RESUMO
The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.
Assuntos
Anticorpos/farmacologia , Anticorpos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores Notch/antagonistas & inibidores , Inibidores da Angiogênese/imunologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos/efeitos adversos , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Biblioteca de Peptídeos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/imunologia , Receptor Notch2/antagonistas & inibidores , Receptor Notch2/imunologia , Receptores Notch/genética , Receptores Notch/imunologia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Olho/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Olho/irrigação sanguínea , Feminino , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Pressão Intraocular , Masculino , Melanoma , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Especificidade de Órgãos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Retina/anatomia & histologia , Retina/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Capeamento Imunológico/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/imunologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/imunologia , UbiquitinaçãoRESUMO
Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and ß-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce ß-cell regeneration in humans. Here, we discover the ß-cell regenerative effects of glucagon receptor antibody (anti-GcgR). Treatment with anti-GcgR in mouse models of ß-cell deficiency leads to reversal of hyperglycemia, increase in plasma insulin levels, and restoration of ß-cell mass. We demonstrate that both ß-cell proliferation and α- to ß-cell transdifferentiation contribute to anti-GcgR-induced ß-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from ß-cell regeneration after antibody clearance from the body. Importantly, we are able to show that anti-GcgR-induced ß-cell regeneration is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases ß-cell mass in a mouse model of autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Glucagon , Hiperglicemia , Células Secretoras de Insulina , Animais , Modelos Animais de Doenças , Glucagon , Hiperglicemia/tratamento farmacológico , Camundongos , Receptores de GlucagonRESUMO
Krüppel-like factor 6 (Klf6) belongs to a family of zinc finger transcription factors known to play a role in development and tumor suppression. Although Klf6 is highly mutated in prostate cancer, its function in prostate development is unknown. We have generated a prostate-specific Klf6-deficient mouse model and report here a novel role for Klf6 in the regulation of prostate branching morphogenesis. Importantly, our study reveals a novel relationship between Klf6 and the Shh pathway. Klf6-deficiency leads to elevated levels of hedgehog pathway components (Shh, Ptc, and Gli) and loss of their localized expression, which in turn causes impaired lateral branching.
Assuntos
Inativação Gênica , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Morfogênese , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Animais , Proteína Morfogenética Óssea 4/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Músculo Liso/crescimento & desenvolvimento , Especificidade de Órgãos , Próstata/anormalidades , Próstata/patologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima/genética , beta-Galactosidase/metabolismoRESUMO
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.
Assuntos
Anticorpos Monoclonais/farmacologia , Glucose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteassoma , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.
Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Musculares/química , Proteínas Musculares/fisiologia , Animais , Apoptose , Western Blotting , Proliferação de Células , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Glicerol/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Fígado/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Microscopia Eletrônica , Proteínas Mitocondriais/fisiologia , Plasmídeos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retroviridae/genética , Relação Estrutura-Atividade , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.
Assuntos
Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas B-raf/fisiologia , Neoplasias Cutâneas/patologia , Animais , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Melanoma/genética , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Interferência de RNA , Transdução de Sinais , Neoplasias Cutâneas/genética , Transplante HeterólogoRESUMO
Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Deleção de Genes , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento/genética , Processamento Alternativo , Animais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação para Baixo , Ativação Enzimática , Éxons , Feminino , Humanos , Íntrons , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-met , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
BACKGROUND: Immuno-oncology and cancer immunotherapies are areas of intense research. The numbers and locations of CD8+ tumor-infiltrating lymphocytes (TILs) are important measures of the immune response to cancer with prognostic, pharmacodynamic, and predictive potential. We describe the development, validation, and application of advanced image analysis methods to characterize multiple immunohistochemistry-derived CD8 parameters in clinical and nonclinical tumor tissues. METHODS: Commercial resection tumors from nine cancer types, and paired screening/on-drug biopsies of non-small-cell lung carcinoma (NSCLC) patients enrolled in a phase 1/2 clinical trial investigating the PD-L1 antibody therapy durvalumab (NCT01693562), were immunostained for CD8. Additional NCT01693562 samples were immunostained with a CD8/PD-L1 dual immunohistochemistry assay. Whole-slide scanning was performed, tumor regions were annotated by a pathologist, and images were analyzed with customized algorithms using Definiens Developer XD software. Validation of image analysis data used cell-by-cell comparison to pathologist scoring across a range of CD8+ TIL densities of all nine cancers, relying primarily on 95% confidence in having at least moderate agreement regarding Lin concordance correlation coefficient (CCC = 0.88-0.99, CCC_lower = 0.65-0.96). RESULTS: We found substantial variability in CD8+ TILs between individual patients and across the nine types of human cancer. Diffuse large B-cell lymphoma had several-fold more CD8+ TILs than some other cancers. TIL densities were significantly higher in the invasive margin versus tumor center for carcinomas of head and neck, kidney and pancreas, and NSCLC; the reverse was true only for prostate cancer. In paired patient biopsies, there were significantly increased CD8+ TILs 6 weeks after onset of durvalumab therapy (mean of 365 cells/mm2 over baseline; P = 0.009), consistent with immune activation. Image analysis accurately enumerated CD8+ TILs in PD-L1+ regions of lung tumors using the dual assay and also measured elongate CD8+ lymphocytes which constituted a fraction of overall TILs. CONCLUSIONS: Validated image analysis accurately enumerates CD8+ TILs, permitting comparisons of CD8 parameters among tumor regions, individual patients, and cancer types. It also enables the more complex digital solutions needed to better understand cancer immunity, like analysis of multiplex immunohistochemistry and spatial evaluation of the various components comprising the tumor microenvironment. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Processamento de Imagem Assistida por Computador , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors.
RESUMO
Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G2-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4-8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G2-checkpoints in p53 proficient cells.
Assuntos
Caspases/metabolismo , Cromossomos Humanos/genética , Fragmentação do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Carbolinas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ativação Enzimática , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Rad51 Recombinase/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , GencitabinaRESUMO
PURPOSE: In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. EXPERIMENTAL DESIGN: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. RESULTS: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment. CONCLUSIONS: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/biossíntese , Quinazolinas/administração & dosagem , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Intervalo Livre de Doença , Receptores ErbB/biossíntese , Cloridrato de Erlotinib , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/biossínteseRESUMO
The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.
Assuntos
Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Subunidades Proteicas/química , Animais , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Terminologia como AssuntoRESUMO
PURPOSE: Increased hepatocyte growth factor/MET signaling is associated with poor prognosis and acquired resistance to epidermal growth factor receptor (EGFR) -targeted drugs in patients with non-small-cell lung cancer (NSCLC). We investigated whether dual inhibition of MET/EGFR results in clinical benefit in patients with NSCLC. PATIENTS AND METHODS: Patients with recurrent NSCLC were randomly assigned at a ratio of one to one to receive onartuzumab plus erlotinib or placebo plus erlotinib; crossover was allowed at progression. Tumor tissue was required to assess MET status by immunohistochemistry (IHC). Coprimary end points were progression-free survival (PFS) in the intent-to-treat (ITT) and MET-positive (MET IHC diagnostic positive) populations; additional end points included overall survival (OS), objective response rate, and safety. RESULTS: There was no improvement in PFS or OS in the ITT population (n = 137; PFS hazard ratio [HR], 1.09; P = .69; OS HR, 0.80; P = .34). MET-positive patients (n = 66) treated with erlotinib plus onartuzumab showed improvement in both PFS (HR, .53; P = .04) and OS (HR, .37; P = .002). Conversely, clinical outcomes were worse in MET-negative patients treated with onartuzumab plus erlotinib (n = 62; PFS HR, 1.82; P = .05; OS HR, 1.78; P = .16). MET-positive control patients had worse outcomes versus MET-negative control patients (n = 62; PFS HR, 1.71; P = .06; OS HR, 2.61; P = .004). Incidence of peripheral edema was increased in onartuzumab-treated patients. CONCLUSION: Onartuzumab plus erlotinib was associated with improved PFS and OS in the MET-positive population. These results combined with the worse outcomes observed in MET-negative patients treated with onartuzumab highlight the importance of diagnostic testing in drug development.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Estudos Cross-Over , Intervalo Livre de Doença , Método Duplo-Cego , Cloridrato de Erlotinib , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-met/biossíntese , Quinazolinas/administração & dosagem , Quinazolinas/efeitos adversos , Resultado do TratamentoRESUMO
To examine the potential of combining Bcl-2 family inhibitors with chemotherapy in ovarian cancer, we evaluated a panel of 27 ovarian cancer cell lines for response to the combination of navitoclax (formerly ABT-263) and paclitaxel or gemcitabine. The majority of cell lines exhibited a greater than additive response to either combination, as determined by the Bliss independence model, and more than 50% of the ovarian cell lines exhibited strong synergy for the navitoclax/paclitaxel combination. To identify biomarkers for tumors likely to respond to this combination, we evaluated the protein levels of intrinsic apoptosis pathway components. Bcl-x(L) seems necessary, but not sufficient, for navitoclax/paclitaxel synergy in vitro, suggesting that exclusion of patients whose tumors have low or undetectable Bcl-x(L) would enrich for patients responsive to the combination. We evaluated Bcl-x(L) levels in ovarian cancer tumor tissue from 40 patients (20 taxane responsive and 20 with poor response to taxane) and found that patients with high Bcl-x(L) were less sensitive to taxane treatment (10 of 12) Bcl-x(L) positive patients, P = 0.014). These data support the use of navitoclax in combination with taxane-based therapy in ovarian cancer patients with high levels of Bcl-x(L).
Assuntos
Compostos de Anilina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Sulfonamidas/farmacologia , Proteína bcl-X/metabolismo , Compostos de Anilina/administração & dosagem , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Paclitaxel/farmacologia , Sulfonamidas/administração & dosagem , GencitabinaRESUMO
Breast cancer has been redefined into three clinically relevant subclasses: (i) estrogen/progesterone receptor positive (ER+/PR+), (ii) HER2/ERRB2 positive, and (iii) those lacking expression of all three markers (triple negative or basal-like). While targeted therapies for ER+/PR+ and HER2+ tumors have revolutionized patient treatment and increased lifespan, an urgent need exists for identifying novel targets for triple-negative breast cancers. Here, we used integrative genomic analysis to identify candidate oncogenes in triple-negative breast tumors and assess their function through loss of function screening. Using this approach, we identify lactate dehydrogenase B (LDHB), a component of glycolytic metabolism, as an essential gene in triple-negative breast cancer. Loss of LDHB abrogated cell proliferation in vitro and arrested tumor growth in fully formed tumors in vivo. We find that LDHB and other related glycolysis genes are specifically upregulated in basal-like/triple-negative breast cancers as compared with other subtypes, suggesting that these tumors are distinctly glycolytic. Consistent with this, triple-negative breast cancer cell lines were more dependent on glycolysis for growth than luminal cell lines. Finally, we find that patients with breast cancer and high LDHB expression in their tumors had a poor clinical outcome. While previous studies have focused on the ubiquitous role of LDHA in tumor metabolism and growth, our data reveal that LDHB is upregulated and required only in certain cancer genotypes. These findings suggest that targeting LDHB or other components of lactate metabolism would be of clinical benefit in triple-negative breast cancer.
Assuntos
Neoplasias da Mama/genética , Lactato Desidrogenases/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lactato Desidrogenases/biossíntese , Células MCF-7 , Camundongos , Camundongos Nus , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transplante HeterólogoRESUMO
The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non-small cell lung cancer and is mutually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non-small cell lung cancer (NSCLC). We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mechanism of EML4-ALK inhibition by a small molecule inhibitor.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/fisiologia , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para CimaRESUMO
PURPOSE: To explore the potential of navitoclax in combination with taxane-based chemotherapy in the treatment of non-small cell lung cancer (NSCLC) by defining mechanism of synergy and identifying correlative biomarkers. EXPERIMENTAL DESIGN: We treated a panel of NSCLC lines with a dose matrix of paclitaxel and navitoclax (formerly ABT-263), an inhibitor of Bcl-2, Bcl-x(L), and Bcl-w (1), and evaluated synergy. We next used time-lapse microscopy to explore mechanism of synergy. Finally, we developed an immunohistochemical assay and assessed prevalence of Bcl-x(L) in NSCLC tumor tissues. RESULTS: All cell lines exhibit greater than additive response to the combination of navitoclax and a taxane. These results were extended to mouse xenograft tumor models, in which the combination is more efficacious than either single-agent docetaxel or navitoclax. Addition of navitoclax to paclitaxel decreases the time from mitotic entry to cell death and changes cell fate from mitotic slippage to death during mitotic arrest. The relative levels of Bcl-x(L) and Mcl-1 correlate with the extent of synergy, suggesting that cancers with elevated levels of Bcl-x(L) will be relatively resistant to taxane-based therapy but could benefit from the addition of navitoclax to taxane treatment. Finally, a significant percentage of NSCLC patient samples exhibit relatively high Bcl-x(L) levels. CONCLUSIONS: The addition of navitoclax to taxane-based chemotherapy in NSCLC has the potential to increase efficacy, particularly in patients whose tumors express high levels of Bcl-x(L).
Assuntos
Compostos de Anilina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Sulfonamidas/farmacologia , Taxoides/química , Compostos de Anilina/administração & dosagem , Animais , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Ciclo Celular , Linhagem da Célula , Sobrevivência Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Mitose , Transplante de Neoplasias , Paclitaxel/administração & dosagem , Sulfonamidas/administração & dosagem , Fatores de Tempo , Proteína bcl-X/antagonistas & inibidoresRESUMO
UNLABELLED: A 48 year-old female with chemo-refractory metastatic gastric cancer to the liver was treated on a Phase I clinical trial with MetMAb, a monoclonal antibody targeting the Met tyrosine kinase receptor. The primary tumor had high MET gene polysomy and evidence for an autocrine production of HGF, the growth factor ligand of Met. A complete response was obtained lasting two years; the cancer recurred as a peritoneal deposit invading into the transverse colon and a gastrohepatic ligament node. Compassionate use of MetMAb therapy at recurrence achieved a mixed response--a partial response of the two initial lesions, but with development of multiple new foci of carcinomatosis. Tissue and serum studies evaluating the Met signaling pathway did correlate with MetMAb treatment response initially and at the time of recurrence. SIGNIFICANCE: This research brief is the first to describe a durable complete response obtained with a molecularly targeted monoclonal antibody, MetMAb, to the receptor tyrosine kinase, Met, in a patient with chemorefractory metastatic gastric cancer. It is also the first to report biomarkers that predicted therapeutic response to Met inhibition.