RESUMO
A growing number of agents targeting ligand-induced Wnt/ß-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/ß-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/ß-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/ß-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of ß-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas Oncogênicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina , Aciltransferases , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Proteínas de Ligação a DNA/genética , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Ubiquitina-Proteína Ligases , Proteínas Wnt/genética , Via de Sinalização WntRESUMO
Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible ß-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/ß-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon ß-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/ß-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear ß-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/ß-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/ß-catenin pathway.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Genes APC , Mutação , Via de Sinalização Wnt , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Interferente Pequeno/genética , Transdução de Sinais , Transplante Heterólogo , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFR(vIII) do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.
Assuntos
Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Genes Supressores de Tumor , Glioblastoma/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/metabolismoRESUMO
WNT974 is a potent, selective, and orally bioavailable first-in-class inhibitor of Porcupine, a membrane-bound O-acyltransferase required for Wnt secretion, currently under clinical development in oncology. A phase I clinical trial is being conducted in patients with advanced solid tumors. During the dose-escalation part, various dosing regimens, including once or twice daily continuous and intermittent dosing at a dose range of 5-45 mg WNT974 were studied, however, the protocol-defined maximum tolerated dose (MTD) was not established based on dose-limiting toxicity. To assist in the selection of the recommended dose for expansion (RDE), a model-based approach was utilized. It integrated population pharmacokinetic (PK) modeling and exposure-response analyses of a target-inhibition biomarker, skin AXIN2 mRNA expression, and the occurrence of the adverse event, dysgeusia. The target exposure range of WNT974 that would provide a balance between target inhibition and tolerability was estimated based on exposure-response analyses. The dose that was predicted to yield an exposure within the target exposure range was selected as RDE. This model-based approach integrated PK, biomarker, and safety data to determine the RDE and represented an alternative as opposed to the conventional MTD approach for selecting an optimal biological dose. The strategy can be broadly applied to select doses in early oncology trials and inform translational clinical oncology drug development.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Neoplasias/genética , Pirazinas/uso terapêutico , Piridinas/uso terapêutico , Resultado do TratamentoRESUMO
Genetically engineered, Cre/LoxP-conditional mouse models of cancer are designed to investigate the genetic contributors of tumorigenesis and are well suited to assess therapeutic treatment responses. The capacity to serially visualize tumor burden in a noninvasive fashion would greatly strengthen their applications. We report the generation of a bioluminescent reporter strain that allows monitoring of tumor development in preexisting conditional mouse tumor models. We demonstrate that, in a Cre-dependent glioblastoma multiforme model, tumor initiation and progression is readily monitored over time and that luminescent output is related to tumor volume. Our results show that this reporter strain may be combined with various Cre/loxP mouse tumor models to allow for noninvasive longitudinal monitoring of tumor growth and therapeutic response in vivo.
Assuntos
Transformação Celular Neoplásica/patologia , Genes Reporter , Glioblastoma/patologia , Integrases/genética , Luciferases/análise , Medições Luminescentes/métodos , Animais , Transformação Celular Neoplásica/química , Feminino , Glioblastoma/química , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Glioblastoma multiforme (GBM) is an aggressive primary brain cancer that includes focal amplification of PDGFRα and for which there are no effective therapies. Herein, we report the development of a genetically engineered mouse model of GBM based on autocrine, chronic stimulation of overexpressed PDGFRα, and the analysis of GBM signaling pathways using proteomics. We discover the tubulin-binding protein Stathmin1 (STMN1) as a PDGFRα phospho-regulated target, and that this mis-regulation confers sensitivity to vinblastine (VB) cytotoxicity. Treatment of PDGFRα-positive mouse and a patient-derived xenograft (PDX) GBMs with VB in mice prolongs survival and is dependent on STMN1. Our work reveals a previously unconsidered link between PDGFRα activity and STMN1, and highlight an STMN1-dependent cytotoxic effect of VB in GBM.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Estatmina/metabolismo , Vimblastina/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Sobrevivência Celular , Células Cultivadas , Biologia Computacional , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Transplante de Neoplasias , Fosforilação , Proteômica , Transdução de SinaisRESUMO
The initial segment (IS) of the epididymis plays an essential role in male fertility. The IS epithelium is undifferentiated and nonfunctional at birth. Prior to puberty, the epithelium undergoes differentiation that leads to the formation of a fully functional organ. However, the mechanistic details of this program are not well understood. To explore this further, we used genetic engineering to create a kinase dead allele of the ROS1 receptor tyrosine kinase in mice and studied the effects of ROS1 tyrosine kinase activity on the differentiation of the IS epithelium. We show that the expression and activation of ROS1 coincides with the onset of differentiation and is exclusively located in the IS of the maturing and adult mouse epididymides. Here we demonstrate that the differentiation of the IS is dependent on the kinase activity of ROS1 and its downstream effector MEK1/2-ERK1/2 signaling axis. Using genetic engineering, we show that germ line ablation of ROS1 kinase activity leads to a failure of the IS epithelium to differentiate, and as a consequence sperm maturation and infertility were dramatically perturbed. Pharmacological inhibition of ROS1 kinase activity in the developing epididymis, however, only delayed differentiation transiently and did not result in infertility. Our results demonstrate that ROS1 kinase activity and the ensuing MEK1/2-ERK1/2 signaling are necessary for the postnatal development of the IS epithelium and that a sustained ablation of ROS1 kinase activity within the critical window of terminal differentiation abrogate the function of the epididymis and leads to sterility.
Assuntos
Diferenciação Celular , Epididimo/citologia , Epididimo/enzimologia , Células Epiteliais/enzimologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Epididimo/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Masculino , Camundongos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/metabolismoRESUMO
Glioblastoma multiforme (GBM) is characterized by overexpression of epidermal growth factor receptor (EGFR) and loss of the tumor suppressors Ink4a/Arf. Efforts at modeling GBM using wild-type EGFR in mice have proven unsuccessful. Here, we present a unique mouse model of wild-type EGFR-driven gliomagenesis. We used a combination of somatic conditional overexpression and ligand-mediated chronic activation of EGFR in cooperation with Ink4a/Arf loss in the central nervous system of adult mice to generate tumors with the histopathologic and molecular characteristics of human GBMs. Sustained, ligand-mediated activation of EGFR was necessary for gliomagenesis, functionally substantiating the clinical observation that EGFR-positive GBMs from patients express EGFR ligands. To gain a better understanding of the clinically disappointing EGFR-targeted therapies for GBM, we investigated the molecular responses to EGFR tyrosine kinase inhibitor (TKI) treatment in this model. Gefitinib treatment of primary GBM cells resulted in a robust apoptotic response, partially conveyed by mitogen-activated protein kinase (MAPK) signaling attenuation and accompanied by BIM(EL) expression. In human GBMs, loss-of-function mutations in the tumor suppressor PTEN are a common occurrence. Elimination of PTEN expression in GBM cells posttumor formation did not confer resistance to TKI treatment, showing that PTEN status in our model is not predictive. Together, these findings offer important mechanistic insights into the genetic determinants of EGFR gliomagenesis and sensitivity to TKIs and provide a robust discovery platform to better understand the molecular events that are associated with predictive markers of TKI therapy.
Assuntos
Neoplasias Encefálicas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Ativação Enzimática , Receptores ErbB/genética , Gefitinibe , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Ligantes , Proteínas de Membrana/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Glioblastoma multiforme (GBM) has an abysmal prognosis. We now know that the epidermal growth factor receptor (EGFR) signaling pathway and the loss of function of the tumor suppressor genes p16Ink4a/p19ARF and PTEN play a crucial role in GBM pathogenesis: initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. We have recently shown that this genetic combination is sufficient to promote the development of GBM in adult mice. Therapeutic agents raised against single targets of the EGFR signaling pathway have proven rather inefficient in GBM therapy, showing the need for combinatorial therapeutic approaches. An effective strategy for concurrent disruption of multiple signaling pathways is via the inhibition of the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 inhibition leads to the degradation of so-called client proteins, many of which are key effectors of GBM pathogenesis. NXD30001 is a novel second generation Hsp90 inhibitor that shows improved pharmacokinetic parameters. Here we show that NXD30001 is a potent inhibitor of GBM cell growth in vitro consistent with its capacity to inhibit several key targets and regulators of GBM biology. We also show the efficacy of NXD30001 in vivo in an EGFR-driven genetically engineered mouse model of GBM. Our findings establish that the Hsp90 inhibitor NXD30001 is a therapeutically multivalent molecule, whose actions strike GBM at the core of its drivers of tumorigenesis and represent a compelling rationale for its use in GBM treatment.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactonas/farmacologia , Oximas/farmacologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactonas/farmacocinética , Masculino , Camundongos , Camundongos Transgênicos , Oximas/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The proto-oncogene tyrosine kinase c-ROS is an orphan receptor whose normal expression pattern is tightly spatio-temporally restricted during development. In glioma, c-ROS mRNA expression is frequently ectopically up-regulated. In this study, we determined by immunohistochemical means that c-ROS receptor protein is present in 25% of low-grade and 30% of malignant glioma tumor samples from tissue microarrays. We then explored the molecular basis for the up-regulation of c-ROS expression in these tumors. We identified and characterized the c-ROS gene promoter region and report that the ectopic expression of c-ROS in tumors is tied to hypomethylation of a CpG island in the c-ROS promoter. Bisulfite sequencing analysis in glioma tumor samples revealed that demethylation of the CpG island (-384 to -132 bp) correlated with c-ROS expression. Moreover, c-ROS expression could be activated by treatment of c-ROS-negative cells with the demethylating agent 5-aza-2'-deoxycytidine. These results establish a strong link between c-ROS promoter demethylation and gain of c-ROS expression and function in glioma. Our data suggest that epigenetic activation of c-ROS represents an important oncogenic mechanism for glioma initiation and progression and suggest that cautionary measures in the clinical use of 5-aza-dC for the treatment of glioma be taken into consideration. [Cancer Res 2009;69(6):2180-4].