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DNA methylation is an epigenetic modification and has significant effect on the genes. DNA methylation could be found on the Cytosine nucleotide of the CpG dinucleotide making it 5-methyl cytosine. Enzymes involved in catalyzing these reactions are DNMT1, DNMT3A, and DNMT3B. Methylation of DNA renders effect on the gene when it concentrates in the CpG island of the gene promoter. The Somatostatin gene SST, known for its tumor suppressor activity in gastrointestinal cells, pancreas, still more cells is known to be hypermethylated at its promoter region in the cases of cancer. This could be of possible reason that the Hypermethylation of SST promoter region has inhibited its transcription and tumor suppressor activity by silencing the gene. DNA methylation changes on the gene promoter could hence silence the gene transcription bringing in improper regulation within the cell when such gene plays an important role in cell function.
'Epigenetics' refers to heritable changes in gene expression that do not result from alterations in the gene nucleotide sequence. When DNA is methylated in the promoter region of genes, where transcription is initiated, genes are inactivated and silenced.
This event of DNA methylation plays a significant role in the development and progression of cancer. Mutations occurring in oncogenes frequently result in a gain of function, while mutations or deletions associated with tumor suppressor genes cause a loss or inactivation of negative regulators. Loss of function, however, can also occur through epigenetic changes such as DNA methylation.[1,2] In cancer, epigenetic silencing through DNA methylation occurs at least as frequently as mutations or deletions and leads to aberrant silencing of normal tumor-suppressor function.[2,3]
Epigenetic mechanisms (by DNA methylation) could be the leading cause of silencing of Somatostatin expression in gastric cancer. DNA hypermethylation and loss of expression of SST has been shown also in colon and esophageal cancer.[5,6]
In the coding region of a gene CpG dinucleotide occurs in the proportion of 1 in 100 nucleotides while the gene promoter region has denser CpG' s occurring 1 in 10 nucleotides. Hence referred to as "CpG island''. CpG methylation occurs when a methyl group adds to the 5â€² of cytosine catalyzed by enzymes resulting in 5-methyl cytosine. De novoÂ DNA methylation is mediated by methyltransferases enzymes such as DNMT3A and DNMT3B.
The DNA methylation helps in maintaining the transcriptional silence in non-expressed or non-coding regions of the genome (Heterochromatin). For example, the highly condensed and transcriptionally inactive pericentromeric heterochromatin is heavily methylated. By contrast, these sites are generally unmethylated in promoter regions of euchromatin.[1,2,8]
Silencing of Somatostatin (SST), a Tumor suppressor gene (TSG) could be by the hypermethylation of DNA which contributes in the tumor formation of Gastric Cancer, colon and esophageal cancer.[5, 6]
EPIGENETIC GENE SILENCING AND HUMAN CANCER:
According to the widely accepted 'two-hit' hypothesis of carcinogenesis proposed by Knudson,Â "loss of function of both alleles in a given gene (e.g. a tumor suppressor) is required for malignant transformation". In familial cancer mutations in the TSG occurs in germ line resulting in germ line mutations where wild type allele is lost. In sporadic cancer, the existence of one wild type allele is lost by loss of Heterozygosity, deletions or by different mutations.
Similar results can be achieved through epigenetic gene inactivation by aberrant promoter methylation. In cancer cells, methylation within promoters serves to turn off critical genes which normally suppress tumorigenesis. Tumor suppressor genes and those encoding cell adhesion molecules and growth-regulatory proteins are often silenced in hematopoietic malignancies by DNA hypermethylation leading to improper and immature cell division and tumor formation.Â Hence it could be the hypermethylation of the DNA promoter regions (at their CpG's) which initiates the mis-regulation, supporting cancer formation.
Somatostatin (SST) was first identified in ovine hypothalamus as a growth hormone release-inhibitory factor. SST, which is present in the "q" arm of human chromosome 3 is a regulatory peptide, produced by neuroendocrine (Larger amounts), inflammatory, and immune cells (smaller amounts) in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors (termed SSTR1-5) that are encoded by separate genes segregated on different chromosomes. SST producing cells occur at high densities throughout the CNS, in the gastrointestinal tract, pancreas, and in the gut. In the gastrointestinal tract, SST regulates endocrine and exocrine secretion, modulates motor activity, and is the primary inhibitor of gastrin-stimulated gastric acid secretion.
Several in vitro and in vivo studies have suggested that SST functions as a tumor suppressor gene in human cancers. SST suppresses tumor growth indirectly through 3 mechanisms, They are by: regulating the release of mitogenic hormones and growth factors, inhibiting neoplastic angiogenesis, and modulating the immune system.Â SST directly suppresses cell growth in an autocrine manner, principally via SST receptor type 2, which is widely expressed in both normal and cancerous colonic epithelial cells.Â
Aberrant methylation of promoter CpG islands upstream of tumor suppressor genes is established as a major epigenetic mechanism of gene inactivation in tumorigenesis.
SILENCING OF SST BY DNA METHYLATION IN GASTRIC CANCER:
SST is known to suppress tumor growth through distinct mechanisms that involve inhibition of growth factors and hormones, reduction in vascularization, and regulation of the cells immune system.[6, 16, 17]
In Gastric Adenocarcinoma; mRNA expression of SST is frequently down-regulated. TheÂ SSTÂ gene promoter CpG Island (from âˆ’83 to +678Â bp from the transcription start site) is hypermethylated at its promoter CpG' s in the gastric carcinoma compared with normal tissue. DNA hypermethylation of the SST promoter region (CpG Island) reveals that it's the epigenetic mechanism (DNA hypermethylation) which would be the leading cause of silencing of SST expression in gastric cancer.Â
Invitro 5-aza-dC (DNA methylase inhibitor) treatment in AGS Gastric cancer cell line (cell line had no detectable level of SST mRNA) led to reduction of DNA methylation from 95 to 76% and re-expression of SST mRNA. 5-Aza dC treatment combined with Trichostatin A (TSA), Histone deacetylase inhibitor, significantly reduced DNA methylation from 95 to 43% and demonstrated a synergistic potent increase in mRNA expression confirming the epigenetic silencing of SST.
SILENCING OF SST BY DNA METHYLATION IN COLON CANCER:
A Genome-Wide Search identified silencing of SST gene by DNA hypermethylation which resulted in colon cancer. This epigenetic mechanism leads to down regulation of SST mRNA. The CpG Island in SST promoter region was DNA hypermethylated in colon cancer cell lines similar to that of gastric cancer SST gene silencing. The methylationÂ status of SST promoter region was analyzed in both primary cancers and noncancerous colonic mucosae. The result revealed frequent methylation of SST promoter region in primaryÂ colon cancers. Normal colonic mucosae also demonstrated a certain degree ofÂ methylation. However, theÂ methylationÂ levels ofÂ SST gene was significantly lower in normal colonic mucosae than inÂ colon cancersÂ (PÂ <.001). Studies also revealed that there was no significant association of age, gender, tumor site, or histologic differentiation with theÂ methylationÂ levels of SST.
Invitro treatment of colon cancer cell line with 5-Aza dC drug showed considerable up-regulation of m RNA level which was absent when treated without 5-Aza dC molecules.
The biological importance of DNA methylation in the regulation of Somatostatin gene expression and its role in Gastric and colon cancer is highly recognized.
Somatostatin, which is a neuroendocrine gastrointestinal peptide hormone gene is silenced in gastric adenocarcinoma and colon cancer by hypermethylation in its promoter region (at CpG's) of SST gene. This epigenetic mechanism leads to down regulation of SST mRNA finally silencing the SST gene from performing its Tumor Suppressor role.
Treatment with 5-aza-dC, inhibitor of DNA Methylase and Trichostatin A (TSA), inhibitor of histone deacetylase (HDAC) in Gastric cancer cell line, and 5-aza-dC treatment alone in colon cancer cell line can reactivate the epigenetically silenced Somatostatin gene and has been shown to restore normal mRNA expression as well as normal gene function.
These basic facts on SST gene can pave a way for further focused studies on the role of SST gene in other cancer types apart from the gastric and colon cancer. Analyses of SST gene as potential Biomarker which could be used for early diagnosis of gastric carcinoma and in colon cancer. However further studies would be needed to document the biological outcome of loss of SST in gastric cancer and colon cancer.