TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells

SIMPLE SUMMARY: Prostate cancer is the second leading cause of cancer death among men. Its poor prognosis is mainly due to metastases, and therefore, it is crucial to understand the mechanisms by which cancer cells can spread throughout the body. In this context, among others, we demonstrated the TRPM8 channel to be a major player due to its ability to impair prostate cancer cell motility. The present work elucidates the molecular mechanism through which TRPM8 exerts its inhibitory effect on cell migration. We found that this effect is mediated by a direct interaction between residues E207 and Y240 in the N-terminal TRPM8 tail and residue Y32 in the Switch I region of the Rap1-GDP bound inactive form. Since these results have also been validated in breast and cervical cancer cells, the TRPM8-Rap1 interaction could constitute a therapeutic target against metastatic progression in several types of tumors. ABSTRACT: Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.