Role of pericytes in the resistance to sunitinib therapy in renal cell carcinoma
Sotomayor Fahrenkrog, Paula Camila Stefania
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Renal cell carcinoma (RCC) accounts for approximately 3% of all cancer diagnoses in the U.S. each year. In 2012, an estimated 65,000 new cases of RCC were diagnosed in the United States and approximately 13,500 individuals died of this disease. RCC is made up of several different types of cancer, each of which has a distinct histology and molecular pattern that responds differently to treatments. If detected early, renal cell carcinoma (RCC) can be treated surgically. However, 20-30% of patients present with metastasis at diagnosis and 20-30% will relapse with metastasis. Therefore, 40-50% of the patients develop metastatic disease. Metastatic RCC (mRCC) is generally resistant to chemotherapy and hormonal therapy, and only marginally sensitive to immunotherapy. RCC is highly angiogenic and have one of the highest VEGF levels among various solid tumors. Therefore, anti-angiogenic therapies have been evaluated and have shown significant therapeutic benefits. The anti-angiogenic drug sunitinib is a multitargeted tyrosine kinase receptor inhibitor (TKI) that has been used as front-line therapy for the treatment of mRCC and gastrointestinal cancer. Sunitinib treatment induces an initial favorable response; however, resistance to sunitinib occurs in the majority of patients after approximately 11 months of treatment. One of the mechanisms that has been hypothesized to play a role in sunitinib resistance is an increase in pericyte cell coverage in the tumor vasculature. Pericytes are vascular mural cells embedded within the vascular basement membrane of blood vessels, where they make direct focal contact with endothelial cells. Pericytes have multiple roles including regulating endothelial proliferation, maturation, migration, and survival of endothelial cells. The survival signaling from pericytes to endothelial cells has been studied in vitro and in vivo during different stress responses, but the protective role that pericytes may have under anti-angiogenic therapy is unknown. We hypothesize that pericytes facilitate the survival of endothelial cells after sunitinib treatment. Survival of endothelial cells increases survival of tumor epithelial cells and facilitates recurrence after sunitinib treatment. In vitro results using HUVEC (Human Umbilical Vein Endothelial Cells) and hPC-Pl (Human Pericytes from Placenta) indicate that HUVEC cells are more sensitive to sunitnib than hPC-Pl. Moreover, results of co-cultures experiments treated with sunitinib suggest that pericytes do protect endothelial cells under sunitinib treatment in vitro. For in vivo experiment, we used the murine renal adenocarcinoma model RENCA that arises from spontaneous tumor in a BALB/c mouse. These tumors were found to be sensitive to sunitinib. An effect on the vasculature was observed as early as 10 days of treatment and tumors were considerably smaller compared to vehicles. RENCA tumors treated for long periods of time became resistant to sunitinib and these had a resistant vasculature that allowed for the growth of tumor cells. Importantly, this vasculature was covered by pericytes, with an observed increased coverage as compared to vehicle tumors, suggesting a possible vascular protection by pericytes. These aforementioned data suggest that the RENCA model mimics the development of resistance to sunitinib in RCC, and hence is a very valuable model to investigate the molecular changes involved in sunitinib resistance. Initial results in our laboratory have shown that the tumor suppressor PTEN is expressed in tumor associated pericytes in human RCC tissues and in the RENCA model; and that PTEN expression in pericytes is decreased in sunitinib resistant vasculature. The PTEN protein inhibits the PI3K/AKT/mTOR signaling pathway. Interestingly, this pathway may be inhibited by rapamycin and its analogs, which are the second-line treatment for RCC. Immunohistochemistry for PTEN protein expression in RENCA tumors and human RCC tissues showed high levels of PTEN expression in a population of cells associated with tumor vasculature. Co-staining of PTEN with the endothelial marker CD31 and the pericyte marker SMA demonstrated that the population of cells that overexpress PTEN were pericytes. Moreover, these experiments suggest that tumor signaling induces PTEN pericyte expression. In addition, sunitinib resistant vasculature in RENCA tumors had pericytes with low levels of PTEN expression. Finally, we determined that rapamycin when given in short sequential combination with sunitinib, had a greater anti-tumor effect in the RENCA model than single agents. Overall, these results indicate that pericytes may play an important role in resistance to sunitinib, by protecting endothelial cells and allowing subsequent tumor growth. Moreover the molecular changes in PTEN expression in pericytes during sunitinib and rapamycin treatment suggest that these changes may provide valuable information regarding the mechanism(s) of resistance. Finally, our data suggest that sunitinib-rapamycin sequential combination treatments may be a promising therapeutic strategy to overcome the sunitinib resistant phenotype.