WDRPUH, a novel WD-repeat-containing protein, is highly expressed in human hepatocellular carcinoma and involved in cell proliferation.
ABSTRACT: In an attempt to disclose mechanisms of hepatocarcinogenesis and discover novel target molecules for the diagnosis and treatment of hepatocellular carcinomas (HCCs), we previously analyzed expression profiles of HCC tissues by means of human cDNA microarray. Among the genes upregulated in tumor tissues compared with their nontumor counterparts, we focused on a novel gene, termed WDRPUH, and characterized its biologic function. WDRPUH encodes a predicted 620-amino acid protein containing 11 highly conserved WD40-repeat domains. Multiple-tissue Northern blot analysis revealed its specific expression in the testis among 16 normal tissues examined. Transfection of plasmids designed to express WDRPUH-specific siRNA significantly reduced its expression in HCC cells and resulted in growth suppression of transfected cells. Interestingly, we found that WDRPUH associated with HSP70, proteins of the chaperonin-containing TCP-1 (CCT1) complex, as well as BRCA2. These findings have disclosed a novel insight into hepatocarcinogenesis and suggested that WDRPUH may be a molecular target for the development of new strategies to treat HCCs.
Project description:BACKGROUND & AIMS:We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS:We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS:Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS:MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).
Project description:Hepatocellular carcinoma (HCC) remains a major health problem worldwide, and HCC patients have a poor prognostic outcome. In this study, we systematically disclose mechanisms of hepatocarcinogenesis, and also effectively identify and validate novel anticancer targets in HCCs. Keywords: disease state analysis total 58 cDNA microarrays, all experiment samples are hepatocellular carcinoma. RNAs from 33 corresponding noncancerous livers and 5 normal livers were used as the reference, respectively. The tumor samples were labeled with Cy5-dUTP.The nontumor samples were labeled with Cy3-dUTP.
Project description:Background. Previous studies, including ours, have examined the regulation of microRNAs (miRNAs) by DNA methylation, but whether this regulation occurs at a genome-wide level in hepatocellular carcinoma (HCC) is unclear. Subjects/Methods. Using a two-phase study design, we conducted genome-wide screening for DNA methylation and miRNA expression to explore the potential role of methylation alterations in miRNAs regulation. Results. We found that expressions of 25 miRNAs were statistically significantly different between tumor and nontumor tissues and perfectly differentiated HCC tumor from nontumor. Six miRNAs were overexpressed, and 19 were repressed in tumors. Among 133 miRNAs with inverse correlations between methylation and expression, 8 miRNAs (6%) showed statistically significant differences in expression between tumor and nontumor tissues. Six miRNAs were validated in 56 additional paired HCC tissues, and significant inverse correlations were observed for miR-125b and miR-199a, which is consistent with the inactive chromatin pattern found in HepG2 cells. Conclusion. These data suggest that the expressions of miR-125b and miR-199a are dramatically regulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis.
Project description:Hepatocellular carcinoma (HCC) is a prevalent human cancer with rising incidence worldwide. Human HCC is frequently associated with chronic liver inflammation and cirrhosis, pathophysiological processes that are a consequence of chronic viral infection, disturbances in metabolism, or exposure to chemical toxicants. To better characterize the pathogenesis of HCC, we used a human disease-relevant mouse model of fibrosis-associated hepatocarcinogenesis. In this model, marked liver tumor response caused by the promutagenic chemical N-nitrosodiethylamine in the presence of liver fibrosis was associated with epigenetic events indicative of genomic instability. Therefore, we hypothesized that DNA copy number alterations (CNAs), a feature of genomic instability and a common characteristic of cancer, are concordant between human HCC and mouse models of fibrosis-associated hepatocarcinogenesis. We evaluated DNA CNAs and changes in gene expression in the mouse liver (normal, tumor, and nontumor fibrotic tissues). Additionally, we compared our findings to DNA CNAs in human HCC cases (tumor and nontumor cirrhotic/fibrotic tissues) using publicly available data from The Cancer Genome Atlas (TCGA). We observed that while fibrotic liver tissue is largely devoid of DNA CNAs, highly frequently occurring DNA CNAs are found in mouse tumors, which is indicative of a profound increase in chromosomal instability in HCC. The cross-species gene-level comparison of CNAs identified shared regions of CNAs between human fibrosis- and cirrhosis-associated liver tumors and mouse fibrosis-associated HCC. Our results suggest that CNAs most commonly arise in neoplastic tissue rather than in fibrotic or cirrhotic liver, and demonstrate the utility of this mouse model in replicating the molecular features of human HCC.
Project description:Hepatocellular carcinoma (HCC) represents one of the most frequent type of primary liver cancers. Decorin, a small leucine-rich proteoglycan of the extracellular matrix, represents a powerful tumor cell growth and migration inhibitor by hindering receptor tyrosine kinases and inducing p21WAF1/CIP1. In this study, first we tested decorin expression in HCCs utilizing in silico data, as well as formalin fixed paraffin embedded tissue samples of HCC in a tissue microarray (TMA). In silico data revealed that DCN/SMA mRNA ratio is decreased in HCC compared to normal tissues and follows the staging of the disease. Among TMA samples, 52% of HCCs were decorin negative, 33% exhibited low, and 15% high decorin levels corroborating in silico results. In addition, applying conditioned media of hepatoma cells inhibited decorin expression in LX2 stellate cells in vitro. These results raise the possibility that decorin acts as a tumor suppressor in liver cancer and that is why its expression decreased in HCCs. To further test the protective role of decorin, the proteoglycan was overexpressed in a mouse model of hepatocarcinogenesis evoked by thioacetamide (TA). After transfection, the excessive proteoglycan amount was mainly detected in hepatocytes around the central veins. Upon TA-induced hepatocarcinogenesis, the highest tumor count was observed in mice with no decorin production. Decorin gene delivery reduced tumor formation, in parallel with decreased pEGFR, increased pIGF1R levels, and with concomitant induction of pAkt (T308) and phopho-p53, suggesting a novel mechanism of action. Our results suggest the idea that decorin can be utilized as an anti-cancer agent.
Project description:EIF4EBP1 acts as a crucial effector in mTOR signaling pathway. Studies have suggested that EIF4EBP1 plays a critical role in carcinogenesis. However, the clinical significance and biological role of EIF4EBP1 in hepatocellular carcinoma (HCC) have not been elucidated. Therefore, we aimed to investigate the clinical significance of EIF4EBP1 in HCC.Total 128 cases of HCCs were included in this study. EIF4EBP1 expression in HCC tissues was detected by qRT-PCR, Western blot and immunohistochemistry, respectively. Then the relationships between EIF4EBP1 expression and clinical features as well as survival were analyzed.The expression level of EIF4EBP1 mRNA is significantly higher in 60% (24/40) of fresh HCC tissues than that in the matched adjacent nontumor liver (NCL) tissues (P = 0.044). Similarly, EIF4EBP1 protein is notably upregulated in 8 HCC tissues (randomly selected from the 40 HCCs) measured by Western blot and is significantly increased in another 88 paraffin-embedded HCCs (53%, 47/88) by immunohistochemistry compared with the matched NCLs (P < 0.001). EIF4EBP1 protein expression in HCC tissues is significantly correlated with serum AFP (P = 0.003) and marginally significantly associated with pathological grade (P = 0.085), tumor number (P = 0.084), tumor embolus (P = 0.084) and capsulation (P = 0.073). Patients with higher EIF4EBP1 protein expression have a much worse 5-year overall survival (40.3% vs 73.6%) and 5-year disease-free survival (33.0% vs 49.0%) than those with low expression. Furthermore, Cox regression analysis shows that EIF4EBP1 protein is an independent prognostic factor for overall survival (HR, 2.285; 95% CI, 1.154-4.527; P = 0.018) and disease-free survival (HR, 1.901; 95% CI, 1.067-3.386; P = 0.029) in HCC patients.Our results demonstrate for the first time that EIF4EBP1 mRNA and protein are markedly up-regulated in HCC tissues, and the protein overexpression is significantly associated with poor survival and progression, which provide a potential new prognostic marker and therapeutic target for HCC patients.
Project description:BACKGROUND & AIMS:Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. METHODS:We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. RESULTS:We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor ? signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. CONCLUSIONS:We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.
Project description:BACKGROUND: Centromere protein A (CENP-A) plays important roles in cell-cycle regulation and genetic stability. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: CENP-A expression at the mRNA and protein levels was examined in 20 pairs of fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for CENP-A was performed on 80 paraffin-embedded HCC specimens, and the clinical significance of its expression was analyzed. A human HCC cell line HepG2 with high abundance of CENP-A was used to study the effects of manipulating CENP-A on HCC growth. Quantitative real-time polymerase chain reaction arrays and Western blot analysis were employed to identify the cell-cycle control- and apoptosis-related genes regulated by CENP-A. The results showed that CENP-A was aberrantly overexpressed in HCCs relative to adjacent nontumor tissues. This overexpression was significantly associated with positive serum HBsAg status, increased histological grade, high Ki-67 index and P53 immunopositivity. Knockdown of CENP-A in HepG2 cells reduced cell proliferation, blocked cell cycle at the G1 phase, and increased apoptosis. The antiproliferative effects of CENP-A silencing were also observed in vivo. Conversely, CENP-A overexpression promoted HCC cell growth and reduced apoptosis. Furthermore, many genes implicated in cell-cycle regulation and apoptosis, including CHK2, P21waf1, P27 Kip1, SKP2, cyclin G1, MDM2, Bcl-2, and Bax, were deregulated by manipulating CENP-A. CONCLUSIONS/SIGNIFICANCE: Overexpression of CENP-A is frequently observed in HCC. Targeting CENP-A can inhibit HCC growth, likely through the regulation of a large number genes involved in cell-cycle progression and apoptosis, and thereby represents a potential therapeutic strategy for this malignancy.
Project description:AIM:To clarify the location and distribution of Kupffer cells in hepatocellular carcinoma (HCC), and to investigate their role in hepatocarcinogenesis. METHODS:Kupffer cells were immunohistochemically stained by streptavadin-peroxidase conjugated method (S-P). The numbers of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues of 48 HCCs were comparatively examined. RESULTS:The mean number of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues was 12.7+/-6.8, 18.1+/-8.2 and 18.9+/-7.9 respectively. The number of Kuppfer cells in cancerous tissues was significantly lower than that in para-cancerous tissues (t=2.423, P<0.05) and adjacent normal liver tissues (t=2.521, P<0.05). As tumor size increased, the number of Kupffer cells in cancerous tissues significantly decreased (F=4.61, P<0.05). Moreover, there was also a significant difference in the number of Kupffer cells among well-differentiated, moderately-differentiated and poorly-differentiated cases(F=4.49, P<0.05). CONCLUSION:This study suggests that decrease of Kupffer cells in HCCs may play an important role in the carcinogenesis of HCC, the number of Kupffer cells in HCC is closely related to the size and differentiation grade of the tumor.
Project description:Hepatocellular carcinoma (HCC) is one of the most deadly cancers that still lacks effective treatments. Dysregulation of kinase signaling has frequently been reported to contribute to HCC. In this study, we used bioinformatic approaches to identify kinases that regulate gene expression changes in human HCCs and two murine HCC models. We identified a role for calcium/calmodulin-dependent protein kinases II gamma isoform (CAMK2?) in hepatocarcinogenesis. CAMK2?-/- mice displayed severely enhanced chemical-induced hepatocarcinogenesis compared with wild-type controls. Mechanistically, CAMK2? deletion potentiates hepatic activation of mechanistic target of rapamycin complex 1 (mTORC1), which results in hyperproliferation of hepatocytes. Inhibition of mTORC1 by rapamycin effectively attenuates the compensatory proliferation of hepatocytes in CAMK2?-/- livers. We further demonstrated that CAMK2? suppressed growth factor- or insulin-induced mTORC1 activation by inhibiting IRS1/AKT signaling. Taken together, our results reveal a novel mechanism by which CAMK2? antagonizes mTORC1 activation during hepatocarcinogenesis.