Inactivation of both alleles of the DPC4/SMAD4 gene in advanced colorectal cancers: identification of seven novel somatic mutations in tumors from Japanese patients

Abstract

Loss of heterozygosity (LOH) of loci on chromosome 18q occurs in a majority of colorectal cancers. The DPC4/SMAD4 gene, lying in close proximity to the DCC gene at 18q21.1, was recently identified as a candidate tumor suppressor for the genesis of pancreatic cancer as well as a predisposing gene for Juvenile Polyposis Syndrome (JPS). The gene product functions as a cytoplasmic mediator in the signaling pathway of transforming growth factor beta (TGF-β). To investigate the potential role of DPC4/SMAD4 gene in colorectal cancers, we examined 73 tumors of clinical stages II or III from Japanese patients, for LOH at 18q21 and also for subtle mutations anywhere within the coding region of DPC4/SMAD4. LOH was identified in 50 (78%) of the 64 tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in seven of those tumors: two frameshift mutations, a 1-bp deletion (326 del T) in exon 8 and a 1-bp insertion (50–51 ins A) in exon 1; two nonsense mutations, Arg445Ter in exon 10 and Glu538Ter in exon 11; and three missense mutations, Asn129Lys in exon 2, Tyr95Asn in exon 2, and Asp355Glu in exon 8. Three of the seven mutations were observed in the mad homology 1 (MH1) domain encoded by exons 1 and 2. In all of the tumors carrying intragenic mutations of one allele, LOH analysis had shown that the other allele was missing. The results demonstrated that inactivation of both alleles of the DPC4/SMAD4 gene occurs in a substantial proportion of advanced colorectal cancers, and that the DPC4/SMAD4 gene probably exerts a tumor-suppressor effect for colorectal carcinogenesis that fulfills the criterion of the two-hit concept proposed by Knudson [A.G. Knudson, Hereditary cancer, oncogenes, and anti-oncogenes, Cancer Res. 45 (1985) 1437–1443.].

Introduction

The multiple genetic alterations associated with colorectal carcinomas are among the best understood for any common human cancer. Among these,tumor suppressor genes are defined when both alleles have been inactivated in a tumor 1, 2, 3, 4, 5, 6, 7. This `double hit' is often accomplished by an intragenic mutation in one allele that occurs either before or after a chromosomal region containing the other allele is lost during an aberrant recombination event; the latter type of alteration can be detected by loss of heterozygosity (LOH) for DNA markers in the region. We have previously observed frequent LOH at 18q21 in human breast and prostate cancers 8, 9. Others have described frequent LOH at 18q21 also in carcinomas of the pancreas, ovary, colon, brain, and lung 10, 11, 12, 13, 14, 15.

The DCC gene and a nearby gene, DPC4/SMAD4, have been identified as candidate tumor suppressors at 18q21. A decrease in expression of the DCC gene and allelic losses have been correlated with lymphatic invasion and hepatic metastasis of colorectal cancers [16]. Patients with stage II colorectal cancers showing LOH at DCC are considered to have poorer prognoses than patients with more advanced stage III cancers not having LOH at DCC [17]. However, although the DCC gene might play some role in progression of colorectal cancers, the frequency of LOH on 18q in such tumors does not correlate simply with low frequency of mutations in the DCC gene [18].

DPC4/SMAD4 gene encodes a cytoplasmic mediator in the signaling pathway of transforming growth factor beta (TGF-β) 19, 20, 21, 22, a pathway mediates growth-inhibitory signals from the cell surface to the nucleus. Somatic mutations in DPC4/SMAD4 have been reported in approximately half of the pancreatic cancers examined, and to a lesser extent in variety of other cancers 23, 24, 25. Recently, germline mutations in DPC4/SMAD4 were identified in some families affected with Juvenile Polyposis Syndrome (JPS) [26], an inherited condition characterized by predisposition to hamartomatous polyps and cancers of the gastrointestinal tract. However, the polyps in JPS, being composed largely of stromal cells, are less likely to become malignant than epithelium-rich adenomatous polyps. This discovery has raised fundamental questions about the role of, and the relationship between, neoplastic cells and the stromal cells that together constitute a colorectal-tumor mass. Thus, the contribution of the DPC4/SMAD4 gene to colorectal carcinogenesis has become a matter of growing interest.

To determine the role of DPC4/SMAD4 alterations in the development and/or progression of colorectal tumors, we examined this gene for mutations and allelic losses in 73 advanced colorectal cancers. We report evidence that both alleles of the DPC4/SMAD4 gene have been inactivated in about 10% of those tumors.