Yasushi Endoh

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 non-cancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

In Vivo Characterization of Bone Marrow–Derived Fibroblasts Recruited into Fibrotic Lesions

Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrow–derived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP+ fibroblast was found even in nonfibrotic skin (8.7% ± 4.6%) and lung (8.9% ± 2.5%). The proportion of GFP+ fibroblasts was significantly increased after cancer implantation(59.7%±16.3%) and excisional wounding (32.2% ± 4.8%), whereas it was not elevated after bleomycin administration (7.1% ± 2.4%). Almost all GFP+ fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow–derived fibroblasts would contribute to tissue fibrosis. GFP+ fibroblasts expressed CD45, Thy‐1, and α‐smooth muscle actin at various proportions. Our results suggested that bone marrow–derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment.

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.

Cellular phenotypes of differentiated-type adenocarcinomas and precancerous lesions of the stomach are dependent on the genetic pathways

To elucidate the relationship between genetic alterations and cellular phenotypes in differentiated‐type carcinomas and precancerous lesions of the stomach, mutations of p53, APC and K‐ras genes were examined, as well as microsatellite instability (MSI), in 52 tumours of the stomach. Tumours were selected with the following phenotypical features, using mucin histochemical and immunohistochemical analyses, in addition to their morphological features: (1) tumours with an extremely well‐preserved gastric foveolar phenotype (foveolar‐type); (2) tumours with an extremely well‐preserved complete‐type intestinal metaplastic phenotype (CIM‐type); and (3) ordinary tumours without extreme phenotypes (ordinary‐type). MSI occurred in 45% of foveolar‐type, 24% of ordinary‐type, and 0% of CIM‐type tumours. p53 gene alterations occurred in 5% of foveolar‐type, 18% of ordinary‐type, and 31% of CIM‐type. APC gene alterations were detected in 9% of foveolar‐type, 6% of ordinary‐type, and 0% of CIM‐type. No K‐ras gene mutation was detected in any of the three types. These results indicate that the genetic pathways are quite different among the phenotypes of tumours of the stomach. The ‘mutator pathway’, characterized by MSI, plays an important role in the tumourigenesis of foveolar‐type, but not CIM‐type tumours. The ‘suppressor pathway’, represented by p53 alteration, could participate in the tumourigenesis of the CIM‐type, but is rare in foveolar‐type tumours. Copyright

Molecular Characterization of Undifferentiated-Type Gastric Carcinoma

As the great majority of gastric cancers develop histologically differentiated, and a significant proportion of differentiated-type carcinomas progress to become undifferentiated, both histological types are likely to share several common genetic abnormalities, such as p53 mutations at advanced stages. However, a subset of gastric cancers develop as undifferentiated carcinomas, including signet-ring cell carcinoma and poorly differentiated adenocarcinoma, and the molecular pathogenesis of this tumor type remains largely unknown. To characterize the molecular features of undifferentiated-type gastric carcinomas that developed as undifferentiated-type, we examined for p53, APC, and epithelial (E)-cadherin gene mutations, microsatellite alterations including loss of heterozygosity (LOH) and microsatellite instability (MSI), and hypermethylation of the E-cadherin gene promoter in 26 early undifferentiated gastric carcinomas, consisting of 14 signet-ring cell carcinomas and 12 poorly differentiated adenocarcinomas. E-cadherin expression was evaluated immunohistochemically. p53 mutations were detected in only one poorly differentiated adenocarcinoma sample (3.8%; 1/26), whereas no APC or E-cadherin mutations were found. LOH was present only at D8S261 on the short arm of chromosome 8 in 2 of 14 (14%) informative tumors, both of which were poorly differentiated adenocarcinomas, and MSI was not observed in any of the tumors. No signet-ring cell carcinomas have been found to carry gene mutations or microsatellite alterations. In contrast, hypermethylation of the E-cadherin promoter occurred in 69% (18/26) of the tumors; 57% (8/14) of signet-ring cell carcinomas, and 83% (10/12) of poorly differentiated adenocarcinomas, and was significantly associated with loss or reduced expression of E-cadherin. Thus, whereas tumor suppressor gene mutation, LOH, and MSI were less common in undifferentiated-type early gastric carcinomas, epigenetic inactivation of E-cadherin via promoter hypermethylation may be an early critical event in the development of undifferentiated tumors.

Frequent loss of expression without sequence mutations of the DCC gene in primary gastric cancer

Loss of heterozygosity (LOH) on chromosome 18q21 is frequently found in various human cancers, suggesting the presence of tumour suppressor gene(s) in this chromosomal region. DCC is the most likely target of LOH because loss or reduction of DCC expression has been found in many types of cancers. However, few reports have focused on sequence mutations of this gene. We investigated sequence mutations and expression of DCC in primary gastric cancers. We studied mutations in 25 of the 29 DCC exons by PCR-SSCP in 17 primary gastric cancers exhibiting LOH on 18q21. No mutations of DCC were found in any of the tumours, although 78% (47/60) of the primary tumours showed apparent loss or reduction of DCC expression by immunohistochemistry. Analysis of methylation status of DCC revealed that methylation frequently occurred in both primary tumours (75%; 45/60) and corresponding non-cancerous gastric mucosae (72%; 43/60). Methylated status of DCC was significantly correlated with the loss of DCC expression in primary tumours (P < 0.01). These results indicate that DCC is frequently silenced, probably by epigenetic mechanisms instead of sequence mutations in gastric cancer. http://www.bjcancer.com

Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer

Abstract. The PTEN tumor suppressor gene on 10q23.3, responsible for the Cowden and Bannayan-Zonana syndromes, encodes a dual-specificity phosphatase able to dephosphorylate both tyrosine phosphate and serine/threonine phosphate residues. Mutational inactivation of PTEN has been reported in various malignancies, including endometrial cancers, ovarian cancers, and glioblastomas. In this study, we investigated PTEN gene mutations in 10 gastric cancer cell lines and 58 primary gastric cancers by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP). Hypermethylation of promoter region CpG islands, an alternative mechanism of gene inactivation to coding region mutations, was also evaluated by methylation specific PCR (MSP). Only one (1.7%) of the 58 primary tumors carried a somatic 5-bp deletion in intron 7 of PTEN, which did not alter the mRNA sequence, and no mutations were detected in any of the cell lines. Similar levels of PTEN mRNA expression were observed in all cell lines and primary tumors studied by RT-PCR, and PTEN promoter CpG islands remained unmethylated. Therefore, we conclude that PTEN does not participate in gastric carcinogenesis as a tumor suppressor gene.

Frequent hypermethylation of the hMLH1 gene promoter in differentiated-type tumors of the stomach with the gastric foveolar phenotype.

Hypermethylation of the hMLH1 mismatch repair gene promoter has been revealed to lead to microsatellite instability (MSI). Previously, we demonstrated a high prevalence of MSI in differentiated-type gastric tumors showing distinct features of gastric foveolar epithelium (foveolar type). To clarify the significance of hMLH1 promoter hypermethylation in the development of this tumor type, we studied promoter methylation status and expression of hMLH1 in foveolar-type tumors and their surrounding non-neoplastic mucosae, as well as in tumors with other cellular phenotypes. The results were compared to MSI status. After phenotypical analyses using mucin histochemistry and immunohistochemistry, 41 differentiated-type tumors with distinct cellular phenotypes were classified into three categories: foveolar type, intestinal type (tumors with the distinct cellular phenotype of the intestine), and combined type (tumors with both foveolar and intestinal phenotypes). Methylation-specific polymerase chain reaction (MSP) was performed to determine the methylation status of hMLH1 promoter. hMLH1 protein expression was immunohistochemically examined. MSI was detected in 57% of the foveolar type, 8% of the intestinal type, and 67% of the combined-type tumors. Hypermethylation of hMLH1 promoter was found in 74% of the foveolar type, 33% of the intestinal type, and 83% of the combined-type tumors. Of 18 MSI-positive tumors, all but one were hypermethylated. Methylation status of hMLH1 promoter correlated well with protein expression in foveolar-type tumors. Moreover, hypermethylation was also detected frequently (71%) in the non-neoplastic surrounding mucosa of the hypermethylated tumors. Hypermethylation of hMLH1 promoter is an initial, vital event in the development of foveolar-type tumors of the stomach.

Matrix metalloproteinase‐7 triggers the matricrine action of insulin‐like growth factor‐II via proteinase activity on insulin‐like growth factor binding protein 2 in the extracellular matrix

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM‐bound growth factor or cytokine activation have not been well documented. We showed that the insulin‐like growth factor type‐1 receptor (IGF‐1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)‐7 to a serum‐starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF‐II neutralizing antibody. In the ECM of this cell line, IGF‐II and IGF binding protein (BP)‐2 coexisted, but IGFBP‐2 disappeared from the ECM fraction after treatment with MMP‐7 or heparinase III. On the other hand, in a cell line in which IGF‐1R was overexpressed, IGF‐1R was phosphorylated by supernatant from the MMP‐7‐treated ECM fraction of HT29 but not by that from a heparinase‐III‐treated ECM fraction. We also demonstrated that MMP‐7 degrades IGFBP‐2 in vitro at three cleavage sites (peptide bonds E151–L152, G175–L176 and K181–L182), which have not been documented previously. Taken together, these results demonstrate that MMP‐7 generates bioactive IGF‐II by degrading the IGF‐II/IGFBP‐2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF‐II‐induced signal transduction. This evidence indicates that some ECM‐associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action (‘protease‐triggered matricrine’) represents an attractive model for understanding ECM–tumor interactions. (Cancer Sci 2007; 98: 685–691)

In vivo and in vitro characterization of human fibroblasts recruited selectively into human cancer stroma

Fibroblasts, which are a major component of cancer‐induced stroma, can have a significant impact on the progression of adjacent malignant epithelia. To characterize fibroblasts recruited into cancer‐induced stroma, we examined the recruitment efficiency of 9 human fibroblast cell lines into experimental tumors generated in immunodeficient mice. Green fluorescence protein (GFP)–labeled fibroblast cell lines and human pancreatic cancer cell line Capan‐1 were injected i.p. at different sites; the GFP‐labeled cells within xenografts were then analyzed. KM104GFP (bone marrow) and VA‐13GFP (lung) were selectively recruited into cancer stroma more efficiently than the other cell lines. KM104GFP cells did not affect tumor volume; however, VA‐13GFP cells increased tumor volume by about 2‐fold. After 5 cyclic in vivo passages of KM104GFP in Capan‐1, we selected a subpopulation with an 8.4‐fold higher recruitment efficiency (KM104GFP‐5G) compared to parental KM104GFP. KM104GFP‐5G also exhibited higher chemotaxis and chemoinvasion activity compared to KM104GFP in response to cancer‐released chemoattractant(s). Oligonucleotide microarray analysis identified 8 genes with >3‐fold upregulation and 6 genes with >3‐fold downregulation in KM104GFP‐5G. Immunohistochemistry confirmed that fibroblasts recruited into pancreatic cancer stroma strongly expressed carbonic anhydrase IX and keratin‐8, whose transcripts were upregulated in KM104GFP‐5G by oligonucleotide microarray analysis, whereas their expression in fibroblasts within noncancerous pancreatic stroma were under the detection level. Our results indicate that fibroblast recruitment is not selective with respect to organ origin and that particular fibroblast subpopulations with specific phenotypic characteristics could be recruited efficiently into cancer‐induced stroma.