Identification Of A G Alpha G Beta Gamma, Akt And Pkc Alpha Signalome Associated With Invasive Growth In Two Genetic Models Of Human Breast Cancer Cell Epithelial-To-Mesenchyinal Transition

The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ER alpha and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and downregulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including G alpha G beta gamma subunits, PKC alpha, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27(kip1)/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting G alpha G beta gamma subunits (BIM-46187, gallein), PKC alpha (Go6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin:, alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists G alpha G beta gamma, PKC alpha and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.