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Growth Arrest and DNA Damage 45G Downregulation Contributes to Janus Growth Arrest and DNA Damage 45G Down-Regulation Contributes to Janus Kinase/Signal Transducer and Activator of Transcription 3 Activation and Cellular Senescence Evasion in Hepatocellular Carcinoma Li Zhang,1 Zhaojuan Yang,1 Aihui Ma,1 Yulan Qu,1 Suhua Xia,1 Dongxu Xu,1 Chao Ge,1 Bijun Qiu,2 Qiang Xia,2 Jinjun Li,1 and Yongzhong Liu1 Growth arrest and DNA damage 45G (GADD45G), a stress sensor with multiple impli- cations in various biological processes, is down-regulated in a broad spectrum of can- cers. However, little is known about the biological effects of GADD45G on hepatocellular carcinoma (HCC) cells and the related mechanisms. In the present study, we found that GADD45G was commonly down-regulated in oncogene-transformed mouse liver cells and in human and mouse HCC. Ectopic expression of GADD45G robustly elicited senescence in HCC cells and suppressed tumor growth in vivo. Further- more, GADD45G-induced senescence occurred in HCC cells independently of p53, p16INK4a (p16), and retinoblastoma (Rb). Instead, the prompt inhibition of Janus kinase 2 (Jak2), tyrosine kinase 2 (Tyk2), and signal transducer and activator of tran- scription 3 (Stat3) activation was observed in cells undergoing senescence. Impairment of Jak-Stat3 activation caused by GADD45G expression was associated with activation of SH2 domain-containing protein tyrosine phosphatase-2 (Shp2). Expression of consti- tutively activated Stat3 or human telomerase reverse transcriptase (hTERT), as well as knockdown of Shp2f, efficiently counteracted GADD45G-induced senescence. More important, in clinical HCC specimens, we found that GADD45G expression was inver- sely correlated with phosphorylated Stat3 expression in tumor cells and disease progres- sion. Conclusion: GADD45G functions as a negative regulator of the Jak-Stat3 pathway and inhibits HCC by inducing cellular senescence. The decrease or absence of GADD45G expression may be a key event for tumor cells or premalignant liver cells to bypass cellular senescence. (HEPATOLOGY 2014;59:178-189) epatocellular carcinoma (HCC) is one of the cence provides a tumor-suppressive mechanism for pre- most malignant cancers and is listed as the venting liver tumorigenesis through the cell-autonomous second-most frequent cause of cancer deaths in regulation of proliferation or by triggering immune sur- H 1 2-5 men and sixth in women worldwide. HCC generally veillance. Consistently, genetic or functional inactiva- develops in patients with liver cirrhosis and chronic infec- tion of senescence-related proteins, such as p53 and p16/ tion with hepatitis B virus (HBV) and hepatitis C virus retinoblastoma (Rb), has been observed in human HCC (HCV). Emerging evidence has shown that cellular senes- specimens.6,7 Because initiation of the senescence program Abbreviations: Akt, protein kinase; BrdUB, bromodeoxyuridine; DEN, diethylnitrosamine; DOX, doxycycline; GADD45G, growth arrest and DNA damage 45G; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; hTERT, human telomerase reverse transcriptase; IHC, immunohistochemi- cal; IL, interleukin; JAK, Janus kinase; LPCs, liver progenitor cells; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; Rb, reti- noblastoma; SA-b-gal, senescence-associated b-galactosidase; SC, subcutaneous(ly); Shp2, SH2 domain-containing protein tyrosine phosphatase 2; siRNA, small interfering RNA; Stat3, signal transducer and activator of transcription 3; SV40 LT, SV40 large T antigen; Tet, tetracycline; TMA, tissue microarray; Tyk2, tyro- sine kinase 2. From the 1State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; and 2Department of Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. Received February 25, 2013; accepted July 7, 2013. This work received support by grants from the National Natural Science Foundation of China (no. 81201542), the Natural Science Foundation of Shanghai (no. 12ZR1430100), the Chinese National Key Project (no. 2013ZX10002-011), and the State Key Laboratory of Oncogenes and Related Genes (no. 91-12-03). 178 HEPATOLOGY, Vol. 59, No. 1, 2014 ZHANG ET AL. 179 in cells may require a reorganization of multiple events by immunochemistry staining. Another set of 45 paired adjusting their strength or functional presence, it is impor- HCC samples from the Qidong Liver Cancer Institute tant to obtain more information regarding the mecha- (Qidong, China) were processed for RNA and protein nisms by which liver cells are transformed as well as how analysis. All human materials were approved by the liver tumor cells bypass senescence. institutional ethical review committee. The human Growth arrest and DNA damage 45G (GADD45G) HCC cell lines SK-Hep1 and Hep3B were purchased is a member of the GADD45 family, which has been from the American Type Culture Collection (Manassas implicated in various biological processes, including VA). The human HCC cell line SMMC-7721, embry- integration of the cellular response to different stresses, onic kidney 293 cells were from the Chinese Academy regulation of development, cell differentiation, and of Sciences (Shanghai, China). Mouse fetal liver pro- survival.8-10 GADD45G expression is broadly down- genitor cells (LPCs) were isolated from p532/2 mice regulated in a number of cancers, such as lymphoma, by the methods described previously13,14 and cultured nasopharyngeal carcinoma, cervical carcinoma, esopha- for the retroviral delivery of H-Ras V12, myristoylated geal carcinoma, lung carcinoma, and HCC.11,12 Mech- protein kinase B (Akt)1, and c-Myc. anistically, decrease of GADD45G expression in these Animal Model. Male BALB/c nude mice (6-8 tumors is primarily attributed to hypermethylation of weeks of age) received single subcutaneous (SC) flank CpG islands.11 Although a ubiquitous down-regulation injection of 5 or 6 3 106 Sk-Hep1 cells diluted in of GADD45G expression has been observed in various 200 lL of saline. Drinking water was supplemented types of cancer, the molecular mechanisms by with doxycycline (2 mg/mL) to induce GADD45G which GADD45G functions as a tumor suppressor are expression. Tumor growth was monitored by bidimen- still unclear. In the present study, we show that sional measurements using a caliper. Tumor-bearing GADD45G robustly elicits cellular senescence in HCC mice were sacrificed 50 days after inoculation, and cells and remarkably suppresses tumor growth in vivo. then the tumors were removed for further study. All We demonstrate, for the first time, that GADD45G mice studies were conducted in accord with protocols induces HCC cell senescence independently of the approved by the Shanghai Medical Experimental Ani- functional presence of p16, p53, and Rb, and that mal Care Commission. down-regulation of Janus kinase (Jak)/signal transducer Statistical Analysis. Statistical analyses were per- and activator of transcription 3 (Stat3) is the key event formed using SPSS 16.0 statistical software (SPSS, for GADD45G-induced cell senescence and tumor Inc., Chicago, IL). For in vivo studies, the percentage suppression. These findings provide new evidence for of tumor-free mice in each group was analyzed using the involvement of the stress sensor, GADD45G, in Kaplan-Meier’s survival analysis and Tarone-Ware’s sta- the biological connection between cellular senescence tistic was used for comparison of curves between evasion and hepatotumorigenesis. groups. For in vitro studies, a two-tailed t test was used to determine significance. The statistical correla- tion between the clinical parameters of HCC and Materials and Methods GADD45G staining levels in tissue sections was ana- lyzed by the chi-square test, nonparametric test, and Human Tissue Specimens and Cell Lines. A com- one-way analysis of variance. A P value less than 0.05 mercially available tissue microarray (TMA) containing was considered statistically significant. 75 pairs of HCC was used, and the patient informa- Laboratory Methods. See the Supporting Materials tion provided by the manufacturer is listed in the and Methods section for detailed experimental Supporting Data. The TMA sections were used for procedures. Address reprint requests to: Yongzhong Liu, Ph.D., State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Wenxuan Medical Building, 800 Dongchuan Road, 200240 Shanghai, China. E-mail: [email protected]; fax: 86-21-3420-6283. Copyright VC 2013 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26628 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article. 180 ZHANG ET AL. HEPATOLOGY, January 2014 Fig. 1. GADD45G is down-regulated in liver tumorigenic cells and human HCC specimens. (A-C) qRT-PCR analysis of the relative expression of GADD45G in (A) transformed and nontransformed cells, (B) HCC cell lines and the immortalized liver cell line, Thle2, and in (C) nontumor tis- sues (N) and mouse HCC tissues (T) from DEN-treated mice (C57BL/6). Data shown are mean 6 SD from three independent experiments (**P < 0.01; *P < 0.05). (D) Scatter plots of relative expression of GADD45G in nontumor tissues (N) and their paired HCC counterparts (T). Statis- tical data are shown as mean 6 SD (***P < 0.001). (E) Protein levels of GADD45G in
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