RESUMEN
Epithelial to mesenchymal transition (EMT) is associated with resistance during EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. Here, we investigated whether EMT is associated with acquired resistance to 3rd generation EGFR-TKIs, and we explored the effects of cyclin-dependent kinase 7 (CDK7) inhibitors on EMT-mediated EGFR-TKIs resistance in non-small cell lung cancer (NSCLC). We established 3rd generation EGFR-TKI resistant cell lines (H1975/WR and H1975/OR) via repeated exposure to WZ4002 and osimertinib. The two resistant cell lines showed phenotypic changes to a spindle-cell shape, had a reduction of epithelial marker proteins, an induction of vimentin expression, and enhanced cellular mobility. The EMT-related resistant cells had higher sensitivity to THZ1 than the parental cells, although THZ1 treatment did not inhibit EGFR activity. This phenomenon was also observed in TGF-ß1 induced EMT cell lines. THZ1 treatment induced G2/M cell cycle arrest and apoptosis in all of the cell lines. In addition, THZ1 treatment led to drug-tolerant, EMT-related resistant cells, and these THZ1-tolerant cells partially recovered their sensitivity to 3rd generation EGFR-TKIs. Taken together, EMT was associated with acquired resistance to 3rd generation EGFR-TKIs, and CDK7 inhibitors could potentially be used as a therapeutic strategy to overcome EMT associated EGFR-TKI resistance in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Fase G2/efectos de los fármacos , Fase G2/genética , Humanos , Neoplasias Pulmonares/genética , Pirimidinas/farmacología , Factor de Crecimiento Transformador beta1/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
BACKGROUND: Immunotherapy with immune checkpoint inhibitors for non-small cell lung cancer (NSCLC) has emerged as an important treatment option. Although immunotherapy may significantly improve survival and quality of life, response rates are as low as 20% in NSCLC patients. OBJECTIVE: The identification of reliable biomarkers predicting response to immunotherapy is required urgently to determine patient selection guidelines. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from nine NSCLC patients were collected pre- and post-treatment with immunotherapy. The immune cell composition of PBMCs was analyzed using CyTOF with an optimized 32-marker panel. The natural killer (NK) cell activity was assessed with the measurement of interferon (INF)-γ using an NK Vue™ kit. RESULTS: We found that the percentages of NK cell populations in the immune cells of PBMCs were prominently elevated in the immunotherapy responder group when compared with non-responders. While no meaningful differences were observed in other populations of immune cells, consistent with these results, the overall activity of NK cells in responders was highly elevated compared with that of non-responders. From the analysis of NK subsets, although differences in the population of early NK cells were not observed, the functionally differentiated late NK cells were prominently high in responders. CONCLUSIONS: The overall activity or number of NK cells may be a useful biomarker to predict immunotherapy response in patients with NSCLC.
Asunto(s)
Biomarcadores/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Inmunoterapia/métodos , Células Asesinas Naturales/metabolismo , Neoplasias Pulmonares/terapia , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Adulto JovenRESUMEN
Tumor-derived exosomes (TEXs) contain enriched miRNAs that act as novel non-invasive biomarkers for cancer diagnosis and play a role in cancer progression. We investigated the exosomal miRNAs that affect cancer progression in non-small cell lung cancer (NSCLC) and identified the specific molecules involved. We identified that specific miRNAs in NSCLC cell-released exosomes can modulate angiogenesis, among which miR-619-5p was the most potent inducer. RCAN1.4 was identified as a target of miR-619-5p and its suppression promoted angiogenesis. Furthermore, the suppression of RCAN1.4 induced cell proliferation and metastasis in NSCLC cells. In patients with NSCLC, the level of RCAN1.4 expression was significantly lower, and that of miR-619-5p significantly higher, in tumor than normal lung tissues. miR-619-5p expression was higher than normal in exosomes isolated from the plasma of NSCLC patients. Finally, hypoxic conditions induced miR-619-5p upload into NSCLC cell-derived exosomes. Our findings indicate that exosomal miR-619-5p promotes the growth and metastasis of NSCLCs by regulating RCAN1.4 and can serve as a diagnostic indicator for these lung cancers.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/secundario , Proteínas de Unión al ADN/metabolismo , Exosomas/genética , Neoplasias Pulmonares/patología , MicroARNs/genética , Proteínas Musculares/metabolismo , Neovascularización Patológica/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones SCID , Proteínas Musculares/genética , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway blockade is a promising new cancer therapy. Although PD-1/PD-L1 treatment has yielded clinical benefits in several types of cancer, further studies are required to clarify predictive biomarkers for drug efficacy and to understand the fundamental mechanism of PD-1/PD-L1 interaction between host and tumor cells. Here, we show that exosomes derived from lung cancer cells express PD-L1 and play a role in immune escape by reducing T-cell activity and promoting tumor growth. The abundance of PD-L1 on exosomes represented the quantity of PD-L1 expression on cell surfaces. Exosomes containing PD-L1 inhibited interferon-gamma (IFN-γ) secretion by Jurkat T cells. IFN-γ secretion was restored by PD-L1 knockout or masking on the exosomes. Both forced expression of PD-L1 on cells without PD-L1 and treatment with exosomes containing PD-L1 enhanced tumor growth in vivo. PD-L1 was present on exosomes isolated from the plasma of patients with non-small cell lung cancer, and its abundance in exosomes was correlated with PD-L1 positivity in tumor tissues. Exosomes can impair immune functions by reducing cytokine production and inducing apoptosis in CD8+ T cells. Our findings indicate that tumor-derived exosomes expressing PD-L1 may be an important mediator of tumor immune escape.
Asunto(s)
Antígeno B7-H1/fisiología , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/genética , Exosomas/metabolismo , Neoplasias Pulmonares/patología , Escape del Tumor/genética , Células A549 , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Células Cultivadas , Exosomas/genética , Exosomas/patología , Células HEK293 , Xenoinjertos , Humanos , Células Jurkat , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Central nervous system (CNS) metastasis is one of the serious complications of epidermal growth factor receptor (EGFR)-mutant lung cancer, which arises due to poor penetration of the brain-blood barrier by EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Although osimertinib, a third-generation EGFR-TKI, has efficacy against CNS metastases, further treatment modalities are still needed as some of these lesions do not respond to osimertinib, or undergo progression after an initial response to this drug if radiotherapy has already been conducted. Here, we investigated the efficacy of water-soluble erlotinib (NUFS-sErt) against these metastases. This agent was synthesized using a nano-particulation platform technology utilizing fat and supercritical fluid (NUFS™) to resolve the low solubility problem that typically prevents the creation of injectable forms of EGFR-TKIs. The average NUFS-sErt particle size was 236.4 nm, and it showed time-dependent dissolution in culture media. The effects of NUFS-sErt were similar to those of conventional erlotinib in terms of inhibiting the proliferation of EGFR-mutant lung cancer cells and suppressing EGFR signaling. In an intraperitoneal xenograft model of HCC827 cells, intraperitoneal administration of NUFS-sErt produced a dose-dependent inhibition of tumor growth and enhanced survival rate. Notably, the injection of NUFS-sErt into the brain ventricle caused significant tumor growth inhibition in an intracranial xenograft model. Hence, our current findings indicate that NUFS-sErt is a novel, water-soluble form of erlotinib that can be administered using intraventricular or intrathecal injections. The target cases would be patients with a progressive CNS metastasis and no other therapeutic options. This drug could also be given intravenously to patients with swallowing difficulties or an inability to ingest due to a medical condition.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/secundario , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/genética , Clorhidrato de Erlotinib/uso terapéutico , Neoplasias Pulmonares/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Clorhidrato de Erlotinib/administración & dosificación , Clorhidrato de Erlotinib/química , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones SCID , Mutación , Nanopartículas/química , Agua/químicaRESUMEN
Oncogenic EGFR is essential for the development and growth of non-small cell lung cancer (NSCLC), but the precise roles of EGFR in lung cancer metabolism remain unclear. Here, we show that EGFR mutation-mediated enhancement of glycolysis is critical for EGFR stability. EGFR knockdown significantly decreased levels of glycolytic pathway intermediates via transcriptional regulation of glycolytic genes. EGFR mutation-enhanced glycolysis was required for fueling the tricarboxylic acid cycle, a critical component of EGFR stability. Nonsustained ATP production enhanced reactive oxygen species accumulation and subsequent JNK-mediated activation of autophagy, which in turn induced EGFR degradation. Our data show that EGFR-mutant NSCLCs require EGFR mutation-enhanced glycolysis to maintain EGFR stability. This pathway may serve as an attractive therapeutic target for EGFR-mutant NSCLCs.Significance: Enhanced glycolysis by EGFR mutation is required for maintaining EGFR levels via inhibition of JNK-induced autophagy. This provides a promising rationale for use of JNK activators in patients with EGFR-mutated NSCLC. Cancer Res; 78(16); 4482-96. ©2018 AACR.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Autofagia/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Glucólisis/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 4/genética , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Despite remarkable activity in epidermal growth factor receptor (EGFR)-mutant lung cancer patients, the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) is limited by the emergence of acquired resistance, which is mostly caused by a secondary T790M mutation. Fortunately, newly developed, mutant-selective EGFR-TKIs against T790M have been proven as an effective therapeutic approach although only osimertinib has received the FDA approval until now. OBJECTIVE: To determine the in vitro and in vivo efficacy of a new EGFR TKI, OBX1-012 in cells with mutant EGFR. METHODS: Effects of OBX1-012 on cellular viability and EGFR-related signaling were determined in EGFR-mutant non-small cell lung cancer (NSCLC) cells, including cells harboring T790M mutations. In addition, in vivo efficacy of OBX1-012 was evaluated in xenograft models. RESULTS: We report the discovery and preclinical assessment of another novel, mutant-selective EGFR-TKI, OBX1-012. Compared with other mutant-selective EGFR-TKIs such as olumitinib and osimertinib, it showed similar potency and selectivity for mutant EGFR. OBX1-012 treatment was highly effective against human EGFR-mutant lung cancer models with or without EGFR T790M, not only in vitro but also in vivo. However, OBX1-012 like other EGFR-TKIs failed to exhibit efficacy for the exon 20 insertion mutation or C797S mutation, which was generated by site-directed mutagenesis and stable transfection of Ba/F3 cells. CONCLUSIONS: These results identify OBX1-012 as a highly effective, mutant-selective EGFR-TKI for the treatment of T790M-mediated resistance in NSCLC.
Asunto(s)
Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The activation of alternative receptor tyrosine kinases (RTKs) is known to mediate resistance to ALK inhibitors. However, the role of multiple RTK activation in resistance has yet to be determined. Two crizotinib-resistant (H3122/CR-1 and H3122/CR-2) and one TAE684-resistant (H2228/TR) cell lines were established. Multi-RTK arrays and Western blots were performed to detect the activation of bypass signals. There were no secondary mutations in the sequencing. EGFR and MET were activated in H3122/CR-1 cells whereas EGFR and IGF1R were activated in H3122/CR-2 cells. Concomitant activation of MET did not contribute to resistance as crizotinib completely suppressed both p-MET and p-ALK in H3122/CR-1 cells, whose survival was not affected by crizotinib. However, combined inhibition of EGFR and ALK was effective in controlling this resistant cell line. In H3122/CR-2 cells, the inhibition of both ALK and IGF1R could effectively suppress cell growth, whereas simultaneous inhibition of ALK and EGFR brought about a less-effective suppression, indicating that IGF1R activation is the main resistance mechanism. H2228/TR cells showed activation of the HER family (EGFR, ErbB2, and ErbB3). Afatinib, a pan-HER inhibitor, was more potent in suppressing resistant cells than gefitinib when combined with crizotinib, which suggests that coactivation of ErbB2 and ErbB3 also contributes to resistance. Interestingly, all three resistant cell lines responded well to AUY922, which can inhibit ALK, EGFR, and IGF1R activity. Activation of multiple RTKs can occur during acquired resistance to ALK inhibitors, in which case the dominant or significant bypass signal should be identified to provide a more appropriate combination therapy.
RESUMEN
Mutant-selective, 3rd-generation EGFR-TKIs were recently developed to control lung cancer cells harboring T790M-mediated resistance. However, the development of resistance to these novel drugs seems inevitable. Thus, we investigated the mechanism of acquired resistance to the mutant-selective EGFR-TKI WZ4002. We established five WZ4002-resistant cells, derived from cells harboring both EGFR and T790M mutations by long-term exposure to increasing doses of WZ4002. Compared with the parental cells, all resistant cells showed 10-100-folds higher resistance to WZ4002, as well as cross-resistance to other mutant-selective inhibitors. Among them, three resistant cells (HCC827/WR, PC-9/WR and H1975/WR) showed dependency on EGFR signaling, but two other cells (PC-9/GR/WR and PC-9/ER/WR) were not. Notably, insulin-like growth factor-1 receptor (IGF1R) was aberrantly activated in PC-9/GR/WR cells in phospho-receptor tyrosine kinase array, consistently accompanied by loss of IGF binding protein-3 (IGFBP3). Down-regulation of IGF1R by shRNA, as well as inhibition of IGF1R activity either by AG-1024 (a small molecule IGF1R inhibitor) or BI 836845 (a monoclonal anti-IGF1/2 blocking antibody), restored the sensitivity to WZ4002 both in vitro and xenograft. Taken together, these results suggest that activation of the IGF1R pathway associated with IGFBP3 loss can induce an acquired resistance to the mutant-selective EGFR-TKI, WZ4002. Therefore, a combined therapy of IGF1R inhibitors and mutant-selective EGFR-TKIs might be a viable treatment strategy for overcoming acquired resistance.