Bioinformatics analysis and experimental validation of TTK as a biomarker for prognosis in non-small cell lung cancer.
ABSTRACT: BACKGROUND:Despite the prominent development of medical technology in recent years, the prognosis of non-small cell lung cancer (NSCLC) is still not optimistic. It is crucial to identify more reliable diagnostic biomarkers for the early diagnosis and personalized therapy of NSCLC and clarify the molecular mechanisms underlying NSCLC progression. METHODS:In the present study, bioinformatics analysis was performed on three datasets obtained from the Gene Expression Omnibus to identify the NSCLC-associated differentially expressed genes (DEGs). Immunohistochemistry-based tissue microarray of human NSCLC was used to experimental validating the potential targets obtained from bioinformatics analysis. RESULTS:By using protein-protein interaction (PPI) network analysis, Kaplan-Meier plotter, and Gene Expression Profiling Interactive Analysis, we selected 40 core DEGs for further study. Then, a re-analysis of 40 selected genes via Kyoto Encyclopedia of Genes and Genomes pathway enrichment showed that nine key genes involved in the cell cycle and p53 signaling pathway participated in the development of NSCLC. Then, we checked the protein level of nine key genes by semi-quantitative of IHC and checked the distribution at a single-cell level. Finally, we validated dual-specificity protein kinase TTK as a biomarker for prognosis in a tissue microarray. High TTK expression associated with a higher histological stage, advanced TNM stage, high frequency of positive lymph nodes, and worse 5-year overall survival. CONCLUSIONS:We found nine key genes were enriched in the cell cycle and p53 signaling pathway. TTK could be considered as a potential therapeutic target and for the prognosis biomarker of NSCLC. These findings will provide new insights for the development of individualized therapeutic targets for NSCLC.
Project description:The X-linked deubiquitinase, USP9X, is implicated in multiple cancers by targeting various substrates. Increased expression of USP9X is observed in non-small-cell lung cancer (NSCLC) and is correlated with poor prognosis. However, the molecular mechanism for USP9X regulation of tumor cell survival and tumorigenesis in NSCLC is less defined. Methods: In this study, chemical labeling, quantitative proteomic screening was applied to analyze A549 cells with or without USP9X RNA interference. Functional in vitro and in vivo experiments were performed to confirm the oncogenic effects of USP9X in NSCLC and to investigate the underlying mechanisms. Results: The resulting data suggested that dual specificity protein kinase TTK is a potential substrate of USP9X. Further experimental evidences confirmed that USP9X stabilized TTK via direct interaction and efficient deubiquitination of TTK on K48 ubiquitin chain. Moreover, knockdown of USP9X or TTK inhibited cell proliferation, migration and tumorigenesis, and the immunohistochemical analysis of clinical NSCLC samples showed that the protein expression levels of USP9X and TTK were significantly elevated and positively correlated in tumor tissues. Conclusions: In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as a potential therapeutic target.
Project description:The X-linked deubiquitinase, USP9X, is implicated in multiple cancers by targeting various substrates. Increased expression of USP9X is observed in non-small cell lung cancer (NSCLC) and is correlated with poor prognosis. However, the molecular mechanism for USP9X regulating tumor cell survival and tumorigenesis in NSCLC is less defined. In this study, chemical labeling quantitative proteomic screening was applied to analyze A549 cells with or without USP9X RNA interference, and the resulting data suggested that TTK is a potential substrate of USP9X. Further experimental evidences confirmed that USP9X stabilized TTK via direct interaction and deubiquitination of TTK. Moreover, knockdown of USP9X or TTK inhibited cell proliferation, migration and tumorigenesis, and the immunohistochemical analysis of clinical NSCLC samples showed that the protein expression levels of USP9X and TTK were significantly elevated and positively correlated in tumor tissues. In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as the potential therapeutic target.
Project description:Accumulating evidence has proved that glioma stem-like cells (GSCs) are responsible for tumorigenesis, treatment resistance, and subsequent tumor recurrence in glioblastoma (GBM). In this study, we identified dual specificity protein kinase TTK (TTK) as the most up-regulated and differentially expressed kinase encoding genes in GSCs. Functionally, TTK was essential for in vitro clonogenicity and in vivo tumor propagation in GSCs. Clinically, TTK expression was highly enriched in GBM, moreover, was inversely correlated with a poor prognosis in GBM patients. Mechanistically, mitochondrial fission regulator 2 (MTFR2) was identified as one of the most correlated genes to TTK and transcriptionally regulated TTK expression via activation of TTK promoter. Collectively, MTFR2-dependent regulation of TTK plays a key role in maintaining GSCs in GBM and is a potential novel druggable target for GBM.
Project description:Breast cancer is one of the most common malignancies among females, and its prognosis is affected by a complex network of gene interactions. In this study, we constructed free-scale gene co-expression networks using weighted gene co-expression network analysis (WGCNA). The gene expression profiles of GSE25055 were downloaded from the Gene Expression Omnibus (GEO) database to identify potential biomarkers associated with breast cancer progression. GSE42568 was downloaded for validation. A total of 9 modules were established via the average linkage hierarchical clustering. We identified 3 hub genes (ASPM, CDC20, and TTK) in the significant module (R 2 = 0.52), which were significantly correlated with poor prognosis both in test and validation datasets. In the datasets GSE25055 and GSE42568, higher expression levels of ASPM, CDC20, and TTK correlated with advanced tumor grades. Immunohistochemistry data from the Human Protein Atlas also demonstrated that their protein levels were higher in tumor samples. According to gene set enrichment analysis, 4 commonly enriched pathways were identified: cell cycle pathway, DNA replication pathway, homologous recombination pathway, and P53 signaling pathway. In addition, strong correlations were found among their expression levels. In conclusion, our WGCNA analysis identified candidate prognostic biomarkers for further basic and clinical researches.
Project description:OBJECTIVE:To screen the key genes related to the prognosis of lung adenocarcinoma through big data analysis and explore their clinical value and potential mechanism. METHODS:We analyzed GSE18842, GSE27262, and GSE33532 gene expression profile data obtained from the Gene Expression Omnibus (GEO). Bioinformatics methods were used to screen the differentially expressed genes in lung adenocarcinoma tissues and KEGG and GO enrichment analysis was performed, followed by PPI interaction network analysis, module analysis, differential expression analysis, and prognosis analysis. The expressions of MAD2L1 and TTK by immunohistochemistry were verified in 35 non-small cell lung cancer specimens and paired adjacent tissues. RESULTS:We identified a total of 256 genes that showed significant differential expressions in lung adenocarcinoma, including 66 up-regulated and 190 down-regulated genes. Thirty-two up-regulated core genes were screened by functional analysis, and among them 29 were shown to significantly correlate with a poor prognosis of patients with lung adenocarcinoma. All the 29 genes were highly expressed in lung adenocarcinoma tissues compared with normal lung tissues and were mainly enriched in cell cycle pathways. Seven of these key genes were closely related to the spindle assembly checkpoint (SAC) complex and responsible for regulating cell behavior in G2/M phase. We selected SAC-related proteins TTK and MAD2L1 to test their expressions in clinical tumor samples, and detected their overexpression in lung adenocarcinoma tissues as compared with the adjacent tissues. CONCLUSIONS:Seven SAC complex-related genes, including TTK and MAD2L1, are overexpressed in lung adenocarcinoma tissues with close correlation with the prognosis of the patients.
Project description:The present study aimed to explore specific molecular targets for the diagnosis and treatment of non?small cell lung cancer (NSCLC). The expression profiles of microRNAs (miRNAs) and mRNAs were downloaded from the GEO (GSE102286 and GSE101929) and TCGA databases. After data preprocessing, differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) in cancer and normal tissues were selected and used to construct a DEM?DEG regulatory network and a protein?protein interaction (PPI) network. The genes and miRNAs in these networks were subjected to functional enrichment and survival analyses. Several key DEMs and DEGs were verified using RT?qPCR, and the results were statistically interpreted using a multivariate logistic regression analysis. In this study, 25 DEMs and 789 DEGs common to all datasets were identified, which were then used for the construction of a DEM?DEG regulatory network and a PPI network module. Survival analyses of 19 DEMs in the DEM?DEG regulatory network and 36 DEGs in the PPI network module revealed that 34 DEGs (including TOP2A, CCNB1, BIRC5, and TTK) and two miRNAs (miR?21?5p and miR?31?5p) were significantly associated with NSCLC prognosis. Moreover, RT?qPCR analysis identified three DEGs and five DEMs that had changes in expression consistent with those observed in the bioinformatic analysis. Finally, a multivariate logistic regression analysis of the data showed that TOP2A, CCNB1, BIRC5, miR?21?5p, miR?193b?3p, miR?210?3p and miR?31?5p could be combined for the diagnosis of NSCLC. In conclusion, TOP2A, CCNB1, BIRC5, miR?21?5p, miR?193b?3p, miR?210?3p and miR?31?5p may therefore serve as important biomarkers and diagnostic targets for NSCLC.
Project description:Hepatocellular carcinoma (HCC) is one of the most malignant cancers with poor clinical outcome. The protein kinase human monopolar spindle 1 (hMps1/TTK) gene expression is significantly increased in HCCs. However, its contributions to hepatocarcinogenesis remain unclear. In this study, we found that TTK was overexpressed in 77.63% (118/152) HCC specimens. Elevated TTK expression positively correlated with large tumor size and presence of the portal vein tumor thrombus (PVTT). Demethylation in its promoter increased TTK expression in HCC. In vitro assays revealed that TTK not only promoted cell proliferation and anchorage-independent growth, but also cell migration. Subsequent investigations revealed that TTK activated Akt/mTOR pathway in a p53 dependent manner. We also found that TTK specific kinase inhibitor AZ3146 could decrease HCC cell growth. In conclusion, TTK contributes to HCC tumorigenesis via promoting cell proliferation and migration. It may serve as a novel biomarker and a potential target in HCC cancer therapy.
Project description:RATIONALE:Pulmonary nontuberculous mycobacterial disease (PNTM) often affects white postmenopausal women, with a tall and lean body habitus and higher rates of scoliosis, pectus excavatum, mitral valve prolapse, and mutations in the CFTR gene. These clinical features and the familial clustering of the disease suggest an underlying genetic mechanism. OBJECTIVES:To map the genes associated with PNTM, whole-exome sequencing was conducted in 12 PNTM families and 57 sporadic cases recruited at the National Institutes of Health Clinical Center during 2001-2013. METHODS:We performed a variant-level and a gene-level parametric linkage analysis on nine PNTM families (16 affected and 20 unaffected) as well as a gene-level association analysis on nine PNTM families and 55 sporadic cases. MEASUREMENTS AND MAIN RESULTS:The genome-wide variant-level linkage analysis using 4,328 independent common variants identified a 20-cM region on chromosome 6q12-6q16 (heterogeneity logarithm of odds score?=?3.9), under a recessive disease model with 100% penetrance and a risk allele frequency of 5%. All genes on chromosome 6 were then tested in the gene-level linkage analysis, using the collapsed haplotype pattern method. The TTK protein kinase gene (TTK) on chromosome 6q14.1 was the most significant (heterogeneity logarithm of odds score?=?3.38). In addition, the genes MAP2K4, RCOR3, KRT83, IFNLR1, and SLC29A1 were associated with PNTM in our gene-level association analysis. CONCLUSIONS:The TTK gene encodes a protein kinase that is essential for mitotic checkpoints and the DNA damage response. TTK and other genetic loci identified in our study may contribute to the increased susceptibility to NTM infection and its progression to pulmonary disease.
Project description:Background:Hepatocellular carcinoma (HCC) is the most common liver cancer and the mechanisms of hepatocarcinogenesis remain elusive. Objective:This study aims to mine hub genes associated with HCC using multiple databases. Methods:Data sets GSE45267, GSE60502, GSE74656 were downloaded from GEO database. Differentially expressed genes (DEGs) between HCC and control in each set were identified by limma software. The GO term and KEGG pathway enrichment of the DEGs aggregated in the datasets (aggregated DEGs) were analyzed using DAVID and KOBAS 3.0 databases. Protein-protein interaction (PPI) network of the aggregated DEGs was constructed using STRING database. GSEA software was used to verify the biological process. Association between hub genes and HCC prognosis was analyzed using patients' information from TCGA database by survminer R package. Results:From GSE45267, GSE60502 and GSE74656, 7583, 2349, and 553 DEGs were identified respectively. A total of 221 aggregated DEGs, which were mainly enriched in 109 GO terms and 29 KEGG pathways, were identified. Cell cycle phase, mitotic cell cycle, cell division, nuclear division and mitosis were the most significant GO terms. Metabolic pathways, cell cycle, chemical carcinogenesis, retinol metabolism and fatty acid degradation were the main KEGG pathways. Nine hub genes (TOP2A, NDC80, CDK1, CCNB1, KIF11, BUB1, CCNB2, CCNA2 and TTK) were selected by PPI network and all of them were associated with prognosis of HCC patients. Conclusion:TOP2A, NDC80, CDK1, CCNB1, KIF11, BUB1, CCNB2, CCNA2 and TTK were hub genes in HCC, which may be potential biomarkers of HCC and targets of HCC therapy.
Project description:Upon prolonged arrest in mitosis, cells undergo adaptation and exit mitosis without cell division. These tetraploid cells are either eliminated by apoptosis or arrested in the subsequent G(1) phase in a spindle checkpoint- and p53-dependent manner. p53 has long been known to be activated by spindle poisons, such as nocodazole and Taxol, although the underlying mechanism remains elusive. Here we present evidence that stabilization and activation of p53 by spindle disruption requires the spindle checkpoint kinase TTK/hMps1. TTK/hMps1 phoshorylates the N-terminal domain of p53 at Thr18, and this phosphorylation disrupts the interaction with MDM2 and abrogates MDM2-mediated p53 ubiquitination. Phosphorylation at Thr18 enhances p53-dependent activation of not only p21 but also Lats2, two mediators of the postmitotic checkpoint. Furthermore, a phospho-mimicking substitution at Thr18 (T18D) is more competent than the phospho-deficient mutant (T18A) in rescuing the tetraploid checkpoint defect of p53-depleted cells. Our findings therefore provide a mechanism connecting the spindle checkpoint with p53 in the maintenance of genome stability.