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Publicaciones científicas más recientes (desde 2010)

Autores: Ajona, Daniel; et al.
Revista: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
ISSN 1073-449X  Vol. 197  Nº 9  2018  págs. 1164 - 1176
RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.
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: 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: Moreno-Artero, Ester; Querol-Cisneros, E; Ivars, Marta; et al.
Revista: JOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VENEREOLOGY
ISSN 0926-9959  Vol. 31  Nº 6  2017  págs. e301 - e302
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: Berraondo, Pedro; Minute,L.; Ajona, Daniel; et al.
Revista: IMMUNOLOGICAL REVIEWS
ISSN 0105-2896  Vol. 274  Nº 1  2016  págs. 290 - 306
Chronic inflammation in the tumor microenvironment and evasion of the antitumor effector immune response are two of the emerging hallmarks required for oncogenesis and cancer progression. The innate immune system not only plays a critical role in perpetuating these tumor-promoting hallmarks but also in developing antitumor adaptive immune responses. Thus, understanding the dual role of the innate system in cancer immunology is required for the design of combined immunotherapy strategies able to tackle established tumors. Here, we review recent advances in the understanding of the role of cell populations and soluble components of the innate immune system in cancer, with a focus on complement, the adapter molecule Stimulator of Interferon Genes, natural killer cells, myeloid cells, and B cells.
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: 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: 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: Alegre, Estíbaliz; Restituto, Patricia; et al.
Revista: TUMOR BIOLOGY
ISSN 1010-4283  Vol. 37  Nº 10  2016  págs. 13687 - 13694
Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p < 0.01). There was high agreement for activating mutations between baseline cfDNA and tumor samples, especially for L858R mutation (kappa index = 0.679; p = 0.001). In 34 % of advanced NSCLC patients, we detected mutations in cfDNA not previously detected in tumor samples and double mutations in 17 %. Patients with baseline total EGFR copy levels above the median presented decreased overall survival (OS) (341 vs. 870 days, p < 0.05) and progression-free survival (PFS) (238 vs. 783 days; p < 0.05) compared with those with total EGFR copy levels below the median. Patients with baseline concentrations of activating mutations above the median (94 copies/mL) had lower OS (317 vs. 805 days; p < 0.05) and PFS (195 vs. 724 days; p < 0.05). During follow-up, T790M resistance mutation was detected in 53 % of patients. Total and mutated EGFR analysis in cfDNA seems a relevant tool to characterize the molecular profile and prognosis of NSCLC patients harboring EGFR mutations.
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: 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: 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: Pio, R; Corrales, L.; Lambris, J. D.;
Revista: ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
ISSN 0065-2598  Vol. 772  2014  págs. 229 - 262
Complement is a central part of the immune system that has developed as a first defense against non-self cells. Neoplastic transformation is accompanied by an increased capacity of the malignant cells to activate complement. In fact, clinical data demonstrate complement activation in cancer patients. On the basis of the use of protective mechanisms by malignant cells, complement activation has traditionally been considered part of the body's immunosurveillance against cancer. Inhibitory mechanisms of complement activation allow cancer cells to escape from complement-mediated elimination and hamper the clinical efficacy of monoclonal antibody-based cancer immunotherapies. To overcome this limitation, many strategies have been developed with the goal of improving complement-mediated effector mechanisms. However, significant work in recent years has identified new and surprising roles for complement activation within the tumor microenvironment. Recent reports suggest that complement elements can promote tumor growth in the context of chronic inflammation. This chapter reviews the data describing the role of complement activation in cancer immunity, which offers insights that may aid the development of more effective therapeutic approaches to control cancer.
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: 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: 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: Calleja, Patricia; Espuelas, S; et al.
Revista: NANOMEDICINE
ISSN 1743-5889  Vol. 9  Nº 14  2014  págs. 2109 - 2121
Aim: The authors report a novel approach for enhancing the oral absorption of paclitaxel (PTX) by encapsulation in poly(anhydride) nanoparticles (NPs) containing cyclodextrins and poly(ethylene glycol). Materials & methods: Formulations were prepared using the solvent displacement method. Subsequently, pharmacokinetics and organ distribution assays were evaluated after oral administration into C57BL/6J mice. In addition, antitumor efficacy studies were performed in a subcutaneous tumor model of Lewis lung carcinoma. Results: PTX-loaded NPs displayed sizes between 190-300 nm. Oral NPs achieved drug plasma levels for at least 24 h, with an oral bioavailability of 55-80%. Organ distribution studies revealed that PTX, orally administered in NPs, underwent a similar distribution to intravenous Taxol® (Bristol-Myers Squibb, NJ, USA). For in vivo antitumor assays, oral strategy maintained a slower tumor growth than intravenous Taxol. Conclusion: PTX orally administered in poly(anhydride) NPs, combined with cyclodextrins and poly(ethylene glycol), displayed sustained plasma levels and significant antitumor effect in a syngenic tumor model of carcinoma in mice.
Autores: Pio, R; Ajona, Daniel; Lambris J;
Revista: SEMINARS IN IMMUNOLOGY
ISSN 1044-5323  Vol. 25  Nº 1  2013  págs. 54-64
For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively new and deserves closer attention. In this article, we summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer.
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: 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: 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: Zabaleta, V.; Calleja, Patricia; Espuelas, S; et al.
Revista: ANNALES PHARMACEUTIQUES FRANCAISES
ISSN 0003-4509  Vol. 71  Nº 2  2013  págs. 109 - 118
Paclitaxel is an anticancer drug used as solution for perfusion for the treatment of certain types of cancers. In the last years, a number of strategies have been proposed for the development of an oral formulation of this drug. However, this task is quite complicated due to the poor aqueous solubility of paclitaxel as well as the fact that this compound is substrate of the intestinal P-glycoprotein and the cytochrome P450 enzymatic complex. In this work, we have developed pegylated nanoparticles with mucopenetrating properties in order to conduct paclitaxel onto the surface of the enterocyte. These nanoparticles displayed a size of about 180 nm and a drug loading close to 15% by weight. The pharmacokinetic study in mice has shown that these nanoparticles were capable to offer therapeutic plasma levels of paclitaxel up to 72 hours. In addition, the oral relative bioavailability of paclitaxel when loaded in nanoparticles pegylated with poly(ethylene glycol) 2000 (PEG) was found to be 85%. In a subcutaneous model of tumour in mice, these pegylated nanoparticles administered orally every 3 days have demonstrated a similar efficacy than Taxol® administered intravenously every day during 9 days. All of these results suggested that these pegylated nanoparticles were capable to cross the mucus layer of the gut and, then, reach the surface of the enterocytes. The PEG molecules would facilitate the adhesion of nanoparticles to this epithelial surface, minimise the pre-systemic metabolism of paclitaxel and, thus, promote its absorption.
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: Fayolle, C.; Casares, N; et al.
Revista: VACCINE
ISSN 0264-410X  Vol. 30  Nº 18  2012  págs. 2848 - 2858
The complement system and Toll-like receptors (TLR) are key innate defense systems which might interact synergistically on dendritic cells (DC) to reinforce adaptive immunity. In a previous work, we found that the extra domain A from fibronectin EDA (an endogenous ligand for TLR4) can favour antigen delivery to DC and induce their maturation. Given the potential of anaphylatoxins to cause inflammation and activation of myeloid cells, we hypothesized that a fusion protein between EDA, and anaphylatoxins C3a, C4a or C5a together with an antigen might improve the immunogenicity of the antigen. Naked DNA immunization with a construct expressing the fusion protein between C5a, EDA and the cytotoxic T cell epitope SIINFEKL from ovalbumin, induced strong antigen specific T cell responses. The purified recombinant fusion protein EDA-SIINFEKL-C5a induced activation of dendritic cells, the production of proinflammatory cytokines/chemokines and stimulated antigen presenting cell migration and Nit cell activation. As compared to EDA-SIINFEKL, the fusion protein EDA-SIINFEKL-C5a did not induce the production of the immunosuppressive molecules IL-10, CCL17, CCL1, CXCL12 or XCL1 by DC. Moreover, EDA-SIINFEKL-C5a induced strong specific T cell responses in vivo and protected mice against E.G7-OVA tumor growth more efficiently than EDA-SIINFEKL or SIINFEKL-C5a recombinant proteins. Our results suggest that fusion proteins containing EDA, the anaphylatoxin C5a and the antigen may serve as a su
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: Lozano, María D; Zulueta, Javier J; Echeveste, José Ignacio; et al.
Revista: The Oncologist
ISSN 1083-7159  Vol. 16  Nº 6  2011  págs. 877 - 885
The mutation status was identical in patients who had both biopsies and cytological samples analyzed. Conclusion. Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment
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: 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: 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: Pajares, María Josefa; Agorreta, J; et al.
Revista: Molecular Cancer
ISSN 1476-4598  Vol. 28  Nº 9  2010  págs. 130
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: Pio, R; García, José Javier; et al.
Revista: Cancer epidemiology, biomarkers & prevention
ISSN 1055-9965  Vol. 19  Nº 10  2010  págs. 2655 - 2672
Autores: Aymerich, MS; García, M.; Suburo, A. M.; et al.
Libro:  Biología Celular Biomédica
2015  págs. 173-195