RESUMEN
Missense mutations in p53 generate aberrant proteins with abrogated tumour suppressor functions that can also acquire oncogenic gain-of-function activities that promote malignant progression, invasion, metastasis and chemoresistance. Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumours, which is the key requisite for the acquisition of gain-of-functions activities. Although currently 11 million patients worldwide live with tumours expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo. Here we use a novel mutp53 mouse model expressing an inactivatable R248Q hotspot mutation (floxQ) to show that tumours depend on sustained mutp53 expression. Upon tamoxifen-induced mutp53 ablation, allotransplanted and autochthonous tumours curb their growth, thus extending animal survival by 37%, and advanced tumours undergo apoptosis and tumour regression or stagnation. The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared with normal tissues, is a major determinant of mutp53 stabilization. We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/- (R248Q allele) and H/H (R172H allele) mice by 59% and 48%, respectively, but not their corresponding p53(-/-) littermates. This mutp53-dependent drug effect occurs in H/H mice treated with 17DMAG+SAHA and in H/H and Q/- mice treated with the potent Hsp90 inhibitor ganetespib. Notably, drug activity correlates with induction of mutp53 degradation, tumour apoptosis and prevention of T-cell lymphomagenesis. These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target.
Asunto(s)
Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas Mutantes/antagonistas & inhibidores , Estabilidad Proteica , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Alelos , Aloinjertos , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Humanos , Linfoma/genética , Linfoma/patología , Masculino , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Trasplante de Neoplasias , Estabilidad Proteica/efectos de los fármacos , Tasa de Supervivencia , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Triazoles/farmacología , Triazoles/uso terapéutico , Proteína p53 Supresora de Tumor/genéticaRESUMEN
HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/enzimología , Glucuronosiltransferasa/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Triazoles/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/fisiopatología , Glucuronosiltransferasa/genética , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , HumanosRESUMEN
In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/- glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/- glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/- mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa(LSL-LacZ);WAP-Cre transgenics (LSL-LacZ, lox-stop-lox ß-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/- glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/- ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.
Asunto(s)
Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Transactivadores/metabolismo , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Células Epiteliales/citología , Femenino , Heterocigoto , Humanos , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoproteínas/genética , Embarazo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transactivadores/genéticaRESUMEN
Overexpression of the human epidermal growth factor receptor-2 (HER2) in breast cancer strongly correlates with aggressive tumors and poor prognosis. Recently, a positive correlation between HER2 and MIF (macrophage migration inhibitory factor, a tumor-promoting protein and heat-shock protein 90 (HSP90) client) protein levels was shown in cancer cells. However, the underlying mechanistic link remained unknown. Here we show that overexpressed HER2 constitutively activates heat-shock factor 1 (HSF1), the master transcriptional regulator of the inducible proteotoxic stress response of heat-shock chaperones, including HSP90, and a crucial factor in initiation and maintenance of the malignant state. Inhibiting HER2 pharmacologically by Lapatinib (a dual HER2/epidermal growth factor receptor inhibitor) or CP724.714 (a specific HER2 inhibitor), or by knockdown via siRNA leads to inhibition of phosphoactivated Ser326 HSF1, and subsequently blocks the activity of the HSP90 chaperone machinery in HER2-overexpressing breast cancer lines. Consequently, HSP90 clients, including MIF, AKT, mutant p53 and HSF1 itself, become destabilized, which in turn inhibits tumor proliferation. Mechanistically, HER2 signals via the phosphoinositide-3-kinase (PI3K)-AKT- mammalian target of rapamycin (mTOR) axis to induce activated pSer326 HSF1. Heat-shock stress experiments confirm this functional link between HER2 and HSF1, as HER2 (and PI3K) inhibition attenuate the HSF1-mediated heat-shock response. Importantly, we confirmed this axis in vivo. In the mouse model of HER2-driven breast cancer, ErbB2 inhibition by Lapatinib strongly suppresses tumor progression, and this is associated with inactivation of the HSF1 pathway. Moreover, ErbB2-overexpressing cancer cells derived from a primary mouse ErbB2 tumor also show HSF1 inactivation and HSP90 client destabilization in response to ErbB2 inhibition. Furthermore, in HER2-positive human breast cancers HER2 levels strongly correlate with pSer326 HSF1 activity. Our results show for the first time that HER2/ErbB2 overexpression controls HSF1 activity, with subsequent stabilization of numerous tumor-promoting HSP90 clients such as MIF, AKT and HSF1 itself, thereby causing a robust promotion in tumor growth in HER2-positive breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Receptor ErbB-2/metabolismo , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Femenino , Proteínas HSP90 de Choque Térmico/genética , Factores de Transcripción del Choque Térmico , Humanos , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor ErbB-2/genética , Factores de Transcripción/genéticaRESUMEN
The p53 tumor suppressor network plays a fundamental surveillance role in both homeostatic and adaptive cell biology. p53 is one of the most important barriers against malignant derailment of normal cells, orchestrating growth arrest, senescence, or cell death by linking many different pathways in response to genotoxic and non-genotoxic insults. p53 is the key broadband sensor for numerous cellular stresses such as DNA damage, hypoxia, oxidative stress, oncogenic signaling, and nucleolar stress. The crucial tumor suppressive and tissue homeostasis activity of p53 is its ability to activate cell death via multiple different pathways. A well-characterized biochemical function of p53 in the regulation of apoptosis is its role as a potent transcriptional regulator. p53 activates a panel of proapoptotic genes from the mitochondrial apoptotic and death receptor programs while repressing antiapoptotic Bcl2 family genes. In addition, over the last 10 y a growing body of evidence has also defined direct extranuclear non-transcriptional p53 activities within mitochondria-mediated cell death pathways that are based on p53 protein accumulation in cytosolic and mitochondrial compartments and protein-protein interactions. To date, transcription-independent p53-mediated cell death regulation has been described for apoptosis, necrosis, and autophagy. Because mitochondrial dysregulation is central to the development of a number of pathologic processes such as cancer and neurodegenerative and age-related diseases, understanding the direct roles of p53 protein in mitochondria has high translational impact and could facilitate the development of novel drug targets to combat these diseases. In this review we will mainly focus on mechanisms of p53-mediated transcription-independent cell death pathways at mitochondria.
RESUMEN
Somatic cells can be converted into induced pluripotent stem cells (iPSCs) by forced expression of various combinations of transcription factors, but the molecular mechanisms of reprogramming are poorly understood. Specifically, evidence that the reprogramming process can take many distinct routes only begins to emerge. It is definitively established that p53 deficiency greatly enhances reprogramming, revealing p53's barrier function for induced pluripotency, but the role of its homologs p63 and p73 are unknown. Here we report that in stark contrast to p53, p73 has no role in reprogramming. However, p63 is an enabling (rather than a barrier) factor for Oct4, Sox2 and Klf4 (OSK) and Oct4 and Sox2 (OS), but not for Oct4 and Klf4 (OK) reprogramming of mouse embryonic fibroblasts. Specifically, p63 is essential during reprogramming for maximum efficiency, albeit not for the ability to reprogram per se, and is dispensable for maintaining stability and pluripotency of established iPSC colonies. ΔNp63, but not TAp63, is the principal isoform involved. Loss of p63 can affect reprogramming via several mechanisms such as reduced expression of mesenchymal-epithelial transition and pluripotency genes, hypoproliferation and loss of the most reprogrammable cell populations. During OSK and OS reprogramming, different mechanisms seem to be critical, such as regulation of epithelial and pluripotency genes in OSK reprogramming versus regulation of proliferation in OS reprogramming. Finally, our data reveal three different routes of reprogramming by OSK, OS or OK, based on their differential p63 requirements for iPSC efficiency and pluripotency marker expression. This supports the concept that many distinct routes of reprogramming exist.
Asunto(s)
Reprogramación Celular , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Animales , Proliferación Celular , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosfoproteínas/deficiencia , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transactivadores/deficiencia , Regulación hacia ArribaAsunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Activación Transcripcional/fisiología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/fisiología , Humanos , Proteína Tumoral p73RESUMEN
Mutant p53 proteins not only lose their tumor-suppressor function but some acquire oncogenic gain of function (GOF). The published mutp53 knock-in (KI) alleles (R172H, R270H, R248W) manifest GOF by broader tumor spectrum and more metastasis compared with the p53-null allele, but do not shorten survival. However, whether GOF also occurs with other mutations and whether they are all biologically equal is unknown. To answer this, we created novel humanized mutp53 KI mice harboring the hot spot alleles R248Q and G245S. Intriguingly, their impact was very different. Compared with p53-null mice, R248Q/- mice had accelerated onset of all tumor types and shorter survival, thus unprecedented strong GOF. In contrast, G245S/- mice were similar to null mice in tumor latency and survival. This was associated with a twofold higher T-lymphoma proliferation in R248Q/- mice compared with G245S/- and null mice. Moreover, R248Q/- hematopoietic and mesenchymal stem cells were expanded relative to G245S/- and null mice, the first indication that GOF also acts by perturbing pretumorous progenitor pools. Importantly, these models closely mirror Li-Fraumeni patients who show higher tumor numbers, accelerated onset and shorter tumor-free survival by 10.5 years when harboring codon R248Q mutations as compared with Li-Fraumeni patients with codon G245S mutations or p53 deletions/loss. Conversely, both KI alleles caused a modest broadening of tumor spectrum with enhanced Akt signaling compared with null mice. These models are the first in vivo proof for differential oncogenic strength among p53 GOF alleles, with genotype-phenotype correlations borne out in humans.
Asunto(s)
Transformación Celular Neoplásica/genética , Mutación/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología , Adulto , Alelos , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/patología , Síndrome de Li-Fraumeni/fisiopatología , Linfoma de Células T/genética , Linfoma de Células T/patología , Linfoma de Células T/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/deficienciaAsunto(s)
Proteínas de Unión al ADN/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Células-Madre Neurales/citología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
In response to intense stress, the tumor protein p53 (p53) tumor suppressor rapidly mounts a direct mitochondrial death program that precedes transcription-mediated apoptosis. By eliminating severely damaged cells, this pathway contributes to tumor suppression as well as to cancer cell killing induced by both genotoxic drugs and non-genotoxic p53-reactivating molecules. Here we have explored the role had in this pathway by the prolyl-isomerase Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), a crucial transducer of p53's phosphorylation into conformational changes unleashing its pro-apoptotic activity. We show that Pin1 promotes stress-induced localization of p53 to mitochondria both in vitro and in vivo. In particular, we demonstrate that upon stress-induced phosphorylation of p53 on Ser46 by homeodomain interacting protein kinase 2, Pin1 stimulates its mitochondrial trafficking signal, that is, monoubiquitination. This pathway is induced also by the p53-activating molecule RITA, and we demonstrate the strong requirement of Pin1 for the induction of mitochondrial apoptosis by this compound. These findings have significant implications for treatment of p53-expressing tumors and for prospective use of p53-activating compounds in clinics.
Asunto(s)
Mitocondrias/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Furanos/farmacología , Células HCT116 , Humanos , Ratones , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ectopic expression of defined sets of transcription factors in somatic cells enables them to adopt the qualities of pluripotency. Mouse embryonic fibroblasts (MEFs) are the classic target cell used to elucidate the core principles of nuclear reprogramming. However, their phenotypic and functional heterogeneity represents a major hurdle for mechanistic studies aimed at defining the molecular nature of cellular plasticity. We show that reducing the complexity of MEFs by flow cytometry allows the isolation of discrete cell subpopulations that can be efficiently reprogrammed to pluripotency with fewer genes. Using these FACS-sorted cells, we performed a systematic side-by-side analysis of the reprogramming efficiency with different two- and three-factor combinations of Oct4, Sox2 and Klf4. We show that introduction of exogenous Oct4 with either Sox2 or Klf4 does not directly convert MEFs to a pluripotent state. Instead, each combination of factors disrupts the normal cellular homeostasis and establishes transient states characterized by the concurrent expression of mixed lineage markers. These cells convert into induced pluripotent stem cells in a stochastic fashion. Our data suggest that there is a partial functional redundancy between Sox2 and Klf4 in the disruption of cellular homeostasis and activation of regulatory networks that define pluripotency.
Asunto(s)
Reprogramación Celular/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Pluripotentes/fisiología , Factores de Transcripción SOXB1/metabolismo , Células Madre/fisiología , Animales , Diferenciación Celular/fisiología , Reprogramación Celular/genética , Perfilación de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/biosíntesis , Factores de Transcripción SOXB1/genética , Células Madre/citología , Células Madre/metabolismo , Transfección/métodosRESUMEN
Mutant p53 (mutp53) cancers are surprisingly dependent on their hyperstable mutp53 protein for survival, identifying mutp53 as a potentially significant clinical target. However, exploration of effective small molecule therapies targeting mutp53 has barely begun. Mutp53 hyperstabilization, a hallmark of p53 mutation, is cancer cell-specific and due to massive upregulation of the HSP90 chaperone machinery during malignant transformation. We recently showed that stable complex formation between HSP90 and its mutp53 client inhibits E3 ligases MDM2 and CHIP, causing mutp53 stabilization. Histone deacetylase (HDAC) inhibitors (HDACi) are a new class of promising anti-cancer drugs, hyperacetylating histone and non-histone targets. Currently, suberoylanilide hydroxamic acid (SAHA) is the only FDA-approved HDACi. We show that SAHA exhibits preferential cytotoxicity for mutant, rather than wild-type and null p53 human cancer cells. Loss/gain-of-function experiments revealed that although able to exert multiple cellular effects, SAHA's cytotoxicity is caused to a significant degree by its ability to strongly destabilize mutp53 at the level of protein degradation. The underlying mechanism is SAHA's inhibition of HDAC6, an essential positive regulator of HSP90. This releases mutp53 and enables its MDM2- and CHIP-mediated degradation. SAHA also strongly chemosensitizes mutp53 cancer cells for chemotherapy due to its ability to degrade mutp53. This identifies a novel action of SAHA with the prospect of SAHA becoming a centerpiece in mutp53-specific anticancer strategies.
Asunto(s)
Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Proteínas Mutantes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Regulación hacia Abajo , Activación Enzimática , Histona Desacetilasa 6 , Humanos , Concentración 50 Inhibidora , Leupeptinas/farmacología , Terapia Molecular Dirigida , Proteínas Mutantes/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Estabilidad Proteica/efectos de los fármacos , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismo , VorinostatRESUMEN
Strategies to induce p53 activation in wtp53-retaining tumors carry high potential in cancer therapy. Nutlin, a potent highly selective MDM2 inhibitor, induces non-genotoxic p53 activation. Although Nutlin shows promise in promoting cell death in hematopoietic malignancies, a major roadblock is that most solid cancers do not undergo apoptosis but merely reversible growth arrest. p53 inhibition by unopposed MDMX is one major cause for apoptosis resistance to Nutlin. The Hsp90 chaperone is ubiquitously activated in cancer cells and supports oncogenic survival pathways, many of which antagonize p53. The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) is known to induce p53-dependent apoptosis. We show here that in multiple difficult-to-kill solid tumor cells 17AAG modulates several critical components that synergize with Nutlin-activated p53 signaling to convert Nutlin's transient cytostatic response into a cytotoxic killing response in vitro and in xenografts. Combined with Nutlin, 17AAG destabilizes MDMX, reduces MDM2, induces PUMA and inhibits oncogenic survival pathways, such as PI3K/AKT, which counteract p53 signaling at multiple levels. Mechanistically, 17AAG interferes with the repressive MDMX-p53 axis by inducing robust MDMX degradation, thereby markedly increasing p53 transcription compared with Nutlin alone. To our knowledge Nutlin+17AAG represents the first effective pharmacologic knockdown of MDMX. Our study identifies 17AAG as a promising synthetic lethal partner for a more efficient Nutlin-based therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Imidazoles/farmacología , Lactamas Macrocíclicas/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Sinergismo Farmacológico , Activación Enzimática , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.
Asunto(s)
Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Proteínas de Unión al ADN/fisiología , Células-Madre Neurales/citología , Neurogénesis , Neuronas/citología , Proteínas Nucleares/fisiología , Proteínas Supresoras de Tumor/fisiología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Supervivencia Celular , Senescencia Celular , Sistema Nervioso Central/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Hidrocefalia/patología , Ratones , Ratones Noqueados , Mitosis , Células-Madre Neurales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Fase S , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The activity of p53 as an inducible transcription factor depends on its rapid nuclear stabilization after stress. However, surprisingly, mechanism(s) that regulate nuclear p53 accumulation are not well understood. The current model of stress-induced nuclear accumulation holds that a decrease in p53 nuclear export leads to its nuclear stabilization. We show here that regulated nuclear import of p53 also has a critical function. p53 import is mediated by binding to the importin-alpha3 adapter and is negatively regulated by ubiquitination. p53 harbors several nuclear localization signals (NLS), with the major NLS I located at amino-acids 305-322. We find that direct binding of p53 to importin-alpha3 depends on the positive charge contributed by lysine residues 319-321 within NLS I. The same lysines are also targets of MDM2-mediated ubiquitination. p53 ubiquitination occurs primarily in unstressed cells, but decreases dramatically after stress. Importin-alpha3 preferentially interacts with non-ubiquitinated p53. Thus, under normal growth conditions, ubiquitination of Lys 319-321 negatively regulates p53-importin-alpha3 binding, thereby restraining p53 import. Conversely, stress-induced accumulation of non-ubiquitinated p53 in the cytoplasm promotes interaction with importin-alpha3 and rapid import. In later phases of the stress response, blocked nuclear export also takes effect. We propose that p53 nuclear import defines an important novel level of regulation in the p53-mediated stress response.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Adenocarcinoma/genética , Adenocarcinoma/patología , División Celular/fisiología , Núcleo Celular/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Citoplasma/metabolismo , Células HCT116 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Lisina/metabolismo , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Estrés Fisiológico/fisiología , Proteína p53 Supresora de Tumor/genética , Ubiquitinación/fisiología , alfa Carioferinas/genéticaRESUMEN
Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.
Asunto(s)
Muerte Celular , Apoptosis , Células Eucariotas/citología , Citometría de Flujo , Guías como Asunto , Humanos , Immunoblotting , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Espectrometría de FluorescenciaRESUMEN
p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile alpha-motif) and post-SAM domains that are specific for the alpha and beta isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63gamma isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63alpha/beta isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63alpha/beta but not p63gamma are absolutely required for proper skin and limb development.