RNF2/Ring1b Negatively Regulates P53 Expression in Selective Cancer Cell Types to Promote Tumor Development

RNF2/Ring1b negatively regulates p53 expression in selective cancer cell types to promote tumor development

Wen-jing Sua,b, Jun-shun Fanga, Feng Chenga,b, Chao Liua, Fang Zhoua, and Jian Zhanga,1

aState Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology and bGraduate School, Chinese Academy of Sciences, Beijing 100101, China

Edited by Carol Prives, Columbia University, New York, NY, and approved December 19, 2012 (received for review July 15, 2012)

Large numbers of studies have focused on the posttranslational evolution in some invertebrates, such as Caenorhabditis elegans regulation of p53 activity. One of the best-known negative regu- and Drosophila melanogaster, suggesting that other factors (in- lators for p53 is MDM2, an E3 ubiquitin ligase that promotes p53 cluding other E3 ligases) might be involved in controlling p53 degradation through proteasome degradation pathways. Additional activities in these animals. For example, Bonus is a homolog of E3 ligases have also been reported to negatively regulate p53. How- TRIM 24, a p53 ligase in vertebrates. The results from loss-of- ever, whether these E3 ligases have distinct/overlapping roles in function studies indicated that Bonus is critical for maintaining the regulation of p53 is largely unknown. In this study, we identify p53 activity in Drosophila, suggesting that Bonus might be an RNF2 (ring finger protein 2) as an E3 ligase that targets p53 for evolutionarily conserved E3 ligase for p53 (14). The existence of multiple E3 ligases for p53 strongly suggests that specialization degradation. The E3 ligase activity of RNF2 requires Bmi1 protein, among them must be required for controlling p53 at multiple a component of the polycomb group (PcG) complex. The up-reg- levels in different regulatory programs. ulation of p53 does not affect RNF2 expression. Unlike Mdm2, In addition to genomic alterations, epigenetic changes have RNF2 only degrades p53 in selective cell lines, such as those from been increasingly associated with tumor development. Cross-talk RNF2 germ-cell tumors. The knockdown of induces apoptosis, which between genetic and epigenetic regulations has also been docu- can be rescued through the reduction of p53 expression. Moreover, mented. The polycomb group (PcG) proteins have long been the down-regulation of RNF2 expression in germ-cell tumors signif- considered as one of the epigenetic regulators for gene silencing icantly reduces tumor cell growth, while the simultaneous down-reg- during embryonic development and tissue homeostasis in adult ulation of both genes restores tumorcellgrowthinvitroandintumor life (15, 16). Polycomb proteins exist in at least two multimeric xenograft models. Furthermore, a reverse correlation between RNF2 nuclear protein complexes: polycomb repressor complex 1 (PRC1) and p53 expression was detected in human ovarian cancer tissues. and PRC2 (17). The core human PRC1 consists of Polycomb Together, these results indicate that RNF2 is an E3 ligase for p53 (PC), polyhomeotic (PH), Bmi1, Ring1a and Ring1b (Rnf2) (18), degradation in selective cells, implicating RNF2 as a therapeutic target while PRC2 contains EED, EZH2, and SUZ12 (19). The gene to restore tumor suppression through p53 in certain tumor cells. regulation through PC complexes involves the trimethylation of histone H3 at lysine 27 catalyzed through the activity of PRC2 (20), which in turn recruits the PRC1 complex to chromatin to nder deleterious conditions or during DNA damage, p53 mediate histone H2A monoubiquitination (21). As the primary E3 Uactivity will be up-regulated to inhibit cell cycling or to ligase responsible for H2A modification in the PRC1 complex, promote apoptosis, two major events associated with the tumor Rnf2 plays important roles in both early development and ES cell suppressor function of p53 (1, 2). These two activities ensure that maintenance (22). In addition to its monoubiquitination activity, an individual organism will either initiate the repair of damaged the PRC1 complex also has poly-ubiquitination activity. For ex- cells or eliminate them. During normal cell growth, p53 is main- ample, PRC1 poly-ubiquitinates Geminin, a DNA replication in- tained at extremely low levels through various negative regulatory hibitor, to sustain adult hematopoietic stem cell activity (23). Pro- mechanisms. One of these mechanisms occurs through the 26S tein structure analysis revealed that within the PRC1 complex, proteasome in which a number of ubiquitin-protein ligases are Bmi1 and RNF2 heterodimerize via their N-terminal RING do- involved. Poly- and monoubiquitin can be covalently conjugated to mains to form an active E3 ubiquitin ligase (24–26). The over- p53 through the lysine amino acid residues (K48 versus K63) of expression of Rnf2 has also been documented in clinical gas- ubiquitin (3, 4). The principal E3 ligase for p53 is Mdm2, which trointestinal tumors and lymphomas (27). Despite these findings, itself is a product of p53-inducible genes. The importance of the the mechanisms of Rnf2 in tumor formation remain poorly p53–Mdm2 autoregulatory loop was clearly demonstrated in a understood. study in which the embryonic lethality induced through an Mdm2 Here, we report that p53 is an endogenous binding partner for deletion was rescued through the simultaneous knocking-out of Rnf2 and that Rnf2 functions as an E3 ubiquitin ligase for p53. the p53 gene (5, 6). Additional studies showed that Mdm2 also Rnf2 ubiquitinates and promotes p53 degradation both in vitro affects the transcriptional potential and subcellular distribution of and in vivo. Moreover, the ubiquitination activity of Rnf2 re- p53 (7–11), highlighting the complex nature of p53 regulation. quires the presence of Bmi1. In contrast to reported E3 ligases Furthermore, subsequent investigations uncovered ∼20 additional for p53, Rnf2 only regulates p53 levels in selected tumor cell E3 ligases that regulate p53 activity, including COP1, Pirh2, ARF- types. Furthermore, in a xenograft mouse model, the reduction BP1, CHIP, TOPORS, Synoviolin, CARP1, CARP2, and Trim24, of Rnf2 significantly reduced tumor cell growth, while the which negatively regulate p53 stability through degradation, and ICP0, CUL7, MSL2, WWP1, Ubc13, and E4F1, which affect p53 subcellular localization, transcription activity, tetramerization, Author contributions: W.-j.S. and J.Z. designed research; W.-j.S. performed research; and so forth (3, 12, 13). These findings suggest that both Mdm2- W.-j.S., J.-s.F., F.C., C.L., and F.Z. contributed new reagents/analytic tools; W.-j.S. and dependent and Mdm2-independent mechanisms are involved J.Z. analyzed data; and W.-j.S. and J.Z. wrote the paper. in regulating p53 activity. However, whether these ligases act re- The authors declare no conflict of interest. dundantly and regulate p53 in specific cell types or function under This article is a PNAS Direct Submission. particular contexts remains unclear. Moreover, although p53 1To whom correspondence should be addressed. E-mail: [email protected]. orthologs and the structural conservation of p53 exist across This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. invertebrates, the Mdm2 gene might have been lost during 1073/pnas.1211604110/-/DCSupplemental.

1720–1725 | PNAS | January 29, 2013 | vol. 110 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1211604110 Downloaded by guest on September 29, 2021 simultaneous depletion of p53 restored tumor growth. Our study of p53 messenger RNA (mRNA) (Fig. 1 D and E and Fig. S1B). A not only reveals a p53 E3 ligase but also provides a potential number of genes, including p21, Mdm2, Bax, and Puma, were therapeutic target in therapies for tumors in which the p53 level positively regulated at the transcriptional level through p53 (29). is specifically regulated through Rnf2. Consistent with this result, the mRNA levels of the target genes were greatly increased in Rnf2 knock-down cells (Fig. 1E). The Results p53-mediated up-regulation of the target genes was further con- Identification of Rnf2 as a Negative Regulator of p53 in Selective firmed through the reversion of the increased gene transcriptions Cells. Previous studies have indicated that Rnf2 expression is upon the depletion of p53 in Rnf2 knock-down cells (Fig. 1E). increased in a large variety of tumors (27). As a first step to To further determine the role of Rnf2 in regulating p53 levels, characterize the molecular mechanisms of Rnf2 in tumor for- we overexpressed Rnf2 in Tera-1 cells and the colon cancer cell mation, we attempted to identify the binding partners of this line HCT116, which has greatly decreased endogenous p53 protein. In a yeast two-hybrid screen using Rnf2 as the bait, we protein levels. As a negative control, the overexpression of an identified p53 as a major binding partner. We confirmed the Rnf2 mutant, lacking the Ring domain, did not affect the level of interaction of these proteins in Tera-1 cells, a human testicular p53 (Fig. 1F and Fig. S1C). Moreover, the reduction of the p53 germ-cell-tumor-derived cell line, by coimmunoprecipitation expression could be blocked using the proteasome inhibitor (Fig. 1 A and B). MG132, suggesting that Rnf2 regulates p53 levels through pro- To determine whether the interaction between Rnf2 and p53 fi teasome-dependent degradation. We also examined the degra- is direct, we generated and puri ed recombinant Rnf2 and p53 dation kinetics of p53 in the presence of the protein translation and tested their direct binding in vitro. Purified His-p53 bound to – inhibitor cycloheximide (CHX) in Tera-1 cells. The rate of p53 GST Rnf2 in a cell-free system, indicating a direct interaction protein degradation was clearly reduced in Rnf2 knockdown between Rnf2 and p53 (Fig. 1C). Next, we used an immuno- cells (Fig. 1 G and H). To extend this observation of the negative precipitation approach to map the regions of p53 required for Rnf2-mediated regulation of p53, we repeated the Rnf2 RNAi Rnf2 binding through combining various fragments of p53 with full-length Rnf2 in an in vitro translated system. The results experiments in additional cell lines. Although the p53 expression – increased when the Rnf2 expression was down-regulated in Tera-1, revealed that the DNA binding domain of p53 (AA94 AA292) fi contributes to the Rnf2–p53 interaction (Fig. S1A). Together, Tera-2, MCF-7 cell lines, and mouse embryonic broblasts these results indicate that Rnf2 is a unique p53 binding partner (MEFs), the p53 expression was not affected in transfected HEK and suggest that Rnf2 might regulate p53 activity in vivo. Rnf2 293 cells, HeLa cells, or the human ovary malignant teratoma functions as an E3 ubiquitin ligase to monoubiquitinate sub- cell line PA-1 (Fig. 2 A and B and Fig. S2A). The knockdown of strates, such as H2A (21). More recently, Rnf2 was also shown Rnf2 in Tera-1 cells markedly increased p53 expression in the to poly-ubiquitinate some of its substrates. For example, Rnf2 nucleus, while the overexpression of Rnf2 in Tera-1 and HCT116 mediates PGC-1α degradation in cells under metabolic stress cells reduced the nuclear p53 protein levels (Fig. 2 A, C, and D). to p53-mediated starvation response in vivo (28). To investigate In contrast, Rnf2 overexpression in HEK 293 cells did not affect whether the binding of Rnf2 regulates p53 activity, we used Rnf2- p53 levels. As a control, Mdm2 effectively degraded p53 in HEK specific short hairpin RNAs (shRNAs) to reduce the Rnf2 pro- 293 cells (Fig. 2E). These results demonstrate that, in contrast tein level in Tera-1 cells. The knock-down of Rnf2 using either to the primary p53 E3 ligase Mdm2, Rnf2 only down-regulates CELL BIOLOGY of two independent shRNAs resulted in marked increases in the p53 in specific cell lines, suggesting that Rnf2 has nonredundant levels of endogenous p53 protein, without affecting the expression functions in regulating p53-mediated activities in selective tissues.

Fig. 1. Rnf2 negatively regulates p53 stability. (A and B) Rnf2 interacts with p53 in vivo. Tera-1 cell lysates were subject to immunoprecipitation with anti-IgG, anti-p53 (A), or anti-Rnf2 (B) antibodies. The immunoprecipitates were subsequently blotted with the indicated antibodies. (C) Rnf2 interacts with p53 in vitro. p53 was puriﬁed from bacteria and incubated with GST or GST–Rnf2 coupled to GSH–Sepharose. The proteins retained on the Sepharose beads were subsequently blotted with the indicated antibodies. (D and E) The shRNA- mediated depletion of Rnf2 increases p53 protein levels and induces p53 target gene activation. Tera- 1 cells were transfected with the indicated shRNAs. After 72 h, the proteins and mRNA were extracted and subjected to Western blot or quantitative RT- PCR. The error bars represent the SEM of triplicate experiments. ***P < 0.001 two-tailed Student t test. (F) Overexpression of Rnf2 suppresses p53 protein level. HCT116 cells transfected with the indicated constructs were left untreated or treated with MG132. The proteins were extracted and subjected to Western blot analysis. (G and H) Rnf2 depletion increases p53 stability. Tera-1 cells transfected with the indicated shRNA constructs were treated with CHX (0.1 mg/mL) and harvested at the indicated time. The cell lysate was subjected to Western blot analysis. (G) Immunoblots of p53 and Rnf2. (H) Quantiﬁcation of the p53 protein levels relative to beta-actin.

Su et al. PNAS | January 29, 2013 | vol. 110 | no. 5 | 1721 Downloaded by guest on September 29, 2021 Fig. 2. Rnf2 regulates the p53 levels in selective cells. (A and B) Rnf2 knockdown increases p53 protein levels in Tera-1 cells but not in HEK 293 cells. Tera-1 and HEK 293 cells transfected with the indicated constructs were treated with puromycin (1 μg/μL). After 72 h, the cells were harvested and fractionated into cytosolic and nuclear fractions. The fractions were subsequently blotted with the indicated antibodies. Lamin B, a nuclear marker, and tubulin, a cytosolic marker, were used as loading controls. (C) Rnf2 knockdown causes nuclear p53 up-regulation. Tera-1 cells transfected with the indicated constructs were ﬁxed and stained as described. (D and E) Rnf2 overexpression represses nuclear p53 protein levels in speciﬁc cell types. HCT116 and HEK 293 cells transfected with the indicated constructs were harvested and fractionated. The cellular fractions were subsequently blotted with the indicated antibodies. LE, long exposure; SE, short exposure.

Rnf2 Is an E3 Ligase That Ubiquitinates p53. Previous studies examined whether Rnf2 is essential for tumor cell proliferation revealed that Rnf2 acts as an E3 ubiquitin ligase (21, 23, 28). in vitro, by knocking down Rnf2 in cultured Tera-1 cells. The cell Various E3 ligases regulate p53 activity through direct degra- proliferation results indicated that Rnf2 is indispensable for dation, changes in subcellular localization, or the alteration of Tera-1 cell growth (Fig. 4A and Fig. S4). The requirement of transcription activity (3, 10, 12). We investigated the possibility Rnf2 for colony formation was also confirmed in a separate tu- of Rnf2 to directly degrade p53 through the proteasome path- mor cell growth assay (Fig. 4 B and C). way. First, we examined whether Rnf2 can ubiquitinate p53. To A number of mechanisms might underlie the roles of Rnf2 in this end, we overexpressed Rnf2 in HCT116 cells and detected tumor cell proliferation. Because Rnf2 directly targets p53 for the endogenous p53 ubiquitination levels in the presence of the degradation, we investigated the requirements of Rnf2 for cell proteasome inhibitor MG132. The Rnf2 overexpression indeed cycle arrest and apoptosis, two major events regulated through markedly induced nuclear p53 poly-ubiquitination (Fig. 3A and p53. Indeed, the down-regulation of Rnf2 caused G1 arrest in Fig. S3 A and B). The Ring domain of Rnf2 and phosphorylation Tera-1 cells (28% G0/G1 in Rnf2 shRNA-transfected cells at S41 are indispensable for the activity, as Rnf2ΔRing, Rnf2I53A compared with 17% G0/G1 cells in control shRNA-treated cells) (a Ring domain mutation, lacking the ability to interact with E2 (Fig. 4D). More importantly, silencing Rnf2 and p53 together ligase) and Rnf2S41A mutants (30, 31) failed to increase the through RNA interference showed a marked reversal in G1 ar- ubiquitination levels of p53 (Fig. 3 A and D and Fig. S3A). rest (Fig. 4D). Furthermore, we also examined whether Rnf2 Conversely, the down-regulation of Rnf2 reduced the basal level affects p53-dependent apoptosis. Terminal transferase dUTP of p53 ubiquitination (Fig. 3C). In contrast, Rnf2 overexpression nick-end labeling (TUNEL) and Annexin V staining were used in HEK 293 cells did not up-regulate p53 ubiquitination levels to detect apoptotic cells. Rnf2 depletion greatly increased apo- (Fig. 3B), which is consistent with the previous observation that ptosis in Tera-1 cells, while the coexpression of p53 shRNA re- Rnf2 failed to reduce the p53 protein levels in these cells. versed the number of apoptotic cells to the level of untreated Although Rnf2 ubiquitinates p53, these results did not dis- cells (Fig. 4 E and F). Collectively, these results demonstrate that tinguish the direct or indirect ubiquitination of p53 through Rnf2 antagonizes p53 functions in Tera-1 cells. Rnf2. To resolve this issue, we investigated the ubiquitination activity of Rnf2 toward p53 using a cell-free system. Bmi1 was Rnf2 Is Required for Ovarian Cancer Growth. Our results strongly previously demonstrated to be essential for the E3 ligase activity indicate that Rnf2 plays key roles, through p53, in controlling of the PRC1 complex (32). We used immuno-purified Rnf2 testicular cancer cell proliferation. To further investigate whether or Rnf2I53A translated in vitro and preincubated these samples Rnf2 is required for other types of tumors, particularly those separately with recombinant Bmi1. Similar to previous reported derived from the reproductive system, we also explored whether studies, the purified Rnf2, but not catalytically deficient Rnf2I53A, Rnf2 regulates p53 in ovarian cancer cells. As expected, p53 was promotes p53 ubiquitination in the presence of Bmi1 in vitro greatly up-regulated, even when Rnf2 was partially depleted in (Fig. 3D). The in vitro ubiquitination is less profound compared two ovarian cancer cell lines, A2780 and HO-8910, in which p53 with that observed in vivo, suggesting that additional factors gene is not mutated (Fig. 5A). The depletion of Rnf2 in A2780 might be required for efficient ubiquitination and are lacking in suppressed the cancer cell proliferation, while p53 knockdown the in vitro system. We also observed that the Bmi1 knockdown restored the cell growth rate resulting from the loss of Rnf2 (Fig. in HCT116 cells blocked Rnf2-induced p53 ubiquitination (Fig. S5 B–E). We further evaluated Rnf2 expression levels in clinical S3C). These results indicate that Rnf2, in the presence of Bmi1, ovarian tumor tissues using two different Rnf2 antibodies. Tissue can directly ubiquitinate p53 in vitro and is required for the array analysis revealed that Rnf2 is up-regulated in 88% of the ubiquitination in vivo. ovarian serous cystoadenomas, 90% of ovarian mucinous carcinomas, and 60% of clear cell carcinomas. In contrast, Rnf2 ex- Rnf2 Negatively Regulates p53-Mediated Biological Functions in Tera- pression was barely detectable in normal ovarian tissues (Fig. 5B 1 Cancer Cells. Rnf2 shows increased expression in a high per- and Table S1). Interestingly, we observed a reverse correlation centage of various tumors compared with normal tissue coun- between Rnf2 overexpression and low p53 expression in over terparts (27). Because we identified Rnf2 as a direct negative 60% (44/72) of ovarian serous cystoadenomas and 90% (9/10) of regulator of p53, we postulated that Rnf2 might act as an onco- ovarian mucinous carcinomas (Table S2), suggesting that Rnf2 protein in specific tissue types to antagonize p53 functions and might negatively control p53 levels in these cancer cells. The to promote tumor formation. To test this hypothesis, we first down-regulation of p53 expression in these cells is unlikely

1722 | www.pnas.org/cgi/doi/10.1073/pnas.1211604110 Su et al. Downloaded by guest on September 29, 2021 regulation of p53 affects cell cycle progression and apoptosis controlled by p53; (iii) Rnf2 targets nuclear p53 for degradation in speciﬁc cell types, such as testicular cancer cells and ovarian tumors; and (iv) Rnf2 is overexpressed in most clinical ovarian tumors. Thus, this study illustrates a unique activity for the PcG complex in cancer development. The PcG genes have been implicated in embryonic development and stem cell self-renewal. As a core member of the PRC1 complex, Rnf2 is up-regulated in various tumors. However, the mechanism of Rnf2 in cancer development remains poorly understood. Previously, Rnf2 was shown to repress the transcription of p16Ink4a, which mediates Rnf2 activities in cancer cells

Fig. 3. Rnf2 ubiquitinates p53 both in vivo and in vitro. (A and B) Rnf2 promotes p53 ubiquitination in vivo. HCT116 and HEK 293 cells transfected with the indicated constructs were treated with MG132 for 4 h before har-

vest. p53 was immunoprecipitated with anti-p53 polyclonal antibodies and CELL BIOLOGY immunoblotted with monoclonal anti-HA or anti-p53 antibodies. (C) Rnf2 depletion reduces p53 ubiquitination in vivo. Tera-1 cells transfected with the indicated constructs were treated with MG132 for 4 h before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-HA or anti-p53 antibodies. (D) Phos- phorylation-deﬁcient Rnf2 lacks E3 ligase activity. HCT116 cells transfected with the indicated constructs were treated with MG132 for 4 h before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-HA or anti-p53 antibodies. (E) Rnf2 ubiquitinates p53 in vitro. 35S-labeled p53 was incubated with puriﬁed Rnf2 or Rnf2I53A in the presence of Bmi1 or Bmi1Δ1–44 in vitro. The reactions were subsequently analyzed through autoradiography.

caused by Mdm2 overexpression, as most of the adjacent sections showed no Mdm2 up-regulation compared with normal tissue (Table S3). We established that Rnf2 is critically required for ovarian Fig. 4. Rnf2 regulates p53-mediated cell growth arrest and apoptosis. (A) Rnf2 regulates cell growth. Tera-1 cells transfected with the indicated con- cancer cell proliferation in vitro and that Rnf2 overexpression is structs were trypsinized and plated after puromycin treatment. The cell correlated with tumor formation (Fig. 5B and Fig. S5 A and B). numbers were subsequently counted at the indicated times. (B and C) Col- However, direct evidence that Rnf2 is required for tumor cell ony-formation assay was performed with Tera-1 cells transfected with con- survival in vivo has not yet been provided. Thus, we next in- trol shRNA or Rnf2 shRNA. (B) Tera-1 cells transfected with the indicated vestigated the in vivo activity of Rnf2 on the growth of A2780 constructs were plated at a density of 1×106 cells/plate, and the clone tumors in a xenograft model. The suppression of Rnf2 activity numbers were quantified at 3 wk after puromycin treatment. (C) Quantifi- through RNA interference largely inhibited the growth of xeno- cation of colonies formed in B. The error bars represent the SEM of triplicate graft tumors (Fig. 5 D–F). The reduction of tumor volumes/masses experiments. **P < 0.01 two-tailed Student t test. (D) Down-regulation of was dependent on p53 function, as knocking down both Rnf2 and Rnf2 causes G1 arrest in Tera-1 cells. Tera-1 cells transfected with the in- p53 expression reversed tumor growth to the level of the control dicated constructs were treated with puromycin for 3 d. The cells were – harvested, and the cell-cycle profiles were determined through FACS anal- group (Fig. 5 D F). These results strongly indicate that Rnf2 is ysis. (E and F) Rnf2 knockdown increases apoptosis in Tera-1 cells. Tera-1 cells a p53-dependent key player in ovarian tumor progression. were transfected with the indicated constructs. After 3 d of puromycin treatment, the apoptotic cells were visualized and counted. (E) Apoptotic Discussion cells were detected through TUNEL staining. (F) Quantification of apoptotic This study showed that (i) Rnf2, a binding partner for p53, ubiq- cells detected using FACS analysis. The error bars represent the SEM of uitinates p53 for degradation; (ii) the negative Rnf2-mediated triplicate experiments. **P < 0.01; *P < 0.05 two-tailed Student t test.

Su et al. PNAS | January 29, 2013 | vol. 110 | no. 5 | 1723 Downloaded by guest on September 29, 2021 Fig. 5. Regulation of Rnf2 in ovarian carcinoma cell growth depends on p53. (A) Rnf2 depletion up-regulates p53 protein levels in human ovarian carcinoma cell lines (A2780 and HO-8901 bearing wild-type p53). (B) Immuno- histochemical staining of Rnf2 in normal ovarian tissues and ovarian carcinoma. (C–F) Effects of Rnf2 depletion on A2780 cell growth and tumor development. A2780 cells stably expressing the indicated shRNAs (GFP-labeled) were injected into nude mice. After 4 wk of injection, the animals were euthanized and analyzed for tumor growth. (C) Protein expression in A2780 cells stably expressing the indicated shRNAs. (D, Upper) Representative photographs captured with visible light of the animals corresponding to each treatment group at day 20 after tumor cell injection. (Lower) Noninvasive visualization of GFP-tagged tumor cells by whole-body fluorescence imaging showing a significant reduction in tumor size when Rnf2 is depleted. (E) Comparative analysis of the localized tumor growth; bars, SE. **P < 0.01; ***P < 0.001, statistically significant compared with the control shRNA group. (F) Estimated tumor weight for the indicated groups at day 20 after injection. **P < 0.01, statistically significant compared with the control or rescue groups.

(22, 33, 34). However, in the present study, Rnf2 directly de- ligases for p53, Rnf2 is not a transcriptional target of p53 (Fig. graded p53 through the proteasome pathway. We also showed S2D). Moreover, Rnf2 represses nuclear p53 levels in a cell-type- that Rnf2 targets nuclear p53 for degradation in testicular cancer specific manner. In contrast to Mdm2, the overexpression/de- cells and ovarian tumors. The regulation of p53 through Rnf2 pletion of Rnf2 did not affect p53 degradation in HEK 293 cells, affects cell cycle progression and apoptosis controlled by p53. To although the association of Rnf2 and p53 was detected in these confirm its role in vivo, we also showed that Rnf2 is overex- cells (Fig. S2B). Similarly, cell lines, including PA-1 and HeLa pressed in most clinical ovarian tumors and is required for tumor cells, are insensitive to Rnf2 depletion. Conversely, in addition growth in a mouse xenograft model. to Tera cells, MCF-7, HO-8910, and A2780 are also responsive to Approximately 20 E3 ubiquitin ligases for p53 have been iden- Rnf2 depletion, which resulted in the up-regulation of p53. The tified (12). Thus, overlapping/redundant roles for these E3 liga- Rnf2-mediated degradation of p53 in specific cell lines could be ses have been proposed; however, the contributions of the in- achieved through various mechanisms. For example, the unique dividual ligases to the specific physiological/pathological processes functional composition of the PRC1 complex might exist in the remain undetermined. We showed that Rnf2 cooperates with the reactive cells, while p53 degradation-resistant cells might lack key E2 enzyme UbcH5c to mediate the attachment of ubiquitin to functional components of the PRC1 complex, or these compo- p53 in the presence of Bmi1 (Fig. 3E). Notably, p53 ubiquiti- nents might be inaccessible to p53. However, no obvious negative nation in the in vitro assay is less efficient compared with that correlation was observed between Rnf2 and p53 expression levels in vivo (Fig. 3). We suspect that additional components might be in some cancer cell lines (Fig. S2C), suggesting that the expression required for efficient p53 polyubiquitination mediated by Rnf2. of Rnf2 is not a determining factor in the regulation of p53 levels Similar observations have been previously reported. In the pres- in these cells. ence of Bmi1, Rnf2 activity was enhanced to monoubiquitinate We also investigated whether the regulation of Rnf2 phos- H2A (32). Rnf2 was also shown to promote PGC-1 alpha degra- phorylation plays a role in its E3 ligase activity toward p53. The dation and to indirectly regulate p53 functions in cells responsive phosphorylation of 41Ser through p38/MAPK was previously to stress (28). The results obtained in the present study expand the considered as critical for Rnf2 functions (31). Our results showed current understanding of the molecular functions of PcG complex that a mutated Rnf2 (S41A) did not ubiquitinate p53, suggesting 1. The ability of Rnf2 to regulate p53 through direct ubiquitination that the E3 ligase activity is dependent on the phosphorylation adds another E3 ligase to the array of known negative regulators of 41Ser (Fig. 3D). We used immuno-purified Rnf2 and Bmi1 in for p53. Unlike Mdm2, Pirh2, or other previously identified E3 in vitro ubiquitination experiments (Fig. 3E). The nature of the

1724 | www.pnas.org/cgi/doi/10.1073/pnas.1211604110 Su et al. Downloaded by guest on September 29, 2021 in vitro translation system prevents us from excluding other re- Rnf2 is not significantly involved in regulating p53 activity, while sidual factors in the extract from participating in the ubiquitina- in some tumor cells, the overexpression of Rnf2 antagonizes p53 tion reaction. Further analysis is required to completely address through degradation and thus functions as an oncogene. Our the regulation of Rnf2 E3 activity toward p53. findings might provide an additional option for therapeutic in- Because p53 is the major protector of genome integrity, p53 tervention, which might restore the p53 response in selective activation in cancer cells can trigger tumor regression, whereas tumor cells. the misregulation of the negative regulators of p53 plays a role in tumor formation/progression (35–37). Indeed, small molecules Materials and Methods targeted to the interface of p53 and major negative regulators The Institute of Genetics and Developmental Biology Animal Care and Ethics of p53, such as Mdm2, have been examined for the inhibition of Committee approved all experimental procedures involving the immuno- tumor progression through the restoration of the apoptotic func- deficient mice. The experiments were conducted according to the principles tion of p53 (38, 39). Similar to Mdm2, through its regulation of expressed in the Declaration of Helsinki. BALB/c Nude mice were maintained p53, Rnf2 promotes tumor progression. In this study, we revealed under pathogen-free conditions and used at 6–10 wk of age. To analyze the up-regulation of Rnf2 in a high percentage of human ovarian localized ovarian tumor growth in vivo, 1×106 GFP-expressing A2780 cells carcinoma samples. The p53 down-regulation in these samples stably transfected with the indicated shRNA constructs were injected s.c. in was correlated with Rnf2 up-regulation. Surprisingly, p53 muta- both rear flanks (n = 16 tumors per experimental condition). The in vivo tions did not affect the Rnf2-mediated degradation of p53, as the imaging of tumor cells was performed using an IVIS Spectrum fluorescence overexpression of Rnf2 can ubiquitinate four p53 mutants isolated light system (Caliper), and the light imaging was captured with a Canon EOS from human tumors (Fig. S5F). This result suggests that Rnf2 600D camera. The volume of the s.c. xenograft was calculated as V = L × W2/ negatively regulates both wild-type and mutated p53. We observed 2, where L and W stand for tumor length and width, respectively. that Rnf2 might bind to multiple regions in the DNA binding Detailed descriptions of other experimental procedures are provided in domain of p53, which might partially explain why single point the SI Text. mutants do not significantly diminish the interaction between p53 and Rnf2. The significance of the degradation of mutant p53 ACKNOWLEDGMENTS. The authors thank Drs. Quan Chen, Qinmiao Sun, through Rnf2 remains unknown. Previous studies have shown Dahua Chen, Shengcai Lin, and Michelle Craig Barton for reagents and that the increased expression of Bmi1 and Rnf2 are associated helpful discussions. The authors also thank Dr. Haruhiko Koseki for the Rnf2 conditional knockout mice. This work was financially supported through with tumor cell transformation in a number of tumors (27, 40). grants from the Ministry of Science and Technology of China (2011CB943800; Our results indicate that Rnf2 is required for tumor progression 2013CB945000), the Chinese Academy of Sciences (XDA01010108), and in a p53-dependent manner. We propose that in normal cells, National Natural Science Foundation of China (30425013).

1. Prives C, Hall PA (1999) The p53 pathway. J Pathol 187(1):112–126. 22. Vidal M (2009) Role of polycomb proteins Ring1A and Ring1B in the epigenetic 2. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408(6810): regulation of gene expression. Int J Dev Biol 53(2-3):355–370. 307–310. 23. Ohtsubo M, et al. (2008) Polycomb-group complex 1 acts as an E3 ubiquitin ligase for 3. Brooks CL, Gu W (2006) p53 ubiquitination: Mdm2 and beyond. Mol Cell 21(3): Geminin to sustain hematopoietic stem cell activity. Proc Natl Acad Sci USA 105(30): – 307 315. 10396–10401. 4. Chen ZJ, Sun LJ (2009) Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 24. Bentley ML, et al. (2011) Recognition of UbcH5c and the nucleosome by the Bmi1/ – 33(3):275 286. Ring1b ubiquitin ligase complex. EMBO J 30(16):3285–3297. 5. Jones SN, Roe AE, Donehower LA, Bradley A (1995) Rescue of embryonic lethality in 25. Buchwald G, et al. (2006) Structure and E3-ligase activity of the Ring-Ring complex of CELL BIOLOGY Mdm2-deficient mice by absence of p53. Nature 378(6553):206–208. polycomb proteins Bmi1 and Ring1b. EMBO J 25(11):2465–2474. 6. Montes de Oca Luna R, Wagner DS, Lozano G (1995) Rescue of early embryonic le- 26. Li Z, et al. (2006) Structure of a Bmi-1-Ring1B polycomb group ubiquitin ligase com- thality in mdm2-deficient mice by deletion of p53. Nature 378(6553):203–206. plex. J Biol Chem 281(29):20643–20649. 7. Boyd SD, Tsai KY, Jacks T (2000) An intact HDM2 RING-finger domain is required for 27. Sánchez-Beato M, et al. (2006) Variability in the expression of polycomb proteins in nuclear exclusion of p53. Nat Cell Biol 2(9):563–568. 8. Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of different normal and tumoral tissues. A pilot study using tissue microarrays. Mod – p53. Nature 387(6630):296–299. Pathol 19(5):684 694. α 9. Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 stability by Mdm2. 28. Sen N, Satija YK, Das S (2011) PGC-1 , a key modulator of p53, promotes cell survival Nature 387(6630):299–303. upon metabolic stress. Mol Cell 44(4):621–634. 10. Li M, et al. (2003) Mono- versus polyubiquitination: Differential control of p53 fate by 29. el-Deiry WS (1998) Regulation of p53 downstream genes. Semin Cancer Biol 8(5): Mdm2. Science 302(5652):1972–1975. 345–357. 11. Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogene 30. Elderkin S, et al. (2007) A phosphorylated form of Mel-18 targets the Ring1B histone product forms a complex with the p53 protein and inhibits p53-mediated trans- H2A ubiquitin ligase to chromatin. Mol Cell 28(1):107–120. activation. Cell 69(7):1237–1245. 31. Rao PS, et al. (2009) RNF2 is the target for phosphorylation by the p38 MAPK and ERK 12. Jain AK, Barton MC (2010) Making sense of ubiquitin ligases that regulate p53. Cancer signaling pathways. Proteomics 9(10):2776–2787. Biol Ther 10(7):665–672. 32. Ben-Saadon R, Zaaroor D, Ziv T, Ciechanover A (2006) The polycomb protein Ring1B 13. Lee JT, Gu W (2010) The multiple levels of regulation by p53 ubiquitination. Cell generates self atypical mixed ubiquitin chains required for its in vitro histone H2A – Death Differ 17(1):86 92. ligase activity. Mol Cell 24(5):701–711. 14. Allton K, et al. (2009) Trim24 targets endogenous p53 for degradation. Proc Natl Acad 33. Calés C, et al. (2008) Inactivation of the polycomb group protein Ring1B unveils an – Sci USA 106(28):11612 11616. antiproliferative role in hematopoietic cell expansion and cooperation with tumori- 15. Simon JA, Kingston RE (2009) Mechanisms of polycomb gene silencing: Knowns and genesis associated with Ink4a deletion. Mol Cell Biol 28(3):1018–1028. unknowns. Nat Rev Mol Cell Biol 10(10):697–708. 34. Voncken JW, et al. (2003) Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell 16. Sparmann A, van Lohuizen M (2006) Polycomb silencers control cell fate, de- cycle inhibition. Proc Natl Acad Sci USA 100(5):2468–2473. velopment and cancer. Nat Rev Cancer 6(11):846–856. 35. Martins CP, Brown-Swigart L, Evan GI (2006) Modeling the therapeutic efficacy of p53 17. Otte AP, Kwaks TH (2003) Gene repression by Polycomb group protein complexes: A – distinct complex for every occasion? Curr Opin Genet Dev 13(5):448–454. restoration in tumors. Cell 127(7):1323 1334. 18. Levine SS, et al. (2002) The core of the polycomb repressive complex is compositionally 36. Ventura A, et al. (2007) Restoration of p53 function leads to tumour regression in – and functionally conserved in flies and humans. Mol Cell Biol 22(17):6070–6078. vivo. Nature 445(7128):661 665. 19. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg D (2002) His- 37. Xue W, et al. (2007) Senescence and tumour clearance is triggered by p53 restoration tone methyltransferase activity associated with a human multiprotein complex con- in murine liver carcinomas. Nature 445(7128):656–660. taining the Enhancer of Zeste protein. Genes Dev 16(22):2893–2905. 38. Patel S, Player MR (2008) Small-molecule inhibitors of the p53-HDM2 interaction for 20. Cao R, et al. (2002) Role of histone H3 lysine 27 methylation in Polycomb-group si- the treatment of cancer. Expert Opin Investig Drugs 17(12):1865–1882. lencing. Science 298(5595):1039–1043. 39. Vassilev LT (2007) MDM2 inhibitors for cancer therapy. Trends Mol Med 13(1):23–31. 21. Wang H, et al. (2004) Role of histone H2A ubiquitination in Polycomb silencing. Na- 40. Richly H, Aloia L, Di Croce L (2011) Roles of the Polycomb group proteins in stem cells ture 431(7010):873–878. and cancer. Cell Death Dis 2(9):e204.

Su et al. PNAS | January 29, 2013 | vol. 110 | no. 5 | 1725 Downloaded by guest on September 29, 2021