RESUMO
Recent clinical data in lung cancer suggests that epigenetically targeted therapy may selectively enhance chemotherapeutic sensitivity. There have been few if any studies rigorously evaluating this hypothesized priming effect. Here we describe a series of investigations testing whether epigenetic priming with azacitidine and entinostat increases sensitivity of NSCLC to cytotoxic agents. We noted no differences in chemosensitivity following treatment with epigenetic therapy in in vitro assays of viability and colony growth. Using cell line and patient derived xenograft (PDX) models, we also observed no change in responsiveness to cisplatin in vivo. In select models, we noted differential responses to irinotecan treatment in vivo. In vitro epigenetic therapy prior to tumor implantation abrogated response of H460 xenografts to irinotecan. Conversely, in vitro epigenetic therapy appeared to sensitize A549 xenografts (tumor growth inhibition 51%, vs. 22% in mock-pretreated control). In vivo epigenetic therapy enhanced the response of adenocarcinoma PDX to irinotecan. Taken together, these data do not support broadly applicable epigenetic priming in NSCLC. Priming effects may be context-specific, dependent on both tumor and host factors. Further preclinical study is necessary to determine whether, and in which contexts, priming with epigenetic therapy has potential to enhance chemotherapeutic efficacy in NSCLC patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Azacitidina/farmacologia , Benzamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Piridinas/farmacologia , Animais , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular , Metilação de DNA , Avaliação Pré-Clínica de Medicamentos , Epigênese Genética , Feminino , Histonas/química , Humanos , Irinotecano , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de NeoplasiasRESUMO
Overexpression of the antiapoptotic protein Bcl-2 is observed in the majority of small cell lung cancer (SCLC) cases and is associated with resistance to chemotherapy. While targeting Bcl-2 in hematologic malignancies continues to show signs of promise, translating the BH3 mimetic ABT-737 (or ABT-263; navitoclax) to the clinic for solid tumors has remained problematic, with limited single-agent activity in early-phase clinical trials. Here, we used patient-derived xenograft (PDX) models of SCLC to study ABT-737 resistance and demonstrated that responses to ABT-737 are short lived and coincide with decreases in HIF-1α-regulated transcripts. Combining the mTOR inhibitor rapamycin with ABT-737 rescued this resistance mechanism, was highly synergistic in vitro, and provided durable tumor regressions in vivo without notable hematologic suppression. In comparison, tumor regressions did not occur when ABT-737 was combined with etoposide, a gold-standard cytotoxic for SCLC therapy. Rapamycin exposure was consistently associated with an increase in the proapoptotic protein BAX, whereas ABT-737 caused dose-dependent decreases in BAX. As ABT-737 triggers programmed cell death in a BAX/BAK-dependent manner, we provide preclinical evidence that the efficacy of ABT-737 as a single agent is self-limiting in SCLC, but the addition of rapamycin can maintain or increase levels of BAX protein and markedly enhance the anticancer efficacy of ABT-737. These data have direct translational implications for SCLC clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos de Bifenilo/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Nitrofenóis/farmacologia , Sirolimo/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Compostos de Bifenilo/administração & dosagem , Linhagem Celular Tumoral , Sinergismo Farmacológico , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos SCID , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Distribuição Aleatória , Sirolimo/administração & dosagem , Carcinoma de Pequenas Células do Pulmão/metabolismo , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We assessed the efficacy of Seneca Valley virus (SVV-001), a neuroendocrine cancer-selective oncolytic picornavirus, in primary heterotransplant mouse models of small cell lung cancer (SCLC), including three lines each of classic and variant SCLC. Half-maximal effective concentrations for cell lines derived from three variant heterotransplants ranged from 1.6×10(-3) (95% confidence interval [CI] = 1×10(-3) to 2.5×10(-3)) to 3.9×10(-3) (95% CI = 2.8×10(-3) to 5.5×10(-3)). Sustained tumor growth inhibition in vivo was only observed in variant lines (two-sided Student t test, P < .005 for each). Doses of 10(14) vp/kg were able to completely and durably eradicate tumors in a variant SCLC heterotransplant model in two of six mice. Gene expression profiling revealed that permissive lines are typified by lower expression of the early neurogenic transcription factor ASCL1 and, conversely, by higher expression of the late neurogenic transcription factor NEUROD1. This classifier demonstrates a sensitivity of .89, specificity of .92, and accuracy of .91. The NEUROD1 to ASCL1 ratio may serve as a predictive biomarker of SVV-001 efficacy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Carcinoma de Células Pequenas/terapia , Neoplasias Pulmonares/terapia , Vírus Oncolíticos , Picornaviridae , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Pequenas/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Neoplasias Pulmonares/química , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Valor Preditivo dos Testes , Análise Serial de Proteínas , Transplante Heterólogo , TropismoRESUMO
New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). Insulin-like growth factor 1 receptor (IGF-1R) inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways, including phosphatidylinositol-3-kinase-Akt and mitogen-activated protein kinase. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R, and the closely related insulin receptor. Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 µmol/L. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (P = 0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell-cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared with mock-treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptor IGF Tipo 1/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/administração & dosagem , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
BACKGROUND: New therapies are urgently needed for patients with small cell lung cancer (SCLC). Chemotherapy and targeted therapies, including the Bcl-2 inhibitor ABT-737, may induce tumor cell autophagy. Autophagy can promote survival of cancer cells under stress and comprise a pathway of escape from cytotoxic therapies. METHODS: We explored the combination of ABT-737 and chloroquine, an inhibitor of autophagy, in preclinical models of SCLC. These included cell culture analyses of viability and of autophagic and apoptotic pathway induction, as well as in vivo analyses of efficacy in multiple xenograft models. RESULTS: Combination treatment of SCLC lines with ABT-737 and chloroquine decreased viability and increased caspase-3 activation over treatment with either single agent. ABT-737 induced several hallmarks of autophagy. However, knockdown of beclin-1, a key regulator of entry into autophagy, diminished the efficacy of ABT-737, suggesting either that the effects of chloroquine were nonspecific or that induction but not completion of autophagy is necessary for the combined effect of ABT-737 and chloroquine. ABT-737 and chloroquine in SCLC cell lines downregulated Mcl-1 and upregulated NOXA, both of which may promote apoptosis. Treatment of tumor-bearing mice demonstrated that chloroquine could enhance ABT-737-mediated tumor growth inhibition against NCI-H209 xenografts, but did not alter ABT-737 response in three primary patient-derived xenograft models. CONCLUSION: These data suggest that although ABT-737 can induce autophagy in SCLC, autophagic inhibition by choroquine does not markedly alter in vivo response to ABT-737 in relevant preclinical models, arguing against this as a treatment strategy for SCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteína Beclina-1 , Compostos de Bifenilo/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Cloroquina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , RNA Interferente Pequeno/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Sulfonamidas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK-driven NSCLCs, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene-driven NSCLCs. We recently showed that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene-driven human NSCLCs. Silencing of TWIST1 in KRAS-mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or, in some cases, apoptosis. Similar effects were observed in EGFR mutation-driven and c-Met-amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS-mutant, EGFR-mutant, and c-Met-amplified NSCLCs. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS-mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver-dependent NSCLCs represents a novel and promising therapeutic strategy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Oncogenes , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Proteína 1 Relacionada a Twist/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Senescência Celular/genética , Inativação Gênica , Células HEK293 , Xenoenxertos , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
The Notch signaling pathway is a critical embryonic developmental regulatory pathway that has been implicated in oncogenesis. In non-small cell lung cancer (NSCLC), recent evidence suggests that Notch signaling may contribute to maintenance of a cancer stem or progenitor cell compartment required for tumorigenesis. We explored whether intact Notch signaling is required for NSCLC clonogenic and tumorigenic potential in vitro and in vivo using a series of genetically modified model systems. In keeping with previous observations, we find that Notch3 in particular is upregulated in human lung cancer lines and that downregulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003) is associated with decreased proliferation and clonogenic capacity in vitro. We show that this phenotype is rescued with the expression of NICD3, a constitutively active cleaved form of Notch3 not affected by γ-secretase inhibition. Using an inducible LSL-KRAS(G12D) model of lung cancer in vivo, we show a transient upregulation of Notch pathway activity in early tumor precursor lesions. However, a more rigorous test of the requirement for Notch signaling in lung oncogenesis, crossing the LSL-KRAS(G12D) mouse model with a transgenic with a similarly inducible global dominant-negative suppressor of Notch activity, LSL-DNMAML (dominant-negative mastermind-like), reveals no evidence of Notch pathway requirement for lung tumor initiation or growth in vivo. Distinct Notch family members may have different and potentially opposing activities in oncogenesis, and targeted inhibition of individual Notch family members may be a more effective anticancer strategy than global pathway suppression.
Assuntos
Receptores Notch/genética , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Ensaio de Unidades Formadoras de Colônias , Óxidos S-Cíclicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Receptor Notch3 , Transdução de Sinais , Tiadiazóis/farmacologiaRESUMO
The antiangiogenic agent bevacizumab has been approved for the treatment of non-small cell lung cancer (NSCLC), although the survival benefit associated with this agent is marginal, and toxicities and cost are substantial. A recent screen for selective inhibitors of endothelial cell proliferation identified the oral antifungal drug itraconazole as a novel agent with potential antiangiogenic activity. In this article, we define and characterize the antiangiogenic and anticancer activities of itraconazole in relevant preclinical models of angiogenesis and lung cancer. Itraconazole consistently showed potent, specific, and dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to both VEGF- and basic fibroblast growth factor-mediated angiogenic stimulation. In vivo, using primary xenograft models of human NSCLC, oral itraconazole showed single-agent growth-inhibitory activity associated with induction of tumor hypoxia-inducible factor 1 alpha expression and marked inhibition of tumor vascularity. Itraconazole significantly enhanced the antitumor efficacy of the chemotherapeutic agent cisplatin in the same model systems. Taken together, these data suggest that itraconazole has potent and selective inhibitory activity against multiple key aspects of tumor-associated angiogenesis in vitro and in vivo, and strongly support clinical translation of its use. Based on these observations, we have initiated a randomized phase II study comparing the efficacy of standard cytotoxic therapy with or without daily oral itraconazole in patients with recurrent metastatic NSCLC.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Itraconazol/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Idoso , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Cisplatino/administração & dosagem , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Itraconazol/administração & dosagem , Itraconazol/farmacologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of small cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. We compared primary SCLC xenografts prepared from de novo human tumors with standard cell line-based xenografts in the evaluation of a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent dichotomy may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significant decreases in tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, we isolated resistant derivatives of an initially sensitive cell line-based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling reveals 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. Taken together, these data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the testing of novel anticancer strategies in relevant preclinical models.