Androgen Activates PEG10 to Promote Carcinogenesis in Hepatic Cancer Cells
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Oncogene (2007) 26, 5741–5751 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Androgen activates PEG10 to promote carcinogenesis in hepatic cancer cells X Jie1,2,8, C Lang1,8, Q Jian1,8, L Chaoqun2,3,8, Y Dehua2,4,8,SYi2, J Yanping1, X Luokun1, Z Qiuping1, W Hui5, G Feili6, J Boquan7, J Youxin2 and T Jinquan1 1Department of Immunology, and Laboratory of Allergy and Clinical Immunology, Institute of Allergy and Immune-related Diseases and Medical Research Center, Wuhan University School of Medicine, Wuhan, China; 2The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China; 3Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai, China; 4College of Life Sciences, South China Normal University, Guangzhou, China; 5Department of Pharmacology, Wuhan University School of Medicine, Wuhan, China; 6Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China and 7Department of Immunology, Fourth Military Medical University, Xian, China The molecular mechanism of striking higher prevalence of Keywords: hepatocellular carcinoma; androgen; androgen hepatocellular carcinoma (HCC) in male subjects has not receptor; PEG10; apoptosis yet been fully elucidated. Here, we report that androgen receptor (AR) is differentially expressed in different HCC cell lines. AR agonist dihydrotestosterone (DHT) en- hances HCC cell growth and apoptotic resistance. Introduction Antagonist flutamide (FLU) blocks the effects of DHT on the HCC cell lines. Paternally expressed gene 10 Hepatocellular carcinoma (HCC) is the fifth most (PEG10) is expressed in HCC cell lines at substantial high common malignancy with a striking higher prevalence level. Using small interfering RNAs against AR and in men subjects than in women subjects throughout the PEG10 in AR- and PEG10-expressing BEL-7404 hepa- world (Yu et al., 2001). Endocrinological and epide- toma cells and HuH7 hepatoma cells (HuH7) cells, and miological studies indicate that HCC has a significantly AR-transfection technique in AR-lacking and PEG10- higher concentration of androgen receptor (AR) than expressing HepG2 cells, we have confirmed that through the surrounding liver tissue (Nagasue et al., 1995), and is upregulation and activation of PEG10, DHT enhances an androgen-dependent tumor. However, the molecular HCC cell growth and apoptotic resistance. We have mechanisms underlying the cellular regulation of AR further demonstrated that DHT upregulates expression of expression in these cells still remain to be fully human telomerase reverse transcriptase (hTERT) in HCC investigated. cell lines in a PEG10-dependent manner. Moreover, AR Paternally expressed gene 10 (PEG10) is newly directly interacts in vivo with androgen-responsive ele- identified as a paternally expressed gene at human ments in the regions of promoter and exon 2 of PEG10 chromosome 7q21 (Ono et al., 2001), functioning as gene in HCC cell lines. DHT promotes the hepatoma a transcriptionalfactor (Steplewski et al., 1998). An formation in vivo nude mice through PEG10 activation. elevated PEG10 expression in the majority of the human AR antagonists (FLU and valproate) inhibit the hepatoma HCC cells and G2/M phase of regenerating mouse liver formation. These findings suggest that PEG10 plays are indicating that this gene has growth-promoting an essential role in hepatocarcinogenesis. The PEG10 activity (Okabe et al., 2001; Tsou et al., 2003). The inhibition can be a novel approach for therapy of HCC. imbalance between expression of PEG10 and SIAH1, a Oncogene (2007) 26, 5741–5751; doi:10.1038/sj.onc.1210362; mediator of apoptosis, may be involved in hepatocarci- published online 19 March 2007 nogenesis through inhibition of apoptosis (Okabe et al., 2003; Tsou et al., 2003). PEG10 is also overexpressed in the embryonic form of biliary atresia, a disease associated with cell proliferation (Zhang et al., 2004). PEG10À/À mice show early embryonic lethality owing to Correspondence: Professor T Jinquan, Department of Immunology, defects in the placenta, indicating a critical role for Wuhan University Schoolof Medicine, Dong Hu Road 115, Wuchang, Wuhan, Hubei 430071, China or Professor J Youxin, The State Key mouse parthenogenetic development (Ono et al., 2006). Laboratory of Molecular Biology, Institute of Biochemistry and Cell PEG10 knockdown inhibits the proliferation of pan- Biology, Shanghai Institutes for Biological Sciences, Chinese Academy creatic carcinoma and HepG2 HCC cells (Li et al., of Science, Shanghai, China. 2006). Being activated by CCR7 and CXCR5, over- E-mail: [email protected] or [email protected] 8These authors contributed equally to this work. expressed PEG10 is involved in apoptotic resistance in þ þ Received 28 April2006; revised 15 January 2007; accepted 5 February B-acute lymphoblastic leukaemia CD23 CD5 B cells 2007; published online 19 March 2007 (Chunsong et al., 2006). Hepatocarcinogenesis and PEG10 activation X Jie et al 5742 The human telomerase reverse transcriptase (hTERT) is the key regulator of telomerase activity. Activation of telomerase enzyme and telomere stabilization is an important step in carcinogenesis (Greider, 1999). A high telomerase activity can be detected in HCC (Komine et al., 2000; Youssef et al., 2001). A connection between telomerase activity and resistance to apoptosis has been reported (Ramirez et al., 2003). Telomerase inhibiting and telomere shortening trigger apoptotic death in various cell types (Greider, 1999). Telomerase has therefore been proposed as a marker for carcinogenesis. Results Differential expression of AR in HCC cell lines Real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) assay data for messenger ribonucleic acid (mRNA) revealed that AR was rarely expressed in normalhepatocytes. It was expressed in BEL-7404 hepatoma cells (BEL) cells at substantial high level, in HuH7 hepatoma cells (HuH7) cells at moderate level, and in HepG2 cells at very low level (Figure 1a). The data by Northern and Western blotting assays confirmed the observation (Figure 1b). Figure 1 Expression of AR. AR expression in normalhepatocytes Effects of DHT on HCC cell lines (NML), HepG2 hepatoma cells (HepG2), HuH7 and the BEL were We next examined effects of AR agonist dihydrotestos- examined by Q-PCR (a), Northern blot (b, upper panels) and terone (DHT) on cell growth, apoptosis and PEG10 Western blot (b, lower panels). In (a), the showing bars were mean values7s.d. of eight similar experiments conducted. Statistically expression in different HCC cells. There was no significant differences were indicated compared with NML statistically significant proliferation of normal hepato- (*Po0.05; **Po0.001, normal hepatocytes versus hepatoma cells). cytes in culture without or with DHT (Figure 2a). There In upper panels of (b), mRNA of AR in NML, HepG2, HuH7 and were no statisticaldifferences on cellnumbersof tested BEL cells were detected by Northern blot. The hybridization three HCC cell lines in culture without DHT signals for AR mRNA from different cells were shown in upper images. The 28S rRNAs in lower images confirmed that compar- (Figure 2a). The growth rates of BEL cells and HuH7 able amounts of total RNA were used. In lower panels of (b), the cells in culture with DHT were significantly increased in AR protein was examined using Western blot analysis. Actins in comparison with that of the normalhepatocytes and lower images indicated the quantity of total cellular protein from HepG2 cells (Figure 2a). AR antagonist flutamide the tested samples loaded in each lane. Arrows indicated markers used to verify equivalent molecular weights of appropriate proteins (FLU) completely blocked the effects of DHT on the in each lane. cell growth (Figure 2a). The number of apoptotic cells after 5-FU treatment was significantly decreased in culture of BEL cells and HuH7 cells in the presence of comparison with that in the normalhepatocytes and DHT in comparison with that of the normalhepatocytes HepG2 cells (Figure 3c and d). FLU completely blocked and HepG2 cells, whereas, there were no statistical the effects of DHT on the upregulation of PEG10 differences on cell numbers of the tested three HCC cell expression (Figure 3c and d). FLU alone did not affect lines in culture without DHT (Figure 2b and c). FLU on PEG10 expression in normalhepatocytes (data not completely blocked the effects of DHT on the cell shown) and different tested HCC cell lines (Figure 3b, c apoptosis. FLU alone in culture did not affect either on and d). cell growth or on resistance to apoptosis in normal hepatocytes and different HCC cell lines (data not shown). Necessity of PEG10 to DHT-induced HCC cell growth To determine whether PEG10 was responsible for cell and apoptotic resistance growth and resistance to apoptosis induced by stimula- To further confirm roles of AR and PEG10 in DHT- tion with DHT, the cells were pretreated without or with induced HCC cell growth and apoptotic resistance, we DHT or/and FLU before further assays. PEG10 was chose the high AR- and PEG10-expressing BEL cells as expressed in tested three HCC cell lines at high level and investigating target cells. We first applied small inter- in normal hepatocytes at rather low level (Figure 3a). fering RNA (siRNAAR) to knockdown AR expression DHT or/and FLU did not alter PEG10 expression