PO-217 Tumour suppressor function of the oxidoreductase p66Shc in BRAFV600E-transformed cells

Introduction Elevated levels of reactive oxygen species (ROS) frequently observed in tumours are part of the metabolic reprogramming, which is critical for cancer initiation and progression. However, cancer cells remain sensitive to treatments which further increase or decrease ROS. Tailored modulation of ROS levels thus may become a new strategy in cancer therapy. In contrast to other tumours, melanoma have low ROS levels and we have shown previously that RAF kinases are able to prevent excessive mitochondrial ROS production. To understand the underlying reasons for impaired ROS production in BRAFV600E transformed cells, we studied a possible link to the oxidoreductase p66Shc, a protein that is directly involved in the generation of mitochondrial ROS and frequently overexpressed in tumours. Material and methods Mitochondrial ROS production was analysed in fibroblasts singly transformed by mutant BRAFV600E, the melanoma cell lines A375 and M238 carrying the identical BRAF mutation, and the vemurafenib resistant variant M238R. Mitochondrial ROS production was analysed by microscopic imaging or FACS analysis of MitoTracker Red CM-H2XRos stained cells. Intracellular signalling was monitored through the use of phosphorylation-specific and control antibodies. Results and discussions We show that following treatment with the cell death inducing agent phenethyl isothiocyanate (PEITC) oncogenic BRAFV600E renders cells refractory to phosphorylation of S36 in p66Shc, which is essential for p66Shc activation, mitochondrial translocation and ROS production. Consistent with this observation, the activation of JNK1/2, the kinases phosphorylating S36, as demonstrated by us previously, was blunted, while other MAPKs were not affected. Use of a JNK1/2 inhibitor most efficiently prevented ROS production while inhibition of BRAF or MEK increased ROS levels, without affecting S36 phosphorylation. Melanoma cells, which had become resistant to the mutant BRAF-specific inhibitor vemurafenib, were impaired in S36 phosphorylation and ROS production after PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA-mediated knockdown of p66Shc led to increased colony size of BRAFV600E transformed cells in soft agar. Taken together these data suggest that phoshpo-p66Shc functions as a tumour suppressor in melanoma. Conclusion Our findings demonstrate that interference with cytoplasmic signalling pathways regulating p66Shc activation may be therapeutically exploited to alter ROS levels in tumour cells.