Par-4 mediated Smad4 induction in PDAC cells restores canonical TGF-β/Smad4 axis driving the cells towards lethal EMT

Deregulation of TGF-beta signaling is intricately engrossed in the pathophysiology of pancreatic adenocarcinomas (PDACs). The role of TGF-beta all through pancreatic cancer initiation and progression is multifarious and somewhat paradoxical. TGF-beta plays a tumor suppressive role in early-stage pancreatic cancer by promoting apoptosis and inhibiting epithelial cell cycle progression, but incites tumor promotion in late-stage by modulating genomic instability, neo-angiogenesis, immune evasion, cell motility, and metastasis. Here, we provide evidences that Par-4 acts as one of the vital mediators to regulate TGF-beta /Smad4 pathway, wherein, Par-4 induction/overexpression induced EMT which was later culminated in to apoptosis in presence of TGF-beta via positive regulation of Smad4. Intriguingly, Par-4(-/-) cells were devoid of significant Smad4 induction compared to Par-4(-/-) cells in presence of TGF-beta and ectopic Par-4 steadily augmented Smad4 expression by restoring TGF-beta /Smad4 axis in Panc-1 cells. Further, our FACS and western blotting results unveiled that Par-4 dragged the PDAC cells to G1 arrest in presence of TGF-beta byelevating p21 and p27 levels while attenuating Cyclin E and A levels and augmenting caspase 3 cleavage triggering lethal EMT. Through restoration of Smad4, we further establish that in BxPC3 cell line (Smad4(-/-)), Smad4 is essential for Par-4 to indulge TGF-beta dependent lethal EMT program. The mechanistic relevance of Par-4 mediated Smad4 activation was additionally validated by co-immunoprecipitafion wherein disruption of NM23H1-STRAP interaction by Par-4 rescues TGF-beta /Smad4 pathway in PDAC and mediates the tumor suppressive role of TGF-5, therefore serving as a vital cog to restore the apoptotic functions of TGF-beta pathway.