Activation of the canonical TGF-b signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations 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 spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations 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. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were 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 and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-b signaling pathway. Cancer Res; 73(2); 725-35. Ó2012 AACR.

BackgroundPrevalence of colorectal cancer (CRC) in the British Bangladeshi population (BAN) is low compared to British Caucasians (CAU). Genetic background may influence mutations and disease features.MethodsWe characterized the clinicopathological features of BAN CRCs and interrogated their genomes using mutation profiling and high-density single nucleotide polymorphism (SNP) arrays and compared findings to CAU CRCs.ResultsAge of onset of BAN CRC was significantly lower than for CAU patients (p=3.0 x 10-5) and this difference was not due to Lynch syndrome or the polyposis syndromes. KRAS mutations in BAN microsatellite stable (MSS) CRCs were comparatively rare (5.4%) compared to CAU MSS CRCs (25%; p=0.04), which correlates with the high percentage of mucinous histotype observed (31%) in the BAN samples. No BRAF mutations was seen in our BAN MSS CRCs (CAU CRCs, 12%; p=0.08). Array data revealed similar patterns of gains (chromosome 7 and 8q), losses (8p, 17p and 18q) and LOH (4q, 17p and 18q) in BAN and CAU CRCs. A small deletion on chromosome 16p13.2 involving the alternative splicing factor RBFOX1 only was found in significantly more BAN (50%) than CAU CRCs (15%) cases (p=0.04). Focal deletions targeting the 5’ end of the gene were also identified. Novel RBFOX1 mutations were found in CRC cell lines and tumours; mRNA and protein expression was reduced in tumours.ConclusionsKRAS mutations were rare in BAN MSS CRC and a mucinous histotype common. Loss of RBFOX1 may explain the anomalous splicing activity associated with CRC.

Purpose: PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial.Experimental Design: A total of 1,093 stage I-IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12-13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n ¼ 5,594).Results: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status.Conclusion: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAF mut ), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRAS mut ) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes.