Chinigò Giorgia, Grolez Guillaume P, Audero Madelaine, Bokhobza Alexandre, Bernardini Michela, Cicero Julien, Toillon Robert-Alain, Bailleul Quentin, Visentin Luca, Ruffinatti Federico Alessandro, Brysbaert Guillaume, Lensink Marc F, De Ruyck Jerome, Cantelmo Anna Rita, Fiorio Pla Alessandra, Gkika Dimitra
Department of Life Sciences and Systems Biology, University of Torino, 10123 Torino, Italy.
INSERM, U1003-PHYCEL-Physiologie Cellulaire, University of Lille, F-59000 Lille, France.
Cancers (Basel). 2022 Apr 30;14(9):2261. doi: 10.3390/cancers14092261.
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.
新出现的证据表明，瞬时受体电位香草酸亚型8（TRPM8）通道通过损害前列腺癌细胞的运动性，在前列腺癌（PCa）进展中发挥重要作用。在此，我们揭示了通过该通道与小GTP酶Rap1A的直接蛋白质-蛋白质相互作用（PPI）来控制PCa运动性的一个新方面。通过活性Rap1下拉实验和活细胞成像实验评估了这两种蛋白质的功能相互作用。分子建模分析确定了参与TRPM8-Rap1A相互作用的四个假定残基。如GST下拉、免疫共沉淀和PLA实验所示，这些位点的点突变损害了PPI，并揭示了它们在PC3前列腺癌细胞粘附和迁移中的关键功能作用。更确切地说，TRPM8通过将Rap1A捕获在其GDP结合的无活性形式中，从而抑制其在质膜上的激活，进而抑制细胞迁移和粘附。特别是，TRPM8序列中的E207和Y240残基以及Rap1A序列中的Y32残基不仅对PC3细胞，而且对宫颈（HeLa）和乳腺（MCF-7）癌细胞中两种蛋白质之间的相互作用都至关重要。这项研究加深了我们对TRPM8在癌症进展中发挥保护作用机制的认识，并为将TRPM8作为癌症治疗新的治疗靶点的可能应用提供了新的见解。
