The E3 Ligase RING1 Targets P53 for Degradation and Promotes Cancer

Published OnlineFirst November 29, 2017; DOI: 10.1158/0008-5472.CAN-17-1805 Cancer Molecular Cell Biology Research

The E3 Ligase RING1 Targets p53 for Degradation and Promotes Cancer Cell Proliferation and Survival Jiajia Shen1, Pengyu Li2, Xuejing Shao3, Yang Yang4, Xiujun Liu1, Min Feng5, Qiang Yu5, Ronggui Hu4, and Zhen Wang1

Abstract

As a component of the transcriptional repression complex 1 p53-deficient cells. Its growth inhibitory effect was partially res- (PRC1), the ring finger protein RING1 participates in the epige- cued by p53 silencing, suggesting an important role for the netic regulation in cancer. However, the contributions of RING1 RING1–p53 complex in human cancer. In clinical specimens of to cancer etiology or development are unknown. In this study, we hepatocellular carcinoma, RING1 upregulation was evident in report that RING1 is a critical negative regulator of p53 homeo- association with poor clinical outcomes. Collectively, our results stasis in human hepatocellular and colorectal carcinomas. RING1 elucidate a novel PRC1-independent function of RING1 and acts as an E3 ubiquitin (Ub) ligase to directly interact with and provide a mechanistic rationale for its candidacy as a new prog- ubiquitinate p53, resulting in its proteasome-dependent degra- nostic marker and/or therapeutic target in human cancer. dation. The RING domain of RING1 was required for its E3 Ub Significance: These results elucidate a novel PRC1-independ- ligase activity. RING1 depletion inhibited the proliferation and ent function of RING1 and provide a mechanistic rationale for its survival of the p53 wild-type cancer cells by inducing cell-cycle candidacy as a new prognostic marker and/or therapeutic target in arrest, apoptosis, and senescence, with only modest effects on human cancer. Cancer Res; 78(2); 1–13. 2017 AACR.

Introduction quitinate histone H2A at lysine 119 (H2AK119Ub1; refs. 8, 9). Different from the well-studied roles of RNF2 and BMI1 in In mammals, polycomb group (PcG) proteins consist of two carcinogenesis (10–12), limited evidence has been provided major polycomb repressive complexes (PRC): PRC1 and PRC2 with regard to the role of RING1 in cancer. An earlier report (1, 2). The PRC1 core complex includes RING1/RING1A, suggested overexpression of RING1 contributed to cellular RING1B/RNF2, and BMI1, whereas the PRC2 core complex transformation by upregulating the expression of proto- includes EED, EZH2, and SUZ12. PRC1 and PRC2 are known to oncogenes such as c-jun and c-fos (13). However, homeostatic play an important role as transcriptional repressors in embryonic levels of RING1 were found to vary significantly among development, stem cell self-renewal, and cell proliferation different cancer types, including lung cancer, prostate through epigenetic modifications of target genes (3, 4). Deregu- cancer, lymphoma, and kidney cancer (14–20), suggesting a lation of PcG genes is frequently found to be associated with yet unclear role of RING1 in cancer. developmental defects and cancer (3, 5–7). In this study, we have uncovered tumor suppressor p53 protein RING1 is a crucial component of the PRC1 and, along with as a novel bona fide substrate of RING1. RING1 directly interacts RNF2 and BMI1, acts as an E3 ubiquitin ligase to monoubi- with, and ubiquitinates p53, which finally leads to its proteasome- dependent degradation. As a result, RING1 depletion results in p53 stabilization, leading to cell-cycle arrest, apoptosis, senes- 1 Department of Biochemistry, Institute of Medicinal Biotechnology, Chinese cence, and migration inhibition in p53-proficient cells. Moreover, Academy of Medical Sciences and Peking Union Medical College, Beijing, China. we found that RING1 is highly expressed in hepatocellular car- 2Qilu Hospital of Shandong University, Jinan, China. 3Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Institute of Pharmacology and cinoma (HCC) tissues and its expression is associated with poor Toxicology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou, outcomes. China. 4State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. 5Cancer Thera- peutics and Stratified Oncology, Genome Institute of Singapore, Agency for Materials and Methods Science, Technology, and Research (A STAR), Biopolis, Singapore. Plasmids Note: Supplementary data for this article are available at Cancer Research The pCMV-HA-p53, pcDNA3.0-RING1-Flag and deletion or Online (http://cancerres.aacrjournals.org/). point mutants, pGEX-4T-1-GST-p53, pSJ8-MBP-RING1-6His, Corresponding Authors: Zhen Wang, Institute of Medicinal Biotechnology and pGL4.17-p21-luc plasmids were constructed in our lab. Chinese Academy of Medical Sciences, 1# Tian Tan Xi Li, Beijing 100050, The siRNAs duplexes were synthesized by RiboBio Co., Ltd. China. Phone/Fax: 8610-6316-5289; E-mail: [email protected]; and si-RING1-1: 50-GTGGGAACTGAGTCTGTATGA-30,si-RING1-2: Ronggui Hu, E-mail: [email protected] 50-CAGATCAGACCACAACGAT-30. sh-RING1 and sh-p53 (with doi: 10.1158/0008-5472.CAN-17-1805 asequenceof50-GACTCCAGTGGTAATCTAC-30)werecloned 2017 American Association for Cancer Research. into pLKO.1 vector.

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Antibodies, reagents, and cell lines Etoposide treatment The anti-RING1 (D2P4D) (#13069), anti-p21 (12D1; #2947), HepG2 cells were seeded (5 105) into 6-well plates. Seventy- anti-p53 (1C12; #2524), anti-ubiquitin (P4D1; #3936) antibo- two hours after transfection with pcDNA3.0-RING1-Flag or dies were purchased from Cell Signaling Technology. The anti- si-RING1-1, cells were treated with 50 mmol/L etoposide for p53 (DO-1) (#SAB5100001), anti-Flag (#F1804), anti-HA indicated time before harvest and subsequent immunoblotting (#H6908), anti-GAPDH (#G8795), anti-b-actin (#A1978) anti- analyses, using described antibodies. bodies, anti-Flag M2 Affinity Gel (#A2220), and propidium iodide (PI, #P4170) were purchased from Sigma-Aldrich. Annexin Cell growth and colony formation assay V-PI Staining Kit was from Beijing 4A Biotech Co, Ltd. The anti-HA Following plasmid transfection or lentivirus infections, cell Affinity Gel (#B23302) was purchased from Biotool. The Gluta- growth was assessed as described previously (21). For colony thione Sepharose 4B (#17075601) was purchased from GE formation assay, the cells stably expressing sh-RING1 or sh-p53 Healthcare. Etoposide (ETO, #S1225) was purchased from Sell- were seeded (5 103/well) onto 6-well plates. After 3 weeks, the eckchem. HepG2 and HCT116 cell lines were obtained from colonies were stained with hematoxylin and counted. China Infrastructure of Cell Line Resources. HCT116 p53 / cells were gifted from Prof. Bert Volgestein at Johns Hopkins University Cell cycle and apoptosis analysis School of Medicine, Baltimore, MD. HEK293T cell lines were The cells were infected with sh-RING1 for 72 hours, and cell- from ATCC in the United States. All of the cell lines were authen- cycle distribution was performed as described previously (21). ticated by STR profiling at December 2016. Apoptosis was determined by Annexin V/PI Apoptosis Detection Kit. In drug treatment groups, the cells were infected with Cell transfection and infection sh-RING1 for 24 hours, followed by etoposide (50 mmol/L) Cell transfection was carried out using Lipofectamine 2000 treatment for another 48 hours. following the manufacturer's instruction. For lentiviral packaging, psPAX2, pMD2.0G, and pLKO.1-sh-p53, pLKO.1-sh-RING1, or the Senescence-associated b-galactosidase staining pLKO.1 scramble vectors were cotransfected into HEK293T cells, Senescence-associated b-galactosidase (SA-b-gal) staining was and the supernatants harvested at 48 or 96 hours after transfection. performed and quantified as described before (21). Lentivirus infection was transient or followed by screening with appropriate antibiotics to establish cell lines stably expressing the Migration assay indicated shRNAs. Specifically, for stable knockdown of RING1 or Migration capacity was measured by transwell and wound fi p53, the cells were infected with lentivirus expressing RING1 or p53 healing assay. The transwell lter (8-mm pores; Millicell Cell shRNA (sh-RING1 or sh-p53) in the presence of 8 mg/mL polybrene Culture Inserts) was used according to the instructions. Hema- (#107689; Sigma). Stable cell lines were selected and maintained at toxylin was used to stain the cells that migrated to the lower side of 5 mg/mL puromycin (#P8833; Sigma). the top chamber. For wound-healing assay, the cells infected with sh-RING1 were seeded into 6-well plates, and confluent cell Protein array analyses monolayers were wounded by manually scraping the cells, PathScan Cancer Phenotype Antibody Array Kit (#14821, Cell washed with PBS, and further cultured in medium without FBS Signaling Technology) was used to examine the level of cancer for the indicated time. Migration ability was represented by the cell-associated proteins in RING1 knockdown cells. The cells were percentage of the wound-healing area after normalization to infected with shRNA for 72 hours. Cell extracts were prepared and control using ImageJ. analyzed following the manufacturer's instruction. Generation of RNF2 KO cells using CRISPR/Cas9 system RNF2 fi Dual-luciferase assay A CRISPR/Cas9 system was used to establish -de cient The cells infected with sh-RING1 for 24 hours were seeded in HEK293T cells. Single guide RNA (sgRNA) was generated by 96-well plates at 1 104 cells per well for 24 hours before online CRISPR Design tool (http://crispr.mit.edu/). The sgRNA transfection with the pGL4.17-p21-luc plasmid (100 ng per well) sequences were cloned into pX260-U6-Chimeric-BB-CBh- and Renilla (5 ng per well) in triplicate. After 48 hours, the hSpCas9 plasmid and transfected into HEK293T cells. After being luciferase activity was measured as described (21). selected with 5 mg/mL puromycin, the cells were seeded into 96-well plates for monoclonalization. Colonies derived from a RT-PCR analysis signal cell were detected for gene expression. The sgRNA sequences of RNF2 were 50-AATTCACTGTGTAGACTTCG-30 and RNA was extracted using TRIzol (#15596026; Invitrogen). The 0 0 cDNA was synthesized using PrimeScript RT Master Mix 5 -ATCATCACAGCCCTTAGAAG-3 . (#DRR036A; TaKaRa). The reaction conditions used for PCR set for 35 cycles of denaturation at 94C for 30 seconds, annealing at Immunoblotting, immunofluorescence, and 65C for 30 seconds, and elongation at 72C for 1 minute. immunoprecipitation Sequences of the primers were as follows: Standard immunoblotting (IB) was performed with indicated antibodies and scanned with ImageJ software for normal- Table 1. Primer sequences of the indicated genes ization where needed as described before (21). For immuno- fl Gene Forward sequence Reverse sequence uorescence analysis, the cells were seeded on glass coverslips RING1 50-AGAATGCCAGCAAAACGTGG-30 50-AGATAGGGCACATGAGTTCTGA-30 and fixed with 4% paraformaldehyde for 20 minutes at room p53 50-GAGGTTGGCTCTGACTGTACC-30 50-TCCGTCCCAGTAGATTACCAC-30 temperature and stained with indicated antibodies. The cell 0 0 0 0 p21 5 -TGTCCGTCAGAACCCATGC-3 5 -AAAGTCGAAGTTCCATCGCTC-3 nuclei were stained with DAPI. For immunoprecipitation (IP) GAPDH 0 0 0 0 5 -CATGAGAAGTATGACAACAGCCT-3 5 -AGTCCTTCCACGATACCAAAGT-3 analysis, the cells were lysed in Triton X-100 lysis buffer

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(150 mmol/L NaCl, 50 mmol/L Tris, and 1% Triton X-100, identified as statistically significant at three levels: P < 0.05, P < pH 7.5), and incubated with 5 mL respective antibodies and 0.01, and P < 0.001. 20 mL protein G plus-agarose or 20 mL Flag-agarose beads overnight at 4 C. The immunocomplexes were subsequently Results washed with lysis buffer and subjected to IB. P53 protein expression is regulated by RING1 GST/MBP pull-down We first used two siRNAs targeting RING1 to suppress the RING1 and p53 proteins were purified as described previously protein expression in HepG2 cells and HCT116 cells. Both siRNAs (22). Purified GST–p53 fusion protein bound to the Glutathione efficiently knocked down RING1 expression compared with con- Sepharose 4B was incubated with cell lysates. MBP-RING1-His trol group, with si-RING1-1 showing more pronounced effect fusion protein bound to the Amylose resin was incubated with (Supplementary Fig. S1A). This siRNA sequence was thus used to purified His-p53. All the interactions were rotated incubated at construct lentiviral sh-RING1 vector for stable expression in the 4C for 6 hours. After washing with Triton X-100, the Sepharose following work, which markedly depleted endogenous RING1 in beads bound proteins were separated by SDS/PAGE and IB. both cells at 72 hours postinfection (Fig. 1A). To unbiasedly identify possible molecular targets of RING1, a protein antibody Ubiquitination assay array was employed to assess the effects of RING1 knockdown on In vivo ubiquitination assay was performed as described before the static levels of 19 cancer cell–associated proteins, from which (23). Briefly, HEK293T cells were transfected with the indicated p53 level to be apparently upregulated upon RING1 knockdown plasmids or siRNA for 72 hours, followed by MG132 treatment for in both HepG2 and HCT116 cell lines (Fig. 1B). Further IB 6 hours before harvested and lysed. IP was carried out with anti- analyses using two siRNAs targeting RING1 cells confirmed the HA-beads. After rotated incubation, the anti-HA beads were p53 upregulation in these cells (Fig. 1C). Consistently, p21, a well- washed with RIPA lysis buffer and boiled in 1 SDS-PAGE sample known target of p53, was also increased at both mRNA and buffer, followed by IB analyses using indicated antibodies. The protein levels (Fig. 1C; Supplementary Fig. S1B). In addition, in vitro autoubiquitination assay was carried out using an Ubi- RING1 knockdown has little effect on p53 mRNA level in both quitination Kit from Enzo Life Science (#BML-UW9920-0001) as HepG2 and HCT116 cells, as shown by RT-PCR analysis (Fig. 1D). described previously (24). Together, these findings suggested a role for RING1 in negatively regulating the static level of wild-type p53 proteins, apparently at Xenograft tumor model posttranscriptional level. Female BALB/c nude mice (6-week-old) were purchased from SPF Biotechnology Co. Ltd. The animal study was approved by RING1 negatively regulates p53 protein stability with or our Institute's Animal Care and Use Committee. The HepG2- without genotoxic stress GFP cell population with stable RING1 knockdown was ampli- Next, to determine whether RING1 might regulate the fied, mixed with matrigel (50%) and followed by injection stability of p53, a cycloheximide (CHX)-based chase exper- 6 subcutaneously into the mice (1 10 /per mice). Tumor iment was performed to examine the half-life of p53 protein volumes were measured twice a week and calculated. The in the presence of RING1 depleted or at endogenous level. whole-body imaging of tumor-bearing mice was monitored CHX (50 mg/mL) was added at 72 hours post-infection of twice a week by the FluorVivo Model 100 (INDEC BioSystems). sh-RING1 to block protein synthesis, and static levels of All mice were sacrificed after 18 days, and the tumor tissues endogenous p53 proteins were then checked by IB analysis. were collected, photographed, and weighted. Upon RING1 knockdown, the half-life of p53 in both HepG2 and HCT116 cells was significantly extended, com- IHC analysis of human tumor tissue array pared with that of the control groups, whereas RING1 over- IHC was performed in human liver cancer tissue microarrays expression caused marked reduction in p53 protein level (purchased from Shanghai Biochip in Shanghai, China) contain- (Fig. 2A and B). When the proteasome activity was blocked ing 90 pairs of clinical hepatocellular carcinoma with adjacent by the proteasome inhibitor MG132 (20 mmol/L) for 6 normal tissues to compare in situ expression of RING1 as hours, the decrease in p53 protein level upon RING1 over- described previously (21). The staining intensity was classified expression could be reversed (Fig. 2B). This suggested that into five groups with increasing staining intensity from marginal RING1 could negatively regulate p53 protein stability in a () to the strongest (þþþþ; ref. 25). The paired human liver proteasome-dependent manner. tumor tissues were collected from Qilu Hospital in Jinan, China, Given the fact that p53 is stabilized upon DNA damage- with written informed consents obtained from all the patients induced stress, we also checked whether RING1 regulates p53 based on the Declaration of Helsinki. The study was approved by expression in response to DNA damage. As shown in Fig. 2C, Research Ethics Committee of Qilu Hospital and our Institute's when HepG2 cells were treated with DNA damaging agent Ethics Committee. etoposide (ETO), ectopic RING1 expression significantly decreased ETO-induced upregulation of p53 protein and the Statistical analysis downstream hallmarks of DNA damage response such as All experiments were repeated at least three times. Statistical phosphorylated p53, p21, and gH2AX (Fig. 2C, left). Con- analysis was performed using Student t test (between two groups) versely, RING1 depletion upregulated p53 protein level as well or one-way ANOVA analysis (within multiple groups) for data as the hallmarks for DNA damage response upon the ETO comparisons. For IB analysis, one representative result from at exposure (Fig. 2C, right). This finding suggested that RING1 least three experiments was shown. Statistical analysis was per- destabilizes endogenous p53 proteins in cells with or without formed using SPSS 13.0, and differences between the groups were genotoxic stress.

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sh-RING1 – + A sh-RING1 – + B sh-control + – RING1 HepG2 HepG2 p53 b-Actin

RING1 HCT116 HCT116 b-Actin p53

C HepG2 HCT116 Figure 1. RING1 depletion increases the protein si-RING1-2 – – + – – + level of p53. A, Efﬁcient knockdown si-RING1-1 – + – – + – of endogenous RING1 by sh-RING1. B, Changes of 19 cancer cell–associated p53 proteins upon RING1 knockdown for 1.00 1.26 1.09 1.00 2.03 2.36 72 hours. C, Static levels of p53 and p21 p21 proteins upon RING1 knockdown. The cells were transfected with siRNAs for 1.00 1.42 1.10 1.00 1.64 1.19 72 hours before IB analysis. The band RING1 intensities were quantiﬁed with ImageJ 1.00 0.57 0.56 1.00 0.82 0.80 and normalized to respective b-actin in each lane. D, Semiquantitative RT-PCR b-Actin analysis. The cells were infected with shRNA for 48 and 72 hours. The D intensity of the bands was scanned by ImageJ. Data are expressed as mean 48 h 72 h SD, n ¼ 5. , P < 0.05; , P < 0.01 versus sh-control respective control. sh-RING1 – ++– 150 sh-control ++–– sh-RING1 RING1 100 HepG2 p53

GAPDH 50 (% of control) Relative denisity

RING1 0 48 h 72 h 48 h 72 h48 h 72 h 48 h 72 h HCT116 p53 RING1 p53 RING1 p53 GAPDH HepG2 HCT116

RING1 directly binds to p53 to ubiquitinate p53 in vivo and with p53 (Fig. 3C). Pull-down assays with recombinant in vitro MBP-RING1-His and His-p53 proteins further confirmed the As RING1 itself is an E3 Ub ligase (26), we speculated that direct binding of the proteins to each other in vitro (Supple- RING1 might directly interact with, and conjugate Ub to p53, mentary Fig. S2B), suggesting a direct interaction between which could finally lead to faster p53 degradation. As shown RING1 and p53. in Fig. 3A, coimmunoprecipitation (co-IP) analysis indicated that To check whether RING1 did promote p53 ubiquitination, a significant fraction of Flag-tagged RING1 and HA-tagged p53 RING1 was overexpressed in HEK293T cells that were pre- could form a complex in HEK293T cells. Further immunofluo- treated with MG132 to block proteasome-dependent degrada- rescence microscopy analyses indicated that endogenous RING1 tion, which seemed to markedly induce p53 polyubiquitina- and p53 proteins, visualized by respective antibodies, were colo- tion (Fig. 3D). Conversely, RING1 knockdown decreased the calized in the nuclei of HepG2 and HCT116 cells (Fig. 3B). ubiquitination of p53 (Supplementary Fig. S2C). In a recon- Supportively, endogenous binding between RING1 and p53 stituted in vitro ubiquitination assay that included E1, UbcH6 proteins was observed in HCT116 cells by co-IP analysis with (E2), ATP and Ub, RING1 was also found to ubiquitinate RING1 antibody (Supplementary Fig. S2A). p53 (Fig. 3E). Altogether, these data clearly suggested that Furthermore, in an in vitro GST pull-down assay using RING1 not only directly interacts with p53 protein, but is also the recombinant GST-p53 protein and whole-cell lysates capable of conjugating poly-Ub chains onto p53 both in vitro from HEK293T cells expressing RING1-Flag, RING1 bound and in vivo.

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HepG2 A CHX (50 mg/mL) 00.51 24 8(h) 150 sh-control p53 sh-RING1 sh-control 100 b-Actin

50 p53

sh-RING1 % p53 Remaining 0 b-Actin 0 0.5 1 2 4 8 Time post CHX treatment (h)

HCT116

CHX (50 mg/mL) 00.51 24 8(h) 150 sh-control p53 sh-RING1 sh-control 100 b-Actin 50 p53

sh-RING1 % p53 Remaining 0 b-Actin 00.51 2 4 8 Time post CHX treatment (h)

B MG132

RING1-Flag – +– +

RING1

p53 HepG2

GAPDH

C HepG2 VectorRING1-Flag Vector si-RING1-1 ETO 0 4 8 12 0 4 8 12 h 0481204812h

RING1

p53

p-p53(S15)

p21

gH2AX

b-Actin

Figure 2. RING1 destablizes p53 protein. A, Knockdown of RING1 increased the half-life of p53 by pulse-chase assay. B, Reduction of p53 by RING1 overexpression was dependent on proteasomal degradation. The cells transfected with pcDNA3.0-Ring1-Flag were cultured for 72 hours and treated with MG132 (20 mmol/L) for another 6 hours. C, The DNA damage response upon RING1 overexpression or knockdown. After transfection with pcDNA3.0-Ring1-Flag or si-RING1-1, HepG2 cells were cultured for 72 hours before treatment with 50 mmol/L etoposide (ETO) for indicated time. The proteins were probed by IB analyses.

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293T A B RING1-Flag –+ HA-p53 ++ DAPI RING1 p53 Merge

IB: HA IP: Flag HepG2 IB: Flag

Flag Input HCT116

b-Actin Figure 3. RING1 directly interacts with and C Input Pull-down D ubiquitinates p53 ubiquitination. 293T A, Exogenous RING1 bound to p53. The Cell lysate + + + plasmid of pcDNA3.0-Ring1-Flag and GST-p53 – – + RING1-Flag – – + pCMV-HA-p53 were cotransfected – + – GST HA-p53 – + + into HEK293T cells for 72 hours before IP analysis. B, Endogenous RING1 IB: RING1 His-ub + ++ and p53 colocalization by GST-p53 immunoﬂuorescence staining. Scale bar, 15 mm. C, GST pull-down assay indicating the interaction of puriﬁed IB: GST GST-p53 protein from bacteria IB: His (bottom) with RING1. D, RING1 GST ubiquitinated p53 in vivo. HEK293T cells IP: HA were cotransfected with indicated plasmids for 48 hours, followed by GST-p53 MG132 treatment for another 6 hours before IP analysis. E, RING1 promoted IB: HA the ubiquitination of p53 in vitro.The GST ubiquitination assay was carried out as described in Materials and Methods. RING1 E Ubiquitin+E1+UbcH6+ATP Input HA His-p53 – ++ MBP-RING1-His – ++ GAPDH

IB: Ub

IB: RING1 RING1

E3 ubiquitin ligase activity of RING1 is dependent on its RING greatly reduced the E3 ligase activity of RING1, without affecting domain the binding ability with p53 (Supplementary Fig. S2D). Together, We went on to map the p53-interacting regions in RING1. As this suggests the N-terminal RING domain in RING1 is critical for shown in Fig. 4A, co-IP experiments were performed with full- its binding and ubiquitin ligase activity towards p53. length p53 and different deletion mutants of Flag-tagged RING1 that were coexpressed in HEK293T cells. Deletion of RING- RNF2 is not required for RING1-mediated poly-ubiquitination domain–containing area of aa48-88 (RING1-D1) significantly of p53 in vivo reduced the interaction of RING1 with p53. Consistently, RING RNF2 was previously shown to ubiquitinate p53 (27). As a domain deletion totally abolished the ubiquitination state of p53 paralog of RING1, RNF2 can also form a complex with RING1, so compared with the full-length RING1, or either of two other we next checked whether RNF2 is involved or required for the E3 deletion mutants examined (Fig. 4B). To further address the role ligase activity of RING1 toward p53. It has first come to our of RING domain, a same point mutation within the RING domain attention that the shRNA for RING1 used in above sections might as reported before, which is not capable of binding with E2 ligase also interfere with RNF2 expression. However, as shown in (27), was used to compare the ubiquitination activity of RING1 Supplementary Fig. S3A, the knockdown effect of sh-RING1 with that of the wild type. As shown in Fig. 4C, the I50A mutation specifically limited to RING1, thus suggesting that the above

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Full length A RING1-Flag B 89–406aa RING1-D1-Flag RING1-Flag – – D1+ D2 D3 1–88aa 230–406aa HA-p53 ++– ++ + RING1-D2-Flag His-ub ++++++ RING1-D3-Flag 1–229aa

293T RING1-Flag – D1+ D2 D3 IB: His HA-p53 +++++ IP: HA IB: HA

IB: HA IP: Flag

IB:Flag

Figure 4. Flag E3 ligase activity of RING1 is RING domain-dependent, not depending on Input RNF2. A, Interaction of p53 with the different deletion mutants of RING1. Flag HEK293T cells coexpressing p53 and HA full-length or deletion mutants of RING1 Input (with schematic diagram shown above) were cultured for 72 hours, prior to HA harvest and co-IP analysis. B, Ubiquitination assay of p53 after b-Actin cotransfection with different RING1 constructs. C, RING1I50A inhibited the 293T(RNF2–/–) E3 ligase activity of RING1. D, RING1 CDMG132 ubiquitinated p53 in RNF2-knockout RING1-Flag –– + cells. 293T RING1-Flag – – + I50A HA-p53 – + + HA-p53 – ++ + His-ub + ++ His-ub ++++

IB: His IB: His IP: HA IP: HA

IB: HA IB: HA Flag Flag Input Input HA HA

GAPDH b-Actin observed RING1-mediated ubiquitination of p53 is unlikely an knockdown on the cell proliferation or survival were assessed. artifact. Furthermore, two independent HEK293T-derived RNF2 RING1 knockdown dramatically inhibited the proliferation of knocked out cell lines (RNF2 / ) were constructed with the HepG2 or HCT116 cells, compared with that of the control CRISPR/cas9 technique (Supplementary Fig. S3B). We used #2 (Fig. 5A), which was also consistent with the data from further cell line to demonstrate that coexpressed RING1-Flag and HA-p53 colony formation assay (Fig. 5B). were able to form complex that survived the co-IP procedures As cellular senescence constitutes an another major tumor- (Supplementary Fig. S3C), and RING1 interacted with p53 in the suppressing mechanism in cancer, we next asked whether absence of RNF2 and induced p53 degradation independently of RING1 knockdown might affect senescent phenotype using the RNF2 (Fig. 4D). established senescence-associated b-galactosidase (SA-b-Gal) staining assay (21). As shown in Fig. 5C, signiﬁcant induction Knockdown of RING1 inhibits the proliferation and survival of of senescence was observed, as 3- to 7-fold more cells were cancer cells in a p53-dependent manner positively stained in the RING1-deﬁcient group showed than in To further investigate the possible functional consequences of the controls. FACS analysis also indicated that, upon RING1 RING1-mediated ubiquitination of p53, the effects of RING1 depletion, increased cell subpopulations were found to retain

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A 200 500 400 –/– HepG2-shcontrol HCT116-shcontrol HCT116(p53 )/sh-control ) )

) –/– 4 4 4 HepG2-shRING1 400 HCT116-shRING1 HCT116(p53 )/sh-RING1 150 300 300 100 200 200 100 50 100 Cell number (10 Cell number Cell number (10 Cell number Cell number (10 Cell number 0 0 0 01234567 01234567 01234567 Time (day) Time (day) Time (day)

B HepG2 HCT116 HCT116 (p53–/–)

sh-RING1 – + – + – + sh-control + – + –+–

HepG2 HCT116 (p53–/–) 150 150 HCT116 150

100 100 100

50 50 50 (% of control) (% of control) (% of control) Number of colonies Number of colonies Number of colonies 0 0 0 sh-control + – sh-control + – sh-control + – sh-RING1 – + sh-RING1 –+ sh-RING1 –+

C sh-control sh-RING1

HepG2 50

SA- b -gal 20 HCT116 (% of total) 10

0 sh-control ++–– + – sh-RING1 – + ––+ +

HCT116 (p53–/–) HepG2 HCT116 HCT116 (p53–/–)

Figure 5. RING1 depletion suppresses the cancer cell survival and proliferation and induces senescence. A, RING1 knockdown suppressed cell proliferation. The proliferation curve was measured at 24-hour intervals up to 7 days. Data are presented as mean SD, n ¼ 3. , P < 0.05. B, Colony formation upon RING1 knockdown. Data are presented as mean SD, n ¼ 3. , P < 0.01 versus control. C, Cell senescence increased upon RING1 knockdown. SA-b-gal staining was performed with the cells 120 hours after transfection with the shRNA. Representative images from three experiments are shown. Data are presented as mean SD. , P < 0.05; , P < 0.01 versus control.

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AB HepG2/sh-control HepG2/sh-RING1 40 HepG2 HCT116 150 HepG2/sh-RING1/sh-p53

) 30 4 120 20 90 10 60 of Percentage apoptotic cells (%) 30 0 sh-control ++++–– –– Figure 6. (10 Cellular number 0 sh-RING1 –– –– ++++ 012345 6 Knockdown of RING1 induces ETO ––++ –– ++ apoptosis, suppresses migration, and Time (day) inhibits tumor growth in vivo. A, knockdown of p53 partially rescued C D the growth inhibitory effect in RING1- depleted cells. Data are presented as HepG2/control HepG2 mean SD, n ¼ 3. , P < 0.05. B, RING1 HepG2/RING1-Flag

) 100 4 knockdown induced apoptosis. Data Time (h) 24 48 72 were presented as mean SD. 80 , P < 0.05. C, Overexpression of RING1 60 enhanced cell proliferation. HepG2 sh-control cells were transfected with pcDNA3.0- 40 RING1-Flag and growth curve was plotted. , P < 0.05. D, RING1 20 knockdown inhibited migration (10 Cellular number sh-RING1 capacity of HepG2 cells. E, RING1 0 01234567 knockdown suppressed HepG2 tumor Time (day) growth in the xenograft cancer model. The tumor growth was recorded twice a week as described in Materials and Methods. , P < 0.05. E

Day 1 4 8 11 15 18 ) 250 2 sh-control 200 sh-RING1

sh-control 150

100 50

sh-RING1 Area of tumor (mm 0 14 811 15 18 Days

in G1 phase (increased from 60.0% to 68.4% in HepG2 cells, (Fig. 6B). In addition, when cells were subjected to both RING1 and from 53.1% to 58.9% in HCT116 cells), suggesting that knockdown and ETO treatment, significantly more apoptosis RING1 deficiency might at least cause partial G1 arrest (Sup- were observed with both HepG2 and HCT116 cells (Fig. 6B), plementary Fig. S4A). suggesting that RING1 depletion did potentiate cellular response However, RING1 knockdown in HCT116 p53 / cells caused to DNA damage response. Markedly more proliferation was asignificantly less inhibitory effect on the proliferation and recorded with HepG2 cells overexpressing Flag-tagged RING1, survival, when compared those with p53 wild-type cells when compared to the cells expressing RING1 endogenously, (Fig. 5A and B). Moreover, little changes of senescence or G1 consistently suggesting a proliferation-promoting function of arrest was observed in HCT116 p53 / cells upon RING1 RING1 (Fig. 6C). depletion (Fig. 5C; Supplementary Fig. S4A). Consistently, RING1 knockdown caused little effect on p21 expression in Knockdown of RING1 inhibits the migration ability of cancer HCT116 p53 / cells (Supplementary Fig. S4B). These data cells clearly suggested a prominent role of p53 in RING1 actions to Metastasis is a key feature of cancer and p53 also plays a pivotal regulate cell cycle, proliferation, or senescence. Supportively, role in regulating metastasis (28). We further used transwell and knockdown of p53 partially and significantly rescued the wound healing assay to investigate the effect of RING1 on tumor growth inhibitory effect of RING1 knockdown in HepG2 cells migration capacity. In Fig. 6D, RING1 depletion significantly (Fig. 6A; Supplementary Fig. S4C). reduced the migration capacity within 72 hours by transwell Furthermore, by FACS analysis after PI/Annexin V staining, assay, and the wound healing ability reduced to 31.5% and 8.3% or 11.9% of HepG2 or HCT116 cells, respectively, were 14.7% at 24 and 72 hours, respectively, in HepG2 cells (Supple- found to undergo apoptosis upon RING1 knockdown, in com- mentary Fig. S5A). A similar result was found in HCT116 cells parison with 1.96% or 4.05% in the respective control groups (Supplementary Fig. S5A).

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RING1 knockdown suppresses the growth of tumor xenograft from some E3 Ub ligases like MDM2 whose expression was in mice transcriptionally regulated by p53, our data indicated that expres- We then assessed the possible effects of RING1 knockdown on sion of human RING1 gene was not affected by p53 (Supple- tumor growth in vivo, by using a HepG2 tumor xenograft mouse mentary Fig. S6A). model. In Fig. 6E, the growth of the tumor deficient in RING1 was Previous work has established that PcG proteins are critically significantly suppressed, as judged by whole-body florescence- involved in regulating tumorigenesis by regulating expression of imaging of the tumor-bearing mice. Final imaging of all the target genes through both polycomb-dependent and -indepen- groups on the 18th day since implanting of tumor cells along dent manners (5). By identifying p53, along with TOP2a (35), as with tumor mass was shown as well (Supplementary Fig. S5B). another non-histone substrate for the E3 Ub ligase activity of Consistent with above observed effect of RING1 depletion on cell RING1, our work has significantly deepened the current under- proliferation, these data from mouse model clearly suggested that standing about the non-polycomb function of RING1 in cancer. knockdown of RING1, which certainly stabilized tumor suppres- Previously, RNF2, another crucial component of PRC1, has been sor p53, significantly inhibits tumor proliferation both in vitro and shown to regulate the homeostatic level of p53, by either directly in vivo. degrading p53, or promoting MDM2-mediated p53 ubiquitination in cancer cells (27, 36). In this work, our data have clearly RING1 is upregulated in clinical hepatocellular carcinoma indicated that RNF2, although a paralog of RING1 that may form specimens compared with adjacent liver tissues complex with RING1, is not absolutely required for the E3 Ub With IHC assay with human hepatocellular carcinoma tissue ligase activity of RING1 towards p53 in the cells (Supplementary array containing 90 cases of paired specimens, as shown in two Fig. S3C; Fig. 4D). Obviously, one could not exclude the possi- representative images in Fig. 7A, much stronger staining for bility that other components such BMI, which was shown to RING1 was observed in the tumors (left) compared with that in promote RING1- and RNF2-mediated ubiquitination (27, 35, adjacent tissues (right). On the basis of the intensities in IHC 37), could also modulate the E3 Ub ligase activity of RING1 staining signals, all samples were classified into five groups with towards p53 in vivo. increasing intensities, with the strongest staining (þþþþ) for Our data demonstrated that depletion of RING1 results in the RING1 in approximately 50% tumor specimens whereas 30% in suppression of cell proliferation and survival, causing G1-phase adjacent tissues (Fig. 7B). RING1 was also assessed at both protein arrest, apoptosis, and senescence, in dependence of endogenous and mRNA levels in eight paired tissues obtained from cancer wild-type p53 (Figs. 5 and 6A). Given the fact that p53, upon patients. As shown in Fig. 7C, both protein and mRNA of RING1 activation, suppresses tumor through regulating the expression of expression were significantly higher in tumor tissues than that of a set of genes in controlling cell-cycle arrest, apoptosis, and adjacent ones. Therefore, it seemed that the static levels of RING1 senescence (38), our findings have strongly suggested that the were predominantly higher in tumors when compared to adjacent biological functions of RING1 could be, at least partially, medi- tissues. ated by p53 pathway. Certainly, this does exclude the possibility Consistently, bioinformatics analysis with hepatocellular car- that, on some occasions, RING1 depletion might lead to cell-cycle cinoma data retrieved from Oncomine database also indicated arrest independent of p53 function, especially when RING1, significantly higher abundance of RING1 in the tumor tissues than along with BMI1, was reported to promote ubiquitination of the in controls (Fig. 7D; ref. 29). Meanwhile, the patients of higher chromatin at the promoters of specific genes to stimulate cell-cycle levels of RING1 (P < 0.001) were also recorded with poorer 5-year progression (39). In addition, the PcG proteins were also shown posttreatment survival, whereas RING1 abundances were also to promote carcinogenesis through bypassing cellular senescence positively correlated with the Barcelona Clinic Liver Cancer Stage due to the transcriptional repression of the INK4b-ARF-INK4a progression (P < 0.05; Fig. 7D). All these data strongly suggested locus (40). Therefore, apart from the direct involvement of p53, that RING1 abundance might be closely associated with liver there is possibility that these epigenetic changes might also cancer progression and poor prognosis. contribute to the observed G1 arrest upon RING1 depletion. This may also account for the partial rescue of growth inhibition upon Discussion p53 and RING1 double knockdown in HepG2 cells, compared with RING1 depletion alone (Fig. 6A). Tumor suppressor p53 plays a pivotal role in the maintenance We made novel observation that RING1 depletion markedly of genome integrity, cell metabolism, and cellular responses to abolished the migration ability of both cancer cells (Fig. 6D; stresses, and primarily undergoes proteasome-mediated degrada- Supplementary Fig. S5A), indicating RING1 might be involved in tion (30). In this study, we have identified RING1 as a novel E3 the regulation of tumor metastasis. Despite the possibly involved ligase to ubiquitinate p53 and target it for degradation, based on role of p53 during the metastasis regulation (28), another piece of the observations: (i) p53 and RING1 protein expression is inverse- work suggested the possible involvement of Snail, a master ly correlated either with or without genotoxic stress (Figs. 1 and 2); regulator of epithelial–mesenchymal transition and metastasis (ii) RING1 negatively regulates p53 protein half-life (Fig. 2); (iii) in cancer, based on observations that RING1, along with RNF2, is By interacting with p53, RING1 promotes its polyubiquitination capable of interacting with Snail and is required for Snail-medi- and degradation in vivo and in vitro (Figs. 3 and 4). Our work has ated target gene repression and cell migration in pancreatic cancer thus added RING1 to the already existed list of E3 Ub ligases for cells (41). p53 ubiquitination, including MDM2, COP1, and Pirh2 (30–34). Notably, high levels of RING1 proteins were widely observed in It is both intriguing and important to understand how the hepatocellular carcinoma specimens but not in the normal tis- activities of these E3 Ub ligases are delicately orchestrated to sues, suggesting that potential association between RING1 ampli- control the homeostasis and functionality of p53 in both tumoral fication and hepatocellular carcinoma pathology, based on Onco- and nontumoral settings. It is interesting to note that, different mine database analysis (Fig. 7). It is interesting to note that, high

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RING1 Degrades p53 and Promotes Cancer Survival

A Liver cancer B Tumor tissues Adjacent tissues

20× 200× 20× 200× RING1 60 Tumor tissues Adjacent tissues 45/90 1# 40 30/90 27/90 22/90 20 15/90 11/90 11/90 Percentage (%) 2# 7/90 7/90 5/90

0 + / – + ++ +++ ++++ 300 mm

600 C 1 2 34 5 678 TATATATA TATATATA 400

RING1 200

GAPDH 0 Relative density (% of control) 1 234 56 78 TA T A T A T A T A T A T A T A T A 100 RING1 80 60 GAPDH 40 20 0 TA Relative density (% of control)

D 1.0 1.0

0.5 0.5

0.0 0.0

–0.5 –0.5

median centered intensity Liver (75) median centered intensity Alive at 5 years (12) 2 2 –1.0 Hepatocellular carcinoma (138) –1.0 Dead at 5 years (40) Log Log

0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 –0.2 –0.2 –0.4 –0.4 Barcelona Clinic Liver Cancer Stage A (16)

Liver (86) median centered intensity median centered intensity 2

2 Barcelona Clinic Liver Cancer Stage B (4) Hepatocellular carcinoma (99) –0.6

Log Barcelona Clinic Liver Cancer Stage C (4) Log

Figure 7. Overabundance of RING1 in human hepatocarcinorma specimens. A, Representative images for IHC staining of RING1 in tumor and adjacent tissues, left and right panels, respectively. B, Classification of RING1 staining intensities in tissue assay of human HCC tumor array. C, Protein (top) and mRNA (bottom) levels of RING1 in paired samples from eight HCC patients. The band intensities were quantified with ImageJ and plotted. T, tumor tissue; A, adjacent tissue. , P < 0.05. D, Stratified data analysis of RING1 copy number retrieved from Oncomine datasets. , P < 0.05; , P < 0.001.

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Shen et al.

RING1 levels seem to be more enriched in tumors with wild-type Acquisition of data (provided animals, acquired and managed patients, p53 according to the data retrieved from Oncomine database provided facilities, etc.): J. Shen, P. Li, X. Liu, Z. Wang (Supplementary Fig. S6B). In light of our data that overexpression Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Shen, P. Li, X. Shao, Y. Yang, Q. Yu, R. Hu, Z. Wang of RING1 indeed promoted the proliferation of liver cancer cells Writing, review, and/or revision of the manuscript: J. Shen, Q. Yu, R. Hu, (Fig. 6C), it is possible that RING1-targeted therapeutic interven- Z. Wang tions might help restore the function of p53 to suppress tumor. Administrative, technical, or material support (i.e., reporting or organizing Moreover, as RING1 destabilizes endogenous p53 protein level data, constructing databases): M. Feng, Z. Wang upon genotoxic stress and RING1 depletion potentiates DNA Study supervision: R. Hu, Z. Wang damage responses (Figs. 2C and 6B), RING1-targeted therapeutic Acknowledgments strategy may also be employed in combination with DNA-dam- This project is sponsored by the National Natural Science Foundation of aging agents to potentiate the antitumor effect of chemotherapy. China (nos. 81572752, 81373438, 81321004 to Z. Wang) and CAMS Innova- Taken together, here we have presented strong evidence that tion Fund for Medical Sciences (2016-I2M-2-002 to Z. Wang). We thank Prof. RING1 might serve as a yet another E3 Ub ligase for p53 in Tan Jing in Zhongshan University (Guangzhou, China) for helpful discussions regulating human cancer. RING1 is thus emerging as a potential in the project. biomarker and target for cancer prognosis and treatment.

Disclosure of Potential Conﬂicts of Interest The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ﬂ No potential con icts of interest were disclosed. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: J. Shen, R. Hu, Z. Wang Received June 22, 2017; revised October 11, 2017; accepted November 21, Development of methodology: J. Shen, X. Shao, Y. Yang, Z. Wang 2017; published OnlineFirst November 29, 2017.

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www.aacrjournals.org Cancer Res; 78(2) January 15, 2018 OF13

The E3 Ligase RING1 Targets p53 for Degradation and Promotes Cancer Cell Proliferation and Survival

Jiajia Shen, Pengyu Li, Xuejing Shao, et al.

Cancer Res Published OnlineFirst November 29, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-1805

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