title: SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway.
abstract: Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.
authors.affiliation: Molecular and Population Genetics Laboratory, Imperial Cancer Research Fund, London, United Kingdom. woodford@icrf.icnet.uk
authors.forename: K L
authors.initials: KL
authors.lastname: Woodford-Richens
authors.forename: A J
authors.initials: AJ
authors.lastname: Rowan
authors.forename: P
authors.initials: P
authors.lastname: Gorman
authors.forename: S
authors.initials: S
authors.lastname: Halford
authors.forename: D C
authors.initials: DC
authors.lastname: Bicknell
authors.forename: H S
authors.initials: HS
authors.lastname: Wasan
authors.forename: R R
authors.initials: RR
authors.lastname: Roylance
authors.forename: W F
authors.initials: WF
authors.lastname: Bodmer
authors.forename: I P
authors.initials: IP
authors.lastname: Tomlinson
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D015153:Blotting, Western; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002872:Chromosome Deletion; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D016163:Genes, DCC; D006801:Humans; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D011003:Ploidies; D018807:Polymorphism, Single-Stranded Conformational; D015398:Signal Transduction; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000502:physiology; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta