RESUMEN
OBJECTIVE: Nuclear receptors (NRs) play a vital role in the development and progression of several cancers including breast and prostate. Using TCGA data, we sought to identify critical nuclear receptors in high grade serous ovarian cancers (HGSOC) and to confirm these findings using in vitro approaches. METHODS: In silico analysis of TCGA data was performed to identify relevant NRs in HGSOC. Ovarian cancer cell lines were screened for NR expression and functional studies were performed to determine the significance of these NRs in ovarian cancers. NR expression was analyzed in ovarian cancer tissue samples using immunohistochemistry to identify correlations with histology and stage of disease. RESULTS: The NR4A family of NRs was identified as a potential driver of ovarian cancer pathogenesis. Overexpression of NR4A1 in particular correlated with worse progression free survival. Endogenous expression of NR4A1 in normal ovarian samples was relatively high compared to that of other tissue types, suggesting a unique role for this orphan receptor in the ovary. Expression of NR4A1 in HGSOC cell lines as well as in patient samples was variable. NR4A1 primarily localized to the nucleus in normal ovarian tissue while co-localization within the cytoplasm and nucleus was noted in ovarian cancer cell lines and patient tissues. CONCLUSIONS: NR4A1 is highly expressed in a subset of HGSOC samples from patients that have a worse progression free survival. Studies to target NR4A1 for therapeutic intervention should include HGSOC.
Asunto(s)
Neoplasias Glandulares y Epiteliales/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Neoplasias Ováricas/metabolismo , Animales , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Genoma , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones SCID , Neoplasias Glandulares y Epiteliales/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Neoplasias Ováricas/genética , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
MET amplification/mutations are important targetable oncogenic drivers in NSCLC, however, acquired resistance is inevitable and the majority of patients with targetable MET alterations fail to respond to MET tyrosine kinase inhibitors (TKIs). Furthermore, MET amplification is among the most common mediators of TKI resistance. As such, novel therapies to target MET pathway and overcome MET TKI resistance are clearly needed. Here we show that the epithelial-mesenchymal transition (EMT) transcription factor, TWIST1 is a key downstream mediator of HGF/MET induced resistance through suppression of p27 and targeting TWIST1 can overcome resistance. We found that TWIST1 is overexpressed at the time of TKI resistance in multiple MET-dependent TKI acquired resistance PDX models. We have shown for the first time that MET directly stabilized the TWIST protein leading to TKI resistance and that TWIST1 was required for MET-driven lung tumorigenesis as well as could induce MET TKI resistance when overexpressed. TWIST1 mediated MET TKI resistance through suppression of p27 expression and genetic or pharmacologic inhibition of TWIST1 overcame TKI resistance in vitro and in vivo. Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome resistance in MET-driven NSCLC as well as in other oncogene driven subtypes in which MET amplification is the resistance mechanism.
Asunto(s)
Resistencia a Antineoplásicos , Factor de Crecimiento de Hepatocito , Neoplasias Pulmonares , Proteínas Nucleares , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-met , Proteína 1 Relacionada con Twist , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Resistencia a Antineoplásicos/genética , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transición Epitelial-Mesenquimal/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Oncogenes/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Acrilamidas , Sustitución de Aminoácidos , Compuestos de Anilina , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación Missense , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Purpose: TMEM16A is a calcium-activated chloride channel that is amplified in a variety of cancers, including 30% of head and neck squamous cell carcinomas (HNSCCs), raising the possibility of an anti-apoptotic role in malignant cells. This study investigated this using a multimodal, translational investigation.Experimental Design: Combination of (i) in vitro HNSCC cell culture experiments assessing cell viability, apoptotic activation, and protein expression (ii) in vivo studies assessing similar outcomes, and (iii) molecular and staining analysis of human HNSCC samples.Results: TMEM16A expression was found to correlate with greater tumor size, increased Erk 1/2 activity, less Bim expression, and less apoptotic activity overall in human HNSCC. These findings were corroborated in subsequent in vitro and in vivo studies and expanded to include a cisplatin-resistant phenotype with TMEM16A overexpression. A cohort of 41 patients with laryngeal cancer demonstrated that cases that recurred after chemoradiation failure were associated with a greater TMEM16A overexpression rate than HNSCC that did not recur.Conclusions: Ultimately, this study implicates TMEM16A as a contributor to tumor progression by limiting apoptosis and as a potential biomarker of more aggressive disease. Clin Cancer Res; 23(23); 7324-32. ©2017 AACR.
Asunto(s)
Anoctamina-1/genética , Apoptosis/genética , Proteína 11 Similar a Bcl2/genética , Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/genética , Animales , Anoctamina-1/metabolismo , Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Regulación hacia Abajo , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.