MAD-related Genes on 18q21.1, Smad2 and Smad4, Are Altered Infrequently in Esophageal Squamous Cell Carcinoma

The MAD (mothers against decapentaplegic)-related genes, Smad2 (former name MA DR2 or JV18-1 ) and Smad4 (former name DPC4 ), have been identified on chromosome 18q21.1, We analyzed 30 primary esophageal squamous cell carcinomas (ESCC) and 7 cell lines derived from ESCC for intragenic mutations and loss of heterozygosity (LOH) of the Smad2 and Smad4 genes. LOH was detected in 5 of 14 (35%) informative cases. However, no mutations in either gene were detected in either the primary carcinomas or the cell lines, and only a G-to-A base transition within the 3'-untranslated region of the Smad4 gene was observed in a carcinoma. There were no homozygous deletions in either of the genes in the cell lines. MAD -related genes on chromosome 18q21.1 are altered infrequently in ESCC.     

Mutational analysis of TGF-beta type II receptor,Smad2, Smad3, Smad4, Smad6 and Smad7 genes in colorectal cancer

Transforming growth factor-beta(TGF-beta) is known to play an important role in controlling embryonal development, cell proliferation and homeostasis. The purpose of this study is to elucidate the involvement of the TGF-beta pathway in colorectal carcinogenesis. DNA was extracted from 100 patients with colorectal cancer. Then, all coding regions of the TGF-beta type II receptor (TRII) and the genes for Smad2, Smad3, Smad4, Smad6, and Smad7 were analyzed by PCR-SSCP and direct sequencing. Also, a LOH analysis of 18q21, where the Smad2 and Smad4 genes are located, was performed. We detected 11 cases of frameshift mutation in the TRII gene (11%) and 5 cases of point mutations in the Smad4 gene (5.0%); LOH at 18q21 was detected with 33% frequency. No abnormalities were found in the genes for Smad2, Smad3, Smad6, and Smad7. These results suggest that the abnormalities of TRII and Smad4 play an important role inhibiting TGF-beta signaling in colorectal carcinogenesis.     

Frequent microsatellite instability in primary esophageal carcinoma associated with extraesophageal primary carcinoma

Patients with esophageal squamous cell carcinoma (ESCC) frequently develop other primary cancers, such as gastric cancer and head and neck cancer. Details of carcinogenesis in patients with multiple primaries that include esophageal carcinoma with other primary carcinoma (ECOPC) remain uncertain. We examined microsatellite instability (MSI) status, frameshift mutation in target genes of MSI, mismatch repair protein expression and hypermethylation of the hMLH1 promoter region in ECOPC patients to better understand the underlying carcinogenic processes. High frequency MSI (MSI-H) was found in 15 (44.1%) of 34 patients with ECOPC, but in only 6 (14.3%) of 42 patients with esophageal cancer alone (p < 0.01). Frameshift mutations in TGFbetaRII, BAX, MSH3 and MSH6 genes respectively were present in 4, 1, 2 and 2 of 34 ECOPC patients. Immunohistochemical study showed that 12 (80.0%) of 15 MSI-H tumors showed loss of expression of either hMLH1 or hMSH2. In addition, 6 of 9 tumors (66.7%) that showed reduced hMLH1 expression also had hypermethylation of the hMLH1 promoter region. Our findings suggested that carcinogenesis in ECOPC was closely associated with the MSI pathway because of mismatch repair protein deficiency.     

Allelotype analysis of the PTEN,Smad4 and DCC genes in biliary tract cancer

Aberrations of the PTEN, Smad4 and DCC genes have not been determined in biliary tract cancers. We performed allelotype analysis to screen for alterations of these genes. We looked for the presence of allelic imbalance (AI) at the PTEN and Smad4 genes in extrahepatic bile duct (EHBD) and ampullary cancers using polymorphic microsatellite markers. These tumors were also examined for AI at the DCC gene using polymerase chain reaction amplification of variable numbers of tandem repeats. AI at the PTEN, Smad4 and DCC genes was observed in 5.3%, 8.3% and 20.7%, respectively, of EHBD tumor cases. AI at the PTEN, Smad4 and DCC genes was detected in 13.3%, 50% and 8.3%, respectively, of ampullary cancer cases. Our results suggest that (a) alteration of the Smad4 gene is a major factor in the development of ampullary cancer and (b) PTEN, Smad4 and DCC genes are altered infrequently in EHBD cancers.     

Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis.

We have previously detected an increased frequency of loss of heterozygosity (LOH) on chromosome 18q during progression of colorectal carcinomas. To clarify the target of 18qLOH, mutation of Smad4 and Smad2 genes was analysed in 176 colorectal tumors with different stages, including liver metastasis, from 111 sporadic, 52 familial adenomatous polyposis (FAP) and nine hereditary nonpolyposis colorectal cancer (HNPCC) patients. Mutation of other Smad gene families in the TGF-beta signaling pathway was also examined. Twenty-one Smad4 mutations and one Smad2 mutation were detected, whereas mutation of Smad3, 6 and 7 genes was not detected. Smad4 mutations included seven frameshift, one inframe deletion, four nonsense and nine missense mutations, 95% of which resulted in alteration of Smad4 protein regions included in homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44 (7%) in primary invasive carcinoma without distant metastasis, 6/17 (35%) in primary invasive carcinoma with distant metastasis, and 11/36 (31%) in distant metastasis (metastatic/non-metastatic: P=0.006 approximately 0.01). Loss of the other allele was observed in 19 of 20 (95%) invasive and metastasized carcinomas with Smad4 mutations. In four cases both primary and metastasized carcinomas in the same patients showed the same mutations. The present results suggest that Smad4 gene is one of true targets of 18qLOH, and that its inactivation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis.     

Loss of the Smad3 expression increases susceptibility to tumorigenicity in human gastric cancer

Loss of the tumor suppressive effect of transforming growth factor-beta (TGF-beta) has been commonly found at later stages in carcinogenic progression. Although the genes encoding TGF-beta receptors and Smads have been found genetically altered in certain human cancers, no mutation in Smad3 has been observed. Therefore, suppression of Smad3 expression may mediate key oncogenic properties of TGF-beta. First, we observed that 37.5% of human gastric cancer tissues showed low to undetectable levels of Smad3 and that in nine human gastric cancer cell lines examined, two showed deficient Smad3 expression. Introduction of Smad3 into human gastric cancer cells that did not express Smad3, restored TGF-beta responsiveness: induction of p21 and p15 gene expression, and growth inhibition in response to TGF-beta. Furthermore, these Smad3-expressing cells showed markedly decreased and delayed tumorigenicity in vivo. These findings suggest that Smad3 expression may have a critical role in tumor suppression in the early stages of gastric carcinogenesis.     

Mutation analysis of the Smad6 and Smad7 gene in human ovarian cancers

The Smad6 and Smad7 genes are members of the Smad family, involved in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad6 and Smad7 gene mutations in 30 human ovarian cancers and 4 ovarian cancer cell lines, and found that 12 cases (35.3%) had a polymorphism in intron 2 of the Smad6 gene and that 8 cases (23.5%) had a polymorphism at codon 208 in the Smad7 gene. Because these polymorphisms were not accompanied by amino acid substitution, the present results show that the mutations in the Smad6 and Smad7 genes are unlikely to be involved in human ovarian cancers.     

三种食管鳞癌细胞系的染色体异常研究

目的:分析食管鳞癌(ESCC)细胞系的染色体异常,为将来寻找食管癌相关基因提供线索。方法:采用比较基因组杂交法(CGH)分析3种ESCC细胞系的染色体DNA拷贝数改变情况。结果:9q(3/3)、3q(2/3)、5q(2/3)、5p(2/3)、8q(2/3)、12p(2/3)和20q(2/3)为常见的染色体增加区。4q(2/3)和6q(2/3)是常见的染色体丢失区。结论:这些常见的染色体异常有助于寻找和定位食管癌相关基因。     

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.     

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.     

Proteolytic degradation of Smad4 in extracts of AML blasts

Loss of transforming growth factor (TGF) β signaling has been implicated in malignant transformation of various tissues. To investigate a potential role of Smad4 in acute myeloid leukemia (AML), the expression of Smad4 was determined in blast cells from AML patients. Western analysis of nuclear extracts of nine AML samples indicated the absence of Smad4 protein in two cases. Smad4 RT-PCR analysis of these cases indicated normal Smad4 mRNA expression, and sequencing of one of these cases revealed no mutations as compared to wild type Smad4. Next, it was investigated whether Smad4 protein from these AML cases was subject to proteolytic degradation by incubating cell extracts of these Smad4-negative AML cells with extracts from COS-7 cells in which a tagged Smad4 was overexpressed. Inhibitor studies indicated that the extracts of AML blasts lacking Smad4 possessed a serine-dependent proteolytic activity, capable of degrading Smad4. Transfection studies using an SBE containing reporter construct as well as RT-PCR analysis of endogenous TGFβ1 responsive genes indicated that the AML blasts were still able to respond to TGFβ1, despite the observed degradation of Smad4. It was, therefore, concluded that the degradation of Smad4 was possibly AML subtype-dependent, in vitro phenomenon, occurring during the preparation of nuclear and cellular extracts despite the addition of a protease inhibitor cocktail. The results indicate that care should be taken when interpreting data obtained from protein expression studies using AML blast cells.     

Profiling of differentially expressed cancer-related genes in esophageal squamous cell carcinoma (ESCC) using human cancer cDNA arrays: overexpression of oncogene MET correlates with tumor differentiation in ESCC.

PURPOSE: To examine the global gene expression of cancer-related genes in esophageal squamous cell carcinoma (ESCC) through the use of Atlas Human Cancer Array membranes printed with 588 well-characterized human genes involved in cancer and tumor biology. EXPERIMENTAL DESIGN: Two human ESCC cell lines (HKESC-1 and HKESC-2) and one morphologically normal esophageal epithelium tissue specimen from the patient of which the HKESC-2 was derived were screened in parallel using cDNA expression arrays. The array results were additionally validated using semiquantitative PCR. The overexpression of oncogene MET was studied more extensively for its protein expression by immunohistochemistry in the two ESCC cell lines and their corresponding primary tissues and 61 primary ESCC resected specimens. Sixteen of these 61 ESCC cases also had available the corresponding morphologically normal esophageal epithelium tissues and were also analyzed for MET expression. The clinicopathological features associated with overexpression of the MET gene were also correlated. RESULTS: The results of cDNA arrays showed that 13 cancer-related genes were up-regulated > or =2-fold (CDC25B, cyclin D1, PCNA, MET, Jagged 2, Integrin alpha3, Integrin alpha6, Integrin beta4, Caveolin-2, Caveolin-1, MMP13, MMP14, and BIGH3) and 5 genes were down-regulated > or =2-fold (CK4, Bad, IGFBP2, CSPCP, and IL-1RA) in both ESCC cell lines at the mRNA level. Semiquantitative RT-PCR analysis of 9 of these differentially expressed genes, including the MET gene, gave results consistent with cDNA array findings. The immunostaining results of the expression of MET gene showed that MET was overexpressed in both ESCC cell lines and their corresponding primary tumors at the protein level, validating the cDNA arrays findings. The results of the clinical specimens showed that the MET gene was overexpressed in ESCC compared with normal esophageal epithelium in 56 of 61 cases (92%). Moreover, the overexpression of MET protein was more often seen in well/moderately differentiated than in poorly differentiated ESCC. CONCLUSIONS: Multiple cancer-related genes are differentially expressed in ESCC, the oncogene MET is overexpressed in ESCC compared with normal esophageal epithelium, and its protein overexpression correlates with tumor differentiation in ESCC.