RESUMEN
Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8+ T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Células 3T3 , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Inflamación/genética , Inflamación/metabolismo , Neoplasias Renales/genética , Espectrometría de Masas , Ratones , Proteómica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis, and its early rise in patients with chronic kidney disease is independently associated with all-cause mortality. Since inflammation is characteristic of chronic kidney disease and associates with increased plasma FGF23 we examined whether inflammation directly stimulates FGF23. In a population-based cohort, plasma tumor necrosis factor (TNF) was the only inflammatory cytokine that independently and positively correlated with plasma FGF23. Mouse models of chronic kidney disease showed signs of renal inflammation, renal FGF23 expression and elevated systemic FGF23 levels. Renal FGF23 expression coincided with expression of the orphan nuclear receptor Nurr1 regulating FGF23 in other organs. Antibody-mediated neutralization of TNF normalized plasma FGF23 and suppressed ectopic renal Fgf23 expression. Conversely, TNF administration to control mice increased plasma FGF23 without altering plasma phosphate. Moreover, in Il10-deficient mice with inflammatory bowel disease and normal kidney function, plasma FGF23 was elevated and normalized upon TNF neutralization. Thus, the inflammatory cytokine TNF contributes to elevated systemic FGF23 levels and also triggers ectopic renal Fgf23 expression in animal models of chronic kidney disease.
Asunto(s)
Factores de Crecimiento de Fibroblastos/sangre , Enfermedades Inflamatorias del Intestino/inmunología , Insuficiencia Renal Crónica/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Línea Celular , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/inmunología , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Interleucina-10/deficiencia , Interleucina-10/genética , Riñón/inmunología , Riñón/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Cultivo Primario de Células , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/patología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel-Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Cilios/genética , Modelos Animales de Enfermedad , Células Epiteliales , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Estimación de Kaplan-Meier , Túbulos Renales Proximales/citología , Ratones , Mutación , ARN Mensajero/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Tasa de Supervivencia , Ubiquitina-Proteína Ligasas/genética , Microtomografía por Rayos XRESUMEN
One of the earliest requirements for the formation of a solid tumor is the establishment of an adequate blood supply. Clear cell renal cell carcinomas (ccRCC) are highly vascularized tumors in which the earliest genetic event is most commonly the biallelic inactivation of the VHL tumor suppressor gene, leading to constitutive activation of the HIF-1α and HIF-2α transcription factors, which are known angiogenic factors. However it remains unclear whether either or both HIF-1α or HIF-2α stabilization in normal renal epithelial cells are necessary or sufficient for alterations in blood vessel formation. We show that renal epithelium-specific deletion of Vhl in mice causes increased medullary vascularization and that this phenotype is completely rescued by Hif1a co-deletion, but not by co-deletion of Hif2a. A physiological consequence of changes in the blood vessels of the vasa recta in Vhl-deficient mice is a diabetes insipidus phenotype of excretion of large amounts of highly diluted urine. This constitutive diuresis is fully compensated by increased water consumption and mice do not show any signs of dehydration, renal failure or salt wasting and blood electrolyte levels remain unchanged. Co-deletion of Hif1a, but not Hif2a, with Vhl, fully restored kidney morphology and function, correlating with the rescue of the vasculature. We hypothesize that the increased medullary vasculature alters salt uptake from the renal interstitium, resulting in a disruption of the osmotic gradient and impaired urinary concentration. Taken together, our study characterizes a new mouse model for a form of diabetes insipidus and non-obstructive hydronephrosis and provides new insights into the physiological and pathophysiological effects of HIF-1α stabilization on the vasculature in the kidney.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Riñón/metabolismo , Neovascularización Patológica , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/orina , Carcinoma de Células Renales/orina , Diuresis , Electrólitos , Células Endoteliales/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Homeostasis , Hidronefrosis/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/orina , Neoplasias Renales/metabolismo , Neoplasias Renales/orina , Ratones , Ratones Transgénicos , Mutación , Fenotipo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Orina , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/orina , Microtomografía por Rayos XRESUMEN
The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the majority of clear cell renal cell carcinomas (ccRCC), but genetic ablation of Vhl alone in mouse models is insufficient to recapitulate human tumorigenesis. One function of pVHL is to regulate the stability of the hypoxia-inducible factors (HIF), which become constitutively activated in the absence of pVHL. In established ccRCC, HIF1α has been implicated as a renal tumor suppressor, whereas HIF2α is considered an oncoprotein. In this study, we investigated the contributions of HIF1α and HIF2α to ccRCC initiation in the context of Vhl deficiency. We found that deleting Vhl plus Hif1a or Hif2a specifically in the renal epithelium did not induce tumor formation. However, HIF1α and HIF2α differentially regulated cell proliferation, mitochondrial abundance and oxidative capacity, glycogen accumulation, and acquisition of a clear cell phenotype in Vhl-deficient renal epithelial cells. HIF1α, but not HIF2α, induced Warburg-like metabolism characterized by increased glycolysis, decreased oxygen consumption, and decreased ATP production in mouse embryonic fibroblasts, providing insights into the cellular changes potentially occurring in Vhl mutant renal cells before ccRCC formation. Importantly, deletion of either Hif1a or Hif2a completely prevented the formation of renal cysts and tumors in Vhl/Trp53 mutant mice. These findings argue that both HIF1α and HIF2α exert protumorigenic functions during the earliest stages of cyst and tumor formation in the kidney. Cancer Res; 76(7); 2025-36. ©2016 AACR.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , RatonesRESUMEN
The combinations of genetic alterations that cooperate with von Hippel-Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro-proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development.