RESUMEN
We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/patología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Proteasome inhibitors have distinct properties and the biochemical consequences of suppressing ubiquitin E1 enzymes and the proteasome differ. We compared the effects of the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 enzyme inhibitor MLN7243/TAK-243 on cell viability and cell death in normal keratinocytes and cutaneous squamous cell carcinoma (cSCC) cell lines. The effects of both a pulse of treatment and more extended incubation were investigated. This is relevant to directly-delivered therapy (topical treatment/intratumoral injection) where the time of exposure can be controlled and a short exposure may better reflect systemically-delivered inhibitor pharmacokinetics. These agents can selectively kill cSCC cells but there are variations in the pattern of cSCC cell line sensitivity/resistance. Variations in the responses to proteasome inhibitors are associated with differences in the specificity of the inhibitors for the three proteolytic activities of the proteasome. There is greater selectivity for killing cSCC cells compared to normal keratinocytes with a pulse of proteasome inhibitor treatment than with a more extended exposure. We provide evidence that c-MYC-dependent NOXA upregulation confers susceptibility to a short incubation with proteasome inhibitors by priming cSCC cells for rapid BAK-dependent death. We observed that bortezomib-resistant cSCC cells can be sensitive to MLN7243-induced death. Low expression of the ubiquitin E1 UBA1/UBE1 participates in conferring susceptibility to MLN7243 by increasing sensitivity to MLN7243-mediated attenuation of ubiquitination. This study supports further investigation of the potential of proteasome and ubiquitin E1 inhibition for cSCC therapy. Direct delivery of inhibitors could facilitate adequate exposure of skin cancers.
RESUMEN
We show that suppression of the spliceosome has potential for the treatment of cutaneous squamous cell carcinoma (cSCC). The small-molecule inhibitors of the spliceosome at the most advanced stage of development target the splicing factor SF3B1/SF3b155. The majority of cSCC cell lines are more sensitive than normal skin cells to death induced by the SF3B1 inhibitor pladienolide B. Knockdown of SF3B1 and a range of other splicing factors with diverse roles in the spliceosome can also selectively kill cSCC cells. We demonstrate that endogenous c-MYC participates in conferring sensitivity to spliceosome inhibition. c-MYC expression is elevated in cSCC lines and its knockdown reduces alterations in mRNA splicing and attenuates cell death caused by interference with the spliceosome. In addition, this study provides further support for a key role of the p53 pathway in the response to spliceosome disruption. SF3B1 inhibition causes wild-type p53 upregulation associated with altered mRNA splicing and reduced protein expression of both principal p53 negative regulators MDMX/MDM4 and MDM2. We observed that wild-type p53 can promote pladienolide B-induced death in tumour cells. However, p53 is commonly inactivated by mutation in cSCCs and p53 participates in killing normal skin cells at high concentrations of pladienolide B. This may limit the therapeutic window of SF3B1 inhibitors for cSCC. We provide evidence that, while suppression of SF3B1 has promise for treating cSCCs with mutant p53, inhibitors which target the spliceosome through SF3B1-independent mechanisms could have greater cSCC selectivity as a consequence of reduced p53 upregulation in normal cells.
RESUMEN
TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Recurrencia Local de Neoplasia/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Invasividad Neoplásica/genética , Recurrencia Local de Neoplasia/patología , Isoformas de Proteínas/genética , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
The TP53 gene expresses at least nine different mRNA variants (p53 isoform mRNAs), including the one encoding the canonical p53 tumor suppressor protein. We have developed scientific tools to specifically detect and quantify p53 isoform expression at mRNA level by nested RT-PCR (reverse transcription-polymerase chain reaction) and quantitative real-time RT-PCR (RT-qPCR using the TaqMan(®) chemistry). Here, we describe these two methods, while highlighting essential points with regard to the analysis of p53 isoform mRNA expression.
Asunto(s)
ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína p53 Supresora de Tumor/genética , Línea Celular Tumoral , Genes p53 , Humanos , Isoformas de Proteínas/genéticaRESUMEN
The human p53 protein isoforms are expressed in several cell lines and modulate p53 tumor suppressor -activity, mainly through modulation of gene expression (1-4). Thus, identifying the pattern of p53 isoforms expression in cell lines is a key step for future studies of the p53 network (5). At the moment, the detection of p53 protein isoforms is based on the use of a panel of antibodies allowing their identification by comparing their molecular weights and their detection pattern by different antibodies (6). Here, classical protocols supplemented with technical know-how are described to detect p53 protein isoforms at protein level by Western blotting and immunoprecipitation. Furthermore, a simple method to study the impact of p53 protein isoforms on p53 transcriptional activity through luciferase reporter gene assays is provided.
Asunto(s)
Western Blotting/métodos , Inmunoprecipitación/métodos , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular , Drosophila , Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Modelos Animales , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Activación Transcripcional , Pez CebraRESUMEN
The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53ß. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Humanos , Leupeptinas/química , Leupeptinas/farmacología , Isoformas de Proteínas/metabolismo , Proteolisis/efectos de los fármacos , UbiquitinaciónRESUMEN
INTRODUCTION: Normal function of the p53 network is lost in most cancers, often through p53 mutation. The clinical impact of p53 mutations in breast cancer remains uncertain, especially where p53 isoforms may modify the effects of these p53 mutations. METHODS: Expression of p53ß and p53γ isoforms, the isoforms identified in normal breast tissue, was detected by reverse transcription polymerase chain reaction from a cohort of 127 primary breast tumours. Expression of p53ß and p53γ isoforms was analysed in relation to clinical markers and clinical outcomes (5 years) by binary logistic regression, Cox proportional hazards regression and Kaplan-Meier survival analyses. RESULTS: p53ß and p53γ were not randomly expressed in breast cancer. p53ß was associated with tumour oestrogen receptor (ER) expression, and p53γ was associated with mutation of the p53 gene. The patient group with the mutant p53 breast tumour-expressing p53γ isoform had low cancer recurrence and an overall survival as good as that of patients with wild-type p53 breast cancer. Conversely, patients expressing only mutant p53, without p53γ isoform expression, had a particularly poor prognosis. CONCLUSIONS: The determination of p53γ expression may allow the identification, independently of the ER status, of two subpopulations of mutant p53 breast cancer patients, one expressing p53γ with a prognosis as good as the wild-type p53 breast cancer patients and a second one not expressing p53γ with a particularly poor prognosis. The p53γ isoform may provide an explanation of the hitherto inconsistent relationship between p53 mutation, treatment response and outcome in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , Isoformas de Proteínas/genética , Recurrencia , Análisis de SupervivenciaRESUMEN
p53 gene expresses several protein isoforms modulating p53-mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site-directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53.
Asunto(s)
Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Perros , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Ratas , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Activation of apoptosis is believed to be critical for the role of p53 as a tumor suppressor. Here, we report a new mouse strain carrying a human p53 transgene in the mouse p53-null background. Expression of human p53 in these mice was comparable with wild-type murine p53; however, transactivation, induction of apoptosis, and G(1)-S checkpoint, but not transrepression or regulation of a centrosomal checkpoint, were deregulated. Although multiple functions of p53 were abrogated, mice carrying the human p53 transgene did not show early onset of tumors as typically seen for p53-null mice. In contrast, human p53 in the p53-null background did not prevent accelerated tumor development after genotoxic or oncogenic stress. Such behavior of human p53 expressed at physiologic levels in transgenic cells could be explained by unexpectedly high binding with Mdm2. By using Nutlin-3a, an inhibitor of the interaction between Mdm2 and p53, we were able to partially reconstitute p53 transactivation and apoptosis in transgenic cells. Our findings indicate that the interaction between p53 and Mdm2 controls p53 transcriptional activity in homeostatic tissues and regulates DNA damage- and oncogene-induced, but not spontaneous, tumorigenesis.
Asunto(s)
Apoptosis/genética , Genes p53 , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Transgenes , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53beta can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while Delta133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes p53 , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo , Animales , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Drosophila/genética , Evolución Molecular , Genes de Insecto , Genes Reporteros , Variación Genética , Células HT29 , Humanos , Intrones , Luciferasas/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/químicaRESUMEN
In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.