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DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss Chieh-Hsiang Chan1, Chun-Ming Chen2,3, Yan-Hwa Wu Lee4,5, and Li-Ru You1,3

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DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss Chieh-Hsiang Chan1, Chun-Ming Chen2,3, Yan-Hwa Wu Lee4,5, and Li-Ru You1,3 Published OnlineFirst October 8, 2018; DOI: 10.1158/1541-7786.MCR-18-0551 Oncogenes and Tumor Suppressors Molecular Cancer Research DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss Chieh-Hsiang Chan1, Chun-Ming Chen2,3, Yan-Hwa Wu Lee4,5, and Li-Ru You1,3 Abstract The pleiotropic roles of DEAD-box helicase 3, X-linked double-strand breaks in liver indicated that the replicative (DDX3X), including its functions in transcriptional and trans- stress occurred in female mutants. Further chromatin immu- lational regulation, chromosome segregation, DNA damage, noprecipitation analyses demonstrated that DDX3X bound to and cell growth control, have highlighted the association promoter regions and regulated the expression of DNA repair between DDX3X and tumorigenesis. However, mRNA tran- factors, DDB2 and XPA, to maintain genome stability. Loss of scripts and protein levels of DDX3X in patient specimens have Ddx3x led to decreased levels of DNA repair factors, which shown the controversial correlations of DDX3X with hepato- contributed to an accumulation of unrepaired DNA damage, cellular carcinoma (HCC) prevalence. In this study, generation replication stress, and eventually, spontaneous liver tumors flox/flox of hepatocyte-specific Ddx3x-knockout mice revealed that loss and DEN-induced HCCs in Alb-Cre/þ;Ddx3x mice. of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensa- Implications: These data identify an important role of DDX3X tory proliferation in female mutants at 6 weeks of age. The in the regulation of DNA damage repair to protect against sustained phosphorylation of histone H2AX (gH2AX) and replication stress in liver and HCC development and progres- significant accumulation of DNA single-strand breaks and sion. Mol Cancer Res; 1–12. Ó2018 AACR. Introduction feature during HCC progression is repeated cycles of cell death and compensatory proliferation, which causes an inflammatory Liver cancer is the fifth most common cancer and the third most microenvironment in the liver, leading to fibrosis, cirrhosis, and frequent cancer-related of deaths worldwide. The majority of liver ultimately, malignant transformation of the hepatocytes. malignancies are hepatocellular carcinoma (HCC) and cholan- DEAD-box helicase 3, X-linked (DDX3X; also known as DDX3) giocarcinoma that have high morbidity and mortality in devel- belongs to the DEAD-box RNA helicase family and was initially oping countries, but their incidences are also increased in devel- identified as a cellular factor that bound to hepatitis C viral core oped countries. Etiological studies of liver cancer have identified protein. Accumulated evidences showed that DDX3X is involved four major risk factors, including chronic infection with hepatitis in diverse cellular processes, including cell growth, cell-cycle B or C viruses, alcohol use, and the intake of aflatoxin-contam- control, mitotic chromosome segregation, innate immunity, inated food (1–3). In recent years, obesity-associated nonalco- tumorigenesis, and virus replication (6). Our previous studies holic fatty liver disease has continuously increased (4). It is showed that DDX3X acted as a tumor suppressor in various cancer generally accepted that hepatocarcinogenesis is a multistep pro- cell lines. We found that lower DDX3X levels were significantly cess with an accumulation of the genetic and epigenetic altera- correlated with higher HCC prevalence, particularly in male and tions of critical genes involving hepatocyte growth and prolifer- HBV-positive patients (7). Knockdown of DDX3X in nontrans- ation, cell death, cell adhesion, and metabolism (5). A common formed NIH3T3 cells led to a premature entry into S phase and an increase of cell proliferation, and enhanced the oncogenic v-ras– induced anchorage-independent growth. In addition, overexpres- 1 WAF1/Cip1 Institute of Biochemistry and Molecular Biology, National Yang-Ming sion of DDX3X activated the p21 promoter through University, Taipei, Taiwan. 2Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan. 3Cancer Progression its physical interaction with Sp1, and consequently, negatively Research Center, National Yang-Ming University, Taipei, Taiwan. 4Department of regulated cyclin D1 protein levels and tumor cell proliferation (8). Biological Science and Technology, National Chiao Tung University, Hsinchu, A recent study further showed that DDX3X level was inversely Taiwan. 5Center For Intelligent Drug Systems and Smart Bio-devices (IDS2B), associated with the tumor grade and predicted poor prognoses for National Chiao Tung University, Hsinchu, Taiwan. HCC patients. Epigenetic regulation of a subset of HCC-associ- Note: Supplementary data for this article are available at Molecular Cancer ated tumor-suppressor miRNAs, including miR-200b, miR-200c, Research Online (http://mcr.aacrjournals.org/). miR-122, and miR-145, by DDX3X induced stem cell–like prop- Corresponding Authors: Li-Ru You, National Yang-Ming University, No. 155, Sec. erties and promoted tumorigenic potential (9). In contrast to our 2, Li-Nong Street, Taipei 112, Taiwan. Phone: 886-2-28267368; E-mail: studies, Huang and colleagues showed by qRT-PCR analysis that [email protected]; and Yan-Hwa Wu Lee, Department of Biological Science and DDX3X was overexpressed in 64% of HCC tissues (10), and Su Technology, National Chiao Tung University, No. 75, Bo-ai St., East Dist., Hsinchu and colleagues found no association between DDX3X RNA level 300, Taiwan. Phone: 886-3-5712121, ext. 56986; E-mail: [email protected]. and survival in liver cancer patients (11). Therefore, the patho- doi: 10.1158/1541-7786.MCR-18-0551 logic role of DDX3X in liver cancer remains to be clarified. In a Ó2018 American Association for Cancer Research. previous study, we generated a conditional knockout allele for www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 8, 2018; DOI: 10.1158/1541-7786.MCR-18-0551 Chan et al. Ddx3x gene and identified the essential roles of DDX3X in pla- monitoring liver injury and functions, were measured routinely cental and early embryonic development (12). Here, hepatocyte- every 3 months from 12 months of age. TG and TCHO levels in the specific Ddx3x-knockout mice were generated. We showed that mutant mice were not different from their littermate controls at all loss of Ddx3x led to DNA damage and replicative stress at a young time periods (Supplementary Fig. S2). However, ALT levels were age, and mice developed spontaneous liver tumors with aging. elevated in some, but not all, of the hepatocyte-specific Ddx3x- Ddx3x-deficiency females were more susceptible to diethylnitro- knockout mice with visible tumors, when dissected (Fig. 1A). samine (DEN)-induced HCC than littermate female controls. The results showed that 10.7% (3/28) and 42.1% (8/19) of flox flox/flox Moreover, DDX3X regulated DNA damage repair factors DDB2 dissected Alb-Cre/þ;Ddx3x /Y male and Alb-Cre/þ;Ddx3x and XPA, in the liver and in vitro, which are critical to maintain female mutants, respectively, developed liver tumors after approx- genome stability. imately 12 to 27 months. Among these mice, we noted 4 of 5 flox/flox (80%) Alb-Cre/þ;Ddx3x females developed liver tumors at Materials and Methods 24 months of age. None of the littermate controls and Alb-Cre/þ; flox/þ Ddx3x heterozygous females had a tumor (Fig. 1B and C). Mice, DEN treatment, and serum biochemical analysis Hematoxylin & eosin (H&E) staining and immunohistochemical The floxed Ddx3x mice were generated previously (12) and had analysis of hepatocyte marker HNF4a and cholangiocyte markers been backcrossed into the C57BL/6 background for at least 10 K19 and Sox9 in tumor sections revealed that the most common generations. The hepatocyte-specific Ddx3x-knockout mice were flox/flox histopathology was the clear cell type HCC (16), which was generated through breeding Ddx3x mice with Alb-Cre [B6. flox/flox detected in both Alb-Cre/þ;Ddx3x female and Alb-Cre/þ; Cg-Tg(Alb-cre)21Mgn/J] transgenic mice (13). The serum levels of flox Ddx3x /Y male. Other types of liver tumors, including adenoma alanine aminotransferase (ALT), triacylglycerol (TG), and total flox/flox and cholangiocarcinoma, were observed in Alb-Cre/þ;Ddx3x cholesterol (TCHO) were monitored using biochemical slides flox female and Alb-Cre/þ;Ddx3x /Y male, respectively (Fig. 1D). (Fuji DRY-CHEM 400i; Fujifilm) according to the manufacturer's Western blot analysis confirmed that DDX3X protein levels were instructions. To induce HCC, DEN (25 mg/kg body weight; significantly decreased in tumor and nontumor liver tissues from N0756-10ML, Sigma-Aldrich) was injected i.p. into 2-week-old flox flox/flox aged Alb-Cre/þ;Ddx3x /Y male and Alb-Cre/þ;Ddx3x mice. To induce acute liver injury, 10-week-old mice were injected female mice compared with their gender-matched littermate with DEN (100 mg/kg body weight) and sacrificed 1 or 3 days controls (Fig. 1E). Given the higher tumor incidence was observed thereafter. The experimental procedures using mice were flox/flox in aged Alb-Cre/þ;Ddx3x females, the effects of DDX3X on approved by the Institutional Animal Care and Use Committee liver tumorigenesis in female mice were extensively characterized. (IACUC) of National Yang-Ming University, Taiwan. The animal care and experimental procedures were performed in accordance Hepatocyte-specific deletion of Ddx3x leads to liver injury, with the Guidelines of the IACUC of National Yang-Ming Uni- ductular reactions, and inflammation versity, Taiwan. To further determine the pathologic role of DDX3X in tumor progression, serum ALT levels were closely monitored at young Histologic analysis and molecular techniques ages. The mean ALT level of 3- to 20-week-old littermate controls Further details are provided in the Supplementary Information. was 13.4 Æ 0.7 U/l (Fig. 2A). We found significantly elevated ALT flox/flox levels in Alb-Cre/þ;Ddx3x mutants at 6 and 10 weeks of age Statistical analysis (115.1 Æ 12.0 and 26.9 Æ 2.0 U/l, respectively).
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