The Oncoprotein SS 18-SSX 1 Promotes p 53 Ubiquitination and Degradation by Enhancing HDM 2 Stability

Mutations of the p53 gene are uncommon in synovial sarcoma, a high-grade tumor genetically characterized by the chromosomal translocation t:(X;18), which results in the fusion of SS18 with members of SSX gene family. Although implicated in tumorigenesis, the mechanisms by which SS18-SSX promotes tumor growth and cell survival are poorly defined. Here, we show that SS18-SSX1 negatively regulates the stability of the tumor suppressor p53 under basal conditions. Overexpression of SS18-SSX1 enhanced p53 ubiquitination and degradation in a manner dependent on the ubiquitin ligase activity of HDM2. The negative effect of SS18-SSX1 expression on p53 was mediated by its ability to promote HDM2 stabilization through inhibition of HDM2 autoubiquitination. Furthermore, SS18-SSX1 expression altered the induction of p53-regulated genes in response to cellular stress by abrogating the transactivation of HDM2, PUMA, and NOXA but not p21. Our data uncover a novel mechanism whereby SS18-SSX1 can negatively regulate p53 tumor-suppressive function by increasing the stability of its negative regulator HDM2 and suggest that chemical compounds that target the p53-HDM2 regulatory axis may be of therapeutic benefit for the treatment of synovial sarcoma. (Mol Cancer Res 2008;6(1):127–38) Introduction Disruption of p53-mediated tumor surveillance is a common motif in the development of cancer. In response to a variety of cellular stresses, p53 acts as an intracellular mediator of cell fate by inducing cell cycle arrest, DNA repair, or apoptosis, thus allowing cells to cope with situations that could otherwise lead to enhanced tumorigenesis (1). The potent tumor-suppressive function of p53 is kept in check by the action of its negative regulator HDM2, which catalyzes the ubiquitination of conserved lysine residues in the COOH-terminal domain of p53, promoting nuclear export, inhibiting p53 transcriptional activity, and promoting proteasome-mediated degradation (2-4). Intracellular p53 levels are regulated by an elegant feedback loop whereby increased levels of p53 result in enhanced transcription of HDM2, leading to the subsequent downregulation of p53 protein levels by proteasomal-mediated degradation (5). The discovery that HDM2 was commonly overexpressed in a variety of tumors, particularly sarcomas, further provided a mechanism of deactivating p53 in the absence of discernible p53 mutations (6). Consequently, disruption of the HDM2-p53 feedback loop has been suggested as a novel target for cancer therapy particularly in tumors where the tumor-suppressive function of p53 is dampened by enhanced HDM2 expression. Synovial sarcoma, a high-grade soft tissue tumor, displays a reciprocal chromosomal translocation t(X;18), which results in the fusion of two disparate genes: SS18 from chromosome 18 and SSX from the X chromosome. This event results in the expression of a putative fusion protein, which contains all but the last eight amino acids of SS18 and the COOH terminus of SSX (7-10). SS18 is a transcriptional coactivator, whereas SSX has been implicated in transrepression (11-13). In its role as a transcriptional activator, SS18 interacts with an array of transcription factors implicated in gene regulation, such as the histone acetyl transferase p300, the BRM and BRG1 components of the SWI/SNF complex, the leucine zipper transcription factor AF10, and the histone deacetylase Sin3a (13-17). Conversely, members of the SSX gene family are potent corepressors and have been shown to interact with polycomb group proteins, core histones, and matrix metalloproteinase-2 (11, 13, 18-20). In synovial sarcoma, the chromosomal translocation results in the production of a proto-oncogene with dual features of a transactivator and repressor. The resultant fusion protein is presumed to function as an aberrant transcription factor through the misdirected targeting of interacting partners and alteration of epigenetic control (21, 22). Interestingly, in comparison with other tumor types, synovial sarcomas display a remarkably low number of mutations in the p53 gene, implying that defects in upstream pathways may be responsible for loss of p53-mediated tumor suppression (23, 24). In this study, we show that SS18-SSX expression promotes the ubiquitination and degradation of p53 in an HDM2dependent manner. Expression of SS18-SSX1 reduced p53 half-life, promoted cell survival, enhanced colony formation ability, and abrogated the transactivation of p53 target genes in response to cellular stress. The negative effects of SS18-SSX1 on p53 stability were due to enhanced ubiquitination of p53 in a Received 4/15/07; revised 10/15/07; accepted 10/23/07. Grant support: Swedish Cancer Foundation and Swedish Children Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Bertha Brodin, Department of Oncology-Pathology, Cancer Center Karolinska R8:04, Karolinska Institute, Stockholm 17176, Sweden. Phone: 46-8-51775244; Fax: 46-8-321047. E-mail: Bertha.Brodin@ki.se Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0176 Mol Cancer Res 2008;6(1). January 2008 127 on June 16, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from manner dependent on the activity of HDM2. Our results highlight an important oncogenic function of SS18-SSX1 as a positive regulator of HDM2 stability. Results SS18-SSX Expression Negatively Attenuates p53 Stability Previous reports have shown that synovial sarcomas harbor a remarkably low number of mutations in the p53 gene in comparison with other tumor types (23, 24). Consistent with this, we analyzed the p53 status in a set of 20 primary synovial sarcoma tumors and found no discernible mutations of the p53 gene (results not shown). In light of this, we investigated whether a potential oncogenic mechanism of SS18-SSX expression is the abrogation of p53 function. Transfection of increasing amounts of SS18-SSX1 into U2OS or cotransfection with p53 in p53-null Saos-2 cells resulted in a concomitant decrease in the levels of p53 (Fig. 1A and B). Because no reliable antibodies for the detection of SS18-SSX expression are available, SS18-SSX expression was determined using realtime PCR. Cotransfection with a green fluorescent protein (GFP) vector was used as a determinant of transfection efficiency. Transfection with SS18-SSX1 resulted in increased levels of SS18-SSX1 mRNA with minimal changes in the levels of p53 transcripts, indicating that the negative effect of SS18-SSX is a posttranscriptional mechanism (Fig. 1A and B, bottom). The relationship between the expression of SS18-SSX and p53 was further investigated following transfection of a synovial sarcoma cell line CME1 with doxycycline-inducible small interfering RNA (siRNA) vector containing a short hairpin RNA cassette targeting endogenous SS18-SSX. Knockdown of SS18-SSX was confirmed by real-time PCR (Fig. 1C). siRNA targeting of endogenously expressed SS18-SSX in a synovial sarcoma cell line increased p53 levels following induction of siRNA expression with doxycycline (Fig. 1D). To determine if SS18-SSX expression could affect p53 stability, the half-life of p53 was determined by a pulse-chase analysis by immunoprecipitating p53 from [S]methionine-labeled U2OS cells expressing SS18-SSX1 or a control vector. As seen in Fig. 1E, the half-life of p53 in SS18-SSX1–expressing cells was significantly reduced when compared with control