RESUMO
Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian cancer (HGSOC), one of the most chromosomally unstable tumour types, has a 5-year survival rate of only ~30% - largely due to late diagnosis and rapid development of drug resistance, e.g., via CIN-driven ABCB1 translocations. However, CIN is also a cell cycle vulnerability that can be exploited to specifically target tumour cells, illustrated by the success of PARP inhibitors to target homologous recombination deficiency (HRD). However, a lack of appropriate models with ongoing CIN has been a barrier to fully exploiting disease-specific CIN mechanisms. This barrier is now being overcome with the development of patient-derived cell cultures and organoids. In this review, we describe our progress building a Living Biobank of over 120 patient-derived ovarian cancer models (OCMs), predominantly from HGSOC. OCMs are highly purified tumour fractions with extensive proliferative potential that can be analysed at early passage. OCMs have diverse karyotypes, display intra- and inter-patient heterogeneity and mitotic abnormality rates far higher than established cell lines. OCMs encompass a broad-spectrum of HGSOC hallmarks, including a range of p53 alterations and BRCA1/2 mutations, and display drug resistance mechanisms seen in the clinic, e.g., ABCB1 translocations and BRCA2 reversion. OCMs are amenable to functional analysis, drug-sensitivity profiling, and multi-omics, including single-cell next-generation sequencing, and thus represent a platform for delineating HGSOC-specific CIN mechanisms. In turn, our vision is that this understanding will inform the design of new therapeutic strategies.
Assuntos
Transtornos Cromossômicos , Neoplasias Ovarianas , Humanos , Feminino , Proteína BRCA1/genética , Bancos de Espécimes Biológicos , Proteína BRCA2 , Neoplasias Ovarianas/genética , Translocação Genética , Instabilidade CromossômicaRESUMO
A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analysed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork stalling, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential. Importantly nucleoside supplementation restores PARG inhibitor resistance despite the continued presence of PAR chains, indicating that sensitivity does not correlate with PAR levels. In addition, we show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances.
RESUMO
Accurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31(comet) is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31(comet) antagonises the checkpoint is unclear. A prevalent model is that p31(comet) acts as a 'cap' by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1-C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31(comet) localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31(comet) downstream of kinetochores. We show that p31(comet) binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31(comet) results in more Mad2 bound to BubR1-Cdc20, and conversely, overexpression of p31(comet) results in less Mad2 bound to BubR1-Cdc20. Addition of recombinant p31(comet) to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31(comet) mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31(comet) prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31(comet) negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células/citologia , Mitose , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao Cálcio/genética , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células/metabolismo , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Repressoras/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply 'switch' upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Antígenos CD , Caderinas/genética , Caderinas/metabolismo , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Cinetocoros/metabolismo , Camundongos , Microscopia , Prenilação , Interferência de RNA , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
High-grade serous ovarian cancer (HGSOC) is an aggressive disease that typically develops drug resistance, thus novel biomarker-driven strategies are required. Targeted therapy focuses on synthetic lethality-pioneered by PARP inhibition of BRCA1/2-mutant disease. Subsequently, targeting the DNA replication stress response (RSR) is of clinical interest. However, further mechanistic insight is required for biomarker discovery, requiring sensitive models that closely recapitulate HGSOC. We describe an optimized proliferation assay that we use to screen 16 patient-derived ovarian cancer models (OCMs) for response to RSR inhibitors (CHK1i, WEE1i, ATRi, PARGi). Despite genomic heterogeneity characteristic of HGSOC, measurement of OCM proliferation was reproducible and reflected intrinsic tumour-cell properties. Surprisingly, RSR targeting drugs were not interchangeable, as sensitivity to the four inhibitors was not correlated. Therefore, to overcome RSR redundancy, we screened the OCMs with all two-, three- and four-drug combinations in a multiple-low-dose strategy. We found that low-dose CHK1i-ATRi had a potent anti-proliferative effect on 15 of the 16 OCMs, and was synergistic with potential to minimise treatment resistance and toxicity. Low-dose ATRi-CHK1i induced replication catastrophe followed by mitotic exit and post-mitotic arrest or death. Therefore, this study demonstrates the potential of the living biobank of OCMs as a drug discovery platform for HGSOC.
RESUMO
The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , MAP Quinase Quinase Quinases/metabolismo , Mitose , Fuso Acromático/metabolismo , Anáfase , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Fuso Acromático/genéticaRESUMO
Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro, we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo, and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.
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BACKGROUND: Epithelial ovarian cancer (OC) is a heterogenous disease consisting of five major histologically distinct subtypes: high-grade serous (HGSOC), low-grade serous (LGSOC), endometrioid (ENOC), clear cell (CCOC) and mucinous (MOC). Although HGSOC is the most prevalent subtype, representing 70-80% of cases, a 2013 landmark study by Domcke et al. found that the most frequently used OC cell lines are not molecularly representative of this subtype. This raises the question, if not HGSOC, from which subtype do these cell lines derive? Indeed, non-HGSOC subtypes often respond poorly to chemotherapy; therefore, representative models are imperative for developing new targeted therapeutics. METHODS: Non-negative matrix factorisation (NMF) was applied to transcriptomic data from 44 OC cell lines in the Cancer Cell Line Encyclopedia, assessing the quality of clustering into 2-10 groups. Epithelial OC subtypes were assigned to cell lines optimally clustered into five transcriptionally distinct classes, confirmed by integration with subtype-specific mutations. A transcriptional subtype classifier was then developed by trialling three machine learning algorithms using subtype-specific metagenes defined by NMF. The ability of classifiers to predict subtype was tested using RNA sequencing of a living biobank of patient-derived OC models. RESULTS: Application of NMF optimally clustered the 44 cell lines into five transcriptionally distinct groups. Close inspection of orthogonal datasets revealed this five-cluster delineation corresponds to the five major OC subtypes. This NMF-based classification validates the Domcke et al. analysis, in identifying lines most representative of HGSOC, and additionally identifies models representing the four other subtypes. However, NMF of the cell lines into two clusters did not align with the dualistic model of OC and suggests this classification is an oversimplification. Subtype designation of patient-derived models by a random forest transcriptional classifier aligned with prior diagnosis in 76% of unambiguous cases. In cases where there was disagreement, this often indicated potential alternative diagnosis, supported by a review of histological, molecular and clinical features. CONCLUSIONS: This robust classification informs the selection of the most appropriate models for all five histotypes. Following further refinement on larger training cohorts, the transcriptional classification may represent a useful tool to support the classification of new model systems of OC subtypes.
Assuntos
Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transcriptoma , Algoritmos , Álcoois Benzílicos , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Patrimônio Genético , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Aprendizado de Máquina , Mutação , Gradação de TumoresRESUMO
BACKGROUND: Patients with ovarian cancer often present at advanced stage and, following initial treatment success, develop recurrent drug-resistant disease. PARP inhibitors (PARPi) are yielding unprecedented survival benefits for women with BRCA-deficient disease. However, options remain limited for disease that is platinum-resistant and/or has inherent or acquired PARPi-resistance. PARG, the PAR glycohydrolase that counterbalances PARP activity, is an emerging target with potential to selectively kill tumour cells harbouring oncogene-induced DNA replication and metabolic vulnerabilities. Clinical development of PARG inhibitors (PARGi) will however require predictive biomarkers, in turn requiring an understanding of their mode of action. Furthermore, differential sensitivity to PARPi is key for expanding treatment options available for patients. METHODS: A panel of 10 ovarian cancer cell lines and a living biobank of patient-derived ovarian cancer models (OCMs) were screened for PARGi-sensitivity using short- and long-term growth assays. PARGi-sensitivity was characterized using established markers for DNA replication stress, namely replication fibre asymmetry, RPA foci, KAP1 and Chk1 phosphorylation, and pan-nuclear γH2AX, indicating DNA replication catastrophe. Finally, gene expression in sensitive and resistant cells was also examined using NanoString or RNAseq. RESULTS: PARGi sensitivity was identified in both ovarian cancer cell lines and patient-derived OCMs, with sensitivity accompanied by markers of persistent replication stress, and a pre-mitotic cell cycle block. Moreover, DNA replication genes are down-regulated in PARGi-sensitive cell lines consistent with an inherent DNA replication vulnerability. However, DNA replication gene expression did not predict PARGi-sensitivity in OCMs. The subset of patient-derived OCMs that are sensitive to single-agent PARG inhibition, includes models that are PARPi- and/or platinum-resistant, indicating that PARG inhibitors may represent an alternative treatment strategy for women with otherwise limited therapeutic options. CONCLUSIONS: We discover that a subset of ovarian cancers are intrinsically sensitive to pharmacological PARG blockade, including drug-resistant disease, underpinned by a common mechanism of replication catastrophe. We explore the use of a transcript-based biomarker, and provide insight into the design of future clinical trials of PARGi in patients with ovarian cancer. However, our results highlight the complexity of developing a predictive biomarker for PARGi sensitivity.
Assuntos
Glicosídeo Hidrolases/metabolismo , Neoplasias Ovarianas/fisiopatologia , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
High-grade serous ovarian cancer (HGSOC) originates in the fallopian tube epithelium and is characterized by ubiquitous TP53 mutation and extensive chromosomal instability (CIN). However, direct causes of CIN, such as mutations in DNA replication and mitosis genes, are rare in HGSOC. We therefore asked whether oncogenic mutations that are common in HGSOC can indirectly drive CIN in non-transformed human fallopian tube epithelial cells. To model homologous recombination deficient HGSOC, we sequentially mutated TP53 and BRCA1 then overexpressed MYC. Loss of p53 function alone was sufficient to drive the emergence of subclonal karyotype alterations. TP53 mutation also led to global gene expression changes, influencing modules involved in cell cycle commitment, DNA replication, G2/M checkpoint control and mitotic spindle function. Both transcriptional deregulation and karyotype diversity were exacerbated by loss of BRCA1 function, with whole-genome doubling events observed in independent p53/BRCA1-deficient lineages. Thus, our observations indicate that loss of the key tumour suppressor TP53 is sufficient to deregulate multiple cell cycle control networks and thereby initiate CIN in pre-malignant fallopian tube epithelial cells. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Instabilidade Cromossômica , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Células Epiteliais/metabolismo , Tubas Uterinas/metabolismo , Tubas Uterinas/patologia , Feminino , Humanos , Mutação/genética , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.
Assuntos
Bancos de Espécimes Biológicos , Mitose/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Instabilidade Cromossômica , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas Histológicas/métodos , Humanos , Imageamento Tridimensional , Hibridização in Situ Fluorescente , Técnicas In Vitro , Cariotipagem , Modelos Biológicos , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Análise de Célula Única , Imagem com Lapso de Tempo , Proteína Supressora de Tumor p53/genética , Sequenciamento do ExomaRESUMO
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.
Assuntos
Anáfase/genética , Segregação de Cromossomos/genética , Células Eucarióticas/enzimologia , Cinetocoros/metabolismo , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Anáfase/efeitos dos fármacos , Aurora Quinase B , Aurora Quinases , Benzamidas/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/ultraestrutura , Genes cdc/efeitos dos fármacos , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Proteínas Mad2 , Mitose/efeitos dos fármacos , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genética , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
Inhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%-20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Feminino , Glicosídeo Hidrolases/antagonistas & inibidores , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Quinazolinonas/farmacologiaRESUMO
Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1α and that inhibiting Hif-1α restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1α to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.
Assuntos
Aneuploidia , Apoptose , Cromossomos Humanos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Sistemas CRISPR-Cas , Neoplasias do Colo , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/genética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
BACKGROUND: Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC) may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 - a protein kinase, which in concert with APC, targets beta-catenin for proteolysis - and ask whether GSK-3 is required for accurate chromosome segregation. RESULTS: To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3beta. Cells deficient for GSK-3beta exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3beta repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation. CONCLUSION: Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability.
Assuntos
Instabilidade Cromossômica , Segregação de Cromossomos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Interferência de RNA , Fuso Acromático/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Cell fate in response to an aberrant mitosis is governed by two competing networks: the spindle assembly checkpoint (SAC) and the intrinsic apoptosis pathway. The mechanistic interplay between these two networks is obscured by functional redundancy and the ability of cells to die either in mitosis or in the subsequent interphase. By coupling time-lapse microscopy with selective pharmacological agents, we systematically probe pro-survival Bcl-xL in response to various mitotic perturbations. Concentration matrices show that BH3-mimetic-mediated inhibition of Bcl-xL synergises with perturbations that induce an SAC-mediated mitotic block, including drugs that dampen microtubule dynamics, and inhibitors targeting kinesins and kinases required for spindle assembly. By contrast, Bcl-xL inhibition does not synergize with drugs which drive cells through an aberrant mitosis by overriding the SAC. This differential effect, which is explained by compensatory Mcl-1 function, provides opportunities for patient stratification and combination treatments in the context of cancer chemotherapy.
Assuntos
Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Moduladores de Tubulina/farmacologia , Proteína bcl-X/antagonistas & inibidores , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologiaRESUMO
The quest to develop novel antimitotic chemotherapy agents has led to the generation of several small molecule inhibitors targeting Plk1, a protein kinase required for multiple aspects of cell division. Previous studies have shown that upon exposure to Plk1 inhibitors, cells enter mitosis, delay briefly in prophase and then arrest in mitosis due to an inability to undergo centrosome separation. Here, we show that four different classes of Plk1 inhibitor block mitotic entry in several cancer cell lines and non-transformed RPE-1 cells. The proportion of cells that arrest in G2 is cell line and concentration dependent, and is subject to non-genetic heterogeneity. Following inhibitor washout, the G2 block is alleviated and cells enter mitosis but then fail to complete cell division indicating that most Plk1 inhibitors are not fully reversible. An exception is CYC140844; in contrast to five other inhibitors examined here, this novel Plk1 inhibitor is fully reversible. We discuss the implications for developing Plk1 inhibitors as chemotherapy agents and research tools.
Assuntos
Antimitóticos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Heterogeneidade Genética , Células HeLa , Humanos , Mitose/genética , Mitose/fisiologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Bibliotecas de Moléculas Pequenas/farmacologia , Quinase 1 Polo-LikeRESUMO
Aneuploidy is a common feature of cancer, with human solid tumour cells typically harbouring abnormal chromosome complements. The aneuploidy observed in cancer is often caused by a chromosome instability phenotype, resulting in genomic heterogeneity. However, the role aneuploidy and chromosome instability play in tumour evolution and chemotherapy response remains poorly understood. In some contexts, aneuploidy has oncogenic effects, whereas in others it is anti-proliferative and tumour-suppressive. Dissecting fully the role aneuploidy plays in tumourigenesis requires tools and facile assays that allow chromosome missegregation to be induced experimentally in cells that are otherwise diploid and chromosomally stable. Here, we describe a chemical biology approach that induces low-level aneuploidy across a large population of cells. Specifically, cells are first exposed to GSK923295, an inhibitor targeting the mitotic kinesin Cenp-E; while the majority of chromosomes align at the cell's equator, a small number cluster near the spindle poles. By then driving these cells into anaphase using AZ3146, an inhibitor targeting the spindle checkpoint kinase Mps1, the polar chromosomes are missegregated. This results in, on average, two chromosome missegregation events per division, and avoids trapping chromosomes in the spindle midzone, which could otherwise lead to DNA damage. We also describe an efficient route for the synthesis of GSK923295 that employs a novel enzymatic resolution. Together, the approaches described here open up new opportunities for studying cellular responses to aneuploidy.
Assuntos
Aneuploidia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Sarcosina/análogos & derivados , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos/ultraestrutura , Dano ao DNA , Citometria de Fluxo , Células HCT116 , Células HeLa , Humanos , Cinesinas , Mitose , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Interferência de RNA , Sarcosina/síntese química , Sarcosina/química , Fuso AcromáticoRESUMO
Taxol and other antimitotic agents are frontline chemotherapy agents but the mechanisms responsible for patient benefit remain unclear. Following a genome-wide siRNA screen, we identified the oncogenic transcription factor Myc as a taxol sensitizer. Using time-lapse imaging to correlate mitotic behavior with cell fate, we show that Myc sensitizes cells to mitotic blockers and agents that accelerate mitotic progression. Myc achieves this by upregulating a cluster of redundant pro-apoptotic BH3-only proteins and suppressing pro-survival Bcl-xL. Gene expression analysis of breast cancers indicates that taxane responses correlate positively with Myc and negatively with Bcl-xL. Accordingly, pharmacological inhibition of Bcl-xL restores apoptosis in Myc-deficient cells. These results open up opportunities for biomarkers and combination therapies that could enhance traditional and second-generation antimitotic agents.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteína bcl-X/genética , Animais , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Mitose/efeitos dos fármacos , Dados de Sequência Molecular , Neoplasias/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologiaRESUMO
The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD-ZWILCH-ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome-microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery.