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Co-administering a monoclonal antibody that neutralizes tumor-released soluble MHC I chain-related molecule (sMIC) improves anti-CTLA4 antibody therapy effectiveness and reduces treatment-related colitis, report Medical University of South Carolina (MUSC) investigators in an article published online May 17, 2017 by Science Advances.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) can be thought of as an 'off switch' or 'immune checkpoint' for effector T cells that are activated to fight cancer tumors. Researchers have developed a therapy to block CTLA4, specifically, anti-CTLA4 antibody immunotherapy, to help sustain T-cell activation and improve patient survival.

Unfortunately, while anti-CTLA4 therapy is highly effective in animals, response rates in humans vary widely and serious adverse events such as colitis (gastrointestinal inflammation) are common. For example, Ipilimumab, an FDA-approved anti-CTLA4 antibody therapy for advanced melanoma, is highly effective in controlling tumors in mice but has a response rate of only fifteen percent in humans. Clinical investigators have tried to improve the efficacy and safety of anti-CTLA4 therapy by combining it with other agents but response rates and toxicity remain suboptimal.

MUSC cancer immunologist Dr. Jennifer WuA team of MUSC researchers led by Jennifer Wu, Ph.D., professor of Microbiology and Immunology, suspected that response disparities between humans and animals may be due to differences in immune modulators that human tumors express.

"When we use animals to study therapies for humans we often neglect certain human-specific biological pathways simply because they don't exist in the animal," explains Wu. "MIC is one of those molecules that is expressed in human tumors but is absent in mice. We knew that the soluble form, sMIC, is highly immunosuppressive in humans and we knew it was important, but we had no way to study it. We had to create a new model."

Their work to unravel these molecular-level differences has now paid off, with the discovery of a new combination therapy that dramatically improves CTLA4 therapy effectiveness and avoids therapy-induced colitis.

The team developed a clinically relevant, MIC-transgenic spontaneous mouse tumor model that closely resembled the onco-immune dynamics seen in human cancers. Using this model, they investigated how tumor-derived, human sMIC affected anti-CTLA4 therapy. They found that high blood levels of sMIC not only reduced the antitumor efficacy of anti-CTLA4 therapy but also directly evoked colitis.

"It was a total surprise, and we were a little nervous about the results," says Wu. "So, we repeated the test using multiple models and antibodies and different batches of animals to make sure it was reproducible and that what we were seeing was real. Sometimes knowledge itself gets in the way. Because there are certain accepted beliefs that make you think what you're seeing can't be true-that it's impossible. But when multiple experiments are all coming up with the same results, what can you say? That's when we have to let go and come to a new understanding."

Next, the team co-administered an antibody called B10G5 that neutralizes sMIC, alongside anti-CTLA4 therapy. The new combination therapy not only remarkably improved anti-CTLA4 immunotherapy effectiveness, but also alleviated therapy-induced colitis.

"We've been studying B10G5 for a while and published a paper in Clinical Cancer Research in 2015 demonstrating that using B10G5 to target sMIC can alleviate tumor-induced immune suppression and also has a huge immune-stimulating ability," explains Wu. "So those results led us to think it would be a good strategy to combine B10G5 with antibodies that target immune checkpoint molecules, the 'off-switch' of an ongoing immune response."

Finally, inspired by a case study published in 2006, the team decided to try to validate its current findings in humans. The original case reported a melanoma patient who developed anti-MIC autoantibody during anti-CTLA4 therapy and had a superior therapeutic response. So, they contacted a long-time collaborator at Oregon Health and Science University and Knights Cancer Center to obtain plasma samples collected during a clinical trial they were conducting in metastatic prostate cancer patients.

Wu's team looked for anti-sMIC autoantibodies in samples from ten patients receiving anti-CTLA4 therapy with Ipilimumab. One sample showed high levels of anti-MIC autoantibody. When the team followed up with their colleague, it turned out that this particular cancer patient not only demonstrated a remarkable therapeutic response (his prostate-specific antigen fell from 191 to 4.6 ng/ml after eight treatment cycles) but also did not develop autoimmune colitis.

That case reinforced the team's preclinical in vivo findings that coadministering the sMIC-neutralizing antibody (B10G5; CanCure, LLC) enhances anti-CTLA4 therapy and deters colitis. Overall, these results indicate a new, powerful combination immunotherapy for cancer. They also suggest that pre-screening serum sMIC levels might help clinicians to identify patients who are most likely to have a positive therapeutic response.

"I hope that our findings will inspire investigators to revisit other cancer immunotherapies that were successful in animals but presented no efficacy in humans," says Wu. "Anti-CTLA4 therapy is approved for melanoma and also is still in trials for other cancers. Maybe our study will inspire clinical investigators to think about screening their patients to identify who will be a better responder versus a poor responder to anti-CTLA4 therapy."

Primary colorectal tumors secrete VEGF-A, inducing CXCL1 and CXCR2-positive myeloid-derived suppressor cell (MDSC) recruitment at distant sites and establishing niches for future metastases, report Medical University of South Carolina (MUSC) investigators in an article published online ahead of print on April 28, 2017 by Cancer Research. Liver-infiltrating MDSCs help bypass immune responses and facilitate tumor cell survival in the new location. This research illuminates mechanisms by which primary tumors contribute to premetastatic niche formation and suggests CXCR2 antagonists may reduce metastasis.

Recent cancer research shows that premetastatic 'niches' form at sites far from the original tumor before new tumors occur. In colorectal cancer (CRC), these supportive microenvironments form in preferred secondary organs, such as the liver and lung, and facilitate the colonization, survival, and growth of metastasizing tumor cells. However, the mechanisms responsible for the formation of these premetastatic 'niches,' including what role(s) the primary tumor may play, are not well understood. It is critical to better understand the mechanics of CRC metastasis, as it is the second leading cause of cancer deaths in the US and patients with advanced cases often die because current treatments for widely metastasized disease are not effective.

MUSC investDr. Raymond N. DuBois, dean of the Medical University of South Carolina College of Medicineigators led by Raymond N. DuBois, M.D., Ph.D., dean of the MUSC College of Medicine and professor of Biochemistry and Molecular Biology, have now illuminated how primary CRC tumors contribute to premetastatic 'niche' formation.

"The idea that some sort of 'priming' needs to take place for metastasis to occur in distant organs - that there is some sort of activity in the future tumor location - is not new. But most research has focused on growth factors, chemokines and pro-inflammatory cytokines. There hasn't been much work looking at immune cell activity in distant organs prior to metastasis," explains DuBois. "We knew that the type and density of immune cells in the primary tumor plays a role in progression. For example, when more immature myeloid cells are present in the tumor, it becomes resistant to immune attack. But we didn't know what to expect in a metastatic model."

To explore this area, the team first evaluated whether the presence of a primary tumor affected immune cell profiles in premetastatic liver and lung tissues of mice. They found that the presence of a primary cecal tumor caused MDSCs to begin infiltrating the liver before metastasis began. Working backward from this finding, they used a series of experiments to reveal the chain of events that led up to MDSC infiltration.

Because CXCR2 is essential for drawing MDSCs out of the bloodstream and toward CRC tumors and colonic mucosa, the team began looking for CXCR2 and its ligands (CXCL1, CXCL2, and CXCL5) in mouse liver tissue. The team not only found that the ligand, CXCL1, attracted MDSCs from the bloodstream into premetastatic liver tissue, but also that administering a CXCR2 antagonist inhibited CXCL1 chemotaxis. This demonstrated that CXCR2 is required for CXCL1 to induce MDSC liver infiltration. In other words, the CXCL1-CXCR2 axis is required to recruit MDSCs to the liver. Importantly, they also found that liver- infiltrating MDSCs secrete factors that promote cancer cell survival and metastatic tumor formation without invoking the innate and adaptive immune responses.

Next, because VEGF is known to induce CXCL1 expression in lung cancer, the research team examined whether VEGF secreted by CRC tumors also regulated CXCL1 expression. Their results demonstrated that VEGF-A secretion by primary CRC tumor cells stimulates macrophages to produce CXCL1. Interestingly, although VEGF-A knockdown inhibited liver metastasis, it did not affect the growth of the primary tumor.

"We did not expect to find that a primary tumor could affect a distant organ before any of the cancer cells arrived on site," says DuBois. "We were surprised to see these changes before a single metastatic cell took up residence."

Together, these studies reveal that VEGF-A secreted by the primary CRC tumor stimulates macrophages to produce CXCR1, which recruits CXCR2-expressing MDSCs from the bloodstream into healthy liver tissue. The MDSCs then create a premetastatic 'niche' or micro-environment where cancer cells can grow to form new tumors. These results demonstrate for the first time that cells in the primary tumor contribute to forming distant pre-metastatic 'niches' which facilitate the spread of disease.

"Now that we know the primary tumor puts things in motion remotely prior to metastasis, we should be able to inhibit this process and have a positive impact on survival," explains DuBois. "We now know which molecules and immune cells are involved and that if we disrupt the CXCL1-CXCR2 axis we can possibly reduce the spread of disease. Both antibodies and small molecules can inhibit this pathway, but they have not yet been optimized. I hope these findings will speed up the development of inhibitors of the CXCR2 pathway."

"Juicing" Th17 cells with FDA-approved small molecule beta-catenin and p110 delta inhibitors during in vitro expansion for adoptive T cell therapy (ACT) profoundly improves their therapeutic properties, report investigators at the Medical University of South Carolina (MUSC) in an article published online ahead of print on April 20, 2017 by JCI Insight.

MUSC cancer immunologist Dr. Chrystal M. PaulosACT involves harvesting T cells, rapidly amplifying and/or modifying them in the laboratory to boost their cancer-fighting ability, and then reinfusing them back to the patient to boost anticancer immunity. One challenge for ACT has been that the rapid expansion of T cells in the laboratory can cause them to age and wear out, decreasing their longevity after reinfusion.

"Juicing" Th17 cells with the FDA-approved small molecules enhanced their potency, function and stem-like (less differentiated) quality, suggesting that they would persist better after reinfusion into patients, and also reduced regulatory T cells in the tumor microenvironment, which can blunt the immune response. These findings highlight novel investigative avenues for next-generation immunotherapies, including vaccines, checkpoint modulators, and ACT.

"This is exciting because we might be able to overcome some of the delays and disadvantages of rapid expansion in the laboratory," explains senior author Chrystal M. Paulos, Ph.D., associate professor of immunology and Endowed Peng Chair of Dermatology at MUSC and a member of the MUSC Hollings Cancer Center. "We might be able to use fewer cells (for ACT) because we can pharmaceutically 'juice' these T cells to make them more fit in the oppressive tumor microenvironment."

Building upon their previous findings that ICOS costimulation is critical for generating human Th17 cells and for enhancing their antitumor activity, an MUSC research team led by Paulos and including postdoctoral fellow Kinga Majchrzak report for the first time that repurposing FDA-approved small molecule drugs that inhibit two ICOS-induced pathways greatly enhances the antitumor potency of T cells.

Several biologic properties of the Wnt/ beta-catenin and P13K delta pathways led the team to suspect that they supported the antitumor activities of Th17 cells. For example, these pathways are active in both regulating T cell cytokine production during the immune response and in promoting self-renewal of hematopoietic stem cells (HSCs) and sustaining HSCs in an undifferentiated state. So, they designed a series of experiments to determine whether these two pathways were also active in enhancing Th17 antitumor memory and effectiveness.

To test this idea, they pharmaceutically inhibited PI3K delta and beta-catenin in Th17 cells (using idelalisib [CAL-101] to block the PI3K delta pathway and indomethacin [Indo] to inhibit beta-catenin)-anticipating that this would weaken Th17 cells' antitumor activity. To their surprise, the exact opposite occurred. ICOS-stimulated Th17 cells that were treated in vitro with CAL-101 plus Indo elicited a more potent antitumor response against melanoma in mice.

"My post-doc student came to me and said, 'I think I made a mistake because the data are going in the opposite direction to what we originally predicted!" says Paulos. "So, she repeated the experiment several times but we kept getting the same result. The data showed that using drugs to inhibit these pathways actually made the Th17 cells even better at killing tumors."

The team found that Th17 cells treated with CAL-101 express less FoxP3, suggesting that the drug suppresses Treg conversion while sustaining central memory-like Th17 cells. This finding is highly important because the phenotypic plasticity of Th17 cells in vivo allows their conversion to Tregs or Th1 cells with weak antitumor properties. These data suggest that treatment with CAL-101 can halt the development of these poorly therapeutic phenotypes and, thus, enhance the T cells' antitumor activity.

While the findings were initially counterintuitive and perplexing from a mechanistic perspective, in retrospect Paulos sees that they make sense. "Essentially, the T cells are younger," explains Paulos. "We know that T cells used for ACT age and wear out over time. Somehow these drugs sustain their youth and function. They're able to keep all the properties of their youth-they expand better and they're more functional and handle the oppressive tumor microenvironment better."

The discovery that existing FDA-approved drugs that block p110 delta and beta-catenin can make T cells more efficient tumor killers in vivo is an exciting prospect for Paulos' team. "From a clinical standpoint, this finding indicates that the therapeutic effectiveness of ACT could be improved by simple treatments with readily available drugs. It opens a lot of new investigative avenues for next-generation immunotherapy trials," she says.

"This research offers tremendous promise for the treatment of patients with serious forms of skin cancer," says Dirk M. Elston, M.D., chair of the Department of Dermatology and Dermatologic Surgery at MUSC.

Paulos has a patent on ICOS signaling in adoptive T cell transfer therapy (US 9133436), and Paulos, Majchrzak, and J.S. Bowers have a patent on pharmaceutical drug combinations or genetic strategies that instill durable antitumor T cell memory and activity (patent application P1685).

T cell attacking a tumor

T cell attacking a tumor



Stylized image of a T cell attacking a tumor. Illustration by Emma Vought.













Release Summary: The protein moesin could be a target for cancer immunotherapy, report Medical University of South Carolina (MUSC) investigators in an article in the Journal of Clinical Investigation. Their data suggest that moesin promotes conversion of naive T cells into regulatory T cells that suppress the immune response against cancer. Inhibiting moesin could help restore the anti-tumor T cell response and also improve the survival of cancer-killing CD8+ cells after adoptive T cell transfer.

In an article published online ahead of print on March 13, 2017 by the Journal of Clinical Investigation, Medical University of South Carolina (MUSC) investigators report preclinical research showing that moesin, a membrane-domain organizing protein, controls regulatory T cell (Treg) function as well as the abundance and stability of transforming growth factor-beta (TGF-beta) receptors on the surface of cells, providing a potential therapeutic target for cancer immunotherapy.

Their findings show that TGF-beta acts at the protein level to generate Tregs in the tumor microenvironment. Although the human immune system is capable of eradicating cancer, Tregs dampen the immune response and protect cancer cells against tumor-killing (i.e., cytotoxic) T cells. The MUSC study is the first to show that eliminating moesin reduces TGF-beta receptor expression and subsequent Treg generation to restore anti-tumor immunity.

T cells, a subtype of white blood cells, can effectively attack and kill tumor cells when activated by the protein TGF-beta. However, the immune system has a sophisticated network of checks and balances to ensure that the body does not produce so many of these cytotoxic T cells that it harms its own cells and tissues. When the immune reaction is complete, TGF-beta signals naive T cells to become Tregs that suppress and degrade the activated, inflammatory T cells, ensuring that they do not overproduce the immune factors that can lead to autoimmune disease.

Cancer cells have learned to hijack this system of checks and balances to hide from the tumor-killing T cells. Many cancers produce TGF-beta that binds the receptors on the tumor-killing helper T cells so they can’t be recruited to fight the tumor. The T cells convert instead to Tregs, which suppress the immune response against the cancer.

Inhibiting moesin could help prevent conversion of naive T cells into Tregs, thereby restoring the anti-tumor immune response. 

"Because moesin supports greater Treg production, we could design moesin inhibitors to halt or slow active TGF-beta signaling and slow down Treg conversion so that anti-tumor T cells can have a chance to see the cancer and eradicate it,” explains Zihai Li, M.D., Ph.D., chair of the Department of Microbiology and Immunology at MUSC and senior author on the paper.

Earlier studies by Philip Howe, Ph.D., chair of MUSC's Department of Biochemistry and Molecular Biology and a co-author on the paper, demonstrated that many TGF-beta-mediated epithelial mesenchymal transition genes, including moesin, were repressed by an RNA-binding protein in healthy epithelial cells and that moesin expression could be restored through TGF-beta stimulation.

This ability of TGF-beta to dramatically increase moesin expression led the team to investigate moesin’s role in Treg generation. Jointly with other colleagues at MUSC, the team compared the abilities of helper T cells with and without moesin to become Tregs. They found that moesin promotes Treg generation by interacting with a TGF-beta receptor to make it more available, thereby enhancing TGF-beta signaling. Conversely, TGF-beta signaling was reduced in the absence of moesin, impairing the development and function of Tregs.

Perhaps the most compelling results were provided by studies involving adoptive T cell therapy in a mouse model of melanoma. In adoptive T cell therapy, tumor-killing T cells are “harvested” from a human or animal with cancer and amplified or otherwise “supercharged” before being reinfused into the donor. Although these reinfused cells can be very effective at killing tumors, they do not always survive long-term, setting the stage for recurrence. 

The MUSC research team showed that these reinfused anti-cancer CD8+ T cells not only underwent rapid activation and expansion in mice lacking moesin, but that they also survived longer, reducing the likelihood of recurrence. Indeed, after adoptive T cell transfer, all of the mice having moesin relapsed while most of the mice lacking moesin were cured.

"When the mice lacking moesin had no recurrence, this was really exciting. We were not only deleting moesin but, when we gave T cells to the active tumors, those T cells could control the cancer for a very long time,” explains Ephraim Ansa-Addo, Ph.D., a postdoctoral fellow in the Department of Microbiology and Immunology and lead author on the paper.

These findings suggest that moesin could be a therapeutic target in developing new treatments for cancer and Treg-related immune disorders. Chemical modulators of moesin could control the function of T cells by inhibiting moesin in cancers or inducing it to treat autoimmune diseases. Moesin modulators could also be combined with current immunotherapy regimens.

“These findings are very interesting for the field and provide a lot of directions for further research into alternative therapies," says Li.

Dr. Betty Pei-tie Tsao and colleagues

Betty Pei-tie Tsao, Ph.D. (front, center), Richard M. Silver Endowed Chair for Inflammation Research at MUSC and senior author on the Nature Genetics article, with first author Jian Zhao, Ph.D. (to Dr. Tsao's left) and second author Yun Deng, M.D. (to Dr. Tsao's right).

Investigators at the Medical University of South Carolina (MUSC)  report pre-clinical research showing that a genetic variant encoded in neutrophil cytosolic factor 1 (NCF1)  is associated with increased risk for autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren's syndrome, in the January 2017 issue of Nature Genetics. Data indicate that increased NCF1 protects against SLE while decreased NCF1 raises SLE risk and highlight the pathogenic role of reduced reactive oxygen species in autoimmune disease development.

Single-nucleotide polymorphisms (SNPs – pronounced 'snips') are the most common type of human genetic variation; each one represents a small difference in a nucleotide – the building blocks of our DNA. The Immunochip for fine-mapping is an important tool for conducting genome-wide association studies of the genetic components of disease. Researchers use the Immunochip to investigate DNA samples from people with a particular disease for linkage disequilibrium (LD) signals that illuminate associations between specific SNPs and the disease. Autoimmune diseases such as SLE are known to have a strong genetic component and, to date, dozens of SNPs associated with SLE have been identified and included on the Immunochip.

The Achilles heel is, of course, that the Immunochip cannot identify associations with SNPs that it does not include.

When MUSC researchers genotyped DNA samples from Chinese, European-American, and African-American SLE patients, they found a strong signal in the Chinese sample at the rs73366469 locus in the GTF2IRD1–GTF2I intergenic region at 7q11.23. This was puzzling because that locus was not consistent with SLE loci identified by other genome-wide association studies. Furthermore, the very strong signal in the Chinese sample appeared as a modest signal in the European-American sample and did not appear at all in the African-American sample.

"A true risk gene should be the same in all populations,” explained Betty Pei-tie Tsao, Ph.D., Richard M. Silver Endowed Chair for Inflammation Research at MUSC and senior author on the article. “And for such a strong signal, we wondered, 'why hasn't anyone else seen it?' We wanted to find out if what we were seeing was true and explain it." 

The team confirmed their finding using a different genotyping platform in an independent Asian sample provided by Nan Shen, M.D., Ph.D., professor of medicine and director of the Shanghai Institute of Rheumatology at Shanghai Jiao Tong University's School of Medicine. But, because rs73366469 did not show LD with any SNPs in the Immunochip, the researchers hypothesized that the SNP containing the true underlying risk factor was not included in it.

"We sort of came into the study from our Asian samples and then started looking for this signal in other populations,” said Tsao. “Every ethnic group has a different ancestral background and different LD patterns. We used the LD signal strength as a guide to find our way to the true risk gene – the particular variant that actually caused the increased risk for lupus."

Because the SNP they were looking for was most likely not included in the Immunochip, the team turned to the 1000 Genomes Project dataset, where they found two SNPs that were not only not on the Immunochip, but also produced stronger LD signals with rs73366469 in Asian patients than European or African patients. One of these two, rs117026326 located on intron 9 of GTF2I, showed a stronger association with SLE than either the original or the other locus from the 1000 Genomes Project.

As the researchers focused in on rs117026326, they saw that the NCF1 gene was nearby. This was important because NCF1, which encodes a subunit of NOX2, is thought to be related to SLE due to its role in activating the phagocytic complex NOX2.

Preclinical studies have shown that non-functional NOX2 exacerbates lupus in mice. Furthermore, NCF2, which encodes another subunit of NOX2, is associated with SLE risk in European Americans. The strong association of rs117026326 with SLE and the functional implications of nearby NCF1 took the team to their next hypothesis: that the rs117026326 SNP might tag causal variants of NCF1 that were not present in the 1000 Genomes Project database.

But unraveling this mystery was not going to be easy.

 "This is a very complex genomic region,” explained Tsao. “The NCF1 gene has two nearly identical twins – NCF1B and NCF1C – that are 98% the same. But they are non-functional pseudo-genes. This makes working in this region of the human genome very difficult. That's why the next-generation sequencing method that the 1000 Genomes Project has been doing doesn't pertain to this region."

The researchers believed that mapping techniques commonly used by the larger projects, while efficient, limited their ability to find unique sequences among all the copies and duplications in this region. So, they decided to set up their own, novel PCR assay.

"You can't easily sequence this region using the next-generation techniques,” said Tsao. “So, we had to do it the old-fashioned way, which was very time consuming and labor intensive. To genotype the region correctly, we used PCR to selectively amplify the NCF1 copies and conduct copy number variation tests. Then we only used samples with no copy number variation to examine the NCF1 variant. This method ensured that what we identified as an NCF1 variant was truly a variant."

Using this strategy, the team identified 67 SNPs, four of which had a strong association with rs117026326. After conducting a long series of multiple tests in samples from various ethnic populations, they gradually eliminated three of the four SNPs and determined that the one called p.Arg90His was the likely genetic variant causing SLE susceptibility across all populations.

In addition, p.Arg90His was associated with increased risk for other autoimmune diseases, including rheumatoid arthritis and Sjögren's syndrome. The team also found that having only one copy of NCF1 was associated with a higher SLE risk, but having three or more  NCF1 copies was associated with reduced SLE risk. Finally, while the underlying mechanism is unclear, the team found that having reduced NOX2-derived reactive oxygen species also raised the risk for these autoimmune diseases.

Tsao notes that perseverance was a critical component of this work. This work was started years ago when the team was at the University of California Los Angeles  and was completed after moving to MUSC.

"We just stuck with it as a labor of love. Our lead author, Jian Zhao, devoted several years of his life to this project,” explained Tsao.” At the time we started, we didn't know it was going to be so complex. We just wanted to explain what we were seeing. It turned out to be quite a chase and very interesting and rewarding to finally bring this project to this point."

This work also points out an important unmet need in the field of genetic mapping.

"We need a more efficient platform to screen complex genome regions for variants. For a lot of diseases we've identified some, but not all, of the variants. There may be more variants hiding in these complex regions," said Tsao. "You have to sort it out like a puzzle. Autoimmune diseases share certain risk factors but also have unique genetic variants that drive the molecular pathogenesis of the disease. Each time you find a variant, you get more puzzle pieces and you can start to understand more about that disease and other autoimmune diseases as well."

Dr. Robert Gemmill (front) and Dr. Patrick Nasarre (back) were authors on the article.

Medical University of South Carolina (MUSC) investigators report preclinical research showing that the tumor-promoting properties of neuropilin (NRP)-2 reside predominantly on isoform NRP2b, while NRP2a has the opposite effects in non-small cell lung cancer (NSCLC), in the January 17, 2017 issue of Science Signaling. In mouse models, NRP2a inhibited tumor cell proliferation, while NRP2b promoted metastasis and progression. This new understanding may lead to improved therapies that specifically target NRP2b, while sparing the tumor-inhibiting functions of NRP2a.

Lung cancers are highly invasive, metastatic, and drug resistant—accounting for one fifth of cancer deaths worldwide each year. In part, the malignant properties of lung cancer are driven by the epithelial-mesenchymal transition (EMT), which is primarily induced by transforming growth factor beta (TGF-beta), and results in the proliferation of cancer stem cells.

It is known that the two human NRPs—NRP1 and NRP2—are often up-regulated in tumors and associated with poor patient prognosis. Previous work by MUSC Hollings Cancer Center researchers Robert Gemmill, Ph.D. and Harry Drabkin, M.D., first and senior authors on the Science Signaling article, demonstrated that NRP2, in particular, was up-regulated in cultivated NSCLC lines during TGF beta-mediated EMT. Furthermore, inhibiting NRP2 was found to reduce TGF-beta-mediated responses, including invasive tumor growth.

However, to date, nearly all studies of NRP2 have focused on the 2a isoform. Until now, NRP2b, the alternatively spliced isoform, has been largely uninvestigated and poorly understood.

"Most research has been focused on understanding the major effects of NRP2 overall and wasn't really concerned with breaking down the roles of its component parts—the 2a and 2b isoforms,” explained Gemmill, who holds the Melvyn Berlinsky Endowed Chair for Cancer Research at MUSC. “So, we know that NRP2a and NRP2b are nearly identical – only about the last 100 amino acids at the C terminus are different. There was some speculation that they might have different functions, but most of us assumed those differences were minor."

"It's turning out that there are lots of molecule variants that people just haven't looked at before and that we've only recently been discovering are important,“said Drabkin, who holds the Mary Gilbreth Endowed Chair in Clinical Oncology at MUSC. “We usually find the big stuff first and then work down into the deeper layers and that takes time. As they say, 'the devil is in the details' and there's lots of details." 

The team was drawn into their investigation of NRP2b by the results of experiments they were conducting on a potent tumor suppressor, semaphorin 3F (SEMA3F), which uses NRP2 as its receptor. When analyzing NRP2 expression, they unexpectedly observed a double band and that induction appeared to affect one band more than the other. This led them to question why one band was altered more than the other. In addition, they found that, during the progressive changes that lead to tumor metastasis, SEMA3F was lost.

 "So, we asked, what happens to it? Where does it go? And we found that, during lung cancer progression, SEMA3F is lost and NRP2b is induced,” explained Gemmill. “That led us to investigate the 2b isoform to see what it does because no one knew."

To this end, the team designed a series of experiments. Real-time, reverse transcription polymerase chain reaction assays revealed that TGF-beta stimulation substantially increased NRP2b but not NRP2a. This was the first time that NRP2 up-regulation by TGF-beta had been shown to preferentially involve the uninvestigated 2b isoform. They then looked at tumor cell migration and invasion patterns and found that cancer cell migration across Matrigel-coated membranes was inhibited in NRP2b knockdown models and enhanced by NRP2a knockdown. Repeated experiments confirmed that TGF-beta-mediated cancer cell migration and invasion was dependent on NRP2b.

That's when the team realized that the two isoforms had very different functions in terms of cancer progression, leading them to extend their studies to in vivo animal models.

 "As soon as we saw the migration results, we knew we had to put it in an animal model using cancer cell lines where we could control the isoform expression. We thought, 'this is just too good to be true,' but it was true,” explained Gemmill. ”We got the same results time after time. Whenever we expressed NRP2b the cancer metastasized, and whenever we expressed NRP2a progression and metastasis were suppressed. Clearly, with the 2b isoform, we have found something that promotes metastasis."

"Other people had looked at NRPs as co-receptors but never got into the details of which isoform was playing what role,” said Drabkin. “It's sort of like detective work. You follow leads and ask questions. Sometimes you follow a false lead—but in this case we found something new that turned out to be important."

Additional experiments showed that cancer stem cell tumor-spheres, which are highly tumorigenic and resistant to chemo- and radiation-therapies, were substantially reduced in NRP2b knockdown models.

Specifically, significantly fewer cells developed gefitinib chemotherapy resistance in models that knocked out NRP2b (i.e., tumor environments with very low levels of 2b) and significantly more cells developed resistance in NRP2a knockout (i.e., tumor environments with very low levels of 2a).

Finally, the researchers looked at human tissue samples and found that NRP2b abundance in human lung tumors was correlated with a higher cancer stage and more advanced progression.

"EMT produces a wholesale change in the repertoire of growth factors and receptors. In tumors where EMT is underway, there's more resistance to treatment,” explained Drabkin. “What we found was that, in these epithelial tumors, if we block NRP2—especially the 2b protein—on the cell surface, they just did not respond in any way like control cells in terms of their ability to take on the EMT phenotype and migrate. By inhibiting that receptor we'd made a big dent in their ability to become resistant."

"Honestly, I was very, very surprised at how distinctly different the two isoforms were,” said Gemmill. “When we first realized that there was differential expression, I thought we would have to look really hard to find some very minor differences. The fact that they were opposites—one inhibiting and one promoting cancer cell migration—and that this difference fell out so quickly was astounding because there's such a short list of things that are different about these 2 isoforms."

Although there is still a lot to learn about how NRP2a and NRP2b function in both normal tissue as well as cancers, these discoveries open new avenues for potential therapies. Possibilities range from developing monoclonal antibodies to target the 2b isoform, to immunotherapies, to using NRP2b as a biomarker for predicting patients' responses to particular therapies.

Gemmill anticipates that, in the wake of their findings, researchers will start looking at the role of NRP2b in other disease areas.

"I think a lot of people are going to sit up when they read this article and say, 'I wonder what it does in my system?'” said Gemmill. “For example, fibrosis is often associated with a TGF-beta response and we now know that TGF-beta induces NRP2b. So, maybe, NRP2b plays a role in fibrosis that affects the kidneys, liver, and lungs.”   

Both researchers agree that this work exemplifies the importance of not dismissing small but bothersome findings.

Gemmill notes that many of the biggest breakthroughs in science start with one person sitting in a laboratory, pursuing a single, seemingly minor, phenomenon.

"If you see something that isn't quite right, don't dismiss it,” said Gemmill. “This is an exciting finding that came from not letting a detail slip by.”

"That's how we find out what's really going on in a system,” said Drabkin. “If you work out enough of the details, you start to see how things interact. The more you learn, the more you see how things connect and where the pivot points are that can have biologic consequences. Progress happens little-by-little until we find a weakness where we can direct therapy."

The team recently received a four-year Veteran's Administration Merit Award to further fund their work.

A pericytePictured Above: A fluorescent pericyte cell body (red) with processes
extending along adjacent capillaries (green). Courtesy of Dr. Andy Shih and Robert Underly of the Medical University of South Carolina.


Pericytes are the primary locus of matrix-mellaproteinase-9-dependent (MMP-9) capillary damage and blood leakage during ischemia, according to preclinical findings reported by Medical University of South Carolina (MUSC) investigators in an article published online on November 14, 2016 by The Journal of Neuroscience. In vivo two-photon microscopy revealed MMP-9 activity and plasma leakage disproportionately occurred at locations where pericyte somata were attached to the endothelium. These results suggest that pericytes, normally essential for blood-brain barrier (BBB) function, contribute to capillary damage during stroke.

The BBB—a highly specialized vascular structure—prevents the entry of blood-borne substances that can harm the brain (e.g., neurotransmitters such as glutamate, clotting factors such as fibrin, and free radical–generating substances such as iron). During ischemic stroke and related cerebrovascular diseases, the BBB is damaged, allowing incursion of blood plasma that injures neurons and other structures essential for normal cerebral function. 

The role of pericytes as builders and custodians of the BBB is well recognized, but how pericytes respond to blood flow loss in the adult brain has largely been a mystery, until now.

MUSC researchers recently harnessed cutting-edge technology to image pericytes in the intact brains of live mice and to spatially and temporally track the proteolytic enzyme, MMP-9, as the BBB degraded and blood began leaking into the brain.

Findings from this novel study not only provide critical new information about pericytes as a potent source of MMP-9 during BBB leakage but also open new discovery pathways for future therapies in neurological conditions involving ischemia.

It all began when Robert Underly, a PhD candidate in the MUSC College of Graduate Studies Neuroscience Program and first author on the article, noticed that, when a laser was used to induce ischemic strokes in the laboratory, BBB leakage occurred at very specific sites along the capillaries. "I'd assumed that blood leakage occurred along the entire capillary length,” said Underly. “But it wasn't like that. There were hotspots that leaked first and more than the rest of the capillary bed. That was really unexpected."

Andy Y. Shih, Ph.D., Assistant Professor of Neurosciences and senior author on the article, and his team followed up on Underly's observation.  "We found a very close association between where the round cell bodies of pericytes were located and where the leaks occurred,” said Shih. “So that was our first clue that the pericytes were possibly doing something harmful in the early stages of an ischemic stroke."

It is well known that the BBB becomes dysfunctional when genetic defects disrupt pericyte-endothelial signaling from birth. However, very little is known about how normally developed pericytes in the adult brain respond during acute ischemia, and only one or two studies have investigated this in vivo.

"Pericytes have a lot of potential functions—they seem to be a sort of a jack-of-all-trades,” said Shih. “We've had an idea of what these cells do, but we haven't been able to visualize and track them in vivo until recently."


The team was also intrigued by a handful of published studies showing that various inflammatory signaling cascades can induce pericyte MMP-9 expression.

"The problem is that, like pericytes, MMPs are hard to study in vivo—most of what we know about them is from studies on cultured cells or extracted brains,” said Underly. “We wanted to probe this process in live animals so we could see the spatiotemporal relationship between pericytes and MMP activation in vivo—in the acute stroke time frame."

To do this, the team combined several novel tools to create a unique study protocol using transgenic mice, two-photon fluorescent microscopy, and a fluorescent gelatin-based reporter of MMPs that only one other research group had ever used to study the intact brain.

The researchers also used photothrombosis to block blood flow in a small area of the capillary bed and imaged transgenic mouse lines expressing bright fluorescent reporters specifically in the pericytes to clearly identify them.

"The successful combination of technologies is certainly one of the innovations of this project,” said Shih. “It's the first study to combine these tools to look at the relationship among pericytes, MMP activity, and BBB leakage in ischemia."

Their findings revealed that ischemia resulted in extremely rapid (within tens of minutes), localized activation of MMP-9 and plasma leakage. Furthermore, plasma leakage occurred preferentially where the pericyte somata adjoined the capillary wall—not homogenously along the length of the capillaries as previously imagined by the group. These results provide strong evidence that pericytes—normally protectors of the BBB—contribute to early BBB degradation during ischemic stroke.

Using the new technology, the team did not have to extract and cut the brain and so did not lose the structure of the vasculature and blood flow.

“We had an intact system and could see where things were coming from and we were very surprised,” said Shih. “I thought, 'I've been looking at this for years and I never knew that there was this beautiful co-localization.' It told us we were looking at something really interesting. The pericytes seem to nurture or damage the BBB depending on the conditions they're put in."

This discovery opens many directions for further study and could eventually lead to new therapeutic options for patients experiencing an acute stroke.

"The very rapid reaction we saw to ischemia is really important and provides clues to potential mechanisms by which MMPs may be up-regulated,” explained Underly. “This is a future direction for our research—to define upstream regulators of this process that can be therapeutically targeted."

"The findings raise so many new research questions,” said Shih. “For example, why do pericytes have so much MMP? What are they doing with it? What happens to pericytes days to weeks after an ischemic event? There's so much still to be understood about the acute phenomena—we're focused on that for now. In the future, we could look at later, post-injury, events to see what happens next in the life of the pericyte."

Indeed, in vivo cellular-level imaging research has a bright future.

"There's a renaissance happening with the development of new molecular tools to image and modify cell function in vivo," said Shih. "We're going to see a lot more integration between tool-makers and in vivo imaging groups in the next decade or two. There are going to be many more studies looking at the intact brain."

"It's important to fund projects like this because with in vivo imaging we're able to track exactly what happens when brain function breaks down,” said Underly. “The disease state occurs in front of our eyes."

Summary: Boosting the immune system’s cancer-fighting ability is the aim of adoptive T cell immunotherapy, in which the patient’s T cells are harvested, expanded, and then reinfused. Ensuring T cell persistence after reinfusion has been a challenge. In the October 15, 2016 Cancer Research, investigators at the Medical University of South Carolina and Loyola University report that culturing T cells in N-acetyl cysteine (NAC) significantly improves persistence. Adding NAC to melanoma immunotherapy protocols could improve outcomes.

A collaborative team of investigators at the Medical University of South Carolina (MUSC) and Loyola University have demonstrated for the first time that culturing T cells in N-acetyl cysteine (NAC) before they are infused as immunotherapy improves effectiveness and outcomes in a preclinical model of melanoma. These findings were reported in the October 15, 2016 issue of Cancer Research.

Both incidence and mortality rates for metastatic melanoma continue to rise. Only about 15% of Stage IV melanoma patients receiving standard treatment can expect to survive for five years. By contrast, clinical trial data show that up to 40% of Stage IV melanoma patients survive for five years when treated with adoptive cell therapy (ACT), a form of immunotherapy that calls for infusion of autologous, melanoma-specific T cells.

ACT aims to boost a patient’s own immune responses against the cancer. To do this, the patient’s own T cells are harvested, genetically modified with a therapeutic T cell receptor, activated, and then rapidly expanded to generate large numbers of T cells for therapeutic re-infusion. Unfortunately, patient responses vary. Better outcomes are positively correlated with persistence of the transferred cells. The rapid expansion of harvested T cells before reinfusion increases their susceptibility to activation-induced cell death (AICD), prompting the authors to hypothesize that AICD reduces ACT’s overall effectiveness.

Researchers have long known that factors limiting T cell persistence also limit ACT efficacy but, until now, no one knew that something as simple as changing the culture condition by supplementing with NAC could improve survival of the reinfused T cells. The research team showed that adding NAC to the in vitro T cell expansion culture prevents increases in the DNA damage marker ?H2AX and significantly improves T cell persistence and immunotherapy outcomes, including reduced tumor growth and enhanced survival.  

The team found that nearly 40% of NAC-cultured T cells were detectable in tumors after transfer compared to approximately 1.2% of standard-culture T cells. They also found that mice receiving NAC-cultured cells experienced significantly delayed tumor growth compared to mice receiving standard-culture cells (P<.0001).

"We were really surprised by the number of adoptively transferred T cells we saw in the tumor,” said Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC's Hollings Cancer Center and the senior author on the article. “Given the harsh environment T cells encounter within tumors, we did not expect that the number of NAC-cultured T cells would be 33-fold higher than T cells not cultured in NAC.”

Adding NAC to existing protocols should pose little risk to patients, according to Voelkel-Johnson, because NAC is already in clinical use for many indications and the culturing of T cells in NAC would occur outside of the patient. “The only difference would be a change to the cell culture protocol in an effort to generate cells with an improved phenotype," said Voelkel-Johnson.

The road to discovering the protective role of NAC began with the MUSC/Loyola research team hypothesizing that preventing T cells from becoming susceptible to AICD during in vitro expansion might improve their persistence and effectiveness upon reinfusion.

The team started by examining the role of p53, which is crucial for coordinating cellular stress responses and determining the fates of damaged cells (i.e., whether they will be repaired or allowed to die). While it is known that inhibiting p53 protects T cells from AICD, the molecular-level mechanisms that provide this protection were unknown. The investigators created conditions similar to those that occur during in vitro T cell expansion by re-stimulating the T cell receptors (TCR) to assess p53 status and cell death. They found that AICD following TCR re-stimulation is accompanied by phosphorylation of p53 on Ser15 and accumulation of p53 in the cell nucleus.

The next set of experiments clarified that the PI3K-like serine kinase, ataxia telangiectasia mutated (ATM), is necessary for the phosphorylation of p53 on Ser15 after TCR restimulation. They also found that inhibiting ATM almost completely prevents cell death (99%) after TCR restimulation. Thus, ATM appears to be a required upstream factor for AICD onset.

While activation of ATM and p53 are parts of a known DNA damage response pathway, ATM also responds to oxidative stress or hypoxia. So, the team needed to determine whether the changes in ATM and p53 were being caused by DNA damage or oxidative stress/hypoxia. They looked at two established DNA damage markers, gH2AX and p-SMC-1, and found that, within 15 minutes of TCR restimulation, both gH2AX and p-SMC-1 increased three-fold. This suggested that DNA damage and ATM activation occur in parallel, leading the authors to conclude that TCR restimulation causes DNA damage, which then triggers the ATM/p53 DNA damage response pathway.

Because it is known that AICD depends on the generation of reactive oxygen species (ROS), the investigators focused on ROS as a potential cause of DNA damage. They incubated some T cells with the antioxidant NAC prior to restimulation and compared them to T cells that were expanded using a standard protocol. Results showed that ROS levels were significantly lower in the NAC cultures than in the standard cultures. In addition, pretreatment with NAC reduced activation of the DNA damage markers, ?H2AX and p-ATM, by 94% and 69%, respectively. These results confirmed that ROS generated during TCR restimulation play a central role in damaging the DNA.

This is the first study to show that expanding therapeutic T cells in the presence of NAC prior to adoptive transfer improves their ability to resist AICD. Most importantly, using these "AICD-resistant" T cells improves therapeutic outcomes in a preclinical model by enhancing T cell persistence, increasing tumor control, and improving survival.

"Now we are looking at studies to help us understand exactly how NAC changes the phenotype of T cells,” said Voelkel-Johnson. “How does it make these cells survive? How is trafficking to tumors improved? There may be benefits to culturing T cells in NAC aside from generating AICD resistance that we haven't yet recognized.”

MUSC Health Rheumatologist Dr. Jim Oates


Summary: Medical University of South Carolina (MUSC) investigators report preclinical research showing that prognostic models for lupus nephritis that include novel biomarkers have significantly improved predictive power over models using only traditional markers, in the August 2016 issue of Arthritis & Rheumatology. Data reveal that chemokines, cytokines, and markers of cellular damage were most predictive of patients' therapeutic response. This is a critical first step to developing clinically meaningful, decision-support tools in lupus nephritis.



Caption: MUSC Health rheumatologist Jim C. Oates, M.D.

Results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in the August 2016 issue of Arthritis & Rheumatology demonstrate for the first time that including novel biomarkers in lupus nephritis (LN) prognostic models significantly increases their power to predict therapeutic efficacy. Identifying biomarker models with sufficient predictive power is a critical step toward developing clinical decision-making tools that can rapidly identify patients who require a change in therapy and potentially reduce onset of renal fibrosis during induction therapy.

Approximately half of all patients with systemic lupus erythematosus (SLE) develop LN, an immune complex-mediated glomerulonephritis. Lupus nephritis, in turn, leads to renal failure in up to 50% of patients within five years. American College of Rheumatology guidelines recommend changing LN treatment after six months of induction therapy if response to therapy is not achieved. However, 'response to therapy' is not clearly defined and renal damage can occur during the six-month induction period.

Currently, clinicians monitor response to treatment via blood pressure measurements, serum complement levels, anti-double-stranded DNA (anti-dsDNA) antibody levels, urinary sediment, urinary protein-to-creatinine ratios, and surrogates of renal function. Unfortunately, predicting disease progression is difficult using these traditional biomarkers due to their low sensitivity and high LN heterogeneity at presentation. Even when machine learning models are employed, traditional biomarkers are only 69% accurate in predicting a LN diagnosis among SLE patients. There is a need for individualized, decision-support tools that can better define 'therapeutic response' at the start of therapy and allow clinicians to tailor induction therapy to disease severity to prevent renal damage and unnecessary drug toxicity.

"We saw our colleagues' frustration in trying to come up with predictive models,” said Jim C. Oates, M.D., Associate Director of the MUSC Clinical and Translational Research Center, Associate Professor of Rheumatology, and senior author on the article. “The traditional markers we use in clinic today have quite limited predictive capacity. All lupus patients have varying degrees of kidney damage and levels of involvement of the different kidney structures. So, we wanted to account for this heterogeneity and the stages of disease progression. We wanted to include markers for pathways of inflammation as well as for damage."

The research team hypothesized that a targeted panel of urinary biomarkers reflecting initial resident and inflammatory cell activation (cytokines), signals for homing to the kidney (chemokines), activation of inflammatory cells (growth factors), and damage to resident cells, combined with artificial intelligence/machine learning modeling, might provide an early LN decision-support tool that could predict outcomes better than standard biomarkers alone. The team also chose to assess urine biomarkers rather than serum/plasma markers to increase the tool's sensitivity and specificity to signals of renal (rather than systemic) processes.

Urine samples from 140 patients with biopsy-proven LN who had not yet started induction therapy were analyzed for a panel of novel biomarkers using pre-mixed, commercially available kits. Univariate, receiver operating characteristic (ROC) curves were generated for each biomarker and compared to ROC area under the curve (AUC) values from machine learning models developed using random forest algorithms. Outcome models using novel biomarkers plus traditional clinical markers demonstrated greater AUC and significance compared to models developed with traditional markers alone ([AUC 0.79; P<0.001] vs. [AUC 0.61; P=0.05], respectively). The combined models also demonstrated greater power to correctly predict LN therapy outcomes (responder versus non-responder) than models using only traditional markers (76% vs. 27%, respectively [p<0.002]).

The team identified chemokines, cytokines, and markers of cellular damage as most predictive of LN therapy response. Race, anti-double-stranded DNA antibodies, and induction medication did not significantly contribute to the model.

"We were somewhat surprised by some of the analytes that were important in the model,” said Oates. "One traditional marker, protein-to-creatinine ratio, was the third most important, and a standard kidney function measure was the ninth. I was also surprised to see interluekin-8 so high. This is in keeping with recent publications highlighting the importance of neutrophils in the pathogenesis of lupus, however."

Including multiple mechanisms of disease pathogenesis and cellular damage likely provides a more effective diagnostic approach by better reflecting the multi-stage, heterogeneous nature of LN. This is the first study to combine a broad biomarker panel with machine learning techniques to optimize disease outcome models. "This could apply to any model where there is kidney inflammation leading to damage,” said Oates. "It's proof of concept for other kidney diseases that you can take a discovery model and incorporate machine learning to develop and validate predictive models."

The team is now testing other biomarkers and applying the model in a larger patient population to ensure external validity and improve power. They are also exploring other inputs.

"Our next approach is to harness existing data in the medical record to enhance predictions,” said Oates. “This is much more immediately translatable in the clinic than getting through a long FDA validation process and the industry pipeline. Using medical record data is cheaper, and there are patient and system factors in the medical record that you can't measure with an assay, such as economic and societal disparities, which affect outcomes. This approach could also be used to enhance biomarker predictive models”

Summary: Medical University of South Carolina (MUSC) investigators report preclinical research showing that Krüppel-like factor 12 (KLF12) promotes colorectal cancer (CRC) cell growth by activating early growth response protein 1 (EGR1), in the July 2016 issue of PLOS One. Data also reveal that levels of KLF12 and EGR1 correlate synergistically with a poor CRC prognosis. Results indicate that KLF12 plays an important role in CRC progression and provides a potential novel prognostic marker and therapeutic target.

Results of preclinical studies by MUSC investigators reported in the July 2016 issue of PLOS One (doi:10.1371/journal.pone.0159899) demonstrate for the first time that the transcription factor Krüppel-like factor 12 (KLF12) promotes poor colorectal cancer (CRC) cell growth, in part, by activating EGR1. Furthermore, data demonstrate that KLF12 and early growth response protein 1 (EGR1) levels synergistically correlate with  CRC prognoses.

CRC is the third most common and third deadliest cancer in the US. Like most cancers, CRC development is spurred by a series of genetic mutations and epigenetic changes that alter gene expression. In turn, this altered gene expression initiates tumors and supports their progression. Thus, transcription factors that regulate gene expression and signaling pathways during carcinogenesis have long been studied as potential therapeutic targets.

Dr. Raymond DuBois, Dean of the MUSC College of Medicine, Professor of Biochemistry and Molecular Biology, and senior author on the article is focused on understanding the role of inflammation in cancer. "We've been studying the connections between inflammation and cancer in my lab for some time now and have determined that some inflammatory mediators stimulate the progression of cancer,” DuBois said. “We found that KLF12 was increased dramatically in the presence of inflammation in certain cancers, so we were trying to determine the specific molecular mechanisms responsible for these effects."

Other researchers who were studying kidney development previously identified transcription factor KLF12 as a transcriptional repressor of the AP-2? gene. It was then discovered that AP-2? expression is also reduced in advanced CRC tumor tissue compared to matched normal tissue and that loss of AP-2? promoted CRC invasion. This connection illuminated a potential link between KLF12 and CRC. In vitro studies show that KLF12 promotes gastric cancer (GC) cell proliferation and invasion, and that KLF12 levels are elevated in about 40% of poorly differentiated GCs and correlate with tumor size. Furthermore, recent genome-wide analyses find high KLF12 levels in approximately 40% of esophageal adenocarcinomas and in 45% of salivary tumors. Until now, however, the role of KLF12 in CRC remained unclear.

The MUSC research team designed a series of in vitro and in vivo experiments to clarify the role of KLF12 in CRC. The first set of studies examined KLF12 expression in seven human CRC cell lines. They found not only that KLF12 was expressed in six of the seven cell lines, but also that its overexpression led to increased cell numbers and KLF12 knockdown led to reduced cell numbers. In addition, they also found that overexpression of KLF12 led to the formation of larger cecal tumors while KLF12 knockdown led to formation of smaller cecal tumors, compared to controls. Thus, this set of experiments indicates that KLF12 promotes CRC growth by enhancing cancer cell proliferation and/or survival.

The next set of experiments focused on clarifying which KLF12 target genes may be involved in regulating CRC growth. Using microarray assays, the researchers found that KLF12 overexpression altered multiple genes including EGR1. It has been previously reported that KLF12 regulates expression of some target genes by binding to the CACCC motif.  They found that the EGR1 promoter contains two possible KLF12 DNA-binding motifs located at -1488bp (motif 1) and -808bp (motif 2) relative to the transcription start site. Using ChIP assay, the MUSC researcher team found that KLF12 does, indeed, bind strongly to the EGR1 promoter motif 2 but not to motif 1. In vitro experiments demonstrated that, at both the mRNA and protein levels, CRC cells with undetectable levels of KLF12 expressed the lowest levels of EGR1 compared to cells expressing high levels of KLF12. In vivo studies using mice implanted with CRC tumor cells that overexpressed KLF12 showed that EGR1 expression was up-regulated compared to mice implanted with control cells. Furthermore, staining of human CRC tissue specimens produced the same pattern. Taken together, these results indicate that KLF12 directly activates EGR1 in CRC.

The third set of experiments looked at whether EGR1 mediated the effects of KLF12 on tumor cell growth. Results showed that EGR1 knockdown reduced KLF12-induced tumor cell growth, whereas EGR1 overexpression promoted CRC cell growth in vitro as well as tumor growth in the mouse model. The results of this set of studies, thus, indicate that KLF12 enhances CRC cell growth by activating EGR1.

The final set of experiments evaluated whether KLF12 and EGR1 levels correlate with CRC patients' prognoses. Using gene expression data from publicly available microarray databases (Moffitt [n = 177]; Vanderbilt Medical Center [n = 55]), CRC patients were stratified by level of KLF12 and/or EGR1 expression. These data showed that patients with high levels of either KLF12 or EGR1 had worse outcomes compared to those with low levels of these genes, and that those with high levels of both KLF12 and EGR1 had the lowest survival rates.

This is the first study to clarify the role of KLF12 in CRC tumor growth and progression, which appears to occur, at least in part, through EGR1 activation. The finding that synergistic contributions of KLF12 and EGR1 produce the worst outcomes among CRC patients illuminates their potential in developing novel therapies. More studies are needed to further clarify the role of KLF12 in CRC progression and its potential as a novel prognostic marker and therapeutic target. 

Vitamin D Illustration

Summary: Investigators at the Medical University of South Carolina and Ralph H. Johnson VA Medical Center report clinical research showing that African-American and European-American men with prostate cancer exhibit significantly different expression of genes associated with immune response and inflammation, in the July 2016 issue of  Pharmacogenomics. Systems-level, RNA analyses support the concept that inflammatory processes may contribute to racial disparities in disease progression and that vitamin D3 supplementation can modulate pro-inflammatory transcripts.

The results of clinical studies by investigators at the Medical University of South Carolina (MUSC) and the Ralph H. Johnson VA Medical Center (VAMC), reported in the July 2016 issue of Pharmacogenomics (doi:10.2217/pgs-2016-0025) , demonstrate transcriptome-level linkages between racial disparities in circulating levels of vitamin D and expression of pro-inflammatory genes in African American (AA) patients with prostate cancer compared to European American (EA) patients.

Racial disparities in prostate cancer are well documented with AA men having significantly higher risk of developing prostate cancer and significantly higher mortality rates than EA men. In addition, among patients presenting at the same disease stage, AA men often have higher prostate-specific antigen (PSA) levels and higher-grade tumors than EA men. However, the biological mechanisms underlying these substantial and persistent disparities are unclear.

Researchers at MUSC and VAMC noticed that racial disparities in prostate cancer mirror differences in circulating levels of vitamin D between AA and EA patients. Vitamin D3 is known to have multiple anti-cancer actions including suppression of cyclo-oxygenase-II (an independent predictor of cancer recurrence) and inhibition of IL-8 (an angiogenic, pro-inflammatory cytokine). Prostate cells express the vitamin D receptor, vitamin D-25-hydroxylase, 25 hydroxyvitamin D-1-alpha-hydroxylase, and 25-hydroxyvitamin D-24-hydroxylase. Thus, normal prostate cells can synthesize 25(OH)D3 (calcidiol) from vitamin D (cholecalciferol), and 1,25(OH)2D3 (calcitriol) from 25(OH)D3. 1,25(OH)2 D3 (calcitriol) is the bioactive, hormonal, and most potent form of vitamin D and facilitates cell-to-cell communication via paracrine/autocrine pathways.

Sebastiano Gattoni-Celli, M.D., Professor of Radiation Oncology at MUSC, and senior author on the article, explains how his team came to explore the therapeutic potential of vitamin D supplementation in prostate cancer, "A lot of previous work shows that D3 levels are much lower in African Americans than in European Americans and it's well established that prostate cells are very sensitive to vitamin D levels. So this raised the possibility that long-term vitamin D deficiency may contribute to the progression of prostate cancer, especially among African American men. We began to wonder whether eliminating racial disparities in circulating levels of vitamin D, through supplementation, could help reduce the disparities we see in prostate cancer outcomes."

The team designed a prospective, placebo-controlled, clinical study to investigate the effects of a daily 4,000 IU vitamin D3 supplementation over a two-month period among 27 men (10=AA, 17=EA) who had elected to treat their prostate cancer via prostatectomy. A trial length of two months was chosen to leverage the recommended, standard-of-care recovery period between their biopsy and surgery procedures. Using high-throughput RNA sequencing, they performed a series of genome-wide expression profiling experiments to generate transcriptional profiles of patients' prostate tissue samples and assessed them using systems-level analyses. Their primary aims were to: (1) illuminate any molecular differences in gene expression that may be related to prostate cancer disparities between AA and EA men; and (2) investigate any effects vitamin D supplementation may have on the prostate transcriptome.

Not only did the team find significant differences in gene expression between AA and EA men but also between AA men receiving vitamin D supplements and AA men receiving placebo. Due to the size of the RNA sequencing dataset, results are reported as adjusted p-values (or q-values) which represent the smallest 'false discovery rate' at which a result can be called significant. A total of 3,107 prostate genes were differentially expressed between the AA and EA groups (q<0.1) with 8,238 differentially expressed transcripts between AA and EA subjects (q<0.4). Analyses of these found that AA study patients had substantially elevated expression of transcripts related to immune response and inflammation.

"The number of genes expressed differently in AA and EA was a really big surprise—we found differences in over 8,000 genes,” said Gattoni-Celli. “I expected something but not this massive difference and it was not a fluke. When we compared our results with previous studies using a less advanced technology, we saw that they, too, found these differences, but not as many.”

“Our findings captured all of the differences observed in previous studies but also many more because newer RNAseq technology and Big Data analytical approaches allowed us to see the transcriptome in greater detail,” noted Gary Hardiman, Ph.D., Professor of Medicine and Public Health Sciences and Bioinformatics Director for the Center for Genomic Medicine at MUSC, and co-senior author on the article. “This analysis was performed using the OnRamp BioInformatics Genomics Research Platform we deployed at MUSC a little over a year ago. Our approach converged advanced genomics analysis, comprehensive data management, big data analytics and hyperscale servers. A ‘Big Data’ analytical pipeline that utilized Hadoop software was implemented. This enabled an automated RNAseq workflow to process the patient data and explore differential prostate gene expression analysis between AA and EA men and sensitively interrogate the effects of vitamin D supplementation with robust statistical power.”

When comparing AA men receiving vitamin D supplementation to AA men receiving placebo,  the team found 817 differentially expressed genes (q<0.4). However, no similar difference in gene expression was observed between EA men receiving vitamin D supplementation versus placebo. Comparing the 8,238 differentially expressed transcripts between AA and EA subjects with the 817 genes that were differentially expressed among AA men receiving vitamin D supplementation and AA men receiving placebo, the team found an overlap of 346 genes. This finding suggests that a substantial number of genes that are differentially expressed across racial groups can be affected by a brief (2-month) course of vitamin D3 supplementation in AA patients.

This research is an important step in understanding the molecular underpinnings of health disparities in prostate cancer. Further clarification of race-based transcriptome differences and the role of vitamin D in prostate tissue may lead to use of vitamin D3 supplementation as an early-stage therapeutic option in prostate cancer. Furthermore, results from studies among AA and EA women with breast cancer could extend these findings because breast cancer, like prostate cancer, is an endocrine cancer, with many similarities including sensitivity to vitamin D.

An accompanying editorial by Batai K and Kittles RA, "Can vitamin D supplementation reduce prostate cancer disparities?" was published in the same issue of Pharmacogenomics (volume 17, number 11, 2016, pages 1117-1120).   

Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days.










Summary: In an article published online in Nature Cell Biology on July 11, 2016, investigators at the Medical University of South Carolina report preclinical findings suggesting that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis. Maintaining Dab2 by inhibiting cathepsin B could prevent tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in an article published online on July 11, 2016 in Nature Cell Biology (doi: 10.1038/ncb3388) demonstrate that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis.

While expression of Dab2—an endocytic adaptor and tumor suppressor—is known to occur during transforming growth factor-beta (TGF-beta)–mediated epithelial-mesenchymal transition (EMT), the mechanisms by which it regulates apoptosis were, until now, poorly understood.

Exploring the pathways by which Dab2 is degraded and the effects of maintaining Dab2 levels reveals its pivotal role in preventing tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

TGF-beta induces EMT—a process by which cells transform from a polarized epithelial phenotype to a fibroblastic or mesenchymal one. Dab2 is expressed during TGF-beta–mediated EMT. While EMT is essential for normal cellular growth and homeostasis, it is abnormally activated in tumor cells and contributes to their chemo-resistance and metastasis.

TGF-beta has also been reported to regulate autophagy, which, in established tumors, ensures tumor cell survival through times of stress, as for example during chemotherapy. In other words, autophagy supports the chemo-resistance, growth, and metastasis of tumor cells.

Researchers focused on the Dab2 protein after noticing that, in cells treated with TGF-beta, Dab2 levels rose over the initial 24-48 hours as they went through EMT but then fell with continued TGF-beta treatment. By day 7, the tumor cells had transitioned to a morphological state suggestive of either autophagy or apoptosis. Furthermore, the mesenchymal markers N-cadherin and vimentin, which like Dab2 initially rose during EMT, began to decline with longer exposure to TGF-beta.

"This was an unexpected finding that we followed,” explains senior author Philip Howe, Ph.D., Professor and Chair of Biochemistry and Molecular Biology and Hans and Helen Koebig Chair in Clinical Oncology at MUSC. “We knew that if you give cells TGF-beta they go through EMT, and we knew you needed Dab2 for TGF-beta–mediated EMT. But, when we kept adding TGF-beta for more sustained periods (after EMT took place), cells took on a different morphology and we noticed a loss of Dab2. We investigated this loss of Dab2 and discovered that it was being cleaved and that the cells were undergoing autophagy. Upon sustained TGF-beta treatment, the cells had lost their mesenchymal phenotype they'd gained in EMT and entered into an autophagic state.”

The team began to explore how prolonged TGF-beta treatment led to loss of Dab2 and the mesenchymal phenotype.

First, they found that longer TGF-beta exposure significantly increased cathepsin B (CTSB) expression and promoted its co-localization with Dab2. The team then not only demonstrated that CTSB is responsible for cleaving Dab2 but also that it recognizes the cleavage site by the flanking amino acids Val499 and Gly500. Thus, while an unaltered Dab2 sequence (Leu-Val-Gly-Leu) was degraded by CTSB, it did not cleave a mutant Dab2 sequence (Leu-Val-Leu).

Second, findings showed that, after 7 days, continuous TGF-beta treatment induced autophagy and down-regulated markers of apoptosis. This was particularly notable because these conditions promote tumor cell chemo-resistance and metastasis.

Third, they found that CTSB inhibition or expression of a mutant Dab2 without the CTSB cleavage site (i.e., the Leu-Val-Leu mutant) led to time-dependent increases in pro-apoptotic markers. When TGF-beta was withdrawn, cells in which Dab2 had been preserved underwent cell death. This series of experiments show not only how Dab2 is modulated by CTSB but also that it serves as a switch for regulating TGF-b–induced autophagy and apoptosis.

Another series of experiments were undertaken to clarify exactly how Dab2 functions to prevent autophagy and promote apoptosis. These findings show that Dab2 inhibits TGF-beta–induced autophagy by blocking the Vps-Beclin-1 interaction and promotes apoptosis by attenuating ERK-Bim interactions.

Finally, the team used the chemotherapeutic agent doxorubicin (DOXO) to determine whether the role of Dab2 in inhibiting autophagy might affect tumor cell chemo-sensitivity. They found that cells in which CTSB was overexpressed had increased survival in the presence of DOXO. However, cells with high Dab2 levels due to CTSB inhibition or expression of the CTSB-resistant Dab2 mutant were more chemo-sensitive and underwent apoptotic changes. Thus, Dab2 was shown to promote chemotherapeutic drug–induced cell death by attenuating drug-induced autophagy. In vivo tumor studies in mice further found that Dab2 both enhanced DOXO-mediated cell death and attenuated tumor cell metastasis.   

These direct insights into molecular mechanisms supporting tumor cell survival and death are crucial for maximizing the effectiveness of existing chemotherapeutic agents. "This is important because there aren't a whole lot of drugs out there," explains Howe. "Most of what we use today has been around for 20 or 30 years because of a lack of investment in basic science." 

The team's next steps are to investigate in vivo models for combination therapies using DOXO and a CTSB inhibitor to further illuminate the potential for targeting Dab2 as a means of reducing tumor recurrence and metastasis.

Image Caption: In the absence of Disabled-2 (Dab2), Vps34/Beclin-1 Interactions are maintained. Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days. Photos were taken by confocal microscope. Scale bars, 10 mm. The data show that in the absence of Dab2, due to CTSB overexpression, Vsp34/Beclin-1 interactions are maintained and autophagy is initiated. Adapted from a figure originally published in an article by Jiang Y, Woosley AN, Sivalingam N, Natarajan S, and Howe PH in Nature Cell Biology (doi: 10.1038/ncb3388).


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