Non-neoplastic Gastric Tissues Research Articles

Pan-Histone Deacetylase Inhibitor Panobinostat Sensitizes Gastric Cancer Cells to Anthracyclines via Induction of CITED2

Chemotherapy modestly prolongs survival of patients with advanced gastric cancer, but strategies are needed to increase its efficacy. Histone deacetylase (HDAC) inhibitors modify chromatin and can block cancer cell proliferation and promote apoptosis. Gastric cancer cell lines were incubated with the HDAC inhibitor LBH589 (Panobinostat, Novartis, Germany); levels of proliferation, apoptosis, histone acetylation, and gene expression were determined. We identified factors downstream of HDAC that regulated chemoresistance. The effects of combination chemotherapy of HDAC inhibitors and anthracyclines were studied in CEA424/SV40 T-antigen (CEA/Tag) transgenic mice, which develop gastric tumors. We analyzed gastric tumor samples from patients using immunohistochemistry. HDAC2 was expressed in human gastric cancer cell lines and tumor samples, as well as in gastric tumors from CEA/Tag mice, compared with non-neoplastic gastric tissue. LBH589 inhibited proliferation of cancer cells in vitro. LBH589 down-regulated expression of genes that mediate anthracycline resistance by activating expression of Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain 2 (CITED2), a gene that mediates sensitivity to chemotherapeutics. Pre-incubation of cells with an HDAC inhibitor and overexpression of CITED2-sensitized gastric cell lines to anthracycline-mediated cell death. In CEA/Tag mice, LBH589 induced tumor-cell expression of CITED2 and increased the efficacy of anthracycline to reduce tumor growth. Levels of CITED2 were increased in gastric tumor samples from patients who had complete responses to epirubicin. The HDAC inhibitor LBH589 can overcome the resistance of mouse gastric cancer cells to anthracyclines by inducing expression of CITED2. Levels of CITED2 in gastric tumors correlate with patients' response to epirubicin. LBH589 might be used to increase the response of patients to anthracyclines.

The prognostic implication of phosphoinositide 3-kinase/Akt pathway in gastric carcinomas.

49 Background: The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway plays a critical role in cell growth and survival. Dysregulation of this pathway has been found in a variety of cancer cells. Recently, constitutively active PI3K/Akt signaling has been firmly established as a major determinant for cell growth and survival in an array of cancers. Blocking the constitutively active PI3K/AKT signaling pathway provides a new strategy for targeted cancer therapy. The purpose of our present study was to investigate the expression of activated mTOR (p-mTOR), PTEN, and pAKT in non-neoplastic gastric tissue and in gastric cancer patients to determine their pattern of expression in gastric tumors and their prognostic significance. Methods: The expression of p-mTOR, pAKT, and PTEN was detected in specimens of 239 gastric cancers who underwent radical resection (R0) and in 200 non-neoplastic gastric tissues by immunohistochemistry. Slides were scanned and analyzed with an automated cellular imaging system (ACIS III, Dako). The correlation of p-mTOR, pAKT, and PTEN expression to clinicopathologic features and survival of gastric cancer was studied. Results: Overexpression of pMTOR in the cytoplasmic pattern, nuclear pAKT, and nuclear PTEN was observed in the non-neoplastic tissues when compared with gastric tumor tissues (all, p<0.001). Overexpression of pmTOR, pAKT, and PTEN in gastric tumors was closely correlated with pTNM stage (p<0.001, p=0.008, p=0,036, respectively). Patients with p-AKT positive showed significantly longer disease-free survival (DFS) and overall survival (OS) rates than those with pAKT-negative tumors in univariable analyses. In multivariable analyses, higher expression rates of pMTOR and losses of PTEN were significantly associated with worse overall survival. Conclusions: Taken together, the observations indicate the PI3K/Akt pathway plays an important role in the gastric carcinogenesis. A new strategy for combined chemotherapy of gastric cancer should be designed to more specifically block PI3K/Akt pathway for selected patients.

Clinical implication of 14-3-3 epsilon expression in gastric cancer

To evaluate for the first time the protein and mRNA expression of 14-3-3ε in gastric carcinogenesis. 14-3-3ε protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. Authors observed a significant reduction of 14-3-3ε protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue. Reduced levels of 14-3-3ε were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3ε may have a role in gastric carcinogenesis process. Our results reveal that the reduced 14-3-3ε expression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcinogenesis process.

More attention should be paid on the interpretation of gene expression data

Molecular profiling of gene expression is important for determining signatures in cancer progression and diagnosis. For this purpose, polymerase chain reaction-based techniques are preferentially used as a feasible and sensitive approach. Nevertheless, when relative quantitative analyses are performed on gene expression, the interpretation of mathematical equations must be carefully done. This letter to the editor is focused on recently published gene expression data in World Journal of Gastroenterology by Ozmen et al demonstrating increased levels of LYVE-1, VEGFR-3 and CD44 genes in gastric cancer samples compared to non-neoplastic gastric tissues. However, there are major concerns about misinterpretation of the gene expression data obtained with the 2(-ΔΔCt) relative quantitative method. In the study, 2(-ΔΔCt) values calculated for many samples were smaller than 1 (2(-ΔΔCt) < 1) which indicate decreased levels of LYVE-1, VEGFR-3 and CD44 gene expression in the gastric cancer tissues. This unfortunate mistake is an important example showing how a simple error in the interpretation of relative-quantitative gene expression data may result in misleading scientific conclusions. In this letter, a brief explanation of the 2(-ΔΔCt) method is given. In addition, the importance of technical quality and interpretation in gene expression studies is discussed.

Systematic evaluation of the miRNA‐ome and its downstream effects on mRNA expression identifies gastric cancer progression

We investigated the differential expression of Dicer and Drosha, as well as that of microRNA (miRNA), in adjacent normal and tumour samples of patients with gastric cancer. The expression of Dicer and Drosha was studied by immunohistochemistry in 332 gastric cancers and correlated with clinico-pathological patient characteristics. Differential expression of miRNAs was studied using the Invitrogen NCode(™) Multi-Species miRNA Microarray Probe Set containing 857 mammalian probes in a test set of six primary gastric cancers (three with and three without lymph node metastases). Differential expression was validated by RT-PCR on an independent validation set of 20 patients with gastric cancer. Dicer and Drosha were differentially expressed in non-neoplastic and neoplastic gastric tissue. The expression of Drosha correlated with local tumour growth and was a significant independent prognosticator of patient survival. Twenty miRNAs were up- and two down-regulated in gastric carcinoma compared with non-neoplastic tissue. Six of these miRNAs separated node-positive from node-negative gastric cancers, ie miR-103, miR-21, miR-145, miR-106b, miR-146a, and miR-148a. Five miRNAs expressed differentially in node-positive cancers had conserved binding sites for mRNAs differentially expressed in the same set of tumour samples. Gastric cancer shows a complex derangement of the miRNA-ome, including Dicer and Drosha. These changes correlate independently with patient prognosis and probably influence local tumour growth and nodal spread.

SALL4 Represents Fetal Gut Differentiation of Gastric Cancer, and is Diagnostically Useful in Distinguishing Hepatoid Gastric Carcinoma From Hepatocellular Carcinoma

The novel stem cell marker SALL4 has been identified as a diagnostic marker of germ cell tumors, especially yolk sac tumors, in gonadal organs. To clarify the significance of SALL4 as an oncofetal protein, we investigated SALL4 expression by immunohistochemistry in non-neoplastic stomach and gastric carcinoma with particular emphasis on á-fetoprotein (AFP)-producing gastric carcinoma, as AFP-producing gastric carcinoma shares expression of AFP and glypican 3 (GPC3) with yolk sac tumors and hepatic neoplasms. A total of 338 gastric carcinomas, 60 hepatocellular carcinomas, and 48 cholangiocellular carcinomas were studied by immunohistochemistry on tissue microarrays. In addition, more detailed whole tissue section immunohistochemistry was performed on non-neoplastic gastric tissue from 5 adult and 8 fetal specimens, 6 hepatoblastomas, and 31 cases of AFP-producing gastric carcinomas. SALL4 expression was observed in the neofetal stomach in gestational week 9 and disappeared thereafter. It was also identified by tissue microarray study in a fraction of gastric carcinomas (51 of 338, 15%), associated with older age (P=0.0001), male sex (P=0.0033), intestinal-type histology (P=0.0001), and synchronous liver metastasis (P=0.0047). AFP and GPC3 were closely associated with SALL4 expression in gastric carcinoma (both, P<0.0001), and a full-section study indicated that SALL4 was positive in all 31 cases of AFP-producing gastric carcinoma with diffuse staining in 24 cases (78%). Diffuse SALL4 expression was observed in the histologic patterns of hepatoid (89%), glandular (57%), and clear cell (39%) AFP-producing gastric carcinoma. In addition, SALL4 expression was completely negative in hepatoblastoma (n=6) and hepatocellular carcinoma (n=60). SALL4 is an oncofetal protein similar to AFP and GPC3, but it represents fetal gut differentiation in gastric carcinoma. SALL4 is a sensitive marker for AFP-producing gastric carcinoma and is especially useful to distinguish hepatoid gastric carcinoma from hepatocellular carcinoma.

P-cadherin expression in gastric carcinoma: its regulation mechanism and prognostic significance

P-cadherin is a member of the cadherin family and is expressed in several solid tumors. This molecule was recently highlighted with the development of a new targeted compound being studied in a clinical trial on solid tumors. In the present study, we examined the protein and messenger RNA (mRNA) expression status of P-cadherin and its promoter methylation in gastric carcinoma cell lines and tissues. Of the 10 cell lines, 4 were found to express P-cadherin protein and mRNA, and the P-cadherin gene was found to be hypomethylated in its promoter region in these cell lines. Nonneoplastic gastric mucosal tissues from gastric carcinoma patients were negative for P-cadherin protein evaluated by immunohistochemistry and Western blotting and had a methylated P-cadherin promoter region. In carcinoma tissues, 70.8% (749/1058) of cases showed P-cadherin protein expression, and P-cadherin positive cases had a well or moderately differentiated histology according to the World Health Organization classification, intestinal-type histology by Lauren classification, and an earlier pT class. Furthermore, patients with P-cadherin expressing tumors had a favorable prognosis by univariate and multivariate survival analyses. In addition, P-cadherin protein expression was found to be significantly correlated with promoter hypomethylation. In summary, P-cadherin is silenced in nonneoplastic gastric mucosa, and P-cadherin expressing tumors constitute a subset of gastric carcinoma with intestinal-type histology and a favorable prognosis. In addition, our findings suggest that P-cadherin promoter methylation underlies the regulation of its expression. These findings may aid patient selection and the interpretation of P-cadherin targeted therapy and clinical trial results.

Enhancer of zeste homolog 2 expression is associated with tumor cell proliferation and metastasis in gastric cancer

The enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins, plays an important role in cell proliferation and cell cycle regulation. EZH2 is overexpressed in aggressive forms of prostate, breast, bladder, and endometrial cancers. However, the role of EZH2 expression in gastric cancer has not been fully determined. This study was conducted to investigate the correlation between EZH2 and cell cycle-related molecules, and the clinical value of EZH2 expression in gastric cancer. We analyzed EZH2 expression using Western blotting in AGS, MKN-28, SNU-16, SNU-484, SNU-601, and SNU-638 gastric cancer cell lines. After transfection of EZH2 siRNA into MKN-28 cells, the change in cell cycle-related molecules was assessed by Western blot analysis. Expression of EZH2, Ki-67, and p53 was determined by immunohistochemical staining of tissue microarrays from specimens of 137 cases of resected gastric cancer. We found high expressions of EZH2 in all of the tested gastric cancer cell lines. RNA interference of EZH2 induced upregulation of p53 and HDAC1 and downregulation of cyclin D1 and cyclin E. High EZH2 expression was observed in 60.6% of gastric cancers and in 6.7% of non-neoplastic gastric tissues (p < 0.01); 40.1% were positive for p53 in gastric cancers. High EZH2 expression was correlated with Ki-67 and p53 expressions and was significantly associated with distant metastases and non-signet ring cells. Our results suggest that high EZH2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

Methylation ofGATA-4andGATA-5and development of sporadic gastric carcinomas

To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis. Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed. GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides). Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

Gene expression analysis identifies over‐expression of CXCL1, SPARC, SPP1, and SULF1 in gastric cancer

To elucidate gene expression signatures associated with gastric carcinogenesis, we performed a genome-wide expression analysis of 46 Finnish and 20 Japanese gastric tissues. Comparative analysis between Finnish and Japanese datasets identified 58 common genes that were differentially expressed between cancerous and non-neoplastic gastric tissues. Twenty-six of these genes were up-regulated in cancer and 32 down-regulated. Of these genes, 64% were also differentially expressed in another unrelated publicly available dataset. The expression levels of four of the up-regulated genes, CXCL1, SPARC, SPP1 and SULF, were further analyzed in 82 gastric tissues using quantitative real-time RT-PCR. This analysis validated the results from the microarray analysis as the expression of these four genes was significantly higher in the cancerous tissue compared with the normal tissue (fold change 3.4-8.9). Over-expression of CXCL1 also positively correlated with improved survival. To conclude, irrespective of the microarray platform or patient population, a common gastric cancer gene expression signature of 58 genes, including CXCL1, SPARC, SPP1, and SULF, was identified. These genes represent potential biomarkers for gastric cancer.

M1642 IGFI, IGFBP3 and ALS Levels in Colon and Rectal Cancer Before Surgery: Relation to One Year Patients'Survival

were established. Result: In non-neoplastic gastric tissues, TFF1 was strongly expressed in the deep mucous glands of the antrum, neck cells of the fundus and intestinal metaplasia. In diffuse-type gastric carcinoma, TFF1 was found to have rare staining in tumor cells (n= 18). For intestinal-type gastric carcinoma, TFF1 stained more intensely in well-differentiated tumors (100%) when compared to poorly differentiated areas (41.4%). Both early and advanced stage carcinomas showed a similar staining pattern. TFF3 was identified in the deep glands of the antrum and in goblet cells of intestinal metaplasia in the non-neoplastic tissues. In diffuse-type carcinoma, TFF3 was moderate to strongly positive in all cases (n= 18). For intestinal-type carcinoma, TFF3 was moderately expressed in well-differentiated tumors (64.5%), while low to absent expression was detected in poorly differentiated cases (19.5%). Conclusion: TFF1 is expressed as a function of tumor cell differentiation irrespective of stage, with highest levels observed in intestinal metaplasia and in well-differentiated tumor cells. In contrast, TFF3 is strongly expressed in diffuse-type gastric carcinoma. These findings suggest that TFF1/TTF3 can be utilized as a marker for aberrant protein expression and may help to distinguish diffuse and intestinal types of carcinoma.

Up‐regulated expression of ADAM17 in gastrointestinal stromal tumors: coexpression with EGFR and EGFR ligands

Metalloproteinase activities of a disintegrin and metalloproteinases (ADAMs), matrix metalloproteinases (MMPs), and membrane type (MT-)MMPs are involved in many aspects of tumor biology. ADAMs are transmembrane proteins that cleave membrane-anchored proteins to release soluble factors, and thereby mediate important biological phenomena in tumors. The aim of this study was to analyze histopathology, expression and roles of metalloproteinases, especially ADAMs, in gastric gastrointestinal stromal tumor (GIST). Histopathology and immunohistochemical expression of ADAMs were examined in 89 gastric GISTs. In 11 GISTs, ADAM expression was examined at mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting, respectively. RT-PCR analysis showed frequent expression of ADAM9 (91%), ADAM10 (64%), ADAM17 (82%), MMP-2 (82%), and MT1-MMP (73%). However, ADAM17 and MMP-2 were the only metalloproteinases that were up-regulated in GISTs at the protein level compared with non-neoplastic gastric tissues. ADAM17 was immunohistochemically expressed in 93% of GIST versus 16% of normal gastric tissues. Furthermore, CD117-positive interstitial cells of Cajal in normal gastric tissues were all negative for ADAM17 with double immunostaining. Expressions of epidermal growth factor receptor (EGFR) and several EGFR ligands such as amphiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin, and epiregulin were also demonstrated in GIST by RT-PCR. Protein expression of EGFR, phosphorylated EGFR, amphiregulin, and HB-EGF, both of which can be shed by ADAM17, was confirmed in tumors coexpressing ADAM17 by immunoblotting. Moreover, proteolytically cleaved soluble forms of amphiregulin were identified in tumor extracts. Considered together, the results suggest that ADAM17 may contribute to the progression and growth of GIST through shedding of EGFR ligands and consequent EGFR stimulation. ADAM17, as a major sheddase in GIST, could be potentially a suitable target in anticancer treatment of imatinib-resistant GISTs.

Immunohistochemical detection of the Raf kinase inhibitor protein in nonneoplastic gastric tissue and gastric cancer tissue

Expression of the Raf kinase inhibitor protein (RKIP), a metastasis suppressor, is high in normal tissues, low in primary cancers, and lowest or absent in metastatic cancers. Here, we studied the expression of RKIP in nonneoplastic gastric tissue and gastric cancer tissue by immunohistochemistry (IHC) and evaluated its role in the genesis and metastasis of gastric cancer. RKIP immunoreactivity was evaluated in 40 samples of nonneoplastic gastric tissues and 75 samples of gastric cancer tissues. Among the 40 samples of nonneoplastic gastric tissue, 35 (87.5%) were positive for RKIP expression and 5 (12.5%) were negative; in the 75 samples of primary gastric cancer tissue, 39 (52%) were positive for RKIP expression and 36 (48%) were negative. Among 26 samples of metastatic lymph node tissues, 5 (19.2%) were positive for RKIP expression and 21 (80.8%) were negative. RKIP expression level was highest in nonneoplastic gastric tissue, low in primary gastric cancer tissue, and lowest or undetectable in metastatic gastric cancer tissue. Our data suggest that RKIP may play a role in the genesis and metastasis of gastric cancer.

Identification of transgelin as a potential novel biomarker for gastric adenocarcinoma based on proteomics technology

To find a biomarker for gastric adenocarcinomas (GA). Ten protein expression profiles of GA and paired non-neoplastic mucosa tissues were analyzed by two-dimensional gel electrophoresis. Forty-two protein spots that were differentially expressed by twofold or greater between cancer and normal mucosa tissue were excised and identified by MALDI-TOF/TOF MS. One of the over-expressed proteins identified in GA was transgelin, which was chosen for further verification by immunohistochemistry and western blotting. Forty-two distinct proteins that were differentially expressed at least twofold between the tissues were identified. Expression of 29 of these proteins was decreased (ratio >or= 2, P < 0.01), including adenosine deaminase; and 13 proteins displayed over-expression in cancer tissue (ratio >or= 2, P < 0.01), including transgelin. The results of immunohistochemistry confirmed that transgelin was indeed over-expressed in 22 cases of GA (22/41, 53.66%), with strong cytoplasmic staining in cancer cells of positive samples, this was absent in most paired non-neoplastic mucosa cells or gastric ulcer tissues (n = 20). Transgelin was found over-expressed in 21 samples of cancer tissue (21/41, 51.22%) when detected by western blot. This work demonstrates that differentially expressed proteins can be identified by proteomics technology combined with immunohistochemistry and western blot analyses. We have identified one such protein, transgelin, as a novel biomarker for GA.

Differential expression and localisation of gasdermin-like (GSDML), a novel member of the cancer-associated GSDMDC protein family, in neoplastic and non-neoplastic gastric, hepatic, and colon tissues

Gasdermin-like (GSDML) is a novel member of the cancer associated gasdermin-domain containing (GSDMDC) protein family. The GSDMDC family has been linked to cancer development and progression, and this is the first study analysing the expression and intracellular localisation of GSDML. GSDML gene transcription was analysed using quantitative real-time RT-PCR. Anti-peptide antibodies against GSDML were developed in rabbits, and an in vitro transcription-translation reaction was used to verify specificity. The Protein-G affinity purified antibodies were used in immunohistochemistry and immunoblotting on hepatocellular, gastric, and colorectal carcinomas and non-lesional tissues. The GSDML gene was transcribed in human gastric, liver and colon cell lines, carcinomas and non-lesional tissues. The GSDML protein was localised to the cytoplasm of cells in both tumour and non-lesional tissues. The GSDML protein splicing variants range in molecular weight from 35 to 50 kDa, and the expression profile varies between tumour and non-tumour. A distinctive vesicular staining pattern was exhibited by GSDML in the apical region of gastric chief cells and colonic surface mucous cells, and the basal region of neuroendocrine cells. GSDML may be a secretory or metabolic product involved in a secretory pathway, and changes in the regulation of GSDML splicing variant transcription and translation may be seen in the development and/or progression of gastrointestinal and hepatic cancers.

Correlation of Vascular Endothelial Growth Factor-D Expression and VEGFR-3-Positive Vessel Density with Lymph Node Metastasis in Gastric Carcinoma

Lymph node metastasis is an important prognostic factor in gastric cancer. Vascular endothelial growth factor-D (VEGF-D) is a lymphangiogenic growth factor that activates VEGF receptor (VEGFR)-3, a receptor expressed in the lymphatic endothelium. We investigated the clinical value of VEGF-D expression and VEGFR-3 positive vessel density in gastric carcinoma with regard to lymphangiogenesis. Immunohistochemical staining was used to determine the expression of VEGF-D and VEGFR-3 in specimens from 104 cases of resected gastric cancer. VEGF-D expression was observed in 62.5% of the gastric cancers and in 9.6% of the non-neoplastic gastric tissue. The VEGFR-3-positive vessel density was significantly greater in the VEGFD positive group than the negative group. VEGF-D expression was significantly associated with lymph node metastasis, increased serum CEA levels, and the non-signet ring cell type. The VEGFR-3-positive vessel density was correlated with tumor size, lymphatic invasion, and lymph node metastasis. The VEGF-D expression and high VEGFR-3-positive vessel density were significant poor prognostic factors for relapse-free survival. These results suggest that VEGF-D and VEGFR-3-positive vessel density are potential molecular markers that predict lymphatic involvement in gastric carcinoma.

The Hippo Pathway in Human Upper Gastrointestinal Dysplasia and Carcinoma: A Novel Oncogenic Pathway

The Hippo (Hpo) pathway is highly conserved in humans and was originally uncovered in Drosophila as a potent regulator of inhibiting cell growth and promoting apoptosis. The Hippo pathway consists of a tumor suppressor kinase cascade that negatively regulates growth and results in inactivation of a transcriptional co-activator, Yorkie (yki). The human ortholog of Yki, the yes-associated protein (YAP), has a 31% sequence identity and similar biologic activity. The potential role of YAP in tumorigenesis was also reported in a murine genetic screen which identified a genomic amplification of YAP in hepatocellular carcinoma. Given this pathway's critical control of cell growth, survival, proliferation, and amplification in malignancy, we wanted to explore the possible role of the Hippo pathway in human esophageal and gastric tumorigenesis. The expression of YAP was evaluated with immunolabeling of esophageal and gastric tissue microarrays from 169 patients, with nondysplastic, dysplastic, and malignant foci represented. Cytoplasmic and nuclear staining were scored as 0 = none, 1 < 10%, 2 = 10-50%, and 3 > 50% for the nonneoplastic, dysplastic, and malignant epithelium. Multiple scores were averaged for each patient. Expression of YAP could be seen in the proliferating compartments of nonneoplastic tissue. Compared to nonneoplastic epithelium, there was a significant increase in YAP cytoplasmic and nuclear localization in high-grade dysplastic epithelium and adenocarcinoma of the esophagus. There was also a significant increase in YAP cytoplasmic and nuclear staining of gastric carcinoma and metastatic gastric disease compared to nonneoplastic gastric tissue. YAP expression in the cytoplasm and nucleus is significantly increased in high-grade dysplasia and adenocarcinoma of the esophagus as well as gastric adenocarcinoma and metastatic gastric disease, suggesting a role for this recently uncovered pathway in esophageal and gastric epithelial tumorigenesis.

Regulatory role of hepatocyte nuclear factor-4α on gastric choriocarcinoma function

Gastric choriocarcinoma is a highly aggressive carcinoma, most probably originating from somatic cells in the gastric mucosal layer. We herein investigated the regulatory role of hepatocyte nuclear factor (HNF)-4α, a transcriptional regulator expressed in non-neoplastic and neoplastic gastric tissues, on functions of gastric choriocarcinoma cells. HNF-4α cDNA was stably transfected to a gastric choriocarcinoma cell line, SCH. Alterations in SCH cell functions such as histology, ultrastructure, proliferation, production of trophoblast-specific proteins, and chemosensitivity to methotrexate (MTX) were examined. Neither in vitro and in vivo proliferations nor HLA-G expression differed significantly between the mock-transfected and HNF-4α-transfected SCH cells, while suppressed human chorionic gonadotropin (hCG) secretions, increased human placental lactogen (hPL) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) immunoreactivity, and decreased chemosensitivity to MTX were seen in HNF-4α-transfected SCH cells. General histologic features in xenograft nodules were unaltered, but, ultrastructurally, fascicles of paranuclear filaments were significantly more numerous in HNF-4α-transfected SCH cells. These results indicated an HNF-4α-rendered functional regulation in SCH cells, suggesting a role of transcriptional factors abundant in gastric but not in trophoblastic tissues/cells on the functional modulation of gastric choriocarcinoma cells.

Multidrug resistance‐associated protein (MRP1) gene is strongly expressed in gastric carcinomas. Analysis by immunohistochemistry and real‐time quantitative RT‐PCR

To assess MRP1 protein and MRP1 mRNA levels in gastric carcinomas and in non-neoplastic mucosa remote from the tumours. MRP1 gene expression may play a role in the complex pattern of chemoresistance present in gastric carcinomas. A total of 57 carcinomas and respective gastric tissues were included for immunohistochemical assessment with the anti-MRP1 monoclonal antibodies MRPr1 and QCRL-1. Of these, 35 tumour and gastric mucosa tissues were also assessed by real-time quantitative reverse transcriptase-polymerase chain reaction. Medium or high MRP1 protein expression was detected in 89% and 77% of carcinomas and in 96% and 93% of non-neoplastic gastric mucosa by MRPr1and QCRL-1, respectively. No difference in MRP1 mRNA levels was detected between carcinomas and non-neoplastic gastric mucosa tissues in 77% of the patients. A significant correlation was found between MRP1 mRNA level and protein expression detected in carcinomas related to non-neoplastic gastric mucosa, although they were non-concordant in 29% of the patients. MRP1 gene is usually expressed in most gastric carcinomas and does not differ substantially from that observed in non-neoplastic gastric mucosa remote from the tumour. However, a decrease in MRP1 gene expression is found in some carcinomas. For accurate assessment of changes in MRP1 expression between tumour and normal tissues both protein and mRNA detection are necessary.

Demethylation of the synuclein gamma gene CpG island in primary gastric cancers and gastric cancer cell lines.

Whereas synuclein gamma (SNCG) gene expression is usually highly tissue-specific and restricted to the nervous system, SNCG is expressed in advanced-stage breast and ovarian cancers. When overexpressed, SNCG stimulates cancer cell proliferation and metastasis. It is thought that the molecular mechanism of CpG island demethylation may underlie aberrant SNCG expression. To determine whether aberrant SNCG expression and demethylation play a role in gastric carcinogenesis, we examined the expression and methylation status of SNCG in primary gastric cancers, gastric cancer cell lines, and non-neoplastic gastric mucosal tissues. Ten gastric cancer cell lines, 105 primary gastric cancers, and 10 non-neoplastic gastric mucosal tissues were examined. SNCG expression and methylation status were examined by reverse transcription-PCR and bisulfite-single-strand conformational polymorphism followed by direct sequencing, respectively. The relationship between SNCG methylation status and various clinicopathological factors of the primary gastric cancers was then analyzed. SNCG mRNA expression was observed in 5 of 10 cell lines. Analysis of cell lines positive for SNCG expression revealed that most of the SNCG CpGs were demethylated. SNCG mRNA was not expressed in the 10 non-neoplastic gastric mucosal tissues, although several CpGs were demethylated. Of the 105 primary gastric cancers, 40 (38.1%) showed apparent SNCG demethylation, similar to the result obtained using cell lines. SNCG demethylation was more frequent in primary gastric cancers positive for lymph node metastasis (51%; 26 of 51) than in cancers without lymph node involvement (26%; 14 of 54; P < 0.05), and also more common in stage II-IV (48%; 27 of 56) than in stage I (27%; 13 of 49) cancers (P < 0.05). Aberrant SNCG gene expression can occur via CpG island demethylation, and tends to occur during the more progressive stages of gastric carcinogenesis.