Molecular and Cellular Pathobiology SMAD 2 , SMAD 3 and SMAD 4 Mutations in Colorectal Cancer

Activation of the canonical TGF-b signaling pathwayprovides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but towhat extentmutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP)microarray analysis.Mutation spectrawere compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4mutations were associated with mucinous histology. Themutation spectra of SMAD2 and SMAD3were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-XSer motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2–SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented andmutually exclusive to SMAD4mutation, underlining the critical roles of these three proteins within the TGF-b signaling pathway. Cancer Res; 73(2); 725–35. 2012 AACR.