Skip Navigation
request an appointment my chart notification lp musc-logo-white-01 facebook twitter youtube blog find a provider circle arrow
MUSC mobile menu

STAT

An MUSC blog
Keyword: hollings cancer center

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.

SUMMARY: A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Drug treatments for breast cancer patients might soon be designed based on the unique genetic autograph of their tumor.

A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Dr. Stephen EthierCertain oncogenes drive solid tumor growth in some breast cancer patients but are just passenger genes in others—expressed but not essential for growth. As a result, tumors in different breast cancer patients may respond differently to the same treatment depending on which oncogenes are active and which are just along for the ride. Identifying the panel of active genes in a patient’s tumor—called the functional oncogene signature—could help an oncologist select therapies that target its growth, according to Stephen P. Ethier, Ph.D., Interim Director of the Center for Genomic Medicine at MUSC and senior author on the study.  

“In order to move the field forward, we now need to be able to use oncogene signatures to tailor therapies using combinations of targeted drugs so that multiple driving oncogenes can be targeted at once,” said Ethier.  “Doing this successfully requires the identification of the oncogenes to which the cancer cells are addicted, as this allows the use of targeted drugs at very low doses. Low doses are essential if we are to use combinations of different targeted drugs.”

Ethier’s group identified unique functional oncogene signatures in four different human breast cancer cell types. Using next-generation genome sequencing and genome silencing as each cancer cell type grew and multiplied, they assembled a list of genes for each cell type’s functional oncogene signature—those genes that were copy number amplified or point mutated, and most essential to cancer cell survival. Although thousands of candidate oncogenes were screened during experimentation, only a handful made the list—fewer than 20 for each cell type.

The brevity of each list facilitated selection of the best oncogene for pharmaceutical targeting. Because lower doses of targeted drugs can be highly effective, side effects could be reduced. For example, Ethier’s group found that targeting two or more members of a signature with much lower total drug concentrations in combination still killed cancer cells better than one higher-concentration drug alone.

Remarkably, a BCL2L1-targeted drug  that worked in one cell line also then worked in a fifth breast cancer cell line with a similar oncogene signature containing BCL2L1, an oncogene not normally associated with breast cancer. This work demonstrates that one signature-targeting treatment can be extended to more than one cancer cell type. This means that patients with other types of cancer who have a similar functional oncogene signature might benefit from drugs that target BCL2L1, which are already in development.

Ethier thinks that oncogenes identified in a tumor biopsy might one day soon provide a rational and individualized approach to pharmaceutical treatment with targeted drug combinations. Meanwhile, these findings from his laboratory—showing the importance of considering a patient’s functional oncogene signature before testing a new drug— could provide a rationale for redesigning clinical trials for breast cancer.

Stephen T. Guest, Ph.D., of the MUSC Department of Pathology & Laboratory Medicine, was first author on the study.

leddy and surgery team preparing prosthesisIn May 2015, an 8-year-old boy from Columbia, SC became the second child in the state to receive an extendible implant that replaced the leg bone that osteosarcoma had destroyed.  Orthopaedic oncologist Lee Leddy, M.D., Associate Professor in the Department of Orthopaedics at MUSC Health, performed the surgery, removing the cancerous bone (and its growth plate) and replacing it with a device designed to be lengthened over time to ensure that both legs will be of equal length. During follow-up visits every four to six weeks, the boy will place his leg into a doughnut-shaped magnet that will drive a gearbox to extend the prosthesis nine centimeters, the remainder of the boy’s projected growth.

Prior to this technology, options for a child whose growth plate had to be removed due to cancer were amputation; rotationplasty, in which the child’s ankle is substituted for the knee joint; or implants that required repeated surgeries to lengthen the prosthesis.  With this device, future operations are not necessary. More than 100 procedures have been completed in the U.S. with this device, but only two in South Carolina, both by Leddy at MUSC Health.

Leddy says this prosthesis is a dramatic improvement over the ways doctors previously met the challenges of limb salvage surgery in the skeletally immature patient. “Being able to reliably lengthen the extremity without surgery is a major advantage,” he says. “However, it is important to realize how critical the team approach is when treating these complex problems.”

The team of specialists who collaborated on these complex cases included musculoskeletal radiologists who interpreted radiographs and magnetic resonance imaging reports,  pathologists who evaluated biopsy tissues, sarcoma-trained surgical oncologists who helped resect the cancer and reconstruct the extremity, operating room nurses, oncologists who made recommendations regarding chemotherapy, and physical therapists who worked with the patients to help return them  to their active lives.

Leddy says that assuming a good response to chemotherapy and physical therapy, these patients can expect a full recovery. 

Photo provided by Sarah A. Pack

Dr. Robert StuartDr. Azizul Haque

The body’s own immune system could be a potent weapon in the war on cancer if the cloaking mechanisms tumor cells use to elude it could be deactivated. In an article published in the February 15 issue of the Journal of Immunology, one of those cloaking mechanisms was identified in B cell tumors by a team of MUSC immunologists led by Azizul Haque, PhD (above right), MUSC Health hematologist/oncologist Robert Stuart, M.D. (above left), and their colleagues at the University of Indiana and German Research Center for Environmental Health. They reported that overexpression of the c-MYC protein, one of the most commonly activated genes in human cancers that is implicated in the cancer-related deaths of about 100,000 people worldwide, is linked to the ability of B cell tumors to “hide” from the immune system.1 Specifically, they showed for the first time that overexpression of the c-MYC protein in Burkitt’s lymphoma interferes with human leucocyte antigen (HLA) class II antigen presentation. T cells can mount an immune response against antigens only if they can “see” them; they “see” them when TCRs (T cell receptors) on their surface recognize antigen fragments bound to HLA class II molecules on the surface of antigen-presenting cells. When tumor antigen is not presented properly due to c-MYC overexpression, it remains invisible to the T cells. The article also provided evidence that treatment of c-MYC-overexpressing cells with a c-MYC inhibitor decreased c-MYC expression and partially restored HLA class II-mediated antigen presentation. These results suggest that c-MYC inhibitors could help “unmask” B cell lymphomas and promote a more robust immune response. According to Haque, “This study uncovers a mechanism by which c-MYC impairs immune detection of malignant tumors, which could be targeted in future treatments for B cell lymphomas and other malignancies.” The article by Haque and colleagues was highlighted in the “In This Issue” section of the Journal of Immunology, reserved for the top 10% of articles published in the journal.

References

1 God JM, Cameron, C, Figueroa J, Amria S, Hossain A, Kempkes B, Bornkamm GW, Stuart RK, Blum JS, Haque A. Elevation of c-MYC Disrupts HLA Class II–Mediated Immune Recognition of Human B Cell Tumors. The Journal of Immunology 2015;194:1434–1445.

Subscribe to Progressnotes

Submit a Story Idea


Current Issue of Progressnotes

Digital EditionPDF | Home

Progressnotes - Spring 2017 cover thumb

Back Issues of Progressnotes

past issues of progressnotes