Inhibition of multiple MKK signaling pathway suppressed renal cell carcinoma growth and angiogenesis in vivo

5413 Advanced renal cell carcinoma (RCC) is refractory to current therapies. The MKKK/MKK/MAPK signaling pathway plays an essential role in cell proliferation and differentiation. Recent studies also show activation of MAPK signaling pathway in the tumorigenesis, metastasis and angiogenesis of multiple human malignancies including renal cell carcinoma. We evaluated whether targeting this pathway could be of therapeutic value against human renal cell carcinoma. The activation of MAPK signaling pathway and its role in renal cell carcinoma was accessed in vitro in two RCC cell lines by western blot, cell proliferation assay, cell counting, soft agar assay, and TUNEL staining, using anthrax lethal toxin (LeTx) which cleaves and inactivates multiple MKKs, as well as a specific MKK1/2 inhibitor, U0126, to block MKK signaling. In vivo xenograft studies were carried by intravenous or intraperitoneal injection of LeTx into nude mice bearing RCC tumors. Tumor growth was measured and tumors were subjected to immunohistochemical analysis. Western blot showed phosphorylation levels of ERK, JNK and p38 decreased after LeTx treatment. Exposure to LeTx or U0126 for 72h reduced cell growth by up to 40% without induction of apoptosis. Anchorage-independent growth of RCC cells was totally inhibited by LeTx. Importantly, in vivo tumor growth of RCC xenografts could be totally suppressed by LeTx, as well as tumor neovascularization was inhibited by LeTx. Because of the existence of autocrine and paracrine growth factors in tumors, we also tested the effect of LeTx on VEGF release using ELISA method. LeTx could reduce the serum level of mouse VEGF, but had no effect on human VEGF release by RCC cells. Our results suggest that multiple MKK signaling inhibitors may be a potentially useful treatment for patients with renal cell carcinoma.