Ana Salinas

Identification of proteomic signatures associated with lung cancer and COPD.

UNLABELLED Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. The aim of this study was to identify distinct proteomic profiles able to discriminate these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC without COPD, and control with neither COPD nor LC. Proteins were separated into spots by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 40 proteins were differentially expressed in the LC and/or COPD groups as compared with the control group. Distinct protein profiles were identified and validated for each pathological entity (LC and COPD). The main networks involved were related to inflammatory signalling, free radical scavenging and oxidative stress response, and glycolysis and gluconeogenesis pathways. The most relevant signalling link between LC and COPD was through the NF-κB pathway. In conclusion, the protein profiles identified contribute to elucidate the underlying pathogenic pathways of both diseases, and provide new tools of potential use as biomarkers for the early diagnosis of LC. BIOLOGICAL SIGNIFICANCE Sequence coverage. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Ingenuity Pathways Analysis. Mapping of our proteins onto biological pathways and disease networks demonstrated that 22 proteins were linked to inflammatory signalling (p-value: 1.35 10(-08)-1.42 10(-02)), 15 proteins were associated with free radical scavenging and oxidative stress response (p-value: 4.93 10(-11)-1.27 10(-02)), and 9 proteins were related with glycolysis and gluconeogenesis pathways (p-value: 7.39 10(-09)-1.58 10(-02)).

MicroRNA clusters: dysregulation in lung adenocarcinoma and COPD

Lung adenocarcinoma and chronic obstructive pulmonary disease (COPD) are pulmonary diseases that share common aetiological factors (tobacco smoking) and probable dysregulated pathways. MicroRNAs (miRNAs) play an essential role in regulating numerous physiological and pathological processes. The purpose of this study was to assess global miRNA expression patterns in patients with COPD and/or adenocarcinoma to elucidate distinct regulatory networks involved in the pathogenesis of these two smoking-related diseases. Expression of 381 miRNAs was quantified by TaqMan Human MicroRNA A Array v2.0 in bronchoalveolar lavage fluid samples from 87 patients classified into four groups: COPD, adenocarcinoma, adenocarcinoma with COPD, and control (neither COPD nor adenocarcinoma). 11 differentially expressed miRNAs were randomly selected for validation in an independent cohort of 40 patients. Distinct miRNA expression profiles were identified and validated for each pathological group, involving 66 differentially expressed miRNAs. Four miRNA clusters (the mir-17-92 cluster and its paralogues, mir-106a-363 and mir-106b-25; and the miR-192-194 cluster) were upregulated in patients with adenocarcinoma and one miRNA cluster (miR-132-212) was upregulated in patients with COPD. These results contribute to unravelling miRNA-controlled networks involved in the pathogenesis of adenocarcinoma and COPD, and provide new tools of potential use as biomarkers for diagnosis and/or therapeutic purposes. MicroRNA expression profiles in bronchoalveolar lavage fluid enable discrimination of adenocarcinoma from COPD http://ow.ly/tPaVC

MicroRNA-dependent regulation of transcription in non-small cell lung cancer

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

Spinophilin Loss Correlates with Poor Patient Prognosis in Advanced Stages of Colon Carcinoma

Purpose: The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy. Experimental Design: By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy. Results: Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels. Conclusions: Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. Clin Cancer Res; 19(14); 3925–35. ©2013 AACR.

Impact of DLK1-DIO3 imprinted cluster hypomethylation in smoker patients with lung cancer

DNA methylation is important for gene expression and genome stability, and its disruption is thought to play a key role in the initiation and progression of cancer and other diseases. The DLK1-DIO3 cluster has been shown to be imprinted in humans, and some of its components are relevant to diverse pathological processes. The purpose of this study was to assess the methylation patterns of the DLK1-DIO3 cluster in patients with lung cancer to study its relevance in the pathogenesis of this disease. We found a characteristic methylation pattern of this cluster in smoking associated lung cancer, as compared to normal lung tissue. This methylation profile is not patent however in lung cancer of never smokers nor in lung tissue of COPD patients. We found 3 deregulated protein-coding genes at this locus: one was hypermethylated (DIO3) and two were hypomethylated (DLK1 and RTL1). Statistically significant differences were also detected in two different families of SNORDs, two miRNA clusters and four lncRNAs (MEG3, MEG8, MEG9 and LINC00524). These findings were validated using data from the cancer genome atlas (TCGA) database. We have then showed an inverse correlation between DNA methylation and expression levels in 5 randomly selected genes. Several targets of miRNAs included in the DLK1-DIO3 cluster have been experimentally verified as tumor suppressors. All of these results suggest that the dysmethylation of the imprinted DLK1-DIO3 cluster could have a relevant role in the pathogenesis of lung cancer in current and former smokers and may be used for diagnostic and/or therapeutic purposes.

The FGFR4-388arg Variant Promotes Lung Cancer Progression by N-Cadherin Induction

The FGFR4-388Arg variant has been related to poor prognosis in several types of cancer, including lung cancer. The mechanism underlying this association has not been addressed in detail in patients with this pathology. Here, we report that this FGFR4 variant induces MAPK and STAT3 activation and causes pro-oncogenic effects in NSCLC in vitro and in vivo. This variant induces the expression of EMT-related genes, such as N-cadherin, vimentin, Snail1 and Twist1. Indeed, the induction of N-cadherin protein expression by this variant is essential for its pro-tumorigenic role. The presence of the FGFR4-388Arg variant correlates with higher N-cadherin expression levels in clinical NSCLC samples and with poorer outcome in patients with FGFR expression. These results support the prognostic role of this FGFR variant in lung cancer and show that these effects may be mediated by the induction of N-cadherin expression and an EMT phenotype.

Single nucleotide polymorphisms as prognostic and predictive biomarkers in renal cell carcinoma

Despite major advances in the knowledge of the molecular basis of renal cell carcinoma, prognosis is still defined using clinical and pathological parameters. Moreover, no valid predictive biomarkers exist to help us selecting the best treatment for each patient. With these premises, we aimed to analyse the expression and to determine the prognostic and predictive value of 64 key single nucleotide polymorphisms in 18 genes related with angiogenesis or metabolism of antiangiogenics in two cohorts of patients with localized and advanced renal cell cancer treated at our institution. The presence of the selected single nucleotide polymorphisms was correlated with clinical features, disease free survival, overall survival and response rate. In patients with localized renal cell cancer, 5 of these polymorphisms in 3 genes involved in angiogenesis predicted for worse disease free survival (VEGFR2: rs10013228; PDGFRA: rs2228230) or shorter overall survival (VEGFR2: rs10013228; VEGFR3: rs6877011, rs307826) (p < 0.05). Rs2071559 in VEGFR2 showed a protective effect (p = 0.01). In the advanced setting, 5 SNPs determined inferior overall survival (IL8: rs2227543, PRKAR1B: rs9800958, PDGFRB: rs2302273; p = 0.05) or worse response rate (VEGFA: rs699947, rs3025010 p ≤ 0.01)). Additionally 1 single nucleotide polymorphism in VEGFB predicted for better response rate rs594942 (p = 0.03). Genetic analysis of renal cell carcinoma patients might provide valuable prognostic/predictive information. A set of SNPs in genes critical to angiogenesis and metabolism of antiangiogenics drugs seem to determine post-surgical outcomes and treatment response in our series.

Abstract 4362: Coordinated regulation of several microRNAs in lung cancer patients

Background Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. NSCLCs are mainly classified adenocarcinoma and squamous cell carcinoma. It seems more and more obvious that specific molecular analyses are necessary to elucidate the complexity of clinically relevant phenotypes which determine the lung carcinogenesis. This includes the role of microRNAs (miRNAs) in the disease, involved in the complexity of gene expression regulation. Different modulator mechanisms are involved in the biogenesis and function of miRNAs, one of them is DNA methylation. Alterations in these mechanisms may contribute to the tumor genesis and progression. The purpose of this study was to assess miRNA methylation patterns in patients with NSCLC to study the potential of coordinate regulation of miRNAs as a relevant mechanism involved in this pathogenesis. Materials and Methods DNA methylation of gene clusters was analyzed by Illumina 70 subjects. The samples were divided into two cohorts. A first cohort constituted from 47 patients who had undergone surgical resection for clinical early stage NSCLC. A second group from 23 subjects was used as health cohort. DNA was extracted using the QIAamp DNA Mini Kit. For each assay, 500 ng of DNA was treated with sodium bisulfate using EZ DNA Methylation™ Kit and cleaned with ZR-96 DNA Clean-up Kit™, before standard Illumina amplification, hybridization, and imaging steps. Methylation data were processed using the RnBeads R package. Results We identified two clusters clearly corregulated by methylation in lung cancer, involving 49 miRNAs differentially hypomethylated in tumor samples respect to control tissue. Some of these miRNAs have been implicated in several pathways in cancer. Conclusions Our results strongly imply hypomethylation of two miRNA clusters, which represent key targets in unravelling of the mechanism of lung tumorigenesis. Citation Format: Irene Ferrer, Ana Salinas, Angela Marrugal, Jon Zugazagoitia, Amancio Carnero, Luis Paz-Ares, Sonia Molina-Pinelo. Coordinated regulation of several microRNAs in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4362. doi:10.1158/1538-7445.AM2017-4362

Abstract 5305: Transcriptionalregulation by microRNAs in NSCLC.

Squamous cell lung cancer and adenocarcinoma are the most common subtypes of the lung tumours. The search for cancer-directed treatments has increased the need for understanding molecular features of either histological subtypes. The aim of this study was to identify the transcriptional regulation differences due to miRNA expression profiles between SCC and adenocarcinoma. In this work, a total of 44 patients were evaluated to assess the correlation between the miRNA and messenger RNA expression levels. We detected changes in 56 mRNAs as well as in 9 miRNAs between SCC and adenocarcinoma. Nearly 20% of overall deregulated genes were targeted by at least one of the 9 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a, miR-483-5p, miR-494, miR-601 and miR-708) differentially expressed between SCC and adenocarcinoma (table 1). Genes predicted (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) to be targeted by several miRNAs were individually validated by qRT-PCR. We found genes involved in tight junctions and others involved in resistance to anticancer agents. These genes were reliable biomarkers to detect differences between the two most common histological subtypes of lung cancer. Therefore, transcriptional regulation differences through miRNA expression play an important role in key hallmarks of non-small cell lung cancer. Citation Format: Sonia Molina-Pinelo, Gabriel Gutierrez-Pozo, Maria D. Pastor, Marta Hergueta, Gema Moreno-Bueno, Rocio Garcia-Carbonero, Ana BS Nogal, Rocio Suarez, Ana Salinas, Francisco Pozo-Rodriguez, Fernando Lopez-Rios, Teresa Agullo-Ortuno, Jose Palacios, Amancio Carnero, Luis G. Paz-Ares. Transcriptionalregulation by microRNAs in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2013-5305