RESUMEN
Cis-diamminedichloroplatinum(II) (cisplatin)-induced renal proximal tubular apoptosis is known to be preceded by actin cytoskeleton reorganization, in conjunction with disruption of cell-matrix and cell-cell adhesion. In the present study, we show that the proinflammatory cytokine tumor necrosis factor α (TNF-α) aggravated these cisplatin-induced F-actin and cell adhesion changes, which was associated with enhanced cisplatin-induced apoptosis of immortalized proximal tubular epithelial cells. TNF-α-induced RelB expression and lentiviral small hairpin RNA (shRNA)-mediated knockdown of RelB, but not other nuclear factor κB members, abrogated the synergistic apoptosis observed with cisplatin/TNF-α treatment to the level of cisplatin-induced apoptosis. This protective effect was associated with increased stress fiber formation, cell-matrix, and cell-cell adhesion in the shRNARelB (shRelB) cells during cisplatin/TNF-α treatment, mimicking an epithelial-to-mesenchymal phenotypic switch. Indeed, gene array analysis revealed that knockdown of RelB was associated with upregulation of several actin regulatory genes, including Snai2 and the Rho GTPase proteins Rhophilin and Rho guanine nucleotide exchange factor 3 (ARHGEF3). Pharmacological inhibition of Rho kinase signaling re-established the synergistic apoptosis induced by combined cisplatin/TNF-α treatment of shRelB cells. In conclusion, our study shows for the first time that RelB is required for the cisplatin/TNF-α-induced cytoskeletal reorganization and apoptosis in renal cells by controlling a Rho kinase-dependent signaling network.
Asunto(s)
Apoptosis/fisiología , Cisplatino/farmacología , Transición Epitelial-Mesenquimal/fisiología , Túbulos Renales Proximales/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Células Cultivadas , Sinergismo Farmacológico , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Ratones , FN-kappa B/genética , Transducción de Señal , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/genética , Fibras de Estrés/metabolismo , Factor de Transcripción ReIB/genética , Regulación hacia Arriba/efectos de los fármacos , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signaling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium Wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1lox,lox mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function.
Asunto(s)
Evaluación Preclínica de Medicamentos , Triterpenos Pentacíclicos/uso terapéutico , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Animales , Quistes/patología , Quistes/fisiopatología , Pruebas de Función Renal , Ratones , Triterpenos Pentacíclicos/farmacología , Fenotipo , Enfermedades Renales Poliquísticas/patología , Enfermedades Renales Poliquísticas/fisiopatología , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.
Asunto(s)
Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Transcriptoma , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica/diagnóstico , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Pronóstico , Análisis de SupervivenciaRESUMEN
Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.
Asunto(s)
Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Línea Celular Tumoral , Estudios de Cohortes , Conjuntos de Datos como Asunto , Supervivencia sin Enfermedad , Matriz Extracelular/metabolismo , Femenino , Adhesiones Focales/genética , Humanos , Microscopía Intravital , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Empalme del ARN/genética , ARN Interferente Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Transducción de Señal/genética , Análisis de Supervivencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1-/- kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Terapia Molecular Dirigida , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Línea Celular , Colforsina , Hidrogel de Polietilenoglicol-Dimetacrilato , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/patología , Ratones , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Enfermedades Renales Poliquísticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Adhesiones Focales/genética , Adhesiones Focales/patología , Humanos , Células MCF-7 , Proteínas de Neoplasias/genéticaRESUMEN
Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.