ANÁLISIS DE MICROARNS EN CÁNCER DE PULMÓN NO MICROCÍTICO

Lung cancer is the leading cause of death cancer-related worldwide, and is the third most common cancer type. The non-small cell lung cancer (NSCLC) comprises almost 85% of all lung cancers and the 5-year survival rate remains poor. The microRNAs (miRNAs) are a class of small non-coding RNAs that regulate post-transcriptionally the gene expression, being involved in essential processes of the cell. Deregulated miRNAs have been found in many diseases, including cancer, and as a result, there is a growing interest in studying their role as potencial biomarkers. The main objective of this doctoral thesis is to analyze the differential expression of miRNAs in a patient training group by next generation sequencing (NGS) to later validate the findings in an independent set of patients using a less complex technique, such as RTqPCR and evaluate its possible correlation with clinical-pathological and prognostic variables. Likewise, this will allow us to know in greater depth the profile of miRNAs in patients with NSCLC. The analysis by NGS showed 39 miRNAs differentially expressed in tumoral tissue: 28 upregulated and 11 downregulated. 22 of these miRNAs were validated by RTqPCR in a validation set. Later, survival analyses revealed that two miRNAs, miR-21 and miR-188, were related to prognosis. Furthermore, subgroup of patients with elevated expression levels of both miRNAs (miR-21, miR-188) presented a shorter relapse free survival (RFS) and overall survival (OS) than each miRNA individually. The multivariate Cox regression analysis revealed that combined signature could be considered as an independent prognosis biomarker in this group of patients. Therefore, it can be concluded that NGS technology can specifically identify deregulated miRNAs in resected early-stage NSCLC. Moreover, miR-21 and miR-188 overexpression correlated with a worse prognosis and combined signature for both miRNAs (miR-21high- miR-188high) represent a novel independent prognosis biomarker in NSCLC.El cáncer de pulmón es una de las principales causas de muerte relacionada con cáncer en el mundo, siendo el tercer tipo de cáncer más común. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón y la supervivencia a los 5 años es reducida. Los microARNs (miARNs) son un grupo de ARNs no codificantes de pequeño tamaño que regulan postranscripcionalmente la expresión génica y que por tanto están involucrados en procesos esenciales de la célula, encontrándose desregulados en muchas enfermedades incluida el cáncer. Como consecuencia, existe un creciente interés en estudiar su papel como posibles nuevos biomarcadores en cáncer. Por todo ello, el principal objetivo de esta tesis doctoral es analizar la expresión diferencial de miARNs en un grupo prueba de pacientes mediante secuenciación masiva para posteriormente validar el hallazgo en un grupo independiente de pacientes usando una técnica más sencilla como es la RTqPCR y evaluar su posible correlación con las variables clínico-patológicas y pronósticas en pacientes con CPNM. El análisis mediante secuenciación masiva reveló que 39 miARNs estaban diferencialmente expresados en tejido tumoral: 28 sobreexpresados y 11 infraexpresados. 22 de estos miARNs se validaron mediante RTqPCR en una cohorte independiente. Posteriormente, los análisis de supervivencia revelaron que dos miARNs, miR-21 y miR-188, se relacionaron con pronóstico. Además, el grupo de pacientes con elevada expresión de ambos miARNs (miR-21, miR-188) presentaba una supervivencia libre de enfermedad (SLE) y una supervivencia global (SG) más cortas que cada miARN analizado individualmente. El análisis multivariante de regresión de Cox reveló que la firma combinada puede considerarse como un biomarcador pronóstico independiente en este grupo de pacientes. Por tanto se puede concluir que la tecnología de secuenciación masiva puede identificar específicamente miARNs desregulados en CPNM en estadios resecables. Además la sobreexpresión del miR-21 y el miR-188 se correlacionó con un peor pronóstico, y la firma combinada de ambos (miR-21alto- miR-188alto) representó un nuevo biomarcador pronóstico independiente en CPNM.El càncer de pulmó es una de les principals causes de mort relacionades amb càncer al món, sent a més a més el tercer tipus de cáncer més comú. El càncer de pulmó no microcític (CPNM) representa quasi el 85% de tots els casos de càncer de pulmó i la supervivència als 5 anys continua sent molt baixa. Els microARNs (miARNs) són un grup d'ARNs no codificants de petit tamany que regulen postranscripcionalment l'expressió gènica i que per tant estàn involucrats en procesos fonamentals de la cèl¿lula, trobant-se desregulats en moltes malaties inclosa el cáncer. Com a consequència, existeix un creixent interés en estudiar el seu paper com possibles nous biomarcadors en cáncer. Per tot açò, el principal objectiu d'esta tesi doctoral és analitzar l'expressió diferencial de miARNs en un grup prova de pacients mitjançant seqüenciació massiva per a posteriorment validar la trobada en un grup independent de pacients utilitzant una tècnica més senzilla com és l'RTqPCR i evaluar la seu possible correlació amb les variables clinico-patològiques i pronòstiques en pacients amb CPNM. L'ànalisi mitjaçant seqüenciació masiva revelà que 39 miARNs estaven diferencialment expressats en teixit tumoral: 28 sobreexpressats i 11 infraexpressats. 22 d'aquests miARNs van ser validats mitjançant RTqPCR en un grup independent. Posteriorment, les análisis de supervivència revelaren que dos miARNs, miR-21 i miR-188, es relacionaren amb pronòstic. A més a més, el grup de pacients amb elevada expressió de tots dos miARNs (miR-21, miR-188) presentava una supervivència lliure de recaiguda (SLR) y una supervivència global (SG) més curtes que cadascun dels miARNs individualment. L'anàlisi multivariant de regressió de Cox revelà que la firma combinada pot considerar-se com un biomarcador pronòstic independent en aquest grup de pacients. Per tant, se pot concloure que la tecnologia de seqüenciació masiva pot identificar específicament miARNs desregulats en CPNM en estadis resecables. A més, la sobreexpressió del miR-21 i miR-188 correlacionava amb un pitjor pronòstic, i la firma combinada de tots dos (miR-21alt-miR-188alt) va representar un nou biomarcador pronòstic independent en CPNM.Gallach García, S. (2017). ANÁLISIS DE MICROARNS EN CÁNCER DE PULMÓN NO MICROCÍTICO [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90428TESI

MicroRNAs: Promising New Antiangiogenic Targets in Cancer

[EN] MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulators of signalling pathways in multiple cell types including endothelial and perivascular cells. This review summarises the role of miRNAs in tumour angiogenesis and their potential implications as therapeutic targets in cancer.This study was partially supported by a Grant from Ministerio de Ciencia e Inovacion de Espana (TRA09-0132), Beca Roche en Onco-Hematologia 2009, and Red Tematica de Investigacion Cooperativa en Cancer (RD12/0036/0025).Gallach, S.; Calabuig-Farinas, S.; Jantus Lewintre, E.; Camps, C. (2014). MicroRNAs: Promising New Antiangiogenic Targets in Cancer. BioMed Research International. 2014. https://doi.org/10.1155/2014/878450S201

Analysis of Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes' cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease. Keywords: non-small cell lung cancer; liquid biopsy; exosomes; extracellular vesicles; cell cultures; adenocarcinoma; squamous cell carcinoma; biomarker; tumorsphere

Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens

[EN] Objectives: Oral microbiome plays an important role in oral diseases. Among them, proliferative verrucous leucoplakia (PVL) is an uncommon form of progressive multifocal leukoplakia with a worryingly rate of malignant transformation. Here, we aimed to characterize the oral microbiome of PVL patients and compare it with those of healthy controls. Material and methods: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their microbial communities. The sequence of the V3-V4 region of 16S rRNA gene was used as the taxonomic basis to estimate and analyze the composition and diversity of bacterial populations present in the samples. Results: Our results show that the oral microbial composition and diversity are significantly different among PVL patients and healthy donors. The average number of observed operational taxonomic units (OTUs) was higher for healthy donors than for PVL, proving a loss of diversity in PVL. Several OTUs were found to be more abundant in either group. Among those that were significantly enriched in PVL patients, potential protumorigenic pathogens like Oribacterium sp. oral taxon 108, Campylobacter jejuni, uncultured Eubacterium sp., Tannerella, and Porphyromonas were identified. Conclusion: Oral microbiome dysbiosis was found in patients suffering from PVL. To the best of our knowledge, this is the first study investigating the oral microbiome alterations in PVL and, due to the limited number of participants, additional studies are needed. Oral microbiota-based biomarkers may be helpful in predicting the risks for the development of PVL.This work was supported by PI19/00790 from Fondo de Inves-tigacion Sanitaria, ISCIII (PI: Jose Bagan) , CB16/12/00350 from CIBERONC and IDI-2020-133875-a from MICINN.Herreros-Pomares, A.; Llorens, C.; Soriano, B.; Zhang, F.; Gallach, S.; Bagán, L.; Murillo, J.... (2021). Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens. Oral Oncology. 120:1-9. https://doi.org/10.1016/j.oraloncology.2021.1054041912

JunB Breakdown in Mid-/Late G2 Is Required for Down-Regulation of Cyclin A2 Levels and Proper Mitosis▿

JunB, a member of the AP-1 family of dimeric transcription factors, is best known as a cell proliferation inhibitor, a senescence inducer, and a tumor suppressor, although it also has been attributed a cell division-promoting activity. Its effects on the cell cycle have been studied mostly in G1 and S phases, whereas its role in G2 and M phases still is elusive. Using cell synchronization experiments, we show that JunB levels, which are high in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome. The forced expression of an ectopic JunB protein in late G2 phase indicates that JunB decay is necessary for the subsequent reduction of cyclin A2 levels in prometaphase, the latter event being essential for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects. As these aberrations may cause genetic instability, our findings contrast with the acknowledged tumor suppressor activity of JunB and reveal a mechanism by which the deregulation of JunB might contribute to tumorigenesis

The prognostic value of hTERT expression levels in advanced-stage colorectal cancer patients: a comparison between tissue and serum expression

[EN] Aim: Telomeres are regions of highly repetitive, non-coding DNA located at the termini of chromosomes whose principal function is to maintain the structural stability of these ends. In 90% of human tumours, telomere length is maintained by the expression and activation of telomerase reverse transcriptase. Various studies have demonstrated an increase in telomerase activity in tumour tissue, which suggests its possible prognostic value. The main objective of our study was to study the prognostic value of the expression level of telomerase catalytic component (hTERT) in patients with colorectal cancer (CRC). Methods: We analysed the prognostic value of the ratio of telomerase expression in tumour tissue to telomerase expression in the adjacent healthy mucosa and the prognostic value of the expression level of hTERT in the serum of patients diagnosed with CRC. As secondary objectives of the study, we (1) analysed the correlation between telomerase expression in the serum and that in the tumour tissue and (2) analysed the relationship between telomerase expression and different clinical parameters. Results: Peripheral blood and tissue samples taken from 48 patients with CRC were analysed. No significant differences were observed in disease-free survival (DFS) or overall survival time (OST) between the groups of patients categorised based on the ratio of telomerase expression between tumour tissue and healthy tissue. The correlation index (Pearson's coefficient) between telomerase levels in the serum and those in tissue was 0.32. Our study of the relationship between telomerase levels in the serum and different clinical variables, such as tumour size, ganglion affectation, preoperative carcinoembryonic antigen levels and stage, revealed a higher telomerase expression level in patients with stage IV CRC. There was no significant association between telomerase expression in tumour tissue and the clinical parameters analysed. Conclusions: The results obtained in our study do not allow us to propose that the level of telomerase expression be used as a prognostic factor in colorectal cancer. Thus, we cannot consider telomerase expression in the serum as a surrogate marker of its expression in tumour tissue. © 2011 Feseo.This work was made possible by a grant from the Spanish Society of Medical Oncology (SEOM).Safont, MJ.; Gil, M.; Sirera Pérez, R.; Jantus Lewintre, E.; Sanmartín, E.; Gallach, S.; Caballero, C.... (2011). The prognostic value of hTERT expression levels in advanced-stage colorectal cancer patients: a comparison between tissue and serum expression. Clinical & Translational Oncology. 13(6):396-400. doi:10.1007/s12094-011-0673-2S39640013

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The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients

Qualitative analysis of circulating DNA in the blood is a promising non-invasive diagnostic and prognostic tool. Our aim was to study the association between the presence of KRAS mutations at codon 12 and several clinical variables in advanced non-small cell lung cancer (NSCLC) patients. Methods: We examined 308 stage IIIB and IV NSCLC patients who were treated with cisplatin and docetaxel. Blood samples were collected before chemotherapy, and circulating DNA was extracted from the plasma using commercial adsorption columns. The KRAS mutational status was determined by an RT-PCR method that is based on allelic discrimination. Results: The median age of the patients was 60 years [31-80], 84% were male, 98% had a performance status of 0-1 and 84% of the patients were in stage IV. The histological subtypes were as follows: 30% squamous cell carcinoma (SCC), 51% adenocarcinoma (ADC) and 19% others. Of the 277 response-evaluated patients, 1% achieved a complete response (CR), 26% achieved a partial response (PR), 34% had stable disease (SD) and 39% had progressive disease (PD). Additionally, 27 (8.8%) patients had KRAS mutations; 26 had a KRAS codon 12 TGT mutation, and 1 had a codon 12 GTT mutation. Plasmatic KRAS mutations were found in patients presenting SCC or ADC. Patients with KRAS mutations in plasma DNA had a median progression free survival (PFS) of 5.77 months [3.39-8.14], whereas for patients with wild-type (wt) KRAS, the PFS was 5.43 months [4.65-6.22] (p = 0.277). The median overall survival (OS) in KRAS-mutated patients was 9.07 months [4.43-13.70] vs 10.03 months [8.80-11.26] in wt patients (p = 0.514). Conclusions: In advanced NSCLC patients, there were no significant differences between patients with or without KRAS mutations in plasma-free DNA with respect to the baseline characteristics, response rates, PFS or OS. © 2010 Elsevier Ireland Ltd.This work was sponsored in part by a grant from the Spanish Society of Medical Oncology (SEOM) and by a grant (RD06/0020/1024) from Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation & European Regional Development Fund (ERDF) "Una manera de hacer Europa". None of the funding agencies were involved in the design, data management, data analysis, manuscript preparation and review, or decision to submit.Camps, C.; Jantus Lewintre, E.; Cabrera, A.; Blasco, A.; Sanmartin, E.; Gallach, S.; Caballero, C.... (2011). The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients. Lung Cancer. 72(3):365-369. https://doi.org/10.1016/j.lungcan.2010.09.005S36536972