Study Of Truncation Mutation Of A Regulatory Element In The HGF Gene Promoter Associated With Gastric Carcinogenesis

IntroductionGastric cancer is the second most common malignancy worldwide. It develops as a result of multistep process characterized by the accumulation of genetic alteration involving a variety of oncogenes and tumor suppressor genes. Therefore, better knowledge of the changes in genes occurred during gastric carcinogenesis may lead to improvements in diagnosis, treatment and prevention.HGF, hepatocyte growth factor, is an important paracrine mediator of mesenchymal-epithelial interaction, instructing epithelial cells to undergo processes such as cell proliferation, survival, migration, and morphogenesis. HGF exerts its effects via binding to and activating a tyrosine kinase transmembrane cell surface receptor known as Met. HGF gene expression is highly cell type specific and is restricted to mesenchymal cells such as fibroblasts.Normal epithelial cells do not express this gene, although all epithelial cells express the HGF receptor. Studies have established that HGF/Met signaling actively contributes to the process of neoplastic transformation, tumor metastasis, and tumor maintenance. In the case of breast adenocarcinomas, activation of autocrine HGF expression in the cancer cells and its overexpression are believed to contribute to neoplasia. Clinical studies have reported that high levels of HGF mRNA and protein within the breast carcinoma tissue are associated with poor survival in patients.Studies uncovered that the HGF transcript is expressed in human breast adenocarcinoma cells but not in the normal mammary ductal epithelium. In the case of gastric cancer, high levels of HGF are believed to contribute to the process of neoplastic transformation, tumor metastasis, and tumor maintenance.Study has been established to scan human carcinoma cell lines that aberrantly expressed the HGF gene for promoter mutation. The results led to the identification of a novel 30-bp cis-acting DNA element within the proximal promoter region of the human HGF gene. The element consists of a tract of 30 deoxyadenosines (30As; poly [dA]) which termed "deoxyadenosine tract element" (DATE). Functional studies revealed that wild-type DATE alters chromatin structure and silences the HGF promoter in normal epithelial cells through interactions with several nuclear factors and that DATE deletion mutagenesis (DATE truncation or shortening) distorts this DNA structure, resulting in differential binding of several factors to this element and its adjacent sites. This leads to recruitment of chromatin modifying and transcription factors such as C/EBP-βand poly (ADP-ribose) polymerase (PARP), theculmination of which is activation of the HGF promoter in the tumor cells.In this study,wei evaluated the DATE genotype in samples from 80 cases of human gastric carcinoma and matched normal adjacent tissue and detected HGF expression in tumor cells and tissues The results revealed that DATE is a target of deletion mutagenesis (DATE truncation) in gastric cancer and that truncated DATE significantly associates with high HGF gene expression in tumor cells, Notably, HGF gene expression is significantly higher in the mammary tissues of individuals with truncated DATE than in tissues of individuals with wild-type DATE, These results not only shed light on our understanding of the genetic basis of human gastric cancer tumorigenesis but also led to develop better diagnostic and therapeutic targets for patients with gastric cancer.Materials and methods1. Scanning human gastric carcinoma cell lines, tumor tissues and peripheral blood of normal individuals for DATE truncation.In order to examine DATE truncation, we amplified DATE in tumor tissues, cell lines and peripheral blood of normal individuals DNA by PCR, The amplified products were first analyzed in 8% denaturing polyacrylamide gel containing 8 M urea. In cases in which the DATE size variation seems small, DNA sequencing were performed.2. Detection of HGF gene expression in tumor cells and tissus by RT-PCR.In order to reveal that truncated DATE significantly associates with high HGF gene expression in tumor cells and tumor tissues, HGF gene expression was detected in tumor cells and thirty gastric cancer tissues and matching normal tissues by RT-PCR.3. Study of truncated DATE associated with microsatellite Instability.We used 2 poly(CA) microsatellites D5S396 and D5S2056 to establish the MSI status of six gastric cancer primary tumors with truncated DATE to find that truncated DATE associates with microsatellite instability.Results1. Truncation mutation of DATE in the HGF gene promoter region correlates with activation of HGF gene expression in human gastric carcinoma cell lines.HGF of HeLa cells had a stretch of 30As in their DATE identical to the normal human HGF promoter sequence, indicating that these cells had a wild-type DATE, whereas those exhibited a truncated mutant version of DATE in which the number of As was reduced by 5 bp or more. We screened carcinoma cell lines for truncation mutation of DATE. We found that some gastric cancer cell lines (MGC803, MKN28 and HGC27) had truncated mutation of DATE. In MGC803 cells, DATE length was truncated by 5As (thus, these cells had a DATE genotype of 25As in heterozygous manner) and HGC27cells were heterozygous for short alleles of 27As and 25As, while in MKN-28cells, both alleles were truncated by 5 nucleotides (25As) in DATE. On the other hand, GES-1, SGC7901, BGC823 and AGS cells were homozygous for 30As, 29As,29As, and 28As respectively. Detection of HGF gene expression in tumor cells (GES-1, SGC7901,MGC803,BGC823 and AGS) by RT-PCR, we found HGF gene expression was noted in MGC803 cells with truncated DATE but not in other cells with wild-type DATE.2. DATE is a target of deletion mutagenesis in human gastric cancer tissue, and its truncated version associates with aberrant HGF expression.We evaluated the DATE genotype in samples 80 cases of human gastric carcinoma and matched normal adjacent tissue, we found that approximately 15% (12/80) gastric cancer tissues possessed the truncated DATE variant. To be conservative in these analyses, we designated DATE as a truncated variant if it had 25As or fewer as we described above. The size of truncated DATE among different gastric tumor cases ranged from lOAs (loss of 20As) to 25As (loss of 5As). In 58% (7/12) of the cancer cases with shortened DATE, truncated DATE was present only in the tumor and not in the corresponding adjacent normal tissue, indicating that DATE is unstable and prone to deletion mutagenesis in tumor cells. HGF mRNA was highly expressed in cancer tissues with the truncated DATE, since it was readily detectable by 32 cycles of RT-PCR, but little or no HGF expression was detected in tissues with wild-type DATE under these conditions.3. Truncated DATE significantly associates with gastric cancer.Our data suggest that DATE is most likely polymorphic in nature since in some cases both tumor and the corresponding normal tissue had the truncated DATE variant. Therefore, to address this possibility, we determined the incidence of truncated DATE using peripheral blood lymphocyte genomic DNA from 100 healthy Chinese adult human subjects. We observed an incidence of truncated DATE (DATE with 25As or less) in the general population of approximately 4%(4/100). Comparison of the prevalence of truncated DATE in gastric cancer cases with that of normal subjects indicated that truncated DATE significantly correlates with breast cancer incidence (15%vs.4%, P=0.01).4. Study of truncated DATE associated with microsatellite Instability.We used 2 poly(CA) microsatellites D5S396 and D5S2056 to establish the MSI status of six gastric cancer primary tumors with truncated DATE, we found that 5 gastric cancer tissues showed MSI by the use of D5S396 and D5S2056 microsatellites, indicating that analysis of DATE was able to confirm the MSI status 5 of 6 gastric tumors(83.3% efficiency), therefore DATE can be an indicator of MSI in gastric cancer.Conclusion(1)Gastric cancer harbor a truncated DATE variant in their gastric tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression.(2)Analysis of DATE was able to confirm the MSI status and can be an indicator of MSI in gastric cancer.