CNPY2 Inhibits MYLIP-Mediated AR Protein Degradation in Prostate Cancer Cells

www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 25), pp: 17645-17655 Research Paper CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells Saya Ito1,*, Akihisa Ueno1,*, Takashi Ueda1,2, Hideo Nakagawa1, Hidefumi Taniguchi1, Naruhiro Kayukawa1, Atsuko Fujihara-Iwata1, Fumiya Hongo1, Koji Okihara1 and Osamu Ukimura1 1Department of Urology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto-City, Kyoto 602-8566, Japan 2Department of Urology, Uji Takeda Hospital, Uji-City, Kyoto 611-0021, Japan *These authors equally contributed to this work Correspondence to: Takashi Ueda, email: [email protected] Keywords: CNPY2; prostate cancer; AR; MYLIP; protein degradation Received: September 29, 2017 Accepted: March 01, 2018 Published: April 03, 2018 Copyright: Ito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination. INTRODUCTION system [6]. Protein degradation by the ubiquitin/ proteasome system requires 3 types of enzymes and Signaling by the androgen-induced androgen proceeds through 3 steps. First, ubiquitins are activated receptor (AR) promotes cell growth of prostate cancer by E1 activating enzymes, then, ubiquitins are transferred (PC) cells. AR is a ligand-dependent transcription factor. to E2 conjugating enzymes from E1 enzymes and finally, Androgen-bound ARs exerts both genomic action and E3 ligases function as substrate recognition modules of non-genomic action in PC cells. In genomic pathway, AR the system and are capable of interaction with both E2 transported into the cellular nucleus, bind to chromatin and enzymes and substrate [7]. Three AR ubiquitination control the expression of target genes [1]. Alternatively, in sites have been reported. K845 and K847 are in the AR non-genomic pathway, AR activate MAP kinase signaling C-terminal region (AF-2) [8], whereas K311 is in the AR pathway [2] and Src signaling pathway [3]. N-terminal region (AF-1) [9]. K845 and K847 sites are AR protein levels are controlled by both ubiquitinated by several E3 ligases such as RNF6, Siah2, transcriptional regulation [4, 5] and post-transcriptional SKP2, CHIP and MDM2 [8]. Ubiquitination of the K845 regulation [6]. Several reports showed that AR was marked and K847 by SKP2, CHIP and MDM2 induce ubiquitin- with ubiquitins and degraded by the ubiquitin/proteasome mediated AR protein degradation, while RNF6 and Siah2- www.oncotarget.com 17645 Oncotarget mediated ubiquitination of the K845 and K857 enhance cells that had metastasized to various tissues [17–19]. AR transcriptional activity [8]. The K311 site was recently The 22Rv1 cell line originated from a primary prostate reported to be ubiquitinated by SKP2 [9]. Ubiquitination cancer [20]. The expression levels of CNPY2 transcripts of the K311 is critical for both AR protein stability and AR were higher in 22Rv1 and LNCaP than the other cell lines. transcriptional activity [9]. The E3 ligases that specifically Previous reports and our data showed that both 22Rv1 and recognize AR are attracting attention as therapeutic targets LNCaP cells express AR transcripts [21], whereas DU145 for treating PC [6]. Identification of novel E3 ubiquitin and PC3 do not [22] (Figure 1A). Based on the CNPY2 ligases may provide additional therapeutic targets for PC. and AR expression signature in the different cell lines, Previously, we screened a Drosophila PC model to we hypothesized that AR expression was regulated by identify novel regulators of PC cell growth and invasion CNPY2 expression in prostate cancer. To assess whether [10]. We found that Canopy 2 (CNPY2) markedly AR expression was regulated by CNPY2, immunoblotting promoted cell growth and invasion of PC cells [10]. was performed using CNPY2 knockdown cells with an CNPY2 is a member of the Canopy family (which anti-AR antibody (Figure 1B). As the results, AR protein includes CNPY1, 2, 3 and 4) that contains a four amino level was reduced by CNPY2 knockdown in LNCaP acid sequence at the C-terminus that resembles the cells (Figure 1B) and 22Rv1 cells (data not shown). The classical KDEL motif for endoplasmic reticulum (ER) decrease of AR protein level by CNPY2 knockdown retention [11]. Although the biological function of CNPY2 was abrogated by treatment of the ubiquitin/proteasome is unclear, CNPY2 is also called MIR [myosin regulatory inhibitor MG132 (Figure 1B). On the other hand, AR light chain (MRLC) interacting protein]-interacting transcripts were not decreased by CNPY2 knockdown saposin-like protein (MSAP) based on findings that MSAP in LNCaP cells (Figure 1C) and 22Rv1 cells (data not interacts with MIR in vitro [11]. MIR is also known as shown). These results suggested that CNPY2 could inhibit MYLIP and contains a RING domain in its C-terminal AR protein degradation by the ubiquitin/proteasome region [12]. MYLIP functions as an E3 ubiquitin ligase system. and promotes degradation of MRLC and low-density lipoprotein receptor (LDLR) proteins through the CNPY2 represses MYLIP-mediated ubiquitin/proteasome pathway [12, 13]. MYLIP interacts ubiquitination of AR with the UBE2D family of E2 ubiquitin conjugating enzymes (UBE2D1-4) through its RING domain and CNPY2 was previously reported to decrease MYLIP promotes ubiquitination of its substrate proteins [14, 15]. protein levels and inhibit MYLIP’s ubiquitin E3 ligase The finding that CNPY2 overexpression increased protein activity [11, 16]. The MRLC or LDLR were shown to levels of MRLC and LDLR suggested that CNPY2 can be a MYLIP target substrate [12, 13], although few other regulate the protein stability by preventing MYLIP- target substrates of MYLIP are known. Several E3 ligases mediated ubiquitination [11, 16]. However, the mechanism are known to interact with AR [8], but whether MYLIP by which CNPY2 inhibits E3 ligase activity of MYLIP is interacts with AR awaits investigation. We hypothesized unknown. that CNPY2 could inhibit MYLIP recognition of AR and We found that MYLIP is a novel E3 ubiquitin in turn suppress AR degradation. To examine whether ligase that recognizes and ubiquitinates AR. The MYLIP interacts with AR in prostate cancer cells, we first MYLIP-mediated AR ubiquitination induced AR protein performed immunoprecipitation assay using LNCaP cells degradation by ubiquitin/proteasome system. We also transfected with flag-tagged MYLIP. The results of these showed that CNPY2 repressed the MYLIP-mediated AR assays showed that AR and MYLIP physically interacted in ubiquitination through inhibiting the interaction between prostate cancer cells (Figure 2A). To determine interaction E2 and E3 ubiquitin enzymes. Thus, our study suggested domain of AR with MYLIP, immunoprecipitaion was that CNPY2 promoted cell growth of PC cells through performed using 293T cells which co-expressed with His regulating AR protein degradation via inhibition of tag-fused MYLIP and each of FLAG tag-fused partial ubiquitin/proteasome pathway. ARs (full length, AF-1 and AF-2; Figure 2B and 2C). As the results of immunoprecipitation using anti-His-tag RESULTS beads, physical interaction of MYLIP with the C-terminal region of AR (AF-2) was consistently observed, while its AR protein level is decreased by CNPY2 association with the N-terminal region of AR (AF-1) was knockdown in PC cells not detected (Figure 2B and 2C). Next, to investigate whether MYLIP functions as an To investigate CNPY2 function, we used human E3 ubiquitin ligase that recognizes the AR protein, in vivo prostate cancer cell lines to model the disease. The ubiquitination assays were performed using 293T cells expression levels of the CNPY2 transcript in the cell lines that do not express endogenous AR. Cell lysates of 293T were first quantified by RT-PCR (Figure 1A). DU145, cells transfected with plasmids expressing FLAG tag- PC3 and LNCaP cell lines originated from prostate cancer fused AR (AF-2), MYLIP-His and EGFP-ubiquitin were www.oncotarget.com 17646 Oncotarget extracted and immunoprecipitation using anti-FLAG was AR ubiquitination by MYLIP, in vitro ubiquitination performed. Poly-ubiquitinated AR (AF-2) was increased assays were carried out with recombinant E1 enzyme, in lysates from MYLIP-transfected cells that were treated UBE2D1 (E2 enzyme), AR (AF-2)

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