Exploring the functional impact of transposable elements in human cancer genomes
- Autores: Ana Oitabén Fernández
- Directores de la Tesis: José Manuel Castro Tubio (dir. tes.), Mónica Martínez Fernández (dir. tes.)
- Lectura: En la Universidade de Santiago de Compostela ( España ) en 2025
- Idioma: inglés
- Tribunal Calificador de la Tesis: Jesús María Paramio González (presid.), Diana Guallar Artal (secret.), Davide Seruggia (voc.)
- Programa de doctorado: Programa de Doctorado en Medicina Molecular por la Universidad de Santiago de Compostela
- Materias:
- Enlaces
- Tesis en acceso abierto en: MINERVA
- Resumen
Approximately half of our genome is derived from genomic sequences with potential ability to move from one place to another, being integrated into a new location. These sequences are known as mobile or transposable elements (TEs). Traditionally dismissed as "junk DNA", because they reside in non-coding regions thought to lack biological function, TEs are now recognized for their role in genome evolution and diversity. Their ability to mobilize and integrate into new genomic locations has played a significant role in creating genomic variability over thousands of years, influencing both normal biological processes and disease development. Among them, retrotransposons are able to copy and insert themselves into a new region through an RNA intermediate, in a process known as retrotransposition. Although most of them are likely to be phenotypically silent, some have been identified to lead to severe phenotypic consequences, representing an important source of somatic mutation in disease. In the context of cancer, somatic retrotransposition may have important consequences for the evolution of a tumour. LINE-1 (L1) is the only autonomous type of retrotransposon that remains active in the human genome, being highly active in cancer, specifically in certain tumour types such as oesophagus, head and neck, lung and colorectal tumours. This PhD thesis aimed to explore the structural and functional impact of somatic retrotransposon insertions, particularly L1 retrotransposition, in the context of human cancers.
In the first part of this work, we have developed the "RetroTest", a new straightforward method to measure somatic L1 activity in tumours. This tool can be applied to a broad variety of sample sources, working even in FFPE tumour biopsies, which makes it an attractive method to be applied in clinics. L1 elements can mobilize unique sequences along the genome when its transcription continues beyond 3' adjacent unique regions, in a process called 3'-transduction, which can be used to identify active L1 elements. Based on this mechanism, our tool consists of DNA target sequencing libraries directed to the transductions of the 124 L1 source elements active in cancer cells, according to the Pan-Cancer Analysis of Whole Genomes (PCAWG) Project results. These libraries were sequenced by Illumina paired-end (PE) sequencing and analysed with a new bioinformatic pipeline, to evaluate L1 activation according to the detected transductions. Using this method, we have evaluated L1 activation in a cohort of 96 head and neck squamous cell carcinoma (HNSCC) patients at different stages of the disease. We could detect L1 activity (greater than or equal to1 transduction) in 75% of the patients. We found significant differences in L1 activation between stages T1-T2 and T3-T4 (p = 0.0072), indicating higher activation rates in advanced stages. Interestingly, we detected L1 activation even at the first stages, supporting an early L1 activation during tumour development. Moreover, we also found L1 activation in the tissue adjacent to the tumour characterized as healthy in the histopathological determination. These results evidenced an early activation of L1 in HNSCC and its potential as a candidate biomarker in early diagnosis of this tumour type.
We have also used RetroTest to determine L1 activity in primary tumour samples from 31 advanced Non-Small Cell Lung Cancer (NSCLC) patients and 26 Muscle Invasive Bladder Cancer (MIBC) patients treated with immunotherapy at first line, to investigate whether L1 activity could play a role in immunotherapy response. We found activation in 45% of NSCLC patients and in 58% of MIBC. When we analysed L1 activity related to immunotherapy response, we found a statistically significant association for MIBC patients (p = 0.02564), but not for NSCLC, although a positive trend was found for both tumour types. These observations provide promising results for L1 activation as a predictive response biomarker in immunotherapy treated patients. Notably, we found an association of high L1-retrotransposition rates and tobacco habits in HNSCC (p = 0.0015) and NSCLC (p = 0.04887) patients, suggesting that L1 activation play a role in tobacco-related cancers.
In the second chapter of this PhD project, we further explored the characterization of somatic retrotransposition and its effects on tumour cells. For this purpose, we selected seven NSCLC samples (two cell lines and five primary tumours) with high levels of retrotransposon insertions, encompassing almost 3,000 somatic retrotransposition events among all of them. We characterized these samples with a multi-omic approach. We employed genomic - short-read (SR) and long-read (LR) whole genome sequencing (WGS)-, transcriptomic - SR and LR whole transcriptome sequencing (WTS)- and epigenomic - ATAC-seq, PCHi-C and Micro-C- data to provide a comprehensive analysis of somatic mobile element insertions (MEIs) and their impact in gene regulation and genome architecture in tumour samples.
LR sequencing provided an advantage for the detection and characterization of somatic insertions, particularly in challenging genomic regions associated with assembly gaps and alignment artefacts. Almost half of the identified somatic events were exclusively detected in LR sequencing data. Most of MEIs in this cohort were attributed to L1 elements (86%). We observed that MEIs were genome-wide distributed, but we could identify hotspot regions with dense somatic insertions accumulation. Despite most insertions occurring in non-coding regions, as intergenic and intronic regions, some insertions at gene bodies were linked to gene expression changes, including alterations in cancer-related genes. Besides, analysis of WTS LR data revealed examples where insertions led to aberrant splicing forms, including exonizations and premature transcription termination.
Beyond direct disruptions at insertion sites, somatic MEIs also influenced 3D chromatin organization. They formed tumour-specific interactions between inserted regions and distant gene promoters, some of which were linked to differential gene expression, including noteworthy cases involving cancer-related genes. Furthermore, transduction events could mobilize regulatory elements, such as enhancers or promoter-like sequences. Although new interactions between transduced regions and gene promoters were identified, no direct link to gene expression changes was found in our samples. We also found 80 genomic rearrangements mediated by retrotransposons. Additionally, we detailed how these rearrangements influence 3D genome structure by creating new chromatin interactions.
Overall, this work proposed a new method to measure L1 activation in an easy way applicable to the clinical practice, providing promising results for L1 activity as a biomarker in early diagnosis of HNSCC and in response prediction for immunotherapy treated patients. Moreover, it aimed to highlight the multifaceted role of somatic retrotransposition in altering not only the genomic sequence but also the regulatory landscape and chromatin structure in lung cancer genomes.
