Downregulation of KLF6 is an early event in hepatocarcinogenesis, and stimulates proliferation while reducing differentiation.
ABSTRACT: Hepatocellular carcinoma (HCC) has the most rapidly rising cancer incidence in the US and Europe. The KLF6 tumor suppressor is frequently inactivated in HCC by loss-of-heterozygosity (LOH) and/or mutation.Here we have analyzed 33 HBV- and 40 HCV-related HCCs for mRNA expression of wildtype KLF6 (wtKLF6) as well as the KLF6 variant 1 (SV1), a truncated, growth-promoting variant that antagonizes wtKLF6 function. The HCV-related tumors analyzed represented the full histologic spectrum from cirrhosis and dysplasia to metastatic cancer.Expression of KLF6 mRNA is decreased in 73% of HBV-associated HCCs compared to matched surrounding tissue (ST), with reductions of approximately 80% in one-third of the patients. KLF6 mRNA expression is also reduced in dysplastic nodules from patients with HCV compared to cirrhotic livers (p<0.005), with an additional, marked decrease in the very advanced, metastatic stage (p<0.05). An increased ratio of KLF6SV1/wt KLF6 is present in a subset (6/33, 18%) of the HBV-related HCCs compared to matched ST. Reconstituting KLF6 in HepG2 cells by retroviral infection decreased proliferation and related markers including cyclin D1 and beta-catenin, increased cellular differentiation based on induction of albumin, E-cadherin, and decreased alpha fetoprotein.We conclude that reduced KLF6 expression is common in both HBV- and HCV-related HCCs and occurs at critical stages during cancer progression. Effects of KLF6 are attributable to regulation of genes controlling hepatocyte growth and differentiation.
Project description:KLF6-SV1 (SV1), the major splice variant of KLF6, antagonizes the KLF6 tumor suppressor by an unknown mechanism. Decreased KLF6 expression in human hepatocellular carcinoma (HCC) correlates with increased mortality, but the contribution of increased SV1 is unknown. We sought to define the impact of SV1 on human outcomes and experimental murine hepatocarcinogenesis and to elucidate its mechanism of action. In hepatitis C virus (HCV)-related HCC, an increased ratio of SV1/KLF6 within the tumor was associated with features of more advanced disease. Six months after a single injection of diethylnitrosamine (DEN), SV1 hepatocyte transgenic mice developed more histologically advanced tumors, whereas Klf6-depleted mice developed bigger tumors compared to the Klf6fl(+/+) control mice. Nine months after DEN, SV1 transgenic mice with Klf6 depletion had the greatest tumor burden. Primary mouse hepatocytes from both the SV1 transgenic animals and those with hepatocyte-specific Klf6 depletion displayed increased DNA synthesis, with an additive effect in hepatocytes harboring both SV1 overexpression and Klf6 depletion. Parallel results were obtained by viral SV1 transduction and depletion of Klf6 through adenovirus-Cre infection of primary Klf6fl(+/+) hepatocytes. Increased DNA synthesis was due to both enhanced cell proliferation and increased ploidy. Coimmunoprecipitation studies in 293T cells uncovered a direct interaction of transfected SV1 with KLF6. Accelerated KLF6 degradation in the presence of SV1 was abrogated by the proteasome inhibitor MG132.An increased SV1/KLF6 ratio correlates with more aggressive HCC. In mice, an increased SV1/KLF6 ratio, generated either by increasing SV1, decreasing KLF6, or both, accelerates hepatic carcinogenesis. Moreover, SV1 binds directly to KLF6 and accelerates its degradation. These findings represent a novel mechanism underlying the antagonism of tumor suppressor gene function by a splice variant of the same gene.
Project description:Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown.In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2.In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation.Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.
Project description:Chronic infections by hepatitis B virus (HBV) and hepatitis C virus (HCV) appear to be the most significant causes of hepatocellular carcinoma (HCC). Aberrant promoter methylation is known to be deeply involved in cancer, including HCC. In this study, we analyzed aberrant promoter methylation on genome-wide scale in 6 HCCs including 3 HBV-related and 3 HCV-related HCCs, 6 matched noncancerous liver tissues and 3 normal liver tissues by methylated DNA immunoprecipitation-on-chip analysis. Candidate genes with promoter methylation were detected more frequently in HCV-related HCC. Candidate genes methylated preferentially to HBV-related or HCV-related HCCs were detected and selected, and methylation levels of the selected genes were validated using 125 liver tissue samples, including 61 HCCs (28 HBV-related HCCs and 33 HCV-related HCCs) and matched 59 matched noncancerous livers, and 5 normal livers, by quantitative methylation analysis using MALDI-TOF mass spectrometry. Among analyzed genes, preferential methylation in HBV-related HCC was validated in 1 gene only. However, 15 genes were found methylated preferentially in HCV-related HCC, which was independent from age. Hierarchical clustering of HCC using these 15 genes stratified HCV-related HCC as a cluster of frequently methylated samples. The 15 genes included genes inhibitory to cancer-related signaling such as RAS/RAF/ERK and Wnt/b-catenin pathways. It was indicated that genes methylated preferentially in HCV-related HCC exist, and it was suggested that DNA methylation might play an important role in HCV-related HCC by silencing cancer-related pathway inhibitors. we analyzed aberrant promoter methylation in 6 HCC clinical samples (including 3 HBV-related HCCs and 3 HCV-related HCCs) and their matched noncancerous tissues on genome-wide scale by the method. Candidate regions of promoter methylation preferentially to HBV-related HCC and HCV-related HCC were selected, and the methylation levels of these genes were measured quantitatively using MALDI-TOF mass spectrometry. Expression levels of these 6 pairs of HCC and 4 more pairs of HCCs and surrounding noncancerous tissues were analyzed by expression array and are reported in this Series. <br><br>This experiment was reloaded in November 2010 after additional curation. this dataset is part of the TransQST collection.
Project description:Chronic infections by hepatitis B virus (HBV) and hepatitis C virus (HCV) appear to be the most significant causes of hepatocellular carcinoma (HCC). Aberrant promoter methylation is known to be deeply involved in cancer, including HCC. In this study, we analyzed aberrant promoter methylation on genome-wide scale in 6 HCCs including 3 HBV-related and 3 HCV-related HCCs, 6 matched noncancerous liver tissues and 3 normal liver tissues by methylated DNA immunoprecipitation-on-chip analysis. Candidate genes with promoter methylation were detected more frequently in HCV-related HCC. Candidate genes methylated preferentially to HBV-related or HCV-related HCCs were detected and selected, and methylation levels of the selected genes were validated using 125 liver tissue samples, including 61 HCCs (28 HBV-related HCCs and 33 HCV-related HCCs) and matched 59 matched noncancerous livers, and 5 normal livers, by quantitative methylation analysis using MALDI-TOF mass spectrometry. Among analyzed genes, preferential methylation in HBV-related HCC was validated in 1 gene only. However, 15 genes were found methylated preferentially in HCV-related HCC, which was independent from age. Hierarchical clustering of HCC using these 15 genes stratified HCV-related HCC as a cluster of frequently methylated samples. The 15 genes included genes inhibitory to cancer-related signaling such as RAS/RAF/ERK and Wnt/b-catenin pathways. It was indicated that genes methylated preferentially in HCV-related HCC exist, and it was suggested that DNA methylation might play an important role in HCV-related HCC by silencing cancer-related pathway inhibitors. Overall design: we analyzed aberrant promoter methylation in 6 HCC clinical samples (including 3 HBV-related HCCs and 3 HCV-related HCCs) and their matched noncancerous tissues on genome-wide scale by the method. Candidate regions of promoter methylation preferentially to HBV-related HCC and HCV-related HCC were selected, and the methylation levels of these genes were measured quantitatively using MALDI-TOF mass spectrometry. Expression levels of these 6 pairs of HCC and 4 more pairs of HCCs and surrounding noncancerous tissues were analyzed by expression array and are reported in this Series.
Project description:Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection, thereby abrogating its normal antiproliferative activity in liver cells. The aim of the study was to evaluate the impact of KLF6 depletion on human HCC and experimental hepatocarcinogenesis in vivo. In patients with surgically resected HCC, reduced tumor expression of KLF6 was associated with decreased survival. Consistent with its role as a tumor suppressor, KLF6+/- mice developed significantly more tumors in response to the chemical carcinogen diethyl nitrosamine (DEN) than wild-type animals. Gene expression signatures in both surrounding tissue and tumors of KLF6+/- mice closely recapitulated those associated with aggressive human HCCs. Expression microarray profiling also revealed an increase in Mdm2 mRNA in tumors from KLF6+/- compared with KLF6+/+ mice, which was validated by way of quantitative real-time polymerase chain reaction and western blot analysis in both human HCC and DEN-induced murine tumors. Moreover, chromatin immunoprecipitation and cotransfection assays established the P2 intronic promoter of Mdm2 as a bona fide transcriptional target repressed by KLF6. Whereas KLF6 overexpression in HCC cell lines and primary hepatocytes led to reduced MDM2 levels and increased p53 protein and transcriptional activity, reduction in KLF6 by small interfering RNA led to increased MDM2 and reduced p53.Our findings indicate that KLF6 deficiency contributes significantly to the carcinogenic milieu in human and murine HCC and uncover a novel tumor suppressor activity of KLF6 in HCC by linking its transcriptional repression of Mdm2 to stabilizing p53.
Project description:BACKGROUND:Hepatitis B virus (HBV), hepatitis C virus (HCV), and alcohol consumption are predominant causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying how differently these causes are implicated in HCC development are not fully understood. Therefore, we investigated differential alternative splicing (AS) regulation among HCC patients with these risk factors. METHODS:We conducted a genome-wide survey of AS events associated with HCCs among HBV (n?=?95), HCV (n?=?47), or alcohol (n?=?76) using RNA-sequencing data obtained from The Cancer Genome Atlas. RESULTS:In three group comparisons of HBV vs. HCV, HBV vs. alcohol, and HCV vs. alcohol for RNA seq (?PSI>?0.05, FDR?<?0.05), 133, 93, and 29 differential AS events (143 genes) were identified, respectively. Of 143 AS genes, eight and one gene were alternatively spliced specific to HBV and HCV, respectively. Through functional analysis over the canonical pathways and gene ontologies, we identified significantly enriched pathways in 143 AS genes including immune system, mRNA splicing-major pathway, and nonsense-mediated decay, which may be important to carcinogenesis in HCC risk factors. Among eight genes with HBV-specific splicing events, HLA-A, HLA-C, and IP6K2 exhibited more differential expression of AS events (?PSI>?0.1). Intron retention of HLA-A was observed more frequently in HBV-associated HCC than HCV- or alcohol-associated HCC, and intron retention of HLA-C showed vice versa. Exon 3 (based on ENST00000432678) of IP6K2 was less skipped in HBV-associated in HCC compared to HCV- or alcohol-associated HCC. CONCLUSION:AS may play an important role in regulating transcription differences implicated in HBV-, HCV-, and alcohol-related HCC development.
Project description:Recurrent somatic mutations in the promoter region of telomerase reverse transcriptase (TERT) gene and in the exon 3 of CTNNB1 gene have been recognized as common events in hepatocellular carcinoma (HCC) with variable frequencies depending on etiology and geographical region. We have analyzed TERT promoter and CTNNB1 gene mutations in 122 cases of hepatitis B (HBV) and hepatitis C (HCV) related HCCs, in 7 cases of cholangiocarcinoma (CC) and hepatocholangiocarcinoma (HCC-CC) as well as in autologous cirrhotic tissues. Overall, 50.4% and 26% of HCC as well as 14.3% and none of CC and HCC-CC were mutated in TERT promoter and in CTNNB1 exon 3, respectively. TERT and CTNNB1 mutations were found more frequently in HCV related (53.6% and 26.4%, respectively) than HBV related (41.7% and 16.7%, respectively) HCCs and coexisted in 57.6% of CTNNB1 mutated tumors. Mutations in TERT and CTNNB1 were not associated with the functional promoter polymorphism rs2853669. No mutations were detected in the 129 non-HCC cirrhotic tissues. In conclusion, mutations in TERT promoter and in CTNNB1 gene represent specific cancer signatures in the pathogenesis of viral related HCC and could be promising early biomarkers as well as targets for tailored therapies.
Project description:Hepatocellular carcinoma (HCC) is a global health problem, although developing countries are disproportionally affected: over 80% of HCCs occur in such regions. About three-quarters of HCCs are attributed to chronic HBV and HCV infections. In areas endemic for HCV and HBV, viral transmission occurs at an early age, and infected individuals develop HCC in mid-adulthood. As these are their most productive years of life, HCC accounts for a substantial burden on the health-care system and drain of productive capacity in the low-income and middle-income countries most affected by HCV and HBV infections. Environments with disparate resource levels require different strategies for the optimal management of HCC. In high-resource environments, guidelines from the American Association for the Study of Liver Diseases or European Association for the Study of the Liver should be applied. In intermediate-resource or low-resource environments, the fundamental focus should be on primary prevention of HCC, through universal HBV vaccination, taking appropriate precautions and antiviral treatments. In intermediate-resource and low-resource environments, the infrastructure and capacity for abdominal ultrasonography, percutaneous ethanol injection, radiofrequency ablation and surgical resection should be established. Programs to provide targeted therapy at low cost, similar to the approach used for HIV therapy in the developing world, should be pursued.
Project description:Background:The onset of hepatocellular carcinoma (HCC) ranked fifth malignancies all over the world. Increasing evidences showed that the distribution of HCC was related to the incidence of chronic hepatitis B virus (HBV) infection and other factors, such as alcoholism, aflatoxin B1 ingestion and obesity. Recent studies demonstrated that gut dysbiosis plays an important role in liver diseases. However, the researches on gut microbiota of HBV and non-HBV non-HCV related HCC have not been reported. In this study, we investigated the differences between the gut microbiota of HBV related HCC (B-HCC) and non-HBV non-HCV related HCC (NBNC-HCC), finally found some potential bacteria, linking different pathological mechanism of both types of HCCs. Results:We carried out 16S rRNA analyses in a cohort of 33 healthy controls, 35 individuals with HBV related HCC (B-HCC) and 22 individuals with non-HBV non-HCV (NBNC) related HCC (NBNC-HCC). We found that the species richness of fecal microbiota of B-HCC patients was much higher than other two groups. Interestingly, the feces of NBNC-HCC patients harbored more potential pro-inflammatory bacteria (Escherichia-Shigella, Enterococcus) and reduced levels of Faecalibacterium, Ruminococcus, Ruminoclostridium which results in decrease potential of anti-inflammatory short-chain fatty acids. The feces of NBNC-HCC patients had relatively fewer abundance of multiple biological pathways related to amino acid and glucose metabolism, but high level of transport and secretion in some types. However, the B-HCC patients had opposite results of bacterial composition and associated multiple biological pathways versus NBNC-HCC patients. Meanwhile, we found that aberrant network of gut microbiota occurred differently in B-HCC and NBNC-HCC patients. Conclusions:Our study indicated that B-HCC and NBNC-HCC patients showed differential abundance of bacteria involved in different functions or biological pathways. We suggested the modification of specific gut microbiota may provide the therapeutic benefit for B-HCC and NBNC-HCC.
Project description:The Krüppel like factor 6 (KLF6) gene encodes multiple protein isoforms derived from alternative mRNA splicing, most of which are intimately involved in hepatocarcinogenesis and tumor progression. Recent bioinformatics analysis shows that alternative mRNA splicing of the KLF6 gene produces around 16 alternatively spliced variants with divergent or even opposing functions. Intriguingly, the full-length KLF6 (KLF6-FL) is a tumor suppressor gene frequently inactivated in liver cancer, whereas KLF6 splice variant 1 (KLF6-SV1) is an oncogenic isoform with antagonistic function against KLF6-FL. Compelling evidence indicates that miRNA, the small endogenous non-coding RNA (ncRNA), acts as a vital player in modulating a variety of cellular biological processes through targeting different mRNA regions of protein-coding genes. To identify the potential miRNAs specifically targeting KLF6-FL, we utilized bioinformatics analysis in combination with the luciferase reporter assays and screened out two miRNAs, namely miR-210 and miR-1301, specifically targeted the tumor suppressive KLF6-FL rather than the oncogenic KLF6-SV1. Our in vitro experiments demonstrated that stable expression of KLF6-FL inhibited cell proliferation, migration and angiogenesis while overexpression of miR-1301 promoted cell migration and angiogenesis. Further experiments demonstrated that miR-1301 was highly expressed in liver cancer cell lines as well as clinical specimens and we also identified the potential methylation and histone acetylation for miR-1301 gene. To sum up, our findings unveiled a novel molecular mechanism that specific miRNAs promoted tumorigenesis by targeting the tumor suppressive isoform KLF6-FL rather than its oncogenic isoform KLF6-SV1.