Maria Dolores Pastor

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.

Association between the miRNA signatures in plasma and bronchoalveolar fluid in respiratory pathologies

The identification of new less invasive biomarkers is necessary to improve the detection and prognostic outcome of respiratory pathological processes. The measurement of miRNA expression through less invasive techniques such as plasma and serum have been suggested to analysis of several lung malignancies including lung cancer. These studies are assuming a common deregulated miRNA expression both in blood and lung tissue. The present study aimed to obtain miRNA representative signatures both in plasma and bronchoalveolar cell fraction that could serve as biomarker in respiratory diseases. Ten patients were evaluated to assess the expression levels of 381 miRNAs. We found that around 50% miRNAs were no detected in both plasma and bronchoalveolar cell fraction and only 20% of miRNAs showed similar expression in both samples. These results show a lack of association of miRNA signatures between plasma and bronchoalveolar cytology in the same patient. The profiles are not comparable; however, there is a similarity in the relative expression in a very small subset of miRNAs (miR-17, miR-19b, miR-195 and miR-20b) between both biological samples in all patients. This finding supports that the miRNAs profiles obtained from different biological samples have to be carefully validated to link with respiratory diseases.

Identification of Oxidative Stress Related Proteins as Biomarkers for Lung Cancer and Chronic Obstructive Pulmonary Disease in Bronchoalveolar Lavage

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. Cigarette smoke causes oxidative stress and an inflammatory response in lung cells, which in turn may be involved in COPD and lung cancer development. The aim of this study was to identify differential proteomic profiles related to oxidative stress response that were potentially involved in 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, and control (neither COPD nor LC). Proteins were separated into spots by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 16 oxidative stress regulatory proteins were differentially expressed in BAL samples from LC and/or COPD patients as compared with the control group. A distinct proteomic reactive oxygen species (ROS) protein signature emerged that characterized lung cancer and COPD. In conclusion, our findings highlight the role of the oxidative stress response proteins in the pathogenic pathways of both diseases, and provide new candidate biomarkers and predictive tools for LC and COPD diagnosis.

Down-regulation of spinophilin in lung tumours contributes to tumourigenesis.

The scaffold protein spinophilin (Spn, PPP1R9B) is one of the regulatory subunits of phosphatase‐1a (PP1), targeting it to distinct subcellular locations and to its target. Loss of Spn reduces PPP1CA levels, thereby maintaining higher levels of phosphorylated pRb. This effect contributes to an increase in p53 activity. However, in the absence of p53, reduced levels of Spn increase the tumourigenic properties of cells. In addition, Spn knockout mice have a reduced lifespan, an increased number of tumours and increased cellular proliferation in some tissues, such as the mammary ducts. In addition, the combined loss of Spn and p53 activity leads to an increase in mammary carcinomas, confirming the functional relationship between p53 and Spn. In this paper, we report that Spn is absent in 20% and reduced in another 37% of human lung tumours. Spn reduction correlates with malignant grade. Furthermore, the loss of Spn also correlates with p53 mutations. Analysis of miRNAs in a series of lung tumours showed that miRNA106a* targeting Spn is over‐expressed in some patients, correlating with decreased Spn levels. Proof‐of‐concept experiments over‐expressing miRNA106a* or Spn shRNA in lung tumour cells showed increased tumourigenicity. In conclusion, our data showed that miRNA106a* over‐expression found in lung tumours might contribute to tumourigenesis through Spn down‐regulation in the absence of p53. Copyright

Elevated Levels of the Complement Activation Product C4d in Bronchial Fluids for the Diagnosis of Lung Cancer

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 = 0.56–0.83). In conclusion, C4d is consistently elevated in bronchial fluids from lung cancer patients and may be used to improve the diagnosis of the disease.

PDGFRα/β and VEGFR2 polymorphisms in colorectal cancer: incidence and implications in clinical outcome

BackgroundAngiogenesis plays an essential role in tumor growth and metastasis, and is a major target in cancer therapy. VEGFR and PDGFR are key players involved in this process. The purpose of this study was to assess the incidence of genetic variants in these receptors and its potential clinical implications in colorectal cancer (CRC).MethodsVEGFR2, PDGFRα and PDGFRβ mutations were evaluated by sequencing their tyrosine kinase domains in 8 CRC cell lines and in 92 samples of patients with CRC. Correlations with clinicopathological features and survival were analyzed.ResultsFour SNPs were identified, three in PDGFRα [exon 12 (A12): c.1701A>G; exon 13 (A13): c.1809G>A; and exon 17 (A17): c.2439+58C>A] and one in PDGFRβ [exon 19 (B19): c.2601A>G]. SNP B19, identified in 58% of tumor samples and in 4 cell lines (LS174T, LS180, SW48, COLO205), was associated with higher PDGFR and pPDGFR protein levels. Consistent with this observation, 5-year survival was greater for patients with PDGFR B19 wild type tumors (AA) than for those harboring the G-allele genotype (GA or GG) (51% vs 17%; p=0.073). Multivariate analysis confirmed SNP B19 (p=0.029) was a significant prognostic factor for survival, independent of age (p=0.060) or TNM stage (p<0.001).ConclusionsPDGFRβ exon 19 c.2601A>G SNP is commonly encountered in CRC patients and is associated with increased pathway activation and poorer survival. Implications regarding its potential influence in response to PDGFR-targeted agents remain to be elucidated.

VeriStrat: a prognostic and/or predictive biomarker for advanced lung cancer patients?

The role of EGF receptor (EGFR) inhibitors in the treatment of lung cancer without activating EGFR mutations has been a controversial issue, particularly their relative efficacy over the available chemotherapy in the second- and third-line setting. VeriStrat is a serum/plasma proteomic test developed using matrix-assisted laser desorption/ionization methodology, aiming at predicting benefit from EGFR treatment. The VeriStrat algorithm has been interrogated retrospectively and prospectively in samples from randomised trials, such as the PROSE study, confirming the prognostic information associated with the signature. In addition, the test appeared to be predictive of erlotinib impact on survival, as only VeriStrat Good patients benefited from such a treatment. Additional studies should confirm and further define its role in predicting EGFR tyrosine kinase inhibitor benefit, and to establish its better use in terms of clinical efficiency identifying which patients are candidates for the test, at which time on the history of the disease, and lastly at what extra cost.

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

Abstract 4644: PDGFRα/β and VEGFR2 SNPs incolorectal cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Angiogenesis plays an essential role in tumor growth and metastasis, and is a major target in cancer therapy. VEGFR and PDGFR are key players involved in this process. The purpose of this study was to assess the incidence of genetic variants in these receptors and its potential clinical implications in colorectal cancer (CRC). VEGFR2, PDGFRα and PDGFRβ mutations were evaluated by sequencing their tyrosine kinase domains in 8 CRC cell lines and in 92 samples of patients with CRC. Correlations with clinicopathological features and survival were analyzed. Four SNPs were identified, three in PDGFRα [exon 12 (A12): c.1701A>G; exon13 (A13): c.1809G>A; and exon 17 (A17): c.2439+58C>A) and one in PDGFRβ (exon19 (B19): c.2601A>G) (figure 1). SNP B19 was present in 58% of tumor samples and in 4 cell lines (LS174T, LS180, SW48, COLO205), and this was associated with higher PDGFR and pPDGFR protein levels. 5-year survival was greater for patients with PDGFR B19 wild type tumors (AA) than for those harboring the G-allele genotype (GA or GG) (51%vs17%; P=0.073). Multivariate analysis confirmed SNP B19 (P=0.029) was a significant prognostic factor for survival, independent of age (P=0.060) or TNM stage (P G SNP is commonly encountered in CRC patients and is associated with increased pathway activation and poorer survival. Implications regarding its potential influence in response to PDGFR targeted agents remain to be ellucidated. Citation Format: Purificacion Estevez-Garcia, A Castano, Ana Martin, Fernando Lopez-Rios, J Iglesias, Sandra Munoz-Galvan, Iker Lopez-Calderero, Sonia Molina-Pinelo, Maria D. Pastor, Amancio Carnero, Luis Pa-Ares, Rocio Garcia-Carbonero. PDGFRα/β and VEGFR2 SNPs incolorectal cancer. [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 4644. doi:10.1158/1538-7445.AM2013-4644

Abstract 5292: MicroRNA signatures predictive of response to chemotherapy in mCRC.

MicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biological and pathological processes including colorectal cancer (CRC). From a clinical perspective, several studies have identified groups of miRNAs with potential utility for early diagnosis or prognostic stratification of CRC patients. However, very few have evaluated the potential ability of miRNA to predict response to selected chemotherapy regimens. The aim of our study was to evaluate the ability of miRNA expression patterns to predict chemotherapy response in a cohort of 78 patients with metastatic CRC (mCRC). We examined tumor expression levels of 667 miRNAs in the training cohort by TaqMan Low Density Arrays (TLDA). In training cohort (N=39), we identified a miRNA molecular signature with significant association in relevant clinical endpoints (objective response, progression-free and overall survival). In validation cohort (N=39), we validated significantly higher expression levels of miR-107, and miR99a* according to objective response to fluoropyrimidine-based regimens. These results support a substantial role for miRNAs in determining drug response, and highlight their potential value as biomarkers for personalized treatment strategies and as a novel class of therapeutic targets. Citation Format: Sonia Molina-Pinelo, Amancio Carnero, Fernando Rivera, Purificacion Estevez-Garcia, Jose M. Bozada, Maria L. Limon, Javier Gomez, Maria D. Pastor, Rocio Suarez, Luis G. Paz-Ares, Fernando de la Portilla, Andres Carranza, Isabel Sevilla, Luis Vicioso, Rocio Garcia Carbonero. MicroRNA signatures predictive of response to chemotherapy in mCRC. [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 5292. doi:10.1158/1538-7445.AM2013-5292