In vivo effects in melanoma of ROCK inhibition-induced FasL over-expression

Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes, and tumor rejection though inflammatory and immune responses. Our team has previously shown that RhoA GTPase and its effector ROCK kinases negatively control FasL membrane expression in murine melanoma B16F10 cells.In this study we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane over-expression. Although H1152 treatment did not reduce tumor growth rate in vitro, the inhibitor delayed tumor appearance and slowed tumor growth in C57BL/6 immunocompetent mice in vivo. Thanks to the use of mice bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL over-expression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFNγ-KO mice highlighted a major role for T lymphocytes and IFNγ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intra-tumoral infiltration of lymphocytes. Cytofluorometric analysis showed that these were mainly activated CD8 lymphocytes, which were responsible for the reduction in tumor growth. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation, but this effect was not dependent on the T cell-mediated immune response.Altogether our data show that B16F10 melanoma cell treatment with ROCK kinase inhibitors, reduces tumor growth and metastasis development and through increased FasL expression, promotes an anti-tumor immune response. These inhibitors could therefore become interesting pharmacological molecules for melanoma immunotherapy

Statins reduce melanoma development and metastasis through MICA overexpression

Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MICA membrane expression. PPARγ is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin-treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments