RESUMEN
Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10-20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Pronóstico , Recurrencia Local de Neoplasia , Vesículas Extracelulares/metabolismo , Transición Epitelial-Mesenquimal/genéticaRESUMEN
Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings. These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway. Inhibiting this signalling pathway sensitises cells to killing by replication stress-inducing drugs. Here, we investigated the effect of low-level replication stress induced by low concentrations (> 0.2 mm) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S-phase progression but has little effect on cell viability or proliferation. We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in > 70% of melanoma and non-small-cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo. Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with submicromolar concentrations of gemcitabine exhibited complete loss of proliferative potential. The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR. The combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo. These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anticancer efficacy but with reduced normal tissue toxicity. These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Hidroxiurea/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antimetabolitos Antineoplásicos/efectos adversos , Antimetabolitos Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Desoxicitidina/efectos adversos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Progresión de la Enfermedad , Femenino , Humanos , Hidroxiurea/efectos adversos , Hidroxiurea/uso terapéutico , Neoplasias Pulmonares/patología , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , GemcitabinaRESUMEN
Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR.
Asunto(s)
Transformación Celular Neoplásica/genética , Silenciador del Gen , Pulmón/patología , Proteínas de Unión al ARN/genética , Factores de Transcripción de la Familia Snail/fisiología , Animales , Línea Celular Transformada , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Modelos AnimalesRESUMEN
Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.
Asunto(s)
Bronquios/citología , Movimiento Celular/genética , Proliferación Celular/genética , Células Epiteliales/patología , Neoplasias Pulmonares/genética , Animales , Bronquios/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Organismos Libres de Patógenos Específicos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Exosomas/patología , Neoplasias Pulmonares/patología , Mesodermo/patología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proliferación Celular/efectos de los fármacos , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Células Tumorales CultivadasRESUMEN
Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC-oncogenic alterations commonly found in non-small cell lung cancer (NSCLC). We interrogated at single-cell resolution how these alterations could affect classic readouts (ß-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1, and phospho-ERK1/2) of key pathways commonly affected in NSCLC. All three oncogenic alterations were required concurrently to observe significant signaling changes, and significant heterogeneity arose in this condition. Unexpectedly, we found two mutually exclusive altered subpopulations: one with STAT3 upregulation and another with SMAD2/3 downregulation. Treatment with a STAT3 inhibitor eliminated the upregulated STAT3 subpopulation, but left a large surviving subpopulation with downregulated SMAD2/3. A bioinformatics search identified BCL6, a gene downstream of SMAD2/3, as a novel pharmacologically accessible target of our transformed HBECs. Combination treatment with STAT3 and BCL6 inhibitors across a panel of NSCLC cell lines and in xenografted tumors significantly reduced tumor cell growth. We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (STAT3 and BCL6) downstream of common oncogenes, and tumor suppressors may provide a potent way to defeat intratumor heterogeneity. Cancer Res; 77(11); 3070-81. ©2017 AACR.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Terapia Combinada/métodos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Línea Celular Tumoral , Humanos , Transducción de Señal , TransfecciónRESUMEN
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated deaths worldwide. Given the efficacy of membrane proteins as therapeutic targets in human malignancies, we examined cell-surface receptors that may act as drivers of lung tumorigenesis. Here, we report that the PROTOCADHERIN PCDH7 is overexpressed frequently in NSCLC tumors where this event is associated with poor clinical outcome. PCDH7 overexpression synergized with EGFR and KRAS to induce MAPK signaling and tumorigenesis. Conversely, PCDH7 depletion suppressed ERK activation, sensitized cells to MEK inhibitors, and reduced tumor growth. PCDH7 potentiated ERK signaling by facilitating interaction of protein phosphatase PP2A with its potent inhibitor, the SET oncoprotein. By establishing an oncogenic role for PCDH7 in lung tumorigenesis, our results provide a rationale to develop novel PCDH7 targeting therapies that act at the cell surface of NSCLC cells to compromise their growth. Cancer Res; 77(1); 187-97. ©2016 AACR.
Asunto(s)
Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Transformación Celular Neoplásica/metabolismo , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/fisiología , Animales , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN , Receptores ErbB/metabolismo , Xenoinjertos , Chaperonas de Histonas/metabolismo , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos NOD , Reacción en Cadena de la Polimerasa , Modelos de Riesgos Proporcionales , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Protocadherinas , Transducción de Señal/fisiología , Análisis de Supervivencia , Análisis de Matrices Tisulares , Factores de Transcripción/metabolismoRESUMEN
Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-ß) or oncogenetic (MYC) factors. Both TGF-ß- and MYC-induced EMT required ZEB1, but engaged distinct TGF-ß-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-ß and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.
Asunto(s)
Transición Epitelial-Mesenquimal , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Unión al ARN/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/metabolismo , Línea Celular , Transformación Celular Neoplásica , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microcirculación , Invasividad Neoplásica , Metástasis de la Neoplasia , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Calcitriol/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
INTRODUCTION: The human secretin gene (SCT) encodes secretin, a hormone with limited tissue distribution. Analysis of the 450k methylation array data in The Cancer Genome Atlas (TCGA) indicated that the SCT promoter region is differentially hypermethylated in lung cancer. Our purpose was to validate SCT methylation as a potential biomarker for lung cancer. METHODS: We analyzed data from TCGA and developed and applied SCT-specific bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays. RESULTS: The analyses of TCGA 450K data for 801 samples showed that SCT hypermethylation has an area under the curve (AUC) value greater than 0.98 that can be used to distinguish lung adenocarcinomas or squamous cell carcinomas from nonmalignant lung tissue. Bisulfite sequencing of lung cancer cell lines and normal blood cells allowed us to confirm that SCT methylation is highly discriminative. By applying a quantitative methylation-specific polymerase chain reaction assay, we found that SCT hypermethylation is frequently detected in all major subtypes of malignant non-small cell lung cancer (AUC = 0.92, n = 108) and small cell lung cancer (AUC = 0.93, n = 40) but is less frequent in lung carcinoids (AUC = 0.54, n = 20). SCT hypermethylation appeared in samples of lung carcinoma in situ during multistage pathogenesis and increased in invasive samples. Further analyses of TCGA 450k data showed that SCT hypermethylation is highly discriminative in most other types of malignant tumors but less frequent in low-grade malignant tumors. The only normal tissue with a high level of methylation was the placenta. CONCLUSIONS: Our findings demonstrated that SCT methylation is a highly discriminative biomarker for lung and other malignant tumors, is less frequent in low-grade malignant tumors (including lung carcinoids), and appears at the carcinoma in situ stage.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Neoplasias Pulmonares/genética , Secretina/genética , Línea Celular Tumoral , Islas de CpG , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
In order to identify new cancer-associated metabolites that may be useful for early detection of lung cancer, we performed a global metabolite profiling of a non-small cell lung cancer (NSCLC) line and immortalized normal lung epithelial cells from the same patient. Among several metabolites with significant cancer/normal differences, we identified a unique metabolic compound, N-acetylaspartate (NAA), in cancer cells-undetectable in normal lung epithelium. NAA's cancer-specific detection was validated in additional cancer and control lung cells as well as selected NSCLC patient tumors and control tissues. NAA's cancer specificity was further supported in our analysis of NAA synthetase (gene symbol: NAT8L) gene expression levels in The Cancer Genome Atlas: elevated NAT8L expression in approximately 40% of adenocarcinoma and squamous cell carcinoma cases (N = 577), with minimal expression in all nonmalignant lung tissues (N = 74). We then showed that NAT8L is functionally involved in NAA production of NSCLC cells through siRNA-mediated suppression of NAT8L, which caused selective reduction of intracellular and secreted NAA. Our cell culture experiments also indicated that NAA biosynthesis in NSCLC cells depends on glutamine availability. For preliminary evaluation of NAA's clinical potential as a circulating biomarker, we developed a sensitive NAA blood assay and found that NAA blood levels were elevated in 46% of NSCLC patients (N = 13) in comparison with age-matched healthy controls (N = 21) among individuals aged 55 years or younger. Taken together, these results indicate that NAA is produced specifically in NSCLC tumors through NAT8L overexpression, and its extracellular secretion can be detected in blood. Cancer Prev Res; 9(1); 43-52. ©2015 AACR.
Asunto(s)
Acetiltransferasas/sangre , Ácido Aspártico/análogos & derivados , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Acetiltransferasas/metabolismo , Adulto , Anciano , Ácido Aspártico/sangre , Barrera Hematoencefálica , Carcinoma de Pulmón de Células no Pequeñas/sangre , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Glutamina/metabolismo , Humanos , Neoplasias Pulmonares/sangre , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ARNRESUMEN
Lung cancer is caused by combinations of diverse genetic mutations. Here, to understand the relevance of nuclear receptors (NRs) in the oncogene-associated lung cancer pathogenesis, we investigated the expression profile of the entire 48 NR members by using QPCR analysis in a panel of human bronchial epithelial cells (HBECs) that included precancerous and tumorigenic HBECs harboring oncogenic K-rasV12 and/or p53 alterations. The analysis of the profile revealed that oncogenic alterations accompanied transcriptional changes in the expression of 19 NRs in precancerous HBECs and 15 NRs according to the malignant progression of HBECs. Amongst these, peroxisome proliferator-activated receptor gamma (PPARγ), a NR chosen as a proof-of-principle study, showed increased expression in precancerous HBECs, which was surprisingly reversed when these HBECs acquired full in vivo tumorigenicity. Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs. In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPARγ plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism.
Asunto(s)
Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Neoplasias Pulmonares/genética , Receptores Citoplasmáticos y Nucleares/genética , Bronquios/citología , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Células HEK293 , Humanos , Immunoblotting , Neoplasias Pulmonares/metabolismo , Mutación , PPAR gamma/genética , PPAR gamma/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sumoilación , Tiazolidinedionas/farmacologíaRESUMEN
UNLABELLED: Protein S-palmitoylation is a widespread and dynamic posttranslational modification that regulates protein-membrane interactions, protein-protein interactions, and protein stability. A large family of palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif, catalyzes S-palmitoylation; the role of these enzymes in cancer is largely unexplored. In this study, an RNAi-based screen targeting all 23 members of the DHHC family was conducted to examine the effects on the growth in non-small cell lung cancer (NSCLC). Interestingly, siRNAs directed against DHHC5 broadly inhibited the growth of multiple NSCLC lines but not normal human bronchial epithelial cell (HBEC) lines. Silencing of DHHC5 by lentivirus-mediated expression of DHHC5 shRNAs dramatically reduced in vitro cell proliferation, colony formation, and cell invasion in a subset of cell lines that were examined in further detail. The phenotypes were restored by transfection of a wild-type DHHC5 plasmid but not by a plasmid expressing a catalytically inactive DHHC5. Tumor xenograft formation was severely inhibited by DHHC5 knockdown and rescued by DHHC5 expression, using both a conventional and tetracycline-inducible shRNA. These data indicate that DHHC5 has oncogenic capacity and contributes to tumor formation in NSCLC, thus representing a potential novel therapeutic target. IMPLICATIONS: Inhibitors of DHHC5 enzyme activity may inhibit non-small cell lung cancer growth.
Asunto(s)
Aciltransferasas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , ARN Interferente Pequeño/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Trasplante de NeoplasiasRESUMEN
Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel "druggable" targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Linaje de la Célula , Tumores Neuroendocrinos/genética , Oncogenes , Carcinoma Pulmonar de Células Pequeñas/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Supervivencia Celular , Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Tumores Neuroendocrinos/patología , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)125a-3p (miR125a) contributes to the malignant potential of human bronchial epithelial cells (HBEC) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR125a loss in HBEC K-ras lines and determined that miR125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR125a overexpression significantly reduces K-ras-mediated production of these tumorigenic factors. miR125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR125a loss in lung carcinogenesis and lay the groundwork for future studies to determine whether miR125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR125a in K-ras-mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes ras , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Factores de Transcripción/metabolismo , Bronquios/citología , Línea Celular , Proliferación Celular , Quimiocina CXCL1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epitelio/metabolismo , Humanos , Neoplasias Pulmonares/genética , Mutación , Pioglitazona , Mutación Puntual , Lesiones Precancerosas/metabolismo , Proto-Oncogenes Mas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Tiazolidinedionas/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas ras/metabolismoRESUMEN
Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Indoles/farmacología , Neoplasias Pulmonares/metabolismo , Triazinas/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteína Coatómero/metabolismo , Femenino , Genes ras , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR , Trasplante de Neoplasias , Fosforilación OxidativaRESUMEN
Small-cell lung cancer and other aggressive neuroendocrine cancers are often associated with early dissemination and frequent metastases. We demonstrate that neurogenic differentiation 1 (NeuroD1) is a regulatory hub securing cross talk among survival and migratory-inducing signaling pathways in neuroendocrine lung carcinomas. We find that NeuroD1 promotes tumor cell survival and metastasis in aggressive neuroendocrine lung tumors through regulation of the receptor tyrosine kinase tropomyosin-related kinase B (TrkB). Like TrkB, the prometastatic signaling molecule neural cell adhesion molecule (NCAM) is a downstream target of NeuroD1, whose impaired expression mirrors loss of NeuroD1. TrkB and NCAM may be therapeutic targets for aggressive neuroendocrine cancers that express NeuroD1.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Neoplasias Pulmonares/fisiopatología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptor trkB/metabolismo , Carcinoma Pulmonar de Células Pequeñas/fisiopatología , Análisis de Varianza , Animales , Carbazoles , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Furanos , Humanos , Immunoblotting , Inmunoprecipitación , Luciferasas , Neoplasias Pulmonares/metabolismo , Ratones , Análisis por Micromatrices , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Carcinoma Pulmonar de Células Pequeñas/metabolismoRESUMEN
We used CDK4/hTERT-immortalized normal human bronchial epithelial cells (HBEC) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, and MYC) and followed the stepwise transformation of HBECs to full malignancy. This model showed that: (i) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRAS(V12), and c-MYC) is sufficient for full tumorigenic conversion of HBECs; (ii) genetically identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; (iii) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; (iv) high levels of KRAS(V12) are required for full malignant transformation of HBECs, however, prior loss of p53 function is required to prevent oncogene-induced senescence; (v) overexpression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRAS(V12); (vi) growth of parental HBECs in serum-containing medium induces differentiation, whereas growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); (vii) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; (viii) an mRNA signature derived by comparing tumorigenic versus nontumorigenic clones was predictive of outcome in patients with lung cancer. Collectively, our findings show that this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.
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Bronquios/patología , Carcinogénesis/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Animales , Carcinogénesis/genética , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Senescencia Celular , Transición Epitelial-Mesenquimal , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Modelos Biológicos , Proteínas Mutantes/metabolismo , Adhesión en Parafina , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Fijación del Tejido , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismoRESUMEN
Although cyclooxygenase-2 (COX-2) inhibitors, such as the late stage development drug apricoxib, exhibit antitumor activity, their mechanisms of action have not been fully defined. In this study, we characterized the mechanisms of action of apricoxib in HT29 colorectal carcinoma. Apricoxib was weakly cytotoxic toward naive HT29 cells in vitro but inhibited tumor growth markedly in vivo. Pharmacokinetic analyses revealed that in vivo drug levels peaked at 2-4 µM and remained sufficient to completely inhibit prostaglandin E(2) production, but failed to reach concentrations cytotoxic for HT29 cells in monolayer culture. Despite this, apricoxib significantly inhibited tumor cell proliferation and induced apoptosis without affecting blood vessel density, although it did promote vascular normalization. Strikingly, apricoxib treatment induced a dose-dependent reversal of epithelial-mesenchymal transition (EMT), as shown by robust upregulation of E-cadherin and the virtual disappearance of vimentin and ZEB1 protein expression. In vitro, either anchorage-independent growth conditions or forced EMT sensitized HT29 and non-small cell lung cancer cells to apricoxib by 50-fold, suggesting that the occurrence of EMT may actually increase the dependence of colon and lung carcinoma cells on COX-2. Taken together, these data suggest that acquisition of mesenchymal characteristics sensitizes carcinoma cells to apricoxib resulting in significant single-agent antitumor activity.
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Antineoplásicos/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Transición Epitelial-Mesenquimal , Pirroles/farmacología , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Dinoprostona/biosíntesis , Femenino , Células HT29 , Humanos , Ratones , Ratones Desnudos , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
BACKGROUND: Primary tumor recurrence commonly occurs after surgical resection of lung squamous cell carcinoma (SCC). Little is known about the genes driving SCC recurrence. METHODS: We used array comparative genomic hybridization (aCGH) to identify genes affected by copy number alterations that may be involved in SCC recurrence. Training and test sets of resected primary lung SCC were assembled. aCGH was used to determine genomic copy number in a training set of 62 primary lung SCCs (28 with recurrence and 34 with no evidence of recurrence) and the altered copy number of candidate genes was confirmed by quantitative PCR (qPCR). An independent test set of 72 primary lung SCCs (20 with recurrence and 52 with no evidence of recurrence) was used for biological validation. mRNA expression of candidate genes was studied using qRT-PCR. Candidate gene promoter methylation was evaluated using methylation microarrays and Sequenom EpiTYPER analysis. RESULTS: 18q22.3 loss was identified by aCGH as being significantly associated with recurrence (pâ=â0.038). Seven genes within 18q22.3 had aCGH copy number loss associated with recurrence but only SOCS6 copy number was both technically replicated by qPCR and biologically validated in the test set. SOCS6 copy number loss correlated with reduced mRNA expression in the study samples and in the samples with copy number loss, there was a trend for increased methylation, albeit non-significant. Overall survival was significantly poorer in patients with SOCS6 loss compared to patients without SOCS6 loss in both the training (30 vs. 43 months, pâ=â0.023) and test set (27 vs. 43 months, pâ=â0.010). CONCLUSION: Reduced copy number and mRNA expression of SOCS6 are associated with disease recurrence in primary lung SCC and may be useful prognostic biomarkers.
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Carcinoma de Células Escamosas/genética , Hibridación Genómica Comparativa , Neoplasias Pulmonares/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/cirugía , Cromosomas Humanos/genética , Cromosomas Humanos Par 18/genética , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , Femenino , Estudios de Seguimiento , Dosificación de Gen/genética , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Estudios de Asociación Genética , Genoma Humano/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recurrencia , Reproducibilidad de los Resultados , Células Tumorales CultivadasRESUMEN
BACKGROUND: Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non-small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting. METHODS: We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan-Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided. RESULTS: Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor-dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers. CONCLUSIONS: Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.