APC/C ubiquitin ligase: functions and mechanisms in tumorigenesis

APC/C Structure and Function

Human APC/C is composed of 14 proteins for a total of 19 subunits, which can be categorized into three subcomplexes: the catalytic core (APC2, APC11, and APC10), the platform (APC1, APC4, APC5, and APC15), and the tetratricopeptide repeat (TPR) lobe (APC3, APC6, APC7, APC8, APC12, APC13, and APC16) [3–8] (Figure 1). APC1 is the largest subunit within the complex and acts to bridge the TPR lobe, platform and catalytic core. The catalytic core is responsible for APC/C E3 ubiquitin ligase activity, which allows it to catalyze the generation of poly-ubiquitin chains upon its substrates, triggering their degradation by the 26S proteasome. The attachment of ubiquitin is a multi-step process involving a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). There are two E1 enzymes in the human genome, approximately 40 E2 enzymes, and over 500 E3 ligases. This review focuses on the E3 ligase APC/C, which interacts with two predominant E2s: UbcH10 and Ube2s and to a lesser extent UbcH5 [9–16]. To begin the ubiquitination process, ubiquitin is activated through an ATP-dependent interaction with E1. The activated ubiquitin is then transferred to E2, which is recruited by E3, in this case, APC/C. The APC/C subunit Apc11 RING H2 domain interacts with the E2, which catalyzes the transfer of ubiquitin from the E2 active site to a lysine on the target protein [4, 17–20]. Repeating this process generates a chain of ubiquitins on the substrate, labeling it for degradation by the 26S proteasome.

The ubiquitination of substrates by the APC/C requires the formation of an APC/C-activator-substrate complex. The APC/C is controlled by two main activators, Cdc20 and Cdh1, each targeting APC/C to specific substrates at precise times during the cell cycle. Binding of Cdc20 or Cdh1 to the APC/C holoenzyme induces a conformational change to the active forms, APC/C^Cdc20 and APC/C^Cdh1, respectively. The Cdc20 and Cdh1 WD40 domains located at their C termini provide a binding platform to recruit APC/C substrates, which in concert with APC10 functions as the substrate receptor of the APC/C [19, 21–24]. APC/C^Cdc20 is present at the onset of mitosis until the beginning of anaphase (Figure 2A). In early mitosis, Cdc20 becomes active and associates with phosphorylated APC/C leading to the degradation of prometaphase substrates, such as cyclin A and Nek2 [25–29]. Once all chromosomes achieve proper microtubule biorientation in metaphase, APC^Cdc20 degrades securin and cyclin B to promote cellular transition to anaphase. Degradation of securin releases separase, an enzyme that is responsible for cleaving Scc1, a component of the cohesion complex that holds sister chromatids together, and allows physical separation of sister chromatids. Degradation of cyclin B abolishes cyclin-Cdk activity at anaphase and allows the activation of APC/C^Cdh1, which destroys the remaining mitotic regulators, including Cdc20 [26, 30]. APC/C^Cdh1 is present at the onset of anaphase through G1 and promotes efficient mitotic exit and maintenance of the G1 state (Figure 2A).

Substrates targeted for degradation by the APC/C are as varied as the processes they regulate and include cell cycle regulators such as the mitotic cyclins, Securin, and Geminin, as well as the transcription and differentiation inhibitor Id2, the TGF-β regulator SnoN, the replication checkpoint regulator Claspin, and the adaptive glycolytic enzyme Pfkfb3 [31–38]. E3s recognize substrates that contain cis-acting sequence motifs, also known as degrons. The degrons recognized by the APC/C are the destruction-box (D-box) (RXXLXXI/VXN) and/or KEN-box (KENXXXN/D). However, other APC/C-targeting/interacting sequences have been identified such as Cry-box, C-box, and O-box and are thought to interact with the adaptors in a similar fashion to the KEN and D-box. [39, 40]. APC/C substrate degrons directly interact with Cdc20 and Cdh1 via their WD40 domains, which leads to their ubiquitination by E2s and degradation by the 26S proteasome [4, 22, 23, 40, 41].

Because APC/C contributes to many different cellular pathways depending on which activator is bound, a more in depth examination of APC/C activity in respect to Cdc20 or Cdh1 is necessary to review its diverse pathway contributions to cancer development. The key cancer-related interactions of Cdc20 and Cdh1 detailed in the following sections are summarized in Table 1.

The role of Cdc20 in tumorigenesis

In recent years, overexpression of Cdc20 has been associated with a multitude of different cancers such as prostate, glioblastoma, bladder, breast, oral, and diffuse large B cell and mantle cell lymphomas [65–70]. Overexpression of Cdc20 allows a cell to bypass the SAC and exit mitosis prematurely, thus leading to genomic instability. This overexpression commonly results in a poor prognosis for patients due to Cdc20’s effects on a variety of cellular processes. Cdc20 appears to play a strong role in the maintenance of tumors through its expression in cancer stem-like cells (CSCs)/tumor initiating cells (TICs), as discussed below.

Overexpression of Cdc20 has also been correlated with activation of the WNT/ β-catenin pathway, which is strongly associated with the progression of cancer [71–73]. Canonically, upon activation of WNT, β-catenin is stabilized and translocated into the nucleus. Upon translocation, β -catenin interacts with T-cell factor/lymphoid enhancing factors (TCF/LEF) family of DNA-binding factors and activates genes such as stromelysin, fibroblast growth factor (FGF), epidermal growth factor (EGF), cyclin D1, c-Myc, CD44 and ALDH [67]. Inhibition of Cdc20 in cutaneous squamous cell carcinoma through RNA interference resulted in a reduction in β-catenin. While not showing a direct interaction between Cdc20 and β -catenin, it is apparent that Cdc20 plays a role in regulating β-catenin [72]. Another recent study revealed a distinct mechanism through which β-catenin is stabilized in prostate CSCs. Cdc20 was shown to induce the degradation of Axin1, a key regulator of β-catenin, through its proteasome activity [71]. This provides a direct relationship for Cdc20 contributing to the upregulation of β-catenin and leading to cancer progression.

Another mechanism whereby CSCs/TICs can be maintained by APC/C^Cdc20 is accomplished through the SOX2 signaling pathway. SOX2 is a transcription factor associated with pluripotency [74]. APC/C^Cdc20 was shown to form a complex with nuclear SOX2 and to maintain its stability, contributing to neoplastic cells’ ability for self-renewal in glioblastoma. It remains unclear on how this stability is achieved, however the authors speculate it is possible that APC/C^Cdc20 alters post-translational modifications on SOX2, thus providing stabilization. TICs can also be maintained by Cdc20 through the degradation of p21^CIP1/WAF1, which plays a pivotal role in suppressing TICs. p21^CIP1/WAF1 functions as a CDK inhibitor and therefore plays an important role in cell cycle control [75, 76]. Importantly, upon overexpression of Cdc20, TICs increased their ability to generate tumors in vivo, highlighting the oncogenic potential of Cdc20. [68]

In recent years Cdc20 has appeared to play a role both in the positive and negative regulation of apoptosis. This was thought to be accomplished through APC/C^Cdc20 targeting both Bim and Mcl-1 for destruction. As death elicited by anti-mitotic agents (e.g., taxanes) is modulated by both the cells’ ability to induce apoptosis and the duration of the arrest, these observations suggested that APC/C^Cdc20 plays central roles in the cellular response to anti-mitotics by coordinating both fate-determining pathways [77–79]. Bim was shown to specifically bind to Cdc20’s WD-40 motif, thus allowing for the polyubiquitination of Bim and leading to its subsequent degradation and promoting resistance to apoptosis induction by anti-mitotic agents [80]. In contrast, APC/C^Cdc20 was also implicated to play a role in the degradation of Mcl-1, a pro-survival protein, due to evidence of Mcl-1 interacting with APC/C^Cdc20 [81, 82]. However, further study failed to elucidate a clear relationship of Mcl-1 and Cdc20-mediated degradation. Rather, it appears that Mcl-1 may not be dependent on ubiquitin ligases for degradation and that Mcl-1 may function to regulate Cdc20 activity toward substrates such as Cyclin B, thus altering the rate of mitotic slippage and affecting induction of death by prolonged mitotic arrest [83]. Cdc20’s role in regulating apoptosis has clear implications for therapeutic response and it will be important to understand further whether cell or tissue-specific differences may influence its ability to coordinate the mechanisms regulating mitotic slippage and cellular survival.

Cdc20 also plays a role in the regulation of SMAR1, a tumor suppressor that functions to regulate the genome, the cell cycle, and apoptosis [84–87]. SMAR1 works by activating p53 to arrest cells at the G1 and G2/M transition. It is also involved in the DNA damage repair pathway, as well as ensuring the proper splice variant of CD44, a putative cancer stem cell marker and modulator of metastasis, is produced [70, 88, 89]. In higher grades of cancer, SMAR1 levels are often reduced, in contrast to high levels of Cdc20. Further research, using breast cancer, ovarian, melanoma, cervical, and colon cancer cell lines, elucidated that Cdc20 regulates the degradation of SMAR1 through ubiquitination. It is important to note that under stress, Cdc20 did not interact with SMAR1, possibly due to SMAR1 playing an active role in the DNA damage repair response [70].

Given the numerous cancers with reported overexpression of Cdc20, it is important to know how this upregulation occurs. While upregulation of Cdc20 transcription by deregulation of the Rb-E2F pathway or amplification have been described, post translational control of protein stability or function may also contribute to enhanced APC/C^Cdc20 activity in cancer. One potential route for post-translational upregulation is through Speckle-type POZ protein (SPOP). SPOP functions as a tumor suppressor through its function as a substrate-interacting adaptor protein of CRL3, an E3 ubiquitin ligase, specifically by targeting known oncogenic proteins such as ci/Gli, androgen receptor, SRC-3, DEK, TRIM24, MacroH2A, SENP7, and ERG for degradation. 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.

Overall, Cdc20 allows for the progression of cancer through several pathways, allowing for increased tumor proliferation and metastasis. Importantly, Cdc20’s role in cancer development and progression is not limited to a specific type of cancer. The diverse roles Cdc20 plays in tumorigenesis makes it a potent oncogenic factor and a potential target for therapeutic intervention.

APC/C^Cdh1 and Cell Cycle Regulation

Although there have been copious amounts of research focused on how APC/C^Cdh1 regulates the cell cycle, new insights continue to be uncovered. One of the most significant recent findings demonstrated how Cdh1 inactivation is a critical step during S-phase entry [113]. Normal human cells will spend a majority of their time in a quiescent state. Therefore, advancement into S-phase is a significant progression that commits the cell to division and proliferation. Earlier understanding of S-phase entry focused on the importance of the tumor suppressor retinoblastoma (Rb) and the transcription factor E2F. During G1, Rb represses E2F by binding to the transcription factor [114]. As cells move into S-phase cyclin E accumulates and activates Cdk2 which phosphorylates Rb and releases E2F [115]. Once E2F is released it localizes to the nucleus and increases the expression of S-phase proteins. It was previously understood that the phosphorylation of Rb was the point at which cells were committed to S-phase, however recent work on APC/C^Cdh1 has challenged this theory. It was observed that Cdk2 temporally phosphorylates Rb followed by Cdh1, which inactivates APC/C^Cdh1 by displacing the coactivator [113]. Interestingly, human cells were able to return to a quiescent state when Rb was phosphorylated but not when APC/C^Cdh1 was inactivated. This suggests that APC/C^Cdh1 inactivation is the point at which cells are committed to the cell cycle. Furthermore, it has been shown that once APC/C^Cdh1 is inactivated by Cdk2, Emi1 transitions from an APC/C^Cdh1 substrate to an APC/C^Cdh1 inhibitor [116] (Figure 2B). These findings that human cells commit to S-phase entry following the inactivation of APC/C^Cdh1 by Cdk2 phosphorylation and an increase in Emi1 levels further support APC/C^Cdh1 as an important tumor suppressor, which like Rb and p53 , plays a key role in preventing inappropriate entry into the cell cycle.

Although APC/C^Cdh1 activity is greatly reduced beyond the G1/S transition there have been some studies that indicate Cdh1 does contribute to cell cycle control during G2. Several recent studies extend these findings to demonstrate additional interplay between the APC/C and SCF ligases. FBXO31 is a substrate adaptor for the E3 ligase SCF, and it is known as a tumor suppressor that is commonly mutated in breast, ovarian, and prostate cancers [117–121]. Recently, it was identified as an APC/C^Cdh1 substrate, however the ability of APC/C^Cdh1 to recognize FBXO31 is dependent on two different kinases, AKT and ATM [122]. It was observed that in unstressed HEK293T cells, the pro-survival kinase and oncogene AKT would phosphorylate FBXO31 and target it for ubiquitination by APC/C^Cdh1 (Figure 4). Conversely, when HEK293T cells were stressed with ionizing radiation, ATM would phosphorylate FBXO31 and rescue it from APC/C^Cdh1 targeting [122] (Figure 2B). These results suggest Cdh1 plays an important role during ATM-mediated cell cycle arrest and DNA damage repair. AKT activity also regulates the ability of APC/C^Cdh1 to recognize other SCF components, Skp2 [123] and cyclin F [103] (Figure 4). Skp2 recognizes and ubiquitinates the cyclin dependent kinase inhibitor p27 [124]. AKT stabilizes Skp2 by phosphorylating it and disrupting binding between Skp2 and APC/C^Cdh1 [123]. Therefore, once AKT is active Skp2 degrades p27 and the cell cycle progresses. Interestingly, SCF^{Cyclin F} ubiquitinates Cdh1, and conversely APC/C^Cdh1 ubiquitinates Cyclin F. This regulatory loop is broken when AKT phosphorylates and stabilizes Cyclin F [125]. Once Cyclin F is stable, it then begins to ubiquitinate and destabilize Cdh1 leading to cell cycle progression. All together these experiments detail a novel mechanism in which AKT activity modulates Cdh1 activity and consequently regulates the cell cycle (Figure 4).

APC/C^Cdh1 Regulates Genome Stability

Cdh1 has been characterized as a tumor suppressor mainly because mice with heterozygous Cdh1 knock-out develop tumors [96]. Similarly, cultured melanocytes are transformed when Cdh1 and PTEN are knocked down [126], immortalized breast epithelial cells and colonic fibroblasts are transformed by loss of Cdh1, and loss of Cdh1 increases the proliferation of breast cancer cell lines [127]. Recent work has tried to elucidate why these Cdh1 deficient mice develop tumors and has identified that Cdh1 is important in regulating genome stability and the DNA damage response [93, 128–135]. Specifically, APC/C^Cdh1 has an important role regulating chromosomal instability and DNA replication. Mechanistic studies have shown that APC/C^Cdh1 is capable of regulating levels of the centrosome factor STIL in human cells [136]. The dysregulation of STIL is known to cause chromosomal rearrangement and acute lymphoblastic leukemia [137]. Therefore the mechanisms that control the levels of STIL are highly important. Interestingly, it has been observed that disrupting APC/C^Cdh1 control of STIL, by deleting the KEN box degron, caused an increase in centriole numbers [138]. This observation has since been reproduced by studying the interaction between Cdh1 and the STIL homolog, SAS-5, in C. elegans [139]. Other studies have identified the histone H3 variant, CENP-A, as a novel substrate of APC/C^Cdh1 in Drosophila which further connects APC/C^Cdh1 to chromosomal instability [140]. CENP-A localizes to and identifies centromeres. It has been observed that overexpression, and consequently mislocalization, of CENP-A contributes to aberrant kinetochore formation and chromosomal missegregation [141]. Therefore, dysregulation of APC/C^Cdh1 activity in cancer could be leading to more CENP-A expression and increased genome instability.

Cdh1 also protects genome integrity by regulating replication stress. The loss of Cdh1 is associated with replication errors leading to DNA damage and genomic instability [93, 142]. More specifically, there were slower replication forks, and consequently DNA breakage, when Cdh1 was knocked out [143]. While the shortened G1 phase of Cdh1-deficient cells may contribute to replication stress by multiple mechanisms, recent research in mouse embryonic fibroblasts suggests that the most likely cause of replication stress in the absence of Cdh1 is a decrease in the abundance of dNTPs [143].There are several reasons why loss of Cdh1 causes reduced dNTP levels. First, Cdh1 knock-down cells have a shorter G1, enter S-phase prematurely, [144] and may simply not have enough time to accumulate the appropriate amount of dNTPs as suggested by the ability of prolonging G1 in these cells to improve genomic integrity [143]. Second, it is known that a decrease in Cdh1 can lead to an increase of Cyclin F (which is an APC/C^Cdh1 substrate) [103]. As mentioned previously, Cyclin F is an E3 ligase substrate adaptor and it is known to negatively regulate critical subunits of ribonucleotide reductase (which is an essential enzyme in dNTP synthesis) [145]. Therefore, loss of Cdh1 could indirectly reduce the ability of cells to synthesize dNTPs by destabilizing subunits of ribonucleotide reductase. It will be interesting in future work to understand how the replication stress caused by loss of Cdh1 contributes to that induced by oncogenes to generate genomic instability.

The Role of Cdh1 in other Cancer Related Pathways

APC/C^Cdh1 has clear connections to cancer by regulating cell cycle control and genome stability, however Cdh1 has also been connected to other cancer related pathways. For instance, APC/C^Cdh1 can increase the ability of cancer cells to temper the immune response by indirectly regulating the levels of programmed death-ligand 1 (PD-L1) [146] (Figure 5). Cancer cells with increased PD-L1 suppress the adaptive immune response by destroying active T-cells and B-cells. A recent study identified that PD-L1 is ubiquitinated by the E3 ligase Cullin3^SPOP, and that SPOP is ubiquitinated by APC/C^Cdh1 [146]. Therefore, when APC/C^Cdh1 is active PD-L1 levels increase which allows neoplastic cells to subdue the immune response. This is a unique function of Cdh1 because in most instances Cdh1 functions as a tumor suppressor.

For instance, APC/C^Cdh1 is able to regulate the MEK/ERK oncogenic signaling cascade (Figure 5). In normal cells the MEK/ERK pathway is stimulated by an extracellular signal that activates EGFR and stimulates a kinase cascade that leads to cell growth and proliferation. BRAF is an important oncogenic kinase in the MEK/ERK pathway and is constitutively active in many cancer types such as skin, lung, colorectal, and brain [147]. EGFR and BRAF inhibitors can suppress MEK/ERK signaling and are currently being used to treat patients with cancer. Interestingly, APC/C^Cdh1 is also able to suppress MEK/ERK signaling by directly ubiquitinating and destabilizing BRAF in human cells [126]. Further investigation showed that melanocytes have decreased Cdh1 expression, and increased BRAF expression, when exposed to UV radiation [126, 148, 149]. Researchers were also able to identify that Cdh1 interacts with BRAF through the degron D-box 4. According to the Catalog of Somatic Mutations in Cancer, mutations in D-box 4 (R671Q) have been found in patients with skin cancer (mutation Id COSM159405, cancer.sanger.ac.uk). Researchers were able to show that BRAF with a R671Q mutation showed a decrease in Cdh1 binding and increased BRAF stability [126]. This data suggests that when melanocytes are subjected to UV light there is a decrease in APC/C^Cdh1 function and an increase in MEK/ERK signaling because of the increase in BRAF expression. Additionally, patients with BRAF mutations in D-box 4 could have a high risk of developing cancers because APC/C^Cdh1 is unable to regulate BRAF expression. In addition to BRAF, loss of Cdh1 function in melanocytes leads to increased stability of PAX3 an upstream transcriptional regulator of the oncogene MITF that is sufficient to promote melanoma cell proliferation and resistance to chemotherapies [149].

Functions of Cdh1 Independent of APC/C

Most of the research that focuses on the role and regulation of Cdh1 in cancer has concentrated on its APC/C-dependent functions. However, there have been some recent studies that have demonstrated the ability of Cdh1 to regulate cancer associated functions independent of the APC/C (Figure 5). One of these pathways is the PTEN regulation of PI3K/AKT. As previously mentioned AKT is a prosurvival kinase that phosphorylates a number of substrates and can regulate processes such as metabolism, proliferation and cell migration. PI3K is another kinase that is upstream of AKT and initiates its activation. PTEN is a phosphatase and one of the major antagonists of this pathway, and importantly loss of PTEN is common in cancers and leads to increased PI3K/AKT signaling [150]. Novel research has shown that decreasing the amount of Cdh1 in cancer cells caused a decrease in WWP2 substrates such as PTEN [151]. WWP2 is an E3 ligase that ubiquitinates PTEN [152]. Interestingly, the ability of Cdh1 to inhibit WWP2 did not depend on the ability of Cdh1 to interact with APC/C. Conversely, Cdh1 directly binds to WWP2 and locks the E3 ligase in an inactive conformation [151]. Interestingly, another study showed that nuclear PTEN is capable of recruiting Cdh1 to APC/C [153]. This means that there is a positive feedback loop between APC/C^Cdh1 and PTEN. Both are capable of positively regulating each other and this strengthens tumor suppressor capabilities.

Cdh1 has also been show to regulate the non-receptor tyrosine kinase Src [127]. This kinase was one of the first oncogenes identified and is responsible to transducing extracellular signaling. Src targets many substrates and therefore has been connected to numerous pathways and cellular functions. Most commonly, Src has been shown to regulate proliferation, migration, and apoptosis [154]. Given the oncogenic potential of Src it is important to understand how its expression and activity is regulated in human cells. In breast cancer cells lines it was observed that Src activity increased when Cdh1 was knocked down, but there was no change in the abundance of Src [127]. Typical APC/C^Cdh1 substrates have an increase in expression when Cdh1 is knocked down. Through immunoprecipitation experiments it was determined that Cdh1 was binding to the N and C terminus ends of Src and locking it in it inactive conformation [127]. This is a similar mechanism to how Cdh1 inactivates WWP2. Ultimately these studies suggest Cdh1 should not be exclusively characterized as a coactivator for APC/C. It is becoming clear Cdh1 has important functions that are ubiquitin independent (Figure 5).

The diverse nature of the processes that Cdh1 participates in provide a likely explanation of its role as a haploinsufficient tumor suppressor. First, due to the many events that it regulates in the cell cycle alone, it is advantageous for cancer cells to maintain some level of Cdh1 activity. Second, while some of the oncogenic targets of Cdh1 (both APC/C-dependent and -independent) can have enhanced activity by amplification or other Cdh1-independent mechanisms, which may contribute to tumorigenesis, in general it seems that the combined deregulation of multiple pathways by partial reduction in Cdh1 function would be sufficient to create a permissive environment for the evolution of neoplastic cells.

APC/C is essential for normal development and is dysregulated in cancer

Mouse models involving Cdc20 and Cdh1 confirm that APC/C activity is essential for normal development and viability. Mouse embryos with homozygous deletions of Cdc20 are unable to undergo embryongenesis and arrest at the two-cell stage in metaphase [166]. Likewise, loss of Cdh1 leads to embryonic lethality with death occurring by embryonic day 10, due to defects in trophoblast endoreduplication [167] [168]. Lethality of the Cdh1-deficient embryos can be rescued with Cdh1 expression in the placenta, however the mice develop structural and numeric aberrations and proliferate inefficiently. Mouse models also confirm the oncogenic function of Cdc20 and the tumor suppressor function of Cdh1. Cdh1 heterozygous mice have an increased incidence of spontaneous tumor development, confirming the role of Cdh1 as a haploinsufficient tumor suppressor [96, 169]. Cdc20 hypomorphic mice develop considerable aneuploidy [170] and Cre-induced ablation of Cdc20 in mice carrying one null Cdc20 allele and one floxed Cdc20 knockout allele, ablated in vivo tumorigenesis in a skin-tumor mouse model [171]. Importantly, loss of Cdc20 elicited dramatic regression in skin tumors due to apoptotic death, whereas anti-mitotics exhibited more limited efficacy, confirming the rationale for inhibition of Cdc20 as a cancer therapy.

TAME and apcin directly inhibit APC/C and can be used in combination with microtubule-inhibiting drugs

Because APC/C is essential for mitotic exit during cellular division, its inhibition remains an attractive approach for cancer therapies. Indeed SAC-mediated inhibition of APC/C^Cdc20 is an important component of the cellular response to anti-mitotic agents such as taxanes and vinca alkaloids. Tosyl-L-Arginine Methyl Ester (TAME) is an APC/C inhibitor that was first identified as an inhibitor of cyclin proteolysis in a phenotypic Xenopous egg extract screen [172]. Because TAME is not cell permeable, the pro-drug form, pro-TAME, was developed in 2010 by the same lab and was found to inhibit Cdc20 and Cdh1 binding to APC/C, thereby preventing APC/C activation [173]. Later work revealed that TAME preferentially suppresses APC/C^Cdc20 rather than APC/C^Cdh1 [28]. Cells exposed to proTAME exhibit a prolonged mitotic arrest, which causes SAC reactivation and cohesion fatigue, resulting in cell death [174]. A second inhibitor, apcin, was described in 2014 [175]. Apcin binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates through occupation of the D-box-binding pocket within the Cdc20 WD40 domain [175]. Treatment of cells with apcin alone does not significantly affect cell survival because it is an inefficient inhibitor of APC/C. However, apcin is synthetically lethal in cohesion-defective cells and cancers [176].

Because apcin and TAME inhibit APC/C via different protein-protein interactions, a combination treatment synergizes to prolong mitotic duration in human cancer cell lines. For instance, the combination treatment apcin and proTAME has been shown to decrease tumor cell growth in multiple myeloma and osteosarcoma cancer cell lines, as well as in GBM cancer stem cells [177–179]. APC/C inhibitors are also being tested in combination with microtubule-inhibiting drugs to further sustain mitotic arrest and enhance cell death. Crawford et al. show that proTAME in combination with the topoisomerase II inhibitors, etoposide and doxorubicin, and vincristine, a microtubule destabilizing agent, were effective at decreasing cell survival in multiple myeloma cells [180]. In addition, siRNA knockdown of Cdc20 in combination with docetaxel, a microtubule stabilizing agent, decreased castration-resistant prostate cancer cell growth [181]. In a recent study, Raab et al (2019) employed a combination treatment of paclitaxel and Plk1 inhibitor (BI6727) followed by proTAME in a ‘two-punch’ strategy to cause strong mitotic arrest followed by blocking mitotic exit to induce apoptosis in ovarian cancer primary cells and cell lines [182]. In addition, the Malumbres lab found that chemical inhibition of APC/C with proTAME caused increased sensitivity to Eg5 inhibitors and dramatic sensitivity to topoisomerase II inhibitors [183].

Other drugs are being explored as APC/C inhibitors

Numerous studies have implicated APC/C involvement in the response to newly emerging drugs. For example, Liu et al. show that treatment of colorectal cancer cell lines with Indoleamine 2, 3-dioxygenase (IDO), an enzyme that is the rate-limiting step in tryptophan metabolism, coincides with decreased Cdc20 expression and G2/M arrest [184]. Zhang et al. report that curcumin, a substance in turmeric, causes decreased pancreatic cell proliferation presumably due to downregulation of Cdc20 [185]. Rottlerin, polyphenol natural product isolated from the Asian tree Mallotus philippensis, was also shown to decrease glioma cell growth via many pathways [186]. Although the exact mechanism is not known, the authors show decreased Cdc20 expression is glioma cells treated with Rottlerein as well as treatment rescue with Cdc20 overexpression. Another study demonstrated that triterpenes from Poria cocos inhibit the migration of pancreatic cancer cells in correlation with decreased CDC20 [187]. These studies underscore the potential importance of Cdc20 inhibition as a therapeutic strategy in cancer.