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Background: Expression of caveolin-1 (Cav-1) is frequently altered in many human cancers and both tumor suppression and promotion functions of Cav-1 have been suggested based on its expression status. However, it remains unanswered how Cav-1 provokes opposite effects in different cancers or different phases of tumor progression., Methods: To explore the implication of Cav-1 alteration in gastric tumorigenesis, the expression and mutational status of Cav-1 and its effects on tumor cell growth were characterized., Results: A substantial fraction of primary tumors and cell lines displayed abnormally low or high Cav-1 mRNA expression, indicating the bidirectional alteration of Cav-1 in gastric cancers. While allelic imbalance and mutational alterations of the Cav-1 gene were rarely detected, aberrant promoter hyper- or hypo-methylation showed a tight correlation with bidirectional alteration of its expression. Abnormally low and high Cav-1 expression was more frequently observed in early and advanced cancers, respectively, suggesting the oncogenic switch of its function in tumor progression. Cell cycle progression, DNA synthesis, and colony forming ability were markedly decreased by Cav-1 transfection in low-expressing tumor cells but by its depletion in high-expressing cells. Interestingly, Cav-1 exerted opposite effects on MEK-ERK signaling in these two cell types through the reciprocal regulation of the RAF-ERK negative feedback loop. A feedback inhibition of RAF by ERK was stimulated by restoration of Cav-1 expression in low-expressing cells but by it depletion in high-expressing cells. As predicted, the opposite effects of Cav-1 on both tumor cell growth and inhibitory RAF phosphorylation were abolished if ERK is depleted., Conclusion: Bidirectional alteration of Cav-1 is linked to its opposite effects on gastric tumor cell growth, which stem from the reciprocal control on the RAF-ERK negative feedback loop.

Background: CD81 is a transmembrane protein that serves as a putative receptor for hepatitis C virus. In addition, CD81 has been suggested to be involved in a broad range of other cellular functions. Its putative implication in tumorigenesis has so far, however, remained largely unexplored. To assess the candidacy of CD81 as a tumor suppressor in gastric cancer development, we investigated its expression and function in a series of primary gastric tumors and gastric tumor-derived cell lines., Methods: The expression and concomitant methylation status of the CD81 gene and its effect on tumor development and cellular signaling were evaluated., Results: CD81 mRNA levels were found to be low in 16 of 40 (40 %) primary tumors and 9 of 14 (64.2 %) cell lines, and these low expression levels were found to correlate with the stage and grade of the tumors. Genomic alterations of CD81 were not encountered, whereas its expression could be re-activated in low expressing cells upon 5-aza-dC treatment. Bisulfite DNA sequencing analysis of 10 CpG sites within the 5' proximal region of the CD81 gene promoter revealed that the observed transcriptional silencing was tightly associated with aberrant hypermethylation. Subsequent restoration of CD81 expression induced a G1 cell cycle arrest and apoptosis, whereas siRNA-mediated CD81 down-regulation promoted cell proliferation and attenuated cellular responses to various apoptotic stress stimuli. Also the colony-forming ability of the tumor cells could be inhibited and enhanced through CD81 up- and down-regulation, respectively. CD81 was found to inhibit p38 (but not ERK, JNK and AKT) phosphorylation and its growth suppressive effect could be abolished through p38 up- and down-regulation., Conclusion: From our data we conclude that epigenetic inactivation of CD81 is a common feature of gastric tumors and that this inactivation may render growth and survival advantages to the tumor cells, at least partially through p38 signaling.

Background & Aims: HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis., Methods: HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling., Results: Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR., Conclusions: HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. To explore the candidacy of hSRBC as a suppressor of gastric tumorigenesis, we analyzed the expression and mutation status of hSRBC in gastric tissues and cell lines. hSRBC transcript was expressed in all normal and benign tumor tissues examined, but undetectable or very low in 73% (11/15) cancer cell lines and 41% (46/111) primary tumors. Loss or reduction of hSRBC expression was tumor-specific and correlated with stage and grade of tumors. While allelic loss or somatic mutations of the gene were infrequent, its expression was restored in tumor cells by 5-aza-2'-deoxycytidine treatment and aberrant hypermethylation of 23 CpG sites in the promoter region showed a tight association with altered expression. Transient or stable expression of hSRBC led to a G(1) cell cycle arrest and apoptosis of tumor cells, and strongly suppresses colony forming ability and xenograft tumor growth. In addition, hSRBC elevated apoptotic sensitivity of tumor cells to genotoxic agents, such as 5-FU, etoposide and ultraviolet. Interestingly, hSRBC increased the protein stability of p53 and expression of p53 target genes, such as p21(Waf1), PUMA and NOXA, while hSRBC-mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function. Our findings suggest that hSRBC is a novel tumor suppressor whose epigenetic inactivation contributes to the malignant progression of gastric tumors, in part, through attenuated p53 response to stresses., ((c) 2007 Wiley-Liss, Inc.)

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. To explore the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor in gastric tumorigenesis, we investigated the expression and mutation status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas Smac/DIABLO and HtrA2 transcripts were normally expressed in all cancer specimens we examined, XAF1 transcript was not expressed or present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 expression showed a strong correlation with stage and grade of tumors, and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detectable alteration in cancers. Whereas loss of heterozygosity within the XAF1 region or somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. The 5' upstream region of the XAF1 gene encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Moreover, transcriptional silencing of XAF1 was tightly associated with hypermethylation of seven CpGs located in the 5' proximal region (nucleotides -23 to -234). Additionally, loss or abnormal reduction of XAF1 expression was found to inversely correlate with p53 mutations, suggesting that epigenetic inactivation of XAF1 and mutational alteration of p53 might be mutually exclusive events in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human gastric tumors.

Mutational alterations of PTEN and PIK3CA, which negatively and positively regulate PI3-kinase activity, respectively, have been observed in many types of human cancer. To explore the implication of PTEN and PIK3CA mutations in gastric tumorigenesis, we characterized the expression and mutation status of the genes in 126 gastric tissues and 15 cell lines. Expression of PTEN transcript was abnormally low in 5 of 15 (33%) cell lines and 20 of 55 (36%) primary carcinomas, whereas 0 of 71 noncancerous tissues including 16 benign tumors showed altered expression. Allelotyping analysis using an intragenic polymorphism (IVS4+109) revealed that 14 of 30 (47%) informative cases carried LOH of the gene, which is closely linked to low expression. The LOH rate was significantly higher in advanced tumors [12 of 19 (63%)] compared to early-stage tumors [2 of 11 (18%)] and more frequent in poorly differentiated tumors [9 of 13 (69%)] than well- or moderately differentiated tumors [5 of 17 (29%)]. Interestingly, however, none of the LOH tumors carried mutational disruption of the remaining allele, suggesting haploinsufficiency of PTEN in gastric tumorigenesis. Methylation studies revealed that PTEN pseudogene, but not PTEN, is methylated in cell lines and primary tumors, indicating that PTEN is not a target of epigenetic silencing in gastric cancers and that the pseudogene should be considered more carefully in methylation analysis of the PTEN promoter. Genomic amplification of PIK3CA was found in 9 of 15 (60%) cell lines and 20 of 55 (36.4%) primary tumors but in no noncancerous tissues. Furthermore, PIK3CA amplification was predominantly detected in tumors with no PTEN alterations, suggesting that mutations of PTEN and PIK3CA are mutually exclusive events in gastric tumorigenesis. Amplification of PIK3CA was strongly associated with increased expression of PIK3CA transcript and elevated levels of phospho-AKT. Collectively, our data reveal that 13 of 15 (87%) gastric cell lines and 31 of 55 (56%) primary carcinomas harbored either amplification of PIK3CA or abnormal reduction of PTEN. Mutually exclusive alterations of PTEN and PIK3CA also suggest that mutations of either gene could activate the PI3-kinase/AKT signaling pathway, which is directly linked to the malignant progression of gastric tumor cells., (Copyright 2003 Wiley-Liss, Inc.)

Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non-cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis.

p53R2, a recently identified putative tumor suppressor located at 8q23.1, encodes a protein with striking similarity to a small subunit of ribonucleotide reductase. p53R2 is directly induced by wild-type p53 and involved in the p53 checkpoint for repair of damaged DNA, raising the possibility that mutational inactivation of p53R2 may contribute to the development and progression of human malignancies. To explore the p53R2's candidacy for a suppressor in gastric tumorigenesis, we examined the expression and mutation status of p53R2 in 166 gastric specimens including 90 primary adenocarcinomas and 15 cell lines. In response to genotoxic damages, p53R2 transcription was clearly activated in wild-type but not mutant-type p53-carrying cells while basal expression of p53R2 in undamaged cells showed no association with the mutational status of p53. Host cell reactivation assay revealed that p53R2 enhances DNA repair efficiency and plays a role in the p53-mediated repair of damaged DNA, whereas no significant effect of p53R2 on cell growth and apoptosis was detected in flow cytometry and [(3)H]thymidine incorporation assays. p53R2 transcript was expressed in all normal and tumor tissues and its expression levels were not significantly different between normal and malignant carcinoma tissues. p53R2 expression showed no correlation with stage, grade and histological types of tumors. Moreover, no tumor-specific reduction of p53R2 was detected in 30 matched sets. Mutational analysis of p53R2 in 105 carcinomas including 15 cell lines also failed to detect any evidences for genomic deletion or somatic mutations leading to amino acid substitutions or frameshift whereas 31% (28 of 90) of the same primary tumors showed p53 alterations. Whereas 82% (23 of 28) of the mutant p53-carrying primary tumors expressed abnormally low p21(Waf1), no association of p53R2 expression with the p53 status was recognized, suggesting that basal transcription of p53R2 is regulated through the p53-independent mechanism. Collectively, our study indicates that although p53R2 is induced in a p53-dependent manner and involved the p53-mediated DNA repair in gastric epithelial cells, it is not a critical target of genetic inactivation in gastric tumorigenesis., (Copyright 2002 Wiley-Liss, Inc.)