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Background: Small extracellular vesicles (sEVs) in the blood of cancer patients contain higher amounts of tumor markers than those identified as free-circulating. miRNAs have significant biomedical relevance due to their high stability and feasible detection. However, there is no reliable endogenous control available to measure sEVs-miRNA content, impairing the acquisition of standardized consistent measurements in cancer liquid biopsy. Results: In this study, we identified three miRNAs from a panel of nine potential normalizers that emerged from a comprehensive analysis comparing the sEV-miRNA profile of six lung and ovarian human cancer cell lines in the absence of or under different conditions of chemotherapy. Their relevance as normalizers was tested in 26 additional human cancer cell lines from nine different tumor types undergoing chemotherapy or radiotherapy treatment. The validation cohort was comprised of 172 prospective plasma and ascitic fluid samples from three different human tumor types. Variability and normalization properties were tested in comparison to miR-16, the most used control to normalize free-circulating miRNAs in plasma. Conclusion: Our results indicate that miR-151a is consistently represented in small extracellular vesicles with minimal variability compared to miR-16, providing a novel normalizer to measure small extracellular vesicle miRNA content that will benefit liquid biopsy in cancer patients.

Information from proteomics, microscopy, and structural biology are integrated to create structural models of exosomes, small vesicles released from cells. Three visualization methods are employed and compared: 2D painting of a cross section using traditional media, manual creation of a cross section using the mesoscale 2.5D digital painting software cellPAINT, and generation of a 3D atomic model using the mesoscale modeling program cellPACK.

Background: Non-small cell lung cancer (NSCLC) represents the 80-85% of the lung cancer. Cisplatin-based chemotherapy is the paradigm of NSCLC treatment; however, it also induces de novo DNA hyper methylation that is associated with gene expression regulation. We have previously reported that the loss of IGFBP-3 expression by promoter hypermethylation results in reduced NSCLC cells sensitivity to cisplatin. Liquid biopsy has gained increasing attention in recent years, as it is a convenient and minimally invasive alternative to interrogate tumor free DNA (ctDNA). Objective: To study the correlation between the IGFBP-3 gene promoter methylation levels in ctDNA and paired tumor tissues from NSCLC patients at advanced stages. Methods: 66 FFPE/plasma prospective paired samples from stages III-IV NSCLC patients have been collected in a prospective study at Hospital La Paz, together with the associated clinical history. ctDNA and tumor tissue DNA was isolated and bisulfite modified. Methylation levels at IGFBP3-promoter was assessed by qMSP following the next equation: Cmeth = 100/[1+2(CTCG - CTTG)]. A validation cohort of 56 retrospective samples from national biobanks was included. Results: Our first result indicate that mIGFBP-3 assessment in DNA from tumor tissue is an indicator of a worse prognosis, since 75% of patients with stages IIIC/IV present hipermethylated levels in comparison with 25% of patients in stages IIIA/IIIB, (p = 0.035). When analyzing the ctDNA, we found that 87% of patients with mIGFBP-3 progress to the disease (p=0.001), although those patients with mIGFP-3 ≤ 35.5%, present a higher Overall Survival (OS) (p=0.026). In terms of therapy response we observed that those patients with methylation levels in tissue ≤ P75 (74.7%) have a greater survival when they receive QT vs QT + RT (n = 35) (p=0.364; HR = 0.514), in fact, 80% of patients receiving only QT are still alive at 365 days versus those receiving QT + RT. Merged data including the validation cohort reached the statistical significance (p=0.042). We also found a reduced in the risk of death by 82.2% (p=0.01) and 85.9% (p=0.002) respectively, in the patients that following a specific RECITS criteria (patients who progress or have stable disease), presented mIGFBP3 levels in tissue ≤ P50 ( 12,2 %) and in ctDNA ≤ P75 (15,6%). Conclusions: Our results indicate that mIGFBP-3 is a good prognostic biomarker in patients with NSCLC at advanced stages in both, tissue and circulating tumor DNA. Furthermore, its presence in liquid biopsy may be useful as a biomarker for bad prognosis. In summary, methylation status of IGFBP3 may provide a useful clinical tool for deciding on a concomitant radiotherapy applied in both, tissue and in liquid biopsy. This tool also provides a non-invasive and novel approach for the monitoring of NSCLC patients to support the use of the RECIST criteria in order to indicate continuity in QT treatment. Citation Format: Olga Pernía, Rocío Rosas, Julia Jiménez, Itsaso Losantos, Isabel Esteban, Patricia Cruz Castellanos, Oliver Higuera, Tatiana Juste, Olga Vera Puente, Ángel Diaz Lagares, Javier de Castro, Inmaculada ibañez de Caceres. Role of mIGFBP-3 in the clinical monitoring of non-small cell lung cancer patients at advanced stages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2095.

Background: Non-small cell lung cancer (NSCLC) is one leading cause of death worldwide, with a 5-year survival rate of only 24%. Late diagnosis and the innate or acquired anti-cancer drug resistance are the main causes of this high mortality. In recent years, targeted drugs and immunotherapies have substantially increased the survival in aprox 15% of patients. However, the vast majority continue to receive the standard platinum-based chemotherapy (CDDP), although they present poor response to the treatment. Therefore, it is extremely important to identify new biomarkers for survival, prognosis and drug-resistance in NSCLC. Liquid biopsy represents a source of potentially actionable biomarkers, being minimally invasive and allowing to track tumor heterogeneity and detecting very early emergence of therapy resistance and recurrence. Among them, exosomal miRNAs are highly implicated in cancer progression and have great biomedical relevance due to their stability, feasible detection and their functional capacity to regulate gene expression. Methods: To study the implication of exosomal miRNAs in the induction of CDDP resistance, we analyzed the exosomal content of 3 paired CDDP sensitive and resistant lung cancer (H23S/R), and ovarian cancer (A2780S/R and 41M/R) cell lines by small RNAseq and we compared the profiles of the two phenotypes looking for candidates over-represented in the resistant cells-exosomes. Then, we performed a qRT-PCR validation of the candidates and studied their implication in the development of CDDP-resistance through its overexpression in our cell models and subsequent exposure to CDDP. Finally, we evaluated the clinical relevance of the validated candidates in lung, studying their expression levels in a prospective cohort of 51 advanced NSCLC patients and 10 healthy controls with a 3 years follow-up. Results: We identified 2 known miRNAs (miR1 miR2) and 1 novel miRNA (miR3) that were overexpressed in the exosomes of the resistant subtypes and validated in the lung cancer cell line H23. Among them, miR1 overexpression led to an increased sensitivity to CDDP in the H23 R phenotype. This could mean that resistant cancer cells are eliminating miR1 through exosomes to become more aggressive. Regarding exosomal miRNA expression in patients, the only miRNA that was differentially elevated in NSCLC patients vs controls was miR3, showing that it could be involved in the establishment of NSCLC. Then, we divided NSCLC patients in high and low miRNA expression levels, finding that bearing high levels of exosomal miR1 and miR2 extremely increased the risk of recurrence and death in NSCLC patients. Conclusions: We demonstrated that exosomal levels of miR1 and 2 would allow monitoring the prognosis and response to platinum in patients with advanced NSCLC, facilitating the choice of alternative therapies; and that miR3 in liquid biopsy could be useful as a biomarker for early diagnosis. Citation Format: Miranda Burdiel, Julia Jimenez, Carlos Rodriguez-Antolin, Olga Pernia, Rocio Rosas, Dario Sanchez-Cabrero, Patricia Cruz, Itsaso Losantos, Javier de Castro, Inmaculada Ibanez de Caceres. miRNome exosomal characterization reveals a distinct signature able to predict cisplatin-response in NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2381.

Gender-based violence is a social problem that has accompanied man since ancient times. Currently there are many strategies implemented by countries to try to mitigate it and give to the women their rightful place in terms of gender equality and equal rights. Nevertheless, in the municipality of Santiago de Cuba the problem of gender violence is latent, which is evident from the systematicity that women, youth, adolescents, couples and families in general come to the House of Orientation the Women and Family (COMF) for help. In this sense, this paper aims to characterize gender violence in santiagueras females and visible intervention strategy implemented to minimize this reality. Research is based on the triangulation of quantitative and qualitative data and methodological strategy implemented.

This study aims to develop a novel method to synthesise site-wide heat recovery, distribution, cogeneration systems with optimum operating conditions of the steam mains. Previous approaches have simplified the problem to the extent that many important practical issues have been neglected and restricted the scope of the options included. The proposed methodology uses a combination of total site analysis and mathematical programming for a holistic approach to the steam system, which accounts for interactions between site utility system and processes. The optimisation problem involves the selection of more realistic operating conditions of the steam mains (superheating and pressure). The model will also account for water preheating, and superheating and desuperheating for process steam generation and use. Deaerators and let-down stations are also included in the analysis. The application of this methodology to a case study yielded a 7.6 % reduction in total energy requirement, compared to conventional utility system design method. The proposed approach addresses severe shortcomings in previous research on this topic and provides a foundation for future work to explore the next generation of sustainable utility systems.

Adjuvant chemotherapy for solid tumors based on platinum-derived compounds such as cisplatin is the treatment of choice in most cases. Cisplatin triggers signaling pathways that lead to cell death, but it also induces changes in tumor cells that modify the therapeutic response, thereby leading to cisplatin resistance. We have recently reported that microRNA-7 is silenced by DNA methylation and is involved in the resistance to platinum in cancer cells through the action of the musculoaponeurotic fibrosarcoma oncogene family, protein G (MAFG). In the present study, we first confirm the miR-7 epigenetic regulation of MAFG in 44 normal- and/or tumor-paired samples in non–small-cell lung cancer (NSCLC). We also provide translational evidence of the role of MAFG and the clinical outcome in NSCLC by the interrogation of two extensive in silico databases of 2019 patients. Moreover, we propose that MAFG-mediated resistance could be conferred due to lower reactive oxygen species production after cisplatin exposure. We developed specifically selected aptamers against MAFG, with high sensitivity to detect the protein at a nuclear level probed by aptacytochemistry and histochemistry analyses. The inhibition of MAFG activity through the action of the specific aptamer apMAFG6F increased the levels of reactive oxygen species production and the sensitivity to cisplatin. We report first the specific nuclear identification of MAFG as a novel detection method for diagnosis in NSCLC, and then we report that MAFG modulates the redox response and confers cell protection against free radicals generated after platinum administration, thus also being a promising therapeutic target., This study was supported by the “Fondo de Investigación Sanitaria-Instituto de Salud Carlos III” [PI15/00186 and CP 08/000689 to I.I.C. ] ; and the European Regional Development Fund/European Social Fund FIS [FEDER/FSE, Una Manera de Hacer Europa] . MINECO funds support O.V., C.R.A. and O.P.contracts through RTC-2015-4362-1 and RTC-2016-5314-1 projects.

One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance. Methods: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled “in silico” miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls. Results: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells. Conclusion: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors.

One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance. [Methods]: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled “in silico” miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls. [Results]: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells. [Conclusion]: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors., FIS (ISCIII) and FEDER/FSE funds (PI12/00386, PI12/01463, PI14/01495, PI15/00186, and FEDER/FSE, Una manera de hacer Europa support the study and contracts of I.I.C. and O.V. PTA2012/7141-I funds support OP contract and I.I.C. was financed by the ''Miguel Servet'' program (CP 14/00008). The authors gratefully acknowledge the Colombian Department for Science, Technology and Innovation (COLCIENCIAS), Cod.568-2012, for partially funding this work to J.S.

One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance. Experimental procedures: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled “in silico” miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls. Results: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells. Conclusion: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors. Citation Format: Olga Vera, Julia Jimenez, Carlos Rodriguez-Antolin, Olga Pernia, Carmen Rodriguez, Javier Soto, Rocio Rosas, Isabel Esteban-Rodriguez, Rosario Perona, Javier de Castro, Inmaculada Ibáñez-de-Cáceres. DNA methylation of miR-7 is a mechanism involved in platinum response through MAFG overexpression in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4413.

Olga Pernía et al., © 2014 Taylor & Francis Group, LLC. The methylation status of the IGFBP-3 gene is strongly associated with cisplatin sensitivity in patients with non-small cell lung cancer (NSCLC). In this study, we found in vitro evidence that linked the presence of an unmethylated promoter with poor response to radiation. Our data also indicate that radiation might sensitize chemotherapy-resistant cells by reactivating IGFBP-3-expression through promoter demethylation, inactivating the PI3K/AKT pathway. We also explored the IGFBP-3 methylation effect on overall survival (OS) in a population of 40 NSCLC patients who received adjuvant therapy after R0 surgery. Our results indicate that patients harboring an unmethylated promoter could benefit more from a chemotherapy schedule alone than from a multimodality therapy involving radiotherapy and platinum-based treatments, increasing their OS by 2.5 y (p =.03). Our findings discard this epi-marker as a prognostic factor in a patient population without adjuvant therapy, indicating that radiotherapy does not improve survival for patients harboring an unmethylated IGFBP-3 promoter., Supported by FIS (ISCIII), PI12/00386, PI12/01463, PI11/00949; PI11/00537; PTA2012/7141-I, all supported by FEDER funds. IIC was supported by the “Miguel Servet” program (CP 08/000689)

Introduction: The radioresistance of non-small cell lung cancer cells is a less explored and poorly defined field compared with drug resistance. Our group has previously reported that the loss of IGFBP-3 expression by promoter hypermethylation results in reduced tumor cell sensitivity to cisplatin in NSCLC, however the role of the IGF-I/IGBP-3 axe on radiosensitivity in cancer is controversial because of the differing results when various tumor types are evaluated. The purpose of the present study is to investigate the role of radiotherapy on the biology of IGFBP-3 promoter methylation and its clinical value as a potential tool for deciding on a concomitant radiotherapy after NSCLC surgery. Experimental procedure: In the present study we have worked with 5 human cancer cell lines, 40 NSCLC surgical sample patients with known response to CDDP and radiotherapy treatments and 10 control samples with non neoplastic pathology. We have study the relationship between a dose-response radiotherapy treatment and the IGFBP-3 gene expression regulated by promoter methylation measured by Radiation-clonogenic cell survival assays, RT-PCR, bisulfite modification and quantitative methylation specific PCR. We have also studied, the activation of the IGFIR, ERK and PI3K/AKT pathways through the analysis of the AKT, pAKT, pERK, ERK, pIGFIR and IGFIR protein levels by Western-Blot analysis. Additionally, in order to provide a helpful tool that enables clinicians to identify patients with a potential response to radiotherapy, we have used The TCGA annotation to correlate the IGFBP-3 methylation score of the NSCLC patients with their clinical-pathological parameters. Results: Our results suggest in vitro evidence that linked the presence of an IGFBP-3 unmethylated promoter with poor response to radiation. Radiation might sensitize chemotherapy-resistant cells by reactivating IGFBP-3-expression through promoter demethylation, inactivating the PI3K/AKT pathway. Our translational results indicate that patients harboring an unmethylated promoter could benefit more from a chemotherapy schedule alone than from a multimodality therapy involving radiotherapy and platinum-based treatments, increasing their OS by 2.5 years (p = .03). Conclusion: Our findings indiucate that radiotherapy does not improve survival for patients harboring an unmethylated IGFBP-3 promoter. Citation Format: Maria I. Ibáñez-de-Cáceres, Olga Pernía, Cristobal Belda-Iniesta, Olga Vera, Julia Jimenez, Carlos Rodriguez, Javier Soto, Javier de Castro, Teresa Macias, Federico Rojo, Joan Albanell, Rosario Perona. A new medical tool to discriminate on a radiotherapy concomitant treatment for non-small cell lung cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3443. doi:10.1158/1538-7445.AM2015-3443