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. 2018 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 2018 American Association for Cancer Research. previous study, we generated a conditional knockout allele for

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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 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 12.0 and 26.9 2.0 U/l, respectively). There was no Hepatocytes stained positive for different markers in livers were difference in ALT levels between controls and mutants at 3 and 20 determined by the positive hepatocytes/total hepatocytes in each weeks of age. Histologic analyses of H&E-stained liver sections high-power (200 magnification) field. For each sample, at least revealed increased numbers of basophilic cells [i.e., ductular four fields were randomly selected and counted. Quantitative flox/flox reactions (DRs)] in 6- and 10-week-old Alb-Cre/þ;Ddx3x results are expressed as the mean SEM. The Student t test was mice, in accordance with increased serum ALT levels, when used to determine P values, unless stated otherwise. compared with controls (Fig. 2B). The DRs (17) were further verified by significant expansion of liver progenitor cell (LPC)/ Results cholangiocyte marker–positive subsets, including A6-, EpCAM-, Hepatocyte-specific Ddx3x ablation leads to the development of cytokeratin 19 (K19)-, and Sox9-positive cells as assessed by IHC flox/flox hepatocellular tumors in aged mice in 6-week-old Alb-Cre/þ;Ddx3x livers (Fig. 2C). DRs were To investigate the physiopathologic role of DDX3X in liver, notably reduced to a lesser extent in 10-week-old Alb-Cre/þ; flox/flox floxed Ddx3x mice were crossed with Alb-Cre transgenic mice. The Ddx3x livers and disappeared thereafter (Supplementary Alb-Cre transgene is specifically activated in the liver from late Fig. S3). qRT-PCR analyses confirmed a decrease in Ddx3x expres- embryonic stage, and Cre-mediated gene recombination occurs sion and significant increases of LPC/cholangiocyte gene levels, progressively with age (14, 15). PCR of genomic DNA from liver including CD133, EpCAM, and K19 in 6-week-old Alb-Cre/þ; flox/flox confirmed the progressive loss of floxed Ddx3x allele and the Ddx3x livers compared with those of littermate controls. presence of null allele. The reduction of DDX3X protein was Nevertheless, the increasing trend of Sox9 levels did not reach specifically observed in liver. The presence of nonparenchymal statistical significance (Fig. 2D). The liver injury was also associ- cells, which do not have Cre activity, may explain the low levels of ated with significant immune cell infiltration consisting of F4/80 DDX3X protein in livers of mutant mice (Supplementary Fig. S1). (macrophages)-, CD3 (T cells)-, and B220 (B cells)-positive The hepatocyte-specific Ddx3x-knockout mice were born at cells and increased Tnfa levels (Supplementary Fig. S4), which expected Mendelian ratios and were morphologically indistin- coincided with the significant elevation of ALT levels in 6-week- flox/flox guishable from their littermate controls at young ages. Therefore, old Alb-Cre/þ;Ddx3x livers compared with their controls. serum ALT, TG, and TCHO levels, commonly used markers for In aged mice, F4/80-, B220-, and CD3-positive cells were

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DDX3X in DNA Damage and Liver Tumorigenesis

Figure 1. Spontaneous development of liver tumors in hepatocyte-specific Ddx3x knockout mice. The sera and livers were collected from Ddx3x-knockout mice (Alb-Cre/þ; Ddx3xflox/Y males and Alb-Cre/þ;Ddx3xflox/flox females) and their littermate controls (Ddx3xþ/Y, Alb-Cre/þ;Ddx3xþ/Y,orDdx3xflox/Y males and Ddx3xflox/þ or Ddx3xflox/flox females) from 12 to 27 months of age. A, The serum ALT levels. N 15 per group. B, Tumor-free survival of controls and Ddx3x-knockout mice was assessed using Kaplan–Meier analysis. C, The numbers and sizes of tumors in mice. The size of the liver tumor was analyzed by determining the longest diameter of a tumor. D, Gross images of representative livers and H&E and IHC staining of liver sections at indicated ages (M, month). The cholangiocellular carcinoma (CC) and clear cell type HCC were observed in Alb-Cre/þ;Ddx3xflox/Y males. The clear cell type HCC was found in the Alb-Cre/þ;Ddx3xflox/flox females. Expression of hepatocyte marker HNF4a and cholangiocyte markers K19 and SOX9 in tumors of Ddx3x-knockout mice was assessed by IHC. Dashed lines demarcate the boundary between normal liver tissue (N) and tumor (T). E, Expression levels of DDX3X in liver tissues from controls and the tumors (T) and adjacent liver tissues (N) from Ddx3x-knockout mice were evaluated by Western blot analysis. Protein levels were normalized to the loading control GAPDH, expressed as fold changes relative to gender-matched controls (set as 1) and shown under their corresponding panels. N ¼ 4 per group. One-way ANOVA was used to determine P values in A and C. Log-rank (Mantel–Cox) test was used to determine P values in B. , P < 0.05; , P < 0.01; and , P < 0.001. Scale bars, 1 cm (gross view); 200 mm (section).

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ALT levels, large numbers of cleaved Caspase-3 (cCasp3)–positive apoptotic cells were specifically detected in the Alb-Cre/þ; flox/flox Ddx3x livers at 6 weeks of age when compared with their littermate controls (Fig. 3A and B). In 3-week-old mice, cell proliferation was still high in the livers, and proliferation marker Ki67-positive cells were comparable in livers from both controls flox/flox and Alb-Cre/þ;Ddx3x mice. The decrease in cell proliferation was observed in control livers as development proceeded; nevertheless, we detected a significant increase of Ki67-positive cells flox/flox in 6-week-old Alb-Cre/þ;Ddx3x livers (Fig. 3A and C). These results indicated that compensatory proliferation occurred after flox/flox cell apoptosis in 6-week-old Alb-Cre/þ;Ddx3x livers. Our previous study showed that targeted Ddx3x ablation in the epi- blast leads to widespread gH2AX (phosphorylated histone H2AX, as a marker of DNA damage) and apoptosis, which causes embryonic lethality (12). We noted that gH2AX signals were flox/flox significantly increased in Alb-Cre/þ;Ddx3x livers, peaked at 6 weeks of age, and then gradually declined at 10 and 20 weeks of age, compared with littermate controls (Fig. 3A and D). The gH2AX foci were observed in approximately 10% of proliferating flox/flox Ki67-positive hepatocytes in 6-week-old Alb-Cre/þ;Ddx3x þ livers (Supplementary Fig. S6). The significant higher gH2AX / þ flox/flox Ki67 hepatocytes in 6-week-old Alb-Cre/þ;Ddx3x livers imply that loss of Ddx3x causes prolonged DNA damage, and may be linked to genome instability and subsequent liver tumor- flox/flox igenesis in Alb-Cre/þ;Ddx3x mice.

DNA single-strand break and double-strand break signalings flox/flox were induced in Alb-Cre/þ;Ddx3x livers Proper DNA replication and chromosome segregation are pre- requisites for genome integrity. To ensure the normal cellular functions and faithful genome maintenance and transmission, a complex network of DNA damage response (DDR) systems has evolved to sense and respond to different forms of DNA damage Figure 2. and replication stress (19, 20). Along with increased numbers of flox/flox Loss of Ddx3x results in liver injury and DRs in Alb-Cre/þ;Ddx3x mice. A, gH2AX-positive cells, IHC analyses of liver sections revealed that The serum ALT activity was measured in controls (Ddx3xflox/þ and Ddx3xflox/flox) flox/flox enlarged hepatocytes with atypical nuclei were frequently and Alb-Cre/þ;Ddx3x mice at indicated ages (age in weeks). N 5per Alb-Cre/þ;Ddx3xflox/flox group. B, Representative images of H&E-stained liver sections. DRs observed in livers compared with those of (arrowheads) were observed in Alb-Cre/þ;Ddx3xflox/flox livers at 6 and 10 weeks control livers (Supplementary Fig. S7A). Notably, the numbers of of age. C, Representative A6, EpCAM, K19, and Sox9 immunostaining (red) cells with 53BP1 nuclear bodies were significantly increased in 6- flox/flox in 6-week-old controls and Alb-Cre/þ;Ddx3xflox/flox livers. The positively stained week-old Alb-Cre/þ;Ddx3x livers (Supplementary Fig. S7B). cells are shown at higher magnification in the inset. Nuclei (blue) were These results prompted us to speculate that loss of Ddx3x causes Ddx3x counterstained with hematoxylin. D, Relative mRNA levels of and replication stress and may be a driving force for tumorigenesis in cholangiocyte/LPC genes in livers from 6-week-old controls and Alb-Cre/þ; flox/flox flox/flox Alb-Cre/þ;Ddx3x livers. Ddx3x mice were detected by qRT-PCR. N ¼ 5 per group. , P < 0.05; , P < 0.01; and , P < 0.001. Scale bar, 200 mm. Replication protein A (RPA) is a guardian of genome integrity during DNA replication, recombination, and repair events, andalsoactsasakeysensorinDDR(21,22).IHCanalysesof3- significantly increased in nontumor liver tissues of tumor-bearing week-old liver sections showed similar expression levels of flox/flox flox/flox Alb-Cre/þ;Ddx3x mice compared with those of littermate RPA70 and RPA32 in the Alb-Cre/þ;Ddx3x mice and their controls. We found that IL1b-, TNFa-, and NF-kB–positive cells littermate controls (Fig. 4A). As development proceeded, the were significantly increased in both tumors and adjacent non- expression levels of both RPA70- and RPA32-positive cells flox/flox tumor liver tissues of aged tumor-bearing Alb-Cre/þ;Ddx3x declined significantly in 6- and 10-week-old littermate controls livers. Also, Il1b and Tnfa mRNA levels were significantly compared with those of 3-week-old controls. The staining flox/flox increased in tumors from Alb-Cre/þ;Ddx3x livers (Supple- intensities of RPA70 and RPA32 persisted in 6-week-old Alb- flox/flox mentary Fig. S5). Cre/þ;Ddx3x livers (Fig. 4A). The changes of DDX3X, RPA70, RPA32, and phosphorylated RPA32 (the active form Loss of Ddx3x causes DNA damage, cell death, and of RPA32) protein levels in livers from 3-, 6-, and 10-week-old flox/flox compensatory proliferation in young mice controls and Alb-Cre/þ;Ddx3x mice were further con- It has generally been accepted that loss of hepatic mass after firmed by Western blotting. As expected, a significant increase injury activates compensatory proliferation, leading to tumor of gH2AX expression was detected in 6-week-old Alb-Cre/þ; flox/flox development (18). In accordance with the dynamic changes of Ddx3x livers (Fig. 4B; Supplementary Fig. S8A).

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Figure 3. Loss of Ddx3x causes cell death, compensatory proliferation, and DNA damage in Alb-Cre/þ;Ddx3xflox/flox livers at 6 weeks of age. A, IHC analyses of cCasp3 (red), Ki67 (red), and gH2AX (green) in livers from controls (Ddx3xflox/þ and Ddx3xflox/flox)andAlb-Cre/þ;Ddx3xflox/flox mice at indicated ages (age in weeks). Nuclei (blue) were counterstained with hematoxylin and/or 4,6-diamidino-2-phenylindole (DAPI). B–D, Percentages of cCasp3-, Ki67-, and gH2AX-positive flox/flox hepatocytes in livers from controls and Alb-Cre/þ;Ddx3x mice were quantified. N 3 per group. , P < 0.05 and , P < 0.001. Scale bar, 200 mm.

flox/flox It has been demonstrated that RPA binds to ssDNA and Alb-Cre/þ;Ddx3x livers were significantly decreased to triggers activation of the ATR-Chk1 pathway in response to approximately 4% of control hepatocytes. The relative fold DNA damage or replication stress (22, 23). Indeed, significant changes of gH2AX, RPA70, pRPA32, RPA32, and Mre11 proteins increases of phosphorylated ATR (pATR)– and phosphorylated were greater in isolated hepatocytes than those from whole livers flox/flox Chk1 (pChk1)–positive hepatocytes were detected in 6-week-old of 6-week-old Alb-Cre/þ;Ddx3x mice, confirming a hepato- flox/flox Alb-Cre/þ;Ddx3x liver sections compared with those of their cyte-specific effect (Supplementary Fig. S8C compared with Sup- littermate controls (Fig. 4C). These results revealed that loss of plementary Fig. S8A). Taken together, these results showed that flox/flox DDX3X induces replication stress in Alb-Cre/þ;Ddx3x livers not only single-strand break (SSB), but also DSB signaling, was flox/flox at a young age. induced in 6-week-old Alb-Cre/þ;Ddx3x livers. If replication stress persists, ssDNA long-time exposure in cells consequently results in double-strand break (DSB) and apoptosis. Ddx3x loss inhibits the nucleotide excision repair and The presence of the MRN complex (Mre11, Rad50, and NBS1), the downregulates DDB2 and XPA genes through Sp1 functional DSB sensor, in the DNA damage site, subsequently Cells halt cell-cycle progression in the presence of DNA recruits and activates the ATM-Chk2 signaling pathway (22, 23). damage to provide more time for DNA repair. The levels of Cdkn2b Ink4b WAF1/CIP1 Kip1 IHC staining and Western blot analyses showed increased Mre11 (p15 ), Cdkn1a (p21 ), and Cdkn1b (p27 ) were flox/flox levels in 6-week-old Alb-Cre/þ;Ddx3x livers compared with increased by 2-, 3.8-, and 1.4-fold, respectively, in 6-week-old flox/flox their littermate controls (Fig. 4D and E; Supplementary Fig. S8A). Alb-Cre/þ;Ddx3x livers compared with controls, indicating In addition, the pChk2-positive hepatocytes were dramatically that loss of Ddx3x halted cell-cycle progression in response to flox/flox increased in the 6-week-old Alb-Cre/þ;Ddx3x livers (Fig. 4F). DNA damage (Supplementary Fig. S9). Consistent with our To substantiate these findings are hepatocyte specific, the expres- previous study in embryonic cells (12), the Trp53 (p53) level was flox/flox sion levels of DDX3X, gH2AX, and the sensors in DDR in isolated reduced in the Alb-Cre/þ;Ddx3x livers compared with that of flox/flox hepatocytes from 6-week-old controls and Alb-Cre/þ;Ddx3x the controls. livers were examined. In isolated hepatocytes, PCR analysis of The activation of ATR/ATM, upregulation of cell-cycle regula- genomic DNA showed the floxed Ddx3x alleles were almost tors, and the prolonged gH2AX-positive cells in Alb-Cre/þ; flox/flox completely deleted and DDX3X protein levels from 6-week-old Ddx3x livers suggest the possibility that the downstream

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Figure 4. DNA SSB and DSB signalings are induced in 6-week-old Alb-Cre/þ;Ddx3xflox/flox mice. The liver tissues from controls and Alb-Cre/þ;Ddx3xflox/flox mice at 3, 6, and 10 weeks of age were collected and processed for immunostaining and Western blot analysis. A, Representative images of RPA70 and RPA32 (red) immunostaining. B, flox/flox Representative images of DDX3X, gH2AX, RPA70, and RPA32 protein expression in livers from controls (Ctrl) and Alb-Cre/þ;Ddx3x (KO) mice by Western blot analysis. The pRPA32 is the phosphorylated form of RPA32. Protein levels were normalized to the loading control GAPDH, expressed as fold changes relative to 3-week-old controls (set as 1) and shown under their corresponding panels. C, Immunofluorescence costaining of pATR (green) and pChk1 (green) with hepatocyte marker HNF4a (red). Percentages of pATR- and pChk1-positive hepatocytes (HNF4aþ) in livers were quantified. N 3 per group. D, Representative images of Mre11 (red) immunostaining. E, Representative images of Mre11 protein expression in livers from controls (Ctrl) and Alb-Cre/þ;Ddx3xflox/flox (KO) mice by Western blot analysis. Fold changes of Mre11 expression relative to 3-week-old controls (set as 1) are shown under their corresponding panels. F, Immunofluorescence costaining of pChk2 (green) and HNF4a. Percentages of pChk2-positive hepatocytes in livers were quantified. N ¼ 3 per group. The stained cells (A, C, D,andF) are shown at higher magnification in the inset. Nuclei were stained with hematoxylin (A and D) and/or DAPI (C and F). , P < 0.05; , P < 0.01; and , P < 0.001. Scale bar, 200 mm.

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Figure 5. The targets of DDX3X are involved in NER. A, qRT-PCR analyses of NER genes and Sp1 in livers from 6-week-old controls and Alb-Cre/þ; Ddx3xflox/flox mice. N ¼ 4 per group. B, Relative mRNA levels of DDX3X, DDB2, ERCC6, ERCC8, XPA, and Sp1 genes in stable shLuc, shDDX3X#2, and shDDX3X#3 HepG2 cells. N ¼ 3 per group. Representative images of DDX3X, DDB2, XPA, and Sp1 protein expression in shLuc-, shDDX3X#2-,andshDDX3X#3-knockdown HepG2 cells by Western blot analysis were shown (right plot). Protein levels were normalized to the loading control GAPDH, expressed as fold changes relative to controls (set as 1) and shown under their corresponding panels. C–E, Binding of DDX3X and Sp1 to the promoter regions of the DDB2 and XPA genes. ChIP experiments were performed with liver tissues from 6-week-old controls and Alb-Cre/þ;Ddx3xflox/flox mice (C), DDX3X- (shLuc, shDDX3X#2,andshDDX3X#3)(D), and Sp1-(siControl and siSp1) knockdown HepG2 cells (E). The cross-linked protein–DNA complex immunoprecipitated with anti-DDX3X and anti-Sp1 antibodies from liver tissues, and cultured cells were processed for ChIP-qPCR assays. The binding ability on the promoter region was presented in the relative fold change to rabbit IgG, which was normalized with input individually. N 5 per group. Representative images of DDX3X, Sp1, DDB2, and XPA protein expression in siControl- and siSp1-knockdown HepG2 cells by Western blot analysis were shown (E, left plot). The relative levels of proteins were quantified and shown under their corresponding panels. , P < 0.05; , P < 0.01; and , P < 0.001.

functions of DNA repair and signaling may not work efficiently. levels of Ddb2, Ercc6, Ercc8, and Xpa, but not Ddb1 and Xpc, were The nucleotide excision repair (NER) machinery is involved in the significantly decreased (Fig. 5A). The expression of Ercc1, Ercc4, removal of a wide range of DNA lesions either through global Ercc5, and proliferating cell nuclear antigen (Pcna), which are genome NER (GG-NER) or through transcription-coupled NER involved in DNA incision, excision, and de novo synthesis, were (TC-NER). The damage is initially recognized by DDB2 (damage- not affected (Fig. 5A). A survey of the proximal promoter regions specific DNA binding protein 2) and XPC (xeroderma pigmento- identified the confirmed Sp1-binding sites and/or GC-rich ele- sum, complementation group C) in GG-NER, or ERCC6 (excision ments in the responsive genes, Ddb2 (26), Ercc6 (27), and Xpa repair cross-complementation group 6) and ERCC8 in TC-NER. (28). We then found that the expression level of Sp1, a previously After recognition, DDB1 interacts with DDB2 or ERCC8, which identified partner of DDX3X, was decreased in 6-week-old Alb- flox/flox then recruits XPA and its interacting proteins to perform DNA Cre/þ;Ddx3x livers. unwinding (24). Thereafter, sequential and coordinated assembly To gain mechanistic insight into the link between DDX3X and of core repair factors functionally involved in DNA incision, NER, the mRNA levels of the DDB2, ERCC6, ERCC8, and XPA excision, and de novo synthesis proceeds through the NER pathway genes in DDX3X-knockdown (shDDX3X#2 and shDDX3X#3) and flox/flox (25). In 6-week-old Alb-Cre/þ;Ddx3x livers, the expression control (shLuc) HepG2 cells (9) were investigated. Our results

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Figure 6. The percentages of cCasp3-, Ki67-, gH2AX-, 53BP1-, and pATR-positive hepatocytes are significantly increased in tumor samples from Alb-Cre/þ;Ddx3xflox/flox mutants. The liver tissues from 18- to 24-month-old controls and tumor-bearing Alb-Cre/þ;Ddx3xflox/flox mice were processed for immunostaining. A, Representative images of cCasp3 (red), Ki67 (red), gH2AX (green), 53BP1 (green), and pATR (green) immunostaining. The stained cells are shown at higher magnification in the inset. Nuclei were stained with hematoxylin and/or DAPI. B, Percentages of cCasp3-, Ki67-, gH2AX-, 53BP1-, and pATR-positive hepatocytes in liver sections were quantified. N ¼ 3 per group. , P < 0.05; , P < 0.01; and , P < 0.001. Scale bar, 200 mm.

showed that DDX3X, DDB2, XPA, and Sp1, but not ERCC6 and showed that cCasp3 (apoptosis)-, Ki67 (compensatory prolifer- ERCC8, were significantly decreased in DDX3X-knockdown cells ation)-, and gH2AX-positive hepatocytes were significantly (Fig. 5B). Accordingly, the DDX3X, DDB2, XPA, and Sp1 protein increased in tumors compared with adjacent nontumor liver flox/flox levels were significantly reduced in DDX3X-knockdown cells. tissues from Alb-Cre/þ;Ddx3x mutants and their controls. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) In addition, the numbers of cells positive for pATR and 53BP1 assays showed the recruitment of DDX3X proteins to the pro- nuclear bodies were significantly increased in tumors from Alb- flox/flox moter regions of Ddb2 and Xpa was approximately 2.8-fold and Cre/þ;Ddx3x mutants (Fig. 6). Altogether, our data suggest 3.6-fold higher, respectively, in livers from 6-week-old controls that Ddx3x plays a critical role in the maintenance of genome flox/flox than in Alb-Cre/þ;Ddx3x mutants. The recruitment of Sp1 integrity in the cell populations with relatively rapid rates of proteins to the promoter regions of Ddb2 and Xpa showed proliferation. flox/flox decreasing trends in Alb-Cre/þ;Ddx3x livers compared with those of controls (Fig. 5C). Consistently, we detected significantly Loss of Ddx3x predisposes female mice to DEN-induced liver decreased recruitment of DDX3X proteins and slightly decreased injury and tumorigenesis recruitment of Sp1 proteins to the promoter regions of DDB2 and The carcinogen, DEN, which is bioactivated by cytochrome XPA in DDX3X-knockdown HepG2 cells (Fig. 5D). In addition, P450 (Cyp) enzymes in the liver and generates DNA adducts, is the recruitment of DDX3X and Sp1 proteins to the promoter widely used to induce liver injury and tumorigenesis in experi- regions of DDB2 and XPA was decreased in Sp1-knockdown mental animal models (29, 30). To examine the acute liver (siSp1) HepG2 cells (Fig. 5E). These results confirmed that DDX3X injury response, a single i.p. injection of DEN (100 mg/kg) was can transcriptionally regulate the expression of NER factors DDB2 given to 10-week-old mice. We detected significantly increased flox/flox and XPA through cooperation with Sp1, thus suggesting an ALT levels in Alb-Cre/þ;Ddx3x mice compared with those important role of DDX3X in NER. of littermate controls 1 day after DEN injection. At 3 days after DEN injection, the increased ALT levels in controls did not flox/flox Loss of Ddx3x results in genomic stress in rapidly dividing differ from those of the Alb-Cre/þ;Ddx3x mice (Supple- tumor cells mentary Fig. S10A). Increased apoptosis (cCasp3) and com- In normal cells, efficient DDR is crucial for the maintenance of pensatory proliferation (Ki67) matched the degree of liver flox/flox the genome integrity. Previously, we showed inactivation of injury in Alb-Cre/þ;Ddx3x mice when compared with con- Ddx3x results in embryonic lethality due to widespread DNA trols 1 day after DEN injection. Notably, at both 1 and 3 days after damage and apoptosis (12). The data so far showed that inacti- DEN injection, gH2AX-positive hepatocytes were markedly flox/flox vation of Ddx3x in juvenile hepatocytes results in defective increased in Alb-Cre/þ;Ddx3x mice compared with that of DNA repair and sustained DNA damage, which are causally their littermate controls (Supplementary Fig. S10B and S10C). connected to tumor initiation. Given the rapid cell-cycle time is These results demonstrated that loss of Ddx3x facilitates DEN- a common feature of affected Ddx3x-deficient embryonic cells and induced DNA damage accumulation and liver injury. juvenile hepatocytes, we therefore proposed that the reduced NER We next injected a single dose of DEN (25 mg/kg) into flox/flox flox/flox activity in Alb-Cre/þ;Ddx3x hepatocytes may cause genomic Alb-Cre/þ;Ddx3x mice and their littermate controls at stress in the tumor. Indeed, IHC analysis of liver sections from 2 weeks of age. We found that ALT levels were significantly flox/flox flox/flox aged controls and tumor-bearing Alb-Cre/þ;Ddx3x mutants elevated in the Alb-Cre/þ;Ddx3x mice, peaked at 1 month

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Figure 7. Loss of Ddx3x promotes DEN-induced liver tumorigenesis in Alb-Cre/þ;Ddx3xflox/flox mice. Controls and Alb-Cre/þ;Ddx3xflox/flox mice were i.p. injected with a single dose of DEN (25 mg/kg body weight) at 2 weeks of age. The sera and livers were collected at the indicated time intervals (months) after injection. A, The serum ALT levels. N 7 per group. B, Gross images of representative livers from DEN-treated mice. The numbers of tumor-bearing mice in each group are indicated in the bottom-left corners of the images. Tumors are indicated by arrows. The number of visible tumors per liver was counted. Scale bar, 1 cm. C, The average number and size of tumors in tumor-bearing controls and Alb-Cre/þ;Ddx3xflox/flox mice. D, Representative H&E and AFP protein (red) staining of liver sections and quantitative mRNA levels of Afp in livers from control and Alb-Cre/þ;Ddx3xflox/flox mutant 3 and 6 months after DEN injection. The boundaries of basophilic- and AFP-positive nodules, and tumors (T) were demarcated with dashed lines. Nuclei were counterstained with hematoxylin. Both nontumor(N) and tumor tissues were collected from Alb-Cre/þ;Ddx3xflox/flox livers. N 5 per group. E, Representative sirius red staining of liver sections. Liver fibrosis was evaluated by sirius red staining. Quantification of the fibrotic area (4–8 fields, 100x magnification) per liver section was determined. N 6 per group. , P < 0.05; , P < 0.01; and , P < 0.001. Scale bars, 1 cm (B); 200 mm(C and D).

flox/flox after injection, and then gradually declined at 3 and 6 months 58.3% (7/12) of the Alb-Cre/þ;Ddx3x mice exhibited after injection, compared with the controls. ALT levels varied in liver tumors, compared with an approximately 27.3 % (3/11) flox/flox the controls and Alb-Cre/þ;Ddx3x mice 9 months after incidence of tumors in female controls (Fig. 7B). Alb-Cre/þ; flox/flox injection (Fig. 7A). At 3 months after DEN injection, there was Ddx3x mice had more macroscopically visible tumors than flox/flox no perceptible tumor in livers from the Alb-Cre/þ;Ddx3x their controls. We noted the tumor sizes were not further increased flox/flox mice and their controls as assessed by macroscopic examination. in Alb-Cre/þ;Ddx3x livers, and the mean tumor sizes were flox/flox Visible tumors were detected in 50.0% (6/12) of the Alb-Cre/þ; not different between controls and Alb-Cre/þ;Ddx3x livers flox/flox Ddx3x livers, whereas none (0/10) of their controls had (Fig. 7C). Quantitative analyses of cCasp3-, Ki67-, and gH2AX- tumors 6 months after injection. At 9 months after injection, positive cells in immunostained liver sections further showed that

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flox/flox all these indices in Alb-Cre/þ;Ddx3x mice 1 month after DEN Hepatocyte-specific ablation of Ddb1 leads to chronic hepatocyte injection were significantly higher than those of 6-week-old (age- turnover, mild liver damage, and liver tumor formation (36). In flox/flox matched) untreated Alb-Cre/þ;Ddx3x mice (Supplementary experimental models of alcohol-related liver diseases, the trans- Fig. S11 compared with Fig. 3B–D). Notably, basophilic and glutaminase-2 (TG2)–mediated impairment of the Sp1-c-met alpha-fetoprotein (AFP)–positive nodules were readily detected signaling cascade was observed (37). These findings support our flox/flox in the Alb-Cre/þ;Ddx3x livers but not in their controls 3 results that dysregulation of DDR, as evidenced by the decrease of months after DEN injection. The elevated expression of Afp, the Xpa and Ddb2 levels needed for DNA damage repair, in juvenile flox/flox AFP gene, in both nontumor and tumor tissues from Alb-Cre/þ; Alb-Cre/þ;Ddx3x livers is associated with a predisposition to flox/flox Ddx3x livers 6 months after DEN injection was further HCC. This study shows the genomic instability is a driving event flox/flox confirmed by qRT-PCR (Fig. 7D). Sirius red staining of the liver for liver tumorigenesis in Alb-Cre/þ;Ddx3x mutants; how- flox/flox sections showed that DEN-treated Alb-Cre/þ;Ddx3x mice ever, we do not exclude the possibility that DDX3X may also affect exhibited significant increases in fibrosis compared with con- other cellular processes, such as epigenetic alterations, deregula- trols 3 and 6 months after injection (Fig. 7E). In tumor-bearing tion of miRNAs, and overexpression of oncogenes, during pro- flox/flox controls and Alb-Cre/þ;Ddx3x mutants 9 months after gression of HCC. DEN injection, IHC analysis of liver sections showed that the It is wildly accepted that inflammation can increase the risk percentages of cCasp3-, Ki67-, gH2AX-, 53BP1-, and pATR- of cancer by providing the cytokines and chemokines from positive cells were significantly increased in tumors compared infiltrating cells in tumor microenvironment, including HCC with adjacent nontumor liver tissues. We noted the percentages (38, 39). Targeting hepatic inflammation is one of the thera- of gH2AX- and 53BP1-positive cells in tumor sections from peutic opportunities for the treatment of HCC (40). In this flox/flox DEN-treated controls and Alb-Cre/þ;Ddx3x mice were study, we showed that the liver injury in 6-week-old hepato- flox/flox significantly higher than those of spontaneous tumors from cytes and Alb-Cre/þ;Ddx3x mutants was associated flox/flox aged Alb-Cre/þ;Ddx3x mice (Supplementary Fig. S12 com- with inflammation (Supplementary Fig. S4). The infiltrated pared with Fig. 6). All together, these results demonstrated that immune cells and IL1b-, TNFa-, and NF-kB–positive cells were flox/flox DEN-treated Alb-Cre/þ;Ddx3x mice had not only dramat- significantly increased in spontaneous tumor tissues of aged flox/flox ically increased tumor multiplicity but also accelerated tumor Alb-Cre/þ;Ddx3x mutants. Also, Il1b and Tnfa mRNA progression with increased incidence of HCC. Therefore, a role levels were significantly increased in liver tumors from Alb- flox/flox for DDX3X as an important regulator of genome stability in vivo Cre/þ;Ddx3x mutants. In tumor-promoting stimuli, IL1b- was confirmed. and TNFa-positive cells and Il1b levels were significantly higher flox/flox in DEN-treated Alb-Cre/þ;Ddx3x tumors than those of controls (Supplementary Fig. S13). In the context of DNA flox/flox Discussion damage and inflammation, DEN-treated Alb-Cre/þ;Ddx3x Human HCC generally proceeds through a stepwise process mice had not only dramatically increased tumor multiplicity involving hepatic injury and compensatory proliferation, fol- but also accelerated tumor progression with increased inci- lowed by inflammation, fibrosis, and the development of HCC. dence of HCC. These results suggested inflammation-related flox/flox Here, we showed that Alb-Cre/þ;Ddx3x mice recapitulated changes in the microenvironment of liver contribute to liver flox/flox these key features of human HCC pathogenesis. Loss of Ddx3x injury and tumorigenesis in Alb-Cre/þ;Ddx3x mice. affected DNA repair by reducing the expression of NER factors The epidemiologic data of human HCC show that males DDB2 and XPA, which contributed to an accumulation of unre- have a higher risk of developing liver tumors than females (3). paired DNA damage and replication stress in the liver, providing However, we found that the tumor incidence in Alb-Cre/þ; flox/flox further evidence that DDX3X maintains genomic stability in vivo. Ddx3x female mice was higher than in Alb-Cre/þ; flox In general, most adult tissues/organs are in a quiescent state, Ddx3x /Y male mice in this study. DDX3X has a structural and proliferation occurs only when old/dying cells must be homolog, DDX3Y, located on Y chromosome (12, 41). Studies replaced. Nevertheless, the liver is a unique organ with a high have shown that both of the DDX3X and DDX3Y genes are regenerative capacity that can restore its lost mass after resections transcribed in multiple tissues. DDX3X protein was detected in or injury. A cell-cycle kinetic study of the liver showed that the all tissues analyzed; however, the DDX3Y protein was predom- fraction of hepatocytes in a proliferative state, but not the average inantly detected in male germ cells by translational control cell-cycle time, decreased gradually with age, beginning at around (42, 43). Expression and deletion analyses of DDX3Y suggested 2 weeks of age. As development proceeds, individual cells cease aspecific function of DDX3Y in male fertility (44), whereas we division and then increase in cell size until the final size limit is have shown discrete and essential roles of DDX3X protein in reached (31). In this study, we showed that the levels of RPA70, mouse embryonic and placental development (12). These RPA32, pRPA32, and Mre11 were higher in livers from 3-week-old genetic data suggest that DDX3Y may act specifically in testis (juvenile) controls than those of 6-week-old controls, indicating and may have a different function than DDX3X. Although flox that the expression of DDR factors during early liver growth is Ddx3y mRNA levels in Alb-Cre/þ;Ddx3x /Y livers were com- essential to safeguard DNA integrity. Indeed, mutations/inacti- parable with their controls at a young age, decreased Ddx3y flox vation of DDR genes are observed in various diseases and cancers levels were observed in aged Alb-Cre/þ;Ddx3x /Y livers and (32). Among these studies, a higher incidence of spontaneous tumors (Supplementary Fig. S14). We cannot rule out the liver tumors and an increased mutation frequency in livers were possibility that DDX3Y acts as a functional substitute for the / observed in aged Xpa mice compared with their wild-type loss of DDX3X in some contexts. It will be of interest to assess controls (33, 34). In addition to Ultraviolet B (UVB)-induced skin whether basal Ddx3y mRNA and DDX3Y protein are involved in carcinogenesis, Ddb2-deficient mice developed spontaneous the gender differential effect of DDX3X in liver homeostasis tumors at a high rate between 20 and 25 months of age (35). and DEN-induced liver tumorigenesis.

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HCC is a heterogeneous disease that shows high resistance to Writing, review, and/or revision of the manuscript: C.-H. Chan, C.-M. Chen, conventional chemotherapy and radiotherapy. It is generally L.-R. You believed that genomic instability may contribute to poor clinical Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.-R. You outcomes (45, 46). This study suggested a link between DDX3X Study supervision: C.-M. Chen, Y.-H. Wu Lee, L.-R. You function and genome integrity in liver tumorigenesis, at least partly through the control of the expression of NER genes that Acknowledgments mediate DNA repair. In the cell populations with relatively rapid We thank Dr. Hao-Kang Li for technical support. The A6 BCM, TROMA-III rates of proliferation, including juvenile liver and tumor, loss of (K19), and G8.8 (EpCAM) antibodies developed by V.M. Factor, R. Kemler, Ddx3x results in DNA damage, liver injury, cell death–compen- and A.G. Farr, respectively, were obtained from the Developmental Studies satory proliferation, and inflammation. Under the condition of Hybridoma Bank, created by the NICHD of the NIH and maintained at The fl University of Iowa, Department of Biology, Iowa City, IA. We thank the DNA damage and in ammation, aberration functions of DNA Taiwan Animal Consortium (MOST107-2319-B-001-002)–Taiwan Mouse repair could promote the development of dysplastic lesion and Clinic which is funded by the Ministry of Science and Technology (MOST) subsequent HCC. Given that the small-molecule–mediated inhi- of Taiwan for technical support in collagen stain experiment. This work was bition of DDX3X activity may provide new therapeutic opportu- supported by the Ministry of Science and Technology of Taiwan (grant nities to treat various viral infections and cancers (47, 48), the numbers MOST103-2320-B-009-006-, MOST104-2320-B-009-001-, and impact of DDX3X on DNA damage and repair pathways cannot be MOST105-2320-B-009-001- to Y.-H. Wu Lee; MOST105-2320-B-010-002-, MOST106-2320-B-010-029-, and MOST107-2311-B-010-002- to L.-R. You); underscored. A detailed understanding of the biology, pathology, Center For Intelligent Drug Systems and Smart Bio-devices (IDS2B), National and the complexity of the cellular responses to DNA damage in ChiaoTungUniversityandCancerProgression Research Center, National HCC associated with DDR deficiency will allow further preclinical Yang-Ming University from The Featured Areas Research Center Program investigations and provide appropriate treatment strategies. within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan; and the Ministry of Education in Taiwan, Aim for the Top University Plan to L.-R. You. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: C.-H. Chan, C.-M. Chen, L.-R. You this fact. Development of methodology: C.-H. Chan, C.-M. Chen, L.-R. You Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Received May 28, 2018; revised September 9, 2018; accepted September 26, computational analysis): C.-H. Chan, C.-M. Chen, L.-R. You 2018; published first October 8, 2018.

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DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss

Chieh-Hsiang Chan, Chun-Ming Chen, Yan-Hwa Wu Lee, et al.

Mol Cancer Res Published OnlineFirst October 8, 2018.

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