RESUMO
Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has been a longstanding challenge for head and neck oncologists, and current treatments still have limited efficacy. ERK is aberrantly overexpressed and activated in HNSCC. Herein, we aimed to investigate the cause of the limited therapeutic effect of selumetinib, a selective inhibitor of MEK in HNSCC, as MEK/ERK reactivation inevitably occurs. We assessed the effects of combining selumetinib with fibroblast growth factor receptor 3 (FGFR3) inhibitor (PD173074) on tumor growth. Selumetinib transiently inhibited MAPK signaling and reactivated ERK signaling in HNSCC cells. Rebound in the ERK and Akt pathways in HNSCC cells was accompanied by increased FGFR3 signaling after selumetinib treatment. Feedback activation of FGFR3 was a result of autocrine secretion of the FGF2 ligand. The FGFR3 inhibitor PD173074 prevented MAPK rebound and sensitized the response of HNSCC cells to selumetinib. These results provided rational therapeutic strategies for clinical studies of this subtype of patients that show a poor prognosis with selumetinib. Our data provide a rationale for combining a MEK inhibitor with inhibitors of feedback activation of FGFR3 signaling in HNSCC cells. ERK rebound as a result of the upregulation of FGFR3 and the ligand FGF2 diminished the antitumor effects of selumetinib, which was overcome by combination treatment with the FGFR3 inhibitor.
Assuntos
Benzimidazóis/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Pirimidinas/administração & dosagem , Receptor ErbB-3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Pirimidinas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previously, the authors have identified that c-Met mediates reactivation of the PI3K/AKT pathway following BRAF inhibitor treatment in BRAF (V600E) mutant anaplastic thyroid cancer, thereby contributing to the acquired drug resistance. Therefore dual inhibition of BRAF and c-Met led to sustained treatment response, thereby maximizing the specific anti-tumor effect of targeted therapy. The present study goes one step further and aims to investigate the effect of acquired resistance of BRAF inhibitor on epithelial-to-mesenchymal transition (EMT) in BRAF mutant thyroid cancer cells and the effect of dual inhibition from combinatorial therapy. Two thyroid cancer cell lines, 8505C and BCPAP were selected and treated with BRAF inhibitor, PLX4032 and its effect on EMT were examined and compared. Further investigation was carried out in orthotopic xenograft mouse models. Unlike BCPAP cells, the BRAF inhibitor resistant 8505C cells showed increased expressions of EMT related markers such as vimentin, ß-catenin, and CD44. The combinatorial treatment of PLX4032 and PHA665752, a c-Met inhibitor reversed EMT. Similar results were confirmed in vivo. c-Met-mediated reactivation of the PI3K/AKT pathway contributes to the drug resistance to PLX4032 in BRAF (V600E) mutant anaplastic thyroid cancer cells and further promotes tumor cell migration and invasion by upregulated EMT mechanism. Dual inhibition of BRAF and c-Met leads to reversal of EMT, suggesting a maximal therapeutic response.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Humanos , Indóis/farmacologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES/HYPOTHESIS: The development of a simple, reliable, and cost-effective animal model greatly facilitates disease treatment. We aimed to establish a rapid, simple, and reproducible live zebrafish vestibular schwannoma xenograft model for antitumor drug screening. METHODS: We optimized each of the following conditions for tumor cell xenografts in zebrafish larvae: larval stage, incubation temperature, and injected cell number. We used NF2-/-mouse Schwann (SC4) cells and generated mCherry fluorescent protein-expressing cells prior to injection into zebrafish larvae. SC4 cells were counted using a fluorescence microscope, suspended in 10% fetal bovine serum, and injected into the center of the yolk sac using a microinjection system. The injected embryos were transferred to E3 medium (for zebrafish embryos), and subsequent tumor formation was observed by fluorescence microscopy over a 5-day period. To validate our model, xenografted embryos were transferred into 6-well plates (5 embryos per well) and treated with everolimus, a known antitumor drug. RESULTS: mCherry fluorescent protein-expressing SC4 cells were successfully grafted into the yolk sacs of zebrafish embryos without any immunosuppressant treatment. At 2 days postinjection, the xenografted cells had grown into tumor masses. The optimal speed of tumor formation depended on the larval stage (30 hpf), incubation temperature (31°C), and injected cell number (200 cells). In preliminary tests, everolimus treatment yielded a > 20% reduction in the number of SC4 cells in the yolk. CONCLUSION: Our in vivo model has the potential to greatly facilitate vestibular schwannoma treatment because of its speed, simplicity, reproducibility, and amenability to live imaging. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E409-E415, 2016.
Assuntos
Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Neuroma Acústico/tratamento farmacológico , Peixe-Zebra , Animais , XenoenxertosRESUMO
BRAF (V600E) mutation is the most commonly detected genetic alteration in thyroid cancer. Unlike its high treatment response to selective BRAF inhibitor (PLX4032) in metastatic melanoma, the treatment response in thyroid cancer is reported to be low. The purpose of this study is to investigate the resistance mechanism responsible for this low treatment response to BRAF inhibitor in order to maximize the effect of targeted therapy. We examined the expression of feedback regulation mechanisms and alterations in the upper signal transduction pathway in thyroid cancer cell lines harboring BRAF mutation. Also, we investigated the effect of dual inhibition from combinatorial therapy. Two thyroid cancer cell lines, 8505C (anaplastic thyroid cancer) and BCPAP (papillary thyroid cancer) were selected and treated with PLX4032 and its drug sensitivity were examined and compared. Further investigation on the changes in signals responsible for the different treatment response to PLX4032 was carried out and the same experiment was performed on orthotopic xenograft mouse models. Unlike BCPAP cells, 8505C cells presented drug resistance to PLX4032 treatment and this was mainly due to increased expression of c-Met. Effective inhibitions of c-Met, p-AKT, and p-ERK were achieved after dual treatment with BRAF inhibitor (PLX4032) and c-Met inhibitor (PHA665752). Similar results were confirmed by in vivo study with orthotopic xenograft mouse model. c-Met-mediated reactivation of the PI3K/AKT pathway and MAPK pathway contributes to the relative insensitivity of BRAF (V600E) mutant anaplastic thyroid cancer cells to PLX4032. Dual inhibition of BRAF and c-Met leads to sustained treatment response. © 2015 Wiley Periodicals, Inc.
Assuntos
Indóis/administração & dosagem , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Sulfonamidas/administração & dosagem , Sulfonas/administração & dosagem , Neoplasias da Glândula Tireoide/genética , Regulação para Cima , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/metabolismo , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: The intrinsic increase of reactive oxygen species (ROS) production in cancer cells after malignant transformation frequently induces redox adaptation, leading to enhanced antioxidant capacity. Peroxiredoxin I (PrxI), an enzyme responsible for eliminating hydrogen peroxide, has been found to be elevated in many types of cancer cells. Since overexpression of PrxI promoted cancer cells' survival and resistance to chemotherapy and radiotherapy, PrxI has been proposed as a therapeutic target for anticancer drugs. In this study, we aimed to investigate the anticancer efficacy of a small molecule inhibitor of PrxI. RESULTS: By a high-throughput screening approach, we identified AMRI-59 as a potent inhibitor of PrxI. AMRI-59 increased cellular ROS, leading to the activation of both mitochondria- and apoptosis signal-regulated kinase-1-mediated signaling pathways, resulting in apoptosis of A549 human lung adenocarcinoma. AMRI-59 caused no significant changes in ROS level, proliferation, and apoptosis of PrxI-knockdown A549 cells by RNA interference. PrxI overexpression or N-acetylcysteine pretreatment abrogated AMRI-59-induced cytotoxicity in A549 cells. AMRI-59 rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells. Moreover, significant antitumor activity of AMRI-59 was observed in mouse tumor xenograft model implanted with A549 cells with no apparent acute toxicity. INNOVATION: This study offers preclinical proof-of-concept for AMRI-59, a lead small molecule inhibitor of PrxI, as an anticancer agent. CONCLUSIONS: Our results highlight a promising strategy for cancer therapy that preferentially eradicates cancer cells by targeting the PrxI-mediated redox-dependent survival pathways.
Assuntos
Acetofenonas/administração & dosagem , Antineoplásicos/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Peroxirredoxinas/antagonistas & inibidores , Piperidinas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Acetofenonas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Piperidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1α (HIF-1α) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1α-induced invasive characteristics. MATERIALS AND METHODS: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1α, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1α and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1α, tissue analysis was done. RESULTS: Hypoxia induces HIF-1α expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1α via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1α with RNA interference suppressed hypoxia-induced HIF-1α and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1α. These were confirmed in the orthotopic FTC model. CONCLUSION: Hypoxia induced HIF-1α, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.
Assuntos
Adenocarcinoma Folicular/genética , Transição Epitelial-Mesenquimal/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Neoplasias da Glândula Tireoide/genética , Proteína 1 Relacionada a Twist/genética , Adenocarcinoma Folicular/metabolismo , Animais , Caderinas/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfocinas , Camundongos , Invasividade Neoplásica , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/metabolismo , Ativação Transcricional , Proteína 1 Relacionada a Twist/metabolismo , Vimentina/metabolismoRESUMO
Two critical issues in microarray-based gene expression profiling with amplified RNA are its reliability and reproducibility compared to the non-amplified RNA. In this study, the non-linear relationship between the two methods was evaluated with the entropy in addition to the linear relationship using correlation coefficients. The correlation coefficients within the amplification method and between the two methods were significantly high, 0.98 and 0.88, respectively. Comparing the entropy as increasing fold-change difference (k), the average entropy value was reduced to 0.02 in the cell line and 0.09 in the tissue samples, indicating that the number of different genes between the two methods was decreased. In addition, the threshold of k according to the percentage of p estimated from entropy values could be used to provide the cut-off line on gene selection. The quantity discordance rate of 0.3-5.47% and the common outlier proportion of 84.2-94.3% between the two methods were detected, according to the expression levels. In summary, we showed a high similarity between the two methods using non-linear as well as linear comparison. Furthermore, we proved that the entropy as the measure of non-linear relationship is useful for analyzing the similarity of replicated microarray data sets.
Assuntos
Entropia , Perfilação da Expressão Gênica , RNA , Estatística como Assunto , Linhagem Celular Tumoral , Humanos , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/metabolismo , Reprodutibilidade dos TestesRESUMO
Cyclin A1 and cyclin B1 are overexpressed in various tumors but are present at low levels in normal tissues. Cyclin A1 is restricted to germ cells undergoing meiosis. In order to explore the possibility of using cyclin A1 and cyclin B1 as anticancer targets, we knocked them down in two lung cancer cell lines, H157 and H596, using siRNA. As with cyclin A1 siRNA in lung cancer cell lines, cyclin B1, Cdc2 and CDK2 were all significantly downregulated. The S phase fraction increased significantly, and they eventually underwent apoptosis by way of downregulated intrinsic apoptotic pathways and modulators with upregulated extrinsic apoptotic pathways. Our study suggests that cyclin A1 might be a promising anticancer target specific to lung cancer.