Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Renal cell carcinoma (RCC) is hallmarked by metastasis and resistance to radiation and cytotoxic chemotherapy resulting in a mortality rate of nearly 25% within the first year of diagnosis. Approximately, 10% of patients demonstrate improved survival following surgery in combination with cytokine immunotherapy, indirect evidence of an immunosuppressive effect of the tumor. One tumor-derived factor that may limit the efficacy of immunotherapy is prostaglandin E2 (PGE2), a metabolite of the arachidonic acid pathway. In the tumor microenvironment, PGE2 has a dual role: it suppresses anti-tumor immunity by inhibiting NK–cell and T–cell activation and promotes inflammation, angiogenesis and metastasis through combined activation of tumor associated cells. Central to production of PGE2 is cyclooxygenase–2 (COX–2) where elevated expression is associated with poor prognosis for patients with epithelial cancers. Aggregate data from clinical studies indicate that elevated expression of COX–2 is detected in more than 50% of RCC tumors where the frequency and intensity of positive cells increases with advanced tumor stage (26% for stage I, 67% for stage II, and 80% for both stage III and IV). The requirement of COX-2 for PGE2 production suggests a role for this immunosuppressive metabolite in RCC progression and metastasis. Attempts to induce anti-tumor immunity as a treatment modality for RCC have been modestly successful demonstrated by improved survival for patients treated by surgery in combination with interferon–alpha (IFN–α) or interleukin–2 (IL–2) cytokine immunotherapy. Of direct relevance to studies describe here, combinational treatment of RCC patients with IFN–α and selective COX–2 inhibitors has been successfully used to prolong tumor progression and improve survival, suggesting that PGE2 is an important potential target for the treatment of RCC. Herein, I describe the results of studies designed to address this gap in our current knowledge. A relationship between RCC and production of PGE2 was first established using an enzyme immunoassay (EIA) to compare circulating levels of PGE2 in serum samples obtained from patients before and after surgical removal of the tumor–bearing kidney and compared to age–matched, healthy volunteers as a control. Results demonstrated elevated pre–operative levels of PGE2 (72.5 pg/mL; 95%CI= 56.2–88.8) that decreased after removal of the tumor–bearing kidney (40.1 pg/mL; 95%CI= 37.1–43.1) nearly approaching levels observed for healthy volunteers (26.8 pg/mL; 95%CI= 23.3–30.3). This reduction in serum PGE2 suggested that PGE2 was a RCC tumor-derived factor with a potential for limiting treatment efficacy. To study this finding, a mouse model of RCC was established with enforced expression of COX–2 and production of PGE2. For this, Balb/cCr mouse-derived RCC cells (Renca) were transfected with transposon–based plasmids encoding for expression of the mouse COX–2 gene in either sense or antisense orientations both with and without green fluorescent protein (GFP) and firefly luciferase (fLuc) reporter proteins. Evaluation of total cellular protein confirmed that expression of COX–2 was specific to sense cell lines and coincident with elevated production of PGE2(239–old increase over antisense controls). Importantly, this level of PGE2 5969.8 pg/mL± 2771.8) could be potently inhibited following addition of non-specific cyclooxygenase inhibitor, Indomethacin or the COX–2 specific inhibitor, NS398, to the culture media. Examination of cell growth and basal expression of PGE2 cognate receptors (EP1–4), angiogenic growth factors and receptors, chemokine receptors, and basement membrane–degrading enzymes revealed no appreciable change in the modified cell lines when compared to controls. Thus, changes in tumor growth or metastasis in vivo can only be attributed to PGE2 production. In addition to its role in cancer cells, PGE2 has also been associated with recruitment of stromal cells to the tumor environment, and increased production of chemokines and angiogenic factors by these cells. To address this potential, characterization of the PGE2 synthesis pathway, angiogenic growth factors and receptors, basement membrane-degrading enzymes, and chemokine receptors in mouse fibroblasts, endothelial cells, and mesenchymal stem cells was completed. Results from this characterization suggest interactions between stromal cells and the modified cell lines could occur through PGE2–EP receptor signaling or chemokine receptors. In summary, these results suggest COX–2 production of PGE2 could play an important role in RCC carcinogenesis and use of this syngeneic model would allow for a more substantial analysis of this role. Furthermore, it would provide a foundation for testing therapies designed to enhance tumor-specific immunotherapy.
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