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Despite many years of research efforts, lung cancer still remains the leading cause of cancer deaths worldwide. Objective of this study was to set up a platform of non-small cell lung cancer patient derived xenografts (PDXs) faithfully representing primary tumour characteristics and offering a unique tool for studying effectiveness of therapies at a preclinical level. We established 38 PDXs with a successful take rate of 39.2%. All models closely mirrored parental tumour characteristics although a selective pressure for solid patterns, vimentin expression and EMT was observed in several models. An increased grafting rate for tumours derived from patients with worse outcome (p = 0.006), higher stage (p = 0.038) and higher CD133+/CXCR4+/EpCAM- stem cell content (p = 0.019) was observed whereas a trend towards an association with SUVmax higher than 8 (p = 0.084) was detected. Kaplan Meier analyses showed a significantly worse (p = 0.0008) overall survival at 5 years in patients with grafted vs not grafted PDXs also after adjusting for tumour stage. Moreover, for 63.2% models, grafting was reached before clinical recurrence occurred. Our findings strengthen the relevance of PDXs as useful preclinical models closely reflecting parental patients tumours and highlight PDXs establishment as a functional testing of lung cancer aggressiveness and personalized therapies.
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Neoplasias Pulmonares/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Anciano , Animales , Proliferación Celular , Femenino , Estudios de Seguimiento , Humanos , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica , Análisis de SupervivenciaRESUMEN
Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.
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Células Madre Mesenquimatosas/fisiología , Músculo Esquelético/patología , Proteínas Tirosina Quinasas Receptoras/genética , Rabdomiosarcoma/diagnóstico , Rabdomiosarcoma/genética , Adolescente , Adulto , Quinasa de Linfoma Anaplásico , Diferenciación Celular , Línea Celular Tumoral , Niño , Preescolar , Estudios de Cohortes , Femenino , Reordenamiento Génico , Humanos , Lactante , Recién Nacido , Masculino , Metástasis de la Neoplasia , ARN Interferente Pequeño/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Resultado del Tratamiento , Adulto JovenRESUMEN
INTRODUCTION: Genetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of non-small cell lung cancers. METHODS: Ninety-eight PSCs were assessed for MNNG HOS Transforming gene (MET) and anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescence in situ hybridization (FISH) and for relevant protein expression by immunohistochemical analysis, also taking advantage of phosphorylated (p-) antibodies. Moreover, levels of ALK and MET mRNA were also determined by real-time polymerase chain reaction and Western blot analysis for downstream activation pathways involving p-MET, p-protein kinase B, p-mitogen-activated protein kinase, p-SRC proto-oncogene tyrosine-protein kinase, and p-focal adhesion kinase (p-FAK). RESULTS: MET amplification was detected by FISH in 25 of 98 PSCs (25.6%) and ALK amplification (but not the relevant rearrangement) was found in 16 of 98 (16.3%), with all ALK-amplified tumors also showing MET amplification (p < 0.0001). Nine PSCs, however, showed MET amplification without any ALK gene alteration. ALK protein expression was always lacking, whereas MET and p-MET were confined to the relevant amplified tumors only. Increased levels of ALK and MET mRNA were detectable in tumors with no direct relationship between mRNA content, protein expression, or alterations detected by FISH. Western blot assays showed complete activation of downstream signal pathways up to p-SRC proto-oncogene tyrosine-protein kinase, and p-focal adhesion kinase recruitment in MET and ALK-coamplified tumors only, whereas isolated MET amplification, MET and ALK borderline amplification (5%-10% of tumor cells with ≥15 copies of the relevant gene), or negative tumors showing eusomy or chromosome polysomy were confined to p-mitogen-activated protein kinase, p-protein kinase B, and/or p-MET activation. Multivariate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for overall survival (p = 0.140) and disease-free survival (p = 0.060), respectively. CONCLUSIONS: ALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinosarcoma/tratamiento farmacológico , Amplificación de Genes , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundario , Carcinosarcoma/genética , Carcinosarcoma/metabolismo , Carcinosarcoma/secundario , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Mutación/genética , Invasividad Neoplásica , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFß1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFß1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Factores de Transcripción/metabolismo , Microambiente Tumoral , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Plasticidad de la Célula , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Femenino , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Fenotipo , Cultivo Primario de Células , Factores de Transcripción de la Familia Snail , Factor de Crecimiento Transformador beta1/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.
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Antígenos CD/biosíntesis , Antígenos de Neoplasias/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/genética , Moléculas de Adhesión Celular/biosíntesis , Glicoproteínas/biosíntesis , Neoplasias Pulmonares/genética , Receptores CXCR4/biosíntesis , Antígeno AC133 , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD/genética , Antígenos de Neoplasias/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Linaje de la Célula , Cisplatino/administración & dosificación , Supervivencia sin Enfermedad , Molécula de Adhesión Celular Epitelial , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica/genética , Células Madre Neoplásicas/patología , Péptidos/genética , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacosRESUMEN
INTRODUCTION: FHIT and p53 are the two most commonly altered tumor suppressor genes in lung cancer, and their molecular status regulates sensitivity to anticancer drugs. Although their functions are independent, there is evidence that their pathways might be interconnected, but little is known at the molecular level. METHODS: Microarray profiling of FHIT-transduced lung cancer cells and modulation of FHIT levels by RNA interference in human bronchial cells were used to generate a signature of FHIT-regulated transcripts. Expression of these genes was evaluated by real-time polymerase chain reaction in 55 primary lung cancer samples characterized for FHIT and p53 expression by immunehistochemistry. RESULTS: A signature of FHIT-transcripts, particularly enriched in genes involved in cell cycle control, was identified. This signature showed overlap with p53-regulated genes, indicating possible crosstalk between these proteins. Consistently, transcriptional deregulation after FHIT modulation was higher in p53-negative cells. In primary lung cancers, inactivation of either gene was detected in 48 of 55 cases (87%) and both genes in 23 of 55 (42%) cases, confirming the central role of these pathways. Primary tumors with inactivation of both FHIT and p53 displayed the strongest deregulation of growth-related pathways with high levels of expression of CCNB1, BUB1, CDC6, TOP2A, MCM6, and CENPF. CONCLUSIONS: FHIT and p53 seem to rely on common mediators, and inactivation of both genes results in prominent deregulation of growth-related pathways in lung cancer cell lines and primary tumors. This reveals crosstalk between these proteins and suggests a possible distinctive phenotype for tumors with inactivation of both genes.
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Ácido Anhídrido Hidrolasas/fisiología , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/fisiología , Proteína p53 Supresora de Tumor/fisiología , Ácido Anhídrido Hidrolasas/genética , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133(-) counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.