RESUMEN
p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Benzamidas/farmacología , Caspasa 8/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Sinergismo Farmacológico , Regulación de la Expresión Génica , Humanos , Imidazoles/metabolismo , Modelos Biológicos , Piperazinas/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Piridinas/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL-R2 DISC is impaired in the absence of caspase-8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC-mediated apoptosis.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Caspasa 8/metabolismo , Humanos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.
Asunto(s)
Quimioradioterapia , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Macrófagos/patología , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/efectos de la radiación , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Proteínas Inhibidoras de la Apoptosis/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interleucina-8/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/efectos de la radiación , Masculino , Clasificación del Tumor , Pronóstico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Rayos XRESUMEN
TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Cistatina M/genética , Cisteína Endopeptidasas/metabolismo , Proteínas de Dominio T Box/metabolismo , Apoptosis , Western Blotting , Neoplasias de la Mama/genética , Inmunoprecipitación de Cromatina , Cistatina M/metabolismo , Cisteína Endopeptidasas/genética , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas de Dominio T Box/antagonistas & inhibidores , Proteínas de Dominio T Box/genética , Células Tumorales CultivadasRESUMEN
Here, we show for the first time, that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene, an event requiring ΔNp63. We propose that this BRCA1/ΔNp63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells, as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-α (ER-α) expression and other luminal markers. A Notch mimetic peptide could activate an ER-α promoter reporter in a BRCA1-dependent manner, whereas Notch inhibition using a γ-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together, these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue.
Asunto(s)
Proteína BRCA1/fisiología , Neoplasias de la Mama/genética , Mama/metabolismo , Receptores Notch/genética , Animales , Mama/citología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Proteínas de Unión al Calcio/genética , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Células MCF-7 , Proteínas de la Membrana/genética , Ratones , Receptor Notch1/genética , Receptores Notch/biosíntesis , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/genética , Tamoxifeno/farmacología , Factores de Transcripción/fisiología , Transcripción Genética , Proteínas Supresoras de Tumor/fisiología , Regulación hacia ArribaRESUMEN
Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells, siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability, increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together, our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/genética , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Inmunoprecipitación de Cromatina , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Matrices Tisulares , Transactivadores/biosíntesis , Factores de Transcripción , Transfección , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
We carried out a yeast two-hybrid screen using a BRCA1 bait composed of amino acids 1 to 1142 and identified BRD7 as a novel binding partner of BRCA1. This interaction was confirmed by coimmunoprecipitation of endogenous BRCA1 and BRD7 in T47D and HEK-293 cells. BRD7 is a bromodomain containing protein, which is a subunit of PBAF-specific Swi/Snf chromatin remodeling complexes. To determine the functional consequences of the BRCA1-BRD7 interaction, we investigated the role of BRD7 in BRCA1-dependent transcription using microarray-based expression profiling. We found that a variety of targets were coordinately regulated by BRCA1 and BRD7, such as estrogen receptor alpha (ERalpha). Depletion of BRD7 or BRCA1 in either T47D or MCF7 cells resulted in loss of expression of ERalpha at both the mRNA and protein level, and this loss of ERalpha was reflected in resistance to the antiestrogen drug fulvestrant. We show that BRD7 is present, along with BRCA1 and Oct-1, on the ESR1 promoter (the gene which encodes ERalpha). Depletion of BRD7 prevented the recruitment of BRCA1 and Oct-1 to the ESR1 promoter; however, it had no effect on the recruitment of the other Swi/Snf subunits BRG1, BAF155, and BAF57 or on RNA polymerase II recruitment. These results support a model whereby the regulation of ERalpha transcription by BRD7 is mediated by its recruitment of BRCA1 and Oct-1 to the ESR1 promoter.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína BRCA1/genética , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Cromosómicas no Histona/genética , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Perfilación de la Expresión Génica , Genes BRCA1 , Humanos , Inmunoprecipitación , Factor 1 de Transcripción de Unión a Octámeros/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transcripción Genética , TransfecciónRESUMEN
We have developed a versatile and rapid method for the quantitative estimation of cell death kinetics, following direct single-shot activation of the mitochondrial death pathway by a cell permeable BH3 activator peptide (D-R(8)BH3(BID)). This approach employs timelapse epifluorescent imaging of live cells and a machine- vision based feature extraction algorithm, to measure unidirectional stochastic transitions associated with mitochondrial inner membrane potential depolarization and/or permeability transition, at single cell resolution. This data is transformed to enable construction of a right step-wise survival function using the product limit estimator, and estimation of a median latency parameter (lambda), defined for the entire imaged cell population. Estimates of lambda computed for cells exhibiting two-colour fluorescence can be compared statistically using the Mantel-Hansel test. This general method has been applied to measure the kinetics and temporal ordering of BH3 domain induced mitochondrial depolarization and inner membrane permeabilization in cancer cells, and demonstrates the robustness of this technique in resolving temporally distinct intracellular events within individual cells.