Modulating receptor activation, migration and epigenetics of cancer cells using UV light

• Autor(a) principal: Lourenço, Tatiana Filipa Guerreiro
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Texto Completo: http://hdl.handle.net/10400.1/19576
• topic: Cancro; Pulmão; Radiação Ultravioleta; EGFR; hTERT; Epigenética; Metilação do DNA

Cancer is a complex disease characterized by uncontrolled and unorganized proliferation caused by mutations and disruption of epigenetic processes, leading to a malignant phenotype. There are 14 main hallmarks that malignant cells share between them. This work addressed tree of such hallmarks, namely “sustaining proliferative signaling”, “enabling replicative immortality” and “non mutational epigenetic reprogramming”. In the first one, cancer cells are characterized by acquiring the capacity to sustain proliferative signaling by increasing the activation of key receptors, such as the Epidermal Growth Factor Receptor (EGFR) by its cognate ligand EGF. EGFR is a trans-membrane glycoprotein with an extracellular epidermal growth factor binding domain and an intracellular tyrosine kinase domain. EGFR regulates signaling pathways that lead to cellular proliferation and is known to be overexpressed in many cancer types, including lung cancer. Previous studies have demonstrated that photonic modulation with a wavelength of 280 nm alters the 3D structure of EGFR, preventing EGF-EGFR activation and blocking the migration of cancer cells. This wavelength is considered to be safe since it has a low probability for inducing keratitis, skin erythema and skin carcinogenesis. We treated a human lung cancer cell line, A549 EGFR Biosensor, with UV at 280 nm and 295 nm and compared to cells not exposed to UV light as controls, performing wound healing assays to evaluate the migration capacity of these cells and real time fluorescence confocal imaging of cells overexpressing EGFR to determine whether there are changes in morphology and mobility of non-luminated vs. illuminated cells at 280nm and 295nm. Our results show that both the 280nm and 295nm lengths reduce the ability of cancer cells to migrate. Also, the 280nm wavelength decreases the expression of mesenchymal markers and increases the expression of epithelial markers. Furthermore, addressing the second and third hallmark, it is known that cancer cells acquire the capability of dividing indefinitely due to the activation of telomerase, a DNA polymerase that is responsible for the maintenance of telomere length, by adding repetitive sequences at the ends of chromosomes, leading to cell immortalization. We aimed at halting “enabling replicative immortality” and “non mutational epigenetic reprogramming” using the same photonic approach and to investigate if the light assault led to epigenetic changes and possible modulation of the telomerase expression/activity. In addition, we performed RT-qPCR to verify if there is an association between the expression of EGFR and Telomerase Reverse Transcriptase (hTERT)) and found that hTERT expression appears to be altered by radiation when cells were exposed for 30min and harvested at 0h, 24h and 48h. Moreover, we carried out pyrosequencing assays to understand if radiation is involved in epigenetic mechanisms, through altering DNA methylation patterns in a specific promotor region of hTERT. Our data shows that cells irradiated for 30 minutes present a significant reduction in DNA methylation at the studied region and that this pattern is maintained for 24h. Since metastases represent the main cause of death in cancer patients, our data proves to be promising in the development of a new treatment in the future, that improves the quality of life of patients and prevents metastatic progression.