Genetic disruption of transfer RNA modifications in human cancer

[eng] Cancer was reported as the second leading cause of death in 2018 by the World Health Organisation. Cancer is defined as the set of diseases that proceeds in multiple phases generating a transformation lead by an accumulation of genetic (mutations, copy number), epigenetic (CpG methylation, histone modifications), and epitranscriptomic alterations. Lung cancer is the primary cause of cancer-related deaths. It is commonly diagnosed at advanced stages, leading to a 5-year survival of about 21%. Lung cancer is historically subdivided according to resection specimens in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC is an epithelial tumour with neuroendocrine features representing 10-15% of all lung tumours, and the most lethal and aggressive subtype of lung cancer. Epitranscriptomics is the field of study of RNA modifications. RNA modifications are inserted in transcripts derived from both coding and non-coding genes. These modifications are added to ribonucleotide residues on the purine/pyrimidine ring or ribose, and a significant number of enzymes regulates their dynamic. The role of these modifications ranges from provide stability, to be involved in export, maturation, splicing, folding and function of the RNA. Transfer RNAs (tRNAs) tRNA play a fundamental role in protein biosynthesis as an adaptor molecule acting as a biological link between mRNA and protein sequences on the ribosome during the translation process. Transfer RNAs show the highest density of modifications, specifically the anticodon‐loop region is a hotspot of highly diverse modifications. TRIT1 gene encodes the enzyme tRNA-isopentenyltransferase-1 that transfers an isopentenyl group to form the N6‐isopentenyladenosine (i6A) at position 37 of the cytoplasmic tRNA[Ser]Sec and tRNASer(HGA), and mitochondrial (mt) tRNACys(GCA), tRNATyr(GUA), tRNATrp, tRNASer(UGA), and tRNAPhe(GAA). tRNA[Ser]Sec carries the selenocysteine amino acid that is incorporated in 25 proteins in humans called selenoproteins. We investigated genetic and epigenetic alterations that would affect transfer RNAs modifications in cancer. After data-mining the copy number and DNA methylation data for tRNA modifier enzymes in about 1000 cell lines, we identified an outstanding TRIT1 copy number amplification restricted only to small cell lung cancer (SCLC) cell lines (11 of 60). Thus, this was the starting point of an exhaustive study. We focused on evaluating the implication of this event in tumorigenesis, as well as its potential as a drug target candidate. The TRIT1 gene amplification in cancer cell lines was associated to a significant increase at transcriptional and protein levels. To elucidate the functional role of TRIT1 amplification in SCLC, we assessed the consequences of decrease the expression of TRIT1 using the shRNA approach in the TRIT1-amplified DMS-273 SCLC cell line. The level of i6A decreased upon decreasing TRIT1 expression. In vitro assays did not provide keys to elucidate the role of TRIT1 in cancer, and we decided to perform in vivo assays in mice. We observed a significant reduction in tumour growth upon TRIT1 knockdown, providing robust evidence of the impact of TRIT1 amplification in cancer. Moreover, we carried out massive RNA sequencing of the loss-of-function model in the DMS-273 cell line. After computational analysis, 4510 differential expressed mRNA were identified. Notably, 3409 (75.9%) of these mRNAs were found downregulated upon TRIT1 depletion reflecting the significant impact of i6A hypomodification. Gene set enrichment analysis from the downregulated genes showed “regulation of cell differentiation” as the most enriched process upon TRIT1 depletion. Considering that therapeutic options in SCLC are limited, we performed an exhaustive revision of the literature and a computational approach to identify drugs affecting TRIT1-amplified SCLC. The gene amplification-associated overexpression of TRIT1 confers sensitivity to arsenic trioxide and dimethyloxalylglycine (DMOG), as shown in vitro cellular assays and in vivo experiments in mice. Finally, TRIT1 gene amplification was identified in about 10-15% of SCLC primary samples.[spa] Las modificaciones del ARN son claves en multitud de procesos celulares que al verse alteradas pueden participar en el proceso de tumorogénesis. En la presente tesis se aborda el efecto de estas alteraciones implicadas en la transformación celular en a las moléculas de los ARN de transferencia (ARNt). Se identificó la amplificación génica de TRIT1 exclusivamente en líneas celulares de cáncer de pulmón microcítico (SCLC). La amplificación de TRIT1 también fue detectada en pacientes de SCLC. La enzima TRIT1 que se encarga de introducir la modificación i6A la posición A37 de ciertos ARNt. La amplificación de TRIT1 lleva a un aumento de su expresión a nivel transcripcional y traduccional. Se generó un modelo celular de pérdida de expresión empleando una línea celular de SCLC con amplificación de TRIT1, y se realizaron ensayos in vitro. También se evaluaron las consecuencias funcionales in vivo mediante la inyección de las células en ratón, se observó una reducción del crecimiento tumoral en los tumores derivados de las células con pérdida de expresión de TRIT1. El análisis transcriptómico del modelo celular, mediante secuenciación masiva de ARN (RNA-seq), mostro una desregulación de procesos biológicos relacionados con la diferenciación celular debido a la amplificación de TRIT1. Este hallazgo condujo a la identificación del trióxido de arsénico como un posible candidato como tratamiento para SCLC por su acción en la diferenciación celular. Ensayos in vitro e in vivo indican que la amplificación de TRIT1 confiere sensibilidad al trióxido de arsénico, proponiendo un nuevo candidato terapéutico para el tratamiento de una enfermedad con tan pocas opciones terapéuticas como es SCLC