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

Eur J Cell Biol. 2020 May;99(4):151076. doi: 10.1016/j.ejcb.2020.151076. Epub 2020 May 11.

Abstract

Deregulation of TGF-β signaling is intricately engrossed in the pathophysiology of pancreatic adenocarcinomas (PDACs). The role of TGF-β all through pancreatic cancer initiation and progression is multifarious and somewhat paradoxical. TGF-β 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-β/Smad4 pathway, wherein, Par-4 induction/over-expression induced EMT which was later culminated in to apoptosis in presence of TGF-β via positive regulation of Smad4. Intriguingly, Par-4-/- cells were devoid of significant Smad4 induction compared to Par-4+/+ cells in presence of TGF-β and ectopic Par-4 steadily augmented Smad4 expression by restoring TGF-β/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-β 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-β dependent lethal EMT program. The mechanistic relevance of Par-4 mediated Smad4 activation was additionally validated by co-immunoprecipitation wherein disruption of NM23H1-STRAP interaction by Par-4 rescues TGF-β/Smad4 pathway in PDAC and mediates the tumor suppressive role of TGF-β, therefore serving as a vital cog to restore the apoptotic functions of TGF-β pathway.

Keywords: Lethal EMT; NM23H1; Par-4; TGF-β/Smad4.

MeSH terms

  • Carcinoma, Pancreatic Ductal / metabolism*
  • Carcinoma, Pancreatic Ductal / pathology
  • Cell Line, Tumor
  • Epithelial-Mesenchymal Transition
  • G1 Phase Cell Cycle Checkpoints
  • Humans
  • NM23 Nucleoside Diphosphate Kinases / genetics
  • NM23 Nucleoside Diphosphate Kinases / metabolism
  • Pancreatic Neoplasms / metabolism*
  • Pancreatic Neoplasms / pathology
  • Plasmids / genetics
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA, Small Interfering / administration & dosage
  • RNA, Small Interfering / genetics
  • RNA-Binding Proteins / metabolism
  • Receptors, Thrombin / genetics
  • Receptors, Thrombin / metabolism*
  • Signal Transduction
  • Smad4 Protein / biosynthesis
  • Smad4 Protein / genetics
  • Smad4 Protein / metabolism*
  • Transforming Growth Factor beta / metabolism*
  • Up-Regulation

Substances

  • NM23 Nucleoside Diphosphate Kinases
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Receptors, Thrombin
  • SMAD4 protein, human
  • STRAP protein, human
  • Smad4 Protein
  • Transforming Growth Factor beta
  • Proto-Oncogene Proteins c-akt
  • NME1 protein, human
  • protease-activated receptor 4