in intron 2, Shh brain enhancer 1 (SBE1), directs reporter expression to the ventral midbrain and caudal diencephalon. The more distal elements SBE2, SBE3, and SBE4, which are located >400 kb upstream of the Shh transcription start site (TSS) drive reporter expression in the ventral forebrain. The combined activity of these enhancers appears to cover all regions of Shh transcription along the anterior-posterior axis of the mouse neural tube (Jeong et al. 2006).

The enhancer controlling Shh expression in the ZPA of limb buds, mammals–fish conserved sequence 1 (MFCS1), is located even further upstream of the start site, at −1 Mb in intron 5 of the Lmbr1 gene (Sharpe et al. 1999; Lettice et al. 2003;Sagai et al. 2004). This element is the only enhancer in Shh that has been analyzed also by loss-of-function studies (Sagai et al. 2005), which conclusively demonstrate that MFCS1 is necessary for Shh expression in mouse ZPA. Consistently in humans, germline mutations within the conserved MFCS1 element cause congenital limb malformations characterized by preaxial polydactyly (Lettice et al. 2003). Interestingly, the MFCS1 sequence is not conserved in limbless vertebrates such as snake, limbless lizard, and newt (Sagai et al. 2005). Although the SBE2–4 and MFCS1 elements are physically far from Shh, the TSS of the region upstream of Shh contains very few genes, and only one well-described TSS exists between the MFCS1 and the TSS of Shh (Fig. 3). Given the diverse expression pattern of Shh, it is likely that a number of other enhancer-elements remain to be identified in this “gene-poor” region.

Although many enhancers that drive Shh expression have been identified, very little is known about the specific transcription factors that control their activity. The temporal and spatial expression pattern of FoxA2 suggests that it could induce Shh expression (Chang et al. 1997; Epstein et al. 1999) in the midline. Consistently, conserved binding sites for FoxA2 and Nkx6 are required for SFPE2 activity (Jeong and Epstein 2003). The Nkx2.1 homeodomain protein has also been suggested as a likely candidate regulating Shh expression in ventral forebrain (Jeong et al. 2006).

No known consensus binding sites for transcription factors are affected by the mutations in the MFCS1 limb enhancer, and the mutations are not clustered close together. However, the severity of the polydactyly phenotype correlates negatively with the conservation of nucleotide at the mutation sites, suggesting that MFCS1 activity is controlled by conserved transcriptional regulators whose DNA-binding specificity is currently not known.

Hh processing and secretion

After translation, Hh undergoes multiple processing steps that are required for generation and release of the active ligand from the producing cell. The mechanisms involved in Hh processing and secretion are evolutionarily conserved (see Burke et al. 1999; Amanai and Jiang 2001; Chamoun et al. 2001; Ingham and McMahon 2001; Caspary et al. 2002; Dai et al. 2002; Ma et al. 2002).

After the signal sequence is removed, the Hh molecule undergoes a cleavage catalyzed by its own C-terminal domain that occurs between conserved glycine and cysteine residues (Fig. 4; Lee et al. 1994; Porter et al. 1996). First, the peptide bond between these residues is rearranged to form a thioester. Subsequently, a hydroxyl-oxygen of cholesterol attacks the carbonyl of the thioester, displacing the sulfur and cleaving the Hh protein into two parts, a C-terminal processing domain with no known signaling activity and an N-terminal Hh signaling domain (HhN) of ∼19 kDa that contains an ester-linked cholesterol at its C terminus (Porter et al. 1996). The cholesterol modification results in the association of HhN with the plasma membrane. Subsequently, a palmitic acid moiety (Pepinsky et al. 1998) that is required for HhN activity is added to N terminus of Hh by the acyltransferase Skinny hedgehog (Ski, HHAT in humans) (Chamoun et al. 2001; Lee et al. 2001; Buglino and Resh 2008). The resulting fully active HhN signaling molecule is thus modified by cholesterol at its C terminus and palmitate at its N terminus (Chamoun et al. 2001; Lee and Treisman 2001). For clarity, we refer to this protein as Hh hereafter.

Figure 4.

(A) Hedgehog protein maturation. Hh protein undergoes multiple processing steps: (1) the signal sequence is cleaved; (2) the C-terminal domain of the Hh polypeptide catalyzes an intramolecular cholesteroyl transfer reaction, resulting in (3) the formation of a C-terminally cholesterol-modified N-terminal Hh signaling domain (HhN). This causes association of HhN with membranes, which facilitates the final modification step 4, the addition of a palmitic acid moiety to the N terminus by the acyltransferase Skinny hedgehog, resulting in the formation of dually modified Hh signaling domain (HhNp).

Formation of the Hh gradient

Although Hh is tightly associated with the plasma membrane, it is able to act directly over a long range (Roelink et al. 1995; Briscoe et al. 2001; Wijgerde et al. 2002). In both Drosophila and vertebrates, the secretion of Hh from the producing cell requires the activity of the 12-span transmembrane protein, Dispatched (Disp). Disp, like Ptc, belongs to the bacterial RND (Resistance-Nodulation-Division) family of transport proteins. Loss of Disp leads to accumulation of Hh in the producing cells and failure of long-range signaling (Burke et al. 1999; Ma et al. 2002).

Distances over which Hh has been shown to act are ∼50 μm in Drosophila wing imaginal disc and ∼300 μm in vertebrate limb bud (Zhu and Scott 2004). How Hh moves over a such a long distance is still not clear, and could involve passive diffusion, active transport, and/or transcytosis. Genetic evidence points to a role of heparan sulfate proteoglycans in this process, as Hh cannot be transported across a field of cells lacking the heparan sulfate synthesizing enzymes of the EXT/tout velu (ttv)/brother of tout velu (botv)/sister of tout velu (sotv) family (Bellaiche et al. 1998; Lin et al. 2000; Bornemann et al. 2004; Han et al. 2004a;Koziel et al. 2004). The substrates of ttv involved in this process appear to be the glypicans (glycosylphosphatidylinositol-linked HSPGs) Dally and Dally-like (Han et al. 2004b). Dally and Dally-like also affect Hh signaling by facilitating binding of Hh to cell surfaces (Nakato et al. 1995; Lum et al. 2003a; Han et al. 2004b).

Whether Hh is transported as individual molecules or assembled into larger particles prior to transport is not clear. Several lines of evidence support the role of large lipid/protein particles in long-range Hh transport. First, Hh staining of receiving cells displays a punctate pattern (Panakova et al. 2005). In addition, soluble Shh multimers that contain lipids and that have strong signaling potency have been described in mammalian cells (Zeng et al. 2001), and it has been reported that Drosophila Hh is transported in lipoprotein particles (Panakova et al. 2005; Callejo et al. 2006). Recent genetic evidence also suggests that Hh may be secreted in two different forms, the first of which diffuses poorly and acts at a short range. The second form is “packaged” for long-range transport, and its formation requires the cytoplasmic membrane-scaffolding protein Reggie-1/flotillin-2 (Katanaev et al. 2008).

Multiple studies have analyzed the role of cholesterol modification in Hh transport in vivo, with conflicting results suggesting that cholesterol either aids or hinders Hh transport (for example, see Lewis et al. 2001; Dawber et al. 2005; Gallet et al. 2006; Li et al. 2006). These studies are complicated because the protein expression levels of the different mutant forms of Hh need to be constant in order to rule out dose effects. In addition, interpretation of the results is made even more difficult by the fact that Hh protein lacking cholesterol modification is soluble, and thus its secretion does not require Dispatched and it can escape the producing cell without being palmitoylated (Mann and Beachy 2004) and could even become palmitoylated later during transport or at the receiving cell. Thus, genetic experiments alone cannot conclusively determine the role of cholesterol modification in Hh activity and transport. In contrast, analysis of the role of the palmitate modification in Hh transport is more straightforward, as palmitoylation can be selectively prevented either by mutation of Ski, or mutation of the palmitoylated N-terminal cysteine of the Hh proteins. Such experiments indicate that palmitoylation is required for Hh activity in Drosophila (Burke et al. 1999), and for generation of soluble multimeric Hh protein complexes and long-range signaling in vertebrates (Chen et al. 2004).

Several mechanisms are used to control the shape and size of the Hh gradient (for review, see Teleman et al. 2001). Very high levels of Hh can induce Hh expression in responding cells both in Drosophila and in mammals (Tabata et al. 1992;Roelink et al. 1995; Methot and Basler 1999). This increases the local concentration of Hh near the original source. Hh also induces the expression of its receptor Ptc, which internalizes Hh and targets it to the lysosomes for degradation (Chen and Struhl 1996; Incardona et al. 2000; Gallet and Therond 2005). This negative feedback loop restricts the spreading of the Hh signal through tissues. Vertebrates also have an additional transmembrane protein, Hedgehog-interacting protein (HIP), which is also induced by Hh signaling and binds to and further reduces the range of movement of Hh (Chuang and McMahon 1999; Jeong and McMahon 2005).

Hh signal transduction   
Hh receptor

In addition to the glypical dally-like, which acts both in Hh transport and as an accessory receptor, the binding of Hh to responding cells is facilitated by the transmembrane proteins Cdo and Boc (iHog and boi in Drosophila) (Lum et al. 2003a; Tenzen et al. 2006; Yao et al. 2006). These proteins act positively in the pathway, binding to Hh via conserved fibronectin repeats (Yao et al. 2006) and increasing Hh association with its signaling receptor Ptc (Tenzen et al. 2006; Yao et al. 2006). The expression levels of Cdo and Boc are down-regulated in response to Hh signaling, resulting in yet another negative feedback that limits pathway activity (Fig. 1C).

In the absence of Hh ligand, Ptc catalytically inhibits the activity of the seven-transmembrane-span receptor-like protein Smo (Taipale et al. 2002). Binding of Hh to Ptc results in loss of Ptc activity, and consequent activation of Smo. Smo then transduces the Hh signal to the cytoplasm (Stone et al. 1996; Taipale et al. 2002). This general model is based on the genetic observations that loss of Hh or Smo cause similar phenotypes, and that Ptc loss results in a phenotype that is similar to strong overexpression of Hh. Epistasis analyses in turn indicate that Ptc acts downstream from Hh and upstream of or parallel to Smo (Ingham et al. 1991;Alcedo et al. 1996; van den Heuvel and Ingham 1996). Binding of Hh to Ptc, in turn, was determined using purified Shh and cultured cells overexpressing Ptc (Stone et al. 1996; Fuse et al. 1999).

By inferring the protein levels of ligand-bound and unbound Ptc from gene expression, Casali and Struhl (2004) suggested that the activity of the pathway depends on the ratio between these two forms. However, the fact that increasing the level of Ptc protein decreases cellular responsiveness to Hh (see Bailey et al. 2002; Taipale et al. 2002) indicates that it is the absolute amount of unliganded Ptc in a cell that controls pathway activity. This mechanism, together with the induction of Ptc by Hh results in gradual desensitization of cells to Hh and allows cells to accurately interpret the wide range of Hh concentrations present in morphogenetic gradients.

In vertebrates, Ptc exists as two isoforms, Ptc and Ptc2. Mice deficient in Ptc2 are viable, but develop alopecia and epidermal hypoplasia and have increased tumor incidence in the presence of one mutant allele of Ptc (Lee et al. 2006; Nieuwenhuis et al. 2006). Loss of Ptc, in turn, results in complete activation of the Hh pathway (Goodrich et al. 1997), suggesting that Ptc is the functional ortholog of DrosophilaPtc. Ptc has been proposed to function as a permease to affect the transmembrane movement and/or concentration of small molecules that then either agonize or antagonize Smo (Taipale et al. 2002). Supporting this hypothesis, Smo activity can be modulated by many synthetic small molecules (Chen et al. 2002b; Frank-Kamenetsky et al. 2002) and natural products, including the steroidal alkaloids cyclopamine and jervine (Chen et al. 2002a). These compounds were identified byKeeler and Binns (1966) as active ingredients in Veratrum californicum, a plant whose ingestion by sheep led to an outbreak of cyclopia in US midwest in the 1950s. The clue that these compounds antagonize Shh signaling came from the observation that the stillborn lambs have a phenotype that is strikingly similar to that of Shh mutant mouse embryos (Chiang et al. 1996).

The structural similarity between cyclopamine and sterols (Cooper et al. 1998) suggests that endogenous sterols might regulate Smo activity. This hypothesis is also supported by genetic evidence, as disruption of embryonic cholesterol synthesis leads to developmental malformations that strikingly mimic Hh mutants (Kelley et al. 1996; Cooper et al. 1998). Oxysterols (Corcoran and Scott 2006) and vitamin D3 derivatives (Bijlsma et al. 2006) have been suggested to be the endogenous metabolites that modulate Smo activity. Of these, vitamin D3 appears to bind to Smo (Bijlsma et al. 2006) based on its ability to compete against binding of labeled cyclopamine (Chen et al. 2002a).

Based on the fact that increased activity of oncogenically activated Smo proteins correlates with their increased resistance to cyclopamine, it was suggested that Smo exists in active and inactive conformational states (Taipale et al. 2000). Similarly, experiments in Drosophila suggest that dSmo can exist in two conformational states (Zhao et al. 2007). However, the activity of all small molecules found to activate or inhibit Smo appear to be specific for vertebrate Smo proteins, suggesting that mechanisms of action of Drosophila and mammalian Smo may be different. Stronger evidence for this comes from both structural and functional analyses, which indicate that Smo C-terminal domain has evolved differentially in vertebrates and invertebrates.

Several lines of evidence suggest that the cytoplasmic components and the mechanism of Hh signal transduction have diverged between Drosophila and mammals. In the following section, we will first discuss the mechanism of intracellular Hh signal transduction in Drosophila, which is fairly well understood. We will then discuss the evidence suggesting that Drosophila and mammals appear to use different components and mechanisms in transducing the Hh signal between Smo and the Ci/GLI transcription factors.

Intracellular Hh signaling in Drosophila

In the absence of Hh, Ptc keeps Drosophila Smo in an unphosphorylated state. Unphosphorylated Smo is cleared from the cell surface via endocytosis and is degraded in lysosomes (Jia et al. 2004; Zhang et al. 2004). After Hh stimulation, Smo is hyperphosphorylated and its endocytosis and degradation are blocked. Phosphorylation can be mimicked by mutation of the phosphorylation sites to negatively charged residues or by mutating adjacent positively charged arginine clusters to alanine. Based on these observations, Zhao et al. (2007) suggested that phosphorylation neutralizes the positive charge of the dSmo C terminus and induces a conformational switch in the C-terminal cytoplasmic tail and consequent dimerization or multimerization of dSmo. How these events lead to activation of downstream signaling pathway components is not understood (Zhao et al. 2007).

dSmo C terminus binds directly to the kinesin-like protein Cos2, which acts as a scaffolding protein, bringing together multiple cytoplasmic components of the pathway (Jia et al. 2003; Lum et al. 2003b; Ogden et al. 2003; Ruel et al. 2003). These include the full-length transcriptional activator form of Ci, CiA (155 kDa) (Robbins et al. 1997), and multiple serine–threonine kinases, including a kinase that specifically acts on the Hh pathaway, Fused (Fu) (Therond et al. 1996) and the multifunction kinases PKA, GSK3β, CKIα, and CKIε (for review, see Aikin et al. 2008).

In the absence of Hh, CiA is hyperphosphorylated by the combined action of PKA, which acts as a priming kinase, and GSK3β and the casein kinases, which further phosphorylate the primed substrate (Fig. 1B). The hyperphosphorylation promotes recognition of CiA by the ubiquitin E3 ligase Slimb (β-TrCP in vertebrates) (Jiang and Struhl 1998), leading to the generation of a truncated transcriptional repressor form of Ci, CiR (75 kDa) (Y. Chen et al. 1999; Price and Kalderon 1999, 2002;Wang et al. 1999; Jia et al. 2002, 2005). In addition to promoting CiR formation, Cos2 regulates Ci by tethering it to the cytoplasm and preventing its nuclear translocation (C.H. Chen et al. 1999; G. Wang et al. 2000).

In the presence of Hh, Sno accumulates and the binding of Cos2 to Smo prevents conversion of CiA to CiR (Hooper 2003; Jia et al. 2003). However, this mechanism alone is not sufficient to fully activate the pathway, as some CiA is still retained in the cytoplasm by another protein, Supressor of Fused [Su(Fu)] (Pham et al. 1995;Methot and Basler 2000). Genetic evidence from Drosophila indicates that full activation of the pathway in response to Hh requires the Fu protein kinase, which blocks the negative influence of Su(Fu) on Ci (Ohlmeyer and Kalderon 1998; Lefers et al. 2001; Lum et al. 2003b). Upon entering the nucleus, CiA binds to specific sequences (Kinzler and Vogelstein 1990; Hallikas et al. 2006) in promoter and enhancer regions and controls the transcription of the Hh target gene(s).

In Drosophila, cellular responsiveness to Hh is controlled by modulating the expression of Ci. In the posterior compartment of the wing disc, Hh and its receptor components are expressed, but target genes are not activated, as Ci mRNA expression is repressed by Engrailed (Eaton and Kornberg 1990). Cells posterior to the morphogenetic furrow of Drosophila eye, in turn, fail to respond to Hh because Ci levels are post-transcriptionally down-regulated due to the expression of hib (Hh-induced MATH and BTB protein; SPOP in vertebrates), a protein that acts as a substrate recognition subunit for the Cul3 E3 ubiquitin ligase. In contrast to Slimb-mediated ubiquitinylation, which leads to partial Ci degradation, the hib/Cul3-mediated ubiquitinylation causes complete degradation of Ci (L. Zhang et al. 2006). Expression of hib increases in response to Hh, providing another negative feedback mechanism to this pathway (Fig. 1C; Kent et al. 2006; Q. Zhang et al. 2006).

Divergence of pathway components and mechanisms

Despite the conservation of the Hh signaling pathway and many of its roles in development between invertebrate and vertebrate species (Ingham and McMahon 2001; Taipale and Beachy 2001), the mechanisms by which Smo regulates the Ci/GLI transcription factors appears to be distinct between Drosophila and mammals (Huangfu and Anderson 2006; Varjosalo and Taipale 2007).

The relatively rapid evolution of some components of the Hh pathway, including Smo, Cos2, and Fu, is apparent at sequence level. The C-terminal domains of vertebrate Smo proteins are significantly shorter than those of invertebrates and lack the main phosphorylation regions described below. In addition, the two mammalian orthologs of Cos2, Kif27, and Kif7 have none of the unique sequence characteristics of Cos2 that differentiate Cos2 from the kinesin family of motor proteins. Based on sequence, Kif7 and Kif27 appear to be functional molecular motors, whereas Cos2 has apparently lost its ability to bind ATP and function as a motor protein. The closest mammalian homolog of Drosophila Fu is also highly diverged, and significant homology between these proteins can be seen only in the protein kinase domain itself (Murone et al. 2000).

Drosophila Smo activation is coupled to the hyperphosphorylation of 26 serine/threonine residues located within the C-terminal cytoplasmic tail by PKA and CKI (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). None of these PKA or CKI phosphorylation sites are conserved in vertebrate Smo. The vertebrate Smo C termini lacks one of the two known Cos2-binding domains (Jia et al. 2003), and the region homologous to the other domain (Lum et al. 2003b) is dispensable for mouse Smo (mSmo) function (Varjosalo et al. 2006). Drosophila Cos2, or mouse Kif7 or Kif27 do not appear to bind to mSmo or GLI proteins or affect Shh signaling when overexpressed in NIH-3T3 cells (Varjosalo et al. 2006). Furthermore, loss of the Fu protein kinase—which forms a tight complex with Cos2 and is required for Hh signaling in Drosophila—appears not to impair Hh signaling in mice (Chen et al. 2005; Merchant et al. 2005). Taken together, this evidence suggests that the Cos2–Fu complex, which is centrally important inDrosophila, plays little or no role in mammalian Hh signaling. Instead, it appears that mammalian Hh signaling critically depends on Su(Fu) (Svard et al. 2006)—which has a minor role in Drosophila (Ohlmeyer and Kalderon 1998)—and on several components involved in formation of the primary cilia, which either do not have Drosophila orthologs or whose orthologs appear not to function on theDrosophila Hh pathway (Nybakken et al. 2005).

Primary cilium is an organelle that protrudes from the surface of most vertebrate cells. Genetic evidence suggesting a role for primary cilium in mammalian Hh signaling comes from studies that found that mutations of several proteins required for its formation, including Kif3a, Ift88, and Ift172, result in embryonic phenotypes characteristic of the loss of Shh signaling (Huangfu et al. 2003; Park et al. 2006; Caspary et al. 2007; Vierkotten et al. 2007). Subsequent studies have linked these proteins to the processing of the GLI transcription factors (May et al. 2005; Caspary et al. 2007). Some experiments suggest that primary cilium would act as a “signaling center” where the biochemical events of signal transduction take place. It has been reported that activated mammalian Smo accumulates to primary cilia in response to Shh treatment (Corbit et al. 2005); in the absence of Shh, this accumulation is prevented by Ptc (Rohatgi et al. 2007). Other components involved in Hh signaling, including Su(Fu) and unprocessed GLI proteins, have also been localized to the primary cilium (Haycraft et al. 2005).

Drosophila lacking centrioles, and all microtubule-based structures derived from them, including centrosomes, cilia, and flagella develop almost normally, indicating that cilia are not required for Drosophila Hh signaling (Basto et al. 2006). In contrast, the genetic studies described above have clearly established that mammalian Hh signaling depends on a process that requires components involved in formation of primary cilia. However, this evidence is also consistent with a model where some other microtubule-linked process that is critical for Hh signaling is disrupted by loss of these proteins. In addition, the fraction of cellular Hh pathway components found in the primary cilium at any given time appears small. Thus, it remains to be established what role cilia play in mammalian Hh signaling and whether localization of the pathway components to cilia is required for signaling.

The lack of effect of the closest mammalian homolog of Drosophila Fused on Hh signaling suggests that other—mammalian-specific—kinases act on this pathway. We recently identified two such kinases, DYRK2 and MAP3K10, which are required for Shh signaling in NIH-3T3 cells (Varjosalo et al. 2008). Of these, DYRK2 directly phosphorylates GLI2 and GLI3 and induces their degradation. MAP3K10, in turn, appears to act in a more indirect fashion, binding to and phosphorylating multiple other proteins that regulate the Hh pathway, including GSK3β, DYRK2, and Kif3a (Nagata et al. 1998; Varjosalo et al. 2008). Because of the many connections of MAP3K10 to different pathway components, its mechanism of action is likely to be complex, and requires further study. In addition to DYRK2 and MAP3K10, it has been reported that other vertebrate-specific kinases regulate Shh signaling. These include protein kinase C-δ (PKCδ), mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1), Akt, and DYRK1 (Mao et al. 2002; Riobo et al. 2006a,b). From our studies and previous analyses of the Hh pathway, it appears that Hh does not regulate the activity of any known kinase toward a generic substrate. Thus, the mechanism by which Hh signal is transduced appears not to depend on activation of pathway-specific kinases, but on regulation of access of substrates to relatively generic multifunctional kinases.

In conclusion, the mechanisms of mammalian Hh signaling have clearly diverged from those of Drosophila. This suggests that even signal-transduction mechanisms of conserved signaling pathways have not been “locked” early in evolution, and that they can be subject to evolutionary change. The divergence of the Hh pathway—arguably the last major signaling pathway to evolve—is also relevant to the evolution of multicomponent signaling pathways. Some pathways, such as the Notch pathway, where the same protein (Notch) functions as a receptor and a transcriptional coactivator are relatively simple and consist of a small number of pathway-specific components (Artavanis-Tsakonas et al. 1999; Pires-daSilva and Sommer 2003). Other pathways, such as the Hh signaling pathway inDrosophila are more complex. In addition to many multifunctional proteins, the Hh pathway consists of 11 known specific components: Hh, Skinny hedgehog (Ski), Dispatched, iHog/boi, Ptc, Smo, Cos2, Fu, Su(Fu), and Ci (Burke et al. 1999;Chamoun et al. 2001; Lum and Beachy 2004). The emergence of the Cos2–Fu system in invertebrates suggests that such multicomponent pathways may evolve by insertion of novel proteins between existing pathway components.

Regulation of GLI activity

In contrast to the differences in signaling between Smo and GLI, the activities of the GLI proteins themselves are regulated similarly to Ci—with the added complexity that the activator and repressor functions of Ci are divided in mammals to three GLI proteins, GLI1–3 (Jacob and Briscoe 2003; Ruiz i Altaba et al. 2007). GLI1 and GLI2 are responsible for most activator functions and have similar activities at protein level (Bai and Joyner 2001). Whereas loss of GLI2 is embryonic lethal (Mo et al. 1997; Ding et al. 1998; Matise et al. 1998), GLI1 is dispensable for normal development (Park et al. 2000). GLI1 expression is induced by Hh ligands, and its function appears to be primarily to provide positive feedback and to prolong cellular responses to Hh. GLI3, in turn, functions primarily as a repressor (B. Wang et al. 2000; Litingtung et al. 2002), and its loss or mutation leads to limb malformations in mice and humans (Vortkamp et al. 1991; Schimmang et al. 1992).

GLI activity appears to be regulated by Hh in a way that is very similar to that observed in Drosophila. In the absence of Hh, GLI3 is phosphorylated, recognized by β-TrCP, and proteolytically processed to a truncated repressor form (B. Wang et al. 2000; Pan et al. 2006). Whether similar processing of GLI2 results in complete degradation or generation of a truncated repressor form is unclear (Pan et al. 2006; Wang and Li 2006). Addition of Shh leads to inhibition of processing and accumulation of full-length forms of both GLI2 and GLI3.

Dose-, time-, and context-dependent responses to Hh

The developmental processes that the Drosophila and vertebrate Hh signaling pathways regulate appear remarkably conserved (Ingham and McMahon 2001). At the cellular level, the effects of Hh range from growth and self-renewal to cell survival (Liu et al. 1998; Rowitch et al. 1999), differentiation, and/or migration. During embryogenesis, the Hh cascade is used repeatedly and in different tissues to induce a large number of developmental processes. The ability of a single morphogen to affect almost every part of the vertebrate body plan is made possible by the fact that cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time the cell is exposed to Hh (see below). At the molecular level, the diverse cellular responses are effected by induction of different sets of target genes. Among the genes regulated tissue specifically by Hh signaling are those encoding other secreted signaling proteins, including bone morphogenetic protein 4 (BMP4) (Astorga and Carlsson 2007),fibroblast growth factor 4 (FGF4) (Laufer et al. 1994), and vascular endothelial growth factor (VEGF)-A (Pola et al. 2001), genes involved in cell growth and division (e.g., N-Myc) (Oliver et al. 2003), and many transcription factors that are essential for animal development, including members of the Myod/Myf, Pax, Nkx, Dbx, and Irx families (Pierani et al. 1999; Gustafsson et al. 2002; Jacob and Briscoe 2003; Vokes et al. 2007). The total number of genes that Hh regulates is only beginning to be discovered: A number of expression profiling studies have identified several novel target genes (for example, see Xu et al. 2006; Vokes et al. 2007), and our genome-wide in silico analyses identified 42 conserved enhancer modules with two or more GLI sites in the human genome (Hallikas et al. 2006).

The genes that are induced by Hh in many tissues, in turn, are generally involved in positive and negative feedback to the pathway itself and include Hib, GLI1, Ptc, and HIP (Fig. 1C). As Ci and the GLI proteins act as repressors in the absence of Hh and activators in its presence, many of the target genes also behave similarly, being repressed in the absence of Hh and induced in its presence.

Hh acts both directly and indirectly to pattern tissues

During the development of the Drosophila wing imaginal disc, posterior (P) compartment cells express and secrete the Hh protein (Fig. 5A). The secreted Hh then induces the expression of target genes in cells located in the anterior (A) compartment. Hh acts both directly at intermediate range to pattern the anterior wing tissues close to the A–P boundary and indirectly over long range by inducing the BMP family morphogen decapentaplegic (dpp) (Basler and Struhl 1994; Tabata and Kornberg 1994). Dpp diffuses bidirectionally into both A and P compartments and controls the growth and patterning of the entire wing. Dpp expression is normally repressed by CiR, and its activation only requires that this repression is lifted. Therefore, very low levels of Hh suffice to induce dpp expression (Methot and Basler 1999). The short and intermediate range effects of Hh require induction of target genes such as collier (col) and engrailed (en), whose expression require CiA function and higher levels of Hh (Methot and Basler 1999; Hooper 2003).

Figure 5.

(A) Hh acts both directly and indirectly to pattern theDrosophila wing imaginal disc. (Left) Hh activates decapentaplegic (dpp; red) at the anterior side of the A–P boundary of the imaginal disc, which diffuses into and patterns both A and P compartments (red arrow). Hh (blue) also acts directly to pattern the anterior compartment close to the A–P boundary. (Right) Adult wing showing the regions derived from the anterior (A, top) and posterior compartment (P, bottom, shaded), and the regions patterned by Dpp (red arrows) and Hh (blue color, between wing veins 3 and 4). (B) Shh has a similar role in anterior-posterior patterning of the distal elements of vertebrate limbs and in specifying digit identity (roman numerals). (C) Time and dose-dependent action of Shh. The gradient of Shh (blue color) emanating from the notochord (not shown) and floor plate (FP) progressively defines five different neuronal subtypes in the ventral neural tube. The Shh protein gradient is converted to gradient of GLI activities shown on the left. GLI1 and GLI2 (bottom) act as transcriptional activators, whereas GLI3 functions as a repressor (GLI3R, top). (MN) Motoneuron; (V0–V3) interneurons. Dotted line indicates the dorsal limit of the domain patterned by the Shh gradient. Data adapted from Fuccillo et al. (2006).

Shh has an analogous role in controlling vertebrate limb patterning. Shh expressed by the ZPA located at the posterior margin of developing limb buds diffuses to adjacent tissues, forming a temporal and spatial gradient that specifies the anterior–posterior pattern of the limbs (Fig. 5B).

Time and dose dependency of the Hh response

The effect of Hh dose on target tissue responses is best characterized in the specification of cell identities in the ventral neural tube (Jessell 2000; Patten and Placzek 2000; Marti and Bovolenta 2002). During neural tube development, Shh protein diffuses from the notochord and floor plate, creating a concentration gradient across the ventral neural tube (Fig. 5C). Different doses of Shh within this gradient specify five neuronal subtypes at precise positions along the floor plate–roof plate axis. Initially, Shh induces Class II homeodomain (e.g., Nkx2.2, Nkx6.1) (Pierani et al. 1999; Jacob and Briscoe 2003) and represses Class I homeodomain (Pax6, Pax7, Irx3, and Dbx1/2) transcription factors. Cross-repressive interactions between these factors then act to sharpen the expression boundaries and to subsequently direct cells to differentiate into specific lineages (Briscoe and Ericson 2001).

The activity of Shh as a morphogen was initially thought to be due to concentration-dependent responses, but the duration of Shh signal seems also to critically affect cellular responses. Both during neural tube and limb development, the pattern of cellular differentiation is controlled not only by the amount but also by the time of Shh exposure (Briscoe and Ericson 2001; Ahn and Joyner 2004;Harfe et al. 2004). The changing of the concentration or duration of Shh seem to have an equivalent effect on intracellular signaling.

Chick neural cells convert different concentrations of Shh into time-limited periods of signal transduction, such that signal duration is proportional to Shh concentration (Dessaud et al. 2007). This depends on the gradual desensitization of cells to Shh caused by up-regulation of patched (Ptc) (Taipale et al. 2002). Thus, in addition to its role in shaping the Shh gradient (Chen and Struhl 1996; Briscoe et al. 2001; Jeong and McMahon 2005), Ptc participates cell-autonomously in gradient sensing. This mechanism integrates Shh signal strength over time, allowing cells to more accurately determine their distance from the Hh source—resulting in more robust patterning of the nervous system.

Role of Hh signaling in young and adult mammals

The multiple roles of Hh signaling in embryonic patterning are discussed above and reviewed in more detail in McMahon et al. (2003). Much less is known about the roles played by Hh in pupal development and in maintaining homeostasis of tissues during adult life.

During maturation of mouse pups, Ihh signaling is important for bone growth. Permanent deletion of Ihh in chondrocytes of mice carrying conditional and inducible null alleles of Ihh results in permanent defects in bone growth, inhibiting proliferation and promoting differentiation of chondrocytes, leading to dramatic expansion of the hypertrophic zone (Razzaque et al. 2005; Maeda et al. 2007) and truncation of long bones. Interestingly, similar phenotype was observed with treatment of young mice with Smo antagonist for just 48 h (Kimura et al. 2008). In adults, Hh pathway controls bone homeostasis; activation of the Hh pathway in osteoblasts leads to bone resorption, and conversely, Hh inhibition protects aging mice against bone loss (Mak et al. 2008; Ohba et al. 2008). Adult mice seem to tolerate Hh antagonists well, suggesting that short-term Hh pathway inhibition might not interfere with the possible role of Hh as a stem cell factor (Berman et al. 2002; Kimura et al. 2008).

The best-characterized role for Hh signaling in adults is in the reproductive system, and Hh proteins are expressed and required for maturation of the germ cells in multiple species. In Drosophila ovary, Hh acts as a somatic stem cell factor, directly controlling the proliferation and maintenance of ovarian somatic stem cells (Zhang and Kalderon 2001). In mammals, Ihh and Dhh produced by granulosa cells act as paracrine factors to induce target gene expression in the developing theca cell compartment. This suggests that hedgehog signaling regulates the theca cell development in growing follicles (Wijgerde et al. 2005). Dhh also has a role in the regulating the development and function of the somatic cells of the testis (Bitgood et al. 1996; Yao et al. 2002).

Aberrant Hh signaling in disease

Loss of Hh signaling activity during vertebrate embryogenesis causes severe developmental disorders including holoprosencephaly, polydactyly, craniofacial defects, and skeletal malformations (Muenke and Beachy 2000; Hill et al. 2003;McMahon et al. 2003; L. Zhang et al. 2006). It is now also becoming evident that components required for the function of primary cilia are required in mammalian Shh signaling (Huangfu et al. 2003). It is therefore possible that Hh signaling may also be altered in human syndromes caused by defects in cilia, including Meckel, Bardet-Biedl and Kartagener syndromes, polycystic kidney disease, and retinal degeneration (Pan et al. 2005; Kyttala et al. 2006).

On the other hand, aberrant activation of Hh signaling can cause basal cell carcinoma (BCC, the most common type of skin cancer) (Hahn et al. 1996; Johnson et al. 1996), medulloblastoma (a childhood cancer with an invariably poor prognosis) (Goodrich et al. 1997; Berman et al. 2002), and rhabdomyosarcoma (Table 1; Kappler et al. 2004). These tumor types occur at an increased rate in patients or mice with germline mutations in Ptc, and sporadic cases are often associated with mutations in the Hh pathway components Ptc, Smo, or Su(Fu), or more rarely, the amplification of GLI1.

Table 1.

Cancers linked to aberrant Shh signaling

Aberrantly activated Shh signaling has also been suggested to play a role in other cancers, such as glioma, breast, esophageal, gastric, pancreatic, prostate, and small-cell lung carcinoma (see Table 1 for references). With the exception of rare GLI1 amplifications found in gliomas (Kinzler et al. 1987), the mutational basis of Hh pathway activation in these types of cancer has not been ascertained.

Multiple lines of evidence suggest that Hh acts to promote cancer by directly regulating cellular growth and/or survival. Loss of one ptc allele causes larger body size in mice (Goodrich et al. 1997) and in humans (Gorlin 1987). Several common human single nucleotide polymorphisms affecting body height map close to Hh pathway components, including Ihh, Ptc, and Hip (Lettre et al. 2008; Weedon et al. 2008), suggesting that individual variation in height is determined in part by the strength of negative feedback loops that fine-tune Ihh signaling during bone growth. Hh pathway controls growth also during embryonic development—transgenic mice that overexpress ptc are consistently smaller than control mice, but remarkably well proportioned, illustrating that Hh signaling controls growth in many tissues. However, whether this growth effect is direct or indirectly caused by altered placental or vascular development is unclear.

In development of midbrain and forebrain, Shh is crucial in driving the rapid, extensive expansion of the early brain vesicles. The action of Shh is mediated through coordination of cell proliferation and survival (Britto et al. 2002). In addition, Shh has been implicated in regulating cell proliferation and survival in a number of other cell types, including retinal precursor cells (Jensen and Wallace 1997), myoblasts (Duprez et al. 1998), optic nerve astrocytes (Wallace and Raff 1999), cerebellar granule cells (Dahmane and Ruiz i Altaba 1999), and neural crest cells (Ahlgren and Bronner-Fraser 1999).

The molecular mechanisms by which Shh controls growth are beginning to be unraveled. In vitro studies have shown that the Shh protein up-regulates N-myc expression in cerebellar granule neuron progenitor (CGNP) cultures and that N-myc overexpression promotes CGNP proliferation even in the absence of Shh (Kenney et al. 2003). N-myc is thought to promote proliferation of CGNPs synergistically with cyclins D and E (Knoepfler et al. 2002), whose expression is also regulated by Shh (Duman-Scheel et al. 2002).

Direct evidence for the role of N-myc in pathway-associated cancer comes from a study of Shh pathway-induced medulloblastoma formation in mice, where it was shown that the disruption of N-myc, but not c-myc, inhibits cellular proliferative responses to Shh (Hatton et al. 2006). This provides in vivo evidence that N-myc plays a central role in Shh-mediated proliferation in CGNPs and in medulloblastoma cells, which are thought to be derived from CGNPs (Hatton et al. 2006).

Potential for therapeutic intervention

As the Hh pathway in BCC and medulloblastoma is often affected at the level of Ptc or Smo, small molecule antagonists should act at/or downstream from these components (Taipale et al. 2000). Furthermore, several studies have shown that Smo can be targeted by small molecule drugs, and that antagonizing Smo could provide a way to interfere with tumorigenesis and tumor progression. The most commonly used antagonist of the Hh pathway is the plant alkaloid cyclopamine (Taipale et al. 2000). Cell-based high-throughput screening has revealed several distinct classes of antagonists, which, like cyclopamine, bind to Smo. These include SANTs 1–4 (Chen et al. 2002b); KAAD-cyclopamine (Taipale et al. 2000), compound-5 and compound-Z (Borzillo and Lippa 2005), and Cur-61414 (Frank-Kamenetsky et al. 2002). Although one phase I clinical trial has already reported promising results of Hh pathway antagonist in advanced BCC (Garber 2008), further clinical studies are needed to establish which of these antagonists are suitable for therapeutic use. As it has been proposed that autocrine Shh expression is required for growth of some cancers (Dahmane et al. 1997;Karhadkar et al. 2004), and stromal cell-derived Shh can also activate the Hh pathway in tumors (Becher et al. 2008), it might also be possible to treat tumors with Shh-specific monoclonal antibodies. In fact, one such antibody, 5E1, has been shown to block the growth of some tumors, including small-cell lung carcinoma (Watkins et al. 2003). In addition to targeting tumors that themselves have hyperactive Hh pathways, antagonists of the Hh pathway could also affect growth of tumors that use Hh ligands to induce angiogenesis (Pola et al. 2001; Nagase et al. 2008) or recruit other types of stromal cells supporting tumor growth. Further studies are needed to characterize the role that Shh plays in such tumor–host interactions.

Because adults can tolerate inhibition of the Hh pathway (Berman et al. 2002;Kimura et al. 2008), specifically blocking Hh signaling offers an effective treatment for the various cancers originating from aberrant Hh pathway activation. However, systemic treatment of pediatric tumors such as medulloblastoma may not be feasible due to the severe effects that transient inhibition of the Hh pathway has on bone growth (Kimura et al. 2008).

Perspective

The Hh signaling pathway was first identified in Drosophila 16 yr ago. Subsequently, it has taken its rightful place among the major signaling pathways controlling animal development, being found to regulate the morphogenesis of a variety of tissues and organs during the development of organisms ranging fromDrosophila to human (McMahon et al. 2003). In addition, the Hh pathway has been linked to multiple forms of human cancer, and the possibilities for therapeutic intervention are being actively pursued.

The synthesis and processing of the Hh ligand, its release and transport through tissues, and mechanisms of signal transduction in the receiving cells have been studied extensively. However, many aspects of Hh signaling remain incompletely understood. Further research is needed in multiple areas, including the study of Hh target gene responses, which is required to understand in detail how the graded Hh signals are converted to discrete cell-fate decisions, and to decipher the molecular mechanisms that underlie the exquisite specificity of cellular responses to Hh. In addition, the therapeutic potential of Hh pathway agonists and antagonists in human degenerative diseases and cancer should be further investigated.

 

Targeting the Hedgehog pathway in cancer

Sachin GuptaNaoko Takebe, Patricia LoRusso

Wayne State University, Karmanos Cancer Institute, Detroit, MI, USA
Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA
Wayne State University, Karmanos Cancer Institute, 4100 John R, Detroit, MI 48201, USA
Ther Adv Med Oncol. 2010 Jul; 2(4): 237–250.  doi:  10.1177/1758834010366430
The Hedgehog (Hh) pathway is a major regulator of many fundamental processes in vertebrate embryonic development including stem cell maintenance, cell differentiation, tissue polarity and cell proliferation. Constitutive activation of the Hh pathway leading to tumorigenesis is seen in basal cell carcinomas and medulloblastoma. A variety of other human cancers, including brain, gastrointestinal, lung, breast and prostate cancers, also demonstrate inappropriate activation of this pathway. Paracrine Hh signaling from the tumor to the surrounding stroma was recently shown to promote tumorigenesis. This pathway has also been shown to regulate proliferation of cancer stem cells and to increase tumor invasiveness. Targeted inhibition of Hh signaling may be effective in the treatment and prevention of many types of human cancers. The discovery and synthesis of specific Hh pathway inhibitors have significant clinical implications in novel cancer therapeutics. Several synthetic Hh antagonists are now available, several of which are undergoing clinical evaluation. The orally available compound, GDC-0449, is the farthest along in clinical development. Initial clinical trials in basal cell carcinoma and treatment of select patients with medulloblastoma have shown good efficacy and safety. We review the molecular basis of Hh signaling, the current understanding of pathway activation in different types of human cancers and we discuss the clinical development of Hh pathway inhibitors in human cancer therapy.

The Hedgehog (Hh) gene was initially discovered by Christiane Nusslein-Volhard and Eric F. Weischaus in 1980 in their screen for mutations that disrupt the Drosophila larval body plan [Nusslein-Volhard and Wieschaus, 1980]. The name Hedgehog originates from the short and ‘spiked’ phenotype of the cuticle of the Hh mutant Drosophila larvae, which resembled the spikes of a hedgehog [Varjosalo and Taipale, 2008;Ingham and McMahon, 2001]. The Hh family of proteins have since been recognized as key mediators of many fundamental processes in vertebrate embryonic development playing a crucial role in controlling cell fate, patterning, proliferation, survival and differentiation of many different regions. Hh signals have diverse functions in different contexts. They may act as morphogens in the dose-dependent induction of distinct cell fates within a target field, or may act as a mitogen in the regulation of cell proliferation controlling the form of developing organs [Ingham and McMahon, 2001]. The crucial developmental function of Hh signaling is illustrated by the dramatic consequences in human fetuses, with defects in the Hh signaling pathway resulting in fetuses with brain, facial and other midline defects such as holoprosencephaly (failure of forebrain development) or microencephaly, cyclopia, absent nose or cleft palate [Rubin and de Sauvage, 2006; Belloniet al. 1996; Roessler et al. 1996]. In adults, the Hh pathway remains active and is involved in regulation of tissue homeostasis, continuous renewal and repair of adult tissues, and stem cell maintenance [Hooper and Scott, 2005].

The Hh signaling pathway has also recently been recognized to be one of the most important signaling pathways and a therapeutic target in cancer. In adults, mutation or deregulation of this pathway plays a key role in both proliferation and differentiation leading to tumorigenesis or tumor growth acceleration in a wide variety of tissues. Basal cell carcinoma (BCC) and medulloblastoma are two well-recognized cancers with mutations in components of the Hh pathway [Tostar et al. 2006; Taylor et al. 2002; Dahmane et al. 1997]. Inappropriate activation of the Hh signaling pathway has been implicated in the development of several other types of cancer including lung, prostate, breast, and pancreas, as examples. In addition, some recent findings suggest that Hh might also promote tumorigenesis by signaling in a paracrine manner from the tumor to the surrounding stroma or in cancer stem cells (CSCs).

The first Hh pathway inhibitor to be identified was the naturally occurring plant alkaloid, cyclopamine. This was discovered as a teratogenic compound causing cyclopia and holoprosencephaly in lambs whose mothers had ingested corn lilies, a phenotype similar to Sonic Hedgehog (Shh) knockout mice [Bryden et al. 1971]. No untoward effect was seen in the adult sheep. The active chemical identified in the corn lily, cyclopamine, was subsequently shown to inhibit the Hh pathway by binding to and inactivating the Smoothened (SMO) transmembrane receptor protein [Chen et al. 2002; Cooper et al. 1998]. Cyclopamine is of low affinity, has poor oral bioavailability and suboptimal pharmacokinetics and thus more potent derivatives have been synthesized. Several synthetic, small-molecule SMO antagonists with higher potency than cyclopamine such as SANT1–SANT4, CUR-61414, HhAntag-691 and GDC-0449 are now available and have been tested in preclinical models against a variety of solid tumors [Rudin et al. 2009; Scales and de Sauvage, 2009; Von Hoff et al. 2009]. In this review, we provide a brief overview of Hh signaling, discuss the roles of this pathway in solid tumors, and summarize the clinical advances in using therapeutic agents targeting the Hh signaling cascade.

Hedgehog signal transduction

Hh proteins are secreted signaling proteins that were first discovered in Drosophila along with many other components of their signal transduction machinery [Nusslein-Volhard and Wieschaus, 1980]. The mechanism of Hh protein processing, secretion, and signaling appear to be more or less conserved in evolution between Drosophila and higher organisms, although some differences exist. Drosophila has only one Hh gene, whereas vertebrate Hh signal transduction involves three Hh homologues with different spatial and temporal distribution pattern: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog(Dhh) [Ingham and McMahon, 2001; McMahon, 2000]. The Hh proteins undergo multiple processing steps before signaling. The Hh protein is made as a precursor molecule, consisting of a C-terminal protease domain and an N-terminal signaling unit. The precursor Hh molecule is cleaved to release the active signaling domain called HhNp. Then, the C-terminal domain of the Hh polypeptide catalyzes an intramolecular cholesteroyl transfer resulting in a formation of a C-terminal cholesterol modified N-terminal Hh signaling domain. The cholesterol modification results in association of Hh with membranes, facilitating the final processing step in which a palmitoyl moiety is added to the N-terminus of Hh (acylation), generating the fully active HhN [Varjosalo and Taipale, 2007; Porter et al. 1996]. The gene Rasp encodes the enzyme, likely located at the endoplasmic reticulum, required for the Hh acylation and the production of active Hh [Micchelli et al. 2002]. Hh is then released from the secreting cell by a dedicated transmembrane transporterDispatched (Disp) protein. In embryonic development, the cells that synthesize Hh ligands are distinct from the responsive cells. These responsive cells may either be adjacent to, or at a significant distance from, the Hh secreting cell [Varjosalo and Taipale, 2007].

In humans, the Hh signaling cascade is initiated in the target cell by the Hh ligand binding to the Patched 1protein (PTCH), a 12-span transmembrane protein (Figure 1). In the absence of a Hh ligand, PTCH catalytically inhibits the activity of the seven-transmembrane-span receptor-like protein, SMO, potentially by affecting its localization to the cell surface. It is also proposed that an endogenous intracellular small molecule that acts as an agonist for SMO is transported outside the cell by PTCH, preventing its binding to SMO. Binding of Hh to PTCH results in the loss of PTCH activity and the consequent activation of SMO, which transduces the Hh signal to the cytoplasm [Taipale et al. 2002]. The Hh signal is transmitted via an alteration of the balance between the activator and repressor forms of the Ci (cubitus interruptus)/GLI family of zinc-finger transcription factors. In Drosophilia, the Hh signal is transmitted via a protein complex which includes the atypical kinesin-like protein, Costal 2 (Cos2), Fused (Fu) and Suppressor of Fused (SuFu) and the transcription factor, Ci. In higher organisms, the Cos2 and Fu are not conserved, although SuFu still seems to play an important role in signal transduction. In mammals, the Hh signaling takes place in the nonmotile cilia to which the SMO and other downstream pathway components must need to transit to activate the Ci ortholog in mammals, the GLI transcription factors [Rubin and de Sauvage, 2006; Corbit et al. 2005;Huangfu and Anderson, 2005; Huangfu et al. 2003]. The GLI transcription factors exist as three separate zinc-finger proteins, GLI 1 and GLI 2 functioning as transcriptional activators and GLI 3 as a transcriptional repressor [Ruiz i Altaba, 1997]. The expression of GLI 1 is highly dependent upon active Hh signaling and is thus often used as a readout of pathway activation. In the absence of a Hh ligand, PTCH blocks SMO activity and full length GLI proteins are proteolytically processed to generate the repressor GLIR, largely derived from GLI 3, which represses Hh target genes. Hh binding to PTCH relieves SMO inhibition, promotes generation of the activator GLIA, largely contributed by GLI 2 and the subsequent expression of the Hh target genes. Ubiquitous mammalian Hh target genes include GLI 1, PTCH1, Hh interacting protein (Hhip) and other cell-specific genes such as Cyclin D, Myc, Bmi1, Bcl-2, VEGF (vascular endothelial growth factor) and Snail depending upon the cell type [Scales and de Sauvage, 2009; Ferretti et al. 2005]. GLI activation is regulated at several different levels via phosphorylation by inhibitors such as SuFu, Ren, protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and activators such as Dyrk1, Ras and Akt [Varjosalo and Taipale, 2007; Ferretti et al. 2005]. Hh and PTCH are subsequently internalized and degraded in the lysosomes.

An external file that holds a picture, illustration, etc. Object name is 10.1177_1758834010366430-fig1.jpg
Hedgehog signaling pathway in vertebrates. The above model illustrates our current understanding of the vertebrate Hedgehog (Hh) pathway signaling. Hh signaling cascade is initiated in the target cell by the Hh ligand binding to the Patched 1 protein (PTCH), a 12-span transmembrane protein located on the plasma membrane. Smoothened (SMO), a 7-transmembrane-span protein receptor, is located on the membrane of the intracellular endosome. In mammalians, the Hh signaling takes place in the nonmotile cilia to which the SMO and other downstream pathway components transit to in order to activate the GLI transcription factors [Rubin and de Sauvage, 2006; Corbit et al. 2005;Huangfu and Anderson, 2005; Huangfu et al. 2003]. An endogenous small molecule acting as a SMO agonist is transported outside the cell by PTCH, preventing its binding to SMO. In the absence of a Hh ligand, PTCH catalytically inhibits the activity of SMO by affecting its localization to the cell surface. Full-length GLI proteins are thus proteolytically processed to generate the repressor GLIR, largely derived from GLI 3, which represses Hh target genes. Binding of Hh to PTCH, internalizes and destabilizes PTCH, so that it can no longer transport the endogenous SMO agonist molecules outwards. Intracellular accumulation of this agonist molecule activates SMO which translocates to the plasma membrane, apparently concentrating in the cilia. Relief of SMO inhibition promotes generation of the activator GLIA, largely contributed by GLI 2 and the subsequent expression of the Hh target gene [Taipale et al. 2002]. CK1α, casein kinase 1α; GPCR, G-protein-coupled receptor; GSK3β, glycogen synthase kinase 3β; PKA, protein kinase A. Reprinted by permission from Macmillan Publishers Ltd: Rubin, L.L. and de Sauvage, F.J. (2006) Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 5: 1026–1033.

Although the extent of Hh signaling is significantly lower in the adult compared with the embryo, it is still detectable at a few sites such as the central nervous system (CNS) neural stem cells [Palma et al. 2005;McMahon, 2000]. Hh also plays an important role in the maintenance and proliferation of continuously renewing tissues such as the gut epithelium [van den Brink et al. 2004] and is reactivated at sites of tissue damage and repair [Beachy et al. 2004; Mirsky et al. 1999; Parmantier et al. 1999].

Alteration of the Hedgehog pathway and cancer

In recent years, it has become increasingly clear that the aberrant activation of the Hh signaling pathway can lead to cancer. Three basic models have been proposed for Hh pathway activity in cancer (Figure 2AC) [Scales and de Sauvage, 2009; Rubin and de Sauvage, 2006]. The first discovered were the type I cancers harboring Hh pathway-activating mutations which are Hh ligand independent, such as BCCs and medulloblastomas. Type II cancers are autocrine (or juxtacrine) ligand dependent, meaning that Hh is both produced and responded to by the same (or neighboring) tumor cells. Type III cancers, which are paracrine ligand dependent, have been described recently. In paracrine signaling, Hh produced by the tumor cells is received by the stroma, which feeds other signals back to the tumor to promote its growth or survival [Scales and de Sauvage, 2009; Rubin and de Sauvage, 2006].

An external file that holds a picture, illustration, etc. Object name is 10.1177_1758834010366430-fig2.jpg
An external file that holds a picture, illustration, etc. Object name is 10.1177_1758834010366430-fig2a.jpg
 Different models of Hedgehog pathway signaling. (A) Type I ligand-independent cancers harbor inactivating mutations in Patched 1 protein (PTCH) or activating mutations in Smoothened (SMO) leading to constitutive activation of the Hedgehog (Hh) pathway even in the absence of the Hh ligand. (B) Type II ligand-dependent autocrine cancers both produce and respond to the Hh ligand leading to support tumor growth and survival. (C) Type III ligand-dependent paracrine cancers secrete the Hh ligand which is received by the stromal cells leading to pathway activation in the stroma. The stroma in turn feeds back various signals such as IGF, Wnt, VEGF to the tumor tissue leading to its growth or survival. (D) Type IIIb reverse paracrine tumors receive Hh secreted from the stroma leading to pathway activation in the tumor cells and upregulation of survival signals. (E) Cancer stem cells (CSCs): Hh signaling occurs only in the self-renewing CSCs, from the Hh ligand produced either by the CSCs or by the stroma. CSC will give rise to more Hh pathway dependent CSCs or possibly may differentiate into Hh-pathway negative tumor cells comprising the bulk of the tumor. Reprinted from: Scales, S.J. and de Sauvage, F.J. (2009) Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 30: 303–312, with permission from Elsevier.
Type I Hedgehog signaling: ligand independent, mutation driven

The first hint to the involvement of the Hh pathway in human cancer was appreciated when inactivating mutations in PTCH were identified in the rare condition Gorlin’s syndrome [Hahn et al. 1996; Johnson et al. 1996]. Patients with Gorlin’s syndrome develop numerous BCCs during their lifetime and are at an increased risk of other tumors including medulloblastoma, a tumor of the cerebellar progenitor cells, and rhabdomyosarcoma, a muscle tumor. This link was further strengthened when ligand-independent activation of the Hh pathway was observed in a majority of sporadically occurring BCCs [Dahmane et al. 1997]. Most of these tumors either had inactivating mutations in PTCH (85%) or activating mutations in SMO (10%) [Xieet al. 1998]. Furthermore, about one third of all medulloblastomas and occasional rhabdomyosarcomas were shown to have inappropriate Hh pathway activation, often due to PTCH mutations and sometimes due to SuFu mutations [Tostar et al. 2006; Taylor et al. 2002]. Dysregulated Hh signaling led to increased cell proliferation and tumor formation. These observations have been confirmed in various mouse models as well. Mice that are heterozygous for a PTCH mutation have a higher frequency of developing medulloblastoma, and susceptible to formation of UV-induced BCC, similar to patients with the Gorlin’s syndrome [Aszterbaum et al. 1999]. Other mouse models with ectopic expression of various Hh signaling components have been shown to develop skin phenotypes with increased epidermal proliferation and BCC-like tumors as seen in Gorlin’s syndrome [Rubin and de Sauvage, 2006; Svard et al. 2006]. The first clinical trials of Hh pathway inhibitor therapy included several patients with recurrent or metastatic BCC. Since these tumors are ligand independent, Hh pathway inhibitors must act at or below the level of SMO to be effective.

Type II Hedgehog signaling: autocrine, ligand dependent

Constitutive activation of the Hh pathway has been detected in a broad variety of tumors including lung, stomach, esophagus, pancreas, prostate, breast, liver and brain [Clement et al. 2007; Sicklick et al. 2006;Karhadkar et al. 2004; Kubo et al. 2004; Berman et al. 2003; Thayer et al. 2003; Watkins et al. 2003b]. Most of these tumors are dissimilar to BCC or medulloblastomas in that they do not harbor any somatic mutations in the Hh signaling pathway. Rather, they demonstrate an autocrine, ligand-dependent, abnormal Hh pathway activation. Most of these tumors have an elevated expression of the Hh ligand (Shh or Ihh) and/or ectopic PTCH and GLI expression within the epithelial compartment. Ectopic Hh ligand production occurring in all tumor cells or in a small number of tumor stem cells, acts upon itself or the neighboring tumor cells to support tumor growth and survival. This autocrine tumor growth can be effectively suppressed by various pathway inhibitors such as Hh neutralizing antibodies or SMO antagonists.

Type III Hedgehog signaling: paracrine, ligand dependent

A recent report by Yauch and colleagues highlighted that tumor Hh signaling may occur via paracrine mechanisms and emphasized the importance of Hh signaling in promoting the tumor microenvironment [Jiang and Hui, 2008; Yauch et al. 2008]. Paracrine Hh signaling is critical during development and for the maintenance of various epithelial structures such as the small intestine [Theunissen and de Sauvage, 2009;Varjosalo and Taipale, 2008; Ingham and McMahon, 2001]. Hh ligand secreted by the epithelium is received by the mesenchymal stroma and directly affects and stimulates proliferation in the mesenchyme. Upon Hh target gene activation, the mesenchyme produces additional molecules that feed back to the epithelium.

Fan and colleagues first showed that at least one model of prostate cancer signals to the stroma through paracrine mechanisms, with an elevated expression of PTCH and GLI in the murine stroma in response to Hh production by human xenografts [Fan et al. 2004]. These results were extended recently by three reports which showed that the Hh ligand expressing cancers were refractory to the ligand, whereas the surrounding stroma was ligand responsive [Nolan-Stevaux et al. 2009; Theunissen and de Sauvage, 2009; Tian et al. 2009; Yauch et al. 2008]. Yauch and colleagues observed that the tumor-derived Hh from several naturally Hh overexpressing xenografts stimulated expression of GLI 1/GLI 2 and PTCH in the infiltrating stroma but not in the tumor itself. Treatment with both a Hh-blocking antibody 5E1 and a small-molecule SMO inhibitor downregulated these murine stromal genes and slowed tumor growth, implying that the stromal cells send growth or survival signals back to the tumor [Theunissen and de Sauvage, 2009; Yauch et al. 2008]. In addition, Nolan-Stevaux and colleagues recently showed that the genetic deletion of SMO from pancreatic cells did not substantially alter PTCH and GLI expression in the neoplastic ductal cells and more importantly did not affect the development or progression of Kras driven pancreatic adenocarcinoma [Nolan-Stevaux et al. 2009]. Conversely, Tian and colleagues showed that the epithelial expression of mutationally activated SMO, which triggers constitutive, ligand-independent activation of the Hh pathway, was not able to induce neoplastic transformation in murine pancreatic epithelium, nor affect tumor development and progression ofKras driven pancreatic ductal adenocarcinoma models [Theunissen and de Sauvage, 2009; Tian et al. 2009].

These studies support the paracrine model of Hh signaling in which tumor cells activate Hh signaling in the surrounding stroma, resulting in the expression of soluble factors and extracellular matrix components that act upon the tumor epithelium to ultimately promote tumor growth [Theunissen and de Sauvage, 2009]. The exact mechanism of stromal feedback to the tumor remains to be determined but could involve components of the molecular signaling pathways involving insulin-like growth factor (IGF) and Wnt pathways, as IGF and Wnt signaling molecules in the tumor stroma were modulated similar to GLI and other Hh target genes in xenograft tumor models treated with Hh pathway inhibitors [Scales and de Sauvage, 2009; Yauch et al. 2008]. Inhibition of this paracrine signaling in epithelial tumors may be of therapeutic value as specific inhibition of Hh signaling in the stroma did result in growth inhibition of tumor xenografts, although the most effective way of treating these tumors would possibly be to use a combination of a Hh pathway inhibitor to target the stroma and other drugs to target the tumor cells.

Reverse paracrine signaling

Very recently, a ‘reverse paracrine’ signaling model has also been recognized in which Hh is secreted from the stroma and is received by the tumor cells (Figure 2D) [Theunissen and de Sauvage, 2009]. So far, this has only been observed in hematological malignancies such as multiple myeloma, lymphoma and leukemia, in which the Hh secreted from the stroma seems to be essential for the survival of the cancerous B cells via upregulation of the antiapoptotic factor Bcl2 [Scales and de Sauvage, 2009; Hegde et al. 2008; Dierks et al. 2007]. Stromal Hh was also found in high-grade, platelet-derived growth factor (PDGF)-induced gliomas in endothelial cells [Becher et al. 2008]. In the reverse paracrine signaling model, stromal Hh is thought to provide the appropriate microenvironment for potentiating tumor growth and would thus be a suitable therapeutic target as well.

Hedgehog signaling in cancer stem cells

Most renewing tissues are maintained by small populations of stem cells that have the ability to both generate additional stem cells and give rise to all mature cell types of the tissue. Hh signaling is an important regulator of stem cell activity, stimulating self-renewal and proliferation of stem cells in various tissues (Figure 2E) [Taipale and Beachy, 2001; Zhang and Kalderon, 2001]. It is believed that tumor growth and propagation might be dependent on a small population of CSCs that are similar to normal tissue stem cells and are regulated by the same signaling molecules as the normal stem cells [Reya et al. 2001]. Growing evidence suggests that the abnormal formation and expansion of cancer is due to deregulation of the multiple signaling pathways in the stem cells including the Hh, Wnt, Notch and BMP pathways [Rubin and de Sauvage, 2006]. Hh signaling has been shown to regulate the self-renewal of CSCs in breast, glioma and multiple myeloma, and more convincingly in the maintenance of chronic myelogenous leukemia (CML) stem cells [Theunissen and de Sauvage, 2009; Dierks et al. 2008; Clement et al. 2007; Peacock et al. 2007; Liu et al. 2006]. Dierks and colleagues observed that CML stem cells (Bcr-Abl driven Lin/Sca1+/c-Kit+) with SMO knockout had a reduced ability to form tumors in irradiated mice whereas SMOM2 expression enhanced it [Dierks et al. 2008; Peacock et al. 2007]. Furthermore, SMO antagonists such as cyclopamine and Hh blocking antibody 5E1 both inhibited growth of the CML CSCs in vitro and in vivo and enhanced time to relapse after the end of treatment. A recent report showing that Hh signaling is essential for maintenance of CSCs in CML lends further support for this concept. The loss of SMO in the mouse hematopoietic system resulted in decreased induction of CML by the Bcr-Abl oncoprotein and induced Numb, causing depletion of CML stem cells. Cyclopamine treatment inhibited the growth of imatinib-resistant mouse and human CML indicating that Hh signaling may be an important target to avoid induction of imatinib-resistant CML [Zhao et al. 2009].

Tumors contain only a minority of CSCs, which can give rise to more CSCs as well as nontumorigenic cancer cells [Al-Hajj and Clarke, 2004; Beachy et al. 2004]. CSCs are typically resistant to conventional chemotherapy and radiation as they are slow growing and are thought to be the cause of cancer relapse after tumor debulking by conventional therapy. The fact that active Hh signaling has been identified in several types of CSCs makes Hh inhibition a promising therapeutic target to deplete the tumor-forming CSCs, ideally in combination with other tumor debulking agents or radiation to remove the differentiated bulk of the tumor [Scales and de Sauvage, 2009]. Another recent finding that Hh positively regulates the expression of drug transport pumps in stem cells, enabling them to resist uptake of cytotoxic drugs [Sims-Mourtada et al. 2007], makes the strategy of using Hh inhibitors to target the CSCs more rational.

Hh signaling has also been shown to promote tumor metastasis by being actively involved in the epithelial–mesenchymal transition (EMT). EMT involves transforming polarized epithelial cells into motile mesenchymal cells facilitating invasive growth and ultimately causing metastasis. Hh exerts its effects on EMT via the upregulation of transcription factor SNAIL and downregulation of E-cadherin [Rubin and de Sauvage, 2006; Karhadkar et al. 2004]. This observation was first made by Karhadkar and colleagues in prostate cancer cell lines where they showed that the rarely metastasizing clone AT2.1 could be induced to metastasize by overexpression of GLI 1, and that the capacity of another cell line AT6.3 to metastasize to the lung was abrogated by cyclopamine [Karhadkar et al. 2004]. Similar observations in pancreatic cancer cell lines were made by Feldman and colleagues, who showed that ectopic expression of GLI led to increased invasiveness, whereas inhibition of the Hh pathway led to downregulation of Snail expression and reduction in invasive properties [Feldmann et al. 2007].

Targeting Hedgehog pathway signaling in solid tumors

Aberrant Hh signaling can be activated in a variety of cancers through various mechanisms, as discussed earlier. Understanding the specific mechanism of Hh activation in a particular tumor might help in selecting the most appropriate agent and strategy for optimizing the therapeutic benefit to be obtained by Hh pathway inhibition. Tumors such as BCC or medulloblastoma, which have a constitutive, mutation-driven activation of the Hh pathway, may be best treated with single-agent Hh inhibitors acting downstream of the activating mutation. Tumors with predominant autocrine or paracrine Hh signaling and CSCs might be more effectively treated with a combination of Hh antagonists and cytotoxic drugs targeting tumor cells [Scales and de Sauvage, 2009].

The first Hh pathway inhibitor to be identified, cyclopamine, inhibited the Hh pathway by binding to, and inactivating, SMO [Chen et al. 2002; Cooper et al. 1998]. However, cyclopamine has low affinity, poor oral bioavailability and suboptimal pharmacokinetics, and more potent derivatives have been synthesized. Several synthetic, small-molecule SMO antagonists with higher potency than cyclopamine such as SANT1–SANT4, CUR-61414, HhAntag-691, GDC-0449, MK4101, IPI-926 and BMS-833923 as examples, are now available and have been tested in preclinical models [Scales and de Sauvage, 2009]. Hh-blocking antibodies, which act upstream of SMO by preventing the binding of Hh to PTCH like 5E1, are also available and have demonstrated good preclinical activity [Scales and de Sauvage, 2009]. Multiple other drugs targeting different points of the Hh pathway, such as the natural Hh inhibitor Hhip mimetic, SUFU mimetics and GLI activity/transcription blocking agents (Gant 61 and Gant 58) are in various phases of development, as well [Lauth et al. 2007; Lauth and Toftgard, 2007]. Recently, a small molecule that binds the extracellular Shh protein, robotnikin, was isolated from small-molecule microarray-based screens [Stanton et al. 2009]. Targeting Shh ligands may be an interesting approach since the tumor-derived Shh ligands directly activate signaling in stromal cells. So far, only the SMO antagonists have been tested in the humans, and of these the CUR-61414 and GDC-0449 compounds, IPI-926, and BMS-833923 (XL139) are in the most advanced phase of clinical evaluation.

Basal cell carcinoma

BCC is the most common skin cancer in the United States, with an annual incidence of approximately 1,000,000 new cases. BCC was the first group of cancers in which the tumorigenic potential of deregulated Hh signaling was identified. This was based on the identification that patients with Gorlin’s syndrome had a marked susceptibility to develop BCCs [Hahn et al. 1996; Johnson et al. 1996]. Using family-based linkage studies of kindred with Gorlin’s syndrome, the causative mutation was mapped to the Patched 1 gene (PTCH1) on chromosome 9 [Gailani et al. 1992]. It is believed that upregulation of Hh signaling is the sole and pivotal abnormality in all BCCs [Epstein, 2008; Hutchin et al. 2005]. Approximately 90% of the sporadic BCCs have an identifiable mutation in at least one allele of PTCH1 (loss-of-function mutation) and about 10% have activating mutations in SMO (gain-of-function mutation) [Epstein, 2008; Xie et al. 1998;Gailani et al. 1996]. These mutations cause constitutive Hh pathway signaling that mediate unrestrained proliferation of basal cells of the skin, which has been confirmed in various mouse models of BCC, as well [Grachtchouk et al. 2000; Aszterbaum et al. 1999; Xie et al. 1998]. With such strong evidence of dysregulated Hh ‘oncogene addiction’ in BCC, blocking the Hh pathway would theoretically be a useful therapeutic approach for patients with metastatic BCC not controllable by other local therapies.

The first discovered steroidal alkaloid cyclopamine was used as a topical application by one group to induce regression in four BCCs [Tabs and Avci, 2004]. Several other synthetic cyclopamine derivatives have subsequently been developed as Hh pathway inhibitors, with better pharmacological and inhibitory properties than cyclopamine. Cur-61414, one of the earlier synthetic SMO inhibitors, prevented the formation of BCC-like ‘basaloid nests’ in Shh-treated ex vivo skin punches from PTCH+/− mice and also eliminated preformed BCC-like lesions [Scales and de Sauvage, 2009; Athar et al. 2004]. Interestingly, Cur-61414 selectively induced apoptosis and decreased proliferation in the BCC-like lesions, without any deleterious effects on normal surrounding skin [Scales and de Sauvage, 2009; Athar et al. 2004]. Cur-61414 was safe and well tolerated in other preclinical models, as well, and was thus formulated as a topical agent [Scales and de Sauvage, 2009; Flagella, 2006]. It was the first class of Hh antagonists to enter phase I clinical trials for use in sporadic BCC patients. However, it did not produce any clinical changes or reduction in Hh target gene GLI1 transcription when applied topically to BCC lesions, possibly because the formulation did not adequately penetrate the human skin [Fretzin et al. 2006].

GDC-0449, a second Curis-Genentech novel SMO inhibitor, was discovered by high-throughput screening of a library of small-molecule compounds and subsequent optimization through medicinal chemistry. GDC-0449 is a selective Hh pathway inhibitor with greater potency and more favorable pharmaceutical properties than cyclopamine, with good antitumor activity seen in preclinical models [Rudin et al. 2009; Von Hoff et al. 2009; Yauch et al. 2008]. The results of the phase I study of GDC-0449 demonstrating antitumor activity in patients with BCC and medulloblastoma were published recently [Rudin et al. 2009; Von Hoff et al. 2009]. Thirty-three patients with metastatic or locally advanced BCC received oral GDC-0449 at one of three doses, 150, 270 or 540 mg daily for as long as the patients had clinical benefit. Of the 33 patients, 18 had an objective response to GDC-0449, with 2 complete responses and 16 partial responses. Eleven other patients had stable disease with 4 patients having progressive disease. GDC-0449 has an unusual pharmacokinetic profile with high, sustained micromolar plasma concentrations and long terminal half-life. The median time to steady state was 14 days (range, 7–22 days). A consistent steady-state total plasma level of GDC-0449 was maintained throughout the treatment period of the study, with no apparent decline at the time of disease progression. Pharmacodynamic downmodulation in the Hh pathway was shown by a decrease in GLI1 expression as compared with pretreatment biopsy-sample analysis. The extent of GLI1 downmodulation did not correlate with pharmacokinetic levels of GDC-0449 in individual patients. Grade 3 adverse events related to the study drug included fatigue, hyponatremia, muscle spasm and atrial fibrillation. Other milder side effects included hair loss or thinning, altered taste sensation, nausea and vomiting, dyspepsia and weight loss. Interestingly, some of these toxicities might be attributable to the on-target effects of Hh in taste bud papillae formation and hair growth [Scales and de Sauvage, 2009]. High levels of GLI1 mRNA expression were observed in the tumors from responding patients, consistent with constitutive activation of the Hh pathway. Based on these promising results, GDC-0449 has now entered phase II trials in advanced BCC.

Medulloblastoma

Medulloblastoma, an aggressive childhood tumor of cerebellar origin, is another malignancy with a well-recognized dependency on aberrant Hh signaling. The first indication that alteration in the Hh signaling pathway contributes to medulloblastoma was the discovery that patients with Gorlin’s syndrome, who have germline mutations in the PTCH-1 gene, have an increased incidence of medulloblastoma [Goodrich and Scott, 1998; Kimonis et al. 1997]. Although rare, the outcome of medulloblastomas is invariably poor. Primary management consists of surgical resection followed by radiation and chemotherapy, with serious treatment-related morbidity from these modalities. Patients with recurrent disease after primary therapy have a median survival of less than 6 months [Zeltzer et al. 1999].

Hh signaling has a critical role in the developing cerebellum. Shh released by the migrating Purkinje cells delays neuronal differentiation and induces proliferation of granular neuron precursors in the external germinal layer of the cerebellum [Berman et al. 2002; Wechsler-Reya and Scott, 2001; Wallace, 1999]. Although critical during embryogenesis, the Hh pathway is downregulated after early postnatal development in most tissues, including brain, and the constitutive activation of this pathway seems to give rise to medulloblastomas [Romer et al. 2004]. More than 30% of human medulloblastomas demonstrate high levels of GLI1 expression consistent with abnormal activation of the Hh pathway [Lee et al. 2003]. Hh pathway antagonists thus have potential therapeutic value in the treatment of medulloblastomas and have been tested successfully in preclinical models and most recently in the clinic as well.

Cyclopamine was shown to decrease the rate of growth of mouse medulloblastoma cells both in culture and in mouse allograft models [Berman et al. 2002; Dahmane et al. 2001]. Interestingly, cyclopamine inhibited the in vitro growth of all human medulloblastoma cell lines, although only about one third would be expected to harbor Hh pathway mutations, suggesting Hh antagonists could be broadly effective in treating all medulloblastomas [Scales and de Sauvage, 2009; Berman et al. 2002]. Romer and colleagues used another small-molecule SMO-binding Hh antagonist, Hh-Antag to treat endogenous medulloblastomas in PTCH1+/−p53−/− mice models, where tumors develop with 100% incidence [Romer et al. 2004]. Hh-Antag completely eliminated the medulloblastomas by blocking tumor cell proliferation and stimulating apoptosis, without adversely affecting the surrounding cerebellum [Romer et al. 2004]. Rudin and colleagues recently reported a patient with metastatic medulloblastoma, refractory to multiple therapies responding to the novel Hh pathway inhibitor, GDC-0449 [Rudin et al. 2009]. Treatment resulted in rapid regression of the tumor burden and reduction of symptoms, although resistance to drug developed rapidly. Molecular analyses of the patient’s tumor specimens obtained before treatment showed increased expression of Hh target genes including GLI1, PTCH1, PTCH2 and secreted frizzled-related protein 1 (SFRP1), suggesting activation of the Hh pathway. Genomic analysis of the PTCH1 locus in tumor cells showed loss of heterozygosity and somatic mutation with no such alterations seen in the normal skin tissue biopsies [Rudin et al. 2009]. There is currently an ongoing phase II trial evaluating the efficacy and safety of GDC-0449 in the treatment of adults with recurrent or refractory medulloblastoma (see www.clinicaltrials.gov). The use of Hh pathway inhibitors in the treatment of medulloblastomas may offer a more effective therapeutic option and may avoid some of the serious adverse effects of current treatments. Since the Hh pathway also regulates various developmental pathways, it is unclear what the adverse effects of Hh pathway blockade may be in prepubescent children.

Other solid tumors

Multiple other solid tumors that do not harbor any somatic mutations in the Hh signaling pathway, such as BCC or medulloblastoma, also demonstrate a ligand-dependent activation of the Hh pathway. Constitutive activation of the Hh pathway has been detected in a broad variety of tumors including lung, stomach, esophagus, pancreas, prostate, breast, liver and brain [Clement et al. 2007; Sicklick et al. 2006; Karhadkar et al. 2004; Kubo et al. 2004; Berman et al. 2003; Thayer et al. 2003; Watkins et al. 2003b]. Although preclinical xenograft and animal models of many of these Hh overexpressing tumors show tumor growth inhibition on treatment with cyclopamine [Karhadkar et al. 2004; Berman et al. 2003; Thayer et al. 2003;Watkins et al. 2003a; Watkins et al. 2003b], the potential usefulness of Hh pathway inhibitors have yet to be tested in a clinical setting.

In addition to the above effect of Shh signaling in cancer and stromal cells, inhibition of the Shh pathway seems to augment the formation of desmoplasia in pancreas cancer [Olive et al. 2009]. The expression of Shh was found to cause desmoplasia formation in pancreatic cancer [Bailey et al. 2008]. IPI-926, a synthetic, small-molecule SMO antagonist, combined with gemcitabine was shown to improve the gemcitabine delivery to this pancreatic tumor model by depleting tumor-associated stromal tissue.

There are multiple Hh pathway inhibitors in development, including SANT1–SANT4, CUR-61414, HhAntag-691, GDC-0449, MK4101, IPI-926, BMS-833923 and itraconazole [Kim, 2009; Scales and de Sauvage, 2009]. The orally available SMO inhibitor GDC-0449 is the farthest along in development and is the major Hh antagonist actively being tested for use in ligand-dependent cancers. Two trials utilized GDC-0449 as maintenance therapy, one in patients with ovarian cancer in a second or third complete remission and the other for first-line therapy for metastatic colorectal cancer in combination with concurrent chemotherapy and bevacizumab (see www.clinicaltrials.gov and Scales and de Sauvage, 2009). Two other trials evaluating the use of GDC-0449 for the treatment of extensive-stage small cell lung cancer in combination with chemotherapy and unresectable pancreatic cancer in combination with erlotinib have recently been opened and are actively recruiting patients (see www.clinicaltrials.gov).

Conclusions

The last decade has seen extraordinary progress in understanding the roles and mechanism of action of Hh proteins in development and cancer. Targeting the Hh signaling pathway provides a new and exciting therapeutic option for a broad variety of cancers. Novel associations with dysregulated Hh signaling and the formation of cancer continue to emerge. Although all mechanisms of the Hh signaling pathway are not completely understood, it is clear that aberrant Hh signaling causes tumor growth and proliferation, increases tumor aggressiveness and raises the frequency of metastasis. Inhibition of the Hh pathway is thus a promising new approach for the treatment of select advanced malignancies. These include cancers such as BCC and medulloblastoma, which have mutations leading to constitutive activation of the Hh pathway, as well as other tumors which are Hh ligand dependent for tumor growth either by autocrine or paracrine mechanisms. Initial clinical trials of the oral SMO antagonist GDC-0449 show good efficacy and safety in BCC and medulloblastoma [Rudin et al. 2009; Von Hoff et al. 2009]. Although Hh pathway inhibitors seem to be safe in adults, their safety in children, especially for the treatment of medulloblastoma, is yet to be ascertained. The use of Hh antagonists in the treatment of ligand-dependent cancers is also to be determined, with multiple ongoing clinical trials in other solid tumors (see www.clinicaltrials.gov). Hh signaling also seems to be important for regulating stem cells in various tissues and Hh pathway inhibition might represent another method to target these relatively resistant and slow-growing CSCs. Optimally this approach would warrant the combination of systemic Hh pathway inhibition with other cytotoxic inhibitors of tumor growth. To maximally exploit the Hh pathway for therapeutic purposes, a better understanding of the precise Hh signaling mechanisms in various tumors is required.

It has been exciting to follow the advances of Hh pathway inhibitors in the ongoing preclinical and clinical trials including the recently reported use in advanced and metastatic BCC. These preliminary studies have set the stage for using these inhibitors in other cancers. Hh pathway inhibitors truly represent an important new class of therapeutic agents, which are bound to have far-reaching implications in oncology.

Similar articles in PubMed

Cited by other articles in PMC

Read Full Post »

%d bloggers like this: