Semaphorin 5A suppresses the proliferation and migration of lung adenocarcinoma cells.

Semaphorin 5A (SEMA5A), a member of the semaphorin family, plays an important role in axonal guidance. Previously, the authors identified another possible role of SEMA5A as a prognostic biomarker for non­smoking women with lung adenocarcinoma in Taiwan, and this phenomenon has been validated in other ethnic groups. However, the functional significance of SEMA5A in lung adenocarcinoma remains unclear. Therefore, we assessed the function of SEMA5A in three lung adenocarcinoma cell lines in this study. Kaplan­Meier Plotter for lung cancer was conducted for survival analyses. Reverse transcription­quantitative PCR (RT­qPCR) and western blot analysis were performed to investigate the expression and post­translational regulation of SEMA5A in lung adenocarcinoma cell lines. A pre­designed PyroMark CpG assay and 5­aza­2'­deoxycytidine treatment were used to measure the methylation levels of SEMA5A. The biological functions of lung adenocarcinoma cells overexpressing SEMA5A were investigated by microarrays, and validated both in vitro (proliferation, colony formation and migration assays) and in vivo (tumor xenografts) experiments. The results revealed that the hypermethylation of SEMA5A and the cleavage of the extracellular domain of SEMA5A were responsible for the downregulation of the SEMA5A levels in lung adenocarcinoma cells (A549 and H1299) as compared to the normal controls. Functional analysis of SEMA5A­regulated genes revealed that they were involved in cellular growth and proliferation. The overexpression of SEMA5A in A549 and H1299 cells significantly decreased the proliferation (P<0.01), colony formation (P<0.001) and migratory ability (P<0.01) of the cells. The suppressive effects of SEMA5A on the proliferative and migratory ability of the cells were also observed in both in vitro and in vivo experiments using brain metastatic Bm7 lung adenocarcinoma cells. On the whole, the findings of this study suggest a suppressive role for SEMA5A in lung adenocarcinoma involving the inhibition of the proliferation and migration of lung transformed cells.

Identification of Methylation-Driven, Differentially Expressed STXBP6 as a Novel Biomarker in Lung Adenocarcinoma.

DNA methylation is an essential epigenetic marker associated with the silencing of gene expression. Although various genome-wide studies revealed aberrantly methylated gene targets as molecular biomarkers for early detection, the survival rate of lung cancer patients is still poor. In order to identify methylation-driven biomarkers, genome-wide changes in DNA methylation and differential expression in 32 pairs of lung adenocarcinoma and adjacent normal lung tissue in non-smoking women were examined. This concurrent analysis identified 21 negatively correlated probes (r ≤ -0.5), corresponding to 17 genes. Examining the endogenous expression in lung cancer cell lines, five of the genes were found to be significantly down-regulated. Furthermore, in tumor cells alone, 5-aza-2'-deoxycytidine treatment increased the expression levels of STXBP6 in a dose dependent manner and pyrosequencing showed higher percentage of methylation in STXBP6 promoter. Functional analysis revealed that overexpressed STXBP6 in A549 and H1299 cells significantly decreased cell proliferation, colony formation, and migration, and increased apoptosis. Finally, significantly lower survival rates (P < 0.05) were observed when expression levels of STXBP6 were low. Our results provide a basis for the genetic etiology of lung adenocarcinoma by demonstrating the possible role of hypermethylation of STXBP6 in poor clinical outcomes in lung cancer patients.

Aberrant expression of the PRAC gene in prostate cancer.

Identification of aberrant expression patterns of genes in prostate cancer (PCa) is a key step towards the development of effective therapies. Prostate-specific antigen (PSA) levels are commonly measured for the early detection of PCa, but which itself is still not an ideal biomarker. We analysed the expression patterns of prostate cancer susceptibility candidate (PRAC) in prostate cancer. The PRAC gene is known to be commonly expressed in prostate tissue, rectum and colon. To provide clear insights into the expression patterns of PRAC in PCa, we examined the gene expression by quantitative real-time PCR (qRT-PCR), western blot analysis and immunohistochemistry (IHC). The results showed that PRAC expression levels in androgen­insensitive cells (DU145 and PC3) are lower than those in androgen-sensitive cell lines (LNCaP, LNCaP-R and CW22R). However, treatment of the LNCaP cell line with androgen and anti-androgen demonstrated that PRAC is expressed in an androgen-independent manner. Further, PRAC expression was restored upon treatment of DU145 and PC3 cells with the methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR), which indicates the effect of methylation in the control of PRAC expression. In addition, IHC analysis revealed a significantly decreased immunoreactivity of PRAC protein in PCa tissues compared to benign prostatic hyperplasia (BPH) (p<0.0001). Thus, our findings suggest that the pathogenesis of PCa may be due to the expression levels of PRAC protein, and this protein can serve as a potential biomarker for the management of PCa.

cDNA microarray profiling of rat cholangiocarcinoma induced by thioacetamide.

Cholangiocarcinoma (CCA) is a malignant neoplasm affecting thousands of individuals worldwide. CCA develops through a multistep process. In the current study, an oral thioacetamide (TAA)­induced model of rat CCA was established which generates the histological progression of human CCA, particularly the mass­forming type. Seven male Sprague­Dawley rats were treated with TAA for 24 weeks to induce CCA. Following the generation of the rat CCA model, whole rat genomic oligo microarray was performed to examine gene expression profiles in CCA and non­cancerous liver samples. In brief, 10,427 genes were found to be differentially expressed (8,318 upregulated and 3,489 downregulated) in CCA compared with non­tumor liver tissue. The top 50 genes (upregulated or downregulated) were selected and their functional involvement in various pathways associated with cancer progression was analyzed, including cell proliferation, apoptosis, metabolism and the cell cycle. In addition, increased expression of CLCA3, COL1A2, DCN, GLIPr2 and NID1, and decreased expression of CYP2C7 and SLC10A1 were validated by quantitative real­time PCR. Immunohistochemical analysis was performed to determine the protein expression levels of GLIPr2 and SLC10A1. The gene expression profiling performed in this study provides a unique opportunity for understanding the carcinogenesis of TAA­induced CAA. In addition, expression profiling of a number of specific genes is likely to provide important novel biomarkers for the diagnosis of CCA and the development of novel therapeutic strategies for CCA.

Phosphorylated T567 ezrin is associated with merlin expression in KIT-mutant gastrointestinal stromal tumors.

Membrane-cytoskeleton linker organizer ezrin is a member of the ERM (ezrin-radixin-moesin) protein family. It has been suggested as an important element in the oncogenic process, particularly in conferring a metastatic ability on tumor cells. We hypothesized that the KIT oncogenic form is one of the proteins that modulates expression of the ezrin protein via phosphorylated ezrin at different residues; furthermore, it may interact with the protein merlin, and promoting tumor development via the PI3K or MAPK pathway. In the present study, we observed that differential expression of ezrin was a common feature in gastrointestinal stromal tumors (GISTs). We further demonstrated that cases exhibiting expression of phosphorylated Thr567 in the ezrin protein were associated with immunoactivities of KIT and merlin expression (p=0.039 and 0.013, respectively). In conclusion, GISTs harbor activation of KIT protein may induce phosphorylation of the downstream protein ezrin at certain residues, thereby triggering subsequent signal transduction cascades and driving downstream pathways of tumor progression. However, a larger series of tumor samples should be analyzed in future studies, as well as the identification of phosphorylated sites to determine the role of ezrin in tumor progression thus shedding light on clinical outcomes.

Functional promoter polymorphisms govern differential expression of HMG-CoA reductase gene in mouse models of essential hypertension.

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5'-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.