RESUMO
Development of drug responsive biomarkers from pre-clinical data is a critical step in drug discovery, as it enables patient stratification in clinical trial design. Such translational biomarkers can be validated in early clinical trial phases and utilized as a patient inclusion parameter in later stage trials. Here we present a study on building accurate and selective drug sensitivity models for Erlotinib or Sorafenib from pre-clinical in vitro data, followed by validation of individual models on corresponding treatment arms from patient data generated in the BATTLE clinical trial. A Partial Least Squares Regression (PLSR) based modeling framework was designed and implemented, using a special splitting strategy and canonical pathways to capture robust information for model building. Erlotinib and Sorafenib predictive models could be used to identify a sub-group of patients that respond better to the corresponding treatment, and these models are specific to the corresponding drugs. The model derived signature genes reflect each drug's known mechanism of action. Also, the models predict each drug's potential cancer indications consistent with clinical trial results from a selection of globally normalized GEO expression datasets.
Assuntos
Antineoplásicos/farmacologia , Cloridrato de Erlotinib/farmacologia , Regulação Neoplásica da Expressão Gênica , Modelos Estatísticos , Neoplasias/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Ensaios Clínicos Fase II como Assunto , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Redes Reguladoras de Genes , Humanos , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Niacinamida/farmacologia , Transdução de Sinais , Sorafenibe , Análise de SobrevidaRESUMO
The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.
Assuntos
Adaptação Fisiológica , Cílios/fisiologia , Transdução de Sinais , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos , Microscopia de FluorescênciaRESUMO
Mechanical stress is known to modulate postnatal skeletal growth and development. However, the mechanisms underlying the mechanotransduction are not fully understood. Polycystin-1 (PC1) is a promising candidate among proteins that may play a role in the process as it has been shown to function as a flow sensor in renal epithelium and it is known to be important for skeletal development. To investigate whether PC1 is involved in mechanotransduction in skeletal tissues, mice with a conditional deficiency for PC1 in neural crest cells, osteoblasts or chondrocytes were subjected to midpalatal suture expansion. Dynamic bone labeling revealed that new bone formation in response to expansion was significantly reduced in Wnt1Cre;Pkd1 mice, as the suture area containing new bone was 14.0+/-3.4% in mutant mice versus 65.0+/-3.8% in control mice at 2 weeks (p<0.001). In contrast, stress-induced new bone formation was not affected in OsxCre;Pkd1 mice. The increase in cell proliferation and differentiation into osteoblasts, seen in wild-type mice 1 day after force delivery, was not observed until 14 days in Wnt1Cre;Pkd1 mice. TUNEL labeling showed a significant increase in apoptotic suture cells at days 1 and 3 (from 7.0+/-0.5% to 13.5+/-1.4% at day 1 and from 4.6+/-1.1% to 10.5+/-1.7% at day 3, p<0.05). Abnormal ossification of nasal cartilage of Wnt1Cre;Pkd1 mice was accelerated upon suture expansion. Such ossification was also observed, but to a lesser extent in Col2a1-ERCre;Pkd1 mice. Transcript levels of Runx2 and MMP13 were significantly increased in the nasal cartilage of Wnt1Cre;Pkd1 mice compared to controls (p<0.05 and p<0.001, respectively), and in mutant mice with expansion versus without expansion (p<0.05 and p<0.001, respectively). Lack of PC1 in chondroprogenitor cells also resulted in increased cell apoptosis and an altered arrangement of chondrocytes in nasal cartilage. These results indicate that PC1 plays a critical role in the response of osteochondroprogenitor cells to the mechanical tissue stress induced by midpalatal suture expansion. They also suggest that the combination of an in vivo mechanical model, such as midpalatal suture expansion, with conditional deficiency for proteins that play a role in mechanotransduction, represents a powerful experimental strategy to explore underlying mechanisms.
Assuntos
Técnica de Expansão Palatina , Palato/cirurgia , Canais de Cátion TRPP/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Colágeno Tipo I/metabolismo , Suturas Cranianas/fisiologia , Suturas Cranianas/cirurgia , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Cartilagens Nasais/anatomia & histologia , Cartilagens Nasais/metabolismo , Periósteo/citologia , Reação em Cadeia da Polimerase , Estresse Mecânico , Canais de Cátion TRPP/genéticaRESUMO
In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies. Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.
Assuntos
Suturas Cranianas/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPP/metabolismo , Animais , Western Blotting , Bromodesoxiuridina , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Crista Neural/fisiologiaRESUMO
Here we report on the severe defects in renal epithelium induced by the transgenic Col2-Cre line used previously for skeletal tissue-specific gene targeting. We demonstrate that conditional ablation of the Kif3a or Pkd1 genes encoding primary cilium/intraflagellar transport-associated proteins using type II collagen-specific Cre transgenic strain results in a severe form of polycystic kidney disease in mice. We detect Col2-Cre recombinase expression in kidney epithelium, which reflects expression of the endogenous Col1alpha(II) gene in the embryonic renal tubules. We determine the exon 2-containing splice variant of the Col1alpha(II) gene as a major transcript expressed in kidney. Furthermore, the confocal immunocytochemical analysis demonstrates deposition of the type II collagen within the mesenchymal-epithelial renal tissue interfaces and its co-localization with the basement membrane marker collagen IV during embryonic kidney morphogenesis.
Assuntos
Colágeno Tipo II/metabolismo , Epitélio/metabolismo , Integrases/metabolismo , Rim/anatomia & histologia , Doenças Renais Policísticas , Processamento Alternativo , Animais , Colágeno Tipo II/genética , Integrases/genética , Rim/metabolismo , Rim/patologia , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Transgênicos , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Hedgehog signaling plays an essential role in patterning of the vertebrate skeleton. Here we demonstrate that conditional inactivation of the Kif3a subunit of the kinesin-2 intraflagellar transport motor in mesenchymal skeletal progenitor cells results in severe patterning defects in the craniofacial area, the formation of split sternum and the development of polydactyly. These deformities are reminiscent of those previously described in mice with deregulated hedgehog signaling. We show that in Kif3a-deficient mesenchymal tissues both the repressor function of Gli3 transcription factor and the activation of the Shh transcriptional targets Ptch and Gli1 are compromised. Quantitative analysis of gene expression demonstrates that the Gli1 transcript level is dramatically reduced, whereas Gli3 expression is not significantly affected by kinesin-2 depletion. However, the motor appears to be required for the efficient cleavage of the full-length Gli3 transcription factor into a repressor form.