Androgen Activates PEG10 to Promote Carcinogenesis in Hepatic Cancer Cells

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) enhances 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 in in the cells. The siRNAAR at high concentration low-AR-expressing HepG2 cells (Figure 3b). However, (2 mg/ml) sequence specifically abolished AR expression PEG10 expressions were significantly increased in the in BEL cells (Figure 4a). The siRNAAR (2 mg/ml) HuH7 cells and BEL cells in culture with DHT in partially knocked down PEG10 expression in BEL cells

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5743

Figure 2 DHT enhances cell growth and reduces apoptosis in AR-expressing HCC cell lines. Cell growth (a), apoptosis (b) and total dead cells (necrotic and apoptotic) (c) of normal hepatocytes and different HCC lines were examined. The cells were treated without (white bars) or with DHT (100 nM) (black bars) or with FLU þ DHT (gray bars) before assays. In (a), equalnumbers (2 Â 104) of cells with different treatment were plated in complete media and viable cell numbers were determined at 5 days later. Each test was triplicate in three wells. Each bar represents mean values7s.d. of eight experiments. Statistically significant differences were indicated as compared with NML (**Po0.001, untreated versus treated with DHT or with FLU þ DHT). In (b), The cells were treated without or with DHT or with FLU þ DHT before assays. The 5-fluorouracil(1 m M) was added directly into the medium to induce apoptosis. The cells treated without 5-FU were used as negative control (Untreated). After 72 h, the cells were analyzed by flow cytometry for PI (y axis) and FITC-conjugated annexin V (x axis). The percentages of PIÀ annexin V þ cells and PI þ annexin V þ cells were indicated in the figure. The data were from representatives of eight experiments performed. In (c), the cells were treated without (black bars) or with DHT (100 nM) (brick format bars) or with FLU þ DHT (gray bars). The cells treated without 5-FU were used as negative control (white bars). The data for total dead cells (PIÀ annexin V þ þ PI þ annexin V þ ) were mean values7s.d. of eight experiments performed. Statistically significant differences as compared with controls were indicated (**Po0.001, untreated versus treated with DHT or with FLU þ DHT).

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5744 siRNAAR in AR-expressing and PEG10-expressing HuH7 cells (data not shown). The 27-mer-siRNAAR (Kim et al., 2005) had very effective down-knock function. It almost completely abrogated expression of AR, and significantly and indirectly inhibited expression of PEG10, demonstrated by Q-PCR (Figure 4e), Northern and Western blots (data not shown). It more effectively inhibited DHT-induced cell growth and apoptotic resistance (Figure 4e), in comparison with 21-mer-siRNAAR (Figure 4c and d). The siRNAPEG10 at high concentration (2 mg/ml) sequence-specifically and completely abolished expression of PEG10 in BEL cells at mRNA and protein levels (Figure 5a). Functionally, the siRNAPEG10 (2 mg/ml) sequence-specifically blocked the ability of DHT to enhance cell growth and apoptotic resistance in BEL cells (Figure 5b and c). We had also observed a similar effect of siRNAPEG10 in AR-expressing and PEG10- expressing HuH7 cells (data not shown). We chose the low-AR-expressing and high PEG10- expressing HepG2 cells as investigating target cells to transfect additional AR. The cells were successfully transfected with AR-encoding plasmid and the expression level of AR was increased significantly, especially in the cells that a higher dose of AR-encoding plasmid was applied (600 ng) (Figure 6a and b). PEG10 expression, however, was not altered in the cells. HepG2 cells grew significantly faster after a successful AR-transfection at high dose in the presence of DHT (Figure 6c). The number of apoptotic cells was significantly decreased after a successfulAR-transfection within a dose-dependent manner in the presence of DHT (Figure 6d). Generally, DHT could promote cell growth and apoptotic resistance in HCC cells, but had no effect on AR- or PEG10-downknocked cells.

Effect of DHT on hTERT expression in HCC cells is PEG10-dependent To further explore the mechanisms of DHT effects on HCC cells, the chosen BEL cells were first cultured without or with siRNAPEG10, and then treated without Figure 3 DHT upregulates PEG10 expression in AR-expressing or with DHT. The expression level of hTERT was HCC lines. The expression levels of PEG10 in NML, HepG2 (b), HuH7 (c) and BEL (d) HCC lines were examined. The cells were significantly increased when the cells were treated with treated without, or with DHT (100 nM) or with FLU þ DHT and DHT (Figure 7a). However, when BEL cells were before assays. In (a), the expression levels of PEG10 in untreated pretreated with siRNAPEG10, the expression levels of hepatocytes and different HCC cell lines were measured by Q-PCR hTERT were not altered even in the presence of DHT (upper panel), Northern blot (middle panels) and Western blot (Figure 7b). FLU completely blocked the effects of (lower panel) as described in the legend to Figure 1. In (b, c and d), different HCC cell lines as indicated were treated without, or with DHT on the upregulation of hTERT expression (Figure DHT (100 nM) or with FLU þ DHT and then subjected to further 7a and b). FLU alone in culture did not affect on assays (Q-PCR, upper panels; Northern blot, middle panels; hTERT expression in normalhepatocytes (data not Western blot, lower panels, respectively). Statistically significant shown) and BEL cells without or with PEG10-down- differences were indicated (*Po0.05, **Po0.001, normalhepatocytes versus HCCs, or untreated versus treated). The data were knocking (Figure 7a and b). Additionally, we had also mean values7s.d. of eight experiments in each group. observed a similar effect of siRNAPEG10 on hTERT expression in HuH7 cells (data not shown). The similar effects of DHT on hTERT expression were also (Figure 4b), indicating that PEG10 expression was demonstrated in HEK293T cells cotransfected with closely associating with AR expression. Functionally, AR- and PEG10-encoding plasmids (600 ng each) the siRNAAR (2 mg/ml) significantly and sequence (Figure 7c). In HEK293T cells transfected with only specifically blocked the ability of DHT to enhance cell AR, we observed that hTERT expression was constant growth and apoptotic resistance in BEL cells (Figure 4c at low level no matter whether the cells were treated with and d). Additionally, we had observed a similar effect of DHT (Figure 7d).

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5745

Figure 4 siRNAAR downknocks AR expression and functions in BEL cells. The expression levels of AR (a) and PEG10 (b) in BEL cells were examined by Q-PCR (upper panels), Northern blot (middle panels) and Western blot (lower panels). The siRNAAR was transfected with appropriate concentration following the manufacturer’s instructions. Control, negative control siRNA with irrelevant sequence (2 mg/ml); siRNAARl, low concentration (0.02 mg/ml); siRNAARh, high concentration (2 mg/ml). The data were mean values7s.d. of eight experiments in each group. The images were representatives of eight experiments performed in each group. Cell growth (c) and apoptosis (d) were examined after treatment with DHT (100 nM). In (c), BEL cells were transfected with vector (’), negative controlsiRNA ( &), siRNAARl( ) and siRNAARh (J) before assays. The totalcellnumberswere measured to assess the effect of steroid DHT on cell growth by day 5. Each test was triplicate in three wells. Each line represents mean values7s.d. of eight experiments. Statistically significant differences were indicated (*Po0.05, **Po0.001, controlversus siRNA treated). In ( d), BEL cells were untransfected (Untrans.) or transfected with vector, negative controlsiRNA (Control),siRNAARland siRNAARh as indicated before assays. (E), untransfected cells were treated without DHT (100 nM). (J), siRNAAR-transfected cells were treated without DHT. The 5-fluorouracil(1 m M) was added directly into the medium to induce apoptosis. The data were representatives of eight experiments performed. In (e), BEL cells were transfected with vector (white bars), negative control 27-mer-siRNA (black bars) and 27- mer-siRNAAR (2 mg, gray bars) before assays. The expression level of AR and PEG10 were examined by Q-PCR (upper left panel). The total cell numbers were measured to assess the effect of steroid DHT on cell growth in 5 days (lower panel). Cells were transfected with vector (’), negative control27-mer-siRNA ( &) and 27-mer-siRNAAR (J) before assays. After pretreatment with DHT (100 nM), the 5-fluorouracil(1 m M) was added directly into the medium to induce apoptosis (upper right panel). BEL cells were transfected with vector (white bars), negative control27-mer-siRNA (blackbars) and 27-mer-siRNAAR (2 mg/ml, gray bars) before assays. The data were mean values7s.d. of six experiments performed. Statistically significant differences were indicated (**Po0.001, vector versus 27-mer siRNA treated).

AR binds to PEG10 in vivo regions as well as in promoter region. Four primer sets In order to further explore the mechanisms of DHT were designed to cover the found PEG10-AREs. After effects on HCC cells, we applied chromatin immuno- the cells were stimulated with DHT, AR interacted precipitation (ChIP) assay to examine whether DHT in vivo with the ARE in exon 2 and promoter region, but could activate AR directly to bind to PEG10 and its not in intron in the PEG10 gene (Figure 8a and b), promoter region. We searched for androgen response indicating a direct regulation of PEG10 expression. We elements (AREs) in the PEG10 gene (Rokhlin et al., co-transfected a PEG10/luciferase reporter with AR 2005). SeveralAREs had been found in intron and exon expression vector into HEK293T cells, which originally

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5746 Hepatoma formation of HCC cells in vivo nude mice To test whether there were effects of AR agonist and antagonist, siRNAAR and siRNAPEG10 on the carci- nogenicity of HCC cells in vivo, differentially treated HepG2 and BEL cells were subcutaneously injected into nude mice, and tumor growth was monitored. Showing in Table 1, at the end of the study (day 40), tumors had developed at all sites injected with different HCC cells. However, the average tumor volumes for the original and differentially treated HepG2 and BEL cells were signiﬁcantly different. Generally, DHT treatment could promote hepatoma formation, especially where high AR-expressing BEL cells were injected. The tumor growth was faster where AR-transfected HepG2 cells were injected in the presence of DHT. The hepatoma formation was decreased where siRNAPEG10-treated HepG2 or BEL cells were injected even in the presence of DHT. Pretreatment with AR antagonist FLU (interestingly also with valproate (VPA)) signiﬁcantly slowed down hepatoma formation in nude mice.

Discussion

Primary HCC represents the fifth leading cancer and the third most common cause of death from cancer world- wide. HCC is more prevalent in men subjects than it is in female subjects (Yu et al., 2001). The carcinogenesis of original HepG2 cells and BEL cells are significantly different in vitro and in vivo (Figure 2 and Table 1) because of differentialAR expression on the cells Figure 5 siRNAPEG10 down-knocks PEG10 expression and (Figure 1). Our consistent evidences have demonstrated functions in BEL cells. The expression level of PEG10 (a) in BEL that DHT exposure and AR expression level are cells was examined by Q-PCR (left), Northern blot (right, upper responsible for the androgen-related enhancement of panels) and Western blot (right, lower panels). The siRNAPEG10 HCC carcinogenesis. was transfected with appropriate concentration following the Distinct expression of PEG10 is found in the brain, manufacturer’s instructions. Control, negative control siRNA with irrelevant sequence (2 mg/ml); siRNAPEG10l, low concentration kidney, lung, testis and placenta but not in the liver and (0.02 mg/ml); siRNAPEG10h, high concentration (2 mg/ml). The a number of other tissues (Ono et al., 2001; Okabe et al., data were mean values7s.d. of eight experiments in each group. 2003). PEG10 is expressed in three HCC cell lines at The images were representatives of eight experiments performed in high level (Figure 3). The expression level of PEG10 can each group. Cell growth (b) and apoptosis (c) were examined after treatment with DHT (100 nM). In (b), BEL cells were transfected be downregulated as AR expression is reduced by with vector (white bars), negative controlsiRNA (blackbars), siRNAAR (Figure 4). PEG10 expression is elevated in siRNAPEG10l(brick form bars) and siRNAPEG10h (gray bars) the AR-expressing HCC cells by culture with DHT in before assays. The totalcellnumberswere measured to assess the comparison with that in the low AR-expressing HCC effect of steroid DHT on cell growth 5 days later. Each test was cells (Figure 3). The siRNAPEG10 blocks the ability of triplicate in three wells. Each line represents mean values7s.d. of eight experiments. In (c), BEL cells were untransfected or DHT to enhance cell growth and apoptotic resistance in transfected with vector (white bars), negative controlsiRNA HCC cells in vitro (Figure 5) and hepatoma formation of (black bars), siRNAPEG10l (brick form bars) and siRNAPEG10h HCC cells in vivo (Table 1). This is the first time that (gray bars) before assays. The 5-fluorouracil(1 m M) was added PEG10 has been found to directly interact with DHT/ directly into the medium to induce apoptosis. The data were representatives of eight experiments performed. The data were AR increasing HCC cell malignancy. Understanding the mean values7s.d. of eight experiments in each group. Statistically molecular basis of abnormal imprinting of PEG10 in significant differences were indicated (*Po0.05, **Po0.001, human HCC will shed new light into the process and controlversus siRNA treated). mechanism to develop malignant liver disorders. Telomerase has been proposed as an important marker for carcinogenesis and is a potentially highly expressed neither AR nor PEG10. The co-transfected selective target for the development of antiproliferative HEK293T cells were treated with 100 nM DHT for 24 h agents and anti-tumor drugs (Shammas et al., 2005). before harvest for luciferase assays. PEG10 promoter Expression of hTERT is increased when PEG10 activity was significantly increased in DHT-treated cells expressing HCC cells are treated with DHT. The in comparison with the cells treated without the hTERT expression is downregulated as PEG10 is hormone (Figure 8c). downknocked by siRNAPEG10, and upregulated as

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5747 PEG10 is transfected into the low PEG10-expressing down hepatoma formation in nude mice. This clue HEK293T cells in the presence of DHT (Figure 7). The indicates that androgen antagonism of VPA seems likely results strongly suggest that the effect of DHT on to contribute to a new therapeutic usage in patients with hTERT expression in HCC cells is PEG10-dependent, HCC, particularly, in the patients complicated with which may partially explain the effect of PEG10 in cell endocrine disturbances. growth promotion, apoptotic resistance and hepatoma formation. VPA, an anticonvulsant drug, has been recently found Materials and methods as a non-steroidalantagonist for human AR (Death et al., 2005). Pretreatment with VPA signiﬁcantly slows Cell cultures and reagents The BEL-7404 human hepatoma cells (from the cell bank of the Institute of Biochemistry and Cell Biology, SIBS, Shanghai, China. Huang et al., 2003), HuH7 human hepatoma cells, HepG2 human hepatoma cells and HEK293T cells (ATCC, Rockville, MD, USA) were grown in monolayers and cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM), high glucose (Gibco, Carlsbad, CA, USA). Normal primary human hepatocytes were anonymously from the patients without hepatic disease background undergoing partialliver resection for accidentalabdomen trauma. AR agonist dihydrotestosterone (DHT, at 100 nM concentration unless indicated otherwise), AR antagonist FLU and VPA were obtained from Sigma-Aldrich (Saint Louis, MO, USA).

Real-time quantitative RT-PCR assay (Q-PCR) Q-PCR reactions were performed as described elsewhere (Jinquan et al., 2003). Q-PCR was performed with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems, Foster City, CA, USA). By using SYBR Green reagent, ﬂuorescence signals were generated during each PCR cycle to provide Q-PCR information. The data were expressed as relative mRNA (gene) copies in 25 ng complementary DNA, which was normalized with the expression of a target gene to a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The sequences of the speciﬁc primers were as follows:

AR sense: 50-AAGGCTATGAATGTCAGCCCA-30, AR antisense: 50-CATTGAGGCTAGAGAGCAAGGC-30; PEG10 sense: 50-ATGATGACATCGAGCTCCG-30; PEG10 antisense: 50-GCTGGGTAGTTGTGCATCA-30, hTERT sense: 50-CGGAAGAGTGTCTGGAGCAA-30; hTERT antisense: 50-GGATGAAGCGGAGTCTGGA-30;

Figure 6 AR transfection increases cell growth and apoptotic resistance in HepG2 cells. The expression level of AR (a) and PEG10 (b) in HepG2 cells were examined by Q-PCR (upper panels), Northern blot (middle panels) and Western blot (lower panels). The pSVhAR was transfected with appropriate concentration following the manufacturer’s instructions. pSVhARl, low concentration (6 ng); pSVhARh, high concentration (600 ng). The data were mean values7s.d. of eight experiments in each group. The images were representatives of eight experiments performed in each group. Cell growth (c) and apoptosis (d) were examined after treatment with DHT. In (c), HepG2 cells were transfected with vector (J), pSVhARl( ) and pSVhARh (’) before assays. The totalcellnumberswere measured to assess the effect of steroid DHT on cell growth by day 5. Each test was triplicate in three wells. Each line represents mean values7s.d. of eight experiments. Statistically signiﬁcant differences were indicated (*Po0.05, **Po0.001, un-transfected versus transfected). In (d), HepG2 cells were untransfected (Untrans.) or transfected (vector, pSVhARland pSVhARh) and treated with DHT as indicated before assays. (E) Untransfected cells were treated without DHT. The 5-ﬂuorouracil (1 mM) was added directly into the medium to induce apoptosis. The data were from a single experiment, which was representative of eight experiments performed.

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5748 hybridization overnight in a rotating incubator at 681C with PerfectHyb hybridization solution (TOYOBO). Then wash the membrane intensively before being scanned using a Phosphor- Imager (Storm860, Amersham Bioscience Company, USA).

Western blot analysis Western blot analysis was contacted as what described previously (Massari et al., 2000). Briefly, the rabbit anti- human polyclonal to AR was purchased from Abcam Inc. (Cambridge, UK). The rabbit anti-human polyclonal antibody to PEG10 was raised in our lab and purified from sera of immunized rabbits with recombinant PEG10-his tag protein produced in Escherichia coli. Cell extracts were prepared using sodium dodecyl sulfate (SDS) loading buffer. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis gel and transferred to Polyvinylidene fluoride membranes (Milli- pore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk tris buffered saline/Tween-20 and incubated with the antibody. Goat peroxidase-conjugated affinity purified anti-rabbit IgG (Rockland, Gilbertsville, PA, USA) served as the secondary antibody for the SuperSignalWest Pico TrialKit Detection System (Pierce, Rockford, IL, USA). Actin served as an internalcontrol.

Cell growth assays Cell growth measurement in complete media was performed using a hemocytometer (Zhang et al., 2005). To assess the effect of steroid DHT on cell growth, cells (2 Â 104) were plated in 12- well plates in the presence of 100 nM DHT (unless indicated otherwise). The antagonist FLU (1 mM) or VPA (100 mM)was added in blockage experiments. After 24 h, the medium was replaced with steroid DHT medium for 48 h. After 48 h, cells were re-fed with fresh steroid-reduced medium and totalcell numbers were assessed 5 days (or indicated days) later.

Flow cytometry For detection of apoptosis, cells were seeded on six-well plates Figure 7 siRNAPEG10 downregulates hTERT expression in BEL in steroid-free medium and transfected as described above. cells and HEK293T cells. The expression levels of hTERT in BEL After 24 h, the medium was replaced without or with DHT or without (a) or with siRNAPEG10 transfection (b) and HEK293T with FLU þ DHT medium for 48 h. After 24 h of gene cells co-transfected with AR and PEG10 (c) or with only AR (d) transfection, 5-fluorouracilwas directlyadded into the were examined by Q-PCR (all upper panels), Northern blot (all medium to induce apoptosis. The cells were collected after lower panels). BEL cells (b) and HEK293T cells (c) were co- 72 h and washed by cold phosphate-buffered saline twice, transfected with plasmids encoding AR and PEG10 (600 ng each). followed by stain in staining buffer with 1 mg/mlpropidium The cells were then treated without or with DHT (100 nM) or with 1 FLU þ DHT. Statistically significant differences as compared to iodide (PI) for 30 min at 4 C, then stained with fluorescein controls were indicated (*Po0.05, untreated versus treated with isothiocyanate-conjugated annexin V with binding buffer (BD DHT or with FLU þ DHT). In (d), the HEK293T cells were Pharmingen, San Diego, CA, USA) as described previously transfected with plasmids encoding AR (600 ng). The cells were (Jinquan et al., 2003). COULTER XL (Coulter, Miami, FL, treated then without or with DHT (100 nM) or with FLU þ DHT. USA) was used for analyses. The data were mean values7s.d. of six experiments in each group. siRNA gene knockdown assay GAPDH sense: 50-ACAACAGCCTCAAGATCATCAG-30; Sense and antisense strands of siRNA oligonucleotides were GAPDH designed and synthesized by the State Key Laboratory of antisense: 50-GGTCCACCACTGACACGTTG-30; Molecular Biology (the Institute of Biochemistry and Cell Biology, SIBS, CAS). PsiRNA-hH1neo (InvivoGen, San Diego, USA) was used to get the sequence of a siRNA duplex. Northern blot analysis The insert was cloned downstream of human H1 promoter. For mRNA detection, 2 mg of totalRNA obtained from each The siRNAs against AR and PEG10 were named siRNAAR, sample were electrophoresed on 1% agarose formaldehyde gel, siRNAPEG10, respectively. A 27-mer siRNA against AR were followed by blotting onto Nytran membranes (Amersham designed (Kim et al., 2005) in the additionalexperiments. Pharmacia Biotech, Buckinghamshire, UK), and were cross- The sequences of siRNAs were as follows: linked by UV irradiation as described previously (Sica et al., 1997). PEG10 ORF coding sequence was obtained by PCR siRNAAR sense: 50-GGGAAACAGAAGTACCTGTTT-30 amplification of total RNA from BEL cell. Radiolabeled siRNAPEG10 sense: 50-GTCGCTGTCTGCTCTGATTTT-30 probes were made by a random priming reaction with Negative control [a-32P]dATP. The RNA blots were prehybridized 1 h and siRNA sense: 50-TGAAGGAGTTCCTGATCTTTT-30

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5749

Figure 8 The PEG10 contains AREs and binds to the AR in vivo. The ChIP assay was analyzed by conventional PCR (a) and Q-PCR (b). BEL cells were cultured without (DHTÀ) or with DHT (100 nM, DHT þ ). PCR was performed in the presence of [a-32P]-dATP, using primers for AREs. Input, positive control with non-complex DNA before immunoprecipitation; Mock, dialysis buffer instead of chromatin; AbÀ, controlimmunoprecipitation procedure without antibodies; Ab þ , chromatin DNA immuno-complexed with AR antibody; Abi, ChIP with mouse IgG. The data shown were representative of three independent experiments. In (c), effect of DHT on PEG10 gene promoter activity. HEK293T cells were cotransfected with the PEG10/luciferase reporter (1 mg) and AR expression vector (600 ng). At 24 h after transfection, cells were cultured without (DHTÀ) or with DHT (100 nM, DHT þ ) for further 24 h. The cells were then lysed, and the activities of luciferase were assayed. Vector, empty GL3 basic vector; pLUC-ARE, pGL3 basic vector inserted PEG10 promoter sequence; pGL3-pos, Promega pGL3 positive control vector. Fold change was calculated by comparing the normalized luciferase activity from PEG10-transfected cells with that from vector-transfected cells. The means and s.d. from three independent experiments were shown. The normalized luciferase activity from the cells transfected with the vector was set as 1.0.

siRNAAR sense transiently transfected with vectors (pCDNA3.0) encoding (27-mer): 50-AAGGGAAACAGAAGUACCUGU target genes as described elsewhere (Lu et al., 2001; Sathya GCGCCA-30 et al., 2003; Tsou et al., 2003). Cells were cultured with serum siRNAAR antisense free and the complexes were transfected, then supplemented (27-mer): 50-UGGCGCACAGGUACUUCUGUU with 10% FCS 6 h post-transfection, then incubated at 371Cin 0 UCCCUU-3 air containing 5% CO2 for 48 h. The stable transfection were performed using 8 mlof Lipofectamine2000 and 15 mgof The siRNAs were diluted with Opti-MEM I (Invitrogen, pEGFP-target gene was constructed by inserting a cDNA Carlsbad, CA, USA) to appropriate concentration. Cells were into the XhoI and EcoRI site of pEGFP-C1) or 15 mgof cultured with serum free and the complexes of siRNAs with pEGFP empty vector. To produce stable transfectants, the the Lipofectamine2000 were transfected for 48 h. cells were then split at ratios of 1:20 and cultured for 2 weeks in the presence of 400 mg/mlof G418 (Geneticin sulfate,GIBCO). AR and PEG10 transfection Human AR expression plasmid (pSVhAR) was cloned into ChIP assay pCDNA3.0 (Invitrogen) downstream of a cytomegalovirus The ChIP assays were performed essentially as described immediate early promoter (Lu et al., 2001; Sathya et al., 2003). previously (Rokhlin et al., 2005) but with some modiﬁcations. Plasmids encoding PEG10 used in this study have been Brieﬂy, a total1 Â 107 BEL-7404 cells were cultured without or described previously (Tsou et al., 2003). The cells were with DHT for 24 h, crosslinked then with 1% formaldehyde

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5750 Table 1 Effects of AR agonist and antagonist, siRNAAR and siRNAPEG10 on hepatoma formation of hepatic cancer cells Treatment HepG2a BELa

Originalb AR+c PEG10Àc Originalb ARÀc PEG10Àc

Dd Wd DWDWDWDWDW

None 10.5e 1.2e 13.7 2.2 7.5** 0.7** 13.6 2.1 12.6 1.7 8.6** 0.8** DHTf 11.0 1.4 16.2* 3.7* 7.8** 0.8** 18.8* 5.9* 12.8 1.8 9.8** 1.0** FLUf —g — 10.0 1.6 — — 12.2 1.9 — — 8.8** 0.9** FLU+DHTf — — 8.7* 0.7* — — 9.5* 0.9* — — 7.9** 0.8** VPA+DHTf — — 7.9* 0.6* — — 10.4* 1.4* — — 9.5** 1.1**

Abbreviations: AR, androgen receptor; BEL, BEL-7404 hepatoma cells; DHT, dihydrotestosterone; FLU, ﬂutamide; PEG10, paternally expressed gene 10. aHepG2 and BEL hepatoma cells were treated as indicated before nude mice hepatoma formation. b,cThe cells were applied neither stable gene transfection nor siRNAs gene knockdown assays before nude mice hepatoma formation. dD, tumor diameter ( Â mm); W, tumor weight ( Â g). fThe cells were applied either AR agonist DHT (100 nM) for 48 h, or/and antagonist FLU (1 mM) or VPA (100 mM) for 24 h (receptor blocking) as indicated before nude mice hepatoma formation. eData were means7s.d. For simpliﬁcation, s.ds were not shown. g—, no determination. *Po0.001, n ¼ 6, AR agonist or antagonist versus none treated; **Po0.001, n ¼ 6, gene knockdown by siRNAs versus original cell lines.

for 15 min at room temperature. By using a ChIP Assay Kit pGL3 basic vector (Promega, Madison, WI, USA). The (Upstate), cell DNAs were sonicated to an average size of oligonucleotide primers were sense: 50-GGTAC^CACTTC 500 bp by an ultrasonic cell disruptor. Immunoprecipitation GGTTGGTGTGTGTCG-30, kpnI; antisense: 50-C^TCGAG was performed overnight at 41C with an anti-AR (C-19; Santa AAAGCATGGCACTTGGTAGC-30, XhoI. HEK293T cells Cruz, Santa Cruz, CA, USA). Precipitates were washed and were seeded at a density of 2 Â 105/well in a six-well dish and samples were extracted twice with elution buffer, heated at grown to 60–70% confluence. A PEG10/luciferase reporter 651C to reverse crosslinks and DNA fragments were purified was cotransfected with AR expression vectors using with phenol/chloroform. A 5-mlaliquotwas used in the pCDNA3.0 (Invitrogen). Luciferase activities were determined PCR. Four primers sets were designed to cover AREs in using a Luciferase Reporter Assay System in an Analytical promoter, intron and exon regions of androgen-responsive Luminometer Monolight 2010 (PharMingen). The pGL3 gene (PEG10), respectively. ARE sequences were searched controlvector was used as a positive control(Promega). using nGnnCnnnnnGnn (in promoter region), or nGnn Luciferase activities were calculated as the ratio of luciferase CnnnnnGnnCn (in intron and exon regions, Rokhlin et al., activity/protein concentration. 2005) as consensus DNA sequences. The chosen primers for AREs were as follows. Primer set for ARE1 (in promoter region, product: 190 bp): Nude mice hepatoma formation HCC tumors were produced in nude mice (CBy. Cg-Foxn1nu, forward 50-GCTTCAACTTCGGTTGGTGT-30; Jackson Laboratory) and maintained in animalfacilityin reverse 50-CTGCGAAGACAGGAAGGATG-30. Institute of Biochemistry and Cell Biology, SIBS, CAS Primer set ARE2 (in intron region, product: 207 bp): (Shanghai, China) in the specific pathogen-free condition. forward 50-ACCAAGTGCCATGCTTTA-30; Different human hepatoma cells were subcutaneously reverse 50-GCTCCTCGTTCTGGATTGT-30. injected at left thigh of mice (5 Â 106 cells per mouse). Tumors Primer set ARE3 (in exon 2 region, product: 469 bp): were excised from all the mice at around 40 days after the forward 50-AAATAAGCGGGTTTTGAA-30; injection, when the tumor diameter and volume were reverse 50-GAGGTCTTCTGGGCACTC-30. measured. Primer set ARE4 (in exon 2 region, product: 399 bp): Acknowledgements forward 50-CCAAACGCAAGATCAGACG-30; reverse 50-TTCCACAGTAGAGGCACAGG-30. This work was supported by NationalNaturalScience Foundation of China (no. 39870674, 30572119, 30030130, PEG10/luciferase reporter constructs and transient 30471509, 30670937). Dr Tan Jinquan is a Chang Jiang transfection assay Scholar supported by Chang Jiang Scholars Program from Site-directed mutagenesis of the PEG10/luciferase reporter was Ministry of Education, China and Li Ka Shing Foundation, constructed with synthetic oligonucleotide primers using the Hong Kong, China.

References

Chunsong H, Yuling H, Li W, Jie X, Gang Z, Qiuping Z et al. Greider CW. (1999). Telomerase activation: one-step on the (2006). CXC chemokine ligand 13 and CC chemokine ligand road to cancer? Trends Genet 15: 109–112. 19 cooperatively render resistance to apoptosis in B cell Huang R, Xing Z, Luan Z, Wu T, Wu X, Hu G. (2003). A lineage acute and chronic lymphocytic Leukemia speciﬁc splicing variant of SVH, a novel human armadillo CD23+CD5+ B Cells. J Immunol 177: 6713–6722. repeat protein, is up-regulated in hepatocellular carcinomas. Death AK, McGrath KC, Handelsman DJ. (2005). Valproate Cancer Res 63: 3775–3782. is an anti-androgen and anti-progestin. Steroids 70: Jinquan T, Jacobi HH, Jing C, Millner A, Sten E, Hviid L 946–953. et al. (2003). CCR3 expression induced by IL-2 and IL-4

Oncogene Hepatocarcinogenesis and PEG10 activation X Jie et al 5751 functioning as a death receptor for B cells. J Immunol 171: event of DNA damaged-induced apoptosis. J Biol Chem 278: 1722–1731. 836–842. Kim DH, Behlke MA, Rose SD, Chang MS, Choi S, Rossi JJ. Rokhlin OW, Taghiyev AF, Guseva NV, Glover RA, (2005). Synthetic dsRNA Dicer substrates enhance RNAi Chumakov PM, Kravchenko JE et al. (2005). Androgen potency and efficacy. Nat Biotechnol 23: 222–226. regulates apoptosis induced by TNFR family ligands Komine F, Shimojima M, Moriyama M, Amaki S, Uchida T, via multiple signaling pathways in LNCaP. Oncogene 24: Arakawa Y. (2000). Telomerase activity of needle-biopsied 6773–6784. liver samples: its usefulness for diagnosis and judgment Sathya G, Chang CY, Kazmin D, Cook CE, McDonnell DP. of efficacy of treatment of small hepatocellular carcinoma. (2003). Pharmacological uncoupling of androgen receptor- J Hepatol 132: 235–241. mediated prostate cancer cell proliferation and prostate- Li CM, Margolin AA, Salas M, Memeo L, Mansukhani M, specific antigen secretion. Cancer Res 63: 8029–8036. Hibshoosh H et al. (2006). PEG10 is a c-MYC target gene in Shammas MA, Koley H, Batchu RB, Bertheau RC, cancer cells. Cancer Res 66: 665–672. Protopopov A, Munshi NC et al. (2005). Telomerase Lu ML, Schneider MC, Zheng Y, Zhang X, Richie JP. (2001). inhibition by siRNA causes senescence and apoptosis in Caveolin-1 interacts with androgen receptor. A positive Barrett’s adenocarcinoma cells: mechanism and therapeutic modulator of androgen receptor mediated transactivation. potential. Mol Cancer 4: 24–37. J Biol Chem 276: 13442–13451. Sica A, Saccani A, Borsatti A, Power CA, Wells TN, Luini W Massari P, Ho Y, Wetzler LM. (2000). Neisseria meningitidis et al. (1997). Bacterial lipopolysaccharide rapidly inhibits porin PorB interacts with mitochondria and protects cells expression of C-C chemokine receptors in human mono- from apoptosis. Proc Natl Acad Sci USA 97: 9070–9075. cytes. J Exp Med 185: 969–974. Nagasue N, Yu L, Yukaya H, Kohno H, Nakamura T. (1995). Steplewski A, Krynska B, Tretiakova A, Haas S, Khalili K, Androgen and oestrogen receptors in hepatocellular carci- Amini S. (1998). MyEF-3, a developmentally controlled noma and surrounding liver parenchyma: impact on brain-derived nuclear protein which specifically interacts intrahepatic recurrence after hepatic resection. Br J Surg with myelin basic protein proximal regulatory sequences. 82: 542–547. Biochem Biophys Res Commun 243: 295–301. Okabe H, Satoh S, Furukawa Y, Kato T, Hasegawa S, Tsou AP, Chuang YC, Su JY, Yang CW, Liao YL, Liu WK Nakajima Y et al. (2003). Involvement of PEG10 in human et al. (2003). Overexpression of a novelimprinted gene, hepatocellular carcinogenesis through interaction with PEG10, in human hepatocellular carcinoma and in regen- SIAH1. Cancer Res 63: 3043–3048. erating mouse livers. J Biomed Sci 10: 625–635. Okabe H, Satoh S, Kato T, Kitahara O, Yanagawa R, Youssef N, Paradis V, Ferlicot S, Bedossa P. (2001). In situ Yamaoka Y et al. (2001). Genome-wide analysis of gene detection of telomerase enzymatic activity in human expression in human hepatocellular carcinomas using hepatocellular careinogenesis. J Pathol 194: 459–465. cDNA microarray: identification of genes involved in Yu MW, Yang YC, Yang SY, Cheng SW, Liaw YF, Lin SM viralcarcinogenesis and tumor progression. Cancer Res 61: et al. (2001). Hormonalmarkers and hepatitis B virus- 2129–2137. related hepatocellular carcinoma risk: a nested case-control Ono R, Kobayashi S, Wagatsuma H, Aisaka K, Kohda T, study among men. J Natl Cancer Inst 93: 1644–1651. Kaneko-Ishino T et al. (2001). A retrotransposon-derived Zhang DY, Sabla G, Shivakumar P, Tiao G, Sokol RJ, gene, peg10, is a novelimprinted gene locatedon human Mack C et al. (2004). Coordinate expression of regulatory chromosome 7q21. Genomics 73: 232–237. genes differentiates embryonic and perinatalforms of biliary Ono R, Nakamura K, Inoue K, Naruse M, Usami T, atresia. Hepatology 39: 954–962. Wakisaka-Saito N et al. (2006). Deletion of Peg10, an Zhang Y, Wang XW, Jelovac D, Nakanishi T, Yu MH, imprinted gene acquired from a retrotransposon, causes Akinmade D et al. (2005). The ErbB3-binding protein Ebp1 early embryonic lethality. Nat Genet 38: 101–106. suppresses androgen receptor-mediated gene transcription Ramirez R, Carracedo J, Jimenez R, Canela A, Herrera E, and tumorigenesis of prostate cancer cells. Proc Natl Acad Aljama P et al. (2003). Massive telomere loss is an early Sci USA 102: 9890–9895.

Oncogene Oncogene (2011) 30,2798 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc RETRACTIONS Androgen activates PEG10 to promote carcinogenesis in hepatic cancer cells

X Jie, C Lang, Q Jian, L Chaoqun, Y Dehua, S Yi, J Yanping, X Luokun, Z Qiuping, W Hui, G Feili, J Boquan, J Youxin and T Jinquan

Oncogene (2011) 30, 2798; doi:10.1038/onc.2011.66

Retraction to: Oncogene (2007) 26, 5741–5751; doi:10.1038/ sj.onc.1210362; published online 19 March 2007

This paper has been retracted.

Selectively frequent expression of CXCR5 enhances resistance to apoptosis in CD8 þ CD34 þ T cells from patients with T-cell-lineage acute lymphocytic leukemia

Z Qiuping, X Jie, J Youxin, W Qun, J Wei, L Chun, W Jin, L Yan, H Chunsong, Y Mingzhen, G Qingping, L Qun, Z Kejian, S Zhimin, L Junyan and T Jinquan

Oncogene (2011) 30, 2798; doi:10.1038/onc.2011.67

Retraction to: Oncogene (2005) 24, 573–584; doi:10.1038/ sj.onc.1208184; published online 6 December 2004

This paper has been retracted.