APC/C^Cdh1 targets multiple substrates for degradation during mitosis (Figure 1). Foremost among these are the mitotic cyclins, whose degradation ablates cyclin-dependent kinase 1 (CDK1) activity (Brandeis and Hunt, 1996; Irniger and Nasmyth, 1997). APC/C also reduces activity of multiple CDKs by initiating degradation of two components of the SCF ubiquitin ligase, the F-box protein Skp2 and an accessory protein, Cks1 (Bashir et al., 2004; Wei et al., 2004). Degradation of these two substrates raises the levels of the cyclin-dependent kinase inhibitors p27^Kip1 and p21^Cip1, which are normally targeted for degradation in an SCF^Skp2 and Cks1 dependent manner (Ganoth et al., 2001; Bornstein et al., 2003). CDK inhibitors associate with specific cyclins and CDKs, preventing them from binding to ATP, and hence blocking their catalytic activity. Thus, high p27^Kip1 and p21^Cip1 levels maintain an early G1 state by reducing CDK activity. Another mechanism to promote G1 by reducing CDK activity involves APC/C^Cdh1 mediated inhibition of an activator of S phase entry, cyclin D1. APC/C^Cdh1 initiates degradation of the transcription factor Ets2 (Li et al., 2008), which normally controls cyclin D1 levels (Albanese et al., 1995). Consistent with these findings, depletion of Cdh1 by siRNA stabilizes Skp2 and Ets2, resulting in p21^Cip1 and p27^Kip1 degradation and cyclin D1 elevation in G1, followed by premature S phase entry (Wei et al., 2004; Li et al., 2008) and increased proliferation (Bashir et al., 2004). Thus, APC/C^Cdh1 function during mitotic exit is coupled to G1 maintenance. It initiates a sharp decrease in CDK1 activity during mitotic exit by targeting the mitotic cyclins for destruction and subsequently remains active in early G1 to ensure that they remain inactive. In addition, it ensures that other cyclin-dependent kinases, namely CDK2/cyclin E, CDK2/cyclin A, CDK4/cyclin D, and CDK6/cyclin D are inactive since it maintains high p27^Kip1 levels and low cyclin D1 levels through Skp2 and Ets2 degradation.

One question that immediately arises from these studies is that if APC/C keeps CDKs inactive in early G1, how do cells eventually reach S phase. The answer lies in the multiple mechanisms that decrease APC activity (Figure 1). For instance, the ubiquitination of the APC/C-specific ubiquitin-conjugating enzyme (E2) UbcH10 by APC/C^Cdh1 provides a negative feedback mechanism that eventually dampens APC/C^Cdh1 activity (Rape and Kirschner, 2004; Rape et al., 2006). Further, Cdh1 is inactivated by both phosphorylation and degradation (Lukas et al., 1999; Listovsky et al., 2004; Benmaamar and Pagano, 2005). Finally, E2F activates the transcription of the APC/C pseudo-substrate early mitotic inhibitor-1 (Emi1)/Rca1 in late G1, which inhibits APC/C^Cdh1 activity (Hsu et al., 2002). These distinct mechanisms ensure that APC/C^Cdh1 activity remains low from late G1 until the subsequent metaphase when it is activated via reduction of CDK and Emi1 activity (Kotani et al., 1998; Hsu et al., 2002; Figure 1).

One of the main reasons APC/C^Cdh1 activity must remain low during S phase is that it is an inhibitor of pre-replication complex formation required for S phase entry (Diffley, 2004). APC/C^Cdh1 controls the formation of the pre-replication complexes by modulating levels of three major regulators of the formation of the complex: Orc1, Cdc6, and geminin. For DNA replication to proceed, cells need to alternate between periods of low CDK activity and low geminin levels, in which the pre-replicative complexes (preRCs) are assembled; and periods of high CDK activity and high geminin levels in which origin firing and DNA replication occurs (McGarry and Kirschner, 1998; Petersen et al., 2000; Araki et al., 2003). APC/C^Cdh1 is crucial to properly regulate the switch between these two states. For instance, Orc1 and Cdc6 are degraded via APC/C^Cdh1 during early G1 (Petersen et al., 2000; Araki et al., 2003). Further, geminin levels are tightly controlled by APC/C^Cdh1. Geminin prohibits initiation of DNA replication at inappropriate times of the cell cycle by preventing MCM recruitment at the replication origins. During G1 the APC/C is active and as a consequence, geminin concentration is low. At the G1–S transition, APC/C is inactivated and geminin begins to accumulate. However, geminin concentration is not sufficient to inhibit a first wave of preRC formation and DNA replication begins. As S phase progresses, geminin accumulates and inhibits subsequent recruitment of MCMs to the replication origins (McGarry and Kirschner, 1998), and therefore re-duplication is avoided (McGarry and Kirschner, 1998; Wohlschlegel et al., 2000).

Multiple studies suggest that a major APC/C^Cdh1 function outside of the cell cycle is limiting CDK activity required for cell cycle progression (Qiao et al., 2010). APC/C^Cdh1 regulates CDK inhibitors that reduce CDK activity and initiate cell cycle exit. Adding a second layer of regulation to APC/C^Cdh1 activity, Binné et al. (2007) connected retinoblastoma protein (pRb) to APC/C^Cdh1, Skp2, and CDK inhibitor dependent cell cycle exit. These studies demonstrated that hypophosphorylated pRb associates with the APC/C specifically when activated by Cdh1, thus promoting Skp2 degradation and accumulation of p27^Kip1 and p21^Cip1. This important finding linked extracellular signaling mechanisms, which normally control pRb activity to APC/C dependent degradation of Skp2 and initiation of cell cycle exit.

Once cells have exited the cell cycle, APC/C activity is required to maintain quiescence or differentiation. For instance, APC^Cdh1 inactivation by deleting its Apc2 subunit in adult hepatocytes induced these otherwise quiescent cells to re-enter the cell cycle (Wirth et al., 2004). Similarly, APC^Cdh1 has also been proposed to block postmitotic differentiated neurons from inappropriate cycling and apoptosis (Almeida et al., 2005; Jackson, 2006).

APC/C^Cdh1 activity promotes cell cycle exit and differentiation since Cdh1 depletion reduces differentiation of muscle, lens, hematopoietic, and neuronal cells (Lasorella and Iavarone, 2006; Li et al., 2007; Wu et al., 2007; Garcia-Higuera et al., 2008). Although our knowledge of the involvement of APC/C^Cdh1 in the differentiation of other tissues awaits further investigation, its critical function in the degradation of cell cycle proteins suggests it likely plays relevant roles in linking quiescence and differentiation in most cell types. Further, since cancer progression is often thought to involve a dedifferentiation process (Daley, 2008; Trosko, 2009), understanding APC/C’s involvement in normal cell cycle exit and differentiation will give us clues as to how APC/C regulation or substrate targeting may be misregulated during tumorigenesis.

A well-established tumor suppressor protein that positively regulates APC/C activity is the pRB. pRB plays multiple roles during the cell cycle; it blocks cell cycle progression from G1 to S phase, and guards the cell from replicating damaged DNA, thus preventing the integration of mutations into the genome. This tumor suppressor function requires binding and repressing the E2F family transcription factors (Classon and Harlow, 2002; Chen et al., 2009). Reversion of the pRb–E2F interaction occurs after mitogenic stimuli sufficient for the activation of CDKs that phosphorylate pRB in the nucleus. Subsequently, phospho-pRb is released from E2Fs, which can then induce S phase entry (Thoma et al., 2011). Consistent with a direct inhibitory role in S phase entry, loss of various pRBs leads to unscheduled cell proliferation in many tissues (Vidal and Koff, 2000; Cobrinik, 2005; Marx et al., 2010). Accordingly, mutations in Rb are found in a wide variety of cancers mainly in the lung, breast, and eye (Meraldi et al., 1999; Sabado Alvarez, 2008).

Retinoblastoma protein also possesses E2F-independent functions that contribute to cell cycle control. pRB interacts with both Skp2 (Ji et al., 2004) and with APC/C^Cdh1 through distinct surfaces (Binné et al., 2007). pRB needs to interact with both proteins to target Skp2 for ubiquitin-mediated degradation and promote p27^Kip1 accumulation and cell cycle exit. Interestingly, similar to E2F1, Cdh1 only interacts with the hypophosphorylated (active) form of pRb, and phosphorylation of pRb by cyclin A/CDK2 kinase abolished the ability of Rb to interact with both Cdh1 and E2F1. Therefore Cdh1, by competing with pRb interaction, can also regulate the activity of the E2F1 transcription factor (Sorensen et al., 2000; Gao et al., 2009a). Further, given that both pRB and Cdh1 have tumor suppressive functions, whether the pRB–APC/C^Cdh1 interaction is misregulated during tumorigenesis should be investigated (Figure 2).

Recent studies have demonstrated that the well-characterized tumor suppressor protein PTEN is a positive regulator of APC/C activity. PTEN (Cairns et al., 1997; Steck et al., 1997; Feilotter et al., 1998; Gray et al., 1998; Li and Sun, 1998) encodes a lipid phosphatase, which plays a crucial role in adhesion, migration, growth, and apoptosis. Pten-null mice die embryonically, but heterozygous mice survive and develop tumors in the lymphoid system, endometrium, prostate, and the thyroid (Di Cristofano et al., 1998; Podsypanina et al., 1999). Pten somatic mutations occur in a large percentage of human cancers, with the highest numbers found in endometrial, central nervous system, skin, and prostate cancers (Chalhoub and Baker, 2009).

The effects observed after PTEN loss have been attributed to activation of the PI3K/AKT pathway as PTEN can dephosphorylate phosphoinositide-3,4,5-triphosphate (PIP3), a potent activator of Akt1 (Maehama and Dixon, 1998). PTEN inactivation by phosphorylation or oxidation in human cancer results in elevated Akt1 activity and abnormal growth regulation (Silva et al., 2008; Chalhoub and Baker, 2009; McCubrey et al., 2011). However, functions independent of its phosphatase activity have been described (Maier et al., 1999; Georgescu et al., 2000; Koul et al., 2002a,b; Gildea et al., 2004; Blanco-Aparicio et al., 2007). Recently, a novel interaction between PTEN and APC/C^Cdh1 has been reported (Song et al., 2011). Song et al. (2011) demonstrated that nuclear PTEN directly enhances the activity of APC/C by promoting its association with Cdh1 in a phosphatase-independent manner. Conversely, PTEN loss impairs the activity of the APC/C^Cdh1 complex. Therefore, loss of PTEN function decreases APC/C^Cdh1 activity, suggesting novel molecular pathways that may be involved in the progression and initiation of tumorigenesis. This in turn highlights the complexity of the dose-dependent tumor promoting and fail-safe cellular responses evoked by PTEN loss. Accordingly, tumor-derived mutations that do not affect the phosphatase enzymatic activity of PTEN in vitro have also been identified. These were found to affect the compartmentalization of PTEN in the cell, either by sequestering PTEN in the nucleus (Denning et al., 2007) or by interfering with PTEN’s recruitment to the plasma membrane (Lee et al., 1999; Georgescu et al., 2000; Walker et al., 2004), thus possibly negatively impacting the PTEN–APC/C^Cdh1 interaction. Consistent with this possibility, APC/C^Cdh1 substrates are overexpressed in tumors containing PTEN deficiency (Marino et al., 2002; Gao et al., 2009a; Liu et al., 2011b). Further, PTEN overexpression has been found to suppress tumorigenesis by inducing G1 cell cycle arrest in human glioblastoma cells (Li and Sun, 1998). Given the importance of PTEN and APC/C^Cdh1 for cancer progression, it will be crucial to determine whether tumors containing PTEN misregulation have both hyeractivation of the Akt1 pathway and dysregulation of APC/C^Cdh1 function (Figure 2).

The Emi1 is an APC/C inhibitor, which is overexpressed in multiple tumors. Emi1 is a key cell cycle regulator that is required for accumulation of mitotic cyclins and other critical cell cycle regulators during S phase and G2 (Hsu et al., 2002). At the G1/S transition, Emi1 functions as a pseudo-substrate inhibitor of the APC/C (Miller et al., 2006), allowing substrates to accumulate (Guardavaccaro et al., 2003; Miller et al., 2006). In early mitosis, Emi1 is phosphorylated by Plk1 (Hansen et al., 2004), which triggers SCF^βTrCP-dependent ubiquitination and destruction, thus inducing APC/C activation and mitotic progression (Margottin-Goguet et al., 2003). Emi1 overexpression leads to unscheduled cell proliferation, tetraploidy, and chromosomal instability in p53-deficient cells (Lehman et al., 2006; Figure 2). In p53 wild-type cells, the induction of tetraploidy and aneuploidy by overexpressing APC/C inhibitors like Emi1 typically leads to G1 arrest or apoptosis. Indeed, Emi1 overexpression causes mitotic catastrophe and genomic instability through APC/C misregulation, and thus potentially contributes to tumorigenesis (Hsu et al., 2002). Upregulation of Emi1 mRNA has been found in a variety of malignant tumors compared to matched normal and benign tumor tissue. Notably, Emi1 protein is highly expressed in renal cell carcinomas, cervical adenocarcinomas, hepatocellular carcinomas, oligodendrogliomas, lung adenocarcinomas, endometrial cancers, of melanomas, many lymphomas and ovarian clear cell carcinoma (Lehman et al., 2006; Gütgemann et al., 2008). Emi1 expression is also related to the pRB/E2F pathway since Emi1 is a target of the E2F transcription factor. At the G1-S transition Emi1 is transcriptionally induced by E2F, thus accelerating S phase entry (Hsu et al., 2002). The importance of this regulation is underscored by the finding that lack of pRb repression of E2F-mediated transcription causes misregulation of Emi1 and APC/C substrates in malignant tumors (Lehman et al., 2006).

Recently, the histone deacetylase (HDAC) SIRT2 has been shown to positively regulate APC/C activity (Kim et al., 2011). Histone acetyltransferases (HATs) and HDACs are enzymes controlling protein acetylation. Both target histones, whereas HATs catalyze the acetylation of histones and relax chromatin to increase accessibility of transcription factors to promoters of target genes, HDACs remove acetyl groups from histones and repress transcription (Strahl and Allis, 2000). Most of them have been found in transcription factor complexes and, therefore, have been considered to regulate transcription by modulating acetylation levels of chromatin (Kuo and Allis, 1998; Kouzarides, 1999). HDACs are important for cell cycle progression and their inhibition promote cell arrest in G1 and G2 (Marks et al., 2001; Johnstone and Licht, 2003; Noh and Lee, 2003). HDACs also have an important role in heterochromatin formation and maintenance (Olsson et al., 1999; Taddei et al., 2001) and are implicated in chromosome segregation (Nakayama et al., 2003; Silverstein et al., 2003; Kimata et al., 2008).

Several HDACs have a role in tumorigenesis, acting as a suppressors or promoters (Lagger et al., 2002; Glaser et al., 2003; Saunders and Verdin, 2007; Wang et al., 2008a,b; Deng, 2009; Bell et al., 2011; Kim et al., 2011) and HDAC inhibitors are a promising class of anti-cancer agents (Johnstone and Licht, 2003; Yoshida et al., 2003; Bolden et al., 2006). SIRT2 is predominantly localized in the cytoplasm where it deacetylates microtubules (North et al., 2003). During mitosis, SIRT2 is localized to chromosomes regulating chromosomal condensation (Vaquero et al., 2006; Inoue et al., 2007), and is also associated with mitotic structures, including the centrosome, mitotic spindle, and midbody, presumably to ensure normal cell division (North and Verdin, 2007). Kim et al. (2011) demonstrated that SIRT2 regulates APC/C activity through deacetylation of its coactivators Cdh1 and Cdc20. Deacetylation of Cdh1 and Cdc20 by SIRT2 enhances the interaction of these coactivators with Cdc27, leading to activation of APC/C. In Sirt2 mutant mice, reduced APC/C activity results in tumor formation by producing genomic instability associated with centrosome amplification, aneuploidy mitotic cell death, and spontaneous tumor formation. These deficiencies seem to be caused by a combined effect of altered expression of mitotic regulators that are controlled by APC/C (Figure 2). Accordingly, SIRT2 expression is reduced in several human malignancies including breast, liver, brain, kidney, and prostate cancers (Kim et al., 2011).

Mitotic defects associated with chromosomal aberrations are often observed in prostate cancer cells and tissue, suggesting possible misregulation by APC/C (Tribukait, 1991; Beheshti et al., 2001; Pihan et al., 2001). Two regulators of the APC/C have been found to be clearly involved in prostate cancer, prostate specific membrane antigen (PMSA) and male germ cell-associated kinase (MAK). PSMA is a type II transmembrane protein that exhibits both N-acetylated alpha-linked acidic peptidase (NAALADase) and folate hydrolase activities (Ghosh and Heston, 2004; Rajasekaran et al., 2005). PSMA is localized to secretory cells within the prostatic epithelium although its physiological and pathological functions remain unclear. PSMA is upregulated in advanced prostate carcinoma and metastatic disease (Rajasekaran et al., 2005), and is a feature of practically every prostatic tissue examined (Rajasekaran et al., 2005, 2008). PSMA is absent or moderately expressed in hyperplastic and benign tissues, while malignant tissues have high levels, demonstrating that PSMA expression increases proportionally to tumor aggressiveness (Troyer et al., 1995; Kawakami and Nakayama, 1997; Liu et al., 1997; Silver et al., 1997; Sweat et al., 1998; Chang et al., 1999, 2001; Burger et al., 2002; Ross et al., 2003). PSMA is localized to a membrane compartment in the vicinity of centrosomes at the spindle poles and associates with the APC/C subunit Cdc27 leading to premature activation of APC/C, and induction of aneuploidy (Rajasekaran et al., 2008). Increased APC/C activity observed in PSMA expressing cells is sufficient to impair the mitotic spindle checkpoint, which agrees with the finding that PSMA expressing cells exit mitosis prematurely (Figure 2). Therefore, PSMA may have a causal role in the progression of prostate cancer.

Male germ cell-associated kinase belongs to a protein kinase family characterized by a catalytic domain resembling a hybrid of the TXY motif found in mitogen-activated protein kinases (MAPK) and the TY motif in CDKs (Payne et al., 1991; Brown et al., 1999; Xia et al., 2002; Fu et al., 2005). MAK expression is elevated in castration-resistant prostate cancer cell lines and is generally overexpressed in prostate tumors, thus possibly contributing to malignancy via aberrant regulation of mitosis (Wang and Kung, 2011). MAK protein has a dynamic subcellular localization during the cell cycle: it is localized to the mitotic spindle and centrosomes during metaphase and anaphase, and to the mitotic midbody from anaphase to telophase. MAK negatively regulates APC/C^Cdh1 through interaction and phosphorylation of Cdh1 in CDK phosphorylated sites, in a manner reminiscent of CDK-dependent inactivation of Cdh1 (Wang and Kung, 2011; Figure 2). The phosphorylation of MAK increases between S and G2, peaks at early mitosis, and drastically decreases at the end of mitosis. This promotes the dissociation of Cdh1 and APC/C thus decreasing APC/C^Cdh1 activity and promoting the stabilization of the substrates Aurora kinase A and Plk1. As overexpression of Aurora-A is known to induce centrosome amplification (Zhou et al., 1998), the extra-centrosomes observed in MAK overexpressed cells is likely due to cellular accumulation of Aurora-A.

The proteolysis of APC/C substrates starts as cells make the transition from G2 to M phase. One of the earliest targets is the NIMA-related kinase 2 (Nek2) family of serine/threonine protein kinases. They are implicated in the regulation of centrosome separation and spindle formation. Nek2A, along with cyclin A are unique APC/C^Cdc20 substrates that get degraded during prometaphase even when the SAC is active. A C-terminal dipeptide methionine–arginine (MR) tail enables Nek2A to directly bind the APC/C independently of Cdc20 (Hayes et al., 2006). Nek2 levels are upregulated in human breast cancer, pediatric osteosarcoma, and B-cell lymphomas (Wai et al., 2002; de Vos et al., 2003; Hayward et al., 2004).

Cell division cycle protein 20 is an essential coactivator of the APC/C during mitosis. Multiple studies have indicated that maintaining appropriate Cdc20 levels is important for cellular homeostasis. When this is disturbed and Cdc20 is overexpressed, tumorigenesis can occur. For instance, oral squamous cell carcinomas (OSCC) and breast cancer cells overexpress Cdc20 causing premature anaphase and aneuploidy linked to tumor formation (Yuan et al., 2006; Mondal et al., 2007). Importantly, even a twofold overexpression of Cdc20 leads to spindle checkpoint defects and early Pds1 (securin) destruction producing aneuploidy (Pan and Chen, 2004).

One of the major ways Cdc20 protein levels are controlled is via the SAC. When the SAC is active during metaphase, the checkpoint proteins Mad2, BubR1, and Bub3 bind to Cdc20 and convert it into a substrate of APC/C^Cdc20. This allows for the ubiquitination and degradation of Cdc20 by APC/C^Cdc20 (Nilsson et al., 2008; Ge et al., 2009). Thus, SAC activation inhibits Cdc20 and arrests cells in metaphase. Inactivation of SAC at the end of metaphase relieves this inhibition and Cdc20 promotes mitotic exit by degrading key substrates such as the mitotic cyclins. The importance of SAC dependent control of Cdc20 levels is underscored by the finding that anti-mitotic cancer drugs that activate the SAC cause cell cycle arrest. These drugs cause a prolonged metaphase arrest that may eventually lead to cell death. However, during this prolonged metaphase arrest, some Cdc20 escapes inhibition by the SAC and as a result, cyclin B1 levels start to fall and cells exit mitosis, thereby initiating mitotic slippage (Nilsson, 2011). This may be especially true in some cancer subtypes since the frequency of mitotic slippage is hastened in tumor cells lacking p53 and pRb (Depamphilis, 2011). Further, mitotic slippage reduces the sensitivity of tumor cells to chemotherapeutics. Thus, alternative means of inhibiting Cdc20 are needed.

Recent evidence suggests that targeting Cdc20 directly could be a better alternative than targeting the SAC in cancer therapy (Huang et al., 2009; Manchado et al., 2010). Knockdown of Cdc20 by siRNA induces mitotic arrest and apoptosis in various cancer cell lines that are otherwise resistant to apoptosis and prone to mitotic slippage (Huang et al., 2009). Further, genetic ablation of the Cdc20 gene in murine skin tumor and aggressive fibrosarcoma models results in complete tumor regression (Manchado et al., 2010). Cdc20 deletion induces metaphase arrest and apoptosis in tumor cells both in vitro and in vivo. Importantly, the activities of Cdk1 and Mast1 kinases are required for this arrest. When Cdk1 and Mast1 kinases are inhibited, Cdc20 null cells exit mitosis via the activities of the phosphatases PP2A/B55α and PP2A/B55δ (Manchado et al., 2010). Thus, small molecules attenuating Cdc20 coupled with those inhibiting these phosphatases may be effective therapeutically.

Another APC/C substrate degraded at the metaphase–anaphase transition is geminin. It is a small 25 kDa protein that inhibits DNA re-replication during S phase. Geminin levels accumulate during S, G2, and early mitosis. It suppresses the DNA replication factor CDT1 that is required for the formation of preRCs. Depletion of geminin by siRNA in tumor cells leads to DNA re-replication, cell cycle arrest, and apoptosis. However, regulation of CDT1 by geminin is found to be rate limiting for initiation of DNA replication only in cancer cells and not in normal or immortalized cells. Normal cells show the same effect only when cyclin A is co-depleted with geminin (Zhu and Depamphilis, 2009). Thus it has been proposed that inhibition of geminin activity can be used to selectively kill cancer cells (Zhu and Depamphilis, 2009).

Toward the end of anaphase, the pro-mitotic regulatory kinase Plk1, is degraded by APC/C^Cdh1 in a D-box dependent manner (Lindon and Pines, 2004). Plk1 is a major kinase in eukaryotic cells involved in a variety of processes such as activation of the maturation promoting factor (MPF) by phosphorylation of Cdc25C and cyclin B1, bipolar spindle formation, and maturation of the centrosome (Ohkura et al., 1995; Lane and Nigg, 1996; Abrieu et al., 1998; Qian et al., 1998; Eckerdt et al., 2005). Elevated Plk1 levels are present in a broad spectrum of cancers including breast cancer, colorectal cancer, ovarian cancer, melanomas, pancreatic cancer, prostate cancer, lung cancer, and squamous cell carcinomas of the head and neck (Wolf et al., 1997, 2000; Knecht et al., 1999; Strebhardt et al., 2000; Macmillan et al., 2001; Gray et al., 2004; Weichert et al., 2004). This broad range of expression makes Plk1 a prime target in cancer (Strebhardt and Ullrich, 2006).

Another APC/C^Cdh1 substrate during late mitosis is Aurora-A (STK15/BTAK), a serine/threonine kinase localized to the centrosome controlling mitotic spindle assembly and centrosome maturation. It is involved in a variety of human cancers (Bischoff et al., 1998; Zhou et al., 1998; Sen et al., 2002; Mondal et al., 2007). Ectopic expression of Aurora-A leads to chromosome instability and centrosome amplification (Miyoshi et al., 2001). One way it achieves this is by phosphorylating and regulating Centrin, the calcium-binding phosphoprotein located in the centrosome. Phosphorylated Centrin is more stable against APC/C^Cdh1 mediated destruction and an excess of it is thought to promote centrosome amplification in cancer (Lukasiewicz et al., 2011). Aurora-A itself becomes resistant to APC/C^Cdh1 mediated degradation when it is constitutively phosphorylated on Ser 51, and consequently gets overexpressed in head and neck cancers (Kitajima et al., 2007).

High expression in cancer 1 (HEC1), a recently identified APC/C^Cdh1 substrate, is part of the NDC80 complex controlling kinetochore microtubule dynamics. HEC1 levels peak during early mitosis and fall by telophase (Lipkowitz and Weissman, 2011). The mitotic regulatory kinase Nek2 phosphorylates HEC1 on Ser 165 thus activating it during G2/M. Inactivation of HEC1 results in severe chromosome segregation defects (Chen et al., 1997, 2002; Zheng et al., 1999). HEC1 is considered as a candidate marker for breast lesions likely to undergo malignant transformation because it is significantly overexpressed in benign breast tumors (Bieche et al., 2011). A small molecule called INH1, which binds to HEC1 and specifically disrupts the HEC1–Nek2 interaction, is found to suppress human breast cancer cell proliferation in culture as well as tumor growth in nude mice bearing xenografts (Wu et al., 2008).

c-Jun NH₂-terminal kinase (JNKs), a class of MAPK, lead the cell’s response to stress stimuli such as heat shock, radiation, and cytotoxic and genotoxic stress. Recently, nuclear JNK was found to be a substrate of APC/C^Cdh1 during late mitosis and G1 (Gutierrez et al., 2010a). JNK, in turn, controls APC/C^Cdh1 by phosphorylating Cdh1 at three residues (Yu et al., 1998; Summers et al., 2008) to prevent premature Cdh1 association with the APC/C during G2. JNK-induced phosphorylation of key regulators not only controls cell survival and differentiation, but also cell cycle progression. JNK phosphorylates Cdc25C at Ser 168 in G2, downregulating its phosphatase activity required for CDK1 activation (Gutierrez et al., 2010b). This is required for the correct timing of mitotic entry and the proper establishment of G2/M checkpoint upon UV irradiation. However, constitutively active JNK causes defects in cell cycle progression. Indeed, many human tumors have been reported to require JNK activity for their growth and survival (Potapova et al., 2000; Antonyak et al., 2002; Tsuiki et al., 2003; Yang et al., 2003; Lopez-Bergami et al., 2007; Alexaki et al., 2008). In gastrointestinal cancers, a small molecule inhibitor of JNK shows promise as a therapeutic agent, because it induces cell cycle arrest and apoptosis (Xia et al., 2006).

The oncogenic protein Ect2 is ubiquitinated by APC/C^Cdh1 shortly after completion of mitosis (Liot et al., 2011). Ect2 is a positive regulator of the Rho GTPase pathway that controls cell cycle progression and cytokinesis (Bustelo et al., 2007). Many human tumors overexpress Ect2 (Sano et al., 2006; Salhia et al., 2008; Justilien and Fields, 2009; Justilien et al., 2011). Until now, this overexpression was believed to be a result of gene amplification and transcriptional upregulation (Seguin et al., 2009; Liot et al., 2011). But the newly discovered APC/C^Cdh1 dependent proteolysis of Ect2 shows that impaired destruction can also be a reason for increased Ect2 levels in tumor cells, which possibly asserts the role of Cdh1 as a tumor suppressor protein.

Skp2 is one of the most important substrates targeted by APC/C^Cdh1 during mitotic exit. An SCF containing Skp2 targets the essential cell cycle substrates p27^Kip1, p21^Cip1, and FOXO1 for degradation (Yu et al., 1998; Tsvetkov et al., 1999; Huang et al., 2005). Destruction of the CDK inhibitor p27^Kip1 is a prerequisite for entry into mitosis. Once cells exit mitosis, APC/C^Cdh1 targets Skp2 for proteolysis in G1 (Bashir et al., 2004; Wei et al., 2004). Skp2 functions as an oncogene because most of its ubiquitination targets are tumor suppressor proteins. Thus overexpression of Skp2 is common in many cancers including breast cancer, prostate cancer, pancreatic cancer, hepatocellular carcinomas, melanomas, and malignant lymphomas (Lim et al., 2002; Yang et al., 2002; Radke et al., 2005; Lu et al., 2009; Rose et al., 2011; Schuler et al., 2011). Importantly, the Akt1 serine/threonine kinase phosphorylates Skp2 at Ser 72 promoting its cytoplasmic translocation and impairing degradation by APC/C^Cdh1 (Gao et al., 2009b,c; Lin et al., 2009). Since the Akt1 pathway is also found to be hyperactive in cancer cells, it may contribute significantly to overexpression of Skp2 protein found in some cancers (Gao et al., 2009b,c). Opposing the Akt1 pathway is PTEN, which is frequently misregulated in human brain, breast, prostate cancers, and leukemias (Li et al., 1997; Dahia et al., 1999). Thus, PTEN misregulation may contribute to Akt1 hyperactivation and increased levels of Skp2 in tumors.

Ube2C is an E2 enzyme that works exclusively with the APC/C throughout the cell cycle. However, Ube2C levels peak during mitosis and fall as cells enter G1, finally becoming a substrate for APC/C^Cdh1 at the end of G1 (Rape and Kirschner, 2004; Summers et al., 2008). When Ube2C is overexpressed in mouse embryonic fibroblasts (MEFs), precocious cyclin B1 degradation, mitotic slippage, and chromosome abnormalities have been observed (van Ree et al., 2010). Importantly, elevated Ube2C levels is a common feature in a wide variety of human cancers including lung, prostate, breast, bladder, ovarian, uterine, thyroid, esophageal, and gastric carcinomas (Okamoto et al., 2003; Pallante et al., 2005; Berlingieri et al., 2007; Jiang et al., 2008; van Ree et al., 2010). Further, induced expression of high levels of Ube2C hastens tumor formation in transgenic mice (van Ree et al., 2010).

Several APC/C substrates are overexpressed in various cancers, making them prime targets for small molecule inhibition (Table 2). One potential APC/C substrate that could be targeted via such a strategy is the proto-oncogene Skp2 (Fujita et al., 2008, 2009). Downregulation of Skp2 by antisense RNA treatment induces apoptosis in lung cancer cells (Yokoi et al., 2003). Therefore, one possibility is utilizing compounds affecting Skp2 expression. For instance, treatment with EB1089 (vitamin D analog) reduces Skp2 expression in treated cancer cells, thus promoting increased stability of p27^Kip1 protein and subsequent growth arrest (Lin et al., 2003; Figure 3). Skp2 expression is also affected by treatment with retinoic acid, thus altering the ability of p27^Kip1 to be ubiquitinated (Nakamura et al., 2003). Furthermore, the PPARg agonist Troglitazone reduces Skp2 mRNA levels, leading to p27^Kip1 accumulation (Koga et al., 2003).

Since the APC/C mediates Skp2 ubiquitination, enhancing APC/C dependent degradation of Skp2 in cancer would also be therapeutically attractive. One possible means of achieving this is to inhibit casein kinase 1 (CK1), which controls APC/C dependent degradation of Skp2 (Gao et al., 2009b,c). CK1 inhibition induces Skp2 degradation since CK1 dependent phosphorylation normally inhibits Skp2 nuclear translocation and interaction with APC/C^Cdh1 (Gao et al., 2009b,c). An alternative strategy, however, could be increasing the affinity of Skp2 or other oncogenic substrates for APC/C, thus reducing their protein levels after ubiquitination and degradation. The advent of high-throughput screens to study APC/C-dependent degradation and protein–protein interactions will facilitate identification of small molecule agonists of the APC/C–Skp2 interaction (Madoux et al., 2010; Zeng et al., 2010).

Another potential APC/C cancer target is the substrate Aurora-A, since its overexpression is known to induce centrosome amplification and is overexpressed in human cancer cells (Zhou et al., 1998). Recently, several compounds have been found to target Aurora-A (Figure 3). Curcumin, an active compound in turmeric and curry that has been proven to induce tumor apoptosis and inhibit tumor proliferation, invasion, angiogenesis, and metastasis, has been shown to reduce Aurora-A mRNA expression in human bladder cancer cells (Liu et al., 2011a). These curcumin-induced phenomena were similar to those using Aurora-A small interfering RNA and were attenuated by ectopic expression of Aurora-A. Also, several cyclic peptide ligands inhibiting Aurora-A kinase activity have been developed (Shomin et al., 2011), although future studies regarding proliferation of cancer cells still have to be performed.

In addition to Aurora-A inhibition, there is pharmaceutical interest in targeting Plk1 as Plk1 depletion in cancer cells dramatically inhibits cell proliferation and decreases viability (Liu and Erikson, 2003; Xu et al., 2011; Yoon et al., 2011). Depletion of Plk1 perturbs spindle assembly, which leads to activation of the mitotic checkpoint, prolonged mitotic arrest, and eventually apoptosis (Liu and Erikson, 2003). Thus, given the known functions and effects of Plk1 inhibition, coupled with its wide overexpression pattern in cancer, a naturally occurring Plk1 inhibitor with low or no toxicity will be immensely useful in prevention as well as treatment of cancer. Numerous Plk1 inhibitors are in development to evaluate their potential as treatments in oncology, some of them in preclinical and clinical phase I/II development (Schöffski, 2009; Figure 3). It will be interesting to determine whether enhancing APC/C mediated degradation of Plk1 yield similar therapeutic benefits as Plk1 inhibitors currently in clinical trials.

Several lines of evidence suggest that modulation of the APC/C complex is a good anti-cancer therapeutic strategy. Downregulation of the catalytic subunits Apc2 and Apc11 leads to growth arrest and cell death in HeLa cells (Pray et al., 2002). Also, a small molecule, tosyl-l-arginine methyl ester (TAME), which binds to APC/C and prevents its activation by Cdc20 and Cdh1, produces mitotic arrest (Zeng et al., 2010; Figure 3). Furthermore, expression of several APC/C subunits is higher in tumor relative to normal tissue (Wang et al., 2003). Collectively, these studies suggest that pharmacological interference with APC/C activity through disruption of protein–protein interaction is likely to have potent anti-tumor activity (Pray et al., 2002; Huang et al., 2009).

Targeting upstream regulators of the APC/C complex controlling mitosis is also a promising strategy. Overexpression of APC/C inhibitors such as Emi1 in mammalian cells impedes cell cycle progression and results in cell death (Reimann et al., 2001). HDACs might directly target APC/C to ensure proper chromosome segregation and anti-tumor effects of HDAC inhibitors could be attributed to this deregulation (Kimata et al., 2008). There are emerging HDAC inhibitors that have been clinically validated as therapeutic agents in cancer patients with hematologic malignancies. Two HDAC inhibitors, vorinostat (pan-HDAC inhibitor) and depsipeptide (HDAC 1 and II inhibitor) have been approved by the FDA and are under further clinical investigation (Figure 3). HDAC inhibitors are well tolerated and clinically effective against hematologic cancers (Duvic and Vu, 2007; Luu et al., 2008; Modesitt et al., 2008).