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Natasha J Caplen - One of the best experts on this subject based on the ideXlab platform.
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abstract 3204 the identification of aurkb BUB1B and cdk18 as putative molecular targets for breast cancer using kinome focused rnai screens
Cancer Research, 2013Co-Authors: Lihui Ou, Tamara L Jones, Kristen Gehlhaus, Konrad Huppi, Natasha J CaplenAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The signal transduction events regulated by the kinase family of proteins control many cellular processes including normal cell proliferation and survival. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells, including breast cancer, and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. Functional genomic RNAi screens of the kinome conducted within the context of a specific tumor type and/or a specific cancer-associated genetic background have the potential to identify kinases required for cancer cell survival. A comparison of RNAi screening data that we have obtained targeting the human kinome in three breast cancer cell lines with other published RNAi screens led us to perform a detailed analysis of the phenotypic effects of silencing AURKB, BUB1B, and CDK18 in multiple breast cancer cell lines with the aim of further characterizing kinase genes required for the growth of breast tumor cells. The non-tumorigenic mammary epithelial cell line MCF-10A exhibited minimal BUB1B protein expression and no detectable expression of AURKB protein, while all of the breast cancer cell lines studied (a total of eight), exhibited expression of both of these proteins. This differential expression was associated with functional differences in silencing of AURKB or BUB1B resulting in suppression of colony formation in almost all of the breast cancer cell lines tested, but not in MCF-10A cells. Silencing of AURKB also induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468 and HCC-38 cells, but not in MCF-10A cells. These results were phenocopied by an ATP-competitive inhibitor of aurora kinase with selectivity for aurora B kinase ZM447439. BUB1B silencing resulted in cell cycle arrest in both MDA-MB-468 and HCC-38 cells, but caspase 3/7-activation and PARP cleavage were only observed in MDA-MB-468 cells. None of these effects were observed in MCF-10A cells. Little is known as to the functional role of CDK18, but its silencing induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468. In contrast we saw minimal phenotypic effects in HCC-38 cells and MCF-10A cells, even though both of these cell lines express CDK18. To further elucidate the functional role of CDK18, we performed genome-wide expression profiling of MDA-MB-468 cells in which CDK18 had been silenced. Interestingly, one of the transcripts showing the greatest decrease in expression following silencing of CDK18 was CCNE1 that encodes cyclin E1. Another cyclin gene, CCND1, also exhibited a decrease in expression following silencing of CDK18, and we are currently examining this potential link between CDK18 function and cyclin E1 and cyclin D1 in more detail. Citation Format: Lihui Ou, Kristen Gehlhaus, Tamara Jones, Konrad Huppi, Natasha Caplen. The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2013-3204
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abstract 3204 the identification of aurkb BUB1B and cdk18 as putative molecular targets for breast cancer using kinome focused rnai screens
Cancer Research, 2013Co-Authors: Lihui Ou, Tamara L Jones, Kristen Gehlhaus, Konrad Huppi, Natasha J CaplenAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The signal transduction events regulated by the kinase family of proteins control many cellular processes including normal cell proliferation and survival. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells, including breast cancer, and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. Functional genomic RNAi screens of the kinome conducted within the context of a specific tumor type and/or a specific cancer-associated genetic background have the potential to identify kinases required for cancer cell survival. A comparison of RNAi screening data that we have obtained targeting the human kinome in three breast cancer cell lines with other published RNAi screens led us to perform a detailed analysis of the phenotypic effects of silencing AURKB, BUB1B, and CDK18 in multiple breast cancer cell lines with the aim of further characterizing kinase genes required for the growth of breast tumor cells. The non-tumorigenic mammary epithelial cell line MCF-10A exhibited minimal BUB1B protein expression and no detectable expression of AURKB protein, while all of the breast cancer cell lines studied (a total of eight), exhibited expression of both of these proteins. This differential expression was associated with functional differences in silencing of AURKB or BUB1B resulting in suppression of colony formation in almost all of the breast cancer cell lines tested, but not in MCF-10A cells. Silencing of AURKB also induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468 and HCC-38 cells, but not in MCF-10A cells. These results were phenocopied by an ATP-competitive inhibitor of aurora kinase with selectivity for aurora B kinase ZM447439. BUB1B silencing resulted in cell cycle arrest in both MDA-MB-468 and HCC-38 cells, but caspase 3/7-activation and PARP cleavage were only observed in MDA-MB-468 cells. None of these effects were observed in MCF-10A cells. Little is known as to the functional role of CDK18, but its silencing induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468. In contrast we saw minimal phenotypic effects in HCC-38 cells and MCF-10A cells, even though both of these cell lines express CDK18. To further elucidate the functional role of CDK18, we performed genome-wide expression profiling of MDA-MB-468 cells in which CDK18 had been silenced. Interestingly, one of the transcripts showing the greatest decrease in expression following silencing of CDK18 was CCNE1 that encodes cyclin E1. Another cyclin gene, CCND1, also exhibited a decrease in expression following silencing of CDK18, and we are currently examining this potential link between CDK18 function and cyclin E1 and cyclin D1 in more detail. Citation Format: Lihui Ou, Kristen Gehlhaus, Tamara Jones, Konrad Huppi, Natasha Caplen. The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2013-3204
Patrick J Paddison - One of the best experts on this subject based on the ideXlab platform.
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bugz facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis
Journal of Biological Chemistry, 2020Co-Authors: Hazheen K Shirnekhi, Jacob A Herman, Patrick J Paddison, Jennifer G DelucaAbstract:BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.
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sensitivity to BUB1B inhibition defines an alternative classification of glioblastoma
Cancer Research, 2017Co-Authors: Eunjee Lee, Jennifer G Deluca, Jacob A Herman, Patrick J Paddison, Margaret Pain, Huaien Wang, Chad M Toledo, Raymund YongAbstract:Glioblastoma multiforme (GBM) remains a mainly incurable disease in desperate need of more effective treatments. In this study, we develop evidence that the mitotic spindle checkpoint molecule BUB1B may offer a predictive marker for aggressiveness and effective drug response. A subset of GBM tumor isolates requires BUB1B to suppress lethal kinetochore-microtubule attachment defects. Using gene expression data from GBM stem-like cells, astrocytes, and neural progenitor cells that are sensitive or resistant to BUB1B inhibition, we created a computational framework to predict sensitivity to BUB1B inhibition. Applying this framework to tumor expression data from patients, we stratified tumors into BUB1B-sensitive (BUB1BS) or BUB1B-resistant (BUB1BR) subtypes. Through this effort, we found that BUB1BS patients have a significantly worse prognosis regardless of tumor development subtype (i.e., classical, mesenchymal, neural, proneural). Functional genomic profiling of BUB1BR versus BUB1BS isolates revealed a differential reliance of genes enriched in the BUB1BS classifier, including those involved in mitotic cell cycle, microtubule organization, and chromosome segregation. By comparing drug sensitivity profiles, we predicted BUB1BS cells to be more sensitive to type I and II topoisomerase inhibitors, Raf inhibitors, and other drugs, and experimentally validated some of these predictions. Taken together, the results show that our BUB1BR/S classification of GBM tumors can predict clinical course and sensitivity to drug treatment. Cancer Res; 77(20); 5518-29. ©2017 AACR.
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abstract a05 functional genetic approach identifies cancer specific requirement for BUB1B bubr1 in human brain tumor isolates and genetically transformed cells
Molecular Cancer Therapeutics, 2013Co-Authors: Yu Ding, Jennifer G Deluca, Jacob A Herman, Christopher G Hubert, James M Olson, Patrick J PaddisonAbstract:Cancer cells give rise to distinctive and complimentary capabilities that enable them to continuously grow. These capabilities may as well generate additional vulnerabilities and can be targeted for therapy. To identify new candidate therapeutic targets for glioblastoma multiforme, we combined functional genetics and glioblastoma network modeling to identify kinases required for the growth of patient-derived brain tumor-initiating cells (BTIC) but that are dispensable to proliferating human neural stem cells (NSC). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top-scoring glioblastoma lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1B9s GLE2p-binding sequence (GLEBS) domain activity is required to suppress lethal kinetochore-microtubule (KT-MT) attachment defects in glioblastoma isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT-MT instability. These results indicate that glioblastoma tumors have an added requirement for BUB1B to suppress lethal consequences of altered KT function and further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT-MT stability. Significance: Currently, no effective therapies are available for glioblastoma, the most frequent and aggressive brain tumor. Our results suggest that targeting the GLEBS domain activity of BUB1B may provide a therapeutic window for glioblastoma, as the GLEBS domain is nonessential in untransformed cells. Moreover, the results further suggest that sister KT distances at metaphase may predict sensitivity to anticancer therapeutics targeting KT function. Citation Format: Yu Ding, Christopher Hubert, Jacob Herman, Jennifer DeLuca, James Olson, Patrick Paddison. Functional genetic approach identifies cancer-specific requirement for BUB1B/BubR1 in human brain tumor isolates and genetically transformed cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A05.
Marc E Lippman - One of the best experts on this subject based on the ideXlab platform.
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spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival
Breast Cancer Research and Treatment, 2021Co-Authors: Dilara Koyuncu, Erik T Goka, Utsav Sharma, Marc E LippmanAbstract:The study aimed to investigate the role of spindle assembly checkpoint (SAC) in cancer cells with compromised genomic integrity. Chromosomal instability (CIN) gives cancer cells an adaptive advantage. However, maintaining the balance of this instability is crucial for the survival of cancer cells as it could lead them to the mitotic catastrophe. Therefore, cancer cells adapt to the detrimental effects of CIN. We hypothesized that changes in SAC might be one such adaptation mechanism. The focus of the study was BUB1B, an integral part of the checkpoint. Clinical datasets were analyzed to compare expression levels of SAC genes in normal tissue vs. breast carcinoma. The effects of the reduction of BUB1B expression was examined utilizing RNA interference method with siRNAs. In vitro viability, clonogenicity, apoptosis, and SAC activity levels of a variety of breast cancer (BrCa) cell lines, as well as in vivo tumorigenicity of the triple-negative breast cancer (TNBC) cell line MDA-MB-468, were tested. Additionally, the chromosomal stability of these cells was tested by immunofluorescence staining and flow cytometry. In clinical breast cancer datasets, SAC genes were elevated in BrCa with BUB1B having the highest fold change. BUB1B overexpression was associated with a decreased probability of overall survival. The knockdown of BUB1B resulted in reduced viability and clonogenicity in BrCa cell lines and a significant increase in apoptosis and cell death. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown also impaired chromosome alignment and resulted in acute chromosomal abnormalities. We also showed that BUB1B knockdown on the MDA-MB-468 cell line decreases tumor growth in mice. A functional spindle assembly checkpoint is essential for the survival of BrCa cells. BUB1B is a critical factor in SAC, and therefore breast cancer cell survival. Impairment of BUB1B has damaging effects on cancer cell viability and tumorigenicity, especially on the more aggressive variants of BrCa.
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abstract 512 knockdown of the spindle assembly checkpoint gene BUB1B results in increased cell death and cell cycle impairment in breast cancer
Cancer Research, 2017Co-Authors: Dilara Koyuncu, Erik T Goka, P Miller, Marc E LippmanAbstract:Genomic instability is an enabling hallmark of cancer that provides cancer cells a replicative advantage. Accumulation of genomic aberrations can also compromise the genomic integrity and put cells under mitotic stress. Maintaining balance between the instability that gives cancer cells a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial for survival of cancer cells. Mitotic catastrophe can be caused by rapid progression through mitosis before crucial checkpoints are met, resulting in cell death. To overcome this challenge, cancer cells may acquire overexpression of spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if they undergo mitosis prematurely. BUB1B is an important part of SAC and inhibits the onset of anaphase until all chromosomes are aligned at the metaphase plate. Our analysis of clinical datasets show BUB1B expression is elevated in breast cancer, compared to normal breast, and is exemplified by a pattern of increasing overexpression in more aggressive variants, such as triple negative breast cancer (TNBC). BUB1B overexpression also correlates with decreased overall survival in patients. Expression analysis of breast cancer cell lines corroborates this clinical data. We hypothesize that the requirement for BUB1B expression indicates a vulnerability of rapidly proliferating breast cancers cells, and the inhibition of BUB1B will result in cell death through mitotic catastrophe. Using RNA interference with siRNAs we knocked down BUB1B expression in a variety of breast cancer cells. This resulted in significant decrease in cellular viability and clonogenicity in soft agar. Furthermore, analyses of apoptosis using Annexin V and PI costaining showed that BUB1B knockdown results in significantly increased apoptosis and cell death, especially in the TNBC cell line MDA-MB-468. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown resulted in silencing of SAC activity, as measured by Cyclin B levels, and also significantly impaired cell cycle progression in breast cancer cell lines. Additionally, knockdown of BUB1B in breast cancer cell lines demonstrated an additive effect when combined with DNA-damaging agents. Our data support that BUB1B is a critical player in breast cancer cell survival, by enabling them to overcome mitotic stress. We demonstrate that impairment of BUB1B has detrimental effects on cell cycle and progression of mitosis. Further investigation of the role of BUB1B in promoting successful chromosome alignment and proliferation of breast cancer cells with genomic instability could provide a novel pathway on how cancer overcomes the mitotic stress caused by genomic instability. These studies may also lead to novel therapeutic strategies that combine SAC inhibitors with standard genotoxic treatments. Citation Format: Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. Knockdown of the spindle assembly checkpoint gene BUB1B results in increased cell death and cell cycle impairment in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2017-512
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abstract 3588 the spindle assembly checkpoint gene BUB1B is essential for the survival of some breast cancers
Cancer Research, 2016Co-Authors: Dilara Koyuncu, Erik T Goka, P Miller, Marc E LippmanAbstract:As normal breast epithelium evolves towards malignancy, cells accumulate genomic changes that give them a replicative advantage while at the same time increasing their genomic instability. Increased genomic instability results in accumulation of genomic aberrations that compromise the genomic integrity and, therefore, threaten cell viability, thus putting cancer cells under mitotic stress. As a consequence, cancer cells evolutionarily must adapt themselves to compete with the possible detrimental effects of genomic instability. Finding a balance between the instability that gives them a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial. The mitotic stress caused by genomic instability may require overexpression of certain spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if cancer cells undergo mitosis prematurely. Although the full mechanism of action of SAC is yet to be elucidated, BUB1B through its protein BubR1 is an important part of this checkpoint, and inhibits the onset of anaphase until all chromosomes are aligned correctly at the metaphase plate. Our analysis of clinical datasets shows a significant increase in the expression of BUB1B in breast cancer as compared to normal epithelia. Furthermore, BUB1B overexpression correlates with decreased overall survival in patient samples. Our analyses also show a pattern of increasing BUB1B overexpression in more aggressive variants of breast cancer such as triple negative tumors and high-grade tumors, which also tend to be more resistant to current therapies. Expression analyses of breast cancer cell lines reveal that BUB1B overexpression is positively correlated with more aggressive behavior. We postulated that the requirement for BUB1B expression might be a vulnerability of rapidly proliferating cancers; therefore, its inhibition will result in cell death through mitotic catastrophe. Using RNA interference with siRNAs, we reduced BUB1B levels in a variety of breast cancer cells. Our results showed significant decrease in cell viability and clonogenicity in soft agar upon BUB1B knockdown, especially in triple negative breast cancer cell lines. However, the viability of normal breast epithelium cells, MCF12A, was not affected. Our data indicate that BUB1B is a critical player in breast cancer viability, and further investigation of the role of BUB1B in promoting successful proliferation of breast cancer cells with genomic instability could provide a new therapeutic strategy particularly in concert with standard genotoxic treatments such as alkylators, spindle poisons and radiation therapy. Citation Format: Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. The spindle assembly checkpoint gene BUB1B is essential for the survival of some breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3588.
Lihui Ou - One of the best experts on this subject based on the ideXlab platform.
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abstract 3204 the identification of aurkb BUB1B and cdk18 as putative molecular targets for breast cancer using kinome focused rnai screens
Cancer Research, 2013Co-Authors: Lihui Ou, Tamara L Jones, Kristen Gehlhaus, Konrad Huppi, Natasha J CaplenAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The signal transduction events regulated by the kinase family of proteins control many cellular processes including normal cell proliferation and survival. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells, including breast cancer, and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. Functional genomic RNAi screens of the kinome conducted within the context of a specific tumor type and/or a specific cancer-associated genetic background have the potential to identify kinases required for cancer cell survival. A comparison of RNAi screening data that we have obtained targeting the human kinome in three breast cancer cell lines with other published RNAi screens led us to perform a detailed analysis of the phenotypic effects of silencing AURKB, BUB1B, and CDK18 in multiple breast cancer cell lines with the aim of further characterizing kinase genes required for the growth of breast tumor cells. The non-tumorigenic mammary epithelial cell line MCF-10A exhibited minimal BUB1B protein expression and no detectable expression of AURKB protein, while all of the breast cancer cell lines studied (a total of eight), exhibited expression of both of these proteins. This differential expression was associated with functional differences in silencing of AURKB or BUB1B resulting in suppression of colony formation in almost all of the breast cancer cell lines tested, but not in MCF-10A cells. Silencing of AURKB also induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468 and HCC-38 cells, but not in MCF-10A cells. These results were phenocopied by an ATP-competitive inhibitor of aurora kinase with selectivity for aurora B kinase ZM447439. BUB1B silencing resulted in cell cycle arrest in both MDA-MB-468 and HCC-38 cells, but caspase 3/7-activation and PARP cleavage were only observed in MDA-MB-468 cells. None of these effects were observed in MCF-10A cells. Little is known as to the functional role of CDK18, but its silencing induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468. In contrast we saw minimal phenotypic effects in HCC-38 cells and MCF-10A cells, even though both of these cell lines express CDK18. To further elucidate the functional role of CDK18, we performed genome-wide expression profiling of MDA-MB-468 cells in which CDK18 had been silenced. Interestingly, one of the transcripts showing the greatest decrease in expression following silencing of CDK18 was CCNE1 that encodes cyclin E1. Another cyclin gene, CCND1, also exhibited a decrease in expression following silencing of CDK18, and we are currently examining this potential link between CDK18 function and cyclin E1 and cyclin D1 in more detail. Citation Format: Lihui Ou, Kristen Gehlhaus, Tamara Jones, Konrad Huppi, Natasha Caplen. The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2013-3204
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abstract 3204 the identification of aurkb BUB1B and cdk18 as putative molecular targets for breast cancer using kinome focused rnai screens
Cancer Research, 2013Co-Authors: Lihui Ou, Tamara L Jones, Kristen Gehlhaus, Konrad Huppi, Natasha J CaplenAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The signal transduction events regulated by the kinase family of proteins control many cellular processes including normal cell proliferation and survival. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells, including breast cancer, and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. Functional genomic RNAi screens of the kinome conducted within the context of a specific tumor type and/or a specific cancer-associated genetic background have the potential to identify kinases required for cancer cell survival. A comparison of RNAi screening data that we have obtained targeting the human kinome in three breast cancer cell lines with other published RNAi screens led us to perform a detailed analysis of the phenotypic effects of silencing AURKB, BUB1B, and CDK18 in multiple breast cancer cell lines with the aim of further characterizing kinase genes required for the growth of breast tumor cells. The non-tumorigenic mammary epithelial cell line MCF-10A exhibited minimal BUB1B protein expression and no detectable expression of AURKB protein, while all of the breast cancer cell lines studied (a total of eight), exhibited expression of both of these proteins. This differential expression was associated with functional differences in silencing of AURKB or BUB1B resulting in suppression of colony formation in almost all of the breast cancer cell lines tested, but not in MCF-10A cells. Silencing of AURKB also induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468 and HCC-38 cells, but not in MCF-10A cells. These results were phenocopied by an ATP-competitive inhibitor of aurora kinase with selectivity for aurora B kinase ZM447439. BUB1B silencing resulted in cell cycle arrest in both MDA-MB-468 and HCC-38 cells, but caspase 3/7-activation and PARP cleavage were only observed in MDA-MB-468 cells. None of these effects were observed in MCF-10A cells. Little is known as to the functional role of CDK18, but its silencing induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468. In contrast we saw minimal phenotypic effects in HCC-38 cells and MCF-10A cells, even though both of these cell lines express CDK18. To further elucidate the functional role of CDK18, we performed genome-wide expression profiling of MDA-MB-468 cells in which CDK18 had been silenced. Interestingly, one of the transcripts showing the greatest decrease in expression following silencing of CDK18 was CCNE1 that encodes cyclin E1. Another cyclin gene, CCND1, also exhibited a decrease in expression following silencing of CDK18, and we are currently examining this potential link between CDK18 function and cyclin E1 and cyclin D1 in more detail. Citation Format: Lihui Ou, Kristen Gehlhaus, Tamara Jones, Konrad Huppi, Natasha Caplen. The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2013-3204
Stephen S. Taylor - One of the best experts on this subject based on the ideXlab platform.
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regulation of apc c activity in oocytes by a bub1 dependent spindle assembly checkpoint
Current Biology, 2009Co-Authors: Barry E Mcguinness, Stephen S. Taylor, Martin Anger, Anna Kouznetsova, Ana M Gilbernabe, Wolfgang Helmhart, Nobuaki Kudo, Annelie Wuensche, Christer Hoog, Bela NovakAbstract:Summary Background Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility [1]. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. Results Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1 F allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12–13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation. Conclusions We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.
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bub1 and aurora b cooperate to maintain bubr1 mediated inhibition of apc ccdc20
Journal of Cell Science, 2005Co-Authors: Christopher J. Morrow, Anthony Tighe, Victoria L. Johnson, Maria I. F. Scott, Claire Ditchfield, Stephen S. TaylorAbstract:The spindle checkpoint maintains genome stability by inhibiting Cdc20-mediated activation of the anaphase promoting complex/cyclosome (APC/C) until all the chromosomes correctly align on the microtubule spindle apparatus via their kinetochores. BubR1, an essential component of this checkpoint, localises to kinetochores and its kinase activity is regulated by the kinesin-related motor protein Cenp-E. BubR1 also inhibits APC/C(Cdc20) in vitro, thus providing a molecular link between kinetochore-microtubule interactions and the proteolytic machinery that regulates mitotic progression. Several other protein kinases, including Bub1 and members of the Ipl1/aurora family, also regulate anaphase onset. However, in human somatic cells Bub1 and aurora B kinase activity do not appear to be essential for spindle checkpoint function. Specifically, when Bub1 is inhibited by RNA interference, or aurora kinase activity is inhibited with the small molecule ZM447439, cells arrest transiently in mitosis following exposure to spindle toxins that prevent microtubule polymerisation. Here, we show that mitotic arrest of Bub1-deficient cells is dependent on aurora kinase activity, and vice versa. We suggest therefore that the checkpoint is composed of two arms, one dependent on Bub1, the other on aurora B. Analysis of BubR1 complexes suggests that both of these arms converge on the mitotic checkpoint complex (MCC), which includes BubR1, Bub3, Mad2 and Cdc20. Although it is known that MCC components can bind and inhibit the APC/C, we show here for the first time that the binding of the MCC to the APC/C is dependent on an active checkpoint signal. Furthermore, we show that both Bub1 and aurora kinase activity are required to promote binding of the MCC to the APC/C. These observations provide a simple explanation of why BubR1 and Mad2 are essential for checkpoint function following spindle destruction, yet Bub1 and aurora B kinase activity are not. Taken together with other observations, we suggest that these two arms respond to different spindle cues: whereas the Bub1 arm monitors kinetochore-microtubule attachment, the aurora B arm monitors biorientation. This bifurcation in the signalling mechanism may help explain why many tumour cells mount a robust checkpoint response following spindle damage, despite exhibiting chromosome instability.
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bub1 is required for kinetochore localization of bubr1 cenp e cenp f and mad2 and chromosome congression
Journal of Cell Science, 2004Co-Authors: Victoria L. Johnson, Maria I. F. Scott, Deema Hussein, Sarah Holt, Stephen S. TaylorAbstract:During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase Bub1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that Bub1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Repression of Bub1 increases the number of cells with lagging chromosomes at metaphase, suggesting that Bub1 plays a role in chromosome congression. However, repression of Bub1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of Bub1, other mechanisms contribute to spindle checkpoint function.
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kinetochore localisation and phosphorylation of the mitotic checkpoint components bub1 and bubr1 are differentially regulated by spindle events in human cells
Journal of Cell Science, 2001Co-Authors: Stephen S. Taylor, Deema Hussein, Yunmei Wang, Sarah Elderkin, Christopher J. MorrowAbstract:BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known. To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment. Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different ‘spindle assembly signals’ into a single signal which can then be interpreted by downstream cell cycle regulators.
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the human homologue of bub3 is required for kinetochore localization of bub1 and a mad3 bub1 related protein kinase
Journal of Cell Biology, 1998Co-Authors: Stephen S. Taylor, Frank MckeonAbstract:A feedback control mechanism, or cell cycle checkpoint, delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. Previously, we showed that the murine homologue of Bub1 is not only required for checkpoint response to spindle damage, but also restrains progression through a normal mitosis (Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). Here, we describe the identification of a human homologue of Bub3, a 37-kD protein with four WD repeats. Like Bub1, Bub3 localizes to kinetochores before chromosome alignment. In addition, Bub3 and Bub1 interact in mammalian cells. Deletion mapping was used to identify the domain of Bub1 required for binding Bub3. Significantly, this same domain is required for kinetochore localization of Bub1, suggesting that the role of Bub3 is to localize Bub1 to the kinetochore, thereby activating the checkpoint in response to unattached kinetochores. The identification of a human Mad3/Bub1-related protein kinase, hBubR1, which can also bind Bub3 in mammalian cells, is described. Ectopically expressed hBubR1 also localizes to kinetochores during prometaphase, but only when hBub3 is overexpressed. We discuss the implications of the common interaction between Bub1 and hBubR1 with hBub3 for checkpoint control.
