Regulating SQSTM1/P62 and Wnt/β-catenin signaling of EMT to constrain TNBC invasiveness.

Abstract: Purpose: The prevalence of cells with mesenchymal features is one of the well-known characteristics of triple negative breast cancers (TNBCs); where cells have undergone epithelial to mesenchymal transition (EMT) and are found to be poorly differentiated, giving rise to cancer stem cells (CSCs). Wnt/β-catenin and Wnt/PCP signaling pathways are prominent contributor of EMT, stemness and CSC properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT. Understanding these regulatory links proves to be an instrumental approach to prohibit progression of TNBC. Methods: siRNA targeting SQSTM1/P62 and inhibitor of Wnt/β-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Flow cytometric assays reveled CSC and apoptotic population whereas migration and invasion assay assetred alteration of the metastatic property. Genes and proteins involved were studied at molecular level employing confocal microscopy, western blotting and qRT-PCR. Results: Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Co-treatment reduced the colony formation and self-renewal properties. Furthermore, invasion assay revealed 5.22-fold downregulations of the metastatic property. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis and depletion of p-AKT and p-STAT-3. Conclusions: The synergistic impact of inhibition of SQSTM1/P62 and Wnt/β-catenin signaling facilitates the development of a potential theraputic regimen for TNBC.