Nuestros investigadores

Luis Montuenga Badía

Publicaciones científicas más recientes (desde 2010)

Autores: Pérez, José Luis; Sanmamed, M. F.; Bosch, A.; et al.
Revista: CANCER TREATMENT REVIEWS
ISSN 0305-7372  Vol. 53  2017  págs. 79 - 97
The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework the DESIGN guidelines-to-standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field. (C) 2017 The Authors. Published by Elsevier Ltd.
Autores: Pereira,C.; Gimenez-Xavier, P.; Pros, E.; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 23  Nº 12  2017  págs. 3203 - 3213
Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs).Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of ß2-microglobulin (B2M), a component of the HLA class-I complex.Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFN¿/IFN¿. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1.Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies.
Autores: Pérez, José Luis; Sanmamed, M.F.; Bosch, A.; et al.
Revista: CANCER TREATMENT REVIEWS
ISSN 0305-7372  Vol. 53  2017  págs. 79-97
The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework-the DESIGN guidelines-to standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field.
Autores: Ajona, Daniel; Ortiz Espinosa, S.; Moreno, H. ; et al.
Revista: CANCER DISCOVERY
ISSN 2159-8274  Vol. 7  Nº 7  2017  págs. 694 - 703
Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer. SIGNIFICANCE: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. (C) 2017 AACR.
Autores: de Torres, Juan Pablo; Bastarrika, Gorka; et al.
Revista: EUROPEAN RESPIRATORY JOURNAL
ISSN 0903-1936  Vol. 49  Nº 1  2017  págs. 1601521
Autores: Agorreta, J; Garmendia, Irati; Pajares, María Josefa; et al.
Revista: JOURNAL OF THORACIC ONCOLOGY
ISSN 1556-0864  Vol. 12  Nº 1  2017  págs. S446 - S447
Autores: Ajona, Daniel; Moreno, H.; et al.
Revista: JOURNAL OF THORACIC ONCOLOGY
ISSN 1556-0864  Vol. 12  Nº 1  2017  págs. S391 - S391
Autores: Azpilikueta, A.; Agorreta, J; Pérez, José Luis; et al.
Revista: JOURNAL OF THORACIC ONCOLOGY
ISSN 1556-0864  Vol. 11  Nº 4  2016  págs. 524 - 536
INTRODUCTION: Anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non-small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. METHODS: We generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N-nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti-PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. RESULTS: In syngeneic mice, large 12-day-established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti-PD-1, anti-PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti-PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-¿. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. CONCLUSIONS: These promising results advocate the use of combined anti-PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non-small cell lung cancer in the clinical setting.
Autores: Díaz-Lagares, A.; Méndez-González, J.; Sandra Hervas-Stubbs; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 22  Nº 13  2016  págs. 3361-3371
PURPOSE: Lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies. EXPERIMENTAL DESIGN: The identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts. RESULTS: We identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes:BCAT1, CDO1, TRIM58, andZNF177 Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis. CONCLUSION: Minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients.
Autores: Salazar-Degracia, A.; Vilà-Ubach, M.; et al.
Revista: JOURNAL OF TRANSLATIONAL MEDICINE
ISSN 1479-5876  Vol. 14  Nº 1  2016  págs. 244
BACKGROUND: Muscle wasting negatively impacts the progress of chronic diseases such as lung cancer (LC) and emphysema, which are in turn interrelated. OBJECTIVES: We hypothesized that muscle atrophy and body weight loss may develop in an experimental mouse model of lung carcinogenesis, that the profile of alterations in muscle fiber phenotype (fiber type composition and morphometry, muscle structural alterations, and nuclear apoptosis), and in muscle metabolism are similar in both respiratory and limb muscles of the tumor-bearing mice, and that the presence of underlying emphysema may influence those events. METHODS: Diaphragm and gastrocnemius muscles of mice with urethane-induced lung cancer (LC-U) with and without elastase-induced emphysema (E-U) and non-exposed controls (N = 8/group) were studied: fiber type composition, morphometry, muscle abnormalities, apoptotic nuclei (immunohistochemistry), and proteolytic and autophagy markers (immunoblotting) at 20- and 35-week exposure times. In the latter cohort, structural contractile proteins, creatine kinase (CK), peroxisome proliferator-activated receptor (PPAR) expression, oxidative stress, and inflammation were also measured. Body and muscle weights were quantified (baseline, during follow-up, and sacrifice). RESULTS: Compared to controls, in U and E-U mice, whole body, diaphragm and gastrocnemius weights were reduced. Additionally, both in diaphragm and gastrocnemius, muscle fiber cross-sectional areas were smaller, structural abnormalities, autophagy and apoptotic nuclei were increased, while levels of actin, myosin, CK, PPARs, and antioxidants were decreased, and muscle proteolytic markers did not vary among groups. CONCLUSIONS: In this model of lung carcinogenesis with and without emphysema, reduced body weight gain and muscle atrophy were observed in respiratory and limb muscles of mice after 20- and 35-week exposure times most likely through increased nuclear apoptosis and autophagy. Underlying emphysema induced a larger reduction in the size of slow- and fast-twitch fibers in the diaphragm of U and E-U mice probably as a result of the greater inspiratory burden imposed onto this muscle.
Autores: Villalba, M.; López, L.; Redrado M; et al.
Revista: HISTOLOGY AND HISTOPATHOLOGY
ISSN 0213-3911  Vol. 32  Nº 9  2016  págs. 929 - 940
Metastatic spread is responsible for the majority of cancer deaths and identification of metastasis-related therapeutic targets is compulsory. TMPRSS4 is a pro-metastatic druggable transmembrane type II serine protease whose expression has been associated with the development of several cancer types and poor prognosis. To study the role and expression of this protease in cancer, we have developed molecular tools (active recombinant proteins and a polyclonal antibody) that can be used for diagnostic purposes and for testing anti-TMPRSS4 drugs. In addition, we have evaluated TMPRSS4 protein expression in several cancer tissue microarrays (TMAs). Full length and truncated TMPRSS4 recombinant proteins maintained the catalytic activity in two different expression systems (baculovirus and E. coli). Sensitivity of the rabbit polyclonal antisera against TMPRSS4 (ING-pAb) outperformed the antibody most commonly used in clinical settings. Analysis by immunohistochemistry in the different TMAs identified a subset of adenocarcinomas, squamous carcinomas, large cell carcinomas and carcinoids of the lung, which may define aggressive tumors. In conclusion, our biological tools will help the characterization of TMPRSS4 activity and protein expression, as well as the evaluation of anti-TMRSS4 drugs. Future studies should determine the clinical value of assessing TMPRSS4 levels in different types of lung cancer.
Autores: Pajares, María Josefa; Ajona, Daniel; Sharma, Ravi Datta; et al.
Revista: MOLECULAR ONCOLOGY
ISSN 1574-7891  Vol. 10  Nº 9  2016  págs. 1437 - 1449
Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.
Autores: Redrado M; Villalba, M.; et al.
Revista: CANCER LETTERS
ISSN 0304-3835  Vol. 370  Nº 2  2016  págs. 165 - 176
Metastasis involves a series of changes in cancer cells that promote their escape from the primary tumor and colonization to a new organ. This process is related to the transition from an epithelial to a mesenchymal phenotype (EMT). Recently, some authors have shown that migratory cells with an EMT phenotype share properties of cancer stem cells (CSCs), which allow them to form a new tumor mass. The type II transmembrane serine protease TMPRSS4 is highly expressed in some solid tumors, promotes metastasis and confers EMT features to cancer cells. We hypothesized that TMPRSS4 could also provide CSC properties. Overexpression of TMPRSS4 reduces E-cadherin and induces N-cadherin and vimentin in A549 lung cancer cells, supporting an EMT phenotype. These changes are accompanied by enhanced migration, invasion and tumorigenicity in vivo. TMPRSS4 expression was highly increased in a panel of lung cancer cells cultured as tumorspheres (a typical assay to enrich for CSCs). H358 and H441 cells with knocked-down TMPRSS4 levels were significantly less able to form primary and secondary tumorspheres than control cells. Moreover, they showed a lower proportion of ALDH+ cells (examined by FACS analysis) and lower expression of some CSC markers than controls. A549 cells overexpressing TMPRSS4 conferred the opposite phenotype and were also more sensitive to the CSC-targeted drug salinomycin than control cells, but were more resistant to regular chemotherapeutic drugs (cisplatin, gemcitabine and 5-fluorouracil). Analysis of 70 NSCLC samples from patients revealed a very significant correlation between TMPRSS4 expression and CSC markers ALDH (p¿=¿0.0018) and OCT4 (p¿=¿0.0004), suggesting that TMPRSS4 is associated with a CSC phenotype in patients' tumors. These results show that TMPRSS4, in addition to inducing EMT, can also promote CSC features in lung cancer; therefore, CSC-targeting drugs could be an appropriate treatment for TMPRSS4+ tumors.
Autores: Villalba, M.; Redrado M; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 7  Nº 16  2016  págs. 22752 - 22769
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped.
Autores: Vallejo-Diaz, J.; Olazabal-Moran, M.; Cariaga-Martinez, A. E.; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 7  Nº 51  2016  págs. 85063 - 85078
Oncogenic mutations in the PI3K/AKT pathway are present in nearly half of human tumors. Nonetheless, inhibitory compounds of the pathway often induce pathway rebound and tumor resistance. We find that lung squamous cell carcinoma (SQCC), which accounts for similar to 20% of lung cancer, exhibits increased expression of the PI3K subunit PIK3R2, which is at low expression levels in normal tissues. We tested a new approach to interfere with PI3K/AKT pathway activation in lung SQCC. We generated tumor xenografts of SQCC cell lines and examined the consequences of targeting PIK3R2 expression. In tumors with high PIK3R2 expression, and independently of PIK3CA, KRAS, or PTEN mutations, PIK3R2 depletion induced lung SQCC xenograft regression without triggering PI3K/AKT pathway rebound. These results validate the use PIK3R2 interfering tools for the treatment of lung squamous cell carcinoma.
Autores: Romero, J. P.; Aramburu, A.; et al.
Revista: BMC GENOMICS
ISSN 1471-2164  Vol. 17  2016  págs. 467
Background: Alternative splicing (AS) is a major source of variability in the transcriptome of eukaryotes. There is an increasing interest in its role in different pathologies. Before sequencing technology appeared, AS was measured with specific arrays. However, these arrays did not perform well in the detection of AS events and provided very large false discovery rates (FDR). Recently the Human Transcriptome Array 2.0 (HTA 2.0) has been deployed. It includes junction probes. However, the interpretation software provided by its vendor (TAC 3.0) does not fully exploit its potential (does not study jointly the exons and junctions involved in a splicing event) and can only be applied to case-control studies. New statistical algorithms and software must be developed in order to exploit the HTA 2.0 array for event detection. Results: We have developed EventPointer, an R package (built under the aroma. affymetrix framework) to search and analyze Alternative Splicing events using HTA 2.0 arrays. This software uses a linear model that broadens its application from plain case-control studies to complex experimental designs. Given the CEL files and the design and contrast matrices, the software retrieves a list of all the detected events indicating: 1) the type of event (exon cassette, alternative 3', etc.), 2) its fold change and its statistical significance, and 3) the potential protein domains affected by the AS events and the statistical significance of the possible enrichment. Our tests have shown that EventPointer has an extremely low FDR value (only 1 false positive within the tested top-200 events). This software is publicly available and it has been uploaded to GitHub. Conclusions: This software empowers the HTA 2.0 arrays for AS event detection as an alternative to RNA-seq: simplifying considerably the required analysis, speeding it up and reducing the required computational power.
Autores: Rakha, E.; Pajares, María Josefa; Ilie, M.; et al.
Revista: EUROPEAN JOURNAL OF CANCER
ISSN 0959-8049  Vol. 51  Nº 14  2015  págs. 1897 - 1903
Background: Mortality in early stage, resectable lung cancer is sufficiently high to warrant consideration of post-surgical treatment. Novel markers to stratify resectable lung cancer patients may help with the selection of treatment to improve outcome. Methods: Primary tumour tissue from 485 patients, surgically treated for stage I-II lung adenocarcinoma, was analysed for the RNA expression of 31 cell cycle progression (CCP) genes by quantitative polymerase chain reaction (PCR). The expression average, the CCP score, was combined with pathological stage into a prognostic score (PS). Cox proportional hazards regression assessed prediction of 5-year lung cancer mortality above clinical variables. The PS threshold was tested for risk discrimination by the Mantel-Cox log-rank test. Results: The CCP score added significant information above clinical markers (all patients, P = 0.0029; stage I patients, P = 0.013). The prognostic score was a superior predictor of outcome compared to pathological stage alone (PS, P = 0.00084; stage, P = 0.24). Five-year lung cancer mortality was significantly different between the low-risk (90%, 95% confidence interval (CI) 81-95%), and high-risk groups (65%, 95% CI 57-72%), P = 4.2 x 10(-6)). Conclusions: The CCP score is an independent prognostic marker in early stage lung adenocarcinoma. The prognostic score provides superior risk estimates than stage alone. The threefold higher risk in the high-risk group defines a subset of patients that should consider therapeutic choices to improve outcome.
Autores: Aramburu, A.; Pajares, María Josefa; Agorreta, J; et al.
Revista: BMC GENOMICS
ISSN 1471-2164  Vol. 16  2015  págs. 752
Background: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. Results: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). Conclusion: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.
Autores: Pio, R; Agorreta, J; Montuenga, Luis;
Revista: JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
ISSN 0022-5223  Vol. 150  Nº 4  2015  págs. 986 - 992
Objective: The current staging system for lung cancer is not sufficient to accurately identify those patients with early-stage tumors who would benefit from postsurgery chemotherapy. The objective of this study was to validate a prognostic signature based on the expression of 5 RNA (ribonucleic acid) metabolism-related genes. Methods: Five lung cancer microarray datasets, 3 from adenocarcinomas and 2 from squamous cell carcinomas, were analyzed. Kaplan-Meier survival curves and Cox proportional hazards models were used to evaluate the relationship between the classifier and recurrence and survival. Results: Statistically significant differences in relapse-free survival and overall survival were observed when lung adenocarcinoma patients were divided into 3 risk groups. The prognostic information provided by the signature was independent from other demographic and disease variables, including stage. Significant differences in survival were observed between low-and high-risk groups in stage-IB patients: 5-year survival rates ranged from 83% to 100% in the low-risk groups, and from 30% to 71% in the high-risk groups, depending on the dataset. The RNA metabolism score additionally displayed an association with the benefit of adjuvant chemotherapy (P<. 001), suggesting that those patients in the low-risk group are not good candidates for this treatment. Conclusions: The RNA metabolism signature is a prognostic marker that may be useful for predicting survival and optimizing the benefit of adjuvant chemotherapy in patients with lung adenocarcinoma.
Autores: Grbesa, I.; Pajares, María Josefa; Agorreta, J; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 10  Nº 4  2015  págs. e0124670
Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology. Material and Methods Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor. Results High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with nontumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC. Conclusions Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lu
Autores: Monsó, E.; Montuenga, Luis; Sánchez de Cos, J.; et al.
Revista: ARCHIVOS DE BRONCONEUMOLOGIA
ISSN 0300-2896  Vol. 51  Nº 9  2015  págs. 462 - 467
he aim of the Clinical and Molecular Staging of Stage I-IIp Lung Cancer Project is to identify molecular variables that improve the prognostic and predictive accuracy of TMN classification in stage I/IIp non-small cell lung cancer (NSCLC). Clinical data and lung tissue, tumor and blood samples will be collected from 3 patient cohorts created for this purpose. The prognostic protein signature will be validated from these samples, and micro-RNA, ALK, Ros1, Pdl-1, and TKT, TKTL1 y G6PD expression will be analyzed. Tissue inflammatory markers and stromal cell markers will also be analyzed. Methylation of p16, DAPK, RASSF1a, APC and CDH13 genes in the tissue samples will be determined, and inflammatory markers in peripheral blood will also be analyzed. Variables that improve the prognostic and predictive accuracy of TNM in NSCLC by molecular staging may be identified from this extensive analytical panel.
Autores: Ajona, Daniel; Razquin, Cristina; Pastor, M. D.; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 10  Nº 3  2015  págs. e0119878
Molecular markers in bronchial fluids may contribute to the diagnosis of lung cancer. We previously observed a significant increase of C4d-containing complement degradation fragments in bronchoalveolar lavage (BAL) supernatants from lung cancer patients in a cohort of 50 cases and 22 controls (CUN cohort). The present study was designed to determine the diagnostic performance of these complement fragments (hereinafter jointly referred as C4d) in bronchial fluids. C4d levels were determined in BAL supernatants from two independent cohorts: the CU cohort (25 cases and 26 controls) and the HUVR cohort (60 cases and 98 controls). A series of spontaneous sputum samples from 68 patients with lung cancer and 10 controls was also used (LCCCIO cohort). Total protein content, complement C4, complement C5a, and CYFRA 21-1 were also measured in all cohorts. C4d levels were significantly increased in BAL samples from lung cancer patients. The area under the ROC curve was 0.82 (95%CI = 0.71-0.94) and 0.67 (95%CI = 0.58-0.76) for the CU and HUVR cohorts, respectively. In addition, unlike the other markers, C4d levels in BAL samples were highly consistent across the CUN, CU and HUVR cohorts. Interestingly, C4d test markedly increased the sensitivity of bronchoscopy in the two cohorts in which cytological data were available (CUN and HUVR cohorts). Finally, in the LCCCIO cohort, C4d levels were higher in sputum supernatants from patients with lung cancer (area under the ROC curve: 0.7; 95%CI
Autores: Wilson, D. O.; de Torres, Juan Pablo; et al.
Revista: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
ISSN 1073-449X  Vol. 191  Nº 8  2015  págs. 924 - 931
Rationale: Lung cancer (LC) screening using low-dose chest computed tomography is now recommended in several guidelines using the National Lung Screening Trial (NLST) entry criteria (age, 55-74; ¿30 pack-years; tobacco cessation within the previous 15 yr for former smokers). Concerns exist about their lack of sensitivity. Objectives: To evaluate the performance of NLST criteria in two different LC screening studies from Europe and the United States, and to explore the effect of using emphysema as a complementary criterion. Methods: Participants from the Pamplona International Early Lung Action Detection Program (P-IELCAP; n = 3,061) and the Pittsburgh Lung Screening Study (PLuSS; n = 3,638) were considered. LC cumulative frequencies, incidence densities, and annual detection rates were calculated in three hypothetical cohorts, including subjects whometNLST criteria alone, those withcomputed tomography-detected emphysema, and those who met NLST criteria and/or had emphysema. Measurements and Main Results: Thirty-six percent and 59% of P-IELCAP and PLuSS participants, respectively, met NLST criteria. Among these, higher LC incidence densities and detection rates were observed. However, applying NLST criteria to our original cohorts would miss asmany as 39% of all LC. Annual screening of subjects meeting either NLST criteria or having emphysema detected most cancers (88% and 95% of incident LC of P-IELCAP and PLuSS, respectively) despite reducing the number of screened participants by as much as 52%. Conclusions: LC screening based solely on NLST criteria could miss a significant number of LC cases. Combining NLST criteria and emphysema to select screening candidates results in higher LC detection rates and a lower number of cancers missed.
Autores: de Torres, Juan Pablo; et al.
Revista: ARCHIVOS DE BRONCONEUMOLOGIA
ISSN 0300-2896  Vol. 51  Nº 4  2015  págs. 169 - 176
The experience in Spain's longest lung cancer screening program is comparable to what has been described in the rest of Europe, and confirms the feasibility and efficacy of lung cancer screening using LDCT.
Autores: Redrado M; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 6  Nº 29  2015  págs. 27288 - 27303
The spread of lung cancer cells to distant sites represents a common event associated with poor prognosis. A fraction of tumor cells named cancer stem cells (CSCs) have the ability to overcome therapeutic stress and remain quiescent. However, whether these CSCs have also the capacity to initiate and sustain metastasis remains unclear. Here, we used tumor sphere cultures (TSC) isolated from mouse and human lung cancer models to enrich for CSCs, and assessed their metastatic potential as compared to non-CSCs. As expected, TSC overexpressed a variety of stem cell markers and displayed chemoresistance. The CSC phenotype of TSC was confirmed by their higher growth ability in soft agar and tumorigenic potential in vivo, despite their reduced in vitro cell growth kinetics. Surprisingly, the appearance of spontaneous lung metastases was strongly delayed in mice injected with TSC as compared to non-TSC cells. Similarly, this finding was confirmed in several other models of metastasis, an effect associated with a retarded colonization activity. Interestingly, such delay correlated with a quiescent phenotype whose underlined mechanisms included an increase in p27 protein and lower phospho-ERK1/2 levels. Thus, these data suggest that cells enriched for CSC properties display an impaired metastatic activity, a finding with potential clinical implications.
Autores: Ajona, Daniel; Pajares, María Josefa; Chiara, M. D.; et al.
Revista: ORAL DISEASES
ISSN 1354-523X  Vol. 21  Nº 7  2015  págs. 899 - 904
OBJECTIVE: Complement C4d-containing fragments have been proposed as diagnostic markers for lung cancer. The purpose of this study was to evaluate the presence of C4d in oropharyngeal (OPSCC) and oral (OSCC) squamous cell carcinomas. SUBJECTS AND METHODS: C4d staining was analyzed by immunohistochemistry in 244 OPSCC surgical specimens. C4d levels were quantified by ELISA in resting saliva samples from 48 patients with oral leukoplakia and 62 with OSCC. Plasma samples from 21 patients with leukoplakia and 30 with oral carcinoma were also studied. RESULTS: C4d staining in OPSCC specimens was associated with nodal invasion (P = 0.001), histopathologic grade (P = 0.014), disease stage (P = 0.040), and focal-adhesion kinase expression (P < 0.001). No association was found between C4d and prognosis. Saliva C4d levels were higher in patients with oral cancer than in subjects with leukoplakia (0.07 ± 0.07 vs 0.04 ± 0.03 ¿g ml(-1) , P = 0.003). The area under the ROC curve was 0.63 (95%CI: 0.55-0.71). Salivary C4d levels in stage IV patients were higher than in patients with earlier stages (P = 0.028) and correlated with tumor size (P = 0.045). Plasma C4d levels also correlated with salivary C4d levels (P = 0.041), but differences between patients with oral cancer and subjects with leukoplakia were not significant (1.26 ± 0.59 vs 1.09 ± 0.39 ¿g ml(-1) , P = 0.232). CONCLUSION: C4d-containing fragments are detected in oral primary tumors and are increased in saliva from patients with OSCC.
Autores: Pajares, María Josefa; Agorreta, J; Behrens, C.; et al.
Revista: BRITISH JOURNAL OF CANCER
ISSN 0007-0920  Vol. 110  Nº 6  2014  págs. 1545 - 1551
Background: Transforming growth factor beta-induced protein (TGFBI) is a secreted protein that mediates cell anchoring to the extracellular matrix. This protein is downregulated in lung cancer, and when overexpressed, contributes to apoptotic cell death. Using a small series of stage IV non-small cell lung cancer (NSCLC) patients, we previously suggested the usefulness of TGFBI as a prognostic and predictive factor in chemotherapy-treated late-stage NSCLC. In order to validate and extend these results, we broaden the analysis and studied TGFBI expression in a large series of samples obtained from stage I-IV NSCLC patients. Methods: TGFBI expression was assessed by immunohistochemistry in 364 completely resected primary NSCLC samples: 242 adenocarcinomas (ADCs) and 122 squamous cell carcinomas (SCCs). Kaplan-Meier curves, log-rank tests and the Cox proportional hazards model were used to analyse the association between TGFBI expression and survival. Results: High TGFBI levels were associated with longer overall survival (OS, P < 0.001) and progression-free survival (PFS, P < 0.001) in SCC patients who received adjuvant platinium-based chemotherapy. Moreover, multivariate analysis demonstrated that high TGFBI expression is an independent predictor of better survival in patients (OS: P = 0.030 and PFS: P = 0.026). Conclusions: TGFBI may be useful for the identification of a subset of NSCLC who may benefit from adjuvant therapy.
Autores:  et al.
Revista: MOLECULAR ONCOLOGY
ISSN 1574-7891  Vol. 8  Nº 2  2014  págs. 196 - 206
Lung adenocarcinoma (ADC) is the most common lung cancer subtype and presents a high mortality rate. Clinical recurrence is often associated with the emergence of metastasis and treatment resistance. The purpose of this study was to identify genes with high prometastatic activity which could potentially account for treatment resistance. Global transcriptomic profiling was performed by robust microarray analysis in highly metastatic subpopulations. Extensive in vitro and in vivo functional studies were achieved by overexpression and by silencing gene expression. We identified the small GTPase RHOB as a gene that promotes early and late stages of metastasis in ADC. Gene silencing of RHOB prevented metastatic activity in a systemic murine model of bone metastasis. These effects were highly dependent on tumor-host interactions. Clinical analysis revealed a marked association between high RHOB levels and poor survival. Consistently, high RHOB levels promote metastasis progression, taxane-chemoresistance, and contribute to the survival advantage to ¿-irradiation. We postulate that RHOB belongs to a novel class of "genes of recurrence" that have a dual role in metastasis and treatment resistance.
Autores: Romero, O. A.; Torres-Diz, M.; Pros, E.; et al.
Revista: CANCER DISCOVERY
ISSN 2159-8274  Vol. 4  Nº 3  2014  págs. 292 - 303
Our knowledge of small cell lung cancer (SCLC) genetics is still very limited, amplification of L-MYC, N-MYC, and C-MYC being some of the well-established gene alterations. Here, we report our discovery of tumor-specific inactivation of the MYC-associated factor X gene, MAX, in SCLC. MAX inactivation is mutually exclusive with alterations of MYC and BRG1, the latter coding for an ATPase of the switch/sucrose nonfermentable (SWI/SNF) complex. We demonstrate that BRG1 regulates the expression of MAX through direct recruitment to the MAX promoter, and that depletion of BRG1 strongly hinders cell growth, specifically in MAX-deficient cells, heralding a synthetic lethal interaction. Furthermore, MAX requires BRG1 to activate neuroendocrine transcriptional programs and to upregulate MYC targets, such as glycolysis-related genes. Finally, inactivation of the MAX dimerization protein, MGA, was also observed in both non-small cell lung cancer and SCLC. Our results provide evidence that an aberrant SWI/SNF-MYC network is essential for lung cancer development.
Autores: Agorreta, J; Hu, J.; Liu, D.; et al.
Revista: MOLECULAR CANCER RESEARCH
ISSN 1541-7786  Vol. 12  Nº 5  2014  págs. 660 - 669
The TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance and protection from apoptosis in colorectal and prostate cancer. Here, the effect of TRAP1 ablation on cell proliferation, survival, apoptosis, and mitochondrial function was determined in non-small cell lung cancer (NSCLC). In addition, the prognostic value of TRAP1 was evaluated in patients with NSCLC. These results demonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1 downregulation impairs mitochondrial functions such as ATP production and mitochondrial membrane potential as measured by TMRM (tetramethylrhodamine methylester) uptake, but it does not affect mitochondrial density or mitochondrial morphology. The effect of TRAP1 silencing on apoptosis, analyzed by flow cytometry and immunoblot expression (cleaved PARP, caspase-9, and caspase-3) was cell line and context dependent. Finally, the prognostic potential of TRAP1 expression in NSCLC was ascertained via immunohistochemical analysis which revealed that high TRAP1 expression was associated with increased risk of disease recurrence (univariate analysis, P = 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085-6.012; P = 0.03). In conclusion, these results demonstrate that TRAP1 impacts the viability of NSCLC cells, and that its expression is prognostic in NSCLC.
Autores: Freire, Francisco Javier; Pajares, María Josefa; et al.
Revista: INTERNATIONAL JOURNAL OF CANCER
ISSN 0020-7136  Vol. 135  Nº 11  2014  págs. 2516 - 2527
New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition), accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.
Autores: Sharma, Ravi Datta; Pajares, María Josefa; Montuenga, Luis; et al.
Revista: CANCER RESEARCH
ISSN 0008-5472  Vol. 74  Nº 4  2014  págs. 1105 - 1115
Abnormal alternative splicing has been associated with cancer. Genome-wide microarrays can be used to detect differential splicing events. In this study, we have developed ExonPointer, an algorithm that uses data from exon and junction probes to identify annotated cassette exons. We used the algorithm to profile differential splicing events in lung adenocarcinoma A549 cells after downregulation of the oncogenic serine/arginine-rich splicing factor 1 (SRSF1). Data were generated using two different microarray platforms. The PCR-based validation rate of the top 20 ranked genes was 60% and 100%. Functional enrichment analyses found a substantial number of splicing events in genes related to RNA metabolism. These analyses also identified genes associated with cancer and developmental and hereditary disorders, as well as biologic processes such as cell division, apoptosis, and proliferation. Most of the top 20 ranked genes were validated in other adenocarcinoma and squamous cell lung cancer cells, with validation rates of 80% to 95% and 70% to 75%, respectively. Moreover, the analysis allowed us to identify four genes, ATP11C, IQCB1, TUBD1, and proline-rich coiled-coil 2C (PRRC2C), with a significantly different pattern of alternative splicing in primary non-small cell lung tumors compared with normal lung tissue. In the case of PRRC2C, SRSF1 downregulation led to the skipping of an exon overexpressed in primary lung tumors. Specific siRNA downregulation of the exon-containing var
Autores: Larzábal, Leyre; De Aberasturi, A.L.; Redrado M; et al.
Revista: BRITISH JOURNAL OF CANCER
ISSN 0007-0920  Vol. 110  Nº 3  2014  págs. 764 - 774
BACKGROUND: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin ¿5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin ¿5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin ¿5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin ¿5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.
Autores: Larráyoz, Marta; Pio, R; Pajares, María Josefa; et al.
Revista: EMBO MOLECULAR MEDICINE
ISSN 1757-4684  Vol. 6  Nº 4  2014  págs. 539 - 550
The vascular endothelial growth factor (VEGF) pathway is a clinically validated antiangiogenic target for non-small cell lung cancer (NSCLC). However, some contradictory results have been reported on the biological effects of antiangiogenic drugs. In order to evaluate the efficacy of these drugs in NSCLC histological subtypes, we analyzed the anticancer effect of two anti-VEGFR2 therapies (sunitinib and DC101) in chemically induced mouse models and tumorgrafts of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Antiangiogenic treatments induced vascular trimming in both histological subtypes. In ADC tumors, vascular trimming was accompanied by tumor stabilization. In contrast, in SCC tumors, antiangiogenic therapy was associated with disease progression and induction of tumor proliferation. Moreover, in SCC, anti-VEGFR2 therapies increased the expression of stem cell markers such as aldehyde dehydrogenase 1A1, CD133, and CD15, independently of intratumoral hypoxia. In vitro studies with ADC cell lines revealed that antiangiogenic treatments reduced pAKT and pERK signaling and inhibited proliferation, while in SCC-derived cell lines the same treatments increased pAKT and pERK, and induced survival. In conclusion, this study evaluates for the first time the effect of antiangiogenic drugs in lung SCC murine models in vivo and sheds light on the contradictory results of antiangiogenic therapies in NSCLC.
Autores: Ajona, Daniel; Pajares, María Josefa; et al.
Revista: JOURNAL OF THE NATIONAL CANCER INSTITUTE
ISSN 1460-2105  Vol. 105  Nº 18  2013  págs. 1385 - 1393
BACKGROUND: There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. METHODS: We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test. RESULTS: Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 ± 0.87 vs 0.16 ± 0.11 µg/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 ± 2.02 vs 1.86 ± 0.95 µg/mL, P < 0.001; 3.18 ± 3.20 vs 1.13 ± 0.69 µg/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). CONCLUSIONS: Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.
Autores: Garasa, S.; Pajares, María Josefa; et al.
Revista: INTERNATIONAL JOURNAL OF CANCER
ISSN 0020-7136  Vol. 132  Nº 9  2013  págs. 1986 - 1995
Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.
Autores: Sandoval, J.; Mendez Gonzalez, J.; Nadal, E.; et al.
Revista: JOURNAL OF CLINICAL ONCOLOGY
ISSN 0732-183X  Vol. 31  Nº 32  2013  págs. 4140 - 4147
PURPOSE: Non-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. PATIENTS AND METHODS: A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. RESULTS: Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). CONCLUSION: The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.
Autores: de Torres, Juan Pablo; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 4  2013  págs. e60260
Current or former smokers expressing a well-defined disease characteristic such as emphysema, has a specific plasma cytokine profile. This includes a decrease of cytokines mainly implicated in activation of apoptosis or decrease of immunosurveillance. This information should be taken into account when evaluated patients with tobacco respiratory diseases
Autores: Bastarrika, Gorka; Agorreta, J; et al.
Revista: MEDICAL IMAGE ANALYSIS
ISSN 1361-8423  Vol. 17  Nº 8  2013  págs. 1095-1105
We present and evaluate an automatic and quantitative method for the complex task of characterizing individual nodule volumetric progression in a longitudinal mouse model of lung cancer. Fourteen A/J mice received an intraperitoneal injection of urethane. Respiratory-gated micro-CT images of the lungs were acquired at 8, 22, and 37 weeks after injection. A radiologist identified a total of 196, 585 and 636 nodules, respectively. The three micro-CT image volumes from every animal were then registered and the nodules automatically matched with an average accuracy of 99.5%. All nodules detected at week 8 were tracked all the way to week 37, and volumetrically segmented to measure their growth and doubling rates. 92.5% of all nodules were correctly segmented, ranging from the earliest stage to advanced stage, where nodule segmentation becomes more challenging due to complex anatomy and nodule overlap. Volume segmentation was validated using a foam lung phantom with embedded polyethylene microspheres. We also correlated growth rates with nodule phenotypes based on histology, to conclude that the growth rate of malignant tumors is significantly higher than that of benign lesions. In conclusion, we present a turnkey solution that combines longitudinal imaging with nodule matching and volumetric nodule segmentation resulting in a powerful tool for preclinical research.
Autores: Marcos, T. ; Serrano, Diego; et al.
Revista: INTEGRATIVE BIOLOGY
ISSN 1093-4391  Vol. 5  Nº 2  2013  págs. 402-413
Dyskerin is one of the three subunits of the telomerase ribonucleoprotein (RNP) complex. Very little is known about the role of dyskerin in the biology of the telomeres in cancer cells. In this study, we use a quantitative, multiscale 3D image-based in situ method and several molecular techniques to show that dyskerin is overexpressed in lung cancer cell lines. Furthermore, we show that dyskerin expression correlates with telomere length both at the cell population level--cells with higher dyskerin expression have short telomeres--and at the single cell level--the shortest telomeres of the cell are spatially associated with areas of concentration of dyskerin proteins. Using this in vitro model, we also show that exogenous increase in dyskerin expression confers resistance to telomere shortening caused by a telomerase inactivating drug. Finally, we show that resistance is achieved by the recovery of telomerase activity associated with dyskerin. In summary, using a novel multiscale image-based in situ method, we show that, in lung cancer cell lines, dyskerin responds to continuous telomere attrition by increasing the telomerase RNP activity, which in turn provides resistance to telomere shortening.
Autores: Freire, Francisco Javier; Ajona, Daniel; et al.
Revista: NEOPLASIA
ISSN 1522-8002  Vol. 15  Nº 8  2013  págs. 913 - 924
The association between inflammation and lung tumor development has been clearly demonstrated. However, little is known concerning the molecular events preceding the development of lung cancer. In this study, we characterize a chemically induced lung cancer mouse model in which lung cancer developed in the presence of silicotic chronic inflammation. Silica-induced lung inflammation increased the incidence and multiplicity of lung cancer in mice treated with N-nitrosodimethylamine, a carcinogen found in tobacco smoke. Histologic and molecular analysis revealed that concomitant chronic inflammation contributed to lung tumorigenesis through induction of preneoplastic changes in lung epithelial cells. In addition, silica-mediated inflammation generated an immunosuppressive microenvironment in which we observed increased expression of programmed cell death protein 1 (PD-1), transforming growth factor-beta 1, monocyte chemotactic protein 1 (MCP-1), lymphocyte-activation gene 3 (LAG3), and forkhead box P3 (FOXP3), as well as the presence of regulatory T cells. Finally, the K-RAS mutational profile of the tumors changed from Q61R to G12D mutations in the inflammatory milieu. In summary, we describe some of the early molecular changes associated to lung carcinogenesis in a chronic inflammatory microenvironment and provide novel information concerning the mechanisms underlying the formation and the fate of preneoplastic lesions in the silicotic lung.
Autores: Corrales, L; Ajona, D; Rafail, S; et al.
Revista: The Journal of Immunology
ISSN 0022-1767  Vol. 189  Nº 9  2012  págs. 4674 - 4683
The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.
Autores: Vallés, Iñaki; Pajares, María Josefa; Segura, Victoriano; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 7  Nº 8  2012  págs. e42086
Lung cancer is a leading cause of cancer death worldwide. Several alterations in RNA metabolism have been found in lung cancer cells; this suggests that RNA metabolism-related molecules are involved in the development of this pathology. In this study, we searched for RNA metabolism-related genes that exhibit different expression levels between normal and tumor lung tissues. We identified eight genes differentially expressed in lung adenocarcinoma microarray datasets. Of these, seven were up-regulated whereas one was down-regulated. Interestingly, most of these genes had not previously been associated with lung cancer. These genes play diverse roles in mRNA metabolism: three are associated with the spliceosome (ASCL3L1, SNRPB and SNRPE), whereas others participate in RNA-related processes such as translation (MARS and MRPL3), mRNA stability (PCBPC1), mRNA transport (RAE), or mRNA editing (ADAR2, also known as ADARB1). Moreover, we found a high incidence of loss of heterozygosity at chromosome 21q22.3, where the ADAR2 locus is located, in NSCLC cell lines and primary tissues, suggesting that the downregulation of ADAR2 in lung cancer is associated with specific genetic losses. Finally, in a series of adenocarcinoma patients, the expression of five of the deregulated genes (ADAR2, MARS, RAE, SNRPB and SNRPE) correlated with prognosis. Taken together, these results support the hypothesis that changes in RNA metabolism are involved in the pathogenesis of lung cancer, and identify new potential targets for the treatment of this disease.
Autores: Thunnissen, FB; Prinsen, C; Hol, B.; et al.
Revista: Lung Cancer
ISSN 0169-5002  Vol. 75  Nº 2  2012  págs. 156 - 160
Autores: Pajares, María Josefa; Agorreta, J; Larráyoz, Marta; et al.
Revista: Journal of Clinical Oncology
ISSN 0732-183X  Vol. 30  Nº 10  2012  págs. 1129 - 1136
Purpose: Antiangiogenic therapies targeting the vascular endothelial growth factor (VEGF) pathway have yielded more modest clinical benefit to patients with non-small-cell lung cancer (NSCLC) than initially expected. Clinical data suggest a distinct biologic role of the VEGF pathway in the different histologic subtypes of lung cancer. To clarify the influence of histologic differentiation in the prognostic relevance of VEGF-mediated signaling in NSCLC, we performed a concomitant analysis of the expression of three key elements of the VEGF pathway in the earliest stages of the following two principal histologic subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Patients and Methods: We evaluated tumor cell expression of VEGF, VEGF receptor (VEGFR) 1, and VEGFR2 using automatic immunostaining in a series of 298 patients with early-stage NSCLC recruited as part of the multicenter European Early Lung Cancer Detection Group project. A score measuring the VEGF signaling pathway was calculated by adding the tumor cell expression value of VEGF and its two receptors. The results were validated in two additional independent cohorts of patients with NSCLC. Results: The combination of high VEGF, VEGFR1, and VEGFR2 protein expression was associated with lower risk of disease progression in early SCC (univariate analysis, P = .008; multivariate analysis, hazard ratio, 0.62; 95% CI, 0.42 to 0.92; P = .02). The results were validated in two independent patient cohorts, confirming the favorable prognostic value of high VEGF signaling score in early lung SCC. Conclusion: Our results clearly indicate that the combination of high expression of the three key elements in the VEGF pathway is associated with a good prognosis in patients with early SCC but not in patients with ADC.
Autores: Ormazábal, Cristina; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 18  Nº 4  2012  págs. 969 - 980
Purpose: We investigated the role of the collagen-binding receptor discoidin domain receptor-1 (DDR1) in the initiation and development of bone metastasis. Experimental Design: We conducted immunohistochemical analyses in a cohort of 83 lung cancer specimens and examined phosphorylation status in a panel of human lung cancer cell lines. Adhesion, chemotaxis, invasiveness, metalloproteolytic, osteoclastogenic, and apoptotic assays were conducted in DDR1-silenced cells. In vivo, metastatic osseous homing and colonization were assessed in a murine model of metastasis. Results: DDR1 was expressed in a panel of human lung cancer cell lines, and high DDR1 levels in human lung tumors were associated with poor survival. Knockdown (shDDR1) cells displayed unaltered growth kinetics in vitro and in vivo. In contrast, shDDR1 cells showed reduced invasiveness in collagen matrices and increased apoptosis in basal conditions and induced apoptosis in vitro. More importantly, conditioned media of DDR1-knockdown cells decreased osteoclastogenic activity in vitro. Consequently, in a model of tumor metastasis to bone, lack of DDR1 showed decreased metastatic activity associated with reduced tumor burden and osteolytic lesions. These effects were consistent with a substantial reduction in the number of cells reaching the bone compartment. Moreover, intratibial injection of shDDR1 cells significantly decreased bone tumor burden, suggesting impaired colonization ability that was highly dependent on the bone microenvironment. Conclusions: Disruption of DDR1 hampers tumor cell survival, leading to impaired early tumor-bone engagement during skeletal homing. Furthermore, inhibition of DDR1 crucially alters bone colonization. We suggest that DDR1 represents a novel therapeutic target involved in bone metastasis.
Autores:  et al.
Revista: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
ISSN 1073-449X  Vol. 186  Nº 1  2012  págs. 96 - 105
Rationale: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. Objectives: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. Methods: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. Measurements and Main Results: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. Conclusions: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.
Autores: Montuenga, Luis; Ortiz de Solórzano, Carlos;
Revista: INTERNATIONAL JOURNAL OF BIOMEDICAL IMAGING
ISSN 1687-4188  Vol. 2012  2012  págs. 734734
Objective. To define the sensitivity of microcomputed tomography- (micro-CT-) derived descriptors for the quantification of lung damage caused by elastase instillation. Materials and Methods. The lungs of 30 elastase treated and 30 control A/J mice were analyzed 1, 6, 12, and 24 hours and 7 and 17 days after elastase instillation using (i) breath-hold-gated micro-CT, (ii) pulmonary function tests (PFTs), (iii) RT-PCR for RNA cytokine expression, and (iv) histomorphometry. For the latter, an automatic, parallel software toolset was implemented that computes the airspace enlargement descriptors: mean linear intercept (L(m)) and weighted means of airspace diameters (D(0), D(1), and D(2)). A Support Vector Classifier was trained and tested based on three nonhistological descriptors using D(2) as ground truth. Results. D(2) detected statistically significant differences (P < 0.01) between the groups at all time points. Furthermore, D(2) at 1 hour (24 hours) was significantly lower (P < 0.01) than D(2) at 24 hours (7 days). The classifier trained on the micro-CT-derived descriptors achieves an area under the curve (AUC) of 0.95 well above the others (PFTS AUC = 0.71; cytokine AUC = 0.88). Conclusion. Micro-CT-derived descriptors are more sensitive than the other methods compared, to detect in vivo early signs of the disease.
Autores: Han, N.; Dol, Z.; Vasieva, O.; et al.
Revista: INTERNATIONAL JOURNAL OF ONCOLOGY
ISSN 1019-6439  Vol. 41  Nº 1  2012  págs. 242 - 252
Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.
Autores: Montuenga, Luis; Zulueta, Javier J;
Revista: JOURNAL OF THE NATIONAL CANCER INSTITUTE
ISSN 0027-8874  Vol. 104  Nº 3  2012  págs. 254
Autores: Larzábal, Leyre; Nguewa, Paul; Pio, R; et al.
Revista: British Journal of Cancer
ISSN 0007-0920  Vol. 105  Nº 10  2011  págs. 1608-1614
Autores: Ceresa, M; Bastarrika, Gorka; de Torres, Juan Pablo; et al.
Revista: Academic Radiology
ISSN 1076-6332  Vol. 18  Nº 11  2011  págs. 1382-1390
Autores: Ceresa, M; et al.
Revista: European Radiology
ISSN 0938-7994  Vol. 21  Nº 5  2011  págs. 954-962
Autores: Ponz-Sarvise, Mariano; Nguewa, Paul; Pajares, María Josefa; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 17  Nº 12  2011  págs. 4155 -4166
Autores: Pio, R; Pajares, María Josefa; Aibar, E; et al.
Revista: B M C Genomics-(BioMed Central Ltd.)
ISSN 1471-2164  Vol. 3  Nº 11  2010  págs. 352 - 352
Autores: Pérez-Martín, D; et al.
Revista: European Radiology
ISSN 0938-7994  Vol. 20  Nº 11  2010  págs. 2600 - 2608
Objectives To evaluate the feasibility of using automatic quantitative analysis of breath hold gated micro-CT images to detect and monitor disease in a mouse model of chronic pulmonary inflammation, and to compare image-based measurements with pulmonary function tests and histomorphometry. Material and methods Forty-nine A/J mice were used, divided into control and inflammation groups. Chronic inflammation was induced by silica aspiration. Fourteen animals were imaged at baseline, and 4, 14, and 34 weeks after silica aspiration, using micro-CT synchronized with ventilator-induced breath holds. Lung input impedance was measured as well using forced oscillation techniques. Five additional animals from each group were killed after micro-CT for comparison with histomorphometry. Results At all time points, micro-CT measurements show statistically significant differences between the two groups, while first differences in functional test parameters appear at 14 weeks. Micro-CT measurements correlate well with histomorphometry and discriminate diseased and healthy groups better than functional tests. Conclusion Longitudinal studies using breath hold gated micro-CT are feasible on the silica-induced model of chronic pulmonary inflammation, and automatic measurements from micro-CT images correlate well with histomorphometry, being more sensitive than functional tests to detect lung damage in this model.
Autores: Ajona, Daniel; et al.
Revista: Molecular Cancer
ISSN 1476-4598  Vol. 9  2010  págs. 139
We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.
Autores: Larzabal, L; Larráyoz, Marta; Molina, Enrique; et al.
Revista: MOLECULAR CANCER
ISSN 1476-4598  Vol. 9  Nº 320  2010  págs. 1 - 14
BACKGROUND: Different isoforms of VEGF-A (mainly VEGF¿¿¿, VEGF¿¿¿ and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF¿¿¿/¿¿¿b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. RESULTS: Recombinant VEGF¿¿¿/¿¿¿b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF¿¿¿. Furthermore, treatment of endothelial cells with VEGF¿¿¿/¿¿¿b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF¿¿¿. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF¿¿¿/¿¿¿b isoforms. A549 and PC-3 cells overexpressing VEGF¿¿¿b or VEGF¿¿¿b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found. CONCLUSIONS: Our results demonstrate that VEGF¿¿¿/¿¿¿b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addi
Autores: Pio, R, (Autor de correspondencia); Pajares, María Josefa; Aibar, E.; et al.
Revista: BMC GENOMICS
ISSN 1471-2164  Vol. 11  2010  págs. 352
Background: Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results: The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions: This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.
Autores: Ezponda, Teresa; Pajares, María Josefa; Agorreta, J; et al.
Revista: Clinical cancer research
ISSN 1078-0432  Vol. 16  Nº 16  2010  págs. 4113 - 4125
PURPOSE: SF2/ASF is a splicing factor recently described as an oncoprotein. In the present work, we examined the role of SF2/ASF in human non-small cell lung cancer (NSCLC) and analyzed the molecular mechanisms involved in SF2/ASF-related carcinogenesis. EXPERIMENTAL DESIGN: SF2/ASF protein levels were analyzed in 81 NSCLC patients by immunohistochemistry. SF2/ASF downregulation cellular models were generated using small interfering RNAs, and the effects on proliferation and apoptosis were evaluated. Survivin and SF2/ASF expression in lung tumors was analyzed by Western blot and immunohistochemistry. Survival curves and log-rank test were used to identify the association between the expression of the proteins and time to progression. RESULTS: Overexpression of SF2/ASF was found in most human primary NSCLC tumors. In vitro downregulation of SF2/ASF induced apoptosis in NSCLC cell lines. This effect was associated with a reduction in the expression of survivin, an antiapoptotic protein widely upregulated in cancer. In fact, SF2/ASF specifically bound survivin mRNA and enhanced its translation, via a mammalian target of rapamycin complex 1 (mTORC1) pathway-dependent mechanism, through the phosphorylation and inactivation of the translational repressor 4E-BP1. Moreover, SF2/ASF promoted the stability of survivin mRNA. A strong correlation was observed between the expression of SF2/ASF and survivin in tumor biopsies from NSCLC patients, supporting the concept that survivin expression levels are controlled by SF2/ASF. Furthermore, combined expression of these proteins was associated with prognosis. CONCLUSION: This study provides novel data on the mTORC1- and survivin-dependent mechanisms of SF2/ASF-related carcinogenic
Autores:  et al.
Revista: Oncogene
ISSN 0950-9232  Vol. 29  Nº 26  2010  págs. 3758 - 3769
ADAMs (a disintegrin and metalloprotease) are transmembrane proteins involved in a variety of physiological processes and tumorigenesis. Recently, ADAM8 has been associated with poor prognosis of lung cancer. However, its contribution to tumorigenesis in the context of lung cancer metastasis remains unknown. Native ADAM8 expression levels were lower in lung cancer cell lines. In contrast, we identified and characterized two novel spliced isoforms encoding truncated proteins, Delta18a and Delta14', which were present in several tumor cell lines and not in normal cells. Overexpression of Delta18a protein resulted in enhanced invasive activity in vitro. ADAM8 and its Delta14' isoform expression levels were markedly increased in lung cancer cells, in conditions mimicking tumor microenvironment. Moreover, addition of supernatants from Delta14'-overexpressing cells resulted in a significant increase in tartrate-resistant acid phosphatase+ cells in osteoclast cultures in vitro. These findings were associated with increased pro-osteoclastogenic cytokines interleukin (IL)-8 and IL-6 protein levels. Furthermore, lung cancer cells overexpressing Delta14' increased prometastatic activity with a high tumor burden and increased osteolysis in a murine model of bone metastasis. Thus, the expression of truncated forms of ADAM8 by the lung cancer cells may result in the specific upregulation of their invasive and osteoclastogenic activities in the bone microenvironment. These findings suggest a novel mechanism of tumor-induced osteolysis in metastatic bone colonization.
Autores: Pio, R; García, José Javier; et al.
Revista: Cancer epidemiology, biomarkers & prevention
ISSN 1055-9965  Vol. 19  Nº 10  2010  págs. 2655 - 2672
Autores: Esteban Ruiz, F. J.; Calvo, Alfonso; Montuenga, Luis;
Libro:  Técnicas en histología y biología celular
2014  págs. 85 - 101
Autores: Montuenga, Luis; Bodegas, María Elena; de Andrea, CE; et al.
Libro:  Técnicas en histología y biología celular
2014  págs. 35 - 60
Autores: Montuenga, Luis; Agorreta, J;
Libro:  Técnicas en histología y biología celular
2014  págs. 127 - 154
Autores: Montuenga, Luis; Bodegas, María Elena; de Andrea, CE; et al.
Libro:  Técnicas en histología y biología celular
2014  págs. 61 - 84