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1.
J Biol Chem ; 299(7): 104834, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37201585

RESUMEN

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.


Asunto(s)
Cromatina , Quinasas Ciclina-Dependientes , Transducción de Señal , Cromatina/genética , Cromatina/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Mitosis/genética , Inestabilidad Cromosómica/genética , Humanos , Línea Celular Tumoral , Regulación de la Expresión Génica/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Silenciador del Gen
2.
J Biol Chem ; 297(4): 101162, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34481843

RESUMEN

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-ß) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-ß family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-ß/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-ß/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Subunidades beta de Inhibinas/metabolismo , Proteínas de Neoplasias/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Subunidades beta de Inhibinas/genética , Proteínas de Neoplasias/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Quinasa Activadora de Quinasas Ciclina-Dependientes
3.
J Biol Chem ; 295(33): 11707-11719, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32576660

RESUMEN

The phenotypes of each breast cancer subtype are defined by their transcriptomes. However, the transcription factors that regulate differential patterns of gene expression that contribute to specific disease outcomes are not well understood. Here, using gene silencing and overexpression approaches, RNA-Seq, and splicing analysis, we report that the transcription factor B-cell leukemia/lymphoma 11A (BCL11A) is highly expressed in triple-negative breast cancer (TNBC) and drives metastatic disease. Moreover, BCL11A promotes cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 (MBNL1), a splicing regulator that suppresses metastasis. This ultimately increases the levels of an alternatively spliced isoform of integrin-α6 (ITGA6), which is associated with worse patient outcomes. These results suggest that BCL11A sustains TNBC cell invasion and metastatic growth by repressing MBNL1-directed splicing of ITGA6 Our findings also indicate that BCL11A lies at the interface of transcription and splicing and promotes aggressive TNBC phenotypes.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/genética , Proteínas Represoras/genética , Neoplasias de la Mama Triple Negativas/genética , Regulación hacia Arriba , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Neoplasias de la Mama Triple Negativas/patología
4.
J Biol Chem ; 294(3): 875-886, 2019 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-30482844

RESUMEN

Inhibitors of bromodomain and extra-terminal proteins (BETi) suppress oncogenic gene expression and have been shown to be efficacious in many in vitro and murine models of cancer, including triple-negative breast cancer (TNBC), a highly aggressive disease. However, in most cancer models, responses to BETi can be highly variable. We previously reported that TNBC cells either undergo senescence or apoptosis in response to BETi, but the specific mechanisms dictating these two cell fates remain unknown. Using six human TNBC cell lines, we show that the terminal response of TNBC cells to BETi is dictated by the intrinsic expression levels of the anti-apoptotic protein B-cell lymphoma-extra large (BCL-xL). BCL-xL levels were higher in cell lines that senesce in response to BETi compared with lines that primarily die in response to these drugs. Moreover, BCL-xL expression was further reduced in cells that undergo BETi-mediated apoptosis. Forced BCL-xL overexpression in cells that normally undergo apoptosis following BETi treatment shifted them to senescence without affecting the reported mechanism of action of BETi in TNBC, that is, mitotic catastrophe. Most importantly, pharmacological or genetic inhibition of BCL-xL induced apoptosis in response to BETi, and inhibiting BCL-xL, even after BETi-induced senescence had already occurred, still induced cell death. These results indicate that BCL-xL provides a senescent cell death-inducing or senolytic target that may be exploited to improve therapeutic outcomes of TNBC in response to BETi. They also suggest that the basal levels of BCL-xL should be predictive of tumor responses to BETi in current clinical trials.


Asunto(s)
Apoptosis , Senescencia Celular , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteína bcl-X/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína bcl-X/genética
5.
Breast Cancer Res ; 22(1): 66, 2020 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-32552913

RESUMEN

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by high rates of recurrence and poor overall survival. This is due, in part, to a deficiency of targeted therapies, making it essential to identify therapeutically targetable driver pathways of this disease. While epidermal growth factor receptor (EGFR) is expressed in 60% of TNBCs and drives disease progression, attempts to inhibit EGFR in unselected TNBC patients have had a marginal impact on outcomes. Hence, we sought to identify the mechanisms that dictate EGFR expression and inhibitor response to provide a path for improving the utility of these drugs. In this regard, the majority of TNBCs express low levels of the transcription factor, Krüppel-like factor 4 (KLF4), while a small subset is associated with high expression. KLF4 and EGFR have also been reported to have opposing actions in TNBC. Thus, we tested whether KLF4 controls the expression of EGFR and cellular response to its pharmacological inhibition. METHODS: KLF4 was transiently overexpressed in MDA-MB-231 and MDA-MB-468 cells or silenced in MCF10A cells. Migration and invasion were assessed using modified Boyden chamber assays, and proliferation was measured by EdU incorporation. Candidate downstream targets of KLF4, including EGFR, were identified using reverse phase protein arrays of MDA-MB-231 cells following enforced KLF4 expression. The ability of KLF4 to suppress EGFR gene and protein expression and downstream signaling was assessed by RT-PCR and western blot, respectively. ChIP-PCR confirmed KLF4 binding to the EGFR promoter. Response to erlotinib in the context of KLF4 overexpression or silencing was assessed using cell number and dose-response curves. RESULTS: We report that KLF4 is a major determinant of EGFR expression and activity in TNBC cells. KLF4 represses transcription of the EGFR gene, leading to reduced levels of total EGFR, its activated/phosphorylated form (pEGFR), and its downstream signaling intermediates. Moreover, KLF4 suppression of EGFR is a necessary intermediary step for KLF4 to inhibit aggressive TNBC phenotypes. Most importantly, KLF4 dictates the sensitivity of TNBC cells to erlotinib, an FDA-approved inhibitor of EGFR. CONCLUSIONS: KLF4 is a major regulator of the efficacy of EGFR inhibitors in TNBC cells that may underlie the variable effectiveness of such drugs in patients.


Asunto(s)
Antineoplásicos/farmacología , Clorhidrato de Erlotinib/farmacología , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Fosforilación , Transducción de Señal , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología
6.
Cancer Res ; 83(7): 997-1015, 2023 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-36696357

RESUMEN

Breast cancer subtypes and their phenotypes parallel different stages of the mammary epithelial cell developmental hierarchy. Discovering mechanisms that control lineage identity could provide novel avenues for mitigating disease progression. Here we report that the transcriptional corepressor TLE3 is a guardian of luminal cell fate in breast cancer and operates independently of the estrogen receptor. In luminal breast cancer, TLE3 actively repressed the gene-expression signature associated with highly aggressive basal-like breast cancers (BLBC). Moreover, maintenance of the luminal lineage depended on the appropriate localization of TLE3 to its transcriptional targets, a process mediated by interactions with FOXA1. By repressing genes that drive BLBC phenotypes, including SOX9 and TGFß2, TLE3 prevented the acquisition of a hybrid epithelial-mesenchymal state and reduced metastatic capacity and aggressive cellular behaviors. These results establish TLE3 as an essential transcriptional repressor that sustains the more differentiated and less metastatic nature of luminal breast cancers. Approaches to induce TLE3 expression could promote the acquisition of less aggressive, more treatable disease states to extend patient survival. SIGNIFICANCE: Transcriptional corepressor TLE3 actively suppresses SOX9 and TGFß transcriptional programs to sustain the luminal lineage identity of breast cancer cells and to inhibit metastatic progression.


Asunto(s)
Neoplasias , Factores de Transcripción , Diferenciación Celular , Proteínas Co-Represoras/genética , Receptores de Estrógenos/metabolismo , Factor de Crecimiento Transformador beta , Neoplasias de la Mama/metabolismo , Humanos
7.
Cancer Res ; 77(19): 5395-5408, 2017 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28807940

RESUMEN

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR.


Asunto(s)
Mitosis/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
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