Abstract 5159: Smad7 knockdown in colon cancer cells activates protein kinase RNA-associated eIF2α pathway thereby leading to cell death

Abstract

Background. Up-regulation of Smad7, an inhibitor of TGF-β1, occurs in sporadic colorectal cancer (CRC). Knockdown of Smad7 with a specific antisense oligonucleotide (AS) leads to activation of eIF2α, an attenuator of protein synthesis, and arrest of CRC cells in the S phase of the cell cycle with the downstream effect of inducing cell death.

Aim. To investigate the mechanisms by which Smad7 knockdown activates eIF2α.

Methods. Phosphorylation of eIF2α was evaluated in CRC cell lines (i.e. HCT116 and DLD-1) either untreated or treated with Smad7 sense (S) or AS by Western blotting (WB) and immunofluorescence (IF). Expression of ATF4 and CHOP, two downstream targets of EIF2α, were evaluated by IF and activation of PKR, GCN2 and PERK, up-stream kinases that induce eIF2α phosphorylation, was assessed by WB. Wild type or PKR-deficient CRC cells treated with Smad7 AS were monitored for eIF2α activation and induction of death. Finally, we assessed whether enhanced phosphorylation of eIF2α seen in cells treated with Smad7 AS was also associated with reduced interaction between eIF2α and PP1, a phosphatase that normally dephosphorylates eIF2α.

Results. Smad7 knockdown increased ATF4 and CHOP expression thus confirming previous data showing activation of eIF2α phosphorylation in CRC cells treated with Smad7 AS. Among kinases that induce eIF2α phosphorylation, only PKR was activated by Smad7 knockdown. Consistently, silencing of PKR reduced but did not abolish Smad7 AS-induced eIF2α phosphorylation and cell death, thus suggesting the existence of further mechanisms that control eIF2α phosphorylation in Smad7-deficient cells. Indeed, in CRC cells, Smad7 interacted with PP1 and Smad7 knockdown reduced association of PP1 with eIF2α.

Conclusions

Data show that Smad7 is involved in CRC cell survival and suggest that Smad7 is a valid target for therapeutic intervention in CRC.

Citation Format: Veronica De Simone, Gerolamo Bevivino, Silvia Sedda, Roberta Izzo, Massimo Claudio Fantini, Giovanni Monteleone. Smad7 knockdown in colon cancer cells activates protein kinase RNA-associated eIF2α pathway thereby leading to cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5159.