APC/C prevents a noncanonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest.

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.

Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.

Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.

Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatin-induced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.

Mutations in CCNB3 affect its location thus causing a multiplicity of phenotypes in human oocytes maturation by aberrant CDK1 activity and APC/C activity at different stages.

BACKGROUND: Oocyte maturation arrest results in female infertility and the genetic etiology of this phenotype remains largely unknown. Previous studies have proven that cyclins play a significant role in the cell cycle both in meiosis and mitosis. Cyclin B3 (CCNB3) is one of the members of the cyclin family and its function in human oocyte maturation is poorly understood. METHODS: 118 infertile patients were recruited and WES was performed for 68 independent females that experienced oocyte maturation arrest. Four mutations in CCNB3 were found and effects of these mutations were validated by Sanger sequencing and in vitro functional analyses. RESULTS: We found these mutations altered the location of cyclin B3 which affected the function of cyclin dependent kinase 1 (CDK1) and led to mouse oocyte arrested at germinal vesicle (GV) stage. And then, low CDK1 activity influenced the degradation of cadherin 1 (CDH1) and the accumulation of cell division cycle 20 (CDC20) which are two types of anaphase-promoting complex/cyclosome (APC/C) activators and act in different stages of the cell cycle. Finally, APC/C activity was downregulated due to insufficient CDC20 level and resulted in oocyte metaphase I (MI) arrest. Moreover, we also found that the addition of PP1 inhibitor Okadic acid and CDK1 inhibitor Roscovitine at corresponding stages during oocyte in vitro maturation (IVM) significantly improved the maturation rates in CCNB3 mutant cRNAs injected oocytes. The above experiments were performed in mouse oocytes. CONCLUSION: Here, we report five independent patients in which mutations in CCNB3 may be the cause of oocyte maturation arrest. Our findings shed lights on the critical role of CCNB3 in human oocyte maturation.

Structural investigation of CDCA3-Cdh1 protein-protein interactions using in vitro studies and molecular dynamics simulation.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell-cycle proteins and their functions during mitosis. Levels of the protein cell division cycle-associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/CCdh1 . CDCA3 is an intrinsically disordered protein and contains both C-terminal KEN box and D-box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation-dependent non-canonical ABBA-like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA-like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H-bonds, hydrophobic and ionic interactions across the CDCA3 ABBA-like motif in the linker region between KEN and D-box motifs. This linker region adopts a well-defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non-canonical ABBA-like motif to promote binding to the APC/CCdh1 and regulation of CDCA3 protein levels.

The anaphase promoting complex/cyclosome ubiquitylates histone H2B on the promoter during UbcH10 transactivation.

Among various post-translational modifications of histones, ubiquitylation plays a crucial role in transcription regulation. Histone mono-ubiquitylation by RING finger motif-containing ubiquitin ligases is documented in this respect. The RING finger ligases primarily regulate the cell cycle, where the anaphase-promoting complex/cyclosome (APC/C) takes charge as mitotic ubiquitin machinery. Reportedly, APC/C participates in transcriptional activation of the ubiquitin carrier protein UbcH10. However, the ubiquitylation activity of APC/C on the UBCH10 promoter remains elusive. This study shows that APC/C, with its adapter protein Cdc20, catalyses mono-ubiquitylation of Lysine-120 in histone 2B on the UBCH10 promoter. This study also identified a cell-cycle-specific pattern of this modification. Finally, APC/C-driven crosstalk of acetylation and ubiquitylation was found operational on UBCH10 trans-regulation. Together, these findings suggest a role for APC/C catalysed promoter ubiquitylation in managing transcription of cell cycle regulatory genes.

The role of APC/C in cell cycle dynamics, growth and development in cereal crops.

Cereal crops can be considered the basis of human civilization. Thus, it is not surprising that these crops are grown in larger quantities worldwide than any other food supply and provide more energy to humankind than any other provision. Additionally, attempts to harness biomass consumption continue to increase to meet human energy needs. The high pressures for energy will determine the demand for crop plants as resources for biofuel, heat, and electricity. Thus, the search for plant traits associated with genetic increases in yield is mandatory. In multicellular organisms, including plants, growth and development are driven by cell division. These processes require a sequence of intricated events that are carried out by various protein complexes and molecules that act punctually throughout the cycle. Temporal controlled degradation of key cell division proteins ensures a correct onset of the different cell cycle phases and exit from the cell division program. Considering the cell cycle, the Anaphase-Promoting Complex/Cyclosome (APC/C) is an important conserved multi-subunit ubiquitin ligase, marking targets for degradation by the 26S proteasome. Studies on plant APC/C subunits and activators, mainly in the model plant Arabidopsis, revealed that they play a pivotal role in several developmental processes during growth. However, little is known about the role of APC/C in cereal crops. Here, we discuss the current understanding of the APC/C controlling cereal crop development.

Upregulation of anaphase promoting complex (APC) 7 as a prognostic marker for esophageal squamous cell carcinoma: A hospital based study.

Esophageal cancer is the sixth leading cause of cancer death, and esophageal squamous cell carcinoma (ESCC) is the most prevalent type worldwide, with a poor prognosis due to late diagnosis. The search for new molecular prognostic biomarkers revealed that dysregulation of anaphase promoting complex/cyclosome (APC/C) activation due to altered expression of APC molecules might lead to perturbed mitotic progression leading to malignancy. We analyzed the expression of the four different subunits of the APC/C complex-APC3, APC4, APC5 and APC7-by Real Time Polymerase Chain Reaction (RT-PCR). The findings were then correlated with clinicopathological parameters and different lifestyle factors. Significant upregulation of APC7 (tissue and blood: N = 50; 3.72 ± 1.21 and 4.45 ± 1.18, respectively) and APC3 (tissue and blood: N = 52 and 55 and 4.50 ± 1.41 and 4.58 ± 1.06, respectively) suggests their role in uncontrolled cell proliferation. In addition to their association with increasing age, their significant association with tumor size, node stage (only APC7 (p < 0.05)), and dysphagia grade supports a potential role in tumorigenic transformation in ESCC. Furthermore, several exclusive lifestyle-associated factors play a crucial supporting role in the development of ESCC in the Northeast Indian population. Various lifestyle factors, such as the duration of smoking, tobacco and betel nut consumption, and the duration of alcohol consumption, are significantly associated with the expression of APC. Analysis based on Pearson's correlation coefficient indicated a positive correlation among the gene expression levels ofAPC3 (both blood and tissue), APC5 (tissue) and APC3 (tissue), APC7 (tissue) and APC3 (tissue), and APC7 (tissue) and APC3 (blood). Additionally, a positive correlation was found between APC7 expression in blood and tissue samples. However, no significant correlation was found between APC 7 expression and APC4 and APC5 expression in either blood or tissue samples.

Examining the mechanistic relationship of APC/CCDH1 and its interphase inhibitor EMI1.

Proper protein destruction by the ubiquitin (Ub)-proteasome system is vital for a faithful cell cycle. Hence, the activity of Ub ligases is tightly controlled. The Anaphase-Promoting Complex/Cyclosome (APC/C) is a 1.2 MDa Ub ligase responsible for mitotic progression and G1 maintenance. At the G1/S transition, the APC/C is inhibited by EMI1 to prevent APC/C-dependent polyubiquitination of cell cycle effectors. EMI1 uses several interaction motifs to block the recruitment of APC/C substrates as well as the APC/C-associated E2s, UBE2C, and UBE2S. Paradoxically, EMI1 is also an APC/C substrate during G1. Using a comprehensive set of enzyme assays, we determined the context-dependent involvement of the EMI1 motifs in APC/C-dependent ubiquitination of EMI1 and other substrates. Furthermore, we demonstrated that an isolated C-terminal peptide fragment of EMI1 activates APC/C-dependent substrate priming by UBE2C. Together, these findings reveal the multiple roles of the EMI1 C-terminus for G1 maintenance and the G1/S transition.

The Plant Anaphase-Promoting Complex/Cyclosome.

The anaphase-promoting complex/cyclosome (APC/C) represents a large multisubunit E3-ubiquitin ligase complex that controls the unidirectional progression through the cell cycle by the ubiquitination of specific target proteins, marking them for proteasomal destruction. Although the APC/C's role is largely conserved among eukaryotes, its subunit composition and target spectrum appear to be species specific. In this review, we focus on the plant APC/C complex, whose activity correlates with different developmental processes, including polyploidization and gametogenesis. After an introduction into proteolytic control by ubiquitination, we discuss the composition of the plant APC/C and the essential nature of its core subunits for plant development. Subsequently, we describe the APC/C activator subunits and interactors, most being plant specific. Finally, we provide a comprehensive list of confirmed and suspected plant APC/C target proteins. Identification of growth-related targets might offer opportunities to increase crop yield and resilience of plants to climate change by manipulating APC/C activity.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Using Budding Yeast to Identify Molecules That Block Cancer Cell 'Mitotic Slippage' Only in the Presence of Mitotic Poisons.

Research on the budding yeast Saccharomyces cerevisiae has yielded fundamental discoveries on highly conserved biological pathways and yeast remains the best-studied eukaryotic cell in the world. Studies on the mitotic cell cycle and the discovery of cell cycle checkpoints in budding yeast has led to a detailed, although incomplete, understanding of eukaryotic cell cycle progression. In multicellular eukaryotic organisms, uncontrolled aberrant cell division is the defining feature of cancer. Some of the most successful classes of anti-cancer chemotherapeutic agents are mitotic poisons. Mitotic poisons are thought to function by inducing a mitotic spindle checkpoint-dependent cell cycle arrest, via the assembly of the highly conserved mitotic checkpoint complex (MCC), leading to apoptosis. Even in the presence of mitotic poisons, some cancer cells continue cell division via 'mitotic slippage', which may correlate with a cancer becoming refractory to mitotic poison chemotherapeutic treatments. In this review, knowledge about budding yeast cell cycle control is explored to suggest novel potential drug targets, namely, specific regions in the highly conserved anaphase-promoting complex/cyclosome (APC/C) subunits Apc1 and/or Apc5, and in a specific N-terminal region in the APC/C co-factor cell division cycle 20 (Cdc20), which may yield molecules which block 'mitotic slippage' only in the presence of mitotic poisons.

Intricate Regulatory Mechanisms of the Anaphase-Promoting Complex/Cyclosome and Its Role in Chromatin Regulation.

The ubiquitin (Ub)-proteasome system is vital to nearly every biological process in eukaryotes. Specifically, the conjugation of Ub to target proteins by Ub ligases, such as the Anaphase-Promoting Complex/Cyclosome (APC/C), is paramount for cell cycle transitions as it leads to the irreversible destruction of cell cycle regulators by the proteasome. Through this activity, the RING Ub ligase APC/C governs mitosis, G1, and numerous aspects of neurobiology. Pioneering cryo-EM, biochemical reconstitution, and cell-based studies have illuminated many aspects of the conformational dynamics of this large, multi-subunit complex and the sophisticated regulation of APC/C function. More recent studies have revealed new mechanisms that selectively dictate APC/C activity and explore additional pathways that are controlled by APC/C-mediated ubiquitination, including an intimate relationship with chromatin regulation. These tasks go beyond the traditional cell cycle role historically ascribed to the APC/C. Here, we review these novel findings, examine the mechanistic implications of APC/C regulation, and discuss the role of the APC/C in previously unappreciated signaling pathways.

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts.

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Human Immunodeficiency Virus Type 1 Vpr Mediates Degradation of APC1, a Scaffolding Component of the Anaphase-Promoting Complex/Cyclosome.

HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G2/M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G2 cell cycle arrest during infection of CD4+ T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.

LxCxD motif of the APC/C coactivator subunit FZR1 is critical for interaction with the retinoblastoma protein.

Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.

The Role of Anaphase-Promoting Complex/Cyclosome (APC/C) in Plant Reproduction.

Most eukaryotic species propagate through sexual reproduction that requires male and female gametes. In flowering plants, it starts through a single round of DNA replication (S phase) and two consecutive chromosome segregation (meiosis I and II). Subsequently, haploid mitotic divisions occur, which results in a male gametophyte (pollen grain) and a female gametophyte (embryo sac) formation. In order to obtain viable gametophytes, accurate chromosome segregation is crucial to ensure ploidy stability. A precise gametogenesis progression is tightly regulated in plants and is controlled by multiple mechanisms to guarantee a correct evolution through meiotic cell division and sexual differentiation. In the past years, research in the field has shown an important role of the conserved E3-ubiquitin ligase complex, Anaphase-Promoting Complex/Cyclosome (APC/C), in this process. The APC/C is a multi-subunit complex that targets proteins for degradation via proteasome 26S. The functional characterization of APC/C subunits in Arabidopsis, which is one of the main E3 ubiquitin ligase that controls cell cycle, has revealed that all subunits investigated so far are essential for gametophytic development and/or embryogenesis.

The importance of CDC27 in cancer: molecular pathology and clinical aspects.

BACKGROUND: CDC27 is one of the core components of Anaphase Promoting complex/cyclosome. The main role of this protein is defined at cellular division to control cell cycle transitions. Here we review the molecular aspects that may affect CDC27 regulation from cell cycle and mitosis to cancer pathogenesis and prognosis. MAIN TEXT: It has been suggested that CDC27 may play either like a tumor suppressor gene or oncogene in different neoplasms. Divergent variations in CDC27 DNA sequence and alterations in transcription of CDC27 have been detected in different solid tumors and hematological malignancies. Elevated CDC27 expression level may increase cell proliferation, invasiveness and metastasis in some malignancies. It has been proposed that CDC27 upregulation may increase stemness in cancer stem cells. On the other hand, downregulation of CDC27 may increase the cancer cell survival, decrease radiosensitivity and increase chemoresistancy. In addition, CDC27 downregulation may stimulate efferocytosis and improve tumor microenvironment. CONCLUSION: CDC27 dysregulation, either increased or decreased activity, may aggravate neoplasms. CDC27 may be suggested as a prognostic biomarker in different malignancies.

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint.

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.