Huwe1 Sustains Normal Ovarian Epithelial Cell Transformation And

Published OnlineFirst July 7, 2017; DOI: 10.1158/0008-5472.CAN-16-2597

Cancer Molecular and Cellular Pathobiology Research

Huwe1 Sustains Normal Ovarian Epithelial Cell Transformation and Tumor Growth through the Histone H1.3-H19 Cascade Dong Yang1,2, Bin Sun2,3, Xiaohong Zhang4, Daomei Cheng5, Xiaoping Yu5, Lanzhen Yan2,6, Lei Li2,7, Sanqi An2,3, Hua Jiang4, Anna Lasorella8, Antonio Iavarone8, Shu Zhang9, Fangdong Zou1, and Xudong Zhao2,6

Abstract

Ubiquitination-directed protein degradation is important expression of histone H1.3 increased, inhibiting the expression in many cancers for tumor initiation and maintenance, and of noncoding RNA H19. H19 silencing phenocopied the E3 ligases containing HECT domains are emerging as new effects of Huwe1 deficiency, whereas H1.3 silencing partially therapeutic targets. In contrast to many other E3 ligases, the rescued the expression of H19 and the Huwe1-null phenotype. role of HUWE1 in ovarian cancer where HUWE1 is dysregulated Inducible silencing of HUWE1 in human ovarian cancer has been unclear. Here we report that genetic deletion of cells produced a similar phenotype. Mechanistically, HUWE1 Huwe1 in the mouse inhibits transformation of ovary surface bound and ubiquitinated H1.3, which was consequently epithelium cells without significantly affecting cell survival marked for destruction by proteasomes. Our results establish and apoptosis, and that Huwe1 deletion after tumors have that HUWE1 plays an essential role in promoting ovarian been initiated inhibits tumor growth. In Huwe1-deficient cells, cancer. Cancer Res; 77(18); 4773–84. 2017 AACR.

Introduction groups have shown that HUWE1 targets P53 and MCL-1 for degradation and that this process promotes cell apoptosis or Ubiquitination and its control of subsequent protein degrada- survival, respectively, in U2OS cells (2, 3). Furthermore, many tion are essential biological processes in many cells, including of its substrates have been shown to play positive roles in cancer. The specificity of these processes is regulated by the E3 tumorigenesis. These include a variety of tumor-promoting and ligase. The HECT family (homologous to E6-AP carboxyl termi- tumor-suppressing substrates, such as MYC (4), histones (5), nus-type E3s) is an important class of E3 ligases, and many of its CDC6 (6), N-MYC (7), POLB (8), TopBP1 (9), PA2G4 (isoform members are involved in cancer-associated processes, including 2) (10), Miz-1 (11), HDAC2 (12), Pol l (13), MyoD (14), BRCA1 apoptosis and growth arrest (1). HUWE1 is a large HECT E3 ligase (15), TIAM1 (16), and H2AX (17). with a molecular size of 480 kDa, and its role in cancer has been In humans, it has been reported that elevated HUWE1 expres- the subject of debate since it was first cloned. Different research sion is associated with a variety of cancers, such as breast, lung, colon, liver, and pancreatic cancers (4, 18, 19), and high HUWE1 expression is associated with a worse prognosis (19). However, 1Key Laboratory of Bio-resources and Eco-environment, College of Life Sciences, HUWE1 mutations and deletions have also been reported in Sichuan University, Chengdu, Sichuan, P.R. China. 2Key Laboratory of Animal many cancers. These include a deletion in glioblastoma (20) Models and Human Disease Mechanisms of Chinese Academy of Sciences and and mutations in various types of cancer, such as those catalog- Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, 3 ed in The Cancer Genome Atlas (TCGA) data, which include skin Kunming, Yunnan, China. Kunming College of Life Science, University of – Chinese Academy of Sciences, Kunming, Yunnan, China. 4Department of Hema- cutaneous melanoma, squamous non small cell lung cancer, tology/Oncology, Guangzhou Women and Children's Medical Center, Guangz- and uterine cancer. HUWE1 expression has also been shown to hou, Guangdong, China. 5Chengdu Medical College, Chengdu, Sichuan, China. be lower in teratoma cells than in embryonic stem cells that 6Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, possess very similar biological characteristics (21). 7 Yunnan 650223, China. School of Life Sciences, University of Science and HUWE1 inactivation results in the inhibition of cell prolifer- Technology of China, Hefei, Anhui, China. 8Institute for Cancer Genetics, Colum- ation in some human cancer cell lines, but it has no such effects in bia University, New York, New York. 9Renji Hospital, Shanghai Jiaotong Uni- versity, Shanghai, China. other cell lines, including the colon adenocarcinoma cell line SW480 (4). HUWE1 inactivation also arrested proliferation in Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). another colon carcinoma cell line (Ls174T) and inhibited tumor growth in a xenograft model (22). However, Huwe1 knockout in fi D. Yang, B. Sun, and X. Zhang are the co- rst authors of this article. mice accelerated colon tumorigenesis initiated by loss of tumor Correspondence Author: Xudong Zhao, Kunming Institute of Zoology, CAS, 21 suppressor gene Apc (23). In a 7,12-dimethylbenz[a]anthracene/ qingsong Road, Kunming, Yunnan 650223, China, Phone: 86-871-68125430; phorbol myristate acetate (DMBA/PMA)-induced mouse model E-mail: [email protected]; and Fangdong Zou, [email protected] of skin cancer, deleting the Huwe1 gene accelerated tumor devel- doi: 10.1158/0008-5472.CAN-16-2597 opment by inducing the accumulation of the Myc/Miz complex 2017 American Association for Cancer Research. (24). These contradictory data indicate that HUWE1 may play

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different roles in different cell types and in response to different H1.3 shRNA plasmids, the target sequences were cloned into a genetic alterations. Tet-pLKO-puro vector (Addgene) according to the manufac- Ovarian cancer is the fifth leading cause of cancer-related turer's protocol (http://www.addgene.org/21915/). The follow- deathinwomenandthefirst leading cause of cancer-related ing target sequences were used: H1.3,50-CACTTTGTTATGGGTA- death in the female genital organs (25, 26). The mechanisms CATTT-30; H19,50-GTCCTGGGTCCATCAATAAAT-30; and HUW- underlying ovarian cancer remained to be determined (27, 28). E1,50-CGACGAGAACTAGCACAGAAT-30. In one study, HUWE1 expression was increased in approximately 70% of human ovarian cancer samples (29). However, Lentiviral preparation and infection the roles played by HUWE1 in ovarian cancer remain to be Lentiviral DNA vectors were cotransfected into HEK293T clarified. Here, we report that Huwe1 deletion impaired trans- cells with the packaging plasmids pCMVD8.9 and pMD2.G at formation in mouse ovary surface epithelium (MOSE) cells and a ratio of 10:5:2. The culture medium was collected and cen- inhibited tumor formation and growth. These effects were trifuged at 2,500 rpm for 10 minutes, and the supernatant partially dependent on the Huwe1-H1.3-H19 cascade. was filtered. The supernatant containing the lentiviral particle was centrifuged at 25,000 rpm at 4C for 2.5 hours. The lentivirus pellet was resuspended in PBS containing 0.1% BSA Materials and Methods and stored at –80 C in aliquots. The copy number of lentiviral Experimental animals particles was confirmed using quantitative RT-PCR with U5 L/L Conditional Huwe1 knockout mice were obtained from A. primers (U5-F, 50-AGCTTGCCTTGAGTGCTTCA-30 and U5-R, Lasorella (The Institute for Cancer Genetics, Columbia University 50-TGACTAAAAGGGTCTGAGGG-30;ref.33).MOSEcellswere Medical Center, New York, NY; ref. 20). Six-week-old BALB/c nude seeded at 5 105 in 6-cm plates and infected at a multiplicity of mice were purchased from Vital River Laboratory Animal Tech- infection (MOI) of 10. nology Co., Ltd. The animal protocol was approved by the animal ethics committee of the Kunming Institute of Zoology, Chinese RNA isolation and real-time PCR Academy of Sciences. Total RNA was isolated from cell lines or tumor tissues using a PureLink RNA Mini Kit (Life Technologies) according to the Cell culture manufacturer's instructions. Undesired DNA was removed using L/L MOSE cells were isolated from Huwe1 mice following a a TURBO DNA-free Kit (Invitrogen). Total RNA (2 mg) was reverse previously described procedure (30). Ovaries were dissected from transcribed using random primers with a RevertAid First Strand 6- to 8-week-old virgin mice and immediately transferred into cDNA Synthesis Kit (Thermo Scientific). Quantitative PCR was digestion buffer [DMEM/F12 (Life Technologies) containing 4 performed in triplicate using the SYBR Green method (Life mg/mL collagenase-Dispase (Roche), 30 mg/mL bovine albumin Technologies). Relative expression levels were normalized to the (Millipore), and 1 mL/mL DNase I (Sigma)]. The mixture was then level of 18S rRNA. All of the primers used in this study are shown incubated at 37 Cin5%CO2 for 1 hour. The cells were collected in Supplementary Table S1. and cultured in MOSE complete medium on gelatinized plates for 24 hours to eliminate residual non-MOSE cells. SKOV-3 and RNA-seq analysis HEK293T cells were cultured in DMEM containing 10% FBS FastQC (v0.11.2) was used to confirm high-quality reads, (Millipore), 100 U/mL penicillin, and 100 mg/mL streptomycin which were those with Q scores higher than 20 (99.99% accura- (Life Technologies). All of the cells were cultured in a humidified cy). Clean reads were mapped to the mouse genome (version incubator with 5% CO2 at 37 C and confirmed to be mycoplas- GRCm38.p4, which includes 21,936 protein-coding and 3,495 ma-free by PCR (31). The SKOV-3 cell line was purchased from lincRNA genes) using Tophat-2.1.0. Gene expression levels were China Infrastructure of Cell Line Resources in September 2016 calculated as the expected fragments per kilobase of transcript per and validated by the provider using short tandem repeat (STR) million fragments (FPKM) using express Cufflinks2.2.1. Genes profiling. All experiments using this cell line were completed were considered significantly up- or downregulated at a false within 3 months after purchase. discovery rate (FDR)-adjusted P 0.05 and an absolute value of fold change 2 using Cuffdiff (34). Hierarchical clustering of Plasmid construction samples was performed in the R Package (v3.2.2) gplots (v2.17.0; The lentiviral vectors pTomo-pCMV-KRASG12D-2A-MYC-IRES- https://www.R-project.org/; ref. 35). The raw RNA-seq data has CreER-shp53 and pTomo-pCMV-KRASG12D-2A-MYC-IRES-Cre- been deposited into the SRA database on the NCBI website shp53, which were used for MOSE malignant transformation, (accession no. SRP081212). were constructed in a pTomo-Flag-HRAS-H1siMp53 vector back- bone that was obtained from Dr. Inder M. Verma (The Salk Soft agar colony formation assay Institute for Biological Studies, La Jolla, CA; ref. 32). The genetic Complete medium containing 0.6% agarose (Sigma) was mixed elements KRASG12D-2A-MYC-IRES-CreER and KRASG12D-2A- using a pipette and then aliquoted at 1 mL per well in 6-well plates. MYC-IRES-Cre were inserted into the plasmids between the XbaI The plates were then cooled for approximately 5 minutes at 4C and SalI restriction sites to replace Flag-Hras. To construct the to solidify the agarose (base agar). A total of 2 104 cells were H1.3-expressing lentiviral vector pTomo-pCMV-H1.3-HA, mixed with 1 mL complete medium containing 0.3% soft agar, the coding sequences of the mouse H1.3 gene (NM_145713.4) and the mixture was then added to the top of the base agar. or human H1.3 gene (NM_005320.2) were cloned into the The plates were cooled again for approximately 5 minutes at 4C, pTomo vector between the XbaI and BamHI restriction sites and 1 mL of complete medium was then added to each well. After using an HA-Tag sequence (50-TACCCATACGATGTTCCAGAT- 4 weeks, 1 mL of PBS containing 4% formaldehyde and 0.005% TACGC T-30) at the C-terminus. For the Huwe1, H19, and Crystal violet was added to fix and stain the colonies.

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Xenograft mouse models of ovarian cancer Fig. S1; refs. 37, 38). Overexpression of oncogenic KRASG12D and A total of 2 106 cells were resuspended in PBS containing MYC in combination with inactivating p53 induced malignant 30% Matrigel (BD Biosciences) and subcutaneously injected into transformation in vitro and serous carcinoma in nude mice L/L 6-week-old BALB/c nude mice. The tamoxifen (Sigma; 50 mg/kg ) (39, 40). MOSE cells were isolated from a Huwe1 mouse and treatment was administered via intraperitoneal injection every cultured in complete medium, as described previously (30). other day after the subcutaneous tumors had reached a diameter Approximately 93% of the cells were positively labeled for of approximately 5–8 mm. Doxycycline (Sigma) was delivered in CK8, which is a marker of ovarian epithelial cells (Supplementary water containing 5% sucrose at a concentration of 2 mg/mL. Fig. S2). A lentivirus expressing KRASG12D, MYC, and a tp53 Tumor volume was calculated using the formula 0.5 length shRNA was used to transform MOSE cells. Henceforth, we use width width (36). The data are shown as the means SD, and MOSE cells to refer to infected MOSE cells unless otherwise indi- the statistical analyses were performed using GraphPad Prism cated. To delete the Huwe1 gene, a Cre or CreER element was sub- statistical software. cloned into the lentiviral vector (Fig. 1A). The results of Western blot analysis showed that KRASG12D and MYC were expressed Immunoprecipitation and Western blotting in infected cells and that p53 expression was efficiently silenced The use of human cancer tissues was approved by the Institu- (Fig. 1B). Huwe1 expression was similar between MOSE cells tional Review Board at Kunming Institute of Zoology, Chinese infected with the control lentivirus and those infected with the Academy of Sciences (approval ID: SWYX-2012027). Tissues and lentivirus expressing CreER that were not treated with 4-hydro- cells were lysed in RIPA buffer [50 mmol/L Tris-HCl, 150 mmol/L xytamoxifen (4-OHT). Huwe1 expression was not detectable at sodium chloride, 1% IGEPAL CA-630 (Sigma), 0.5% sodium the protein level in cells expressing Cre. Upon treatment with deoxycholate, 0.1% SDS, and 0.1 mg/ml phenylmethylsulfonyl 4-OHT, Huwe1 was deleted in MOSE cells that expressed CreER fluoride (PMSF, Sigma)] for 30 minutes on ice. For immunopre- (Supplementary Fig. S3A and S3B). Consistent with previous cipitation, 500 mg of total protein was incubated with 10 mLof reports, the MYC protein accumulated as a result of Huwe1 anti-HA antibodies and protein G agarose (Santa Cruz Biotech- knockout (4). Cell proliferation was also inhibited by Huwe1 nology, sc-2003), and the procedure was performed according to knockout, as shown in the growth curve and bromodeoxyuridine the manufacturer's instructions. The protein samples were sub- (BrdUrd) incorporation data (Fig. 1C and D; Supplementary jected to standard SDS-PAGE electrophoresis and immuno- Fig. S4). No obvious cell death or clear staining for the apoptosis blotted. The antibodies used in the study are shown in Supple- marker cleaved caspase-3 was observed in these experiments mentary Table S2. (Supplementary Fig. S5). Cre-mediated or 4-OHT–induced Huwe1 knockout significantly blocked colony-forming capacity BrdUrd incorporation assay in MOSE cells (Fig. 1E). Bromodeoxyuridine (BrdUrd; Thermo Scientific) was added to To determine whether MOSE cells were transformed and the cell medium at a final concentration of 10 mmol/L, and the form tumors in vivo,2 106 cells were subcutaneously injected mixture was incubated for 1 hour. The cells were fixed with 4% into immunodeficient mice. MOSE cells with intact Huwe1 paraformaldehyde in PBS and then permeabilized with 0.3% (CreER) rapidly and efficiently formed tumors in all mice, Triton X-100 in PBS. The cells were then treated with 2 mol/L whereas none of the mice that were injected with Huwe1 HCl for 30 minutes and blocked in 10% goat serum in a PBS knockout cells formed tumors (Fig. 1F). The tumors expressed solution for 1 hour at room temperature. The anti-BrdUrd primary CK8 and CA-125, which are markers of ovarian epithelial antibody (Abcam ab6326, 1:250) was then added, and the cancer (Fig. 1G and H), and exhibited characteristics of ade- mixture was incubated first for 1 hour at room temperature and nocarcinoma when stained with hematoxylin and eosin (H then with CY3-labeled secondary antibodies. and E; Fig. 1I).

Immunohistochemical staining Huwe1 is indispensable for tumor maintenance Sections cut from paraffin-embedded samples (5 mm thick) To determine whether Huwe1 plays an essential role in cancer L/L were deparaffinized, rehydrated, and processed for antigen retriev- maintenance, we subcutaneously injected Huwe1 CreER cells al. The sections were blocked with 10% serum and incubated with into mice. Huwe1 deletion was induced by treatment with tamox- primary antibodies at 4C overnight. A horseradish peroxidase ifen, which was transformed into the active metabolite 4-OHT in (HRP)-labeled secondary antibody and tyramide amplification vivo on day 16, when tumors were not yet clearly detectable, and system (Perkin Elmer) were used to fluorescently detect Huwe1 on day 32, when the tumor size reached 5–8 mm in diameter (Fig. expression. Cy3- or DyLight 488–labeled secondary antibodies 2A). Treatment with tamoxifen inhibited tumor growth and (Jackson Immunoresearch Laboratories) were used to label the decreased tumor size (Fig. 2B). When the mice were sacrificed tissue for other markers. The information for all primary anti- on day 50, the mice that received tamoxifen treatment before any bodies used in this study are provided in Supplementary Table S2. tumor mass had clearly formed had only small tumors or no tumors at all, whereas the mice that received tamoxifen treatment after a tumor mass had clearly formed had significantly smaller Results tumor masses than those observed in the control group (Fig. 2C). Huwe1 deletion impairs transformation in MOSE cells Huwe1 was efficiently deleted, as shown by a decrease in Huwe1 To determine the role of Huwe1 in ovarian cancer, we estab- protein expression in tumors (Fig. 2D). The loss of Huwe1 lished an ovarian cancer model by transforming MOSE cells decreased the number of proliferating cells and induced cellular in vitro via the lentiviral expression of KRASG12D, MYC, and tp53 apoptosis in vivo (Fig. 2E and F). Tumors in all of the groups shRNA, which are the genes that are most frequently mutated in expressed the epithelial marker CK8 (Supplementary Fig. S6). ovarian cancer according to the TCGA database (Supplementary Interestingly, the small tumors in the group that was treated with

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Figure 1. Huwe1 deletion impairs MOSE cell transformation. A, The lentiviral vector used to induce the malignant transformation of MOSE cells in vitro. B, Immunoblot showing the detection of the Huwe1, MYC, KRASG12D, and p53 proteins in the lysates of MOSE-Huwe1L/L-CreER and MOSE-Huwe1L/L-Cre cells. b-actin was used as a loading control. C, Growth curves for the Huwe1 wild-type cell line MOSE-Huwe1L/L-CreER, the Huwe1 knockout cell lines MOSE-Huwe1L/L-Cre and MOSE-Huwe1L/L-CreER following treatment with 4-OHT. Huwe1 knockout was induced for 5 days in the MOSE-Huwe1L/L-CreER cells using 4-OHT (0.5 mmol/L). The data are presented as the means SD; P < 0.05 for MOSE-Huwe1L/L-CreER versus MOSE-Huwe1L/L-Cre or MOSE-Huwe1L/L-CreER plus 4-OHT treatment. D, BrdUrd incorporation assay was used to evaluate the DNA synthesis and proliferation rates of the indicated cell lines (MOSE-Huwe1L/L-CreER, MOSE-Huwe1L/L-CreER plus 4-OHT, and MOSE-Huwe1L/L-Cre). The data were quantitated by counting the number of BrdUrd-positive cells in 7 random microscopic views in each sample. The data are presented as the means SD; , P < 0.01; , P < 0.001. E, Soft agar colony formation assay for MOSE-Huwe1L/L-CreER, MOSE-Huwe1L/L-Cre, and MOSE-Huwe1L/L-CreER plus 4-OHT cells. The images show colonies at 4 weeks after the cells were seeded in soft agar. F,MOSE-Huwe1L/L-CreER or MOSE-Huwe1L/L-Cre cells were subcutaneously injected into BALB/c nude mice. The results are presented as the mean volume SD, n ¼ 10. G and H, CK8 and CA-125 staining in parafﬁn sections cut from tumors obtained from MOSE-Huwe1L/L-CreER in BALB/c nude mice. I, Histopathologic assay of tumors formed by MOSE-Huwe1L/L-CreER cells [hematoxylin and eosin (H&E) staining].

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Figure 2. Huwe1 is indispensable for tumor maintenance. A, Diagram of tamoxifen-treated BALB/c nude micethatwereinjectedwithMOSE-Huwe1L/L-CreER. Group A: mice were intraperitoneally injected with corn oil every other day beginning on day 16 after cell injection. Group B: mice were treated with corn oil ﬁrst, and when the tumors reached approximately 5–8 mm in diameter (day 32), the mice were treated with tamoxifen in oil. Group C: mice were treated with tamoxifen beginning on day 16. B, Volume analysis of tumors formed by MOSE-Huwe1L/L-CreER cells in BALB/c nude mice. Tumor sizes were measured every 3 days. The results are presented as the mean volume SD; P < 0.001. C, Tumors are shown, and the quantiﬁcations of tumor volumes are shown at the right. Each dot represents one tumor volume in a single mouse. The results are presented as the mean volume SD for the group (Group A, n ¼ 7; Group B, n ¼ 7; and Group C, n ¼ 5). , P < 0.001. D–F, Huwe1, Ki67, and cleaved caspase-3 staining in cancers obtained from the mice in Groups A, B,andC,asindicated.

tamoxifen early expressed the Huwe1 protein. This finding indi- that the antitumor effect observed in tamoxifen-treated mice cates that these tumors were derived mainly from MOSE cells that was mainly the consequence of tamoxifen itself instead of L/L escaped tamoxifen-induced Huwe1 deletion. Huwe1 deletion, we transformed Huwe1 MOSE cells using alentivirusthatlackedCre or CreER elements. Cell proliferation The effect of tamoxifen treatment in Huwe1CreER xenografts and colony formation capacity were affected by when MOSE was caused by Huwe1 deletion and not tamoxifen cells expressing CreER, were treated with 4-OHT, whereas Tamoxifen can inhibit proliferation in ovarian cancer cells 4-OHT induced no significant effect in MOSE cells that lacked L/L by blocking the estrogen receptor and induce apoptosis by CreER expression (Fig. 3A–C).WhennudemicewithHuwe1 increasing oxidative stress, and its therapeutic effects have been CreER xenograft tumors were treated with tamoxifen, the sizes evaluated in human patients (41). To exclude the possibility of the tumors were significantly decreased. However, only a

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Figure 3. The anticancer effect is principally due to the tamoxifen-induced deletion of Huwe1 and not by tamoxifen itself. A, Growth curves for the indicated cell lines. B, BrdUrd incorporation assay was used to evaluate DNA synthesis and proliferation rates in MOSE-Huwe1L/L and MOSE-Huwe1L/L-CreER cells in response to 4-OHT. The quantitative analysis was performed by counting the proportion of BrdUrd-positive cells in 7 random microscopic ﬁelds in each sample. The data are presented as the means SD; , P < 0.001. C, Soft agar colony formation assay of the response of MOSE-Huwe1L/L and MOSE-Huwe1L/L-CreER cells to 4-OHT. The images were taken 4 weeks after seeding. D, Volume measurement of the xenograft tumors. Tamoxifen or oil was added when the tumors reached approximately 5–8 mm in diameter. The data are presented as the means SD; , P < 0.05; , P < 0.001. E, Tumors were dissected at the end of experiments (18 days after treatment with tamoxifen or oil). The data are presented as the means SD; , P < 0.05; , P < 0.001.

small effect on tumor size was observed when mice with 141 genes were downregulated, and 56 genes were upregulated L/L Huwe1 xenograft tumors (without CreER expression) were by Huwe1 deletion. Among the downregulated genes, H19 treated with tamoxifen (Fig. 3D and E). These results demon- expression was the most significantly altered, and this result strate that the anticancer effect observed in these mice was was confirmed using real-time PCR (Fig. 4A; Supplementary mainly caused by the deletion of Huwe1 that was induced by Fig. S7B). H19 has been shown to enhance tumor properties tamoxifen and not by tamoxifen itself. during the initiation, progression, and metastasis stages in different cancers (42, 43). Furthermore, repressing H19 expres- The H1.3-H19 cascade mediates the tumor-promoting sion using H1.3 in human ovarian cancer cell lines inhibited function of Huwe1 cell proliferation and colony formation (44). To determine To understand the molecular mechanisms involving Huwe1 whether the downregulation of H19 would impair proliferation in ovarian cancer, we performed RNA-seq to analyze the differ- and colony formation in transformed MOSE cells, we investi- L/L L/L ences between MOSE Huwe1 CreER and Huwe1 Cre cells. gated the effect of H19 knockdown in Huwe1-intact MOSE L/L A total of 197 genes were expressed at significantly different (Huwe1 CreER) cells. Doxycycline-induced H19 knockdown levels between the groups (q value 0.05 and fold change 2; inhibited cell proliferation and colony formation in vitro Supplementary Fig. S7A; Supplementary Table S3). Of these, (Fig. 4B and C; Supplementary Fig. S8) and tumor growth

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Figure 4. Huwe1 maintains tumor growth through H19. A, Quantitative RT-PCR analysis of H19 expression levels in Huwe1 wild-type or knockout cells. The H19 expression level was normalized to that of 18S rRNA. The data are presented as the means SD. B, Growth curves for the indicated cell lines. CreER/shH19 and Cre/shH19 indicate the MOSE-Huwe1L/L-CreER/shH19 and MOSE-Huwe1L/L-Cre/shH19 cell lines, respectively. The data are presented as the means SD. C, Soft agar colony formation assay of MOSE-Huwe1L/L-CreER cells treated with doxycycline (DOX, 1 mg/mL) or 4-OHT. The image shows colonies at 4 weeks after seeding. Quantiﬁcations of colony numbers are shown on the right. The data are presented as the means SD; , P < 0.05. D, Volume analysis of tumors formed by MOSE-Huwe1L/L-CreER/shH19 cells in BALB/c nude mice. The data are presented as the mean volume SD, P < 0.001. E, The tumors and the measurements of tumor volume are shown on the right. Each dot represents one tumor volume in a single mouse. The data are presented as the means SD; , P < 0.001.

in vivo (Fig.4DandE).BecauseH19 expression was inhibited by rescue effect in these cells may have been caused by the relatively H1.3, we next sought to determine whether Huwe1 knockout longer half-life of histone proteins. Silencing histone H1.3 would increase H1.3 protein expression. Because no specific also partially rescued the effect of Huwe1 on H19 expression antibodies for mouse H1.3 are currently available, we con- (Fig. 6B) and colony formation capacity (Fig. 6C). To investigate structed a HA-tagged H1.3-expressing lentiviral vector. HA- the interaction between Huwe1 and histone H1.3, HA-tagged L/L H1.3 protein expression was increased in Huwe1 CreER MOSE mouse H1.3 sequence was introduced into MOSE cells through cells by treatment with 4-OHT, which induced Huwe1 deletion lentiviral infection, and coimmunoprecipitation was perform- (Fig. 5A). At the protein level, H19 expression decreased as H1.3 ed using anti-HA antibodies and Western blot analysis. The increased (Fig. 5B). Correspondingly, 4-OHT treatment inhib- results demonstrated that Huwe1 binds to H1.3 in MOSE cells ited cell proliferation and colony formation capacity in vitro (Fig. 6D). Ubiquitinated H1.3 accumulated in response to (Fig.5CandD).Toconfirm that the effect of H19 repression in MG-132 treatment, which inhibited its proteasome-mediated Huwe1 knockout cells was mediated by H1.3 upregulation, degradation, and Huwe1 deletion decreased the amount of L/L Huwe1 CreER MOSE cells infected with a lentivirus expressing ubiquitinated H1.3 (Fig. 6E). These data indicated that Huwe1 tet-on shH1.3 were treated with doxycycline and/or 4-OHT. The maintained transformation in MOSE cells and the induction results of real-time PCR showed that histone H1.3 was efficient- of tumor formation by the Huwe1-H1.3-H19 cascade. ly silenced (Supplementary Fig. S9). Partial rescue of inhibition HUWE1 is overexpressed and associated with prognoses in of cell proliferation by histone H1.3 knockdown was observed human ovarian cancers (29). To confirm the relationship between starting from passage 2 (Fig. 6A). The longer latency of the HUWE1 and H1.3 in human ovarian cancer, the HUWE1 and H1.3

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Figure 5. H1.3 overexpression impairs malignancy in MOSE cells. A, Immunoblot of the indicated proteins in the lysates of MOSE-Huwe1L/L-CreER/ vector cells and MOSE-Huwe1L/L- CreER/H1.3-HA cells treated with or without 4-OHT. b-Actin was used as a loading control. B, Quantitative RT- PCR analysis of H19 expression levels in MOSE-Huwe1L/L-CreER cells infected with a blank lentivirus or a lentivirus expressing histone HA- tagged H1.3. The data are presented as the means SD. C, Growth curves for MOSE-Huwe1L/L-CreER /vector, MOSE-Huwe1L/L-CreER /H1.3-HA, and MOSE-Huwe1L/L-CreER /H1.3-HA plus 4-OHT cells. The data are presented as the means SD (P < 0.05) for MOSE- Huwe1L/L-CreER/vector versus other cell lines. D, Soft agar colony formation assay of MOSE-Huwe1L/L-CreER/ vector and MOSE-Huwe1L/L-CreER/ H1.3-HA cells treated with or without 4-OHT. The data are presented as the means SD; , P < 0.05.

proteins were detected in 13 samples using Western blot analysis, surface epithelial cells are currently regarded as the cells of origin and the results were quantified using Image J. HUWE1 expression of serous adenocarcinoma, the most common type of human was inversely correlated with H1.3 expression (Fig. 7A and B). ovarian cancer although this view has been challenged recently To further explore the phenotype associated with HUWE1 inactiva- (30, 46). Considering the frequency of genetic events in human tion in human cancer cells, SKOV-3 cells were infected with cancer, in this study, we took advantage of the capacity of mouse lentiviral particles that expressed tetracycline-inducible HUWE1 ovary surface epithelium to be transformed by oncogenic shRNA (SKOV-3/teton-shHUWE1). Treatment with doxycycline KRASG12D and MYC expression and TP53 inactivation because decreased HUWE1 expression (Supplementary Fig. S10) and cell these are the most frequently implicated genetic alterations in proliferation (Supplementary Fig. S11). Immunocompromised human ovarian cancers (40). We efficiently transformed MOSE mice were subcutaneously inoculated with SKOV-3/teton- cells by using lentiviral infection to introduce oncogenic shHUWE1 cells and then treated with doxycycline after xenograft KRASG12D, MYC and tp53 shRNA. Our results suggest that the formation to explore the role of HUWE1 in tumor development lentiviral technology is likely to be valuable for establishing in vivo. Following treatment with doxycycline, the tumor growth ovarian cancer models. rate and number of Ki67-positive cells decreased (Fig. 7C and D; The differential roles played by HUWE1 substrates in cancer, Supplementary Fig. S12). HUWE1 silencing was confirmed in the finding that their expression and genetic elements are doxycycline-treated tumors (Supplementary Fig. S12). altered in human samples, and the results of studies using To confirm that H1.3 is regulated by HUWE1 in human cells, different mouse models suggest that HUWE1 may either pro- SKOV-3/teton-shHUWE1 cells were infected with lentiviral par- mote or suppress tumors, depending on cellular context. In ticles that expressed HA-tagged human H1.3. H1.3 expression this study, we deleted the Huwe1 gene in transformed MOSE increased and H19 expression decreased in response to doxycy- cells and found that this blocked tumor initiation and main- cline-induced HUWE1 knockdown (Fig. 7E and F). Coimmuno- tenance. Other recent studies have shown that HUWE1 expres- precipitation experiments demonstrated a binding interaction sion is increased in 68% of human ovary samples and has an between the HUWE1 and H1.3 proteins (Supplementary Fig. adverse effect on patient survival (29). These data, in combi- S13A) and that H1.3 degradation is directed by HUWE1-mediated nation with our data revealing its tumor-promoting role, ubiquitination (Supplementary Fig. S13B), similar to what was indicate that HUWE1 promotes tumorigenesis in most human observed in our experiments with mouse cells. ovarian cancers. We show that HUWE1 promotes ovarian tumorigenesis through a novel mechanism involving the inhibition of histone Discussion H1.3 expression and subsequent inhibition of H19 expression Unlike other cancer types, few mouse models accurately reca- in MOSE cells. HUWE1 has been reported to ubiquitinate core pitulate the features of human ovarian cancer (45). Ovarian histone proteins, including H1, H2A, H2B, H3 and H4, and is

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Oncogenic Role of Huwe1 in Ovarian Cancer

Figure 6. Huwe1 knockout decreases H19 expression by upregulating histone H1.3 expression. A, Relative cell growth in the indicated cells during three passages. CreER/shH1.3 and Cre/shH1.3 indicate MOSE-Huwe1L/L-CreER and MOSE-Huwe1L/L-Cre cells, respectively, which were infected with the shH1.3 lentivirus. The data are presented as the means SD; , P < 0.01; , P < 0.001. B, Quantitative RT-PCR analysis of H19 expression in MOSE-Huwe1L/L-CreER/shH1.3 and MOSE-Huwe1L/L-Cre/shH1.3 cells that were treated with 4-OHT or doxycycline (DOX). The data are presented as the means SD. C, Soft agar colony formation assay of MOSE-Huwe1L/L-CreER/shH1.3 and MOSE-Huwe1L/L-Cre/shH1.3 cells treated with 4-OHT or doxycycline, as indicated. The data are presented as the means SD (, P < 0.05). D, The interaction between Huwe1 and histone H1.3 was tested using coimmunoprecipitation assays in MOSE-Huwe1L/L-CreER/H1.3-HA cells. E, Ubiquitination assays to evaluate the effects of histone H1.3. Huwe1 knockout was induced by treating MOSE-Huwe1L/L-CreER /H1.3-HA cells with 4-OHT. The cells were treated with 5 mmol/L MG-132 for 6 hours.

thought to play an essential role during spermiogenesis when cancer cell line. H19 is overexpressed in almost all cancers and proteins are replaced by protamines (5, 47). The effect of involved in various cancer stages, including initiation, progres- HUWE1 on histone proteins in other cells or tissues is not sion, and metastasis (42). Consistent with the results of studies well-described. Here, we report a mechanism by which H1.3 using human ovarian cancer cell lines (44), we observed that expression is increased in Huwe1 knockout cells. Huwe1 binds decreasing H19 expression in MOSE cells led to reductions in to and ubiquitinates H1.3 to designate it for proteasome- cell proliferation and tumor growth. The p53 protein inhibits dependent degradation in MOSE cells and a human ovarian H19 expression by repressing H19 promoter activity and

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Yang et al.

Figure 7. HUWE1 inactivation inhibits proliferation and tumor growth in human ovarian cancer cells. A, Immunoblots showing HUWE1 and H1.3 levels in human ovarian cancer samples. b-Actin was used as a loading control. B, Quantiﬁcation and correlation between the expression of HUWE1 and H1.3 in experiment in A. Each dot represents one ovarian cancer sample. Western blot results were quantiﬁed using ImageJ, and intensity values were normalized to the b-actin band. C, The effect of HUWE1 silencing on growth in tumors formed by SKOV-3/teton-shHUWE1 cells in BALB/c nude mice. The data are presented as the mean volume SD; P < 0.001. D, An image of tumors and measurements of tumor volume at the end of experiment. Each dot represents one tumor volume in a single mouse. The data are presented as the means SD; , P < 0.001. E, Immunoblot showing HUWE1 and H1.3 levels in SKOV-3/teton-sh HUWE1/HA-H1.3 cells. b-Actin was used as a loading control. F, Quantitative RT-PCR analysis of H19 expression levels in SKOV-3/teton-shHUWE1/HA-H1.3 cells treated with doxycycline (DOX). H19 expression was normalized to that of 18S rRNA. The data are presented as the means SD.

altering the epigenetic status of its upstream imprinting control Disclosure of Potential Conﬂicts of Interest region (48). In addition, the dysregulation of H1.3 caused by No potential conﬂicts of interest were disclosed. HUWE1 knockout is another important regulator of H19 expression in ovarian cancer. H1.3 knockdown rescued the Authors' Contributions HUWE1 knockout phenotype and H19 expression, indicating Conception and design: D. Yang, A. Iavarone, F. Zou, X. Zhao H19 Development of methodology: D. Yang, B. Sun, L. Yan, X. Zhao that the Huwe1-H1.3- cascade is a key mediator of HUWE1 Acquisition of data (provided animals, acquired and managed patients, provid- functions. Whether the Huwe1-H1.3-H19 cascade functions ed facilities, etc.): D. Yang, B. Sun, X. Zhang, D. Cheng, L. Li, S. Zhang, X. Zhao orplaysessentialrolesincancerinothercellsortissuesremains Analysis and interpretation of data (e.g., statistical analysis, biostatistics, to be determined. computational analysis): D. Yang, B. Sun, X. Yu, S. An, H. Jiang, A. Iavarone, In conclusion, the loss of HUWE1 inhibited cell proliferation F. Zou, X. Zhao and tumor formation in human ovarian cancer cells and trans- Writing, review, and/or revision of the manuscript: D. Yang, B. Sun, H19 A. Lasorella, A. Iavarone, F. Zou, X. Zhao formed MOSE cells. The HUWE1-H1.3- cascade is therefore Administrative, technical, or material support (i.e., reporting or organizing an important mechanism that mediates the effects of HUWE1 in data, constructing databases): A. Lasorella, F. Zou, X. Zhao ovarian cancer. Study supervision:F. Zou, X. Zhao

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Oncogenic Role of Huwe1 in Ovarian Cancer

Grant Support The costs of publication of this article were defrayed in part by the payment of advertisement This work was supported by the Strategic Priority Research Program page charges. This article must therefore be hereby marked in of the Chinese Academy of Sciences (XDA 01040403), the National accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Natural Science Foundation of China (NSFC, 81171960), and the Top Talents Program of Yunnan Province, China (2012HA014 to Received September 22, 2016; revised February 9, 2017; accepted June 29, X. Zhao). 2017; published OnlineFirst July 7, 2017.

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4784 Cancer Res; 77(18) September 15, 2017 Cancer Research

Huwe1 Sustains Normal Ovarian Epithelial Cell Transformation and Tumor Growth through the Histone H1.3- H19 Cascade

Dong Yang, Bin Sun, Xiaohong Zhang, et al.

Cancer Res 2017;77:4773-4784. Published OnlineFirst July 7, 2017.

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