RESUMEN
Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/CCdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD.
Asunto(s)
Ciclina E , Duplicación de Gen , Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Línea Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Mitosis , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Compuestos de Anilina/farmacología , Animales , Benzamidas/farmacología , Chlorocebus aethiops , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/aislamiento & purificación , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Sinergismo Farmacológico , Pruebas de Enzimas , Flavinas/farmacología , Transferencia Resonante de Energía de Fluorescencia , Furanos/farmacología , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Naftalenos/farmacología , Fenantrenos/farmacología , Quinonas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
The transcription factor E2F plays crucial roles in tumor suppression by activating pro-apoptotic genes such as the tumor suppressor ARF. The regulation of the ARF gene is distinct from that of growth-related E2F targets, in that it is specifically activated by deregulated E2F activity, induced by over-expression of E2F or forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. The phosphatidyl inositol 3 kinase (PI3K) pathway was reported to suppress expression of some atypical pro-apoptotic genes by over-expressed E2F1. However, the effects of the PI3K pathway on the distinct regulation of typical pro-apoptotic E2F targets have not been elucidated. We examined whether the PI3K pathway suppressed activation of the typical pro-apoptotic E2F targets ARF and BIM. Activation of the PI3K pathway by growth stimulation or introduction of a constitutively active Akt/PKB did not reduce induction of ARF or BIM gene expression or activation of their promoters by over-expressed E2F1. These results suggest that the PI3K pathway does not suppress induction of typical pro-apoptotic genes that are selectively activated by deregulated E2F1.
Asunto(s)
Proteína 11 Similar a Bcl2/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción E2F1/metabolismo , Regulación de la Expresión Génica , Fosfatidilinositol 3-Quinasas/metabolismo , Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Fibroblastos/metabolismo , Humanos , Proteína de Retinoblastoma/metabolismo , Transducción de SeñalRESUMEN
In cancer treatment, specifically targeting cancer cells is important for optimal therapeutic efficacy. One strategy is to utilize a cancer specific promoter to express a cytotoxic gene or a viral gene required for replication. In this approach, the therapeutic window is dependent on the relative promoter activity in cancer cells versus normal cells. Therefore, a promoter with optimal cancer cell-specificity should be used. The tumor suppressor ARF promoter, which specifically responds to deregulated E2F activity, is a potent candidate. Defects in the RB pathway resulting in deregulated E2F activity are observed in almost all cancers. Furthermore, the ARF promoter exhibits greater cancer cell specificity than the E2F1 promoter and consequently, adenovirus expressing HSV-TK under the control of the ARF promoter (Ad-ARF-TK) has more selective cytotoxicity in cancer cells than the analogous E2F1 construct. Ideally, cancer specific gene expression driven by the ARF promoter could be enhanced for optimal therapeutic efficacy, with minimal side effects. We show here that ectopic expression of the CDK inhibitor p21Cip1 enhanced deregulated E2F activity and pro-apoptotic E2F target gene expression in cancer cells. Moreover, ectopic expression of p21Cip1 augmented cancer specific cytotoxicity of Ad-ARF-TK, and apoptosis induced by p21Cip1 was dependent on deregulated E2F activity. These results suggest that p21Cip1 specifically enhances deregulated E2F activity and that a combination of the CDK inhibitor with Ad-ARF-TK could be effectively employed for cancer therapy.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Genes p16 , Apoptosis , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Expresión Génica Ectópica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Regiones Promotoras GenéticasRESUMEN
The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Factores de Transcripción E2F/metabolismo , Factor de Transcripción E2F1/metabolismo , Fibroblastos/metabolismo , Genes Supresores de Tumor , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Apoptosis , Proteína 11 Similar a Bcl2 , Línea Celular , Regiones Promotoras GenéticasRESUMEN
In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.
Asunto(s)
Factor de Transcripción E2F1/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Regiones Promotoras Genéticas/genética , Proteína p14ARF Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/genética , Línea Celular Tumoral , Genes Transgénicos Suicidas/genética , Genes Supresores de Tumor , Terapia Genética/métodos , HumanosRESUMEN
Discrimination of oncogenic growth signals from normal growth signals is crucial for tumor suppression. The transcription factor E2F, the main target of pRB, plays central role in cell proliferation by activating growth-promoting genes. E2F also plays an important role in tumor suppression by activating growth-suppressive genes such as pro-apoptotic genes. The regulatory mechanism of the latter genes is not known in detail, especially in response to normal and oncogenic growth signals. E2F is physiologically activated by growth stimulation through phosphorylation of pRB. In contrast, upon dysfunction of pRB, a major oncogenic change, E2F is activated out of control by pRB, generating deregulated E2F activity. We show here that the tumor suppressor TAp73 gene, which can induce apoptosis independently of p53, responds to deregulated E2F activity, but not to physiological E2F activity induced by growth stimulation in human normal fibroblasts. We identified E2F-responsive elements (ERE73s) in TAp73 promoter that can specifically sense deregulated E2F activity. Moreover, RB1-deficient cancer cell lines harbored deregulated E2F activity that activated ERE73s and the TAp73 gene, which were suppressed by re-introduction of pRB. These results underscore the important role of deregulated E2F in activation of the TAp73 gene, a component of major intrinsic tumor suppressor pathways.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/metabolismo , Fibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Apoptosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Medios de Cultivo/metabolismo , Proteínas de Unión al ADN/genética , Factor de Transcripción E2F1/genética , Etopósido/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Mutación Puntual , Regiones Promotoras Genéticas , Unión Proteica , Suero/metabolismo , Activación Transcripcional , Transfección , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genéticaRESUMEN
The transcription factor E2F, the main target of the RB tumor suppressor pathway, plays crucial roles not only in cell proliferation but also in tumor suppression. The cyclin-dependent kinase inhibitor p27(Kip1) gene, an upstream negative regulator of E2F, is induced by ectopically expressed E2F1 but not by normal growth stimulation that physiologically activates endogenous E2F. This suggests that the gene can discriminate between deregulated and physiological E2F activity. To address this issue, we examined regulation of the p27(Kip1) gene by E2F. Here we show that p27(Kip1) promoter specifically senses deregulated E2F activity through elements similar to typical E2F sites. This E2F-like elements were activated by deregulated E2F activity induced by forced inactivation of pRb but not by physiological E2F activity induced by serum stimulation, contrary to typical E2F sites activated by both E2F activity. The endogenous p27(Kip1) gene responded to deregulated and physiological E2F activity in the same manner to the E2F-like elements. Moreover, the E2F-like elements bound ectopically expressed E2F1 but not physiologically activated E2F1 or E2F4 in vivo. These results suggest that the p27(Kip1) gene specifically senses deregulated E2F activity through the E2F-like elements to suppress inappropriate cell cycle progression in response to loss of pRb function.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Factor de Transcripción E2F1/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Línea Celular Tumoral , Factor de Transcripción E2F4/metabolismo , Humanos , Unión Proteica , Elementos de Respuesta/genética , Proteína de Retinoblastoma/metabolismo , Suero/metabolismoRESUMEN
Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Amplificación de Genes/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Privación de Tratamiento , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.
Asunto(s)
Apoptosis , Sistema de Señalización de MAP Quinasas , Melanoma/patología , Terapia Molecular Dirigida , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Ratones , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteína bcl-X/metabolismoRESUMEN
The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.