Characterization of hypoxia biomarkers in cancer through computational and molecular validation

• Autor(a) principal: Agostinho, Filipe Pontes da Costa
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/164850
• topic: Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The World Health Organization defines cancer, also known as malignant tumours or neoplasms, as a large group of diseases that can affect any part of the body. These diseases are characterized by abnormal, rapid and uncontrolled cellular growth beyond their usual boundaries, which can invade adjacent parts of the tissue and disseminate to distant regions of the body through a process known as metastasis. There are several genetic pathways important for tumor survival. Among them the hypoxia induced factor pathway (HIF), and the nuclear factor kappa B (NF-κB) pathway plays a crucial role. The HIF pathway allows cancer cells to adapt to dwindling oxygen concentrations by regulating angiogenesis, erythropoiesis, energy metabolism and cell growth and survival. NF-κB pathway is related to cell survival, differentiation, inflammation and carcinogenesis. The objective of the present thesis was to characterize the molecular alterations in cancer affecting the hypoxia biomarkers HIF1A, EPAS1 (HIF2A) and NFKB1 through two computational in silico analysis (Pancancer and cancer specific) and by assessing the expression alterations of the hypoxia-related genes in different cancer cell lines. First, a computational survey was performed using the cBioPortal resource to perform a PanCancer survey. Then, a cancer specific analysis was performed for Colorectal Adenocarcinoma (COAD), Uterine Corpus Endometrial Carcinoma (UCEC) and Skin Cutaneous Melanoma (SKCM). After the computational survey, two cell lines were used for laboratory analysis: HCT116 and MNT1. Two types of HCT116 were grown: unaltered HCT116 and HCT116 DOX. Both cell lines were grown in 2D monocultures or 3D spheroids for RNA extraction. cDNA was then synthesised through PCR (polymerase chain reaction). With the obtained samples, qPCR (quantitative polymerase chain reaction) was performed to analyse the expression levels of hypoxia-related genes, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as a housekeeping gene. According to the PanCancer analysis, the genes HIF1A, EPAS1 and NFKB1 are most frequently altered in Endometrial Cancer, Neuroepithelial Tumours, and Melanoma, with the most frequent alterations being genetic mutations, and there is significant co-occurrence in the alterations of the hypoxia-related genes. Overall survival of patient with mutations in at least one hypoxia-related gene had better prognosis than patients without mutations. In the single cancer analysis only UCEC shows significant co-occurrence between all three hypoxia-related genes, though all three genes show significant positive correlation of mRNA expression with each other in the three cancer types. Similar to PanCancer results, Endometrial Cancer patients with alterations in HIF1A have better survival than patients without alterations in this gene. Results of the laboratory culture assays show that 3D samples tend to have higher expression than 2D samples. In 2D samples, MNT1 has highest NFKB1 expression, HCT116 has higher HIF1A and EPAS1 expression. Among 3D samples, HCT116 has highest NFKB1 and HIF1A expression, while MNT1 has highest EPAS1 expression. More studies will be required to determine the viability of these genes as biomarkers for determining patient prognosis, as some studies note inconsistent effects of hypoxia-related genes on cancer development.