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 pancreatic and metastatic colon cancers. Smad4 acts as a cofactor that binds transforming 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 effects of TGF-beta. SW480.7 cells contain only one Smad4 allele, which we found encodes a wild type protein that is not expressed. We generated SW480.7 cells conditionally expressing Smad4 via an ecdysone-inducible system. Smad4 expression in these cells failed to rescue TGF-beta antiproliferative and gene responses (c-myc down-regulation and induction of p21/Cip1 and plasminogen activator inhibitor-1). SW480.7 cells contain an activated Ki-ras oncogene. Hyperactivation of Ras can inhibit Smad nuclear accumulation by their phosphorylation at mitogen-activated protein kinase sites. Co-transfection into SW480.7 cells of Smad4 together with a Ras phosphorylation-resistant Smad3 (but not with wild type Smad2, Smad3, adenomatous polyposis coli (APC), or TGF-beta type II receptor) restored the TGF-beta antiproliferative response. These results suggest that loss of Smad4 function by both deletion and silencing and inhibition of Smad2/3 function by a hyperactive Ras pathway jointly prevent TGF-beta antiproliferative responses in SW480.7 colon cancer cells.
Calonge
M J
MJ
Cell Biology Program, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Massagué
J
J
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000502:physiology*; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D018631:ras Proteins