RESUMEN
BACKGROUND: Ovarian cancer (OC) remains one of the most challenging and deadly malignancies facing women today. While PARP inhibitors (PARPis) have transformed the treatment landscape for women with advanced OC, many patients will relapse and the PARPi-resistant setting is an area of unmet medical need. Traditional immunotherapies targeting PD-1/PD-L1 have failed to show any benefit in OC. The CD47/TSP-1 axis may be relevant in OC. We aimed to describe changes in CD47 expression with platinum therapy and their relationship with immune features and prognosis. METHODS: Tumor and blood samples collected from OC patients in the CHIVA trial were assessed for CD47 and TSP-1 before and after neoadjuvant chemotherapy (NACT) and multiplex analysis was used to investigate immune markers. Considering the therapeutic relevance of targeting the CD47/TSP-1 axis, we used the CD47-derived TAX2 peptide to selectively antagonize it in a preclinical model of aggressive ovarian carcinoma. RESULTS: Significant reductions in CD47 expression were observed post NACT. Tumor patients having the highest CD47 expression profile at baseline showed the greatest CD4+ and CD8+ T-cell influx post NACT and displayed a better prognosis. In addition, TSP-1 plasma levels decreased significantly under NACT, and high TSP-1 was associated with a worse prognosis. We demonstrated that TAX2 exhibited a selective and favorable biodistribution profile in mice, localizing at the tumor sites. Using a relevant peritoneal carcinomatosis model displaying PARPi resistance, we demonstrated that post-olaparib (post-PARPi) administration of TAX2 significantly reduced tumor burden and prolonged survival. Remarkably, TAX2 used sequentially was also able to increase animal survival even under treatment conditions allowing olaparib efficacy. CONCLUSIONS: Our study thus (1) proposes a CD47-based stratification of patients who may be most likely to benefit from postoperative immunotherapy, and (2) suggests that TAX2 is a potential alternative therapy for patients relapsing on PARP inhibitors.
Asunto(s)
Biomarcadores de Tumor , Antígeno CD47 , Neoplasias Ováricas , Trombospondina 1 , Antígeno CD47/metabolismo , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Biomarcadores de Tumor/metabolismo , Animales , Ratones , Trombospondina 1/metabolismo , Pronóstico , Línea Celular Tumoral , Terapia Neoadyuvante , Ensayos Antitumor por Modelo de Xenoinjerto , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. Here, we show that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. Our studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, we show that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, our studies provide key insights into the biological processes regulated by KDM5 and expand our understanding of the transcriptional regulators that coordinate animal development.
Asunto(s)
Relojes Biológicos/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Desarrollo Embrionario/genética , Glándulas Endocrinas/embriología , Histona Demetilasas/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Ecdisona/metabolismo , Embrión no Mamífero , Glándulas Endocrinas/metabolismo , Endorreduplicación/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Larva , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Organogénesis/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factores de TiempoRESUMEN
Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.
Asunto(s)
Corteza Suprarrenal/enzimología , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Transducción de Señal , Corteza Suprarrenal/metabolismo , Animales , Diferenciación Celular , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Esteroides/metabolismo , Zona Fascicular/citología , Zona Fascicular/enzimología , Zona Fascicular/metabolismo , Zona Glomerular/citología , Zona Glomerular/enzimología , Zona Glomerular/metabolismoRESUMEN
BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Factor de Transcripción E2F1/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Neoplásica de la Expresión Génica , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Inmunoprecipitación de Cromatina , Biología Computacional , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Humanos , Indoles/farmacología , Ratones Noqueados , Análisis Multivariante , Modelos de Riesgos Proporcionales , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Ribonucleósido Difosfato Reductasa/genética , Securina/genéticaRESUMEN
Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Bases de Datos de Ácidos Nucleicos , Progresión de la Enfermedad , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Interferencia de ARN , Factores de Riesgo , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.
Asunto(s)
Enfermedades de la Corteza Suprarrenal/metabolismo , Enfermedades de la Corteza Suprarrenal/patología , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteína Letal Asociada a bcl/metabolismo , Enfermedades de la Corteza Suprarrenal/genética , Hormona Adrenocorticotrópica/administración & dosificación , Hormona Adrenocorticotrópica/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/ß-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that ß-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/ß-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/metabolismo , Adenoma Corticosuprarrenal/metabolismo , Aldosterona/biosíntesis , Hiperaldosteronismo/metabolismo , Vía de Señalización Wnt , Neoplasias de la Corteza Suprarrenal/complicaciones , Adenoma Corticosuprarrenal/complicaciones , Adulto , Aldosterona/sangre , Aldosterona/metabolismo , Animales , Línea Celular Tumoral , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperaldosteronismo/etiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismoRESUMEN
Regulated gene expression is necessary for developmental and homeostatic processes. The KDM5 family of transcriptional regulators are histone H3 lysine 4 demethylases that can function through both demethylase-dependent and -independent mechanisms. While loss and overexpression of KDM5 proteins are linked to intellectual disability and cancer, respectively, their normal developmental functions remain less characterized. Drosophila melanogaster provides an ideal system to investigate KDM5 function, as it encodes a single ortholog in contrast to the four paralogs found in mammalian cells. To examine the consequences of complete loss of KDM5, we generated a null allele of Drosophila kdm5, also known as little imaginal discs (lid), and show that it is essential for viability. Animals lacking KDM5 show a dramatically delayed larval development that coincides with decreased proliferation and increased cell death in wing imaginal discs. Interestingly, this developmental delay is independent of the well-characterized Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, suggesting key functions for less characterized domains. Consistent with the phenotypes observed, transcriptome analyses of kdm5 null mutant wing imaginal discs revealed the dysregulation of genes involved in several cellular processes, including cell cycle progression and DNA repair. Together, our analyses reveal KDM5 as a key regulator of larval growth and offer an invaluable tool for defining the biological activities of KDM5 family proteins.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Redes Reguladoras de Genes , Histona Demetilasas/genética , Mutación con Pérdida de Función , Alas de Animales/crecimiento & desarrollo , Animales , Ciclo Celular , Muerte Celular , Proliferación Celular , Reparación del ADN , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas/química , Histona Demetilasas/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Fenotipo , Dominios Proteicos , Análisis de Secuencia de ARNRESUMEN
Mutations in KDM5 family histone demethylases cause intellectual disability in humans. However, the molecular mechanisms linking KDM5-regulated transcription and cognition remain unknown. Here, we establish Drosophila as a model to understand this connection by generating a fly strain harboring an allele analogous to a disease-causing missense mutation in human KDM5C (kdm5A512P). Transcriptome analysis of kdm5A512P flies revealed a striking downregulation of genes required for ribosomal assembly and function and a concomitant reduction in translation. kdm5A512P flies also showed impaired learning and/or memory. Significantly, the behavioral and transcriptional changes in kdm5A512P flies were similar to those specifically lacking demethylase activity. These data suggest that the primary defect of the KDM5A512P mutation is a loss of histone demethylase activity and reveal an unexpected role for this enzymatic function in gene activation. Because translation is critical for neuronal function, we propose that this defect contributes to the cognitive defects of kdm5A512P flies.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Histona Demetilasas/genética , Discapacidad Intelectual/genética , Mutación/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Cognición , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Proteínas de Drosophila/química , Perfilación de la Expresión Génica , Histona Demetilasas/química , Histonas/metabolismo , Lisina/metabolismo , Metilación , Fenotipo , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Transcripción Genética , Activación Transcripcional/genéticaRESUMEN
Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates ß-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/etiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Vía de Señalización Wnt , Zona Fascicular/metabolismo , Zona Glomerular/metabolismo , Animales , Carcinogénesis , Diferenciación Celular , Línea Celular Tumoral , Femenino , Humanos , Ratones , Fosforilación , Zona Fascicular/citología , Zona Glomerular/citología , beta Catenina/metabolismoRESUMEN
The adrenal cortex plays essential roles in the control of sodium and water homeostasis, stress response, inflammation and metabolism, through secretion of glucocorticoids and mineralocorticoids. Coordinated production of these hormones relies on functional zonation of the cortex, characterised by expression of Cyp11b2 under the control of angiotensin II and plasma potassium level in zona glomerulosa (ZG) and Cyp11b1 under the control of ACTH in zona fasciculata (ZF). The mechanisms involved in the establishment of functional zonation and its maintenance during centripetal cortex cell renewal are still poorly understood. Here, we hypothesise that the hormonal and signalling pathways that control adrenal cortex function are also involved in cortical zonation. In particular, we summarise evidence on the role of WNT/ß-catenin signalling in ZG differentiation and how tight control of its activity is required to shape the adult cortex. In this context, we discuss the potential role of known WNT regulators and the possibility of a reciprocal cross-talk between PKA and WNT signalling.
Asunto(s)
Corteza Suprarrenal/metabolismo , Homeostasis , Proteínas Wnt/metabolismo , Corteza Suprarrenal/citología , Animales , Autorrenovación de las Células , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
Adrenal cortical carcinomas (ACC) are rare but aggressive tumours associated with poor prognosis. The two most frequent alterations in ACC in patients are overexpression of the growth factor IGF2 and constitutive activation of Wnt/ß-catenin signalling. Using a transgenic mouse model, we have previously shown that constitutive active ß-catenin is a bona fide adrenal oncogene. However, although all these mice developed benign adrenal hyperplasia, malignant progression was infrequent, suggesting that secondary genetic events were required for aggressive tumour development. In the present paper, we have tested IGF2 oncogenic properties by developing two distinct transgenic mouse models of Igf2 overexpression in the adrenal cortex. Our analysis shows that despite overexpression levels ranging from 7 (basal) to 87 (ACTH-induced) fold, Igf2 has no tumour initiating potential in the adrenal cortex. However, it induces aberrant accumulation of Gli1 and Pod1-positive progenitor cells, in a hedgehog-independent manner. We have also tested the hypothesis that Igf2 may cooperate with Wnt signalling by mating Igf2 overexpressing lines with mice that express constitutive active ß-catenin in the adrenal cortex. We show that the combination of both alterations has no effect on tumour phenotype at stages when ß-catenin-induced tumours are benign. However, there is a mild promoting effect at later stages, characterised by increased Weiss score and proliferation. Formation of malignant tumours is nonetheless a rare event, even when Igf2 expression is further increased by ACTH treatment. Altogether these experiments suggest that the growth factor IGF2 is a mild contributor to malignant adrenocortical tumourigenesis.