Original ArticleProstate cancer-associated mutation in SPOP impairs its ability to target Cdc20 for poly-ubiquitination and degradation
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).
References (48)
- et al.
Functional characterization of anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis
Biochim. Biophys. Acta
(2014) Cdc20: a WD40 activator for a cell cycle degradation machine
Mol. Cell
(2007)- et al.
Targeting mitotic exit leads to tumor regression in vivo: modulation by Cdk1, Mastl, and the PP2A/B55alpha, delta phosphatase
Cancer Cell
(2010) - et al.
Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly
Cancer Cell
(2009) - et al.
Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage
Cancer Cell
(2010) - et al.
APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction
Dev. Cell
(2014) - et al.
Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA
Biochem. Biophys. Res. Commun.
(2011) - et al.
Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases
Mol. Cell
(2009) - et al.
Destruction of full-length androgen receptor by wild-type SPOP, but not prostate-cancer-associated mutants
Cell Rep.
(2014) - et al.
Truncated ERG oncoproteins from TMPRSS2-ERG fusions are resistant to SPOP-mediated proteasome degradation
Mol. Cell
(2015)
SPOP promotes ubiquitination and degradation of the ERG oncoprotein to suppress prostate Cancer progression
Mol. Cell
SPOP E3 ubiquitin ligase adaptor promotes cellular senescence by degrading the SENP7 deSUMOylase
Cell Rep.
SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer
Cancer Cell
Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation
Cell
APC/C(Cdh1)-dependent degradation of Cdc20 requires a phosphorylation on CRY-box by Polo-like kinase-1 during somatic cell cycle
Biochem. Biophys. Res. Commun.
A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor
Dev. Cell
BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase
J. Biol. Chem.
Targeting Cdc20 as a novel cancer therapeutic strategy
Pharmacol. Ther.
Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer
Nat. Rev. Cancer
Roles of F-box proteins in cancer
Nat. Rev. Cancer
Ubiquitin ligases: cell-cycle control and cancer
Nat. Rev. Cancer
The anaphase promoting complex/cyclosome: a machine designed to destroy
Nat. Rev. Mol. Cell Biol.
How proteolysis drives the cell cycle
Science
Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p
J. Cell Biol.
Cited by (40)
Novel insights into the SPOP E3 ubiquitin ligase: From the regulation of molecular mechanisms to tumorigenesis
2022, Biomedicine and PharmacotherapyCitation Excerpt :Later large-sample sequencing studies of prostate cancer (PCa) genomes confirmed that SPOP was the most frequently mutated gene type (approximately 10%) and that the mutations could be individually classified into one of the seven molecular subtypes of PCa [17,18]. A series of studies exploring tumorigenic mechanisms have reported that SPOP substrates mediate various physiological cellular processes including hormone signaling [19–21], the DNA damage response [22–24], cell cycle control [25,26], cell stemness [27], and the immune response [28–30]. However, in tumor cells, SPOP functions abnormally, failing to precisely regulate these cellular processes and exhibiting tumorigenic effects.
SPOP-mutant prostate cancer: Translating fundamental biology into patient care
2022, Cancer LettersCitation Excerpt :Earmarking with ubiquitin residues may in many instances lead to protein destruction through the proteasome [30,31]. Many known SPOP substrates follow this pattern and are thus part of the Ubiquitin-Proteasome Pathway (UPP) [32–44]. The proteasome recognition is typically mediated by K48-linked poly-ubiquitylation.
SPOP promotes CDCA5 degradation to regulate prostate cancer progression via the AKT pathway
2021, Neoplasia (United States)Citation Excerpt :The frequently mutated sites often occur in MATH domain, including Y87, F102, F125, K129, D130, W131, and F133 [2–4]. SPOP exerts a tumor suppressor effect in many tumors including PCa by targeting oncoprotein substrates, such as AR [5], ERG [6], TRIM24 [7], NANOG [8], CDC20 [9], and others [10-12]. Meanwhile, it has been reported that SPOP is overexpressed in nearly 100% of clear cell renal cancer tumor tissues (RCC) and displays a possible oncogenic property [13].
APC/C ubiquitin ligase: Functions and mechanisms in tumorigenesis
2020, Seminars in Cancer BiologyCitation Excerpt :SPOP has been shown to often be mutated in cases of prostate cancer and prostate cancer-associated mutations in the MATH domain of SPOP were shown to abolish the interaction with Cdc20. The lack in interaction resulted in a reduction in the polyubiquitination of Cdc20, stabilizing it and inhibiting its degradation through the proteasome pathway [90]. With the prevalence of Cdc20 overexpression in cancers, it is probable that there are a multitude of mechanisms in which Cdc20 is overexpressed, however these mechanism continue to be elucidated.
Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer
2023, Experimental Hematology and OncologyThe potential role of CDC20 in tumorigenesis, cancer progression and therapy: A narrative review
2023, Medicine (United States)