SMAD2/3 mediate oncogenic effects of TGF-ß in the absence of SMAD4

The tumor suppressive effects of TGF-ß are classically associated with the activation of the “canonical” SMAD-mediated pathway, whereas its oncogenic effects are largely attributed to its “non-canonical signaling”. We herein provide evidence of an oncogenic effect for SMAD2 and 3 in response to TGF-ß in SMAD4-null cancer cells. Using the CRISPR/Cas9 technology, we report that simultaneous knockout of Smad2 and 3 in Smad4-negative pancreatic ductal adenocarcinoma (PDAC) cells compromises TGF-ß-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-ß gene signature related to aggressiveness mediated by SMAD2 and 3 in the absence of SMAD4. Using PDAC patients cohorts, we reveal that SMAD4-negative tumors with high levels of (phospho)-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC is associated with oncogenic gain-of-function of SMAD2 and 3 and the onset of associated deleterious effects. Overall design: SMAD2 and SMAD3 were inactivated in BxPC3 cells via Crispr-Cas9. Control and KO cells were then divided in two groups (4 replicates in each group), one treated with TGFB and one not. The experiment was conducted for 1h or 24h of treatment.