Abstract
In 2020, there were more than 2.3 million new breast cancer cases globally, with 665,684 recorded deaths. Despite advances in diagnoses and treatment of early-stage breast cancer, metastatic breast cancer remains almost incurable and the cause of 90% of breast cancer-related deaths. As ion channels are implicated in processes that promote or inhibit cancer progression, my lab investigated a role for the Epithelial Sodium Channel (ENaC) in breast cancer progression. Preliminary results from bioinformatic analysis of published datasets, followed by in vitro cell line studies, revealed that the alpha subunit of ENaC (αENaC) was important in breast cancer proliferation and metastasis. This current study is therefore aimed at further characterizing the role of αENaC and determining the biological mechanisms underlying its effects in breast cancer progression. We hypothesized that: (1) αENaC protein is highly expressed in breast cancer cells characterized by slower rates of proliferation and migration, (2) higher expression of αENaC protein alters global gene expression, especially those involved in processes underlying cell growth and migration, and (3) expression of αENaC protein changes at different phases of the cell division cycle to alter the regulation and transitioning through the cell cycle phases. To further characterize αENaC’s role in breast cancer, bioinformatic analyses were performed on publicly available gene expression datasets from breast cancer and normal breast tissues. These showed that repression of αENaC expression significantly correlates with increased probabilities for developing metastasis and relapse in breast cancer patients. To investigate the mechanisms underlying αENaC’s effects, RNA sequencing was performed on three monoclonal populations of the MDA-MB-231 breast cancer cell line transfected to stably overexpress αENaC and a control. Analysis of the transcriptomes of these cell lines uncovered 386 genes that were differentially expressed across all three clones due to overexpression of αENaC. Gene Set Enrichment Analysis was then performed, which showed significant underrepresentation of gene sets and corresponding leading-edge genes involved in pathways/processes affecting cell adhesion, migration, immune modulation among others. The MDA-MB-231 breast cancer cell lines were subsequently synchronized to the early S phase of the cell cycle by double thymidine block, then followed through progression along the cell cycle by live fluorescence imaging and western blotting. These show that expression of αENaC oscillates as the cells progress through the different phases of the cell cycle. This study has identified molecular mechanisms and targets which promote breast cancer proliferation and metastasis when αENaC expression is repressed. These could be targeted in the future to improve survival.