Identification of Methylation-Driven, Differentially Expressed STXBP6 as a Novel Biomarker in Lung Adenocarcinoma.
ABSTRACT: 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?
Project description:BACKGROUND:This study was designed to investigate the effects of a novel carcinogenetic molecule, p130cas (breast cancer antiestrogen resistance protein 1 or BCAR1) on proliferation and cell growth in lung adenocarcinoma. The study also aimed to identify the possible underlying signal networks of BCAR1. METHODS:First, we evaluated proliferation, cell colony formation, apoptosis, and cell cycle after BCAR1 was knocked out (KO) using CRISPR-Cas9 technology in H1975 and H1299 human lung adenocarcinoma cells. Subsequently, BCAR1 was upregulated in 293T cells and immunoprecipitation-mass spectrometry (IP-MS) was used with bioinformatics analysis to screen for potential networks of BCAR1 interacting proteins. Ultimately, we validated the correlated expressions of BCAR1 and a selected hub gene, RNA polymerase II subunit A (POLR2A), in 54 lung adenocarcinoma tissues, as well as in H1975 and H1299 cells. RESULTS:Cell proliferation of H1975 and H1299 was significantly inhibited following BCAR1-KO. Colony formation of H1975 cells was also significantly decreased following BCAR1-KO. IP-MS demonstrated 419 potential proteins that may interact with BCAR1. Among them, 68 genes were significantly positively correlated to BCAR1 expression, as verified by TCGA. Six hub genes were revealed by PPI String. High expression of POLR2A, MAPK3, MOV10, and XAB2 predicted poor prognosis in lung adenocarcinoma, as verified by the K-M plotter database. POLR2A and MAPK3 are involved in both catalytic activity and transferase activity. POLR2A and BCAR1 were significantly increased in lung cancer tissues as compared with matched normal tissues. High expression of POLR2A was significantly positively correlated to BCAR1 overexpression and predicted poor prognosis in 54 lung cancer cases. POLR2A expression was significantly decreased following BCAR1-KO in H1975 and H1299 cells. CONCLUSIONS:BCAR1 promotes proliferation and cell growth, probably via upregulation of POLR2A and subsequent enhancement of catalytic and transferase activities. However, additional robust studies are required to elucidate the mechanisms involved.
Project description:BACKGROUND:m6 A modification affects the pathological progress of many diseases by affecting RNA stability and translocation. YTHDC2, a m6 A reader, is associated with multiple cancers; however, little is known of its role in non-small cell lung cancer (NSCLC). METHODS:The GEPIA, Oncomine and GEO databases were analyzed to assess expression of YTHDC2 in NSCLC patients. Quantitative polymerase chain reaction, western blot and immunohistochemistry were used to detect YTHDC2 expression in different NSCLC cell lines (H1299, H460, H292 and A549) and patients. The effects of YTHDC2 on NSCLC cell lines (A549 and H1299) proliferation and migration were employed using CCK8 and transwell assays. The potential target RNAs of YTHDC2 were obtained from the POSTAR database. Functional enrichment analysis of YTHDC2 targeted RNAs was performed using the Metascape database. RESULTS:GEPIA, Oncomine and GEO databases showed low expression of YTHDC2 in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. YTHDC2 expression was significantly decreased in different NSCLC cell lines and our clinical samples. Moreover, low expression of YTHDC2 was significantly associated with poor differentiation, lymph node metastasis, tumor size and stage. In addition, YTHDC2 could suppress the proliferation and migration ability of A549 and H1299 cell lines. Kaplan-Meier Plotter database analysis revealed that patients with low level of YTHDC2 had a significantly poor prognosis. Finally, functional enrichment analysis of YTHDC2 targeted RNAs indicated several enriched pathways related to cancer. CONCLUSIONS:These findings elucidate that YTHDC2 suppresses tumorigenesis in NSCLC, indicating that YTHDC2 may be a promising therapeutic target for NSCLC. KEY POINTS:SIGNIFICANT FINDINGS OF THE STUDY: This study demonstrated that the downregulation of YTHDC2 promotes tumor progression and predicts poor prognosis in non-small cell lung cancer (NSCLC). WHAT THIS STUDY ADDS:YTHDC2 might be a promising therapeutic target for non-small cell lung cancer (NSCLC).
Project description:Piwi-like RNA-mediated gene silencing 1 (PIWIL1) has been identified as a novel extremely highly expressed cancer-testis (CT) gene in lung adenocarcinoma. However, the exact function and mechanism of PIWIL1 in lung adenocarcinoma remains unclear. Herein, we sought to investigate the role of PIWIL1 in the occurrence and development of lung adenocarcinoma. We examined the expression pattern of PIWIL1 in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples, and validated it by Real-Time PCR (RT-PCR) in additional 21 paired lung adenocarcinoma tissues and 16 normal tissues. Subsequently, we explored the biological function of PIWIL1 in A549 and H1299 cell lines by gain and loss-of-function analyses. Using TCGA lung adenocarcinoma data, we further performed coexpression and Gene Ontology (GO) analyses, and analyzed the association of DNA methylation levels in PIWIL1 promoter region with its expression. Finally, we evaluated its expression in different mutation status of significantly mutated genes (SMGs) in TCGA lung adenocarcinoma data. We observed that PIWIL1 was expressed in testis and lung adenocarcinoma but not in other normal tissues, and its high expression was associated with shortened survival of lung cancer patients. Overexpression of PIWIL1 could facilitate the proliferation, invasion and migration of lung adenocarcinoma cells and vice versa. GO analysis revealed that PIWIL1 upregulated genes were enriched in embryonic development, cell proliferation and regulation of transcription. Moreover, promoter DNA hypomethylation of PIWIL1 could contribute to its aberrant expression in tumors. Interestingly, PIWIL1 expression was significantly higher in patients without hepatocyte growth factor (HGF) or serine/threonine kinase 11 (STK11) mutation (P = 0.006 and 0.005, respectively). PIWIL1 is an epidriver gene in lung adenocarcinoma, indicating a potential target for further therapy.
Project description:NCAPG2 is a component of the condensin II complex and contributes to chromosome segregation via microtubule-kinetochore attachment during mitosis. It is well known that NCAPG2 plays a critical role in cell mitosis; however, the role of altered NCAPG2 expression and its transcriptional regulatory function in cancer development remains mostly unknown. Here, for the first time we reported that NCAPG2 was evidently increased in non-small cell lung cancer tissues compared to adjacent normal lung tissues. Clinicopathological data analysis showed that NCAPG2 overexpression was significantly correlated with lymph node metastasis and pathologic-Tumour Nodes Metastasen stages, and was an independent prognostic factor in lung adenocarcinoma patients. Moreover, siRNA-mediated knockdown of NCAPG2 could inhibit tumour cell growth of lung adenocarcinoma cells (A549 and H1299) in vitro and could significantly lead to cell cycle arrest in the G2 phase. Furthermore, we found that NCAPG2 silencing significantly decreased the expression levels of G2/M phase cell cycle-related protein expressions (Cyclin B1, Cdc2) and increased the expression levels of p27 and p21 through Western blot analysis. Taken together, we demonstrated that increased NCAPG2 expression could regulate cell proliferation and identified as a poor prognostic biomarker in lung adenocarcinoma.
Project description:Abstract Background Although autophagy plays a dual role in suppressing or promoting certain cancers, the nature of its involvement in breast cancers remains unclear. Here, we investigated the function of STXBP6, a protein regulating the autophagy?associated SNARE complex, in triple negative breast cancer (TNBC). Results We report that STXBP6 is profoundly downregulated in TNBC specimens in association with reduced overall patient survival. Notably, we found that STXBP6 promoter was specifically hyper?methylated in TNBC specimens. Ectopic expression of STXBP6 inhibited TNBC cell proliferation in cellular and mouse models. Mass spectrometric analysis revealed physical interactions of STXBP6 with a number of autophagy?related proteins including SNX27, a molecule involved in endocytosis of plasma membrane receptors and protein trafficking. Overexpression of STXBP6 elicited autophagy through inhibition of mTORC1 signaling. Reciprocally, induction of autophagy rescued STXBP6 expression by inhibiting EZH2 and altering STXBP6 methylation. The mutual regulation between STXBP6 and autophagy was replicated in luminal breast cancer cells only when estrogen receptor (ER) activation was abrogated. Ectopic expression of STXBP6 significantly reduced TNBC cells’ migratory ability in vitro and tumor metastasis in vivo. Conclusions Our results unveil a role of STXBP6 in TNBC that highlights a new paradigm in autophagy regulation. Our results significantly enhance the understanding of the mechanisms of TNBC aggressiveness, which might help in designing novel therapies targeting TNBC.
Project description:The heterogeneity of lung adenocarcinoma is driven by key mutations in oncogenes. To determine the gene expression, single nucleotide polymorphisms, and co-mutations participating in the initiation and progression of lung adenocarcinoma, we comprehensively analyzed the data of 491 patients from The Cancer Genome Atlas. Using log-rank and Kruskal-Wallis analysis, Oncoprint, Kaplan-Meier survival plots, and a nomogram, we found that EGFRL858R with co-mutation TP53 was significant prognostic determinant versus that with co-wild TP53 (hazard ratio, 2.77, P = 0.012). Further gene co-expression network and functional enrichment analysis indicated that co-mutation of EGFRL858R/TP53 increases the expression of COMP and ITGB8, which are involved in extracellular matrix organization and cell surface receptor signaling pathways, thus contributing to poor prognosis in lung adenocarcinoma. Validation was performed using three GEO profiles along with colony formation and CCK-8 assays for proliferation, transwell and wound-healing for migration in transfected H1299 and A549 cell lines. To the best of our knowledge, these results are the first to indicate that patients harboring the co-mutation of EGFRL858R/TP53 show increased expression of COMP and ITGB8, which participate in extracellular matrix dysfunction and can be used as prognostic biomarkers in patients with lung adenocarcinoma.
Project description:BACKGROUND:Aberrant DNA methylation occurs frequently in cancer. The aim of this study was to identify novel methylation markers in lung cancer in Xuanwei, China, through integrated genome-wide DNA methylation and gene expression studies. METHODS:Differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were detected on 10 paired lung cancer tissues and noncancerous lung tissues by methylated DNA immunoprecipitation combined with microarray (MeDIP-chip) and gene expression microarray analyses, respectively. Integrated analysis of DMRs and DEGs was performed to screen out candidate methylation-related genes. Both methylation and expression changes of the candidate genes were further validated and analyzed. RESULTS:Compared with normal lung tissues, lung cancer tissues expressed a total of 6,899 DMRs, including 5,788 hypermethylated regions and 1,111 hypomethylated regions. Integrated analysis of DMRs and DEGs identified 45 tumor-specific candidate genes: 38 genes whose DMRs were hypermethylated and expression was downregulated, and 7 genes whose DMRs were hypomethylated and expression was upregulated. The methylation and expression validation results identified 4 candidate genes (STXBP6, BCL6B, FZD10, and HSPB6) that were significantly hypermethylated and downregulated in most of the tumor tissues compared with the noncancerous lung tissues. CONCLUSIONS:This integrated analysis of genome-wide DNA methylation and gene expression in lung cancer in Xuanwei revealed several genes regulated by promoter methylation that have not been described in lung cancer before. These results provide new insight into the carcinogenesis of lung cancer in Xuanwei and represent promising new diagnostic and therapeutic targets.
Project description: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 non?smoking 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. Kaplan?Meier Plotter for lung cancer was conducted for survival analyses. Reverse transcription?quantitative PCR (RT?qPCR) and western blot analysis were performed to investigate the expression and post?translational regulation of SEMA5A in lung adenocarcinoma cell lines. A pre?designed PyroMark CpG assay and 5?aza?2'?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 SEMA5A?regulated 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.
Project description:The role of the selenoproteome, which is the collection of all proteins containing selenium in an organism, in cancer development, growth and progression requires further investigation, due to the importance of selenium in both cancer and immune system function. Data about the selenoproteome, including its differential expression, single nucleotide variations, copy number variations, methylation, pathways and overall survival (OS) in five leading types of cancer are available from the GSCALite website. Subsequent to the analysis of these datasets, it was revealed that there was increased expression of GPX3 in stomach adenocarcinoma and lung squamous cell carcinoma, SELENOV in oesophageal carcinoma, GPX8 and GPX4 in colon adenocarcinoma, TXNRD1 and SEPHS1 in hepatocellular carcinoma and GPX8 in lung adenocarcinoma were associated with poor survival. Decreased gene expression of SELENOP was indicated in liver hepatocellular carcinoma and GPX3, and SELENOW, SELENOK, SELENBP1 and SECISBP2 in lung adenocarcinoma were associated with a poor prognosis. OS data suggested that hypermethylation of GPX4 in colon adenocarcinoma, GPX8 in lung squamous cell carcinoma, GPX1 in stomach adenocarcinoma and GPX3 in lung adenocarcinoma was associated with low survival, as is hypomethylation of GPX5 in lung adenocarcinoma. The selenoproteome is heterogeneous, especially in its effect on the OS of patients with cancer. The present study demonstrated that the roles of GPX4 in colon adenocarcinoma, SCLY and SELENOV in oesophageal carcinoma, SEPHS1 in liver hepatocellular carcinoma, SELENOK in lung cancer, as well as SELENOM and SELENOW in stomach adenocarcinoma requires further research. The present study may lead to the identification of novel biomarkers or potential therapeutic targets for use in the treatment of cancers, such as colon adenocarcinoma, oesophageal carcinoma, liver hepatocellular carcinoma, lung cancer and stomach adenocarcinoma.