Ramos, Sofia2025-12-222025-12-222024http://hdl.handle.net/10437/15704Breast cancer (BC) remains the most prevalent cancer among women. The identification of new biological pathways, biomarkers, and therapeutic targets is essential for improving patient survival rates. The levels of reactive oxygen species (ROS) and Calcium ion (Ca2+) fluxes play a crucial role in BC progression. Leveraging gene expression data from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) dataset and bioinformatics tools, this study aimed to identify novel druggable targets or prognostic biomarkers based on individual or combined expression profiles of redox and Ca2+ signalling-related genes with impact in BC progression. The first step was to identify and evaluate the online platforms that allow the analysis of BRCA gene expression and patient survival. The selection criteria defined were: user-friendliness, presenting global gene expression and between stages and patient survival, and providing straightforward methods for data extraction. Among the platforms assessed, GEPIA and UALCAN were deemed the most suitable for our analysis due to their robust features and ease of use. Recognizing the critical interplay between Ca2+ and ROS in BC progression, we aimed to evaluate how the co-dysregulation of Ca2+ and ROS-associated genes influences BC prognosis. This study utilized a bioinformatics approach, using data from the TCGA BRCA, to explore the association between the expression of specific genes involved in redox (LOXL2, LOXL3, PRDX4, TXN, TXNRD1, GLRX2, GLRX3, SOD2, NOX4) and Ca2+ homeostasis (TRPM8, CALM2, CAMK2G, ATP2C2, PLCD1, ORAI1, STIM1) on BC patient survival. Genes were selected based on significant expression differences between normal and tumour tissues and their association with survival, measured by hazard ratios. The cumulative proportion of survival at 5 years post-diagnosis was calculated for each quartile of expression of one gene within the population expressing high (Q4) or low levels (Q1) of a second gene. Gene pairs that consistently impacted cumulative survival were further analysed through enrichment analysis, focusing on biological processes from the Gene Ontology. This analysis revealed significant correlations between the simultaneous dysregulation of specific gene pairs and BC patient survival, such as GLRX3/LOXL3, PRDX4/LOXL2, NOX4/PRDX4, TRPM8/CAM2G, ATP2C2/TRPM8, CAMK2G/STIM1, ATP2C2/GLRX2, and PLCD1/TXNRD1. These gene pairs were consistently co-dysregulated with genes associated with critical processes in BC, such as cell cycle regulation, adhesion, and cellular projection. These findings highlight the potential of these gene pairs as biomarkers or therapeutic targets in BC. An example of redox-active proteins dysregulated in BC is the family of Lysyl oxidases. Lysyl oxidase (LOX) and LOX-like1-4 (LOXL1-4) enzymes impact cell migration and can contribute to iii the metastasis of BC. In this context, understanding the differential impact of each enzyme on BC progression is essential for directing drug development. Utilizing the TCGA-BRCA database and tools like GEPIA 2.0 and TIMER2.0, the study revealed that LOXL1, LOXL2, and LOXL3 are significantly overexpressed in BC tissues. This overexpression is linked to poorer disease-free survival (DFS) and overall survival (OS), particularly in specific BC subtypes. In contrast, LOXL4 expression is notably reduced, especially in advanced stages of BC, and its downregulation is associated with worse DFS and OS in HER2+ patients. The analysis also indicated that high levels of LOXL1, LOXL2, and LOXL3 are correlated with an increase in cancer-associated fibroblasts (CAFs) and a reduction in the infiltration of immune cells, such as B cells and T cells. Additionally, there is a strong correlation between the expression of LOX, LOXL1, and LOXL2 and other extracellular matrix (ECM)-related genes, including TIMP2, PDGFR-β, MMP-2, MMP-14, FN1, ITGA5, and ZEB1. These findings suggest that while inhibiting LOXL3 may have varying effects depending on the BC subtype, targeting LOXL1 and LOXL2 could be a more effective therapeutic strategy. Conversely, inhibiting LOXL4 might not be as beneficial. This study highlights the importance of considering the specific roles of LOX family members and expression patterns in developing targeted therapies for BC. The identification of redox-related genes differentially expressed in HER2+ BC compared to normal tissues, their association with patient survival, and with tumour cell infiltrate can potentially lead to the identification of new prognosis biomarkers. Using Gene Ontology and keywords related to oxidative processes, the study filtered data from the TCGA-BRCA-HER2+ database. GEPIA2 and UALCAN were employed to assess the association between the expression of redox homeostasis-related genes on patient survival and to examine differences in expression between HER2+ BC patients and normal tissues (p <0.05). This approach led to the identification of 55 potentially relevant redox-related genes. To explore the relationship between gene expression and cell infiltration levels in HER2+ BC patients, the TIMER2.0 tool was utilized. Out of the 55 genes identified, only six—EZH2, MAPK13, NFE2L2, HDAC2, MACROH2A1, and TSC2— have been experimentally validated in the context of HER2+ BC. However, several redox-related genes were significantly dysregulated, although their specific roles in this subtype are not fully explored. Among these, 17 genes, including TXNRD1 and EZH2, were overexpressed in HER2+ tumours, while 26 genes, such as NFE2L2 and TSC1, were downregulated and correlated with increased infiltration of macrophages and neutrophils. Additionally, ten redox-related genes demonstrated correlations with either high or low HR, suggesting an influence on tumour infiltrates. These findings underscore the potential of redox-related genes as therapeutic targets iv or biomarkers in HER2+ BC, emphasizing the importance of further research to clarify their roles in cancer progression and therapy resistance. Although experimental validation is needed, all the studies presented in this thesis identified novel potential BC biomarkers and therapeutic targets; and deepened our understanding of the interactions between redox and Ca2+-related genes and other genes, and their association with tumour infiltrates and BC patient outcomes. Thus, contributing to the identification of novel potential prognosis biomarkers or druggable targets.application/pdfengopenAccessDOUTORAMENTO EM CIÊNCIAS DA SAÚDECIÊNCIAS DA SAÚDECANCRO DA MAMATERAPÊUTICASANÁLISE DE SOBREVIVÊNCIAHEALTH SCIENCESBREAST CANCERTHERAPEUTICSSURVIVAL ANALYSISdoctoralThesis