Genetic alterations of the TGF-β signaling pathway in colorectal cancer cell lines: A novel mutation in Smad3 associated with the inactivation of TGF-β-induced transcriptional activation

To investigate genetic alterations involved in the TGF-β signaling pathway in colorectal cancer, we assayed DNA synthesis rates after treating TGF-β and checked for genetic alterations in TGF-βRII, TGF-βRI, Smad2, Smad3, and Smad4 in 12 colorectal cancer cell lines. Eleven lines, except SNU-61, show no significant change in DNA synthesis rate after TGF-β treatment. In these 11 lines, several mutations were found in genes involved in the TGF-β signaling pathway: (i) frameshift deletions in the poly(A)₁₀ tract of the TGF-βRII gene in SNU-407, SNU-769A, SNU-769B, and SNU-1047 cell lines, (ii) a missense mutation of Smad2 (R321Q) in SNU-81, (iii) two missense mutations in TGF-βRI (R487W in SNU-175 and A202V in SNU-1040), and (iv) a monoallelic loss at the Smad4 locus in three cell lines. Interestingly, a missense mutation (R373H) in Smad3 gene was found in SNU-769A. To our knowledge, this is the first report of Smad3 mutation in human malignancy. This mutation was found to result in the inhibition of translocation of Smad3 protein to the nucleus and a reduction in the activity of Smad3 during TGF-β-induced transcriptional activation. These results indicate that the majority of cell lines, which are insensitive to TGF-β, have alterations in genes involved in the TGF-β signaling pathway in colorectal cancer cell lines.