Smad4/DPC4 silencing and hyperactive Ras jointly disrupt transforming growth factor-beta antiproliferative responses in colon cancer cells

Smad4/DPC4 is a tumor suppressor gene frequently mutated or deleted in panc reatic and metastatic colon cancers. Smad4 acts as a cofactor that binds tr ansforming growth factor-beta (TGF-beta) receptor-activated Smad2 and Smad3 generating transcriptional complexes. Using SW480.7 colon carcinoma cells, defective in Smad4 function, we have investigated whether this loss plays a role in the resistance of colon cancer cells to the antiproliferative eff ects of TGF-beta. SW480.7 cells contain only one Smad4 allele, which we fou nd encodes a wild type protein that is not expressed. We generated SW480.7 cells conditionally expressing Smad4 via an ecdysone-inducible system. Smad 4 expression in these cells failed to rescue TGF-beta antiproliferative and gene responses (c-myc down-regulation and induction of p21/Cip1 and plasmi nogen activator inhibitor-1). SW480.7 cells contain an activated Ki-ras onc ogene. Hyperactivation of Ras can inhibit Smad nuclear accumulation by thei r phosphorylation at mitogen-activated protein kinase sites. Co-transfectio n into SW480.7 cells of Smad4 together with a Ras phosphorylation-resistant Smad3 (but not with wild type Smad2, Smad3, adenomatous polyposis coli (AP C), or TGF-beta type II receptor) restored the TGF-beta antiproliferative r esponse. These results suggest that loss of Smad4 function by both deletion and silencing and inhibition of Smad2/3 function by a hyperactive Ras path way jointly prevent TGF-beta antiproliferative responses in SW480.7 colon c ancer cells.