Tumorigenesis Of Gastric Carcinoma Research Articles

MiR-429 regulates gastric cancer cell invasiveness through ZEB proteins.

MicroRNAs (miRNAs) play an essential role in the tumorigenesis of gastric carcinoma (GC). MiR-429 has been recently reported to inhibit GC growth; however, whether miR-429 may also regulate the invasion of GC cells is unknown. Here, we showed that miR-429 levels were significantly decreased and ZEB1 and ZEB2 levels were significantly increased in GC specimens, compared to the paired adjacent non-tumor gastric tissue. Moreover, the levels of miR-429 and ZEB1 or ZEB2 inversely correlated in GC specimens. Bioinformatics analyses showed that miR-429 targeted the 3'-untranslated region of both ZEB1 and ZEB2 mRNA to inhibit their translation, which was confirmed by luciferase reporter assay. Overexpression of miR-429 inhibited ZEB1/2-mediated cell invasiveness, while depletion of miR-429 augmented it. Together, our data suggest that miR-429 suppression in GC promotes ZEB1/2-mediated cancer cell invasion.

MiR-429 Induces Gastric Carcinoma Cell Apoptosis Through Bcl-2

Background/Aims: MicroRNAs (miRNAs) play an essential role in the tumorigenesis of gastric carcinoma (GC). MiR-429 has been recently reported to inhibit GC growth, but the underlying mechanisms are not clear. Methods: Here, we studied the levels of miR-429 and anti-apoptotic protein Bcl-2 in GC specimens. We performed bioinformatics analyses and used luciferase-reporter assay to analyze the relationship between miR-429 and Bcl-2 in GC cells. Cell survival upon Fluorouracil treatment was analyzed in a CCK assay. Cell apoptosis was measured by flow cytometry based FITC Annexin V apoptosis detection assay. Results: MiR-429 levels were significantly decreased and Bcl-2 levels were significantly increased in GC specimens, compared to the paired adjacent non-tumor gastric tissue. Moreover, the levels of miR-429 and Bcl-2 inversely correlated in GC specimens. MiR-429-low subjects had an overall inferior survival, compared to miR-429-high subjects. Bioinformatics analyses showed that miR-429 targeted the 3'-UTR of Bcl-2 mRNA to inhibit its translation, which was confirmed by luciferase-reporter assay. Overexpression of miR-429 inhibited Bcl-2-mediated cell survival against apoptosis induced by Fluorouracil, while depletion of miR-429 augmented it. Conclusion: Our data suggest that miR-429 suppression in GC promotes Bcl-2-mediated cancer cell survival against chemotherapy-induced cell death. Re-expression of miR-429 levels in GC cells may enhance cancer apoptosis during chemotherapy.

Aberrant methylation of the SPARC gene promoter and its clinical implication in gastric cancer.

Secreted protein acidic and rich in cysteine (SPARC) gene has been shown to be epigenetically silenced in several cancers. We investigated the loss of expression and promoter methylation of this tumor suppressor gene in gastric cancers and correlated the data with clinicopathological features. We observed the loss of SPARC mRNA and SPARC protein expression in 7 of 10 (70%) gastric cancer cell lines. Upon treatment of expression-negative cell lines with a demethylating agent, expression of mRNA and protein was restored in all cells. Methylation rate of SPARC gene was 80% in ten gastric cancer cell lines and 74% (163 of 220) in primary tumors, while it was 5% in normal gastric mucosa (n = 40). In intestinal gastric cancer, SPARC methylation correlated with a negative prognosis (P < 0.001; relative risk 2.754, 95% confidence interval 1.780–4.261). Immunostaining revealed that SPARC protein was overexpressed in stromal fibroblasts adjacent to neoplastic epithelium but rarely expressed in the primary gastric cancer cells. These results implicate SPARC promoter methylation as an important factor in the tumorigenesis of gastric carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and the potential clinical importance in human gastric cancers.

Chromosome 8q as the most frequent target for amplification in early gastric carcinoma.

Early gastric carcinoma (GC) is considered to be a curable cancer, as it progresses to the advanced stage following varying durations. Understanding the early stage of GC may provide an insight into its pathogenesis and contribute to reducing the mortality rate of this disease. To investigate the genomic aberrations associated with 22 cases of early GC, high-density microarray comparative genomic hybridization was performed in the present study. The most notable finding was copy number gains (log2 ratio >0.25) on the long arm of chromosome 8, which occurred in 77.3% (17/22) of GC cases, and the delineated minimal common region was 8q22.1-q24.3. More specifically, two amplified (log2 ratio >1) loci in the 8q22.1-q24.3 region were detected in 18.2% (4/22) of GC cases. The first loci covered a region of 102.4–107.9 kb, mapping on 8q22.3-q23.1, and comprised the transcription factor CP2-like 3 gene. The second loci, spanning 128.7–145.7 kb on 8q24.21-q24.3, comprised the representative oncogene of myelocytomatosis. Furthermore, the following possible target genes that were not previously considered to play a pathogenic role in GC were identified: Plasmacytoma variant translocation 1, cysteine/histidine rich 1, kinesin family member C2, forkhead box H1, protein phosphatase 1 regulatory subunit 16A, glutamic-pyruvate transaminase, LOC113655 and RecQ protein-like 4. In the present study, previous findings showing that 8q mutations accumulate early during the multistage pathogenesis of GC were confirmed and expanded upon. The confirmation of previously reported 8q gains and the identification of novel target genes at 8q22.1-q24.3 amplified chromosomal sites should aid in improving our understanding of the molecular mechanisms underlying the tumorigenesis of early GC.

DNA methylation of trefoil factor 1 (TFF1) is associated with the tumorigenesis of gastric carcinoma

Trefoil factor1 (TFF1) is a tumor suppressor gene that encodes a peptide belonging to the trefoil factor family of protease‑resistant peptides. Although TFF1 expression is frequently lost in gastric carcinomas (GCs), the tumorigenic pathways that are affected have yet to be determined. The aim of the current study was to identify the mechanism(s) by which the TFF1 gene is regulated in gastric carcinogenesis. In this study, TFF1 was shown to be silenced or downregulated in gastric tumor tissue compared with matched non‑cancerous tissue. In addition, human gastric cells weakly expressed TFF1. The hypermethylation status in the promoter CpG islands appeared to be correlated with TFF1 expression levels in gastric cell lines or specimen tissue. Further molecular analysis indicated that the CpG islands play a role in the promoter activity of the TFF1 gene. The expression of TFF1 and DNA methylation of its promoter affected cell proliferation and apoptosis. The expression of TFF1 in gastric cell lines was restored with a demethylating agent, 5‑azacytidine. Low expression of TFF1 in gastric cell lines and cancer tissue is associated with TP53. In conclusion, the current study demonstrates that DNA methylation is a key mechanism of silencing TFF1 expression in human gastric cells and TFF1 gene hypermethylation of the CpG islands is a potential biomarker for GC.

Aquaporin 5 promotes the proliferation and migration of human gastric carcinoma cells

Aquaporin 5 (AQP5) promotes the progression and invasion of several cancers, but its role in the tumorigenesis of human gastric carcinoma (GC) has not been clearly defined. Here, we investigated the potential functions of AQP5 in the proliferation and migration of human GC. RT-PCR and western blotting were used to detect the expression of AQP5 in human GC cell lines. Immunohistochemistry was applied to evaluate the expression of AQP5 in human GC tissues and corresponding normal tissues. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its inhibitor, acetazolamide (AZA), cell proliferation and migration of AGS cells were analyzed by MTT assay, colony formation assay, and wound healing assay. Heterogeneous expression of AQP5 mRNA and protein was observed in human GC cell lines MKN45, MKN28, AGS, and SGC7901. AQP5 was up-regulated in GC tissues in comparison to corresponding normal tissues. AQP5 protein was mainly localized in the cell membrane. Overexpression of AQP5 was correlated with enhanced lymph node metastasis. In vitro, overexpression of AQP5 notably enhanced, while inhibition of AQP5 by AZA significantly attenuated the proliferation and migration of AGS cells. Our data indicate that AQP5 may play an important role in the tumorigenesis and progression of human GC and suggest that AQP5 is a potential therapeutic target against GC.

Telomere shortening occurs early during gastrocarcinogenesis

When telomeres are shortened to a critical length, they will initiate chromosomal instability (CIN) and may finally cause tumorigenesis. The purpose of the present study was to evaluate the shortened telomere as a potential biomarker for tumorigenesis in gastric carcinoma. The telomeres in matched cancer and adjacent noncancer mucosa samples from 86 gastric carcinoma patients were measured by real-time polymerase chain reaction (PCR). According to the International Union Against Cancer (UICC), tumor stages were classified into four groups: stage I (n = 23), stage II (n = 20), stage III (n = 23), and stage IV (n = 20). Telomere length decreased with aging in both adjacent noncancer mucosa and cancer tissue (r = -0.261 (P = 0.008) and r = -0.27 (P = 0.012), respectively). The telomere length of UICC stage I tumors was significantly shorter than the average telomere length in adjacent noncancer mucosa (P = 0.023). Telomere length increased gradually with increasing UICC stage (P = 0.032). The telomere length of UICC stage IV tumors was significantly longer, when compared to that in noncancer mucosa (P = 0.019) and stage I tumors (P = 0.002). In summary, telomere length undergoes shortening in early stage gastric carcinoma and lengthening in advanced gastric carcinoma. Additionally, telomere shortening may initiate the tumorigenesis of gastric carcinoma.

Correlations of P21-activated kinase 1 expression to clinicopathological features of gastric carcinoma and patients' prognosis

Recent studies proved that P21-activated kinase 1 (PAK1) is highly expressed in many kinds of tumor and plays an important role in genesis, development, and metastasis of tumor. We aimed to detect the expression of PAK1 in gastric carcinoma and to analyze its relationship with clinicopathological features and prognosis of gastric carcinoma. Tissue microarray and immunohistochemical staining were performed to detect PAK1 in paraffin specimens of 189 gastric carcinomas, 54 paracancer tissues, 40 lymph nodes and 30 healthy tissues. Clinicopathologic features and follow-up data of the patients were analyzed by the Chi2 test and the Kaplan-Meier method. Positive rate of PAK1 was 73.0% in gastric carcinoma, 57.4% in paracancer tissues and 23.3% in healthy controls (Chi2 = 29.364, P < 0.05). Expression of PAK1 was significantly correlated with tumor size, tumor differentiation, lymph node metastasis, Lauren classification and invasive depth (all P < 0.05). The positive rate of PAK1 was significantly higher in primary gastric carcinomas than in metastatic lymph nodes (75.0% vs. 52.5%, Chi2 = 4.381, P < 0.05). Survival analysis using the Kaplan-Meier method showed that the expression of PAK1 was a predictor for poor prognosis of the patients with gastric carcinoma (Chi2 = 6.857, P < 0.01). Expression of PAK1 is an early molecular event in the tumorigenesis of gastric carcinoma. It is also closely correlated the development of gastric carcinoma and the patients' prognosis.

Assessment of the Relationship between Helicobacter pylori and Lung Cancer: A Meta-analysis

Helicobacter pylori infection has been thought to play a critical role in gastric carcinoma tumorigenesis and progression. Several studies have been devoted to the relationship between H. pylori infection and lung cancer risk and have generated inconclusive results. In this study we aimed to evaluate the potential association of H. pylori infection with lung cancer risk. We conducted a search in Medline, OVID, EMBASE and CNKI, covering all published papers until October 2008. The relevant published papers were deliberately selected according to the established inclusion criteria for publications. Essential data were then extracted from the included studies and further analyzed by a systematic meta-analysis. A total of 98 papers were identified. Of these, four case-control studies met the inclusion criteria and thus were finally selected. Lung cancer risk for H. pylori infection was 3.24-fold (95% CI=1.11-9.47) (Z=2.15, p<0.05) compared with the controls. The pooled data suggest infection of H. pylori as a potential risk factor for lung cancer.

Loss of heterozygosity analysis of a candidate gastric carcinoma tumor suppressor locus at 7q31

Gastric carcinoma is one of the most common malignancies in Asia. Although the allelic deletion of 7q has been reportedly associated with primary gastric carcinoma tumorigenesis, no predisposing genes in this region have been identified so far. Here, we report the results of genotype and loss of heterozygosity (LOH) analysis on 7q in this tumor. A panel of nine microsatellite markers distributed over the whole chromosome 7q was used for genotyping primary gastric carcinomas. Of 72 primary tumors LOH of D7S486 occurred in 24.0% (12/50) of cases. Fine mapping with 12 additional markers flanking D7S486 resulted in LOH of 30.36% (17/56) and defined one minimal deleted region in primary gastric carcinomas, a 90-kilobase region bounded by D7S2543 and D7S486 at 7q31.2. The allelic deletion correlates statistically with clinicopathologic variables. Our data suggest a possible link between putative tumor suppressor genes and gastric carcinoma in the 7q31 region.

Monocyte Chemoattractant Protein-1 Transfection Induces Angiogenesis and Tumorigenesis of Gastric Carcinoma in Nude Mice via Macrophage Recruitment

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that has various roles in tumor development and progression. We previously reported that expression of MCP-1 is associated with macrophage infiltration and tumor vessel density in human gastric carcinomas. The present study was undertaken to obtain direct evidence that MCP-1 participates in recruitment of macrophages and induction of angiogenesis. We did transfection experiments to analyze the role of MCP-1 in tumorigenicity and angiogenesis in gastric carcinoma in nude mice. The human MCP-1 gene cloned into the BCMGS-Neo expression vector was transfected into the human gastric carcinoma TMK-1 cell line. We examined tumor volumes with the ectopic s.c. xenograft model and tumorigenicity with the orthotopic gastric xenograft model. We determined intratumor microvessel counts and tumor-infiltrating macrophage counts by immunohistochemical staining. There was no difference in in vitro proliferation between MCP-1-transfected TMK-1 cells and mock-transfected (control) cells; however, MCP-1 transfectants induced tumor growth in ectopic xenografts and increased tumorigenicity and induced lymph node metastases and ascites in orthotopic xenografts. In both ectopic and orthotopic xenograft models, strong infiltration of macrophages was observed within and around the tumors after implantation of MCP-1 transfectants whereas fewer macrophages were seen after inoculation of control cells. The microvessel density was significantly higher in tumors produced by MCP-1 transfectants than in control tumors. MCP-1 produced by gastric carcinoma cells may regulate angiogenesis via macrophage recruitment. MCP-1 may be a potential target for antiangiogenic therapy for gastric carcinoma.

Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas

To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level. One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (chi(2) = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLF1 transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.

Kinase domain mutation of NTRK3 gene is uncommon in gastric carcinomas

Protein kinases regulate intracellular signal-trans-duction pathway mediating cell proliferation, differ-entiation and survival [1]. Protein kinase family isone of the most frequently mutated gene familyfound in human cancers and thus are potentialtherapeutic targets for human cancers [1]. Neuro-trophic tyrosine kinase receptor type 3 (NTRK3),also referred to as TrkC, is a receptor tyrosine kinaseand plays an important role in the development ofneural tissues [2]. NTRK3 expression is not re-stricted to neural tissues, and various types of tissues,including the gastrointestinal epithelia (stomach,small intestine and colon), have also been shown toexpress NTRK3 [3]. These data suggest a role ofNTRK3 in the growth and maintenance of thegastrointestinal cells.Recently, Bardelli et al. [4] analyzed 138 tyrosinekinase genes in 147 colorectal cancer tissues for thedetection of the somatic mutations. They identified46 mutations in 14 genes. Of those, seven genes(NTRK3,FES,KDR,EPHA3,NTRK2, MLK4andGUCY2F) were mutated in more than one tumor.NTRK3 gene mutations were found in six (4.1%) ofthe 147 colorectal cancers. Of note, the NTRKmutations were exclusively identified in the kinasedomain. Because NTRK3 is expressed in gastricepithelium as well as in colorectal epithelium [3], wehypothesized that gastric carcinomas might alsoharbor NTRK3 mutation. To see whether alterationof NTRK3 gene is involved in the tumorigenesis ofgastric carcinoma, we analyzed NTRK3 gene for thedetection of somatic mutations in gastric carcinomasby polymerase chain reaction (PCR)-based singlestrand conformation polymorphism (SSCP) assay.We analyzed methacarn-fixed tissues of 140 gas-tric carcinomas. All of the patients of the cancerswere Asians (Koreans). The gastric carcinomasconsisted of 60 diffuse-type, 49 intestinal-type and31 mixed-type gastric adenocarcinomas by Lauren’sclassification, and 25 early and 115 advanced gastriccarcinomas according to the depth of invasion.Malignant cells and normal cells from the samepatients were selectively procured from hematoxylinand eosin-stained slides using a 30G1/2 hypodermicneedle (Becton Dickinson, Franklin Lakes, NJ)affixed to a micromanipulator, as described pre-viously [5]. DNA extraction was performed by amodified single-step DNA extraction method [5].Because NTRK3 mutations were previously de-tected only in the exons 15

Methylation of specific CpG sites in the promoter region could significantly down-regulate p16INK4a expression in gastric adenocarcinoma

Silencing of p16(INK4a) by methylation of the CpG islands in the promoter region has been found to be an alternative mechanism of inactivation in several tumors. However, in gastric carcinoma, the relationship between methylation status and the transcriptional silencing of the p16 gene remains to be clarified. In this study, we investigated whether methylation of a few specific CpG sites in the promoter region could significantly down-regulate p16 activity in the tumorigenesis of gastric carcinoma. By Southern analysis and bisulfite-modified genomic sequencing of 9 gastric-carcinoma cell lines, we found that the 5 cell lines (55.5%) not expressing p16 mRNA had methylated CpG sites at the promoter region of p16. In addition, we analyzed the p16-protein expression of 28 primary gastric carcinomas and their normal counterparts by immunohistochemical staining (IHC) on paraffin sections. Loss of p16 expression was detected in 6 cases (22%). In 5 out of these 6 (83%), the actual p16 gene was inactivated by de novo methylation of the promoter sites. Taken together, these results suggest a strong correlation between de novo methylation of a few specific CpG sites and transcriptional silencing of the p16 gene in gastric carcinoma.

Molecular studies into the role of CD44 variants in metastasis in gastric cancer.

CD44, an integral membrane glycoprotein expressed by many cell types, serves as the principal transmembrane hyaluronate receptor and might be a determinant of metastatic and invasive behaviour in carcinomas. The...

Mutations of the STK11 gene in sporadic gastric carcinoma.

Gastric carcinoma may occur sporadically or in association with hereditary diseases, such as Peutz-Jehgers syndrome (PJS). The PJS gene (named STK11 or LKB1) was mapped to 19p13.3 and recently cloned. Germ-line mutations of the gene have been detected in familial PJS patients and are predicted to predispose STK11 carriers to the development of a wide range of gastrointestinal and other neoplasms. To elucidate the etiological role of the STK11 gene in sporadic gastric carcinoma tumorigenesis, we analyzed 28 gastric carcinomas (22 of intestinal type and 6 of diffuse type) for STK11 gene mutations. STK11 gene mutations were detected in 3 of 28 gastric carcinomas but were not seen in the corresponding germ-line DNA sequence. In one tumor, a missense mutation, C-to-T transition, was detected at codon 324 resulting in proline to leucine substitution; in the other two, silent mutations were detected at codons 106 and 350, respectively. While these results suggest that somatic STK11 mutations are not common in sporadic gastric carcinomas, they may occur in a subset of these tumors.

Differential expression of the human metastasis adhesion molecule CD44V in normal and carcinomatous stomach mucosa of Chinese subjects.

CD44 is a cell surface adhesion molecule involved in cell-cell and cell-matrix interactions. Tumor cells transfected to overexpress the isoform CD44V readily gain access to lymph nodes and form distant metastases. Monoclonal antibodies directed at epitopes common to known CD44 isoforms were used to investigate CD44V expression in 30 normal gastric mucosa tissues, 64 different gastric adenocarcinomas, 20 metastatic adenocarcinoma lymph nodes and 4 established gastric carcinoma cell lines. In addition, CD44V gene expression in six gastric adenocarcinoma tissues and four gastric cancer cell lines were investigated by northern blotting. Immunohistochemistry screening of 30 subjects with normal gastric mucosa did not reveal expression of CD44 variants. Areas of intestinal metaplasia, a precancerous lesion, were stained with antibodies against either V5- or V6-containing isoforms of CD44. Tubular and signet-ring cell types of adenocarcinoma were strongly positive for epitopes encoded by CD44 variants containing exons V5 (41/49 and 10/10, respectively). Some tubular type adenocarcinomas (15/49) also expressed CD44 variants containing the V6 epitope. Tumor differentiation was closely related to CD44 V5 expression (P < 0.001). In addition, 18 of 20 gastric adenocarcinomas metastatic to lymph nodes expressed the V5 epitope of CD44 and 4 of 20 expressed the V6 epitope. Analysis of four established gastric adenocarcinoma cell lines revealed that two had moderate to strong expression of exons V5 and V6 of CD44. An antibody directed against CD44 variants containing exons V8 to V10 strongly stained all gastric adenocarcinoma cell lines. Northern blotting demonstrated that all four tumor cell lines and six gastric carcinoma mucosa tissues expressed CD44V. Generation of CD44 splice variants may be closely linked with gastric carcinoma tumorigenesis and differentiation. In addition, expression of CD44 variants containing exons V5 and V6 may be used as an indicator of evolving gastric cancer.