Cancer Letters

Cancer Letters

Volume 385, 28 January 2017, Pages 207-214
Cancer Letters

Original Article
Prostate cancer-associated mutation in SPOP impairs its ability to target Cdc20 for poly-ubiquitination and degradation

https://doi.org/10.1016/j.canlet.2016.10.021Get rights and content

Highlights

  • Cullin 3 negatively regulates Cdc20 stability in prostate cancer.

  • SPOP promotes Cdc20 poly-ubiquitination and degradation.

  • Prostate cancer-associated mutants are defective in interaction with Cdc20.

  • SPOP-deficient prostate cancer cells become resistant to Cdc20 inhibitor.

Abstract

Recent studies revealed that mutations in SPOP (Speckle-type POZ protein) occur in up to 15% of patients with prostate cancer. However, the physiological role of SPOP in regulating prostate tumorigenesis remains elusive. Here, we identified the Cdc20 oncoprotein as a novel ubiquitin substrate of SPOP. As such, pharmacological inhibition of Cullin-based E3 ligases by MLN4924 could stabilize endogenous Cdc20 in cells. Furthermore, we found that Cullin 3, and, to a less extent, Cullin 1, specifically interacted with Cdc20. Depletion of Cullin 3, but not Cullin 1, could upregulate the abudance of Cdc20 largely via prolonging Cdc20 half-life. Moreover, SPOP, the adaptor protein of Cullin 3 family E3 ligase, specifically interacted with Cdc20, and promoted the poly-ubiquitination and subsequent degradation of Cdc20 in a degron-dependent manner. Importantly, prostate cancer-derived SPOP mutants failed to interact with Cdc20 to promote its degradation. As a result, SPOP-deficient prostate cancer cells with elevated Cdc20 expression became resistant to a pharmacological Cdc20 inhibitor. Therefore, our results revealed a novel role of SPOP in tumorigenesis in part by promoting the degradation of the Cdc20 oncoprotein.

Introduction

Ubiquitination is characterized as a critical type of post-translational modification that governs various important cellular processes including cell cycle and apoptosis, primarily through regulation of the protein abundance of key cell fate determining factors [1], [2]. Thus far, two related, multi-subunit E3 ubiquitin ligases, the Anaphase Promoting Complex (APC) and the Skp1-Cullin1-F-box complex (SCF) have been considered as the major driving forces governing cell cycle regulation and tumorigenesis [1], [2], [3], [4]. In doing so, APC forms two functional sub-complexes APCCdc20 and APCCdh1 [5], by associating with two substrate adaptor proteins Cdc20 or Cdh1, respectively, to play critical roles in regulating both the M and G1 phases [6]. Cdc20 contains seven WD40 repeats for protein binding, serving as the substrate recognizing subunit of APC, recruiting substrates with the Destruction Box (D-box) motif [7]. However, besides Cdh1 [8], the upstream regulator(s) for APCCdc20 remain largely elusive.

Recent studies began to reveal that Cdc20 might possess oncogenic activity [9] and genetic ablation of Cdc20 blocks in vivo tumorigenesis, largely due to elevated cellular apoptosis [10]. Furthermore, depleting Cdc20 in various cancer cell lines also led to mitotic arrest followed by cell death [11]. These studies suggested that inhibiting APCCdc20 might lead to elevated cellular apoptosis, which advocates for Cdc20 as a novel anti-cancer therapeutic target. In keeping with this notion, inactivating APC by an IR-motif-mimetic inhibitor, pro-TAME, induced cell death in multiple cancer cell lines [12], [13]. Furthermore, a more specific APCCdc20 E3 ligase inhibitor, Apcin, inhibits its oncogenic function through directly interfering with the binding of Cdc20 to its substrates and causes blockade of mitotic exit in human cancer cells [14]. Consistent with its oncogenic role, Cdc20 is highly expressed in many human tumors including prostate [15], breast [16], cervical [17], glioblastoma [18] and ovarian tumors [19]. Notably, high expression of Cdc20 was tightly associated with advanced clinical stages and poor prognosis in human cancers including prostate cancer [15]. These findings thus suggest that Cdc20 expression may be used as a prognostic marker and therapeutic target in treating various human cancers.

The Cullin-Ring ligases (CRLs) are the largest family of ubiquitin E3 ligases, which govern a plethora of vital cellular processes including cell cycle progression [20]. Based on Cullin scaffold proteins (Cullin1, 2, 3, 4A, 4B, 5 and 7), CRLs can be categorized into seven subfamilies (termed CRL1 through CRL7, respectively) [2], [20]. Among these CRLs, the CRL1 complex (also named SCF), is well studied at both biochemical and physiological levels [21], [22]. Recently, the emerging CRL3 subfamily complex is identified as major regulators for different cellular processes and disruption of this degradation pathway has been linked to various human diseases, including nerve degeneration and cancers [23]. Structurally, CRL3 is composed of the scaffold protein Cullin 3, the RING protein RBX1, and one of numerous BTB domain adaptor proteins, which recruit its protein substrates for poly-ubiquitination [23].

Recently, it has been reported that Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP), a substrate-interacting adaptor protein of CRL3, is one of most frequently mutated genes, with up to 15% mutation rate in primary human prostate cancers [24], [25], [26]. SPOP comprises two conserved domains: an N-terminal meprin and TRAF homology (MATH) domain that is primarily involved in substrate recognition and a C-terminal bric-a-brac, tramtrack and broad complex (BTB)/POZ domain that binds the CRL scaffold protein, Cullin 3 [27]. The ongoing list of SPOP substrates includes MacroH2A [28], Ci/Gli [29], androgen receptor (AR) [30], [31], steroid receptor coactivator 3 (SRC-3) [32], DEK [33], TRIM24 [33], ERG [34], [35] and SENP7 [36]. As most of these characterized substrates are well-known oncogenic proteins that are frequently overexpressed in human prostate cancers, SPOP probably functions largely as a tumor suppressor in PrCa that negatively regulates the stability of these oncogenic proteins. In keeping with this model, SPOP is frequently inactivated by genetic mutations in human prostate cancers [24], [25]. However, in other cancer settings including kidney cancer and breast cancer, on the other hand, SPOP is overexpressed and displays a possible oncogenic role in part by promoting the degradation of PTEN [37]. Thus, the physiological role of SPOP in tumorigenesis might also be tissue or context dependent. In support of this notion, the transcription factor E2Fs also can function as an oncogene or tumor suppressor in different cellular context [38]. In this circumstance, this study mainly focuses on understanding the tumor suppressor role of SPOP in the prostate cancer setting. To this end, even though known substrates of SPOP are well documented for their oncogenic roles in prostate cancer development and progression, the exact molecular mechanisms by which SPOP suppresses cancer formation in part by regulating cell cycle progression or chromosomal segregation have not yet been fully elucidated. To further clarify the physiological role of SPOP in regulating tumorigenesis, we identified oncoprotein Cdc20 as a novel ubiquitin substrate of SPOP in the prostate cancer setting, which offers further molecular insights into its tumor suppressive role in PrCa development.

Section snippets

Cell culture

PC3 and DU145 cells were cultured in RPMI 1640 medium (Corning, NY) with 10% FBS, 100 units of penicillin and 100 μg/ml streptomycin. 293T, HeLa, and U2OS cells were cultured in DMEM medium (Life Technologies, CA) supplemented with 10% FBS, penicillin and streptomycin.

Plasmids

Myc-Cullin 1, Cullin 2, Cullin 3, Cullin 4A, Cullin 4B, Cullin 5, Flag-SPOP WT, Y87C, F102C, W131G, pGEX-4T-1-SPOP, Flag-Keap1, Flag-Cop1, shScramble, shCullin 1, shCullin 3, shSPOP, and His-ubiquitin constructs were described

Inhibition of the Cullin-based E3 ligases stabilizes endogenous Cdc20 in cells

MLN4924 is a specific inhibitor of the NEDD8-activating enzyme (NAE) and first-in-class anti-cancer drug, which has entered Phase-I trials for cancer therapy [39], [40]. Mechanistically, MLN4924 could inactivate CRLs E3 ligases through blocking Cullin Neddylation, an essential step in activating CRLs [41], which subsequently led to upregulation of downstream substrates of CRLs [40]. Importantly, we found that, in addition to the proteasome inhibitor MG132, MLN4924 could also stabilize the key

Discussion

Here we have identified Cullin 3SPOP as a novel upstream E3 ubiquitin ligase complex that governs Cdc20 stability and oncogenic functions through promoting the ubiquitination and subsequent destruction of Cdc20. Our results showed that although Cdc20 interacts specifically with Cullin 1 and Cullin 3 of the Cullin-based E3 ligase family members, only Cullin 3 is largely responsible for the stability control of Cdc20 in cells (Fig. 2). More importantly, we also showed that SPOP, one of substrate

Acknowledgements

This work was supported by grants from the National Institute of Health (R01CA177910 and R01GM094777 to W.W.), National Basic Research Program of China (2015CB943003) and National Natural Science Foundation of China (81370753). F.W. received financial support from the China Scholarship Council (CSC) (No. 201506100054).

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