RESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer related deaths and its incidence is highly correlated with cigarette smoking. Nicotine, the addictive component of tobacco smoke, cannot initiate tumors, but can promote proliferation, migration, and invasion of cells in vitro and promote tumor growth and metastasis in vivo. This nicotine-mediated tumor promotion is facilitated through the activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit. More recently, nicotine has been implicated in promoting self-renewal of stem-like side-population cells from lung cancers. This subpopulation of cancer stem-like cells has been implicated in tumor initiation, generation of the heterogeneous tumor population, metastasis, dormancy, and drug resistance. Here we describe the molecular events driving nicotine and e-cigarette extract mediated stimulation of self-renewal of stem-like cells from non-small cell lung cancer. METHODS: Experiments were conducted using A549 and H1650 non-small cell lung cancer cell lines and human mesenchymal stem cells according to protocols described in this paper. 2 µM nicotine or e-cigarette extracts was used in all relevant experiments. Biochemical analysis using western blotting, transient transfections, RT-PCR and cell biological analysis using double immunofluorescence and confocal microscopy, as well as proximity ligation assays were conducted. RESULTS: Here we demonstrate that nicotine can induce the expression of embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties in non-small cell lung adenocarcinoma (NSCLC) cells. We further demonstrate that this occurs through a nAChR-Yap1-E2F1 signaling axis downstream of Src and Yes kinases. Our data suggests Oct4 may also play a role in this process. Over the past few years, electronic cigarettes (e-cigarettes) have been promoted as healthier alternatives to traditional cigarette smoking as they do not contain tobacco; however, they do still contain nicotine. Hence we have investigated whether e-cigarette extracts can enhance tumor promoting properties similar to nicotine; we find that they can induce expression of Sox2 as well as mesenchymal markers and enhance migration and stemness of NSCLC cells. CONCLUSIONS: Our findings shed light on novel molecular mechanisms underlying the pathophysiology of smoking-related lung cancer in the context of cancer stem cell populations, and reveal new pathways involved that could potentially be exploited therapeutically.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Sistemas Electrónicos de Liberación de Nicotina , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Células Madre Neoplásicas/metabolismo , Factores de Transcripción SOXB1/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Autorrenovación de las Células/genética , Simulación por Computador , Factor de Transcripción E2F1 , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Nicotina/farmacología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , Receptores Nicotínicos/metabolismo , Factores de Transcripción , Proteínas Señalizadoras YAP , Familia-src Quinasas/metabolismoRESUMEN
The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Dacarbazina/análogos & derivados , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Antígenos de Neoplasias/genética , Antineoplásicos/farmacología , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidad , Camptotecina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estudios de Cohortes , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Dacarbazina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Glioblastoma/diagnóstico , Glioblastoma/mortalidad , Humanos , Masculino , Proteínas de Unión a Poli-ADP-Ribosa , Pronóstico , ARN Mensajero/metabolismo , TemozolomidaRESUMEN
Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin α2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = <0.0001 and <0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.
Asunto(s)
Glioblastoma/diagnóstico , Haptoglobinas/análisis , Haptoglobinas/fisiología , Proteómica/métodos , Animales , Astrocitos/química , Astrocitos/patología , Biomarcadores de Tumor/sangre , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Haptoglobinas/genética , Humanos , Melanoma/química , Melanoma/patología , Ratones , Regulación hacia ArribaRESUMEN
BACKGROUND: The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM). METHODS: Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients. RESULTS: Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis. CONCLUSIONS: Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs. IMPACT: Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis.
Asunto(s)
Astrocitoma/sangre , Biomarcadores de Tumor/sangre , Neoplasias Encefálicas/sangre , Glioblastoma/sangre , Osteopontina/sangre , Adolescente , Adulto , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Osteopontina/genética , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Adulto JovenRESUMEN
The prognosis of patients with glioblastoma, the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy. Genetic heterogeneity of glioblastoma warrants extensive studies in order to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. Serum biomarkers have the potential to revolutionize the process of cancer diagnosis, grading, prognostication and treatment response monitoring. Besides having the advantage that serum can be obtained through a less invasive procedure, it contains molecules at an extraordinary dynamic range of ten orders of magnitude in terms of their concentrations. While the conventional methods, such as 2DE, have been in use for many years, the ability to identify the proteins through mass spectrometry techniques such as MALDI-TOF led to an explosion of interest in proteomics. Relatively new high-throughput proteomics methods such as SELDI-TOF and protein microarrays are expected to hasten the process of serum biomarker discovery. This review will highlight the recent advances in the proteomics platform in discovering serum biomarkers and the current status of glioma serum markers. We aim to provide the principles and potential of the latest proteomic approaches and their applications in the biomarker discovery process. Besides providing a comprehensive list of available serum biomarkers of glioma, we will also propose how these markers will revolutionize the clinical management of glioma patients.