WWOX controls hepatic HIF1α to suppress hepatocyte proliferation and neoplasia

WWOX controls hepatic HIF1α to suppress hepatocyte proliferation and neoplasia Liver cancer is one of the most lethal malignancies with very poor prognosis once diagnosed. The most common form of liver cancer is hepatocellular carcinoma (HCC). The WW domain-containing oxidoreductase (WWOX) is a large gene that is often perturbed in a wide variety of tumors, including HCC. WWOX has been shown to act as a tumor suppressor modulating cellular metabolism via regulating hypoxia-inducible factor 1α (HIF-1α) levels and function. Given that WWOX is commonly inactivated in HCC, we set to determine whether specific targeted deletion of murine ΔHep Wwox affects liver biology and HCC development. WWOX liver-specific knockout mice (Wwox ) showed more potent liver regeneration potential and enhanced proliferation as compared with their control littermates. Moreover, WWOX deficiency in hepatocytes combined with diethylnitrosamine treatment increased the tumor burden, which was associated with increased HIF1α levels and target gene transactivation. Inhibition of HIF1α by systemic treatment with digoxin significantly delayed HCC formation. Our work suggests that WWOX inactivation has a central role in promoting HCC through rewiring of cellular metabolism and modulating proliferation. Introduction reported at this region in aflatoxin B1 exposed HCC , Hepatocellular carcinoma (HCC) is the most common suggesting that it might harbor a tumor suppressor. In particular, WWOX expression is absent or reduced in type of primary liver cancer, representing the fifth type of commonly diagnosed cancer worldwide and third mor- most of the derived liver cancer cell lines . The gene tality cause among other cancer malignances . HCC encodes a 46 kDa protein comprising of two N-terminal prevalence has been dramatically increasing in the last WW domains, known to mediate protein–protein inter- decay because of the expansion of HCC risk factors, actions and a short-chain dehydrogenase/reductase 2 8–10 including hepatitis infection and obesity . Therapeutic domain whose specific function is unknown yet . options are limited and survival after diagnosis is still poor Moreover, WWOX was suggested as a modulator of β- 11,12 leading to high mortality. Therefore, better understanding catenin protein activity in some HCC cells lines .In of the molecular basis of HCC is urgently needed. addition to its genomic re-arrangement and hyper- WW domain-containing oxidoreducatase (WWOX) methylation of its regulatory region, WWOX is inacti- gene resides in one of the most common fragile sites vated by other proteins or microRNAs in HCC cell 13–15 known as FRA16D, a region that is altered in many types lines . However, no direct in vivo evidence linking 3–5 of cancer . Frequent homozygous deletions were WWOX tumor suppressor function with HCC develop- ment is known so far. WWOX is commonly reported as a tumor suppressor Correspondence: Rami I. Aqeilan (ramiaq@mail.huji.ac.il) not only owing to its common loss in many human The Lautenberg Center for General and Tumor Immunology, Department of malignancies but also due to its anti-tumorigenic effect Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel when overexpressed and susceptibility of tumor formation 8,16–19 Department of Surgery, Hebrew University-Hadassah Medical, Jerusalem, in Wwox-mutant mice . Wwox null mice die by the Israel age of 3–4 weeks owing to severe metabolic disorders, Full list of author information is available at the end of the article Edited by G. Melino © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 2 of 12 Fig. 1 Loss of WWOX expression in liver cancer. a TCGA data analysis showing that WWOX genomic region spanning long arm of chromosome 16 at 16q23.1 (red arrow) is frequently lost in HCC patients (n = 438 cases) with single copy number alteration (SCNA) equal to 96.84%. b Prognostic value of WWOX mRNA in HCC patients (n = 438) showing that patients with low expression had poorer survival compared with those with high WWOX expression. P value < 0.05. c Representative images of a liver cancer tissue microarray (TMA) (LV8011, US Biomax) showing immunohistochemical staining of WWOX in normal and HCC samples. d Quantification of WWOX protein levels from c. * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM mainly lethal hypoglycemia precluding studying impli- interacts with wide variety of proteins regulating their 9,35 cations of WWOX loss in adult mice. To overcome this functions and affecting cellular outcome . Previously, limitation, we have recently generated a conditional WWOX have been shown to physically interact with mouse model in which somatic deletion of Wwox is hypoxia-inducible factor 1α (HIF-1α) and inhibit its 21 33 achieved using a specific Cre recombinase , mimicking activity . Ablation of HIF1α expression in RAS- the alterations frequently observed in human cancers and transformed Wwox-deficient mouse embryonic fibro- allowing study of human cancer intervention, develop- blasts significantly reduced tumourigenicity , suggesting ment and progression, and assessing therapeutic strate- that HIF1α mediates the tumorigenic phenotype of 22,23 gies . In this study, we utilized a Cre recombinase Wwox-depelted cells. Nevertheless, the functional asso- driven by the promoter of albumin (Alb-Cre), which ciation of WWOX-HIF1α has not been demonstrated in a results in somatic deletion of WWOX in hepatocytes; cancer mouse model. Here, we studied the effect of ΔHep ΔHep Wwox mice. Interestingly, Iatan et al. have recently treating Wwox mice with DEN and followed HCC demonstrated that WWOX deletion in a murine mouse development and progression. We demonstrated that model modulates levels of lipoproteins, however, these WWOX dysregulation accelerates HCC development mice did not spontaneously develop HCC . One of the through control of HIF1α and other master proliferation most widely used and accepted models for HCC devel- gene networks implicated in hepatocarcinogenesis. We opment in animal models is the use of N- also demonstrate that WWOX ablation modulates liver nitrosodiethylamine (DEN), a known carcinogen, which regeneration. alkylates DNA bases and results in HCC tumor forma- tion in a defined kinetic manner . Results WWOX anti-tumorigenic functions have been shown WWOX expression is commonly lost in liver cancer 26–28 9,29–31 to affect genome integrity , apoptosis , cell Given that WWOX expression is altered in many 32 36 growth and extracellular matrix signaling , and glucose human malignancies , we set to examine its genomic and 33,34 metabolism . WWOX, through its first WW domain, expression status using different available tools and Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 3 of 12 resources. Analysis of 438 liver cancer samples in the Interestingly, WWOX mRNA and protein was decreased in TCGA database using fire browser (www.firebrowse.org) the tumors of control mice as revealed by qRT-PCR (Fig- revealed that the WWOX locus, spanning chromosomal ure S1A) and immunohistochemistry (Figure S1B). In ΔHep region of 16q23.1, is one of the most significant regions addition, Wwox mice had higher levels of serum alanine harboring copy number loss in HCC patients (Fig. 1a). transaminase, indicating liver dysfunction as a result of Using Xena browser (www.xenabrowser.net) we next tumor formation (Fig. 2f). Histological characterization of evaluated the prognostic value of WWOX expression in liver tissues revealed aggressive, poorly differentiated and HCC and found that cases harboring reduced WWOX highly proliferative tumors resembling HCC (Fig. 2g, h). mRNA levels tend to present with worse survival outcome Altogether, these findings indicate that WWOX loss compared to those having high WWOX expression accelerates HCC development. (Fig. 1b). Consistent with these observations we further found that WWOX levels are absent or reduced in dif- Hepatocyte-specific WWOX ablation is associated with ferent liver pathologies, particularly in HCC, as assessed increased proliferation by immunohistochemical staining of a commercial tissue Our findings so far suggest that hepatocyte-specific microarray (Fig. 1c, d). Notably, a tendency of reduced WWOX deletion promoted HCC development. On one WWOX expression was observed in cancer adjacent liver hand, WWOX overexpression was previously shown to tissue (P< 0.05), suggesting that loss of WWOX could be induce apoptosis and suppress proliferation, whereas its an early event in liver carcinogenesis. loss is associated with enhanced survival in a hepatoma cell line . On the other hand, DEN treatment is known to Hepatocyte-specific WWOX ablation accelerates HCC induce acute hepatic injury followed by compensatory development proliferation . We therefore set to determine whether the ΔHep The fact that WWOX expression is reduced in early liver phenotype observed in DEN-treated Wwox mice is a cancer lesions suggests that WWOX may have a role in consequence of impaired proliferation control. To this suppressing liver carcinogenesis. To determine whether end, we analyzed liver tissues of DEN-treated mice at WWOX loss could contribute to HCC development or different time points starting from 1 to 10 months. progression, we generated a hepatocyte-specific Wwox Hematoxylin and Eosin (H&E) evaluation revealed that ΔHep knockout mouse model and followed liver tumor formation. Wwox livers display hepatocyte morphological chan- Wwox-floxed mice (Wwoxf/f) were bred with Albumin- ges starting from 6-month post DEN treatment, which progressed to HCC at the age of 10 months, as shown Cre transgenic mice to generate Wwoxf/f;Albumin-Cre ΔHep (Wwox ) mice on the C57Bl6/J;SVJ129 mixed genetic earlier (Fig. 3a, d). No histological abnormalities were background. Successful ablation of WWOX was validated observed in the groups of 1 and 3-month post DEN ΔHep using quantitative real-time (qRT)-PCR and western blot treatment of Wwox mice nor in the control mice. To ΔHep analyses (Fig. 2a, b). Follow up of Wwox and controlled further support our findings, we immunostained for Ki67, littermate mice for up to 2-years did not reveal spontaneous a surrogate marker of proliferation. Digital quantification ΔHep tumor development, consistent with previous observa- of Ki67-positive nuclei demonstrated that Wwox liver tions . We therefore decided to examine the effect of sections had significantly higher number of Ki67-positive ΔHep WWOX ablation upon N-nitrosodiethylamine (DEN) cells in the groups of 1, 3, 6, and 10 months Wwox treatment, a widely used chemical carcinogen for studying mice relative to the control littermate groups (Fig. 3e). In 22 ΔHep liver carcinogenesis . Cohorts of Wwox mice and addition, qRT-PCR analysis of proliferative genes impli- control littermates were intraperitoneally (IP) injected with cated in liver cancer, including c-Myc, c-Jun, c-Fos, and ΔHep 5 mg/Kg single injection of DEN at the age of 14 days and Axin, display higher levels in the Wwox mouse groups monitored for HCC development as a function of time from as early as 1-month post DEN treatment (Fig. 3f). ΔHep (Fig. 2c). DEN treatment using this protocol is known to Although, DEN-free Wwox mice display no tumori- lead to 85% HCC development in C57BL6/J strain by the genic phenotype, levels of the previous mentioned pro- age ranging from 10 to 18 months , whereas 129SVJ strain liferative genes were also elevated (Figure S2A). develop HCC later .As shown in Fig. 2d, DEN-treated Consistent with these results, a significant increase in ΔHep Wwox mice developed significantly higher incidence of CTGF levels, one of the main effectors of the Hippo 41 ΔHep HCC as compared with control mice (P= 0.0025). By the pathway , was also noted in livers of Wwox mice age of 10 months, the penetrance of tumor development with a slight increase in liver weight in the pre- ΔHep was 100% in Wwox mice whereas just 20% of the tumorigenic phase (Figure S2B and S2C). These findings control littermates’ mice developed macroscopic tumors suggest that WWOX has a critical role in inhibiting (Fig. 2d). Furthermore, tumor load, as assessed by liver proliferation of DEN-treated hepatocytes, whereas its weight relative to body weight, was significantly higher in inactivation leads to increased proliferation contributing ΔHep Wwox mice as compared with control mice (Fig. 2e). to tumor development. Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 4 of 12 ΔHep Fig. 2 Hepatocyte-specific WWOX ablation accelerates HCC development. a Validation of WWOX depletion in Wwox (cKO: conditional knockout) mice using qRT-PCR; n = 3 for each mouse group. Error bars indicate ± SEM. b Validation of WWOX protein ablation by immunoblotting ΔHep using WWOX antibody; HSP90 was used as loading control. c DEN treatment plan: DEN was IP injected to control and Wwox mice at the age of 2 ΔHep 14 days and mice were analyzed at the age of 10 months. d χ analysis of macroscopic tumor incidence in control mice versus Wwox mice ΔHep treated with DEN. e Tumor load analysis represented by liver weight of control mice (n= 15) versus Wwox cKO mice (n = 14) after 10 months of ΔHep DEN treatment. f Serum ALTL levels of control mice (n = 6) versus Wwox mice (n = 6) after 10 months of DEN injection. g Representative pictures ΔHep of control and Wwox livers of two male littermates (upper). Histological images showing liver sections of 10 months DEN-treated control and ΔHep Wwox mice (lower panel) h Histological images (× 10, × 20, and × 40) of 10 months DEN-treated mice. * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM Enhanced expression of glycolytic genes in livers of DEN- moderate and prominent increase at 6 and 10 months in ΔHep ΔHep Wwox mice DEN-treated Wwox mice, respectively (Fig. 4f). Like- Recently, it was shown that WWOX loss is associated wise, levels of PKM2 and membranous GLUT1 protein ΔHep with Warburg effect and impaired mitochondrial were higher in 6 and 10 months DEN-treated Wwox 33,42,43 respiration . In mammalian cells, WWOX ablation mice (Fig. 4e). is inversely correlated with increased levels and activity of Our recent studies have shown suppression of HIF1α hypoxia-inducible factor 1α (HIF1α-enhancing cell transactivation function by WWOX . Therefore, we next 33,44 transformation and tumor growth . In particular, determined whether WWOX loss modulates HIF1α Wwox-deficient cells were shown to display higher levels recruitment to promoters of these glycolytic genes. To of glycolytic genes known to be regulated by HIF1α. Given address this, we performed a chromatin immunoprecipi- that HIF1α has known roles in HCC development ,we tation (ChIP) experiment using anti-HIF1α antibody on next set to determine whether WWOX-targeted loss in genomic DNA isolated from liver tissues of 10 months ΔHep ΔHep the different DEN-treated Wwox and control mice is DEN-treated Wwox and control littermate mice. associated with impaired HIF1α function. Analysis of QRT-PCR analysis revealed HIF1α enrichment on pro- HIF1α glycolytic target genes mRNA, including Hk2, moters of target genes, including Pkm2, Hk2, Glut1, Aldh, ΔHep Pkm2, Glut1 and others, demonstrated no change at 1 and and Gapdh,in Wwox livers (Fig. 4f). Altogether, these 3-months post treatment (Fig. 4a, b). Intriguingly, a sig- results imply that WWOX loss leads to HIF1α enhanced nificant upregulation of these transcripts was noted in 6 activity and, likely, reprogramming of HCC cells to glu- ΔHep and 10 months DEN-treated Wwox mice compared cose metabolism and Warburg effect. with control mice (Fig. 4c, d). Interestingly, DEN-free ΔHep Wwox mice on 6 months age displayed a tendency of Inhibition of HIF1α rescued the effect of WWOX loss HIF1α glycolytic target genes upregulation (Figure S3A). We have previously shown that digoxin, an inhibitor Moreover, nuclear HIF1α protein levels exhibited of HIF1α, rescued the effect of Wwox-deficient mice Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 5 of 12 Fig. 3 Hepatocyte-specific WWOX ablation is associated with increased proliferation. Histological images (H&E staining and Ki67 ΔHep immunohistochemical staining at × 40) of DEN-treated control and Wwox liver sections at the age of 1 a,3 b,6 c, and 10 months d. e ΔHep Quantification of positive Ki67 nuclei in 1, 3, 6, and 10 months old DEN-treated control and Wwox mice liver. (n = 5 for each group). f mRNA ΔHep expression levels of proliferation genes in DEN-treated control and Wwox mice at the age of 1, 3, 6, and 10 months (n = 3 for each group). * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep displaying hypoglycemia and elevated levels of glycolytic High-fat diet (HFD) increased HCC incidence in Wwox genes . Digoxin is a cardiac glycoside, which has been mice shown to inhibit HIF1α transcriptional activity in vitro Obesity is known to be a major risk factor for HCC and suppress tumor growth in vivo . We therefore set to development. Indeed, combined HFD and DEN treatment ΔHep investigate whether digoxin treatment of DEN-Wwox have been previously shown to strongly enhance HCC mice could reduce the incidence of HCC development. development when compared with DEN mice on normal ΔHep 48 DEN-Wwox and control mice received digoxin (1 mg/ diet . Driven by our previous observations, we decided to kg) or vehicle control (saline) through intraperitoneal investigate the effect of HFD on HCC development in ΔHep injections (3-times/week) for 32 weeks starting at DEN-Wwox mice. DEN-treated cohorts of male ΔHep 6 months post DEN treatment (Fig. 5a). Analysis of mice Wwox and control mice were placed on HFD, in 8-months post digoxin treatment revealed marked sup- which 60% of calories were fat-derived, for 30 weeks. ΔHep pression of liver tumor growth with more prominent Tumors of DEN-treated Wwox HFD mice appeared ΔHep effect in Wwox mice compared with saline-treated 3-months earlier compared with normal chow-diet mice ΔHep Wwox mice (Fig. 5b, c). In contrast, no significant (Fig. 2), consistent with previous reported data showing difference was observed between digoxin and saline- acceleration of HCC formation in mice fed with HFD . treated DEN control mice (Fig. 5e). Consistent with these No difference of mice weight between the two groups of ΔHep ΔHep results, digoxin-treated DEN-Wwox mice showed HFD-Wwox and control mice was noted (Fig- downregulations of HIF1α targets, whereas no significant ure S4A). Nevertheless, the number of tumors in ΔHep changes were observed in digoxin-treated DEN control Wwox mice was significantly higher (Fig. 6a, b) and mice (Fig. 5d, f). We conclude that WWOX loss exerts was accompanied with higher levels of serum ALT (Fig- critical regulation on HIF1α function mediating rewiring ure S4B) and hepatosteatosis (Fig. 6c). Consistent with of glucose metabolism and contributing to enhanced anti-proliferative role of WWOX that we showed pre- ΔHep proliferation. viously (Fig. 3), DEN-treated Wwox mice fed with Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 6 of 12 ΔHep Fig. 4 Enhanced expression of glycolytic genes in livers of DEN-Wwox mice. a mRNA expression levels of HIF1α glycolytic target genes in ΔHep DEN-treated control and Wwox (cKO) mice at the age of 1 a,3 b,6 c and 10 d months (n = 3 for each group). e Immunohistochemical staining of ΔHep HIF1α, PKM2, and GLUT1 in 6 and 10 months of DEN-treated Wwox and control liver tissues. Images were taken at × 40 magnification (Bar = ΔHep 100μm). f ChIP experiment with HIF1α antibody on DEN-treated control and Wwox (cKO) mice followed by qRT-PCR analysis of HIF1α target genes. * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep HFD displayed increased proliferation (Fig. 6d, e). Per- control mice, it continued rising in Wwox mice. sistent with a prominent role of β-catenin pathway in Increased proliferation was associated with increased ratio HCC, we observed significant upregulation of Axin2 of liver to body weight on day 7 post hepatectomy (Fig. 7c). ΔHep transcript in DEN-treated Wwox HFD mice (Fig. 6f). Moreover, qRT-PCR analysis of proliferative gene impli- At a later stage, there was also an upregulation of HIF1α- cated in liver regeneration, including c-Myc, displayed ΔHep target genes (Figure S4C). Altogether, our findings suggest higher levels in Wwox mice group on day 2, 3, and 4 as that combined loss of WWOX and HFD intake accelerate compared with control mice (Fig. 7d). Interestingly, Wwox HCC formation mediated by enhanced proliferation. and c-Myc transcripts showed a trend of inverse pattern of expression in Wwox-control mice (Fig. 7e) suggesting that Wwox ablation is associated with increased proliferation WWOX might negatively modulate c-Myc. These results upon partial hepatectomy are consistent with WWOX function to inhibit hepatocyte The preceding observations demonstrated that WWOX proliferation after liver regeneration. inactivation coupled with DEN treatment and HFD intake leads to enhanced proliferation and tumor formation. We Discussion next asked whether WWOX deletion affects hepatic cell We report here that WWOX loss is a common event in proliferation upon partial hepatectomy, a process known to HCC and that its dysregulation synergizes with DEN acutely induce hepatocyte proliferation to achieve liver treatment and HFD to accelerate HCC development ΔHep regeneration. Wwox mice and their littermate controls through regulation of proliferation genes. At the mole- were subjected to 30% hepatectomy and were analyzed 1, 2, cular level, WWOX, known to act as an adapter protein 3, 4, and 7 days later for Ki67 immunohistochemical through interaction via its WW1 domain, functionally staining. As expected, an increase in the proliferation index associates with HIF1α and probably other transcription ΔHep was observed in Wwox mice compared with control factors involved in proliferation, leading to inhibition of mice (Fig. 7a, b). Intriguingly, whereas the proliferation their transactivation function. For example, when index was reduced 4 and 7 days post hepatectomy in WWOX is lost, HIF1α recruitment and function on its Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 7 of 12 Saline Digoxin ΔHep Fig. 5 Inhibition of HIF1α rescued the effect of WWOX loss. a Digoxin experiment plan, DEN was IP injected to control and Wwox mice at the age of 14 days. Six-month later mice were started to be treated with digoxin (1 mg/kg) or vehicle control (saline) 3-times a week for an additional 8- ΔHep ΔHep months. b Representative images of saline-treated Wwox (cKO) mice versus digoxin-treated Wwox (cKO) mice livers and liver sections c ΔHep ΔHep Tumor load of saline-treated Wwox mice versus digoxin-treated Wwox mice livers as assessed by liver weight at the age of 14 months d ΔHep ΔHep mRNA expression levels of HIF1α glycolytic target genes in saline-treated Wwox (cKO) mice versus digoxin-treated Wwox (cKO) mice at the age of 14 months, (n = 3 for each group). e Χ analysis of macroscopic tumor incidence in saline-treated control mice versus digoxin-treated control mice at the age of 14 months. f mRNA expression levels of HIF1α glycolytic target genes in saline-treated control mice versus digoxin-treated control mice at the age of 14 months, (n = 3 for each group). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM target genes are enhanced leading to increased expression Several studies have suggested that the WWOX gene 3,4,7 of glycolytic genes and proliferation rate. We further functions as a tumor suppressor in liver cancer .Early demonstrate that pharmacological inhibition of HIF1α evidence has revealed the existence of frequent homozygous reduces HCC burden in DEN-treated liver-specific deletions at chromosome 16q23, where WWOX resides, in WWOX-deficient mice. Finally, we show that WWOX aflatoxin B1 exposed HCC . In another study, expression of loss increases liver regeneration after hepatectomy pos- both WWOX and FHIT, another tumor suppressor located sibly via eliminating the termination phase of liver in a fragile site, appeared to be correlated and down- regeneration. These findings underscore the significant regulated in liver tissues in a carcinogen-specificmanner . role of WWOX in rewiring metabolic changes in liver A subsequent study has examined the status of the WWOX cells contributing to liver cancer. gene in human HCC cell lines and found its recurrent Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 8 of 12 ΔHep 2 Fig. 6 High-fat diet (HFD) increases HCC incidence in DEN-treated Wwox mice. a Χ analysis of HCC incidence in DEN-treated control and ΔHep ΔHep Wwox mice (at age of 14 days) and fed with HFD from the age of 8 weeks. b Representative pictures of control and Wwox (cKO) livers fed with HFD at the age of 7 months. Arrows indicate macroscopic tumors. c Histological images (× 4, × 10, × 2,0 and ×40) of 7 months DEN-treated ΔHep control and Wwox livers fed with HFD. d Histological images (Ki67 immunohistochemical staining) of mice from c. e Quantification of positive Ki67 nuclei of mice from d. f mRNA expression of Axin2 (control, n = 3; cKO, n = 3). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM alterations further implicating WWOX in hepatocarcino- We also showed that WWOX anti-proliferative activity genesis . In our study, we further delineate the role of is mediated by suppression of the proto-oncogenes c-Myc, WWOX as a tumor suppressor in liver cancer. First, we c-Fos, and c-Jun.c-Myc is known as an oncogene that 51 52,53 showed that WWOX expression is reduced or absent in promotes HCC in human and murine HCC models . ΔHep large cohorts of human liver pathologies, including HCC. We found that c-Myc is upregulated in Wwox mice Furthermore, WWOX low expression in HCC is correlated on 1, 3, 6, and 10 months continually, raising the point with decreased survival suggesting that WWOX expression that WWOX partially suppresses HCC through con- has a prognostic value in HCC. Second, we provide the first tinuous suppression of c-Myc. Previous reports have in vivo evidence that WWOX loss contributes to HCC shown that WWOX suppresses the AP-1 transcriptional development, in two different models of murine HCC. Our activity through regulating c-Jun localization , consistent data show that DEN treatment alone or in combination with our findings showing enhanced expression of c-Jun with HFD clearly have an advantage for HCC development and c-Fos in WWOX-depleted cells. It is also likely that upon WWOX loss. Both tumor incidence and multiplicity WWOX loss releases its inhibitory effect on a plethora of ΔHep (load) were higher in Wwox mice implying the impor- other proto-oncogenes as have been previously demon- 9,16,55 tance of WWOX alteration for HCC development and strated , contributing to the observed aggressive HCC progression. Our findings also suggest that WWOX loss is phenotype. Although WWOX has been shown as an an important contributor for HCC promotion as earlier hits effector of the Wnt/β-catenin pathway in vitro, through 15,35,56 are required for HCC initiation. WWOX expression could interaction with DVL , our results do not support 6,49 be altered by environmental cues or in consequence of such an effect in DEN-mediated HCC development. On microRNA dysregulation or even as a result of genetic the other hand, when DEN treatment was combined with variations in WWOX . If these alterations were combined HFD, WWOX loss resulted in significant increase in Axin with western HFD then our findings indicate that HCC risk levels, suggesting that under these conditions WWOX increases. plays a more important role in regulating the Wnt/β- Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 9 of 12 Control cKO Control cKO Control cKO cMYC WWOX Fig. 7 Wwox ablation is associated with increased proliferation upon partial hepatectomy. a Histological images (H&E staining and Ki67 ΔHep immunohistochemical staining) of control and Wwox mice liver 3-days post 50% partial hepatectomy (× 10, × 20, and × 40). b Quantification of ΔHep positive Ki67 nuclei in 5 months old control (WT) and Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy (n = 5 for each group). c Liver weight to body weight ratio of 1, 2, 3, 4, and 7 days post hepatectomy (n = 5 for each group). d mRNA expression levels of c-Myc in control and ΔHep ΔHep Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy. e mRNA expression levels of Wwox in control and Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy (n = 4 for each group). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep catenin pathway. It is therefore possible that other cellular the age of 14 months, DEN-Wwox mice did not show pathways are also involved as WWOX function has been macroscopic tumors and had lower expression of glyco- shown to include plethora of effectors lytic genes driven by HIF1α. These results are consistent Our data further present a critical metabolic and anti- with our previous data showing that WWOX inhibits 42–44,65,66 proliferative role of the tumor suppressor WWOX in sup- aerobic glycolysis . Rewiring of glucose metabo- ΔHep pressing HCC through regulation of HIF1α function. The fact lism in Wwox mice provides the needed building that HIF1α protein levels and its target genes were elevated in blocks for cell division and proliferation and hence these the pre-tumor stage (6 months; Fig. 4e) implies that these mice could have more proliferation compared with con- metabolic changes fuel and drive HCC development in DEN- trol mice. Future studies shall include more specific ΔHep treated Wwox mice. HIF1α is significantly elevated in inhibitors of HIF1a and probably Myc to inhibit WWOX- 58,59 human HCC samples and associated with bad prognosis . mediated HCC development. Moreover, HIF1α hepatocytes-specific overexpression in a WWOX anti-proliferative function in HCC might also murine model increases HCC-promoting M2 macrophages , involve modulation of fatty acid/lipid metabolism. In fact, whereas HIF1α liver-specific knockout sensitizes hepatoma WWOX genetic variants in human patients and Wwox 61 24 cells to etoposide treatment . WWOX loss is associated with knockout mouse models display decreased serum HDL- elevated HIF1α target genes starting from the age of 6 months. C. Moreover, microarray analyses of Wwox liver-specific In addition, we show that HIF1α is enriched on its targets knockout mice revealed an increase in plasma triglycer- ΔHep promoters in hepatocytes isolated from Wwox mice. ides and altered lipid metabolic pathways suggesting that Targeting tumor suppressor genes is a major challenge. WWOX disruption indeed alters cellular lipid home- Novel approaches, including synthetic lethality and col- ostasis in the liver. Whether these WWOX effects can lateral vulnerability have been proposed to come over this also contribute to HCC development is unknown. limitation. We therefore, assessed whether targeting the Our findings also present a vital role of WWOX in proliferative/survival signals in WWOX-deficient cancer maintaining healthy liver regeneration. Remarkably, partial cells could help inhibit tumor progression. Several hepatectomy in liver-specific Wwox-deficient mice resulted attempts to target HIF1α in HCC were reported via in increased proliferation (Ki67) upon liver regeneration, at 62 63 64 inhibition of its expression , dimerization , or activity . the later phases of this remarkably controlled homeostatic In our model, we were able to rescue, at least partially, the process. Interestingly, Wwox RNA levels increased 2 days tumor phenotype using an HIF1α inhibitor (digoxin); at post hepatectomy while c-Myc levels showed an inverse Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 10 of 12 pattern. c-Myc levels are reported to be highest in 12–18 h Thermoscientific, dilution 1:200), PKM2 antibody (Cat# post heptectomy and then c-Myc levels usually drop 38237, Abcam, dilution 1:50) and HIF1α antibody (Cat# 69–71 down . Our results might suggest that WWOX,ananti- NB100-105, Novus, dilution 1:20). proliferative gene, is induced during the course of liver regeneration to allow shutting down proliferative genes, ChIP such as c-Myc, and maintaining proper organ size. Inter- Hepatocytes were isolated from liver tissues, and solu- estingly,nodifferenceinHIF1α target genes was observed tions were prepared for ChIP analysis according to a ΔHep 74 in liver regeneration of Wwox .These observations standard protocol . 0.8 mg of HIF1α antibody (mouse suggest that WWOX may regulate liver regeneration by mAB, Cat # NB100-105, Novous Biological, CO, USA) HIF1α-independent mechanism. was used to precipitate HIF1α. Targeted PCR was done In conclusion, our study reveals WWOX as a tumor using list of primers (Supplemental Table 2). suppressor with critical roles in HCC suppression through maintaining moderate glucose metabolism and inhibiting Statistics uncontrolled cell proliferation. Results of the experiments were expressed as mean ± standard deviation or standard error of mean. Student’s t- Materials and Methods test, was used to compare values of test and control Mice and related experiments samples. P < 0.05 indicates significant difference. * P- Wwox-floxed (Wwoxfl/fl) C57BL6/J;129sv mixed value < 0.05, ** P-value < 0.01. For the human data ana- genetic background mice were bred with Albumin-Cre lysis, Single Copy Number Alteration of liver hepatocel- transgenic mice to generate Wwox conditional knockout lular carcinoma (LiHC) TCGA data set (n = 434) was ΔHep in hepatocytes (Wwox mice). Male pups of control analyzed by the website www.firebrowse.org (developed ΔHep and Wwox mice were IP injected with 5 mg/Kg DEN by broad institute of MIT and Harvard). Survival curve of (Sigma Aldrich) at the age of 14 days. Partial hepatectomy LiHC TCGA data set was analyzed by the web site www. (30%) of the liver was done on 4-month-aged males as xenabrowser.net (Developed by UCSC). described . For digoxin treatment, digoxin (1 mg/Kg, Acknowledgements Sigma Aldrich) was IP injected into DEN-treated mice We are grateful to Chaim Preiser, Ben Cohen, and Daniel Steinberg for starting at age of 6.5 months, 3-times/week, for a total of technical help with mice work. The Aqeilan laboratory is supported by Worldwide Cancer Research grant (grant agreement No. 14-1095) and 8-months. The mice in HFD experiment were fed by 60% European Research Council (ERC)-Consolidator Grant under the European kcal fat (Research Diets INC, D12492) for 30 weeks. Union’s Horizon 2020 research and innovation program (grant agreement No. Tumor load was assessed by liver weight in grams. All 682118). experiments involving mice were approved by the Hebrew Author details University Institutional Animal Care and Use Committee. The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical RNA extraction and Real-time PCR School, Jerusalem, Israel. Department of Surgery, Hebrew University-Hadassah Medical, Jerusalem, Israel. Department of Cancer Biology and Genetics, Total RNA was prepared using TRI reagent (Sigma Wexner Medical Center, The Ohio State University, Columbus, OH, USA Aldrich) following instructions of the manufacturer. One microgram of RNA was used for complementary DNA Conflict of interest The authors declare that they have no conflict of interest. synthesis using First-Strand cDNA Synthesis kit (Bio-Rad, Hercules, CA). QRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster Publisher's note City, CA). All measurements were performed in triplicate Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. and standardized to the levels of the Ubc gene. List of primers used is provided as Supplemental Table 1. Supplementary Information accompanies this paper at https://doi.org/ 10.1038/s41419-018-0510-4. Histology and immunohistochemistry Received: 2 January 2018 Revised: 13 February 2018 Accepted: 15 February Tissues were fixed in 4% neutral buffered-formalin and then paraffin embedded, sectioned, and stained with H&E. Immunohistochemical staining was done as previously described . Immunohistochemical staining of WWOX (polyclonal anti-WWOX antibody, dilution, 1:4000 for 1 References h) was done after antigen retrieval with 10 mM citrate 1. El-Serag, H. B. Hepatocellular carcinoma. N. Engl. J. Med. 365,1118–1127 buffer (pH 6.0) in a pressure cooker. 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WWOX controls hepatic HIF1α to suppress hepatocyte proliferation and neoplasia

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Abstract

Liver cancer is one of the most lethal malignancies with very poor prognosis once diagnosed. The most common form of liver cancer is hepatocellular carcinoma (HCC). The WW domain-containing oxidoreductase (WWOX) is a large gene that is often perturbed in a wide variety of tumors, including HCC. WWOX has been shown to act as a tumor suppressor modulating cellular metabolism via regulating hypoxia-inducible factor 1α (HIF-1α) levels and function. Given that WWOX is commonly inactivated in HCC, we set to determine whether specific targeted deletion of murine ΔHep Wwox affects liver biology and HCC development. WWOX liver-specific knockout mice (Wwox ) showed more potent liver regeneration potential and enhanced proliferation as compared with their control littermates. Moreover, WWOX deficiency in hepatocytes combined with diethylnitrosamine treatment increased the tumor burden, which was associated with increased HIF1α levels and target gene transactivation. Inhibition of HIF1α by systemic treatment with digoxin significantly delayed HCC formation. Our work suggests that WWOX inactivation has a central role in promoting HCC through rewiring of cellular metabolism and modulating proliferation. Introduction reported at this region in aflatoxin B1 exposed HCC , Hepatocellular carcinoma (HCC) is the most common suggesting that it might harbor a tumor suppressor. In particular, WWOX expression is absent or reduced in type of primary liver cancer, representing the fifth type of commonly diagnosed cancer worldwide and third mor- most of the derived liver cancer cell lines . The gene tality cause among other cancer malignances . HCC encodes a 46 kDa protein comprising of two N-terminal prevalence has been dramatically increasing in the last WW domains, known to mediate protein–protein inter- decay because of the expansion of HCC risk factors, actions and a short-chain dehydrogenase/reductase 2 8–10 including hepatitis infection and obesity . Therapeutic domain whose specific function is unknown yet . options are limited and survival after diagnosis is still poor Moreover, WWOX was suggested as a modulator of β- 11,12 leading to high mortality. Therefore, better understanding catenin protein activity in some HCC cells lines .In of the molecular basis of HCC is urgently needed. addition to its genomic re-arrangement and hyper- WW domain-containing oxidoreducatase (WWOX) methylation of its regulatory region, WWOX is inacti- gene resides in one of the most common fragile sites vated by other proteins or microRNAs in HCC cell 13–15 known as FRA16D, a region that is altered in many types lines . However, no direct in vivo evidence linking 3–5 of cancer . Frequent homozygous deletions were WWOX tumor suppressor function with HCC develop- ment is known so far. WWOX is commonly reported as a tumor suppressor Correspondence: Rami I. Aqeilan (ramiaq@mail.huji.ac.il) not only owing to its common loss in many human The Lautenberg Center for General and Tumor Immunology, Department of malignancies but also due to its anti-tumorigenic effect Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel when overexpressed and susceptibility of tumor formation 8,16–19 Department of Surgery, Hebrew University-Hadassah Medical, Jerusalem, in Wwox-mutant mice . Wwox null mice die by the Israel age of 3–4 weeks owing to severe metabolic disorders, Full list of author information is available at the end of the article Edited by G. Melino © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 2 of 12 Fig. 1 Loss of WWOX expression in liver cancer. a TCGA data analysis showing that WWOX genomic region spanning long arm of chromosome 16 at 16q23.1 (red arrow) is frequently lost in HCC patients (n = 438 cases) with single copy number alteration (SCNA) equal to 96.84%. b Prognostic value of WWOX mRNA in HCC patients (n = 438) showing that patients with low expression had poorer survival compared with those with high WWOX expression. P value < 0.05. c Representative images of a liver cancer tissue microarray (TMA) (LV8011, US Biomax) showing immunohistochemical staining of WWOX in normal and HCC samples. d Quantification of WWOX protein levels from c. * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM mainly lethal hypoglycemia precluding studying impli- interacts with wide variety of proteins regulating their 9,35 cations of WWOX loss in adult mice. To overcome this functions and affecting cellular outcome . Previously, limitation, we have recently generated a conditional WWOX have been shown to physically interact with mouse model in which somatic deletion of Wwox is hypoxia-inducible factor 1α (HIF-1α) and inhibit its 21 33 achieved using a specific Cre recombinase , mimicking activity . Ablation of HIF1α expression in RAS- the alterations frequently observed in human cancers and transformed Wwox-deficient mouse embryonic fibro- allowing study of human cancer intervention, develop- blasts significantly reduced tumourigenicity , suggesting ment and progression, and assessing therapeutic strate- that HIF1α mediates the tumorigenic phenotype of 22,23 gies . In this study, we utilized a Cre recombinase Wwox-depelted cells. Nevertheless, the functional asso- driven by the promoter of albumin (Alb-Cre), which ciation of WWOX-HIF1α has not been demonstrated in a results in somatic deletion of WWOX in hepatocytes; cancer mouse model. Here, we studied the effect of ΔHep ΔHep Wwox mice. Interestingly, Iatan et al. have recently treating Wwox mice with DEN and followed HCC demonstrated that WWOX deletion in a murine mouse development and progression. We demonstrated that model modulates levels of lipoproteins, however, these WWOX dysregulation accelerates HCC development mice did not spontaneously develop HCC . One of the through control of HIF1α and other master proliferation most widely used and accepted models for HCC devel- gene networks implicated in hepatocarcinogenesis. We opment in animal models is the use of N- also demonstrate that WWOX ablation modulates liver nitrosodiethylamine (DEN), a known carcinogen, which regeneration. alkylates DNA bases and results in HCC tumor forma- tion in a defined kinetic manner . Results WWOX anti-tumorigenic functions have been shown WWOX expression is commonly lost in liver cancer 26–28 9,29–31 to affect genome integrity , apoptosis , cell Given that WWOX expression is altered in many 32 36 growth and extracellular matrix signaling , and glucose human malignancies , we set to examine its genomic and 33,34 metabolism . WWOX, through its first WW domain, expression status using different available tools and Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 3 of 12 resources. Analysis of 438 liver cancer samples in the Interestingly, WWOX mRNA and protein was decreased in TCGA database using fire browser (www.firebrowse.org) the tumors of control mice as revealed by qRT-PCR (Fig- revealed that the WWOX locus, spanning chromosomal ure S1A) and immunohistochemistry (Figure S1B). In ΔHep region of 16q23.1, is one of the most significant regions addition, Wwox mice had higher levels of serum alanine harboring copy number loss in HCC patients (Fig. 1a). transaminase, indicating liver dysfunction as a result of Using Xena browser (www.xenabrowser.net) we next tumor formation (Fig. 2f). Histological characterization of evaluated the prognostic value of WWOX expression in liver tissues revealed aggressive, poorly differentiated and HCC and found that cases harboring reduced WWOX highly proliferative tumors resembling HCC (Fig. 2g, h). mRNA levels tend to present with worse survival outcome Altogether, these findings indicate that WWOX loss compared to those having high WWOX expression accelerates HCC development. (Fig. 1b). Consistent with these observations we further found that WWOX levels are absent or reduced in dif- Hepatocyte-specific WWOX ablation is associated with ferent liver pathologies, particularly in HCC, as assessed increased proliferation by immunohistochemical staining of a commercial tissue Our findings so far suggest that hepatocyte-specific microarray (Fig. 1c, d). Notably, a tendency of reduced WWOX deletion promoted HCC development. On one WWOX expression was observed in cancer adjacent liver hand, WWOX overexpression was previously shown to tissue (P< 0.05), suggesting that loss of WWOX could be induce apoptosis and suppress proliferation, whereas its an early event in liver carcinogenesis. loss is associated with enhanced survival in a hepatoma cell line . On the other hand, DEN treatment is known to Hepatocyte-specific WWOX ablation accelerates HCC induce acute hepatic injury followed by compensatory development proliferation . We therefore set to determine whether the ΔHep The fact that WWOX expression is reduced in early liver phenotype observed in DEN-treated Wwox mice is a cancer lesions suggests that WWOX may have a role in consequence of impaired proliferation control. To this suppressing liver carcinogenesis. To determine whether end, we analyzed liver tissues of DEN-treated mice at WWOX loss could contribute to HCC development or different time points starting from 1 to 10 months. progression, we generated a hepatocyte-specific Wwox Hematoxylin and Eosin (H&E) evaluation revealed that ΔHep knockout mouse model and followed liver tumor formation. Wwox livers display hepatocyte morphological chan- Wwox-floxed mice (Wwoxf/f) were bred with Albumin- ges starting from 6-month post DEN treatment, which progressed to HCC at the age of 10 months, as shown Cre transgenic mice to generate Wwoxf/f;Albumin-Cre ΔHep (Wwox ) mice on the C57Bl6/J;SVJ129 mixed genetic earlier (Fig. 3a, d). No histological abnormalities were background. Successful ablation of WWOX was validated observed in the groups of 1 and 3-month post DEN ΔHep using quantitative real-time (qRT)-PCR and western blot treatment of Wwox mice nor in the control mice. To ΔHep analyses (Fig. 2a, b). Follow up of Wwox and controlled further support our findings, we immunostained for Ki67, littermate mice for up to 2-years did not reveal spontaneous a surrogate marker of proliferation. Digital quantification ΔHep tumor development, consistent with previous observa- of Ki67-positive nuclei demonstrated that Wwox liver tions . We therefore decided to examine the effect of sections had significantly higher number of Ki67-positive ΔHep WWOX ablation upon N-nitrosodiethylamine (DEN) cells in the groups of 1, 3, 6, and 10 months Wwox treatment, a widely used chemical carcinogen for studying mice relative to the control littermate groups (Fig. 3e). In 22 ΔHep liver carcinogenesis . Cohorts of Wwox mice and addition, qRT-PCR analysis of proliferative genes impli- control littermates were intraperitoneally (IP) injected with cated in liver cancer, including c-Myc, c-Jun, c-Fos, and ΔHep 5 mg/Kg single injection of DEN at the age of 14 days and Axin, display higher levels in the Wwox mouse groups monitored for HCC development as a function of time from as early as 1-month post DEN treatment (Fig. 3f). ΔHep (Fig. 2c). DEN treatment using this protocol is known to Although, DEN-free Wwox mice display no tumori- lead to 85% HCC development in C57BL6/J strain by the genic phenotype, levels of the previous mentioned pro- age ranging from 10 to 18 months , whereas 129SVJ strain liferative genes were also elevated (Figure S2A). develop HCC later .As shown in Fig. 2d, DEN-treated Consistent with these results, a significant increase in ΔHep Wwox mice developed significantly higher incidence of CTGF levels, one of the main effectors of the Hippo 41 ΔHep HCC as compared with control mice (P= 0.0025). By the pathway , was also noted in livers of Wwox mice age of 10 months, the penetrance of tumor development with a slight increase in liver weight in the pre- ΔHep was 100% in Wwox mice whereas just 20% of the tumorigenic phase (Figure S2B and S2C). These findings control littermates’ mice developed macroscopic tumors suggest that WWOX has a critical role in inhibiting (Fig. 2d). Furthermore, tumor load, as assessed by liver proliferation of DEN-treated hepatocytes, whereas its weight relative to body weight, was significantly higher in inactivation leads to increased proliferation contributing ΔHep Wwox mice as compared with control mice (Fig. 2e). to tumor development. Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 4 of 12 ΔHep Fig. 2 Hepatocyte-specific WWOX ablation accelerates HCC development. a Validation of WWOX depletion in Wwox (cKO: conditional knockout) mice using qRT-PCR; n = 3 for each mouse group. Error bars indicate ± SEM. b Validation of WWOX protein ablation by immunoblotting ΔHep using WWOX antibody; HSP90 was used as loading control. c DEN treatment plan: DEN was IP injected to control and Wwox mice at the age of 2 ΔHep 14 days and mice were analyzed at the age of 10 months. d χ analysis of macroscopic tumor incidence in control mice versus Wwox mice ΔHep treated with DEN. e Tumor load analysis represented by liver weight of control mice (n= 15) versus Wwox cKO mice (n = 14) after 10 months of ΔHep DEN treatment. f Serum ALTL levels of control mice (n = 6) versus Wwox mice (n = 6) after 10 months of DEN injection. g Representative pictures ΔHep of control and Wwox livers of two male littermates (upper). Histological images showing liver sections of 10 months DEN-treated control and ΔHep Wwox mice (lower panel) h Histological images (× 10, × 20, and × 40) of 10 months DEN-treated mice. * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM Enhanced expression of glycolytic genes in livers of DEN- moderate and prominent increase at 6 and 10 months in ΔHep ΔHep Wwox mice DEN-treated Wwox mice, respectively (Fig. 4f). Like- Recently, it was shown that WWOX loss is associated wise, levels of PKM2 and membranous GLUT1 protein ΔHep with Warburg effect and impaired mitochondrial were higher in 6 and 10 months DEN-treated Wwox 33,42,43 respiration . In mammalian cells, WWOX ablation mice (Fig. 4e). is inversely correlated with increased levels and activity of Our recent studies have shown suppression of HIF1α hypoxia-inducible factor 1α (HIF1α-enhancing cell transactivation function by WWOX . Therefore, we next 33,44 transformation and tumor growth . In particular, determined whether WWOX loss modulates HIF1α Wwox-deficient cells were shown to display higher levels recruitment to promoters of these glycolytic genes. To of glycolytic genes known to be regulated by HIF1α. Given address this, we performed a chromatin immunoprecipi- that HIF1α has known roles in HCC development ,we tation (ChIP) experiment using anti-HIF1α antibody on next set to determine whether WWOX-targeted loss in genomic DNA isolated from liver tissues of 10 months ΔHep ΔHep the different DEN-treated Wwox and control mice is DEN-treated Wwox and control littermate mice. associated with impaired HIF1α function. Analysis of QRT-PCR analysis revealed HIF1α enrichment on pro- HIF1α glycolytic target genes mRNA, including Hk2, moters of target genes, including Pkm2, Hk2, Glut1, Aldh, ΔHep Pkm2, Glut1 and others, demonstrated no change at 1 and and Gapdh,in Wwox livers (Fig. 4f). Altogether, these 3-months post treatment (Fig. 4a, b). Intriguingly, a sig- results imply that WWOX loss leads to HIF1α enhanced nificant upregulation of these transcripts was noted in 6 activity and, likely, reprogramming of HCC cells to glu- ΔHep and 10 months DEN-treated Wwox mice compared cose metabolism and Warburg effect. with control mice (Fig. 4c, d). Interestingly, DEN-free ΔHep Wwox mice on 6 months age displayed a tendency of Inhibition of HIF1α rescued the effect of WWOX loss HIF1α glycolytic target genes upregulation (Figure S3A). We have previously shown that digoxin, an inhibitor Moreover, nuclear HIF1α protein levels exhibited of HIF1α, rescued the effect of Wwox-deficient mice Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 5 of 12 Fig. 3 Hepatocyte-specific WWOX ablation is associated with increased proliferation. Histological images (H&E staining and Ki67 ΔHep immunohistochemical staining at × 40) of DEN-treated control and Wwox liver sections at the age of 1 a,3 b,6 c, and 10 months d. e ΔHep Quantification of positive Ki67 nuclei in 1, 3, 6, and 10 months old DEN-treated control and Wwox mice liver. (n = 5 for each group). f mRNA ΔHep expression levels of proliferation genes in DEN-treated control and Wwox mice at the age of 1, 3, 6, and 10 months (n = 3 for each group). * indicates P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep displaying hypoglycemia and elevated levels of glycolytic High-fat diet (HFD) increased HCC incidence in Wwox genes . Digoxin is a cardiac glycoside, which has been mice shown to inhibit HIF1α transcriptional activity in vitro Obesity is known to be a major risk factor for HCC and suppress tumor growth in vivo . We therefore set to development. Indeed, combined HFD and DEN treatment ΔHep investigate whether digoxin treatment of DEN-Wwox have been previously shown to strongly enhance HCC mice could reduce the incidence of HCC development. development when compared with DEN mice on normal ΔHep 48 DEN-Wwox and control mice received digoxin (1 mg/ diet . Driven by our previous observations, we decided to kg) or vehicle control (saline) through intraperitoneal investigate the effect of HFD on HCC development in ΔHep injections (3-times/week) for 32 weeks starting at DEN-Wwox mice. DEN-treated cohorts of male ΔHep 6 months post DEN treatment (Fig. 5a). Analysis of mice Wwox and control mice were placed on HFD, in 8-months post digoxin treatment revealed marked sup- which 60% of calories were fat-derived, for 30 weeks. ΔHep pression of liver tumor growth with more prominent Tumors of DEN-treated Wwox HFD mice appeared ΔHep effect in Wwox mice compared with saline-treated 3-months earlier compared with normal chow-diet mice ΔHep Wwox mice (Fig. 5b, c). In contrast, no significant (Fig. 2), consistent with previous reported data showing difference was observed between digoxin and saline- acceleration of HCC formation in mice fed with HFD . treated DEN control mice (Fig. 5e). Consistent with these No difference of mice weight between the two groups of ΔHep ΔHep results, digoxin-treated DEN-Wwox mice showed HFD-Wwox and control mice was noted (Fig- downregulations of HIF1α targets, whereas no significant ure S4A). Nevertheless, the number of tumors in ΔHep changes were observed in digoxin-treated DEN control Wwox mice was significantly higher (Fig. 6a, b) and mice (Fig. 5d, f). We conclude that WWOX loss exerts was accompanied with higher levels of serum ALT (Fig- critical regulation on HIF1α function mediating rewiring ure S4B) and hepatosteatosis (Fig. 6c). Consistent with of glucose metabolism and contributing to enhanced anti-proliferative role of WWOX that we showed pre- ΔHep proliferation. viously (Fig. 3), DEN-treated Wwox mice fed with Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 6 of 12 ΔHep Fig. 4 Enhanced expression of glycolytic genes in livers of DEN-Wwox mice. a mRNA expression levels of HIF1α glycolytic target genes in ΔHep DEN-treated control and Wwox (cKO) mice at the age of 1 a,3 b,6 c and 10 d months (n = 3 for each group). e Immunohistochemical staining of ΔHep HIF1α, PKM2, and GLUT1 in 6 and 10 months of DEN-treated Wwox and control liver tissues. Images were taken at × 40 magnification (Bar = ΔHep 100μm). f ChIP experiment with HIF1α antibody on DEN-treated control and Wwox (cKO) mice followed by qRT-PCR analysis of HIF1α target genes. * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep HFD displayed increased proliferation (Fig. 6d, e). Per- control mice, it continued rising in Wwox mice. sistent with a prominent role of β-catenin pathway in Increased proliferation was associated with increased ratio HCC, we observed significant upregulation of Axin2 of liver to body weight on day 7 post hepatectomy (Fig. 7c). ΔHep transcript in DEN-treated Wwox HFD mice (Fig. 6f). Moreover, qRT-PCR analysis of proliferative gene impli- At a later stage, there was also an upregulation of HIF1α- cated in liver regeneration, including c-Myc, displayed ΔHep target genes (Figure S4C). Altogether, our findings suggest higher levels in Wwox mice group on day 2, 3, and 4 as that combined loss of WWOX and HFD intake accelerate compared with control mice (Fig. 7d). Interestingly, Wwox HCC formation mediated by enhanced proliferation. and c-Myc transcripts showed a trend of inverse pattern of expression in Wwox-control mice (Fig. 7e) suggesting that Wwox ablation is associated with increased proliferation WWOX might negatively modulate c-Myc. These results upon partial hepatectomy are consistent with WWOX function to inhibit hepatocyte The preceding observations demonstrated that WWOX proliferation after liver regeneration. inactivation coupled with DEN treatment and HFD intake leads to enhanced proliferation and tumor formation. We Discussion next asked whether WWOX deletion affects hepatic cell We report here that WWOX loss is a common event in proliferation upon partial hepatectomy, a process known to HCC and that its dysregulation synergizes with DEN acutely induce hepatocyte proliferation to achieve liver treatment and HFD to accelerate HCC development ΔHep regeneration. Wwox mice and their littermate controls through regulation of proliferation genes. At the mole- were subjected to 30% hepatectomy and were analyzed 1, 2, cular level, WWOX, known to act as an adapter protein 3, 4, and 7 days later for Ki67 immunohistochemical through interaction via its WW1 domain, functionally staining. As expected, an increase in the proliferation index associates with HIF1α and probably other transcription ΔHep was observed in Wwox mice compared with control factors involved in proliferation, leading to inhibition of mice (Fig. 7a, b). Intriguingly, whereas the proliferation their transactivation function. For example, when index was reduced 4 and 7 days post hepatectomy in WWOX is lost, HIF1α recruitment and function on its Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 7 of 12 Saline Digoxin ΔHep Fig. 5 Inhibition of HIF1α rescued the effect of WWOX loss. a Digoxin experiment plan, DEN was IP injected to control and Wwox mice at the age of 14 days. Six-month later mice were started to be treated with digoxin (1 mg/kg) or vehicle control (saline) 3-times a week for an additional 8- ΔHep ΔHep months. b Representative images of saline-treated Wwox (cKO) mice versus digoxin-treated Wwox (cKO) mice livers and liver sections c ΔHep ΔHep Tumor load of saline-treated Wwox mice versus digoxin-treated Wwox mice livers as assessed by liver weight at the age of 14 months d ΔHep ΔHep mRNA expression levels of HIF1α glycolytic target genes in saline-treated Wwox (cKO) mice versus digoxin-treated Wwox (cKO) mice at the age of 14 months, (n = 3 for each group). e Χ analysis of macroscopic tumor incidence in saline-treated control mice versus digoxin-treated control mice at the age of 14 months. f mRNA expression levels of HIF1α glycolytic target genes in saline-treated control mice versus digoxin-treated control mice at the age of 14 months, (n = 3 for each group). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM target genes are enhanced leading to increased expression Several studies have suggested that the WWOX gene 3,4,7 of glycolytic genes and proliferation rate. We further functions as a tumor suppressor in liver cancer .Early demonstrate that pharmacological inhibition of HIF1α evidence has revealed the existence of frequent homozygous reduces HCC burden in DEN-treated liver-specific deletions at chromosome 16q23, where WWOX resides, in WWOX-deficient mice. Finally, we show that WWOX aflatoxin B1 exposed HCC . In another study, expression of loss increases liver regeneration after hepatectomy pos- both WWOX and FHIT, another tumor suppressor located sibly via eliminating the termination phase of liver in a fragile site, appeared to be correlated and down- regeneration. These findings underscore the significant regulated in liver tissues in a carcinogen-specificmanner . role of WWOX in rewiring metabolic changes in liver A subsequent study has examined the status of the WWOX cells contributing to liver cancer. gene in human HCC cell lines and found its recurrent Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 8 of 12 ΔHep 2 Fig. 6 High-fat diet (HFD) increases HCC incidence in DEN-treated Wwox mice. a Χ analysis of HCC incidence in DEN-treated control and ΔHep ΔHep Wwox mice (at age of 14 days) and fed with HFD from the age of 8 weeks. b Representative pictures of control and Wwox (cKO) livers fed with HFD at the age of 7 months. Arrows indicate macroscopic tumors. c Histological images (× 4, × 10, × 2,0 and ×40) of 7 months DEN-treated ΔHep control and Wwox livers fed with HFD. d Histological images (Ki67 immunohistochemical staining) of mice from c. e Quantification of positive Ki67 nuclei of mice from d. f mRNA expression of Axin2 (control, n = 3; cKO, n = 3). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM alterations further implicating WWOX in hepatocarcino- We also showed that WWOX anti-proliferative activity genesis . In our study, we further delineate the role of is mediated by suppression of the proto-oncogenes c-Myc, WWOX as a tumor suppressor in liver cancer. First, we c-Fos, and c-Jun.c-Myc is known as an oncogene that 51 52,53 showed that WWOX expression is reduced or absent in promotes HCC in human and murine HCC models . ΔHep large cohorts of human liver pathologies, including HCC. We found that c-Myc is upregulated in Wwox mice Furthermore, WWOX low expression in HCC is correlated on 1, 3, 6, and 10 months continually, raising the point with decreased survival suggesting that WWOX expression that WWOX partially suppresses HCC through con- has a prognostic value in HCC. Second, we provide the first tinuous suppression of c-Myc. Previous reports have in vivo evidence that WWOX loss contributes to HCC shown that WWOX suppresses the AP-1 transcriptional development, in two different models of murine HCC. Our activity through regulating c-Jun localization , consistent data show that DEN treatment alone or in combination with our findings showing enhanced expression of c-Jun with HFD clearly have an advantage for HCC development and c-Fos in WWOX-depleted cells. It is also likely that upon WWOX loss. Both tumor incidence and multiplicity WWOX loss releases its inhibitory effect on a plethora of ΔHep (load) were higher in Wwox mice implying the impor- other proto-oncogenes as have been previously demon- 9,16,55 tance of WWOX alteration for HCC development and strated , contributing to the observed aggressive HCC progression. Our findings also suggest that WWOX loss is phenotype. Although WWOX has been shown as an an important contributor for HCC promotion as earlier hits effector of the Wnt/β-catenin pathway in vitro, through 15,35,56 are required for HCC initiation. WWOX expression could interaction with DVL , our results do not support 6,49 be altered by environmental cues or in consequence of such an effect in DEN-mediated HCC development. On microRNA dysregulation or even as a result of genetic the other hand, when DEN treatment was combined with variations in WWOX . If these alterations were combined HFD, WWOX loss resulted in significant increase in Axin with western HFD then our findings indicate that HCC risk levels, suggesting that under these conditions WWOX increases. plays a more important role in regulating the Wnt/β- Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 9 of 12 Control cKO Control cKO Control cKO cMYC WWOX Fig. 7 Wwox ablation is associated with increased proliferation upon partial hepatectomy. a Histological images (H&E staining and Ki67 ΔHep immunohistochemical staining) of control and Wwox mice liver 3-days post 50% partial hepatectomy (× 10, × 20, and × 40). b Quantification of ΔHep positive Ki67 nuclei in 5 months old control (WT) and Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy (n = 5 for each group). c Liver weight to body weight ratio of 1, 2, 3, 4, and 7 days post hepatectomy (n = 5 for each group). d mRNA expression levels of c-Myc in control and ΔHep ΔHep Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy. e mRNA expression levels of Wwox in control and Wwox mice liver 1, 2, 3, 4, and 7 days post hepatectomy (n = 4 for each group). * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM ΔHep catenin pathway. It is therefore possible that other cellular the age of 14 months, DEN-Wwox mice did not show pathways are also involved as WWOX function has been macroscopic tumors and had lower expression of glyco- shown to include plethora of effectors lytic genes driven by HIF1α. These results are consistent Our data further present a critical metabolic and anti- with our previous data showing that WWOX inhibits 42–44,65,66 proliferative role of the tumor suppressor WWOX in sup- aerobic glycolysis . Rewiring of glucose metabo- ΔHep pressing HCC through regulation of HIF1α function. The fact lism in Wwox mice provides the needed building that HIF1α protein levels and its target genes were elevated in blocks for cell division and proliferation and hence these the pre-tumor stage (6 months; Fig. 4e) implies that these mice could have more proliferation compared with con- metabolic changes fuel and drive HCC development in DEN- trol mice. Future studies shall include more specific ΔHep treated Wwox mice. HIF1α is significantly elevated in inhibitors of HIF1a and probably Myc to inhibit WWOX- 58,59 human HCC samples and associated with bad prognosis . mediated HCC development. Moreover, HIF1α hepatocytes-specific overexpression in a WWOX anti-proliferative function in HCC might also murine model increases HCC-promoting M2 macrophages , involve modulation of fatty acid/lipid metabolism. In fact, whereas HIF1α liver-specific knockout sensitizes hepatoma WWOX genetic variants in human patients and Wwox 61 24 cells to etoposide treatment . WWOX loss is associated with knockout mouse models display decreased serum HDL- elevated HIF1α target genes starting from the age of 6 months. C. Moreover, microarray analyses of Wwox liver-specific In addition, we show that HIF1α is enriched on its targets knockout mice revealed an increase in plasma triglycer- ΔHep promoters in hepatocytes isolated from Wwox mice. ides and altered lipid metabolic pathways suggesting that Targeting tumor suppressor genes is a major challenge. WWOX disruption indeed alters cellular lipid home- Novel approaches, including synthetic lethality and col- ostasis in the liver. Whether these WWOX effects can lateral vulnerability have been proposed to come over this also contribute to HCC development is unknown. limitation. We therefore, assessed whether targeting the Our findings also present a vital role of WWOX in proliferative/survival signals in WWOX-deficient cancer maintaining healthy liver regeneration. Remarkably, partial cells could help inhibit tumor progression. Several hepatectomy in liver-specific Wwox-deficient mice resulted attempts to target HIF1α in HCC were reported via in increased proliferation (Ki67) upon liver regeneration, at 62 63 64 inhibition of its expression , dimerization , or activity . the later phases of this remarkably controlled homeostatic In our model, we were able to rescue, at least partially, the process. Interestingly, Wwox RNA levels increased 2 days tumor phenotype using an HIF1α inhibitor (digoxin); at post hepatectomy while c-Myc levels showed an inverse Official journal of the Cell Death Differentiation Association Abu-Remaileh et al. Cell Death and Disease (2018) 9:511 Page 10 of 12 pattern. c-Myc levels are reported to be highest in 12–18 h Thermoscientific, dilution 1:200), PKM2 antibody (Cat# post heptectomy and then c-Myc levels usually drop 38237, Abcam, dilution 1:50) and HIF1α antibody (Cat# 69–71 down . Our results might suggest that WWOX,ananti- NB100-105, Novus, dilution 1:20). proliferative gene, is induced during the course of liver regeneration to allow shutting down proliferative genes, ChIP such as c-Myc, and maintaining proper organ size. Inter- Hepatocytes were isolated from liver tissues, and solu- estingly,nodifferenceinHIF1α target genes was observed tions were prepared for ChIP analysis according to a ΔHep 74 in liver regeneration of Wwox .These observations standard protocol . 0.8 mg of HIF1α antibody (mouse suggest that WWOX may regulate liver regeneration by mAB, Cat # NB100-105, Novous Biological, CO, USA) HIF1α-independent mechanism. was used to precipitate HIF1α. Targeted PCR was done In conclusion, our study reveals WWOX as a tumor using list of primers (Supplemental Table 2). suppressor with critical roles in HCC suppression through maintaining moderate glucose metabolism and inhibiting Statistics uncontrolled cell proliferation. Results of the experiments were expressed as mean ± standard deviation or standard error of mean. Student’s t- Materials and Methods test, was used to compare values of test and control Mice and related experiments samples. P < 0.05 indicates significant difference. * P- Wwox-floxed (Wwoxfl/fl) C57BL6/J;129sv mixed value < 0.05, ** P-value < 0.01. For the human data ana- genetic background mice were bred with Albumin-Cre lysis, Single Copy Number Alteration of liver hepatocel- transgenic mice to generate Wwox conditional knockout lular carcinoma (LiHC) TCGA data set (n = 434) was ΔHep in hepatocytes (Wwox mice). Male pups of control analyzed by the website www.firebrowse.org (developed ΔHep and Wwox mice were IP injected with 5 mg/Kg DEN by broad institute of MIT and Harvard). Survival curve of (Sigma Aldrich) at the age of 14 days. Partial hepatectomy LiHC TCGA data set was analyzed by the web site www. (30%) of the liver was done on 4-month-aged males as xenabrowser.net (Developed by UCSC). described . For digoxin treatment, digoxin (1 mg/Kg, Acknowledgements Sigma Aldrich) was IP injected into DEN-treated mice We are grateful to Chaim Preiser, Ben Cohen, and Daniel Steinberg for starting at age of 6.5 months, 3-times/week, for a total of technical help with mice work. The Aqeilan laboratory is supported by Worldwide Cancer Research grant (grant agreement No. 14-1095) and 8-months. The mice in HFD experiment were fed by 60% European Research Council (ERC)-Consolidator Grant under the European kcal fat (Research Diets INC, D12492) for 30 weeks. Union’s Horizon 2020 research and innovation program (grant agreement No. Tumor load was assessed by liver weight in grams. All 682118). experiments involving mice were approved by the Hebrew Author details University Institutional Animal Care and Use Committee. The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical RNA extraction and Real-time PCR School, Jerusalem, Israel. Department of Surgery, Hebrew University-Hadassah Medical, Jerusalem, Israel. Department of Cancer Biology and Genetics, Total RNA was prepared using TRI reagent (Sigma Wexner Medical Center, The Ohio State University, Columbus, OH, USA Aldrich) following instructions of the manufacturer. One microgram of RNA was used for complementary DNA Conflict of interest The authors declare that they have no conflict of interest. synthesis using First-Strand cDNA Synthesis kit (Bio-Rad, Hercules, CA). QRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster Publisher's note City, CA). All measurements were performed in triplicate Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. and standardized to the levels of the Ubc gene. List of primers used is provided as Supplemental Table 1. Supplementary Information accompanies this paper at https://doi.org/ 10.1038/s41419-018-0510-4. Histology and immunohistochemistry Received: 2 January 2018 Revised: 13 February 2018 Accepted: 15 February Tissues were fixed in 4% neutral buffered-formalin and then paraffin embedded, sectioned, and stained with H&E. Immunohistochemical staining was done as previously described . Immunohistochemical staining of WWOX (polyclonal anti-WWOX antibody, dilution, 1:4000 for 1 References h) was done after antigen retrieval with 10 mM citrate 1. El-Serag, H. B. Hepatocellular carcinoma. N. Engl. J. Med. 365,1118–1127 buffer (pH 6.0) in a pressure cooker. 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Cell Death & DiseaseSpringer Journals

Published: May 3, 2018

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