DNA Damage, Liver Injury, and Tumorigenesis: …...DNA Damage, Liver Injury, and Tumorigenesis:...

Oncogenes and Tumor Suppressors

DNA Damage, Liver Injury, and Tumorigenesis:Consequences of DDX3X LossChieh-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(DDX3X), including its functions in transcriptional and trans-lational regulation, chromosome segregation, DNA damage,and cell growth control, have highlighted the associationbetween DDX3X and tumorigenesis. However, mRNA tran-scripts and protein levels of DDX3X in patient specimens haveshown the controversial correlations of DDX3X with hepato-cellular carcinoma (HCC)prevalence. In this study, generationof hepatocyte-specificDdx3x-knockout mice revealed that lossof Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led toprofound ductular reactions, cell apoptosis, and compensa-tory proliferation in female mutants at 6 weeks of age. Thesustained phosphorylation of histone H2AX (gH2AX) andsignificant accumulation of DNA single-strand breaks and

double-strand breaks in liver indicated that the replicativestress occurred in female mutants. Further chromatin immu-noprecipitation analyses demonstrated that DDX3X bound topromoter regions and regulated the expression of DNA repairfactors, DDB2 and XPA, to maintain genome stability. Loss ofDdx3x led to decreased levels of DNA repair factors, whichcontributed to an accumulation of unrepaired DNA damage,replication stress, and eventually, spontaneous liver tumorsand DEN-induced HCCs in Alb-Cre/þ;Ddx3xflox/flox mice.

Implications: These data identify an important role ofDDX3X in the regulation of DNA damage repair to protectagainst replication stress in liver and HCC development andprogression.

IntroductionLiver cancer is thefifthmost common cancer and the thirdmost

frequent cancer-related of deaths worldwide. Themajority of livermalignancies are hepatocellular carcinoma (HCC) and cholan-giocarcinoma that have high morbidity and mortality in devel-oping countries, but their incidences are also increased in devel-oped countries. Etiological studies of liver cancer have identifiedfour major risk factors, including chronic infection with hepatitisB or C viruses, alcohol use, and the intake of aflatoxin-contam-inated food (1–3). In recent years, obesity-associated nonalco-holic fatty liver disease has continuously increased (4). It isgenerally accepted that hepatocarcinogenesis is a multistep pro-cess with an accumulation of the genetic and epigenetic altera-tions of critical genes involving hepatocyte growth and prolifer-ation, cell death, cell adhesion, and metabolism (5). A common

feature during HCC progression is repeated cycles of cell deathand compensatory proliferation, which causes an inflammatorymicroenvironment in the liver, leading to fibrosis, cirrhosis, andultimately, malignant transformation of the hepatocytes.

DEAD-box helicase 3, X-linked (DDX3X; also known asDDX3)belongs to the DEAD-box RNA helicase family and was initiallyidentified as a cellular factor that bound to hepatitis C viral coreprotein. Accumulated evidences showed that DDX3X is involvedin diverse cellular processes, including cell growth, cell-cyclecontrol, mitotic chromosome segregation, innate immunity,tumorigenesis, and virus replication (6). Our previous studiesshowed thatDDX3X acted as a tumor suppressor in various cancercell lines. We found that lower DDX3X levels were significantlycorrelated with higher HCC prevalence, particularly in male andHBV-positive patients (7). Knockdown of DDX3X in nontrans-formedNIH3T3 cells led to a premature entry into S phase and anincrease of cell proliferation, and enhanced the oncogenic v-ras–induced anchorage-independent growth. In addition, overexpres-sion of DDX3X activated the p21WAF1/Cip1 promoter throughits physical interaction with Sp1, and consequently, negativelyregulated cyclinD1protein levels and tumor cell proliferation (8).A recent study further showed that DDX3X level was inverselyassociatedwith the tumor grade and predicted poor prognoses forHCC patients. Epigenetic regulation of a subset of HCC-associ-ated tumor-suppressor miRNAs, including miR-200b, miR-200c,miR-122, and miR-145, by DDX3X induced stem cell–like prop-erties and promoted tumorigenic potential (9). In contrast to ourstudies, Huang and colleagues showed by qRT-PCR analysis thatDDX3X was overexpressed in 64% of HCC tissues (10), and Suand colleagues found no association between DDX3X RNA leveland survival in liver cancer patients (11). Therefore, the patho-logic role of DDX3X in liver cancer remains to be clarified. In aprevious study, we generated a conditional knockout allele for

1Institute of Biochemistry and Molecular Biology, National Yang-MingUniversity, Taipei, Taiwan. 2Department of Life Sciences and Institute ofGenomeSciences, National Yang-Ming University, Taipei, Taiwan. 3Cancer ProgressionResearchCenter, National Yang-MingUniversity, Taipei, Taiwan. 4Department ofBiological Science and Technology, National Chiao Tung University, Hsinchu,Taiwan. 5Center For Intelligent Drug Systems and Smart Bio-devices (IDS2B),National Chiao Tung University, Hsinchu, Taiwan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

CorrespondingAuthors: Li-RuYou, National Yang-MingUniversity, No. 155, Sec.2, Li-Nong Street, Taipei 112, Taiwan. Phone: 886-2-28267368; E-mail:[email protected]; and Yan-HwaWu Lee, Department of Biological Science andTechnology, National Chiao TungUniversity, No. 75, Bo-ai St., East Dist., Hsinchu300, Taiwan. Phone: 886-3-5712121, ext. 56986; E-mail: [email protected].

doi: 10.1158/1541-7786.MCR-18-0551

�2018 American Association for Cancer Research.

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Ddx3x gene and identified the essential roles of DDX3X in pla-cental and early embryonic development (12). Here, hepatocyte-specific Ddx3x-knockout mice were generated. We showed thatloss ofDdx3x led to DNA damage and replicative stress at a youngage, and mice developed spontaneous liver tumors with aging.Ddx3x-deficiency females were more susceptible to diethylnitro-samine (DEN)-induced HCC than littermate female controls.Moreover, DDX3X regulated DNA damage repair factors DDB2and XPA, in the liver and in vitro, which are critical to maintaingenome stability.

Materials and MethodsMice, DEN treatment, and serum biochemical analysis

The floxedDdx3xmice were generated previously (12) and hadbeen backcrossed into the C57BL/6 background for at least 10generations. The hepatocyte-specific Ddx3x-knockout mice weregenerated through breeding Ddx3xflox/flox mice with Alb-Cre [B6.Cg-Tg(Alb-cre)21Mgn/J] transgenic mice (13). The serum levels ofalanine aminotransferase (ALT), triacylglycerol (TG), and totalcholesterol (TCHO) were monitored using biochemical slides(Fuji DRY-CHEM 400i; Fujifilm) according to the manufacturer'sinstructions. To induce HCC, DEN (25 mg/kg body weight;N0756-10ML, Sigma-Aldrich) was injected i.p. into 2-week-oldmice. To induce acute liver injury, 10-week-oldmicewere injectedwith DEN (100 mg/kg body weight) and sacrificed 1 or 3 daysthereafter. The experimental procedures using mice wereapproved by the Institutional Animal Care and Use Committee(IACUC) of National Yang-Ming University, Taiwan. The animalcare and experimental procedures were performed in accordancewith the Guidelines of the IACUC of National Yang-Ming Uni-versity, Taiwan.

Histologic analysis and molecular techniquesFurther details are provided in the Supplementary Information.

Statistical analysisHepatocytes stained positive for differentmarkers in livers were

determined by the positive hepatocytes/total hepatocytes in eachhigh-power (200�magnification) field. For each sample, at leastfour fields were randomly selected and counted. Quantitativeresults are expressed as the mean � SEM. The Student t test wasused to determine P values, unless stated otherwise.

ResultsHepatocyte-specificDdx3x ablation leads to the development ofhepatocellular tumors in aged mice

To investigate the physiopathologic role of DDX3X in liver,floxedDdx3xmice were crossed with Alb-Cre transgenic mice. TheAlb-Cre transgene is specifically activated in the liver from lateembryonic stage, and Cre-mediated gene recombination occursprogressively with age (14, 15). PCR of genomic DNA from liverconfirmed the progressive loss of floxed Ddx3x allele and thepresence of null allele. The reduction of DDX3X protein wasspecifically observed in liver. The presence of nonparenchymalcells, which do not have Cre activity, may explain the low levels ofDDX3X protein in livers of mutantmice (Supplementary Fig. S1).The hepatocyte-specific Ddx3x-knockout mice were born atexpected Mendelian ratios and were morphologically indistin-guishable from their littermate controls at young ages. Therefore,serum ALT, TG, and TCHO levels, commonly used markers for

monitoring liver injury and functions, were measured routinelyevery 3months from12months of age. TGandTCHOlevels in themutantmicewere not different from their littermate controls at alltime periods (Supplementary Fig. S2). However, ALT levels wereelevated in some, but not all, of the hepatocyte-specific Ddx3x-knockout mice with visible tumors, when dissected (Fig. 1A).The results showed that 10.7% (3/28) and 42.1% (8/19) ofdissected Alb-Cre/þ;Ddx3xflox/Y male and Alb-Cre/þ;Ddx3xflox/flox

femalemutants, respectively, developed liver tumors after approx-imately 12 to 27 months. Among these mice, we noted 4 of 5(80%) Alb-Cre/þ;Ddx3xflox/flox females developed liver tumors at24 months of age. None of the littermate controls and Alb-Cre/þ;Ddx3xflox/þ heterozygous females had a tumor (Fig. 1B and C).Hematoxylin & eosin (H&E) staining and immunohistochemicalanalysis of hepatocyte marker HNF4a and cholangiocyte markersK19 and Sox9 in tumor sections revealed that the most commonhistopathology was the clear cell type HCC (16), which wasdetected in both Alb-Cre/þ;Ddx3xflox/flox female and Alb-Cre/þ;Ddx3xflox/Ymale. Other types of liver tumors, including adenomaand cholangiocarcinoma,were observed inAlb-Cre/þ;Ddx3xflox/flox

female and Alb-Cre/þ;Ddx3xflox/Y male, respectively (Fig. 1D).Western blot analysis confirmed that DDX3X protein levels weresignificantly decreased in tumor and nontumor liver tissues fromaged Alb-Cre/þ;Ddx3xflox/Y male and Alb-Cre/þ;Ddx3xflox/flox

female mice compared with their gender-matched littermatecontrols (Fig. 1E). Given the higher tumor incidencewas observedin aged Alb-Cre/þ;Ddx3xflox/flox females, the effects of DDX3X onliver tumorigenesis in femalemice were extensively characterized.

Hepatocyte-specific deletion of Ddx3x leads to liver injury,ductular reactions, and inflammation

To further determine the pathologic role of DDX3X in tumorprogression, serum ALT levels were closely monitored at youngages. The mean ALT level of 3- to 20-week-old littermate controlswas 13.4� 0.7 U/l (Fig. 2A). We found significantly elevated ALTlevels in Alb-Cre/þ;Ddx3xflox/flox mutants at 6 and 10 weeks of age(115.1 � 12.0 and 26.9 � 2.0 U/l, respectively). There was nodifference in ALT levels between controls andmutants at 3 and 20weeks of age. Histologic analyses of H&E-stained liver sectionsrevealed increased numbers of basophilic cells [i.e., ductularreactions (DRs)] in 6- and 10-week-old Alb-Cre/þ;Ddx3xflox/flox

mice, in accordance with increased serum ALT levels, whencompared with controls (Fig. 2B). The DRs (17) were furtherverified by significant expansion of liver progenitor cell (LPC)/cholangiocyte marker–positive subsets, including A6-, EpCAM-,cytokeratin 19 (K19)-, and Sox9-positive cells as assessed by IHCin 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers (Fig. 2C). DRs werenotably reduced to a lesser extent in 10-week-old Alb-Cre/þ;Ddx3xflox/flox livers and disappeared thereafter (SupplementaryFig. S3). qRT-PCR analyses confirmed a decrease inDdx3x expres-sion and significant increases of LPC/cholangiocyte gene levels,including CD133, EpCAM, and K19 in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers compared with those of littermate controls.Nevertheless, the increasing trend of Sox9 levels did not reachstatistical significance (Fig. 2D). The liver injury was also associ-ated with significant immune cell infiltration consisting of F4/80(macrophages)-, CD3 (T cells)-, and B220 (B cells)-positivecells and increased Tnfa levels (Supplementary Fig. S4), whichcoincided with the significant elevation of ALT levels in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers compared with their controls.In aged mice, F4/80-, B220-, and CD3-positive cells were

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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, or Ddx3xflox/Y males and Ddx3xflox/þ orDdx3xflox/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-knockoutmice 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 longestdiameter of a tumor. D, Gross images of representative livers and H&E and IHC staining of liver sections at indicated ages (M, month). The cholangiocellularcarcinoma (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 linesdemarcate 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 adjacentliver tissues (N) from Ddx3x-knockout mice were evaluated by Western blot analysis. Protein levels were normalized to the loading control GAPDH, expressedas 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 todetermine 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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significantly increased in nontumor liver tissues of tumor-bearingAlb-Cre/þ;Ddx3xflox/flox mice compared with those of littermatecontrols. We found that IL1b-, TNFa-, and NF-kB–positive cellswere significantly increased in both tumors and adjacent non-tumor liver tissues of aged tumor-bearing Alb-Cre/þ;Ddx3xflox/flox

livers. Also, Il1b and Tnfa mRNA levels were significantlyincreased in tumors from Alb-Cre/þ;Ddx3xflox/flox livers (Supple-mentary Fig. S5).

Loss of Ddx3x causes DNA damage, cell death, andcompensatory proliferation in young mice

It has generally been accepted that loss of hepatic mass afterinjury activates compensatory proliferation, leading to tumordevelopment (18). In accordance with the dynamic changes of

ALT levels, large numbers of cleaved Caspase-3 (cCasp3)–positiveapoptotic cells were specifically detected in the Alb-Cre/þ;Ddx3xflox/flox livers at 6 weeks of age when compared with theirlittermate controls (Fig. 3A and B). In 3-week-old mice, cellproliferation was still high in the livers, and proliferation markerKi67-positive cells were comparable in livers from both controlsandAlb-Cre/þ;Ddx3xflox/floxmice. The decrease in cell proliferationwas observed in control livers as development proceeded; nev-ertheless, we detected a significant increase of Ki67-positive cellsin 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers (Fig. 3A and C). Theseresults indicated that compensatory proliferation occurred aftercell apoptosis in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers. Ourprevious study showed that targeted Ddx3x ablation in the epi-blast leads to widespread gH2AX (phosphorylated histoneH2AX,as a marker of DNA damage) and apoptosis, which causesembryonic lethality (12). We noted that gH2AX signals weresignificantly increased in Alb-Cre/þ;Ddx3xflox/flox livers, peaked at6 weeks of age, and then gradually declined at 10 and 20 weeks ofage, compared with littermate controls (Fig. 3A and D). ThegH2AX foci were observed in approximately 10% of proliferatingKi67-positive hepatocytes in 6-week-old Alb-Cre/þ;Ddx3xflox/flox

livers (Supplementary Fig. S6). The significant higher gH2AXþ/Ki67þ hepatocytes in 6-week-old Alb-Cre/þ;Ddx3xflox/flox liversimply that loss of Ddx3x causes prolonged DNA damage, andmay be linked to genome instability and subsequent liver tumor-igenesis in Alb-Cre/þ;Ddx3xflox/flox mice.

DNA single-strand break and double-strand break signalingswere induced in Alb-Cre/þ;Ddx3xflox/flox livers

Proper DNA replication and chromosome segregation are pre-requisites for genome integrity. To ensure the normal cellularfunctions and faithful genome maintenance and transmission, acomplex network of DNA damage response (DDR) systems hasevolved to sense and respond to different forms of DNA damageand replication stress (19, 20). Along with increased numbers ofgH2AX-positive cells, IHC analyses of liver sections revealed thatenlarged hepatocytes with atypical nuclei were frequentlyobserved in Alb-Cre/þ;Ddx3xflox/flox livers compared with those ofcontrol livers (Supplementary Fig. S7A). Notably, the numbers ofcells with 53BP1 nuclear bodies were significantly increased in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers (Supplementary Fig. S7B).These results prompted us to speculate that loss of Ddx3x causesreplication stress and may be a driving force for tumorigenesis inAlb-Cre/þ;Ddx3xflox/flox livers.

Replication protein A (RPA) is a guardian of genome integ-rity during DNA replication, recombination, and repair events,and also acts as a key sensor in DDR (21, 22). IHC analyses of 3-week-old liver sections showed similar expression levels ofRPA70 and RPA32 in the Alb-Cre/þ;Ddx3xflox/flox mice and theirlittermate controls (Fig. 4A). As development proceeded, theexpression levels of both RPA70- and RPA32-positive cellsdeclined significantly in 6- and 10-week-old littermate controlscompared with those of 3-week-old controls. The stainingintensities of RPA70 and RPA32 persisted in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers (Fig. 4A). The changes of DDX3X,RPA70, RPA32, and phosphorylated RPA32 (the active formof RPA32) protein levels in livers from 3-, 6-, and 10-week-oldcontrols and Alb-Cre/þ;Ddx3xflox/flox mice were further con-firmed by Western blotting. As expected, a significant increaseof gH2AX expression was detected in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers (Fig. 4B; Supplementary Fig. S8A).

Figure 2.

Loss of Ddx3x results in liver injury and DRs in Alb-Cre/þ;Ddx3xflox/flox mice. A,The serumALT activity wasmeasured in controls (Ddx3xflox/þ andDdx3xflox/flox)and Alb-Cre/þ;Ddx3xflox/flox mice at indicated ages (age in weeks). N � 5 pergroup. B, Representative images of H&E-stained liver sections. DRs(arrowheads) were observed inAlb-Cre/þ;Ddx3xflox/flox livers at 6 and 10weeksof age. C, Representative A6, EpCAM, K19, and Sox9 immunostaining (red)in 6-week-old controls andAlb-Cre/þ;Ddx3xflox/flox livers. The positively stainedcells are shown at higher magnification in the inset. Nuclei (blue) werecounterstained with hematoxylin. D, Relative mRNA levels of Ddx3x andcholangiocyte/LPC genes in livers from 6-week-old controls and Alb-Cre/þ;Ddx3xflox/flox 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.

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It has been demonstrated that RPA binds to ssDNA andtriggers activation of the ATR-Chk1 pathway in response toDNA damage or replication stress (22, 23). Indeed, significantincreases of phosphorylated ATR (pATR)– and phosphorylatedChk1 (pChk1)–positive hepatocytes were detected in 6-week-oldAlb-Cre/þ;Ddx3xflox/flox liver sections compared with those of theirlittermate controls (Fig. 4C). These results revealed that loss ofDDX3X induces replication stress in Alb-Cre/þ;Ddx3xflox/flox liversat a young age.

If replication stress persists, ssDNA long-time exposure in cellsconsequently results in double-strand break (DSB) and apoptosis.The presence of theMRNcomplex (Mre11, Rad50, andNBS1), thefunctional DSB sensor, in the DNA damage site, subsequentlyrecruits and activates the ATM-Chk2 signaling pathway (22, 23).IHC staining and Western blot analyses showed increased Mre11levels in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers compared withtheir littermate controls (Fig. 4D and E; Supplementary Fig. S8A).In addition, the pChk2-positive hepatocytes were dramaticallyincreased in the 6-week-oldAlb-Cre/þ;Ddx3xflox/flox livers (Fig. 4F).To substantiate these findings are hepatocyte specific, the expres-sion levels of DDX3X, gH2AX, and the sensors in DDR in isolatedhepatocytes from 6-week-old controls and Alb-Cre/þ;Ddx3xflox/flox

livers were examined. In isolated hepatocytes, PCR analysis ofgenomic DNA showed the floxed Ddx3x alleles were almostcompletely deleted and DDX3X protein levels from 6-week-old

Alb-Cre/þ;Ddx3xflox/flox livers were significantly decreased toapproximately 4% of control hepatocytes. The relative foldchanges of gH2AX, RPA70, pRPA32, RPA32, and Mre11 proteinswere greater in isolated hepatocytes than those from whole liversof 6-week-old Alb-Cre/þ;Ddx3xflox/flox mice, confirming a hepato-cyte-specific effect (Supplementary Fig. S8C compared with Sup-plementary Fig. S8A). Taken together, these results showed thatnot only single-strand break (SSB), but also DSB signaling, wasinduced in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers.

Ddx3x loss inhibits the nucleotide excision repair anddownregulates DDB2 and XPA genes through Sp1

Cells halt cell-cycle progression in the presence of DNAdamage toprovidemore time forDNA repair. The levels ofCdkn2b(p15Ink4b), Cdkn1a (p21WAF1/CIP1), and Cdkn1b (p27Kip1) wereincreased by 2-, 3.8-, and 1.4-fold, respectively, in 6-week-oldAlb-Cre/þ;Ddx3xflox/flox livers compared with controls, indicatingthat loss of Ddx3x halted cell-cycle progression in response toDNA damage (Supplementary Fig. S9). Consistent with ourprevious study in embryonic cells (12), the Trp53 (p53) level wasreduced in theAlb-Cre/þ;Ddx3xflox/flox livers compared with that ofthe controls.

The activation of ATR/ATM, upregulation of cell-cycle regula-tors, and the prolonged gH2AX-positive cells in Alb-Cre/þ;Ddx3xflox/flox livers suggest the possibility that the downstream

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) and Alb-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-positivehepatocytes in livers from controls and Alb-Cre/þ;Ddx3xflox/flox mice were quantified. N � 3 per group. � , P < 0.05 and �� , P < 0.001. Scale bar, 200 mm.






Figure 4.

DNASSB andDSB signalings are induced in 6-week-oldAlb-Cre/þ;Ddx3xflox/floxmice. The liver tissues from controls andAlb-Cre/þ;Ddx3xflox/floxmice at 3, 6, and 10weeks of agewere collected and processed for immunostaining andWestern blot analysis.A,Representative images of RPA70 and RPA32 (red) immunostaining.B,Representative images of DDX3X, gH2AX, RPA70, and RPA32 protein expression in livers from controls (Ctrl) and Alb-Cre/þ;Ddx3xflox/flox (KO) mice byWestern blot analysis. The pRPA32 is the phosphorylated form of RPA32. Protein levels were normalized to the loading control GAPDH, expressed as fold changesrelative to 3-week-old controls (set as 1) and shown under their corresponding panels. C, Immunofluorescence costaining of pATR (green) and pChk1 (green)with hepatocytemarker HNF4a (red). Percentages of pATR- and pChk1-positive hepatocytes (HNF4aþ) in liverswere quantified.N� 3 per group.D,Representativeimages 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 stainedcells (A, C, D, and F) 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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functions of DNA repair and signaling may not work efficiently.The nucleotide excision repair (NER)machinery is involved in theremoval of a wide range of DNA lesions either through globalgenome NER (GG-NER) or through transcription-coupled NER(TC-NER). The damage is initially recognized by DDB2 (damage-specific DNA binding protein 2) and XPC (xeroderma pigmento-sum, complementation group C) inGG-NER, or ERCC6 (excisionrepair cross-complementation group 6) and ERCC8 in TC-NER.After recognition, DDB1 interacts with DDB2 or ERCC8, whichthen recruits XPA and its interacting proteins to perform DNAunwinding (24). Thereafter, sequential and coordinated assemblyof core repair factors functionally involved in DNA incision,excision, and de novo synthesis proceeds through theNERpathway(25). In 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers, the expression

levels of Ddb2, Ercc6, Ercc8, and Xpa, but not Ddb1 and Xpc, weresignificantly decreased (Fig. 5A). The expression of Ercc1, Ercc4,Ercc5, and proliferating cell nuclear antigen (Pcna), which areinvolved in DNA incision, excision, and de novo synthesis, werenot affected (Fig. 5A). A survey of the proximal promoter regionsidentified the confirmed Sp1-binding sites and/or GC-rich ele-ments in the responsive genes, Ddb2 (26), Ercc6 (27), and Xpa(28). We then found that the expression level of Sp1, a previouslyidentified partner of DDX3X, was decreased in 6-week-old Alb-Cre/þ;Ddx3xflox/flox livers.

To gain mechanistic insight into the link between DDX3X andNER, the mRNA levels of the DDB2, ERCC6, ERCC8, and XPAgenes inDDX3X-knockdown (shDDX3X#2 and shDDX3X#3) andcontrol (shLuc) HepG2 cells (9) were investigated. Our results

Figure 5.

The targets of DDX3X are involved in NER. A,qRT-PCR analyses of NER genes and Sp1 in liversfrom 6-week-old controls and Alb-Cre/þ;Ddx3xflox/flox mice. N ¼ 4 per group. B, RelativemRNA 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-, and shDDX3X#3-knockdownHepG2 cells by Western blot analysis wereshown (right plot). Protein levels werenormalized to the loading control GAPDH,expressed as fold changes relative to controls(set as 1) and shown under their correspondingpanels. C–E, Binding of DDX3X and Sp1 to thepromoter regions of the DDB2 and XPA genes.ChIP experiments were performed with livertissues from 6-week-old controls andAlb-Cre/þ;Ddx3xflox/flox mice (C), DDX3X-(shLuc, shDDX3X#2, and shDDX3X#3) (D), andSp1- (siControl and siSp1) knockdown HepG2cells (E). The cross-linked protein–DNAcompleximmunoprecipitated with anti-DDX3X andanti-Sp1 antibodies from liver tissues, andcultured cells were processed for ChIP-qPCRassays. The binding ability on the promoterregion was presented in the relative fold changeto rabbit IgG, which was normalized with inputindividually. N � 5 per group. Representativeimages of DDX3X, Sp1, DDB2, and XPA proteinexpression in siControl- and siSp1-knockdownHepG2 cells by Western blot analysis wereshown (E, left plot). The relative levels ofproteins were quantified and shown under theircorresponding panels. � , P < 0.05; ��, P < 0.01;and �� , P < 0.001.






showed that DDX3X, DDB2, XPA, and Sp1, but not ERCC6 andERCC8, were significantly decreased in DDX3X-knockdown cells(Fig. 5B). Accordingly, the DDX3X, DDB2, XPA, and Sp1 proteinlevels were significantly reduced in DDX3X-knockdown cells.Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR)assays showed the recruitment of DDX3X proteins to the pro-moter regions of Ddb2 and Xpa was approximately 2.8-fold and3.6-fold higher, respectively, in livers from 6-week-old controlsthan in Alb-Cre/þ;Ddx3xflox/flox mutants. The recruitment of Sp1proteins to the promoter regions of Ddb2 and Xpa showeddecreasing trends in Alb-Cre/þ;Ddx3xflox/flox livers compared withthose of controls (Fig. 5C). Consistently, we detected significantlydecreased recruitment of DDX3X proteins and slightly decreasedrecruitment of Sp1 proteins to the promoter regions ofDDB2 andXPA in DDX3X-knockdown HepG2 cells (Fig. 5D). In addition,the recruitment of DDX3X and Sp1 proteins to the promoterregions of DDB2 and XPA was decreased in Sp1-knockdown(siSp1)HepG2 cells (Fig. 5E). These results confirmed thatDDX3Xcan transcriptionally regulate the expression of NER factorsDDB2and XPA through cooperation with Sp1, thus suggesting animportant role of DDX3X in NER.

Loss of Ddx3x results in genomic stress in rapidly dividingtumor cells

In normal cells, efficient DDR is crucial for the maintenance ofthe genome integrity. Previously, we showed inactivation ofDdx3x results in embryonic lethality due to widespread DNAdamage and apoptosis (12). The data so far showed that inacti-vation of Ddx3x in juvenile hepatocytes results in defectiveDNA repair and sustained DNA damage, which are causallyconnected to tumor initiation. Given the rapid cell-cycle time isa common feature of affectedDdx3x-deficient embryonic cells andjuvenile hepatocytes, we therefore proposed that the reducedNERactivity in Alb-Cre/þ;Ddx3xflox/flox hepatocytes may cause genomicstress in the tumor. Indeed, IHC analysis of liver sections fromaged controls and tumor-bearing Alb-Cre/þ;Ddx3xflox/flox mutants

showed that cCasp3 (apoptosis)-, Ki67 (compensatory prolifer-ation)-, and gH2AX-positive hepatocytes were significantlyincreased in tumors compared with adjacent nontumor livertissues from Alb-Cre/þ;Ddx3xflox/flox mutants and their controls.In addition, the numbers of cells positive for pATR and 53BP1nuclear bodies were significantly increased in tumors from Alb-Cre/þ;Ddx3xflox/flox mutants (Fig. 6). Altogether, our data suggestthat Ddx3x plays a critical role in the maintenance of genomeintegrity in the cell populations with relatively rapid rates ofproliferation.

Loss of Ddx3x predisposes female mice to DEN-induced liverinjury and tumorigenesis

The carcinogen, DEN, which is bioactivated by cytochromeP450 (Cyp) enzymes in the liver and generates DNA adducts, iswidely used to induce liver injury and tumorigenesis in experi-mental animal models (29, 30). To examine the acute liverinjury response, a single i.p. injection of DEN (100 mg/kg) wasgiven to 10-week-old mice. We detected significantly increasedALT levels in Alb-Cre/þ;Ddx3xflox/flox mice compared with thoseof littermate controls 1 day after DEN injection. At 3 days afterDEN injection, the increased ALT levels in controls did notdiffer from those of the Alb-Cre/þ;Ddx3xflox/flox mice (Supple-mentary Fig. S10A). Increased apoptosis (cCasp3) and com-pensatory proliferation (Ki67) matched the degree of liverinjury in Alb-Cre/þ;Ddx3xflox/flox mice when compared with con-trols 1 day after DEN injection. Notably, at both 1 and 3 days afterDEN injection, gH2AX-positive hepatocytes were markedlyincreased in Alb-Cre/þ;Ddx3xflox/flox mice compared with that oftheir littermate controls (Supplementary Fig. S10B and S10C).These results demonstrated that loss of Ddx3x facilitates DEN-induced DNA damage accumulation and liver injury.

We next injected a single dose of DEN (25 mg/kg) intoAlb-Cre/þ;Ddx3xflox/flox mice and their littermate controls at2 weeks of age. We found that ALT levels were significantlyelevated in the Alb-Cre/þ;Ddx3xflox/flox mice, peaked at 1 month

Figure 6.

The percentages of cCasp3-, Ki67-, gH2AX-, 53BP1-, andpATR-positive hepatocytes are significantly increasedin tumor samples from Alb-Cre/þ;Ddx3xflox/floxmutants.The liver tissues from 18- to 24-month-old controls andtumor-bearing Alb-Cre/þ;Ddx3xflox/flox mice wereprocessed for immunostaining. A, Representativeimages of cCasp3 (red), Ki67 (red), gH2AX (green),53BP1 (green), and pATR (green) immunostaining. Thestained cells are shown at higher magnificationin the inset. Nuclei were stainedwith hematoxylin and/orDAPI. B, Percentages of cCasp3-, Ki67-, gH2AX-, 53BP1-,and pATR-positive hepatocytes in liver sections werequantified. N ¼ 3 per group. � , P < 0.05; �� , P < 0.01; and�� , P < 0.001. Scale bar, 200 mm.

Chan et al.





after injection, and then gradually declined at 3 and 6 monthsafter injection, compared with the controls. ALT levels varied inthe controls and Alb-Cre/þ;Ddx3xflox/flox mice 9 months afterinjection (Fig. 7A). At 3 months after DEN injection, there wasno perceptible tumor in livers from the Alb-Cre/þ;Ddx3xflox/flox

mice and their controls as assessed by macroscopic examination.Visible tumors were detected in 50.0% (6/12) of the Alb-Cre/þ;Ddx3xflox/flox livers, whereas none (0/10) of their controls hadtumors 6 months after injection. At 9 months after injection,

58.3% (7/12) of the Alb-Cre/þ;Ddx3xflox/flox mice exhibitedliver tumors, compared with an approximately 27.3 % (3/11)incidence of tumors in female controls (Fig. 7B). Alb-Cre/þ;Ddx3xflox/flox mice had more macroscopically visible tumors thantheir controls.Wenoted the tumor sizeswere not further increasedin Alb-Cre/þ;Ddx3xflox/flox livers, and the mean tumor sizes werenot different between controls and Alb-Cre/þ;Ddx3xflox/flox livers(Fig. 7C). Quantitative analyses of cCasp3-, Ki67-, and gH2AX-positive cells in immunostained liver sections further showed that

Figure 7.

Loss ofDdx3x promotes DEN-induced liver tumorigenesis inAlb-Cre/þ;Ddx3xflox/floxmice. Controls andAlb-Cre/þ;Ddx3xflox/floxmice were i.p. injectedwith a singledose of DEN (25mg/kg body weight) at 2 weeks of age. The sera and livers were collected at the indicated time intervals (months) after injection. A, The serumALTlevels. N � 7 per group. B, Gross images of representative livers from DEN-treated mice. The numbers of tumor-bearing mice in each group are indicatedin 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 averagenumber 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 liversections 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 ofbasophilic- 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 wasevaluated 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).






all these indices inAlb-Cre/þ;Ddx3xflox/floxmice 1month afterDENinjection were significantly higher than those of 6-week-old (age-matched) untreated Alb-Cre/þ;Ddx3xflox/flox mice (SupplementaryFig. S11 compared with Fig. 3B–D). Notably, basophilic andalpha-fetoprotein (AFP)–positive nodules were readily detectedin the Alb-Cre/þ;Ddx3xflox/flox livers but not in their controls 3months after DEN injection. The elevated expression of Afp, theAFP gene, in both nontumor and tumor tissues from Alb-Cre/þ;Ddx3xflox/flox livers 6 months after DEN injection was furtherconfirmed by qRT-PCR (Fig. 7D). Sirius red staining of the liversections showed that DEN-treated Alb-Cre/þ;Ddx3xflox/flox miceexhibited significant increases in fibrosis compared with con-trols 3 and 6 months after injection (Fig. 7E). In tumor-bearingcontrols and Alb-Cre/þ;Ddx3xflox/flox mutants 9 months afterDEN injection, IHC analysis of liver sections showed that thepercentages of cCasp3-, Ki67-, gH2AX-, 53BP1-, and pATR-positive cells were significantly increased in tumors comparedwith adjacent nontumor liver tissues. We noted the percentagesof gH2AX- and 53BP1-positive cells in tumor sections fromDEN-treated controls and Alb-Cre/þ;Ddx3xflox/flox mice weresignificantly higher than those of spontaneous tumors fromaged Alb-Cre/þ;Ddx3xflox/flox mice (Supplementary Fig. S12 com-pared with Fig. 6). All together, these results demonstrated thatDEN-treated Alb-Cre/þ;Ddx3xflox/flox mice had not only dramat-ically increased tumor multiplicity but also accelerated tumorprogression with increased incidence of HCC. Therefore, a rolefor DDX3X as an important regulator of genome stability in vivowas confirmed.

DiscussionHuman HCC generally proceeds through a stepwise process

involving hepatic injury and compensatory proliferation, fol-lowed by inflammation, fibrosis, and the development of HCC.Here, we showed that Alb-Cre/þ;Ddx3xflox/flox mice recapitulatedthese key features of human HCC pathogenesis. Loss of Ddx3xaffected DNA repair by reducing the expression of NER factorsDDB2 and XPA, which contributed to an accumulation of unre-paired DNA damage and replication stress in the liver, providingfurther evidence that DDX3X maintains genomic stability in vivo.

In general, most adult tissues/organs are in a quiescent state,and proliferation occurs only when old/dying cells must bereplaced. Nevertheless, the liver is a unique organ with a highregenerative capacity that can restore its lost mass after resectionsor injury. A cell-cycle kinetic study of the liver showed that thefraction of hepatocytes in a proliferative state, but not the averagecell-cycle time, decreased gradually with age, beginning at around2 weeks of age. As development proceeds, individual cells ceasedivision and then increase in cell size until the final size limit isreached (31). In this study, we showed that the levels of RPA70,RPA32, pRPA32, andMre11were higher in livers from3-week-old(juvenile) controls than those of 6-week-old controls, indicatingthat the expression of DDR factors during early liver growth isessential to safeguard DNA integrity. Indeed, mutations/inacti-vation of DDR genes are observed in various diseases and cancers(32). Among these studies, a higher incidence of spontaneousliver tumors and an increased mutation frequency in livers wereobserved in aged Xpa�/� mice compared with their wild-typecontrols (33, 34). In addition toUltraviolet B (UVB)-induced skincarcinogenesis, Ddb2-deficient mice developed spontaneoustumors at a high rate between 20 and 25 months of age (35).

Hepatocyte-specific ablation of Ddb1 leads to chronic hepatocyteturnover, mild liver damage, and liver tumor formation (36). Inexperimental models of alcohol-related liver diseases, the trans-glutaminase-2 (TG2)–mediated impairment of the Sp1-c-metsignaling cascade was observed (37). These findings support ourresults that dysregulation of DDR, as evidenced by the decrease ofXpa and Ddb2 levels needed for DNA damage repair, in juvenileAlb-Cre/þ;Ddx3xflox/flox livers is associated with a predisposition toHCC. This study shows the genomic instability is a driving eventfor liver tumorigenesis in Alb-Cre/þ;Ddx3xflox/flox mutants; how-ever, we donot exclude the possibility thatDDX3Xmay also affectother cellular processes, such as epigenetic alterations, deregula-tion of miRNAs, and overexpression of oncogenes, during pro-gression of HCC.

It is wildly accepted that inflammation can increase the riskof cancer by providing the cytokines and chemokines frominfiltrating cells in tumor microenvironment, including HCC(38, 39). Targeting hepatic inflammation is one of the thera-peutic opportunities for the treatment of HCC (40). In thisstudy, we showed that the liver injury in 6-week-old hepato-cytes and Alb-Cre/þ;Ddx3xflox/flox mutants was associatedwith inflammation (Supplementary Fig. S4). The infiltratedimmune cells and IL1b-, TNFa-, and NF-kB–positive cells weresignificantly increased in spontaneous tumor tissues of agedAlb-Cre/þ;Ddx3xflox/flox mutants. Also, Il1b and Tnfa mRNAlevels were significantly increased in liver tumors from Alb-Cre/þ;Ddx3xflox/flox mutants. In tumor-promoting stimuli, IL1b-and TNFa-positive cells and Il1b levels were significantly higherin DEN-treated Alb-Cre/þ;Ddx3xflox/flox tumors than those ofcontrols (Supplementary Fig. S13). In the context of DNAdamage and inflammation, DEN-treated Alb-Cre/þ;Ddx3xflox/flox

mice had not only dramatically increased tumor multiplicitybut also accelerated tumor progression with increased inci-dence of HCC. These results suggested inflammation-relatedchanges in the microenvironment of liver contribute to liverinjury and tumorigenesis in Alb-Cre/þ;Ddx3xflox/flox mice.

The epidemiologic data of human HCC show that maleshave a higher risk of developing liver tumors than females (3).However, we found that the tumor incidence in Alb-Cre/þ;Ddx3xflox/flox female mice was higher than in Alb-Cre/þ;Ddx3xflox/Y male mice in this study. DDX3X has a structuralhomolog, DDX3Y, located on Y chromosome (12, 41). Studieshave shown that both of the DDX3X and DDX3Y genes aretranscribed in multiple tissues. DDX3X protein was detected inall tissues analyzed; however, the DDX3Y protein was predom-inantly detected in male germ cells by translational control(42, 43). Expression and deletion analyses of DDX3Y suggesteda specific function of DDX3Y in male fertility (44), whereas wehave shown discrete and essential roles of DDX3X protein inmouse embryonic and placental development (12). Thesegenetic data suggest that DDX3Y may act specifically in testisand may have a different function than DDX3X. AlthoughDdx3y mRNA levels in Alb-Cre/þ;Ddx3xflox/Y livers were com-parable with their controls at a young age, decreased Ddx3ylevels were observed in aged Alb-Cre/þ;Ddx3xflox/Y livers andtumors (Supplementary Fig. S14). We cannot rule out thepossibility that DDX3Y acts as a functional substitute for theloss of DDX3X in some contexts. It will be of interest to assesswhether basalDdx3ymRNA and DDX3Y protein are involved inthe gender differential effect of DDX3X in liver homeostasisand DEN-induced liver tumorigenesis.

Chan et al.





HCC is a heterogeneous disease that shows high resistance toconventional chemotherapy and radiotherapy. It is generallybelieved that genomic instability may contribute to poor clinicaloutcomes (45, 46). This study suggested a link between DDX3Xfunction and genome integrity in liver tumorigenesis, at leastpartly through the control of the expression of NER genes thatmediate DNA repair. In the cell populations with relatively rapidrates of proliferation, including juvenile liver and tumor, loss ofDdx3x results in DNA damage, liver injury, cell death–compen-satory proliferation, and inflammation. Under the condition ofDNA damage and inflammation, aberration functions of DNArepair could promote the development of dysplastic lesion andsubsequent HCC. Given that the small-molecule–mediated inhi-bition of DDX3X activity may provide new therapeutic opportu-nities to treat various viral infections and cancers (47, 48), theimpact ofDDX3XonDNAdamage and repair pathways cannot beunderscored. A detailed understanding of the biology, pathology,and the complexity of the cellular responses to DNA damage inHCCassociatedwithDDRdeficiencywill allow further preclinicalinvestigations and provide appropriate treatment strategies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C.-H. Chan, C.-M. Chen, L.-R. YouDevelopment of methodology: C.-H. Chan, C.-M. Chen, L.-R. YouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.-H. Chan, C.-M. Chen, L.-R. You

Writing, review, and/or revision of the manuscript: C.-H. Chan, C.-M. Chen,L.-R. YouAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.-R. YouStudy supervision: C.-M. Chen, Y.-H. Wu Lee, L.-R. You

AcknowledgmentsWe thank Dr. Hao-Kang Li for technical support. The A6 BCM, TROMA-III

(K19), and G8.8 (EpCAM) antibodies developed by V.M. Factor, R. Kemler,and A.G. Farr, respectively, were obtained from the Developmental StudiesHybridoma Bank, created by the NICHD of the NIH and maintained at TheUniversity of Iowa, Department of Biology, Iowa City, IA. We thank theTaiwan Animal Consortium (MOST107-2319-B-001-002)–Taiwan MouseClinic which is funded by the Ministry of Science and Technology (MOST)of Taiwan for technical support in collagen stain experiment. This work wassupported by the Ministry of Science and Technology of Taiwan (grantnumbers MOST103-2320-B-009-006-, MOST104-2320-B-009-001-, andMOST105-2320-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);Center For Intelligent Drug Systems and Smart Bio-devices (IDS2B), NationalChiao Tung University and Cancer Progression Research Center, NationalYang-Ming University from The Featured Areas Research Center Programwithin the framework of the Higher Education Sprout Project by the Ministryof Education (MOE) in Taiwan; and the Ministry of Education in Taiwan,Aim for the Top University Plan to L.-R. You.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 28, 2018; revised September 9, 2018; accepted September 26,2018; published first October 8, 2018.

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2019;17:555-566. Published OnlineFirst October 8, 2018.Mol Cancer Res Chieh-Hsiang Chan, Chun-Ming Chen, Yan-Hwa Wu Lee, et al. DDX3X LossDNA Damage, Liver Injury, and Tumorigenesis: Consequences of

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