Tumor Suppressor Gene In Hepatocellular Carcinoma Research Articles

Hypermethylation and Expression Silencing of PDCD4 Gene in Hepatocellular Carcinoma: A Consort Study.

Programmed cell death 4 (PDCD4) is a novel tumor suppressor, which is involved in the initiation and progression of cancers. However, the role of PDCD4 in hepatocellular carcinoma (HCC) has not been reported. The aim of this study was to investigate the molecular mechanism and clinical significance of PDCD4 inactivation in HCC.The mRNA levels of PDCD4 in HCC tissues and adjacent nontumor tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Bisulfite sequencing PCR was performed to determine the methylation status of PDCD4 promoter. Furthermore, the mRNA expression level and the methylated level of PDCD4 were analyzed with the clinical and pathological characteristics.qRT-PCR analysis showed that PDCD4 mRNA levels in tumor tissues were significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of PDCD4 promoter was significantly higher in HCC tissues than that in adjacent nontumor tissues. PDCD4 mRNA levels and promoter methylation levels were both statistically correlated with metastasis and the degree of differentiation in HCC. In addition, the correlation between PDCD4 hypermethylation, mRNA levels, and overall survival (OS) was statistically significant.Our results indicated that PDCD4 may be a novel candidate of tumor suppressor gene in HCC, and that promoter hypermethylation is an important mechanism for its downregulation and is also a good predictor of OS for HCC.

Clinical significance and therapeutic value of glutathione peroxidase 3 (GPx3) in hepatocellular carcinoma

We aimed to investigate the clinical significance of GPx3 in hepatocellular carcinoma (HCC) and to characterize its tumor suppressive role. HCC patients (113) who underwent hepatectomy were recruited to examine the clinical relevance of GPx3. The tumor suppressive role of GPx3 was studied by administration of recombinant GPx3 (rGPx3) or over-expression of GPx3 in HCC cells in vitro and in vivo. The therapeutic value of GPx3 for HCC was further investigated using human induced pluripotent stem cell derived mesenchymal stem cells (hiPSC-MSCs) as its delivery vehicle. Down-regulation of GPx3 significantly correlated with advanced tumor stage (P = 0.024), venous infiltration (P = 0.043) and poor overall survival (P = 0.007) after hepatectomy. Lower plasma GPx3 in HCC patients was significantly associated with larger tumor size (P = 0.011), more tumor nodules (P = 0.032) and higher recurrence (P = 0.016). Over-expression of GPx3 or administration of rGPx3 significantly inhibited proliferation and invasiveness of HCC cells in vitro and in vivo. Tumor suppressive activity of GPx3 was mediated through Erk-NFκB-SIP1 pathway. GPx3 could be delivered by hiPSC-MSCs into the tumor and exhibited tumor suppressive activity in vivo. GPx3 is a tumor suppressor gene in HCC and may possess prognostic and therapeutic value for HCC patients.

Identification of Epigenetically Inactivated Genes in Human Hepatocellular Carcinoma by Integrative Analyses of Methylation Profiling and Pharmacological Unmasking

Objectives: DNA methylation-dependent transcriptional inactivation of tumor suppressor genes (TSGs) is critical for the pathogenesis of hepatocellular carcinoma (HCC). This study identifies potential TSGs in HCCs using methylation profiling and pharmacological unmasking of methylated TSGs. Methods: Methylation profiling was performed on 22 pairs of HCCs and their corresponding noncancerous liver tissues using the Infinium HumanMethylation27 BeadChip. We also determined the gene reexpression after treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) in 5 HCC cell lines. Results: We selected CpGs that exhibited a significant increase in methylation in HCC tissues compared with that of the noncancerous control group. Two hundred and thirteen CpGs on different gene promoters with a mean difference in the β value ≥0.15 and a value of p < 0.05 were selected. Of the 213 genes, 45 genes were upregulated in 3 or more HCC cell lines with multiplier value of differences ≥2.0 after 5-Aza-dC and TSA treatment. Conclusions: We identified several potential TSGs that participate in transcription inactivation through epigenetic interactions in HCC. The results of this study are important for the understanding of functionally important epigenetic alterations in HCC.

Abstract 575: NKX6.1 functions as a tumor suppressor in hepatocellular carcinoma

Abstract In addition to genetic alterations, epigenetic alterations have been increasingly recognized as a key event for carcinogenesis. Methylation of CpG islands in promoter regions is almost associated with transcriptional silencing and is implicated in tumor suppressor gene (TSG) inactivation in cancer cells. Our previous data showed that NKX6.1 promoter is hypermethylated and downregulated in hepatocellular carcinoma (HCC) cell lines. The goal of this study was to elucidate whether NKX6.1 is a tumor suppressor gene in HCC. All HCC samples with NKX6.1 promoter hypermethylation showed downregulation of NKX6.1 expression using quantitative methylation-specific PCR and quantitative RT-PCR analysis. In addition, the expression of NKX6.1 in HCC cell lines can be restored by treatment with DNA methyltransferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine. These findings indicate that NKX6.1 is frequently silenced in HCC via promoter methylation. We then cloned and determined the putative promoter of NKX6.1 by luciferase reporter assay. The Sp1 site in the NKX6.1 promoter region is important for the promoter activity. When we treated cells with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), the NKX6.1 promoter activity increased. These data suggest that promoter methylation and histone modification may take part in the epigenetic silencing of NKX6.1 in HCC. Overexpression of NKX6.1 significantly suppressed transformation in HepG2 and SK-Hep1 cells; however, there is no significant effect on cell viability. Moreover we use a xenograft model to demonstrate the suppressor effects of NKX6.1 on tumor formation in vivo. Taken together, these data suggest that NKX6.1 might be a candidate tumor suppressor in HCC in vitro and in vivo. Citation Format: Hsin-Jung Li, Yu-Lueng Shih, Ming-De Yan, Pei-Ning Yu, Ya-Wen Lin. NKX6.1 functions as a tumor suppressor in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2014-575

Abstract 3822: Correlation between worse prognosis and lower expression of the TPPP gene in patients with hepatocellular carcinoma, detected by multiarray analysis

Abstract Background. Hepatocellular carcinoma (HCC) patients who undergo liver resection often suffer multicentric occurrence after surgery. Therefore, understanding carcinogenesis in background liver is important. Methods. Control liver samples (super normal [SN]), were taken from 11 cases of metastatic liver cancer. We selected corresponding normal tissue from typical cases of HCC with chronic hepatitis C (corresponding normal [CN]) for comparison. Expression profiling and methylation arrays were performed to compare SN and CN. Array data showed that the tubulin polymerization promoting protein (TPPP) gene showed differences in both array analyses. The relationship between clinicopathological factors of 179 HCC patients and TPPP expression was investigated. Results. The expression array showed that the TPPP gene was decreased by 2.236-fold (log2 ratio) in CN compared with SN. The methylation array showed a higher value of methylation for CN (0.730) than SN (0.384). We studied TPPP gene expression by real-time reverse transcription PCR. When compared expression level of TPPP (TPPP value ×103/GAPDH), among SN (n=11), normal liver tissue of HCC patients (n=179), and tumor tissue of HCC (n=179), there were significant differences (median: 116, 4.60, and 2.63, respectively). The group with lower TPPP expression in tumors compared with normal tissue showed a worse prognosis of overall survival than that in the increased expression group (P=0.0317). Strongly reduced TPPP expression in tumor tissue compared with normal tissue (ratio &amp;lt;0.3, n=57) was detected as an independent prognostic factor by multivariate analysis (P=0.0310). Conclusions. Expression of the TPPP gene in tumor and non-cancerous liver tissue in HCC patients is significantly lower compared with SN. HCC patients with this low TPPP expression show a poor prognosis. This indicates that this gene is a new candidate of tumor suppressor genes in HCC. Low TPPP expression in liver tissue might be a predictor of HCC. Citation Format: Yoshikuni Inokawa, Shuji Nomoto, Mitsuhiro Hishida, Nao Takano, Mitsuro Kanda, Michitaka Fujiwara, Masahiko Koike, Hiroyuki Sugimoto, Tsutomu Fujii, Goro Nakayama, Suguru Yamada, Chie Tanaka, Daisuke Kobayashi, Naoki Iwata, Yasuhiro Kodera. Correlation between worse prognosis and lower expression of the TPPP gene in patients with hepatocellular carcinoma, detected by multiarray analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2014-3822

736P - Identification of Intragenic Methylation in the Tusc1 Gene As a Novel Prognostic Marker of Hepatocellular Carcinoma

ABSTRACT Aim: Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. Methods: TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. Results: A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Conclusions: Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC. Disclosure: All authors have declared no conflicts of interest.

Methylation patterns of estrogen receptor α promoter correlate with estrogen receptor α expression and clinicopathological factors in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the seventh most common type of cancer; notably, the incidence of HCC is four to eight times higher in men than women. Previous studies reported that the estrogen receptor (ER) signaling pathway is involved in the pathogenesis of HCC, although the extent of its involvement is unclear due to several conflicting reports. In the present study, tumor and paired adjacent non-cancerous tissues from 157 HCC patients were collected. Transcriptome sequencing and real-time quantitative polymerase chain reaction were used to quantify the ER α (ESR1) expression levels, and the Sequenom EpiTYPER assay was used to delineate the methylation patterns in the ESR1 promoter. We found that ESR1 expression was significantly reduced in tumor tissues (P < 0.001) compared to adjacent non-cancerous tissues. The CpG sites around the transcription start site were significantly hypermethylated in the tumor (P < 0.0001). This methylation pattern also correlated with the gene expression (P < 0.0001). Additionally, we found that the hypermethylation of ESR1 was associated with the presence of fibrous capsules (P = 1.2 × 10-4), the absence of microvascular invasions (P = 8.0 × 10-4), thin trabecular pattern (P = 0.025), and lower histologic gradings (P = 5.2 × 10-3). Thus, ESR1 expression is a candidate tumor suppressor gene in HCC. Further, promoter hypermethylation may be a mechanism by which expression of ESR1 is repressed, and the extent of hypermethylation of ESR1 may be a marker for HCC status and progression.

Identification of intragenic methylation in the TUSC1 gene as a novel prognostic marker of hepatocellular carcinoma

Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of the TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC.

Downregulation of DENND2D by promoter hypermethylation is associated with early recurrence of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and its prognosis is poor. Novel targets for treating recurrence and progression along with associated biomarkers are urgently required. In this study, the expression and regulatory mechanism of DENN/MADD domain containing 2D (DENND2D) were investigated in an attempt to identify a tumor suppressor gene for HCC regulated by silencing through promoter hypermethylation. The levels of DENND2D expression in HCC cell lines and surgical specimens were determined using a quantitative polymerase chain reaction assay and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanisms of DENND2D expression in HCC. Most HCC cell lines (89%) and surgical specimens (78%) expressed lower levels of DENND2D mRNA compared with normal liver tissue. In contrast, there was no significant difference in the expression levels of DENND2D mRNA between normal tissues of HCC patients with and without cirrhosis. The expression patterns of DENND2D protein and mRNA were consistent. Patients with significantly lower levels of DENND2D mRNA in HCC tissues had remarkably earlier recurrences after hepatectomy and their prognosis worsened. The DENND2D promoter was methylated in eight out of nine HCC cell lines and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in HCC tissues (75%) and was significantly associated with downregulation of DENND2D mRNA expression. DENND2D is a candidate tumor suppressor gene that is inactivated by promoter hypermethylation in patients with HCC and may serve as a novel biomarker of early recurrence of HCC.

Genome-Wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma

Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis). Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.

Alterations of epigenetics and microRNAin hepatocellular carcinoma

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri-methylation of H3K27. Chromatin-modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non-coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.

Estrogen receptor 1 gene as a tumor suppressor gene in hepatocellular carcinoma detected by triple-combination array analysis

Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC remain insufficient to cure unresectable disease or to prevent HCC. Consistent efforts are, therefore, needed to deepen understanding of pathogenesis of the disease. Genome-wide gene expression profile analyses can now detect various candidate genes that are modified by HCC. We have developed a new technique to identify tumor suppressor genes, triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism and methylation arrays to identify genes with altered expression. Using HCC tissue samples, triple-combination array analysis was performed to identify a candidate tumor suppressor gene. Subsequently, samples from 48 HCC patients were subjected to quantitative polymerase chain reaction (qPCR) and methylation-specific PCR to further elucidate clinical relevance of the gene. Estrogen receptor 1 (ESR1) was detected as a candidate tumor suppressor gene. Of the 48 clinical samples, 40 (83.3%) showed ESR1 promoter hypermethylation. In 24 (50%) HCC samples, the expression levels of the ESR1 gene was decreased by >90%. The decreased expression was significantly related to high liver damage score, pathological invasion of the intrahepatic portal vein, the size of tumor (>3 cm in diameter) and hepatitis B virus infection. The present study represents another example that triple-combination array is a convenient technique for detecting genes with altered expression in disease. The ESR1 gene was identified as a candidate tumor suppressor gene in HCC and further validation is warranted.

Abstract 1152: Identification of protein tyrosine kinase 7 gene in hepatocellular carcinoma using a novel triple-combination array analysis.

Abstract Background. Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC still have failed to prevent HCC. Genome-wide gene-expression profile analyses can now detect many candidate genes that are modified by HCC. We have developed a new technique to identify suppressor genes of HCC: triple-combination array analysis, which combines gene expression profile, single nucleotide polymorphism array and methylation array. Methods. Triple-combination array analysis was performed on one HCC sample from a 68-year-old female patient, and one candidate of tumor suppressor gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in samples from 48 HCC patients and in nine cell lines. Results. Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate of tumor suppressor gene. PTK7 gene is located on 6q21.2, and expression was reduced in HCC tissues at the high level of -2.5 compared with normal tissues. PTK7 locus did not show any deletion or amplification in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.825 compared to 0.047 in normal tissues, indicating high methylation in tumor tissues in the methylation array. Of the 48 clinical samples, 30 (62.5%) showed PTK7 promoter hypermethylation. The PTK7 promoter hypermethylated cases were significantly low expressions status (P=0.035), and bad prognosis in both overall survival (P&amp;lt;0.01) and recurrence-free survival (P=0.018) compared to hypomethylated cases. On the other hand, the cases, of which the PTK7 expressions in tumor tissues decreased by more than 20% degrees compared to those in normal tissues, were significantly worse prognosis in overall survival (P=0.0136) than the other cases, but not in recurrence-free survival (P=0.3102). Conclusions. We suggest that the PTK7 gene acts as a tumor suppressor gene in HCC. This present study indicates that triple-combination array is an available and convenient technique for detecting the genes with altered expression in disease. Citation Format: Mitsuhiro Hishida, Shuji Nomoto, Yoshikuni Inokawa, Masamichi Hayashi, Suguru Yamada, Tsutomu Fujii, Hiroyuki Sugimoto, Shin Takeda, Yasuhiro Kodera. Identification of protein tyrosine kinase 7 gene in hepatocellular carcinoma using a novel triple-combination array analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2013-1152

The centrosomal protein tax1 binding protein 2 is a novel tumor suppressor in hepatocellular carcinoma regulated by cyclin-dependent kinase 2

Deregulation of cellular-signaling pathways by the inactivation of tumor-suppressor genes is one of the major causes of hepatocellular carcinoma (HCC). In this study, we identified Tax1 binding protein 2 (TAX1BP2) as a novel tumor-suppressor gene in HCC. TAX1BP2 transcript was frequently underexpressed (42.2% with T/NT <0.5; P < 0.03) in HCCs, and underexpression of TAX1BP2 was associated with poorer overall survival rates in patients after surgical resection. An effector domain (ED) for TAX1BP2 tumor-suppressor activity was mapped to the amino-acid residues 267-756. Transient or stable expression of either full-length or ED of TAX1BP2 significantly suppressed HCC cell tumorigenicity through the activation of the p38/p53/p21 pathway. In contrast, silencing of TAX1BP2 by short interfering RNA remarkably suppressed the activation of the p38/p53/p21 pathway. Finally, phosphorylation of TAX1BP2 at serine-763 by cyclin-dependent kinase (CDK)2 abolished the TAX1BP2-mediated p38 activation and tumor-suppressive activity, indicating that TAX1BP2 can adapt CDK2 signaling to the p38/p53/p21 pathway. Taken together, our data provide the first evidence that TAX1BP2 is a CDK2-regulated tumor-suppressor gene in HCC and is a novel activator of the p38/p53/p21 pathway.

Abstract 1332: Cisd2 haploinsufficiency leads to non-alcoholic fatty liver disease (NAFLD) and accelerates hepatocarcinogenesis in mice

Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Hepatic steatosis (fatty liver) is a vulnerable factor that could lead to liver damage, inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). However, despite the high prevalence of NAFLD, the genetic factor determining the disease progression remains poorly understood. CISD2 is a previously uncharacterized novel gene located within the overlapping region of HCC critical region and human longevity region on chromosome 4q. We have recently demonstrated for the first time that Cisd2 is a mitochondrial outer membrane protein which mediates mitochondrial integrity and lifespan in mammals. Surprisingly, we have observed phenotype similar to NAFLD in the liver of Cisd2 heterozygous (+/−) knockout mice. Cisd2 haplo-insufficiency led to abnormal lipid metabolism accompanying with fatty liver, cell death, severe inflammation and fibrosis in the liver. Moreover, there was a low incidence (1/18; 5.6%) of HCC developed at 16-18 months of age in the Cisd2+/− mice. Interestingly, our results revealed a significant increase of the Srebp1c which is the master regulator of lipogenesis; the increase in Srebp1-c seems to further up-regulate the expression of its target gene, fatty acid synthase (Fas), in the Cisd2+/− livers. In addition, we found that liver fibrosis was associated with activation of hepatic stellate cells, as revealed by increased expression of α-ΣMA, in the Cisd2+/− livers. Furthermore, we provide evidence that Cisd2 haplo-insufficiency promoted hepatocarcinogenesis and accelerated HCC development by 4-6 months in the transgenic mice expressing HBV X protein. In summary, our results suggested that Cisd2 is likely to be a haplo-insufficient tumor suppressor gene for HCC. Heterozygous deficiency of Cisd2 led to NAFLD, and accelerated the progression of HBV-associated HCC. Our data suggest that Cisd2 is a molecular target for the development of chemoprevention and therapeutic strategy for NAFLD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1332. doi:1538-7445.AM2012-1332

Hepatitis B virus X protein inhibits p53-mediated upregulation of mitofusin-2 in hepatocellular carcinoma cells

The hepatitis B virus X (HBx) protein has many significant roles in hepatocellular carcinoma (HCC). Our previous research demonstrated that mitofusion-2 (Mfn2), a potential tumor suppressor gene in HCC, is a novel direct target of p53 that exerts apoptotic effects via the mitochondrial apoptotic pathway. However, the relationship between HBx and Mfn2 expression in the development of HCC is unknown. We found that HBx had little direct effect on the expression of Mfn2 or p53 in HCC cells not treated with doxorubicin. However, HBx inhibited the upregulation of Mfn2 in HBx-transfected HCC cells simultaneously treated with doxorubicin or cotransfected with p53 plasmid, as evidenced by Western Blot and real-time PCR. Through electrophoretic mobility shift analysis, we confirmed that HBx interfered with the binding event of the p53 protein and the p53 binding site-oligo of the Mfn2 promoter. Moreover, luciferase assays revealed that the activity of the Mfn2 promoter did not increase when transfected with HBx plasmid in doxorubicin-treated HepG2 cells. These results indicate that HBx impacts p53-mediated transcription of Mfn2, providing insight into the negative effect of HBx against p53-dependent chemotherapeutic agents, such as doxorubicin, used in the treatment of HCC.

Microsatellite Alterations of Major Tumor Suppressor Genes in Hepatocellular Carcinoma

Purpose : Hepatocellular carcinoma (HCC) shows various molecular and genetic alterations in its development and progression. The major tumor suppressor genes (TSGs) such as p16, PTEN, E-cadherin and p53 on chromosomes 9p21, 10q23, 16q22, 17p13 may play important roles in various regulation pathways as well as tumor suppression. Microsatellite alteration such as loss of heterozygosity (LOH) has been reported is a predominant mechanism of inactivation of TSGs. Also, LOH is known to be related with allelic loss of various TSGs. This study evaluated LOH of 4 TSGs in HCCs and correlated these results with the clinicopathological factors. Methods : LOH analysis was carried out using a polymerase chain reaction with 16 polymorphic microsatellite markers of 4 chromosomes having TSGs in 50 surgically resected tumors and their non-tumorous counterparts. Results : There was no detectable LOH in normal tissue. LOH was detected in 82% of 50 cases of HCCs. LOH of 9p21, 10q23, 16q22, 17p13 was detected in 36%, 54%, 38% and 66%, respectively. Advanced HCCs showed significant LOH results of 9p21, 10q23 & 17p13. According to tumor grade, those of 10q23, 16q22, 17p13 correlated with higher grade III. Vascular invasion & lymphatic emboli showed correlation with those of 9p21, 10q23, 17p13. Cancer recurrence & metastasis significantly correlated with 9p21 LOH. Conclusion : These results suggest that LOH of various chromosomes may be predominantly involved in hepatocarcinogenesis and tumor invasion. Also, those LOH may be useful clinical indicator in determining the poorer prognosis among patients with HCC.

Identification of the a kinase anchor protein 12 (AKAP12) gene as a candidate tumor suppressor of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major health problem, and identification of new tumor-related genes is an urgent task. To detect tumor-related genes effectively, we performed double-combination array analysis, which consisted of an expression array and a single nucleotide polymorphism (SNP) array of a single surgical HCC specimen. Expression array analysis identified AKAP12 as one of the genes with reduced expression in HCC tissues when compared with non-cancerous adjacent hepatic tissues. In addition, AKAP12 expression levels in tumor tissues from 48 HCC samples were significantly lower (P < 0.001) than those in normal tissues, and the downregulation was significantly correlated with poor overall survival rate (P = 0.003). However, SNP array analysis revealed that locus 6q24-q25 where AKAP12 was located did not show chromosomal deletion. In contrast, hypermethylation in the AKAP12 promoter regions was observed in 41 of 48 HCC samples. We then confirmed that AKAP12 gene re-expression occurs after 5-aza-2'-deoxycytidine (5-aza-dC) treatment through direct sequence analysis of the AKAP12 promoter region in HCC cell lines. The current data suggest that AKAP12 is downregulated in cancer tissues through promoter hypermethylation, and may have a role as a candidate tumor suppressor gene for HCC.

MTSS1, a novel target of DNA methyltransferase 3B, functions as a tumor suppressor in hepatocellular carcinoma

DNA methyltransferase 3B (DNMT3B) mediates gene silencing via epigenetic mechanisms during hepatocellular carcinoma (HCC) progression. We aimed to identify novel targets of DNMT3B and their potential regulatory mechanisms in HCC. Metastasis suppressor 1 (MTSS1) was one of the DNMT3B targets and selected for further study. DNMT3B overexpression was detected in 81.25% of clinical HCC specimens and was negatively associated with MTSS1 in HCC cells and clinical samples. The underlying mechanism by which DNMT3B silences MTSS1 was studied using a combination of methylation-specific polymerase chain reaction (PCR) and bisulfite genome sequencing, chromatin immunoprecipitation-PCR and luciferase reporter assays. We found that the MTSS1 promoter region was sparsely methylated, and the methylation inhibitors failed to abolish DNMT3B-mediated MTSS1 silencing. DNMT3B protein bound directly to the 5'-flanking region (-865/-645) of the MTSS1 gene to inhibit its transcription. The functional role of MTSS1 was investigated using in vitro and in vivo tumorigenicity assays. As a result, MTSS1 exerted tumor suppressor effects and arrested cells in the G2/M phase, but not the G1/S phase of the cell cycle when it was depleted or overexpressed in HCC cells. Taken together, MTSS1, a novel target of DNMT3B, is repressed by DNMT3B via a DNA methylation-independent mechanism. MTSS1 was further characterized as a novel tumor suppressor gene in HCC. These findings highlight how DNMT3B regulates MTSS1, and such data may be useful for the development of new treatment options for HCC.

Abstract 323: Use of triple combination array analysis to identify STEAP family member 4 (STEAP4), a novel tumor suppressor gene in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but the molecular mechanisms underlying hepatocarcinogenesis are not clear. Array technology has made it possible to identify novel genes with alterations in liver cancer. In this study, we performed triple combination array analysis, expression profiling, karyotyping analysis using SNP array, and array-based DNA methylation profiling in the same HCC sample and attempted to find a novel tumor suppressor gene as a prognostic marker. Materials &amp; Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC. The tumor and corresponding normal tissues were used for extraction of DNA and RNA. SNP chip array experiments were performed according to the standard protocol of Affymetrix GeneChip Mapping 500K arrays for DNA. mRNA expression profiles were determined using Affimetrix HGU133A and B Gene-Chips. Bisulphite-converted genomic DNA was analyzed using Illumina's Infinium Human Methylation27 Beadchip Kit. We consequently analyzed the identified gene using quantitative RT-PCR, methylation and un-methylation-specific PCR in nine HCC cell lines and in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital, and then compared these results with clinico-pathological data. Results: The STEAP4 gene, is located on 7q21.12, and expression was decreased at a Log2 ratio of -4.2 to -2.0 in cancerous tissue. There were two copies of 7q21.12, indicating no loss of heterozygosity (LOH) in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.870 compared to 0.234 in non-cancerous tissue, indicating high methylation in tumor tissue in the Methylation array. Re-activation of STEAP4 expression was seen after 5-aza-dC treatment in HCC cell lines. Moreover, we found that 32 of 48 (67%) HCC samples showed promoter hypermethylation. In 32 methylated cases, the expression values of STEAP4 gene in tumor tissues by real-time RT-PCR were tended to decrease compared to corresponding normal samples. In addition, methylated cases were significantly correlated with positive results for Hepatitis B and α-feto protein and had a tendency for poor prognosis (p=0.098). Conclusion: The STEAP4 gene is a tumor suppressor gene in HCC and might be a marker of poor prognosis. The triple combination array was a useful technique to identify the novel methylated tumor suppressor gene in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2011-323