Aberrant expression of SOX9 is associated with gastrokine 1 inactivation in gastric cancers.

BACKGROUND: SOX9 belongs to the SOX [sry-related high-mobility group (HMG) box] family and acts as a transcription factor that plays a central role in the development and differentiation of multiple cell lineages. The aim of this study was to determine whether the GKN1 gene is involved in the development of gastric cancer by regulating SOX9. METHODS: The effect of GKN1 and ß-catenin on SOX9 expression was examined in GKN1 and ß-catenin-transfected AGS and MKN-1 gastric cancer cells. SOX9 expression was also determined in gastric cancer tissues and cell lines by Western blot analysis and immunohistochemistry. RESULTS: Ectopic expression of ß-catenin induced increased expression of SOX9 in AGS cells, whereas GKN1 decreased expression of SOX9 in AGS and MKN-1 cells. In addition, we found an inverse correlation between expression of SOX9 and GKN1 in gastric cancer tissues and cell lines. In immunohistochemistry, nuclear SOX9 expression was detected in 64 (34.6 %) of 185 gastric carcinomas and its expression was closely associated with GKN1 immunonegativity. There was no significant relationship between altered expression of SOX9 protein and clinicopathological parameters including overall survival. CONCLUSION: These data suggest that aberrant SOX9 expression by GKN1 inactivation may be involved in the development of sporadic gastric cancers as an early event.

Inactivation of the Gastrokine 1 gene in gastric adenomas and carcinomas.

Gastrokine 1 (GKN1) plays a role in the gastric mucosal defence mechanism and may be a gastric tumour suppressor. We have investigated whether inactivation of the GKN1 gene is involved in the development and/or progression of gastric cancers. GKN1 protein expression was examined in gastric adenomas and cancer and we also analysed GKN1 mutation and epigenetic alteration, DNA copy number change and mRNA transcript expression. The effect of GKN1 on cell proliferation and death was examined in wild-type GKN1-transfected AGS gastric cancer cells. Reduced or loss of GKN1 expression was detected in 36 (90%) and 170 (89.5%) of 40 adenomas and 190 gastric cancers, respectively. Statistically, there was no significant relationship between altered expression of GKN1 protein and clinicopathological parameters, including depth of invasion, location and lymph node metastasis (χ(2) test, p > 0.05). In western blot analysis, absence or reduced expression was found in 21 (84.0%) of 25 gastric carcinomas. No mutation was detected in gastric tumours, and hypermethylation of GKN1 gene was found in two tumours. DNA copy number and mRNA transcript of GKN1 were significantly decreased in gastric cancers. In functional analysis, AGS gastric cancer cells transfected with GKN1 wild-type showed marked inhibition of cell proliferation and induction of cell death. These data suggest that inactivation of the GKN1 gene may play an important role in the development of sporadic gastric cancers, as an early event.

Bioactive Sphingolipids as Major Regulators of Coronary Artery Disease.

Atherosclerosis is the deposition of plaque in the main arteries. It is an inflammatory condition involving the accumulation of macrophages and various lipids (low-density lipoprotein [LDL] cholesterol, ceramide, S1P). Moreover, endothelial cells, macrophages, leukocytes, and smooth muscle cells are the major players in the atherogenic process. Sphingolipids are now emerging as important regulators in various pathophysiological processes, including the atherogenic process. Various sphingolipids exist, such as the ceramides, ceramide-1-phosphate, sphingosine, sphinganine, sphingosine-1-phosphate (S1P), sphingomyelin, and hundreds of glycosphingolipids. Among these, ceramides, glycosphingolipids, and S1P play important roles in the atherogenic processes. The atherosclerotic plaque consists of higher amounts of ceramide, glycosphingolipids, and sphingomyelin. The inhibition of the de novo ceramide biosynthesis reduces the development of atherosclerosis. S1P regulates atherogenesis via binding to the S1P receptor (S1PR). Among the five S1PRs (S1PR1-5), S1PR1 and S1PR3 mainly exert anti-atherosclerotic properties. This review mainly focuses on the effects of ceramide and S1P via the S1PR in the development of atherosclerosis. Moreover, it discusses the recent findings and potential therapeutic implications in atherosclerosis.

Analysis of succinate dehydrogenase subunit B gene alterations in gastric cancers.

Recently, the succinate dehydrogenase subunit B gene, SDHB, has emerged as a novel tumor suppressor. In this study, we have examined the genetic and epigenetic alterations of the SDHB gene in sporadic gastric adenocarcinomas in order to investigate if the SDHB gene is involved in gastric carcinogenesis. The expression of SDHB proteins was also examined with immunohistochemistry and Western blot in 184 and eight gastric cancers, respectively. There was loss or reduced expression of SDHB in 45 (24.5%) of the 184 gastric cancers. Statistically, altered expression of SDHB was not associated with clinicopathological parameters, including tumor differentiation, location, depth of invasion, and lymph node metastasis (P > 0.05). Western blot analysis showed a reduced expression of SDHB in four (50.0%) of the eight paired gastric cancer tissues. Genetic analysis showed one missense mutation, GCC --> ACC (Ala --> Thr) at codon 29. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and protein expression analysis of the SDHB gene in gastric cancers. Our results suggest that genetic, epigenetic, and protein expression pattern alterations of the SDHB gene might play a minor role in the development or progression of gastric cancers.

Ceramide and Sphingosine 1-Phosphate in Liver Diseases.

The liver is an important organ in the regulation of glucose and lipid metabolism. It is responsible for systemic energy homeostasis. When energy need exceeds the storage capacity in the liver, fatty acids are shunted into nonoxidative sphingolipid biosynthesis, which increases the level of cellular ceramides. Accumulation of ceramides alters substrate utilization from glucose to lipids, activates triglyceride storage, and results in the development of both insulin resistance and hepatosteatosis, increasing the likelihood of major metabolic diseases. Another sphingolipid metabolite, sphingosine 1-phosphate (S1P) is a bioactive signaling molecule that acts via S1P-specific G protein coupled receptors. It regulates many cellular and physiological events. Since an increase in plasma S1P is associated with obesity, it seems reasonable that recent studies have provided evidence that S1P is linked to lipid pathophysiology, including hepatosteatosis and fibrosis. Herein, we review recent findings on ceramides and S1P in obesity-mediated liver diseases and the therapeutic potential of these sphingolipid metabolites.

Systemic cell-cycle suppression by Apicidin, a histone deacetylase inhibitor, in MDA-MB-435 cells.

Histone deacetylase (HDAC) inhibitors are emerging as an exciting new class of potential anti-cancer agents for the treatment of solid and hematological malignancies. However, the best characterized HDAC function concerns the control of gene expression via the regulation of transcription activation or repression. To understand the genome-wide effects of HDAC inhibition on gene regulation, we performed serial gene expression analyses from 0 to 48 h after treating MDA-MB-435, a melanoma-derived highly metastatic tumor cell line, with Apicidin, a HDAC inhibitor. Combined-transcriptomic analysis of large-scale molecular changes induced by Apicidin resulted in the identification of 631 outlier genes that were continuously up- or down-regulated during the 48 h study period. When the 631 outlier genes were mapped to known biological processes, cell-cycle suppression emerged as the function most elicited by Apicidin. In addition comprehensive negative cell-cycle regulation by Apicidin was dissected using gene expression data and validated by Western blot analysis. We suggest the 631 outlier genes as a characteristic molecular signature for Apicidin, and propose concurrent transcriptional suppression of major components of cell-cycle regulatory circuit as potent anti-tumor mechanism of Apicidin. Genetic elements identified during this study also provide the possibility of novel therapeutic interventions in tumor metastasis.

Genetic alterations of the Cdx2 gene in gastric cancer.

Gastric atrophy and intestinal metaplasia are generally considered precancerous lesions of the stomach; Cdx2 plays an important role in intestinal metaplasia and gastric carcinogenesis. To elucidate the potential etiological role of the Cdx2 gene in gastric carcinogenesis, we analyzed genetic mutations and allelic loss in the Cdx2 gene of 95 sporadic gastric cancers. We found two somatic missense mutations in the Cdx2 gene, P63L in exon 1 and E204K in exon 2, encoding the caudal-like protein activation region (codon 13-180) and the homeobox domain (codon 188-243) of the gene, in the gastric cancers. In addition, 9 (25.0%) of 36 informative cases showed allelic loss at D13S220 and/or D13S260. In 11 cases with a genetic alteration, Cdx2 nuclear staining was observed only in 8 cases of gastric mucosa with intestinal metaplasia. Loss or reduced expression of the Cdx2 gene in cancer cells was found in two cases with a somatic mutation and in three cases with LOH. Interestingly, all of the cases were intestinal-type gastric cancers. Thus, these results suggest that genetic alterations of the Cdx2 gene may contribute to the loss of Cdx2 expression and to the development of gastric cancer, especially in the intestinal-type.

Genetic analysis of the DBC2 gene in gastric cancer.

The DBC2 (Deleted in breast cancer, RhoBTB2) has been identified as a tumor suppressor gene that has growth inhibitory function. To investigate whether genetic alterations of the DBC2 gene are involved in the development of gastric cancer, we analyzed mutations and allelic loss in the DBC2 gene in 95 primary gastric cancers by PCR-SSCP, sequencing and LOH analysis. In the mutational analysis, we found one missense somatic mutation (CGG-->TGG, R275W) in the BTB/POZ domain of the gene in a patient with advanced gastric cancer and lymph node metastasis. In addition, we found one known polymorphism and three novel polymorphisms in the coding region of DBC2, which showed an amino acid change, and was detected in both the cancer cells and corresponding normal cells. On LOH analysis, 62 cases were heterozygous for at least one marker and 18 cases (29.0%) showed allelic loss at these markers. In conclusion, the mutations and allelic loss in the DBC2 gene are uncommon in gastric cancers in Korean patients. Further studies to identify the target gene at 8q21 responsible for the development of gastric cancer should be explored.

Genetic and epigenetic analysis of the VHL gene in gastric cancers.

The von Hippel-Lindau tumor suppressor gene (VHL), which is located on chromosome 3p25, plays an important role in tumorigenesis, particularly in tumor growth and vascularization. Mutations of the VHL gene have been observed in the hereditary VHL syndrome and a variety of other sporadic cancers. In this study, in order to investigate whether the VHL gene is involved in gastric carcinogenesis, we have examined the genetic alterations, including somatic mutations and allelic loss, with the two microsatellite markers, D3S1038 and D3S1110, as well as promoter hypermethylation of the VHL gene in 88 sporadic gastric adenocarcinomas. No mutation was detected in the coding region of the VHL gene. Allelic loss was found in 20 (33.9%) of 59 informative cancer cases at one or both markers. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and epigenetic alterations of the VHL gene in gastric cancers. Our results suggest that genetic and epigenetic alterations of the VHL gene may be not involved in the development or progression of gastric cancers. The findings also provide evidence for the presence of another gastric cancer specific tumor suppressor gene at the 3p25 region.

Genetic alterations of the ATBF1 gene in gastric cancer.

PURPOSE: Alpha-fetoprotein (AFP)-producing gastric cancers are aggressive tumors with venous and lymphatic invasion and hepatic metastasis. The goal of the present study was to investigate whether somatic changes of the AFP-negative regulator AT motif binding factor-1 (ATBF1) gene are involved in the development or progression of gastric cancers and the production of AFP in gastric cancer cells. EXPERIMENTAL DESIGN: We searched for genetic alterations of the ATBF1 gene by single-strand conformational polymorphism and sequencing methods as well as allelic loss analysis with the microsatellite markers D16S3066 and D16S3139. Immunochemistry for AFP expression in gastric cancer cells was also done. RESULTS: In 81 sporadic gastric cancers, four mutations were detected in seven cases: one was a missense mutation and three were deletions; loss of heterozygosity at the ATBF1 locus was detected in 52.9% of informative samples. Five of the eight cancers with AFP expression showed ATBF1 genetic alterations. CONCLUSIONS: These results suggest that genetic alteration of the ATBF1 gene may contribute to the aggressive nature of gastric cancers and the production of AFP in gastric cancer cells.

Aspartic acid repeat polymorphism of the asporin gene with susceptibility to osteoarthritis of the knee in a Korean population.

Osteoarthritis (OA) is a common disease that is characterized by the degeneration of joint cartilage in the knee, hip and hand. Recently, it has been reported that susceptibility to OA is affected by the number of aspartic acid (D) residues in the amino-terminal of the asporin protein. In order to investigate whether the association found in Japanese OA is also present in the Korean population, the frequency of the polymorphism was examined in 190 OA patients and 376 healthy controls. The D-repeat microsatellite polymorphism was examined by amplifying the asporin gene and sequencing its products. The frequencies of the D13 and D14 alleles were 69.7% (265/380) and 5.8% (22/380), respectively, in OA patients and 64.2% (483/752) and 8.7% (65/752), respectively, in healthy controls. When we adjusted for gender and age, there was no statistically significant difference between Korean OA patients and healthy controls (P=0.1082) in the allele frequency of D13 compared to the other alleles. However, a significant difference between female OA patients and their controls (P=0.0245) in the allele frequency of the D13 allele was found compared to the other alleles. There was no significant difference between Korean OA patients and healthy controls in the frequency of the D14 and other alleles (P=0.2339). These results suggest that asporin may play a role in OA susceptibility of the knee in the Korean female population.

A drug-repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6-thioguanine as an effective therapeutic agent for TPMT-low cancer cells.

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive-compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose-dependent analysis revealed three compounds with a tumor cell-specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6-thioguanine (6-TG) showed an IC50 of 0.39-1.13 µm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6-TG. Lastly, we used a patient-derived xenograft model to confirm that 6-TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6-TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.

Genetic and epigenetic analysis of the KLF4 gene in gastric cancer.

KLF4, which is also known as the gut-enriched Kruppel-like factor, plays important roles during the proliferation and differentiation of gastrointestinal epithelial cells. A loss of KLF4 expression has been observed in human tumors, particularly in the gastrointestinal tract. In this study, the molecular basis of the KLF4 inactivation in gastric cancer was investigated by analyzing the somatic mutation, the allelic loss with two microsatellite markers, D9S53 and D9S105, and hypermethylation of the KLF4 gene in 47 gastric adenomas and 81 gastric adenocarcinomas. Mutational analysis revealed one mutation of the KLF4 gene in a diffuse-type advanced gastric adenocarcinoma, but not in the gastric adenoma. This mutation was a somatic missense mutation, GGG-->AGG (Gly-->Arg) at codon 107 in exon 3, which encodes a transcriptional activation domain of the protein. An allelic loss was found in 7 (22.6%) of the 31 informative gastric adenoma cases and 15 (31.3%) of the 48 informative cancer cases at one or both markers. In addition, promoter hypermethylation of the KLF4 gene was observed in only two gastric cancers. These results suggest that genetic and epigenetic alterations of the KLF4 gene might play a minor role in gastric carcinogenesis.

Somatic mutations of the beta-TrCP gene in gastric cancer.

Beta-TrCP is a component of the ubiquitin ligase complex targeting beta-catenin for proteasomal degradation, and is a negative regulator of Wnt/beta-catenin signaling. To determine whether genetic alterations of the beta-TrCP gene are involved in the development or progression of gastric cancer, we analyzed its somatic mutations in 95 gastric cancers by single-strand conformational polymorphism and sequencing. We found five missense mutations (5.3%): A99V, H342Y, H425Y, C206Y, and G260E. Tissue carrying mutations showed moderate to strong cytoplasmic and/or nuclear staining of beta-catenin by immunohistochemistry. Thus, somatic mutations of the beta-TrCP gene may contribute to the development of gastric cancer through beta-catenin stabilization.

Genetic and epigenetic analysis of the EPHB2 gene in gastric cancers.

EPHB2 is a member of the Eph receptor tyrosine kinase family and a direct transcriptional target of beta-catenin/TCF. EPHB2 plays an important role in maintaining the correct positioning of the proliferative compartment in the crypt-villous axis. A loss of EPHB2 expression has been observed in human tumors, particularly in colonic adenomas and carcinomas. A search was made for mutations at the A9 tract in exon 17, an allelic loss at the EPHB2 gene locus, and promoter hypermethylation of the EPHB2 gene in 81 sporadic gastric cancers in order to determine if genetic or epigenetic alterations of the EPHB2 gene are involved in the development and/or progression of gastric cancer. Unexpectedly, no frameshift mutation was found and there was a low frequency (20.8%) of allelic loss. In addition, promoter hypermethylation was detected in only one gastric cancer tissue sample. Therefore, genetic or epigenetic alterations of the EPHB2 gene might be an uncommon event in the development or progression of gastric cancers.

Activation-induced cytidine deaminase expression in gastric cancer.

Helicobacter pylori increases the risk of gastric cancer development and triggers aberrant expression of activation-induced cytidine deaminase (AID). The goal of the present study was to investigate whether AID expression is involved in the development or progression of gastric cancer and the nuclear expression of p53 protein in cancer cells. We examined the expression pattern of the AID and p53 proteins in 186 gastric adenocarcinomas by immunohistochemistry. In order to investigate the molecular mechanism of AID expression, we also searched for mutations in the AID gene by single-strand conformational polymorphism and sequencing methods. In 186 sporadic gastric cancers, AID expression was detected in the 73 corresponding normal gastric mucosa and in 50 gastric cancers. Statistically, the expression of AID protein was not associated with clinicopathological parameters, including tumor size, location, differentiation and lymph node metastasis (p > 0.05). Interestingly, a significant association was observed between AID and the nuclear expression of p53 (p = 0.0094). Mutational analysis revealed no mutation in the AID gene in the gastric cancers. These results suggest that aberrant expression of the AID protein may contribute to the development of gastric cancers and induce p53 nuclear expression.

Celecoxib-mediated activation of endoplasmic reticulum stress induces de novo ceramide biosynthesis and apoptosis in hepatoma HepG2 cells mobilization.

Ceramides are the major sphingolipid metabolites involved in cell survival and apoptosis. When HepG2 hepatoma cells were treated with celecoxib, the expression of the genes in de novo sphingolipid biosynthesis and sphingomyelinase pathway was upregulated and cellular ceramide was elevated. In addition, celecoxib induced endoplasmic reticulum (ER) stress in a time-dependent manner. SPTLC2, a subunit of serine palmitoyltransferase, was overexpressed by adenovirus. Adenoviral overexpression of SPTLC2 (AdSPTLC2) decreased cell viability of HEK293 and HepG2 cells. In addition, AdSPTLC2 induced apoptosis via the caspase-dependent apoptotic pathway and elevated cellular ceramide, sphingoid bases, and dihydroceramide. However, overexpression of SPTLC2 did not induce ER stress. Collectively, celecoxib activates de novo sphingolipid biosynthesis and the combined effects of elevated ceramide and transcriptional activation of ER stress induce apoptosis. However, activation of de novo sphingolipid biosynthesis does not activate ER stress in hepatoma cells and is distinct from the celecoxib-mediated activation of ER stress. [BMB Reports 2017; 50(3): 144-149].

Adipocyte-Specific Deficiency of De Novo Sphingolipid Biosynthesis Leads to Lipodystrophy and Insulin Resistance.

Sphingolipids have been implicated in the etiology of chronic metabolic diseases. Here, we investigated whether sphingolipid biosynthesis is associated with the development of adipose tissues and metabolic diseases. SPTLC2, a subunit of serine palmitoyltransferase, was transcriptionally upregulated in the adipose tissues of obese mice and in differentiating adipocytes. Adipocyte-specific SPTLC2-deficient (aSPTLC2 KO) mice had markedly reduced adipose tissue mass. Fatty acids that were destined for the adipose tissue were instead shunted to liver and caused hepatosteatosis. This impaired fat distribution caused systemic insulin resistance and hyperglycemia, indicating severe lipodystrophy. Mechanistically, sphingosine 1-phosphate (S1P) was reduced in the adipose tissues of aSPTLC2 KO mice, and this inhibited adipocyte proliferation and differentiation via the downregulation of S1P receptor 1 and decreased activity of the peroxisome proliferator-activator receptor γ. In addition, downregulation of SREBP (sterol regulatory element-binding protein)-1c prevented adipogenesis of aSPTLC2 KO adipocytes. Collectively, our observations suggest that the tight regulation of de novo sphingolipid biosynthesis and S1P signaling plays an important role in adipogenesis and hepatosteatosis.

Pin1 gene mutation is a rare event in gastric cancer.

The peptidyl-prolyl isomerase Pin1 is strikingly overexpressed in human cancers and is a novel regulator of beta-catenin. To determine whether somatic mutation of the Pin1 gene is involved in the development and/or progression of gastric cancer, we searched for mutations of the Pin1 gene in 95 gastric cancer specimens. The effect of Pin1 on beta-catenin expression was further examined in wild- and mutant-type Pin1-transfected HEK 293T cells. We found only one missense mutation that led to the substitution of alanine by aspartic acid at codon 118 of the Pin1 gene. On transfection study, the mutant Pin1 showed an increased expression of beta-catenin. However, the mutation had no effect on expression of the Pin1 protein in the case with Pin1 mutation. These results suggest that Pin1 may not play a role in the development or progression of gastric cancer.

Altered expression of CDX2 in colorectal cancers.

CDX2 is a caudal-related homeobox transcription factor whose expression in the adult is normally restricted to the intestinal epithelium; it is implicated in the development and maintenance of the intestinal mucosa. The specific aim of this study was to elucidate the potential etiological role of CDX2 protein in colorectal carcinogenesis. We have analyzed the expression pattern of CDX2 protein in relation to the phenotype of 123 sporadic colorectal cancers by immunohistochemistry using tissue microarray. Strong CDX2 immunostaining was seen in the nuclei of corresponding normal intestinal epithelium. Interestingly, loss of CDX2 immunostaining was observed in 29 (23.6%) of 123 colorectal adenocarcinomas and its expression was correlated with the differentiation grade of the carcinoma (Chi-Square test, p<0.01). Clinically, CDX2 protein was immunopositive in 11 (91.7%) of 12 cases corresponding to stage A, 40 (85.1%) of 47 corresponding to stage B, 39 (69.6%) of 56 corresponding to stage C, and 4 (50.0%) of 8 corresponding to stage D. Statistically, CDX2 protein expression was related to tumor stage (Bartholomew test, p<0.05) and lymph node metastasis (Chi-Square test, p<0.05). These results indicate that loss of expression of CDX2 protein may play an important role in the tumorigenesis of colorectal cancers and that CDX2 expression represents a highly significant marker, which is able to identify a subset of patients at high risk.