[Show abstract][Hide abstract] ABSTRACT: Hepatoma-derived growth factor (HDGF) is a secreted heparin-binding growth factor that has been implicated in cancer development and progression. Here, we report that HDGF is a critical target for transcriptional repression by the tumor suppressor p53. Endogenous HDGF expression was decreased in cancer cells with introduction of wild-type p53, which also downregulated HDGF expression after DNA damage. In support of the likelihood that HDGF is a critical driver of cancer cell growth, addition of neutralizing HDGF antibodies to culture media was sufficient to block cell growth, migration, and invasion. Similarly, these effects were elicited by conditioned culture medium from p53-expressing cells, and they could be reversed by the addition of recombinant human HDGF. Interestingly, we found that HDGF was overexpressed also in primary gastric, breast, and lung cancer tissues harboring mutant p53 genes. Mechanistic investigations revealed that p53 repressed HDGF transcription by altering HDAC-dependent chromatin remodeling. Taken together, our results reveal a new pathway in which loss of p53 function contributes to the aggressive pathobiological potential of human cancers by elevating HDGF expression.
Cancer Research 11/2011; 71(22):7038-47. DOI:10.1158/0008-5472.CAN-11-1053 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.
DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.
DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8-18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8-50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.
Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.
BMC Cancer 07/2009; 9(1):198. DOI:10.1186/1471-2407-9-198 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic gene inactivation plays a key role in the development of various types of cancer. Using methylated CpG island amplification coupled with representational difference analysis to identify genes inactivated by DNA methylation in gastric cancer, we identified seven DNA fragments corresponding to the 5' CpG islands of the affected genes. One of the clones recovered was identical to the 5' flanking region of DFNA5, a gene previously shown to be associated with deafness and induced by DNA damage. Further analysis revealed that DFNA5 is expressed in normal tissues but is down-regulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Treating gastric cancer cells that lacked DFNA5 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Methylation of DFNA5 was detected in 50% of primary gastric tumors, and was correlated with positivity for Epstein-Barr virus and the absence of metastasis. Moreover, introduction of exogenous DFNA5 into silenced cells suppressed colony formation. Taken together, these data suggest that the silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the disease.
Cancer Science 02/2007; 98(1):88-95. DOI:10.1111/j.1349-7006.2006.00351.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CpG island methylator phenotype (CIMP), which is characterized by simultaneous methylation of the CpG islands of multiple genes, has been recognized as one of the important mechanisms in gastrointestinal carcinogenesis.
Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes in 78 primary gastric carcinomas was examined using combined bisulfite-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors were detected using real-time polymerase chain reaction analysis followed by an evaluation of the correlations between CIMP status, EBV-association, and genetic alteration of p53 and K-ras. The authors compared the clinicopathologic features of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N).
The methylation profiles of 12 genes showed nonrandom methylation, supporting the presence of CIMP in gastric carcinoma. No p53 mutations were detected among CIMP-H tumors, and no EBV association was detected in tumors that showed mutation of p53 and K-ras. In a multiple logistic regression model with CIMP-H as the dependent variable, proximal location (P = .011), diffuse type (P = .019), and less advanced pathologic TNM status (P = .043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly worse survival than patients who had CIMP-H tumors (P = .004) or CIMP-L tumors (P = .012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation.
CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that gastric carcinomas arose through different molecular pathways may affect not only tumor characteristics but also patient prognosis.
Cancer 04/2006; 106(7):1467-79. DOI:10.1002/cncr.21789 · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BNIP3 protein is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. To examine its role in the progression of gastrointestinal cancer, we examined the expression and DNA methylation status of BNIP3 gene in a panel of colorectal and gastric cancer cell lines. BNIP3 was not expressed in 14 of the 24 cell lines tested, and its absence was not caused by gene mutation or by altered expression of hypoxia inducible factor-1, a key transcription factor that regulates BNIP3 expression. On the other hand, methylation of the 5' CpG island of BNIP3 was closely correlated with silencing the gene. Moreover, treating methylated cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored hypoxia-induced expression of BNIP3 mRNA and protein, which in turn led to cell death. Aberrant methylation of BNIP3 was also detected in 66% of primary colorectal and 49% of primary gastric cancers, but not in normal tissue samples collected from areas adjacent to the tumors. Apparently, epigenetic alteration of BNIP3 is a frequent and cancer-specific event, which suggests that inactivation of BNIP3 likely plays a key role in the progression of some gastrointestinal cancers and that it may be a useful molecular target for therapy.
Clinical Cancer Research 03/2005; 11(3):1021-7. · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ectopic (heterotopic) gastric mucosa (EGM) of the upper esophagus, referred as inlet patch, is an asymptomatic benign lesion that is often detected during endoscopic examination. Although it is considered a source of adenocarcinoma in the upper esophagus, only 17 cases of adenocarcinoma have been reported previously. We report a rare case of adenocarcinoma arising in EGM of the cervical esophagus.
American journal of clinical oncology 01/2005; 27(6):644-5. DOI:10.1097/01.coc.0000147808.63442.b5 · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aberrant methylation of a sodium co-transporter, solute carrier family 5 member 8 gene (SLC5A8), has been detected in a subset of colorectal cancers, suggesting SLC5A8 may also serve as a tumor suppressor. To further investigate the role of epigenetic inactivation of SLC5A8 expression in gastric cancer, we determined the methylation status of the SLC5A8 5' CpG island (CGI) in a panel of gastric cancer cell lines and primary gastric cancers. We detected methylation of the 5'CGI in ten of twelve gastric cancer cell lines, and five of those showed dense methylation, which correlated with the absence of SLC5A8 transcription. Aberrant methylation of SLC5A8 was also detected in 23 of 71 (30%) primary gastric cancers, indicating that epigenetic inactivation of SLC5A8 is not a cell-line-specific phenomenon. SLC5A8 expression was restored in methylated cell lines by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, chromatin immunoprecipitation assays showed that acetylation of histone H3 in the 5' region of the gene correlated directly with SLC5A8 expression and inversely with DNA methylation. It thus appears that aberrant methylation of its 5'CGI and histone deacetylation play key roles in silencing SLC5A8 expression in gastric cancers.
[Show abstract][Hide abstract] ABSTRACT: Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.
Cancer Research 01/2004; 63(24):8606-13. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5' CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P<0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (-300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the genetic factors of familial predisposition to gastric cancer, genetic alterations in the surgically resected stomach samples from gastric-cancer-prone families were investigated. Familial gastric cancer (FGC) was defined as gastric cancer occurring in a family with 3 or more gastric cancer patients over at least two successive generations. We examined replication error (RER) of six microsatellite markers and screened mutations of the 10-(A) repeat sequence in the transforming growth factor-beta receptor type II (TGF-betaRII) gene in individuals from seven unrelated FGC families. Three cases showed RER at one of the six (CA)n microsatellite markers but the other 4 cases showed no RER at any of these loci. No mutation was found in the 10-(A) repeat of the TGF-betaRII gene. Additionally, no germline mutation was found by polymerase chain reaction-single strand conformation polymorphism in exons 1-16 of E-cadherin, exons 5-8 of p53 and in the mutation cluster region of APC. These results indicate that disorders in the DNA mismatch repair system, E-cadherin, p53 and APC may be infrequently involved in the carcinogenesis of Japanese FGC.
[Show abstract][Hide abstract] ABSTRACT: Previous studies reported that mutation of the adenomatous polyposis coli (APC) gene was not observed in the majority of gastric cancers. To evaluate the role of the APC/β-catenin/Tcf pathway, we analyzed mutations in the β-catenin gene and the accumulation of β-catenin protein in gastric carcinomas. An interstitial deletion spanning exon 3 of the β-catenin gene was observed in 1 of 13 gastric cancer cell lines. No missense mutation was found in these 13 cell lines. Nuclear and/or cytoplasmic localization of β-catenin was observed in 16 of 70 primary gastric carcinomas by immunohistochemistry, while we found no mutations in exon 3 in 35 carcinoma tissues available for PCR amplification. Our findings suggest that somatic mutations of the β-catenin gene are rare in human gastric carcinomas and that accumulation of normal β-catenin protein in a subset of gastric cancers may be due to other mechanisms of its activation.
[Show abstract][Hide abstract] ABSTRACT: The clinical features of familial gastric cancer are still unknown. To approach this question, we investigated the clinicopathological characteristics of 16 cases of familial gastric cancer. In this study the criteria used to define familial gastric cancer was the existence of three or more family members with gastric cancer in at least two successive generations. The clinicopathological characteristics of cases who fulfilled this criteria were studied. This study contained 16 familial gastric cancer probands. Seven cases (44%) of gastric cancer had developed at the cardiac region of the stomach. This frequency was significantly higher than for gastric cancer in the general population in Japan (15.4%, p < 0.01). Undifferentiated types were dominant in familial gastric cancer (69%, p < 0.05). Furthermore, the frequency of disseminated peritoneal (40%) and liver metastases (20%) in familial gastric cancer was also significantly higher than for gastric cancer in the general population in Japan (10.9%, p < 0.01, and 4.4%, p < 0.05, respectively). Familial gastric cancers were frequently located at the cardiac region and appeared to be more aggressive than sporadic gastric cancers. The unique characteristics of familial gastric cancer suggest a genetic background in their etiology.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-7 (matrilysin) has been implicated in tumor invasion and metastasis as well as tumor initiation and growth. In this study, we analyzed an association between immunohistochemically detected matrilysin expression at the invasive front in esophageal squamous cell carcinomas and clinicopathological characteristics and determined whether matrilysin predicts recurrence and/or survival Matrilysin expression at the invasive front was detected in 49% of 100 carcinoma tissues and was associated with the depth of invasion (P < 0.0001), advanced tumor stage (P = 0.0159), recurrences (P = 0.0002), and recurrences within the first postoperative year (P = 0.002). Patients with matrilysin-positive carcinoma had a significantly shorter disease-free and overall survival time than did those with a matrilysin-negative one (P < 0.0001). Matrilysin remained a significant predictive value for disease-free and overall survival in multivariate analysis, including conventional clinicopathological factors (P = 0.0007 and 0.0004, respectively). Our results suggest that matrilysin may play a key role in the progression of esophageal carcinoma and that its detection may be useful for the prediction of recurrence and poor prognosis and, possibly, for selecting patients for anti-matrix metalloproteinase therapy.
Cancer Research 07/1999; 59(14):3313-6. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We inserted Dumon stent to 13 patients with tracheobronchial stenosis due to advanced or recurrent esophageal carcinoma. Severe dyspnea was improved in 11 patients except for 2 patients with bilateral recurrent nerve palsy. 3 cases who had radiation therapy or chemotherapy lived over 150 days. We inserted esophageal stent in 4 cases. 2 patients died due to hemoptysis after 156 days and 35 days. We conclude that Dumon stent is one of the useful treatments in order to improve quality of life and prognosis of advanced or recurrent esophageal carcinoma.
The Japanese Journal of Thoracic and Cardiovascular Surgery 12/1998; 46(11):1093-6. DOI:10.1007/BF03217882