Starvation-inactivated MTOR triggers cell migration via a ULK1-SH3PXD2A/TKS5-MMP14 pathway in ovarian carcinoma

SH3PXD2A/TKS5 (SH3 and PX domains 2A) is a scaffold protein that promotes invadopodia formation and regulates cell migration; therefore, the overexpression of SH3PXD2A has been reported in various cancers. However, the molecular mechanisms of the SH3PXD2A-mediated cellular migration signaling pathway remain unknown. Here, we showed that the starvation-induced macroautophagy/autophagy or treatment with the MTOR inhibitor RAD001 elevated SH3PXD2A expression in ovarian cancer cell lines. SH3PXD2A formed a complex with ULK1 (unc-51 like autophagy activating kinase 1) and MTOR, as revealed by co-immunoprecipitation assay. Furthermore, ULK1 affected protein stability by phosphorylating SH3PXD2A at serine residues 112, 142, 146, 147, 175, and 348. Mutation of these six residues in SH3PXD2A reduced ULK1-mediated phosphorylation, blocked SH3PXD2A induction by treatment with RAD001, and reduced its binding to the cell membrane phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) versus recruitment of MMP14 (matrix metallopeptidase 14) in ovarian cancer cells. Finally, the administration of RAD001 induced SH3PXD2A expression in tumor tissues, as revealed by the PDX mouse model. The clinical impact of SH3PXD2A was evaluated in ovarian and endometrial cancers using western blotting. The negative correlation between MTOR-mediated phospho-ULK1 and SH3PXD2A proteins was found in clinical specimen. Furthermore, the TCGA database revealed that the cumulative overall survival of ovarian cancer patients with higher SH3PXD2A RNA/protein expression in tumor lesions was reduced compared to those with lower SH3PXD2A expression. Our results suggest that the ULK1-SH3PXD2A-MMP14 axis might regulate the biological aggressiveness of ovarian cancer and serve as a therapeutic target in this malignancy. Abbreviations: AMPK: AMP-activated protein kinase; CHX: cycloheximide; RAD001: everolimus; HBSS: Hanks’ balanced salt solution; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; MMP14: matrix metallopeptidase 14; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology; SH3: Src homology 3; SH3PXD2A/TKS5: SH3 and PX domains 2A; SH3PXD2A-[6A]: S112A S142A S146A S147A S175A S348A mutant; ULK1: unc-51 like autophagy activating kinase 1.