Tumor-Associated Antigen Prame Targets Tumor Suppressor P14/ARF for Degradation As the Receptor Protein of Crl2prame Complex
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Cell Death & Differentiation (2021) 28:1926–1940 https://doi.org/10.1038/s41418-020-00724-5 ARTICLE Tumor-associated antigen Prame targets tumor suppressor p14/ARF for degradation as the receptor protein of CRL2Prame complex 1,2,3 1,3 1 1,3 3 4,5,6 1 7,8 Wenjuan Zhang ● Lihui Li ● Lili Cai ● Yupei Liang ● Junfeng Xu ● Yue Liu ● Lisha Zhou ● Chen Ding ● 4,5,6 4,5,6 7,8 2,3 9 1 Yanmei Zhang ● Hu Zhao ● Jun Qin ● Zhimin Shao ● Wenyi Wei ● Lijun Jia Received: 16 March 2020 / Revised: 30 November 2020 / Accepted: 27 December 2020 / Published online: 27 January 2021 © The Author(s) 2021. This article is published with open access Abstract Protein Preferentially Expressed Antigen in Melanoma (Prame), a tumor-associated antigen, has been found to frequently overexpress in various cancers, which indicates advanced cancer stages and poor clinical prognosis. Moreover, previous reports noted that Prame functions as a substrate recognizing receptor protein of Cullin RING E3 ligases (CRLs) to mediate potential substrates degradation through Ubiquitin Proteasome System (UPS). However, none of the Prame specific substrate has been identified so far. In this study, proteomic analysis of RBX1-interacting proteins revealed p14/ARF, a well-known tumor suppressor, as a novel ubiquitin target of RBX1. Subsequently, immunoprecipitation and in vivo ubiquitination assay 1234567890();,: 1234567890();,: determined Cullin2-RBX1-Transcription Elongation Factor B Subunit 2 (EloB) assembled CRL2 E3 ligase complex to regulate the ubiquitination and subsequent degradation of p14/ARF. Finally, through siRNA screening, Prame was identified as the specific receptor protein responsible for recognizing p14/ARF to be degraded. Additionally, via bioinformatics analysis of TCGA database and clinical samples, Prame was determined to overexpress in tumor tissues vs. paired adjacent tissues and associated with poor prognosis of cancer patients. As such, downregulation of Prame expression significantly restrained cancer cell growth by inducing G2/M phase cell cycle arrest, which could be rescued by simultaneously knocking down of p14/ARF. Altogether, targeting overexpressed Prame in cancer cells inactivated RBX1-Cullin2-EloB-Prame E3 ligase (CRL2Prame) and halted p14/ARF degradation to restrain tumor growth by inducing G2/M phase cell cycle arrest. Introduction CRL is the largest multiunit E3 ubiquitin ligase family that regulates diverse biological processes through mediating the These authors contributed equally: Wenjuan Zhang, Lihui Li, Lili Cai ubiquitination and degradation of a variety of substrates Edited by V. D'Angiolella including signal transducers, cell cycle regulators, tran- scription factors, tumor suppressors and oncoproteins [1–3]. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41418- The CRL complex contains one of Cullin proteins (Cullin 020-00724-5 1, 2, 3, 4A/4B, 5 and 7) that functions largely as molecular * Lijun Jia 5 Shanghai Key Laboratory of Clinical Geriatric Medicine, Fudan [email protected] University, Shanghai 200040, China 6 Research Center on Aging and Medicine, Fudan University, 1 Cancer Institute, Longhua Hospital, Shanghai University of Shanghai 2000402, China Traditional Chinese Medicine, Shanghai 200032, China 7 State Key Laboratory of Proteomics, Beijing Proteome Research 2 Department of Breast Surgery, Key Laboratory of Breast Cancer in Center, Beijing Institute of Radiation Medicine, Beijing 102206, Shanghai, Fudan University Shanghai Cancer Center, China Shanghai 200032, China 8 National Center for Protein Sciences, The PHOENIX Center, 3 Cancer Institute, Fudan University Shanghai Cancer Center, Beijing 102206, China Shanghai 200032, China 9 Department of Pathology, Beth Israel Deaconess Medical Center, 4 Department of Clinical Laboratory, Huadong Hospital, Fudan Harvard Medical School, Boston 02115 MA, USA University, Shanghai 200040, China Tumor-associated antigen Prame targets tumor suppressor p14/ARF for degradation as the receptor. 1927 scaffold, an adaptor protein alone or together with a of ARF is to suppress aberrant cell growth in response to substrate specific receptor protein at the N-terminus oncogenic insults in part through activating the p53 tumor and a RING protein (RBX1/Roc1 or SAG/Roc2) at the suppressive pathway [33–35]. By blocking ubiquitin ligases C-terminus [1–3]. Activation of CRL requires the expres- MDM2 (MDM2 proto-oncogene) and ARF-BP1/Mule sion of those essential components consisting CRL complex (ARF-binding protein1/Mcl1-ubiquitin ligase E3) mediated as well as neddylation modification on Cullin protein [4–6]. p53 degradation, ARF stabilizes and stimulates p53 activity Protein neddylation is a three-step enzymatic cascade, [36, 37]. ARF also has the ability to restrain cell growth which conjugates neural precursor cell expressed devel- independently of p53. To this end, p53-independent func- opmentally downregulated 8 (NEDD8), a ubiquitin-like tion of ARF is thought to attenuate ribosomal RNA tran- molecule, to targeted proteins and thus modulates multiple scription and processing in part by binding to NPM1 biological processes [7–11]. Once NEDD8 is attached to the (nucleophosmin1) [38, 39]. Moreover, ARF interacts with C-terminus lysine residue of Cullin [4, 6], the structure of and antagonizes the transcriptional function of Myc and CRL complex would change from a closed conformation to E2F1 independently of p53 [37]. ARF also prevents an open one via conformational rearrangement, which angiogenesis by limiting the translation of existing VEGFA subsequently leads to CRL activation [5, 12, 13]. mRNAs [40]. As an important tumor suppressor, the Prame is a tumor-associated antigen that was first iden- expression of ARF is tightly controlled at transcriptional tified in metastatic cutaneous melanoma and is subsequently and post-translational levels [41, 42]. Previous study found to frequently overexpress in various cancers, which reported that β-TrCP2, an F-box protein as well as a sub- also associates with advanced stages and poor clinical strate receptor of SKP1–Cullin1–F-box protein (SCF, a outcomes [14–16]. On the contrast, normal healthy tissues subfamily of CRL E3 ligases) complex, regulates the are not known to express Prame except for testis, ovary, degradation of p19/ARF, but not p14/ARF [43]. Therefore, placenta, adrenals and endometrium [17]. Because of its it is unclear whether CRL E3 ligase regulates p14/ARF expression profile, targeting Prame for cancer treatment and degradation. exploring it as a potential biomarker for diagnosis or We reported here that, by utilizing a non-biased pro- prognosis arouse great clinical interest in recent years teomics approach, p14/ARF is identified as a novel ubi- [15, 18, 19]. At the N-terminus of Prame, there is a BC box quitin substrate of CRL2Prame in human cancers. Moreover, or Cul-2 box mediating the interaction with Cullin2 substrate recognizing receptor protein Prame is determined [19, 20]. The C-terminus of Prame is the consensus to be overexpressed in human cancers and negatively cor- LXXLL-binding domain mediating the interaction with- related with patients’ prognosis. Targeting overexpressed potential substrates [19, 20]. However, substrate of Prame in human cancer cell lines blocks p14/ARF degra- CRL2Prame E3 ligase complex has not been reported so dation and induces G2/M phase cell cycle arrest, thus far [21]. inhibits cancer cell proliferation. Our study reveals a pre- As the core structure of CRL, RBX1 is a high evolu- viously unknown regulatory mechanism of p14/ARF in tionarily conserved RING-H2 finger domain-containing human cancer and validates CRL2Prame E3 ligase complex protein, which interacts with all Cullin family members as a promising anti-cancer target. except Cullin 5, and is required for CRL E3 ligase activity [22, 23]. In addition to function as the essential component of CRL, RBX1 also serves as a NEDD8 E3 ligase to pro- Results mote Cullin (1, 2/5, 3, 4A/4B and 7) neddylation [24, 25]. Previous studies reported that RBX1 is overexpressed in P14/ARF is identified as a novel RBX1-interacting several human cancers and associates with poor prognosis protein of cancer patients [26–28]. In contrast, downregulation of RBX1 expression triggers multiple death and growth arrest Given that RBX1, the core subunit of CRL E3 Ligases, has pathways to effectively suppress the malignant phenotypes been well identified as an oncogenic factor, we performed of cancer cells [26–28]. These findings highlight RBX1 as proteomic analysis of RBX1-interacting proteins to reveal an oncogenic factor during tumorigenesis and an attractive potential mechanisms underlying RBX1-regulating biolo- target for cancer therapy. gical effects. Immunoprecipitation with RBX1 Ab followed Alternate Reading Frame (ARF), known as p14/ARF in by mass spectrometry was performed to identify those human and p19/ARF in mouse, is encoded by Ink4a/ARF proteins specifically interacting with RBX1 in cells (CDKN2A) locus that also encodes the p16/INK4A tumor (Fig. 1A). As shown in Fig. 1B, those well-known RBX1- suppressor [29]. ARF null-mice develop spontaneous interacting proteins such as Cullins (Cullin 1–3, 4A, 4B, 7, tumors at an early age, demonstrating its tumor suppressive 9), corresponding adaptor proteins (Skp1, EloB/C, DDB1, functions [30–32]. One of the most well-defined functions etc.) and target recognizing subunits (substrate receptor 1928 W. Zhang et al. A. B. Cullin Adaptor Receptor proteins proteins