RESUMEN
BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal , Aortitis , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteína Quinasa Deficiente en Lisina WNK 1 , Animales , Aortitis/genética , Aortitis/metabolismo , Aortitis/patología , Ratones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Humanos , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Ratones Endogámicos C57BL , Masculino , Células Cultivadas , Ratones Noqueados para ApoE , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Aorta Abdominal/metabolismo , Aorta Abdominal/patologíaRESUMEN
Vascular Ehlers-Danlos syndrome is a rare inherited disorder caused by genetic variants in type III collagen. Its prognosis is especially hampered by unpredictable arterial ruptures and there is no therapeutic consensus. We created a knock-in Col3a1+/G182R mouse model and performed a complete genetic, molecular and biochemical characterization. Several therapeutic strategies were also tested. Col3a1+/G182R mice showed a spontaneous mortality caused by thoracic aortic rupture that recapitulates the vascular Ehlers-Danlos syndrome with a lower survival rate in males, thin non-inflammatory arteries and an altered arterial collagen. Transcriptomic analysis of aortas showed upregulation of genes related to inflammation and cell stress response. Compared to water, survival rate of Col3a1+/G182R mice was not affected by beta-blockers (propranolol or celiprolol). Two other vasodilating anti-hypertensive agents (hydralazine, amlodipine) gave opposite results on aortic rupture and mortality rate. There was a spectacular beneficial effect of losartan, reversed by the cessation of its administration, and a marked deleterious effect of exogenous angiotensin II. These results suggest that blockade of the renin angiotensin system should be tested as a first-line medical therapy in patients with vascular Ehlers-Danlos syndrome.
Asunto(s)
Rotura de la Aorta , Síndrome de Ehlers-Danlos , Animales , Rotura de la Aorta/genética , Rotura de la Aorta/prevención & control , Arterias , Colágeno Tipo III/genética , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos/tratamiento farmacológico , Síndrome de Ehlers-Danlos/genética , Humanos , Masculino , RatonesRESUMEN
Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino-acids 403-459) (CUL3-∆9). Surprisingly, while CUL3-∆9 is hyperneddylated, a post-translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss-of function, we performed comparative label-free quantitative analyses of CUL3 and CUL3-∆9 interactome by mass spectrometry. It was observed that CUL3-∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes' dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3-∆9 complex an inactive BTB-adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3-∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners.
Asunto(s)
Proteínas Cullin/genética , Hipertensión , Seudohipoaldosteronismo , Proteínas Cullin/metabolismo , Exones/genética , Femenino , Humanos , Hipertensión/genética , Masculino , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Mutations in four genes, WNK lysine deficient protein kinase 1 and 4 (WNK1 and WNK4), kelch like family member 3 (KLHL3), or Cullin 3 (CUL3), can result in familial hyperkalemic hypertension (FHHt), a rare Mendelian form of human arterial hypertension. Although all mutations result in an increased abundance of WNK1 or WNK4, all FHHt-causing CUL3 mutations, resulting in the skipping of exon 9, lead to a more severe phenotype. METHODS: We created and compared two mouse models, one expressing the mutant Cul3 protein ubiquitously (pgk-Cul3∆9) and the other specifically in vascular smooth muscle cells (SM22-Cul3∆9). We conducted pharmacologic investigations on isolated aortas and generated stable and inducible HEK293 cell lines that overexpress the wild-type Cul3 or mutant Cul3 (Cul3∆9) protein. RESULTS: As expected, pgk-Cul3∆9 mice showed marked hypertension with significant hyperkalemia, hyperchloremia and low renin. BP increased significantly in SM22-Cul3∆9 mice, independent of any measurable effect on renal transport. Only pgk-Cul3∆9 mice displayed increased expression of the sodium chloride cotransporter and phosphorylation by the WNK-SPAK kinases. Both models showed altered reactivity of isolated aortas to phenylephrine and acetylcholine, as well as marked acute BP sensitivity to the calcium channel blocker amlodipine. Aortas from SM22-Cul3∆9 mice showed increased expression of RhoA, a key molecule involved in regulation of vascular tone, compared with aortas from control mice. We also observed increased RhoA abundance and t1/2 in Cul3∆9-expressing cells, caused by decreased ubiquitination. CONCLUSIONS: Mutations in Cul3 cause severe hypertension by affecting both renal and vascular function, the latter being associated with activation of RhoA.
Asunto(s)
Presión Arterial/genética , Proteínas Cullin/genética , Hipertensión/genética , Mutación , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Humanos , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Distribución Aleatoria , Ubiquitinación/genéticaRESUMEN
OBJECTIVE: Vascular Ehlers-Danlos syndrome (vEDS) is associated with arterial ruptures due to a mutant gene encoding collagen type III (Col-III). To better understand the role of Col-III, we aimed at evaluating aortic stiffness and dynamic stiffening in vEDS mouse models, with either a quantitative (col3KO mice) or a qualitative Col-III defect (col3KI mice). MATERIALS AND METHODS: Abdominal aortic wall pulse wave velocities (PWV) in col3KO and col3KI mice were compared to their respective wild type (WT) littermates using a 15âMHz ultrafast ultrasonic transducer. A carotid catheter continuously monitored pressure changes due to phenylephrine injections. PWV1, generated at diastolic blood pressure (DBP), and PWV2, at systolic blood pressure (SBP) were recorded. Difference between PWV2 and PWV1 (Delta-PWV) normalized by the pulse pressure (PP), corresponding to the aortic stiffening over the cardiac cycle, were compared between mutant and WT mice, as well as the regression slope of PWV as a function of pressure. RESULTS: Delta-PWV/PP was lower in col3KO (pâ=â0.033) and col3KI mice (pâ<â0.001) vs. WT-mice regardless of the pressure level. The slope of PWV1 with DBP increase showed a lower arterial stiffness in mutant mice vs. controls in both models. This difference was amplified when evaluating stiffness at systolic blood pressure levels with PWV2. CONCLUSION: In both vEDS mouse models, aortic stiffening was reduced, mainly driven by a lower stiffness at systolic blood pressure. Defective Col-III may be responsible for this, as it is utilized when pressure rises. These pre-clinical data could explain vascular fragility observed in vEDS patients.
Asunto(s)
Síndrome de Ehlers-Danlos , Hipertensión , Rigidez Vascular , Animales , Presión Sanguínea , Síndrome de Ehlers-Danlos/diagnóstico por imagen , Humanos , Ratones , Fenotipo , Análisis de la Onda del Pulso , UltrasonografíaRESUMEN
A localized left breast ductal invasive triple-negative breast carcinoma (TNBC) was diagnosed in a 44-year-old woman. After surgery, she was treated with chemotherapy and radiation therapy in accordance with national guidelines. At the end of treatment, she had local and metastatic relapse with multiple sub-diaphragmatic lymph nodes.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/terapia , Adulto , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Cetuximab , Cisplatino/uso terapéutico , Ciclofosfamida/uso terapéutico , Docetaxel , Epirrubicina/uso terapéutico , Femenino , Humanos , Ratones , Metástasis de la Neoplasia/genética , Trasplante de Neoplasias , Paclitaxel/uso terapéutico , Taxoides/uso terapéutico , Trasplante Heterólogo , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Cancer is now the most severe complication in the long term in transplant recipients. As most solid-organ or hematopoietic stem-cell transplantations are allogeneic, chimerism studies can be performed on cancers occurring in recipients. We summarize here the different methods used to study chimerism in cancers developing in allogeneic-transplant recipients, analyze their respective advantages and report the main results obtained from these studies. Chimerism analyses of cancers in transplant recipients require methods suited to tissue samples. In the case of gender-mismatched transplantation, the XY chromosomes can be explored using fluorescent in situ hybridization on whole-tissue sections or Y-sequence-specific PCR after the laser microdissection of tumor cells. For cancers occurring after gender-matched transplantation, laser microdissection of tumor cells enables studies of microsatellite markers and high-resolution melting analysis of mitochondrial DNA on genes with marked polymorphism, provided these are different in the donor and the recipient. The results of different studies address the cancers that develop in both recipients and in transplants. The presence of chimeric cells in these two types of cancer implies an exchange of progenitor/stem-cells between transplant and recipient, and the plasticity of these progenitor/stem-cells contributes to epithelial cancers. The presence of chimeric cells in concomitant cancers and preneoplastic lesions implies that the oncogenesis of these cancers progresses through a multistep process.
Asunto(s)
Quimerismo , Neoplasias Glandulares y Epiteliales/genética , Receptores de Trasplantes , ADN Mitocondrial/genética , Femenino , Humanos , Masculino , Repeticiones de Microsatélite/genética , Trasplante HomólogoRESUMEN
The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.
Asunto(s)
Antígenos CD/análisis , Biomarcadores de Tumor/análisis , Células Endoteliales/química , Linfadenopatía Inmunoblástica/metabolismo , Ganglios Linfáticos/química , Linfoma de Células T/química , Microvasos/química , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptores de Superficie Celular/análisis , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/análisis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Endoglina , Células Endoteliales/inmunología , Células Endoteliales/patología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Linfadenopatía Inmunoblástica/genética , Linfadenopatía Inmunoblástica/inmunología , Linfadenopatía Inmunoblástica/mortalidad , Linfadenopatía Inmunoblástica/patología , Linfadenopatía Inmunoblástica/terapia , Estimación de Kaplan-Meier , Captura por Microdisección con Láser , Ganglios Linfáticos/irrigación sanguínea , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Linfoma de Células T/mortalidad , Linfoma de Células T/patología , Linfoma de Células T/terapia , Masculino , Microvasos/inmunología , Microvasos/patología , Persona de Mediana Edad , Análisis Multivariante , Neovascularización Patológica , Paris , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Asunto(s)
Acidosis/metabolismo , Túbulos Renales Distales/metabolismo , Mutación , Seudohipoaldosteronismo/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Acidosis/genética , Acidosis/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Túbulos Renales Distales/patología , Ratones , Ratones Mutantes , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/patología , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Xenopus laevisRESUMEN
The major long-term prognostic factor for breast cancer patients treated by first-line chemotherapy is response to treatment. We have previously shown that complete responses to high doses epirubicin-cyclophosphamide were observed only in human tumors bearing a TP53 mutation. Three xenografted human breast tumors, 2 of them with a TP53 mutation and one of them without, were studied for their immediate response to this drug association. Cell cycle, cellular senescence and cell death were characterized and quantified on tissue section before and after treatment. The TP53 wild-type tumor showed a strong early induction of senescence-like phenotype with overexpression of SA-beta-gal and p21(CIP1). In contrast both TP53 mutated tumors showed no sign of cell cycle arrest or senescence. Conversely, abnormal mitoses strongly increased, only in TP53 mutated tumors. Thus, in these in vivo models, epirubicin-cyclophosphamide treatment induces senescence-like features in TP53 wild-type tumor, likely accounting for cell cycle arrest and subsequent resistance to treatment. Conversely in TP53 mutated tumors, chemotherapy induces mitotic catastrophe and tumor death, accounting for complete response to this association exclusively in patients with TP53 mutated tumors.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Genes p53 , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Mutación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclo Celular , Senescencia Celular , Ciclofosfamida/administración & dosificación , Análisis Mutacional de ADN , ADN de Neoplasias , Epirrubicina/administración & dosificación , Humanos , Ratones , Trasplante de Neoplasias , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: The genesis of all cancers results from an accumulation of mutations, constitutional and/or acquired when induced by external mutagenic factors. High-speed technologies for genome sequencing have completely changed the study of disease genetics, but with limited knowledge of the functional value of most genetic changes. EXPERIMENTAL DESIGN: Here, we proposed an innovative individual approach by studying tissue samples from a young woman with an unusual association of breast cancer, polycythemia vera, and rheumatoid arthritis. We performed genomic analyses for copy number variations and point mutations on laser-microdissected tumor cells from the breast cancer, and on CD34+ cells sorted from bone marrow aspiration, to identify gene abnormalities common to these two types of cell populations. RESULTS: Using ONCOSCAN technology, we identified a constitutional pR988C, c2962C>T mutation of MET. Using CRISPR-Cas9 technology, we established pR988C MET-mutated transgenic mice, which reproduced the autoimmune diseases and myeloproliferation found in our index-case; one of the transgenic mice spontaneously developed a skin squamous cell carcinoma. We also showed that additional mutagenic factors were required to induce cancers, including skin squamous cell carcinoma and thyroid cancer. Using an anti-MET drug, cabozantinib, we demonstrated for the first time the functional role of this mutation in the maintenance of myeloproliferation and rheumatoid arthritis, and in cancer genesis. CONCLUSIONS: Our study opens a considerable field of application in the domain of constitutional genetics, to establish genetic links between cancers and other very different severe diseases.
Asunto(s)
Anilidas/farmacología , Artritis Reumatoide/patología , Enfermedades Autoinmunes/patología , Neoplasias de la Mama/patología , Mutación , Trastornos Mieloproliferativos/patología , Proteínas Proto-Oncogénicas c-met/genética , Piridinas/farmacología , Adulto , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Enfermedad Crónica , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/genética , Policitemia Vera/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Renal Cell Carcinomas (RCCs) are heterogeneous tumors with late acquisition of TP53 abnormalities during their evolution. They harbor TP53 abnormalities in their metastases. We aimed to study TP53 gene alterations in tissue samples from primary and metastatic RCCs in 36 patients followed up over a median of 4.2 years, and in xenografted issued from primary RCCs. In 36 primary RCCs systematically xenografted in mice, and in biopsies of metastases performed whenever possible during patient follow-up, we studied p53-expressing tumor cells and TP53 gene abnormalities.We identified TP53 gene alterations in primary tumors, metastases and xenografts. Quantification of tumors cells with TP53 gene alterations showed a significant increase in the metastases compared to the primary RCCs, and, strikingly, the xenografts were similar to the metastases and not to the primary RCCs from which they were derived.Using laser-microdissection of p53-expressing tumor cells, we identified TP53-mutated tumor cells in the xenografts derived from the primary RCC, and in a lung metastasis later developed in one patient. The mutation enabled us to track back their origin to a minority sub-clone in the primary heterogeneous RCC. Combining in situ and molecular analyses, we demonstrated a clonal expansion in a living patient with metastatic RCC.
Asunto(s)
Carcinoma de Células Renales/genética , Genes p53 , Neoplasias Renales/genética , Mutación , Anciano , Anciano de 80 o más Años , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Femenino , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Developing strategies to overcome resistance to sunitinib is a major challenge in human renal cell carcinoma (RCC). We hypothesized that sunitinib-induced tumor necrosis-associated hypoxia could interact with renal cancer stem cells in patients with metastatic RCC. EXPERIMENTAL DESIGN: We studied tissue samples from 7 patients with primary metastatic RCC, before and after sunitinib treatment, and from six xenograft models derived from human RCC. Two xenograft models were responders to sunitinib, the four others were nonresponders. CD133/CXCR4-coexpressing cells derived from the two responder xenograft models were used for in vitro studies. RESULTS: In the seven primary RCCs, we identified a significantly larger number of CD133/CXCR4-coexpressing cells in perinecrotic versus perivascular areas. Their numbers also significantly increased after treatment, in perinecrotic areas. We reproduced these clinical and pathologic results in all six RCC xenograft models with again a preferential perinecrotic distribution of CD133-expressing cells. Necrosis occurred at day 7 in the two responder models treated with sunitinib, whereas it occurred at day 21 in the untreated controls and in the four nonresponder models. Strikingly, when we studied the six RCC xenograft models at the time necrosis, whether spontaneous or sunitinib-induced, occurred, necrosis area correlated with stem-cell number in all 120 xenografted RCCs. When studied under experimental hypoxia, the number of CD133/CXCR4-coexpressing cells and their tumorigenic potency increased whereas their sensitivity to sunitinib decreased. CONCLUSIONS: In human RCC, sunitinib was able to generate resistance to its own therapeutic effect via induced hypoxia in perinecrotic areas where cancer stem cells were found in increased numbers.
Asunto(s)
Carcinoma de Células Renales/patología , Resistencia a Antineoplásicos/fisiología , Neoplasias Renales/patología , Células Madre Neoplásicas/patología , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Humanos , Indoles/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Ratones , Ratones Desnudos , Necrosis , Células Madre Neoplásicas/efectos de los fármacos , Pirroles/uso terapéutico , Sunitinib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Renal-cell carcinomas (RCC) are often resistant to conventional cytotoxic agents. Xenograft models are used for in vivo preclinical studies and drug development. The validity of these studies is highly dependent on the phenotypic and genotypic stability of the models. Here we assessed the stability of six aggressive human RCC xenografted in nude/NMRI mice. We compared the initial samples (P0), first (P1) and fifth (P5) passages for the following criteria: histopathology, immunohistochemistry for CK7, CD10, vimentin and p53, DNA allelic profiles using 10 microsatellites and CGH-array. Next we evaluated the response to sunitinib in primary RCC and corresponding xenografted RCC. We observed a good overall stability between primary RCC and corresponding xenografted RCC at P1 and P5 regarding histopathology and immunohistochemistry except for cytokeratin 7 (one case) and p53 (one case) expression. Out of 44 groups with fully available microsatellite data (at P0, P1 and P5), 66% (29 groups) showed no difference from P0 to P5 while 34% (15 groups) showed new or lost alleles. Using CGH-array, overall genomic alterations at P5 were not different from those of initial RCC. The xenografted RCC had identical response to sunitinib therapy compared to the initial human RCC from which they derive. These xenograft models of aggressive human RCC are clinically relevant, showing a good histological and molecular stability and are suitable for studies of basic biology and response to therapy.
Asunto(s)
Carcinoma de Células Renales/patología , Modelos Animales de Enfermedad , Neoplasias Renales/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Anciano , Animales , Carcinoma de Células Renales/genética , Femenino , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Masculino , Ratones , Ratones Desnudos , Persona de Mediana EdadRESUMEN
PURPOSE: Sunitinib is an inhibitor of tyrosine-kinase receptors, and no biomarker predictive of sunitinib response is available. The purpose of this preclinical study was to show whether sunitinib molecular targets could be used as biomarkers to assess tumor response to sunitinib in human cancer cell line xenografts of three different tumor types. METHODS: Using mice xenografted with liver, breast and renal carcinoma cell lines, we sequentially analyzed the effect of 7-day sunitinib treatment on tumor and vascular compartments. RESULTS: In all xenografts, microvessel damage occurred from Day 1. Tumor damage also occurred in liver, breast, but not in renal xenografts. Using specific human and mouse probes for genes encoding sunitinib targets, we showed a significant relation between apoptotic tumor cell numbers and human PDGFRΒ and RET mRNA expression in liver cancer and to human VEGFR2 expression in breast cancer xenografts. In contrast, in renal cancer xenografts, vascular effect evaluated by measuring endothelial cell apoptosis was related to mouse Vegfr1, Vegfr2 and Vegfa-164 expression. CONCLUSION: This study identifies sunitinib vascular and tumor effects according to different tumor types and shows that sunitinib molecular targets used as biomarkers enable assessment of therapeutic response.