Missense allele of a single nucleotide polymorphism rs2294008 attenuated antitumor effects of prostate stem cell antigen in gallbladder cancer cells

Background: Prostate stem cell antigen (PSCA), an organ-dependent tumor suppressor, is down regulated in gallbladder cancer (GBC). It is anticipated that the missense allele C of the single nucleotide polymorphism (SNP) rs2294008 (T/C) in the translation initiation codon of the gene affects the gene′s biological function and has some influence on GBC susceptibility. We examined the biological effect of the C allele on the function of the gene and the relation between the C allele and GBC susceptibility. Materials and Methods: Functional analysis of the SNP was conducted by introducing PSCA cDNA harboring the allele to a GBC cell line TGBC- 1TKB and performing colony formation assays in vitro and tumor formation assays in mice. The effect on transcriptional regulation was assessed by reporter assays. The association study was conducted on 44 Japanese GBC cases and 173 controls. Results: The PSCA cDNA harboring the C allele showed lower cell growth inhibition activity (20% reduction) than that with the T allele. Concordantly, when injected into subcutaneous tissues of mice, the GBC cell line stably expressing the cDNA with the C allele formed tumors of almost the same size as that of the control cells, but the cell line expressing the cDNA with the T allele showed slower growth. The upstream DNA fragment harboring the C allele had more transcriptional activity than that with the T allele. The C allele showed positive correlation to GBC but no statistical significant odds ratio (OR = 1.77, 95% confidence interval 0.85-3.70, P value = 0.127 in dominant model). Conclusions: The missense allele was shown to have a biological effect, attenuating antitumor activities of PSCA, and consequently it may be a potential risk for GBC development. An association study in a larger sample size may reveal a significant association between the allele and GBC

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1 alpha and upregulated lactate dehydrogenase A (LDHA) and PDH-E1 alpha, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1 alpha in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN(-/-) mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1 alpha-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p

Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM

Molecular mechanism of peritoneal dissemination in gastric cancer

Peritoneal dissemination (PD) is the most common cause of metastasis in gastric cancer (GC). Because there are no standard treatments for PD, it is associated with a poor prognosis. Although clinicians have performed intraperitoneal chemotherapy for GC with PD, the outcome remains unsatisfactory. Therefore, the development of novel treatments and diagnostic tools for PD is expected to improve the prognosis of GC patients with PD. Notably, it is essential to elucidate the molecular mechanisms involved in the development of PD in GC. In this review, the molecular mechanisms of PD (three steps: detachment from the primary tumor, adaptation to the microenvironment of the peritoneal cavity, and attachment to peritoneal mesothelial cells) and new topics in GC are highlighted