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, DPC4͞ SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-␤ 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 and͞or 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 aneuploid͞polyploid, 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. For many years the allele loss observed around 18q21.1 in colorectal cancer was believed to be targeting DCC (2, 3). It has more recently been shown, however, that mutations of DCC and SMAD2 are very rare in colorectal cancer (4). By contrast, changes in SMAD4 are much more common in the pathogenesis and evolution of colorectal cancer (5, 6), as has also been shown in pancreatic cancer (7). In particular, inactivation of SMAD4 has been associated with late stage or metastatic colorectal cancer (6,8,9). Additionally, it has recently been shown that constitutional mutations in SMAD4 can cause Juvenile Polyposis Syndrome (JPS; refs. 10 and 11), a disorder characterized by the presence of hamartomatous polyps in the gastrointestinal tract and a markedly increased risk of developing a gastrointestinal malignancy.The SMAD4 gene codes for a protein involved as a downstream regulator in the transforming growth factor ␤ (TGF-␤) signal transduction pathway. The SMAD4 protein acts as a trimer and forms complexes with the receptor-phosphorylated SMAD2 and SMAD3; these heteromeric complexes then translocate from the cytoplasm to the nucleus where association with DNA binding factors facilitates the transcription of ...

Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells

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Loss of expression, and mutations of Smad 2 and Smad 4 in human cervical cancer

“…Suitable PCR primers were designed for Smad 4 to amplify the exon regions of both the functional domains along with the 3 0 and 5 0 intron regions that are important for splicing (Woodford-Richens et al, 2001). RT-PCR primers were designed to cover the important functional regions of Smad 2 and variations in the expression of Smads were analysed by SQRT-PCR.…”

“…Consistent with the low expression of Smad 2 in all of the tissue samples (premalignant and SCC) observed by us in human cervical cancer, loss of expression of Smad 2 was reported in head and neck squamous cell carcinoma by immunohistochemical analysis (MuroCacho et al, 2001). Homozygous deletions of Smad 4 were reported in pancreatic cancer and a 9 bp deletion of Smad 2 was reported in a human lung cancer cell line (Uchida et al, 1996), but in other cancers and other Smads usually the mutations reported are point mutations (MacGrogan et al, 1997;Miyaki et al, 1999;Woodford-Richens et al, 2001).…”

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations

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