Anaphase-promoting Complex Activity Research Articles

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Triphenyltin chloride exposure inhibits meiotic maturation of mouse oocytes by disrupting cytoskeleton assembly and cell cycle progression

Triphenyltin chloride (TPTCL) is widely used in various industrial and agricultural applications. This study aimed to elucidate the mechanisms underlying the toxicological effects of TPTCL on oocytes. The obtained findings revealed that TPTCL exposure reduced polar body extrusion (PBE) and induced meiotic arrest. Mechanistically, TPTCL disrupted meiotic spindle assembly and chromosome alignment. Further analysis indicated a significant decrease in p-MAPK expression, and disturbances in the localization of Pericentrin and p-Aurora A in TPTCL exposed oocytes, which suggesting impaired microtubule organizing center (MTOC)function. Moreover, TPTCL exposure enhance microtubule acetylation and microtubule instability. Therefore, the spindle assembly checkpoint (SAC) remained activated, and the activity of the anaphase-promoting complex (APC) was inhibited, thereby preventing oocytes from progressing into the entering anaphase I (AI) stage. TPTCL exposure also augmented the actin filaments in the cytoplasm. Notably, mitochondrial function appeared unaffected by TPTCL, as evidenced indicated by stable mitochondrial membrane potential and ATP content. Furthermore, TPTCL treatment altered H3K27me2, H3K27me3 and H3K9me3 levels, suggesting changes in epigenetic modifications in oocytes. Taken together, our results suggest that TPTCL disrupts cytoskeleton assembly, continuously activates SAC, inhibits APC activity, and blocks meiotic progression, ultimately impair oocyte maturation.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Abstract B006: Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it

Abstract Chemotherapy is often rendered ineffective by the development of multiple drug resistance (MDR). Given time MDR occurs almost universally and once developed applies in a non-specific manner. The lack of MDR specificity means many drugs, including those a patient has no pervious exposure to, are affected which limits treatment options. The goal of this research is to show nuclear localized tissue factor pathway inhibitor 1 (TFPI1) plays a driving role in development of MDR, and that the Anaphase Promoting Complex (APC) is capable of inhibiting MDR by targeting TFPI1 for degradation. A coagulation regulator, TFPI1 normally inhibits the tissue factor pathway to prevent blood clot formation. However, TFPI1 is highly elevated in many types of MDR cancer where we believe it mediates resistance development. Previous work in our lab has shown plasmid overexpression of TFPI1 induces MDR in MCF7 breast cancer cells, in MDR cells TFPI1 does not localize to the cell surface but instead to the nucleus, and a putative TFPI1 nuclear localization sequence (NLS) was identified. To confirm the putative NLS is required for TFPI1’s localization to nucleus, and subsequent MDR development, NLS mutant or wild type TFPI1-GFP were overexpressed in MCF7 cells. Localization of TFPI1-GFP was observed using widefield and confocal microscopy. Wild type TFPI1-GFP localized to the nucleus while the NLS mutant TFPI1 did not. These results help to confirm the putative NLS and its importance to TFPI1s role in MDR development. The APC is a nuclear E3 ubiquitin ligase and highly conserved regulator of cell cycle progression. Defects in APC function are associated with reduced longevity and cancer. Other work in our lab focuses on the APC’s role in longevity using a yeast model. However, we believe the APC also plays a role in TFPI1’s promotion of MDR. Our observations suggest APC dysfunction exists within MDR cancer cells where APC substrates accumulate, as does TFPI1. We hypothesize that nuclear TFPI1 is an APC substrate, and that APC activation will result in TFPI1 degradation slowing MDR development. Our lab has isolated several small peptides we believe bind and activate the APC, and have used these peptides in conjugation with known APC activators such as Mad2 Inhibitor-1 (M2I-1) to show that APC activation is capable of reducing TFPI1 abundance in MCF7 cells treated with the chemotherapeutic Doxorubicin (Dox). These results suggest TFPI1 is an APC substrate. To summarize, we believe TFPI1 drives development of MDR cancer, for which TFPI1’s accumulation within the nucleus is important. We have shown TFPI1’s NLS is required for localization of the protein to the nucleus, and activation of the APC reduces nuclear accumulation of TFPI1. Moving forward our goal is to demonstrate the effect TFPI1 NLS knockout and APC activation have on the development of MDR in MCF7 cells undergoing drug selection with Dox. By exploring TFPI1’s role in MDR we may elucidate means to slow resistance development and extend chemotherapy effectiveness. Citation Format: Mathew Lubachowski, Troy Harkness. Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B006.

Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

Simple SummaryMultiple drug resistant cancers develop all too soon in patients who received successful cancer treatment. A lack of treatment options often leaves palliative care as the last resort. We tested whether the insulin sensitizer, metformin, known to have anti-cancer activity, could impact canines with drug resistant lymphoma when added to chemotherapy. All canines in the study expressed protein markers of drug resistance and within weeks of receiving metformin, the markers were decreased. A microarray was performed, and from four canines assessed, a common set of 290 elevated genes were discovered in tumor cells compared to control cells. This cluster was enriched with genes that stall the cell cycle, with a large component representing substrates of the Anaphase Promoting Complex (APC), which degrades proteins. One canine entered partial remission. RNAs from this canine showed that APC substrates were decreased during remission and elevated again during relapse, suggesting that the APC was impaired in drug resistant canines and restored when remission occurred. We validated our results in cell lines using APC inhibitors and activators. We conclude that the APC may be a vital guardian of the genome and could delay the onset of multiple drug resistance when activated.Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

The role of Anaphase Promoting Complex activation, inhibition and substrates in cancer development and progression.

The Anaphase Promoting Complex (APC), a multi-subunit ubiquitin ligase, facilitates mitotic and G1 progression, and is now recognized to play a role in maintaining genomic stability. Many APC substrates have been observed overexpressed in multiple cancer types, such as CDC20, the Aurora A and B kinases, and Forkhead box M1 (FOXM1), suggesting APC activity is important for cell health. We performed BioGRID analyses of the APC coactivators CDC20 and CDH1, which revealed that at least 69 proteins serve as APC substrates, with 60 of them identified as playing a role in tumor promotion and 9 involved in tumor suppression. While these substrates and their association with malignancies have been studied in isolation, the possibility exists that generalized APC dysfunction could result in the inappropriate stabilization of multiple APC targets, thereby changing tumor behavior and treatment responsiveness. It is also possible that the APC itself plays a crucial role in tumorigenesis through its regulation of mitotic progression. In this review the connections between APC activity and dysregulation will be discussed with regards to cell cycle dysfunction and chromosome instability in cancer, along with the individual roles that the accumulation of various APC substrates may play in cancer progression.

Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

Activating the Anaphase Promoting Complex to Enhance Genomic Stability and Prolong Lifespan.

In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells encounter multiple endogenous and exogenous DNA damaging events that are mostly repaired, but inevitably DNA mutations, chromosome rearrangements, and epigenetic deregulation begins to mount. Now that people are living longer, more and more late life time is spent suffering from age-related disease, in which genomic instability plays a critical role. However, several major questions remain heavily debated, such as the following: When does aging start? How long can we live? In order to minimize the impact of genomic instability on longevity, it is important to understand when aging starts, and to ensure repair mechanisms remain optimal from the very start to the very end. In this review, the interplay between the stress and nutrient response networks, and the regulation of homeostasis and genomic stability, is discussed. Mechanisms that link these two networks are predicted to be key lifespan determinants. The Anaphase Promoting Complex (APC), a large evolutionarily conserved ubiquitin ligase, can potentially serve this need. Recent work demonstrates that the APC maintains genomic stability, mounts a stress response, and increases longevity in yeast. Furthermore, inhibition of APC activity by glucose and nutrient response factors indicates a tight link between the APC and the stress/nutrient response networks.

CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy.

Rationale: Sustained cardiac hypertrophy often leads to heart failure (HF). Understanding the regulation of cardiomyocyte growth is crucial for the treatment of adverse ventricular remodeling and HF. Cell division cycle 20 (CDC20) is an anaphase-promoting complex activator that is essential for cell division and tumorigenesis, but the role of CDC20 in cardiac hypertrophy is unknown. We aimed to test whether CDC20 participates in the regulation of pathological cardiac hypertrophy and investigate the underlying mechanism in vitro and in vivo.Methods: Male C57BL/6 mice were administered a recombinant adeno-associated virus serotype 9 (rAAV9) vector expressing CDC20 or a siRNA targeting CDC20 and their respective controls by tail intravenous injection.Results: Microarray analysis showed that CDC20 was significantly upregulated in the heart after angiotensin II infusion. Knockdown of CDC20 in cardiomyocytes and in the heart reduced cardiac hypertrophy upon agonist stimulation or transverse aortic constriction (TAC). Conversely, enforced expression of CDC20 in cardiomyocytes and in the heart aggravated the hypertrophic response. Furthermore, we found that CDC20 directly targeted LC3, a key regulator of autophagy, and promoted LC3 ubiquitination and degradation by the proteasome, which inhibited autophagy leading to hypertrophy. Moreover, knockdown of LC3 or inhibition of autophagy attenuated Ang II-induced cardiomyocyte hypertrophy after deletion of CDC20 in vitro.Conclusions: Our study reveals a novel cardiac hypertrophy regulatory mechanism that involves CDC20, LC3 and autophagy, and suggests that CDC20 could be a new therapeutic target for patients with hypertrophic heart diseases.

Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.

Chromosome biorientation and APC activity remain uncoupled in oocytes with reduced volume.

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.

FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia

AbstractFZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G1 phase, and activation of the G2-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage–induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest.

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle.

Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound

Background:Anaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20.Materials and Methods:The structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated.Results:It was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown.Conclusion:The findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds.

Abstract PR13: Germ-line mutations in CDC20 result in familial cancers via deregulation of the cell cycle

Abstract Introduction: Cdc20 is a co-activator recruited by the anaphase-promoting complex (APC), a large, multi-subunit complex that functions as an Ubiqutin ligase that targets key substrates for degradation to promote cell cycle progression. During mitosis the APC/Cdc20 complex is inhibited by the spindle assembly checkpoint (SAC) pathway, which ensures the correct chromosome segregation during mitosis. When associated with the mitotic checkpoint complex (MCC), formed by Mad2, BubR1 and Bub3 proteins, Cdc20 cannot activate the APC and cell cycle is arrested. Deregulation of this pathway can result in cells proceeding through mitosis before proper chromosomal attachment to the kinetochore, potentially leading to aneuploidy and cancer. We have found 2 different missense variants in CDC20 (L151R and N331K), in the affected members of two families with malignant ovarian germ cell tumors, a rare non-epithelial neoplastic transformation of ovarian primitive oocyte. Interestingly, several members of L151R family presented with other additional tumors such as colorectal or breast cancers. Purpose: We aim to elucidate the mechanism by which L151R and N331K are able to trigger tumorigenesis, and demonstrate that the disruption of Cdc20 may play an important role in cancer. Methods: The effect of the mutations has been measured through three different cell types: S. cerevisiae carrying the Cdc20N407K (N331K in humans); HeLa cells transfected with Cdc20L151R and Cdc20N331K; and human primary skin fibroblasts from two siblings carrying N331K mutation. Cell cycle progression has been measured in the presence of the spindle poison nocodazole by several methods: checkpoint cell survival assay, western blot of MCC and APC proteins, flow cytometer and time-lapse. The effect of the mutations on the interaction of Cdc20 and its MCC and APC partners has been assessed by immunoprecipitation. Results: S. cerevisiae carrying the Cdc20N407K (N331K in humans) show a clear reduction in viability of the mutant compared to wild-type (wt) after the addition of nocodazole, as a result of mutant cells keep dividing despite microtubule-kinetochore attachment being destabilized, thus causing genomic instability and catastrophe in progeny cells. HeLa cells transfected with Cdc20L151R and Cdc20N331K show a significant increase in the non-phosphorylated forms of Cdc27 and Bubr1 compared to the wt and the empty vector, indicating the abundance of non-mitotic cells increases with the mutated forms of Cdc20. The increased number of polyploidy cells (8n) in the mutants seen by flow cytometry proves that the non-mitotic cells are a result of the mutated Cdc20 overriding mitosis even in the presence of nocodazole. Additionally, time-lapse results fit with a reduced time spent in mitosis by the HeLa cells carrying the mutated forms of Cdc20. Immunoprecipitation of the transfected Cdc20 showed a reduced binding of the mutated proteins to Bubr1, suggesting that the mutated forms of Cdc20 could be over-activating the APC thanks to a reduced affinity with its MCC partner Bubr1. Time-lapse experiments performed in human skin fibroblasts from two patients carrying the N331K mutation in heterozygosity showed similar results in terms of the length of the mitotic arrest in the presence of nocodazole. Last but not least, we are expecting to be able to see the translation of these in vitro functional results at the tumoral level after the WES and cytogenetic analysis of the patient's tumors is finalized. Conclusions: Our preliminary data suggest that L151R and N331K may drive cancer through the insensitivity of Cdc20 to the SAC. To our knowledge this is the first description of CDC20 as a cancer predisposition gene. The rapid division of cancer cells demands that they maintain high APC activity and this may represent an Achilles heel that can be exploited therapeutically. This abstract is also being presented as Poster B25. Citation Format: Ester Castellsague, Jian Carrot-Zhang, Isabelle Gamache, Barbara Rivera, Mohamed Moustafa, David Barford, Jacek Majewski, Jose Teodoro, William David Foulkes. Germ-line mutations in CDC20 result in familial cancers via deregulation of the cell cycle. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR13.

Activation of anaphase-promoting complex by p53 induces a state of dormancy in cancer cells against chemotherapeutic stress.

Cancer dormancy is a stage in tumor progression in which residual disease remains occult and asymptomatic for a prolonged period. Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy. However, cancer dormancy is poorly characterized and the mechanisms of how cancer cells develop dormancy and relapse remain elusive. In this study, 5- fluorouracil (5-FU) was used to induce cancer cell dormancy. We found that cancer cells escape the cytotoxicity of 5-FU by becoming “dormant”. After exposure to 5-FU, residual non-small cell lung cancer (NSCLC) cells underwent epithelial-mesenchymal transition (EMT), followed by mesenchymal-epithelial transition (MET). These EMT-transformed NSCLC cells were in the state of cell quiescence where cells were not dividing and were arrested in the cell cycle in G0-G1. The dormant cells underwent an EMT showed characteristics of cancer stem cells. P53 is strongly accumulated in response to 5-FU-induced dormant cells through the activation of ubiquitin ligase anaphase-promoting complex (APC/C) and TGF-β/Smad signaling. In contrast to the EMT-transformed cells, MET-transformed cells showed an increased ability to proliferate, suggesting that dormant EMT cells were reactivated in the MET process. During the EMT-MET process, DNA repair including nonhomologous end joining (NHEJ) and homologous recombination (HR) is critical to dormant cell reactivation. Our findings provide a mechanism to unravel cancer cell dormancy and reactivation of the cancer cell population.

DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint.

Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.

Regulation of mRNA translation during mitosis.

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

CDC20 maintains tumor initiating cells.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21CIP1/WAF1, a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21CIP1/WAF1, resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes-FOXM1 and p21CIP1/WAF1-elucidating a potential point for therapeutic intervention.