Abstract
Breast cancer is currently the leading cancer and cause of cancer mortality amongst women across the world, including New Zealand. Despite advancement in therapeutic treatments, breast cancer burden remains high, with the majority of deaths reportedly caused by metastasis, where breast cancer cells spread to distant organs. Progression of breast cancer to metastasis requires cancer cells to undergo changes in their phenotypes and this is often achieved through epithelial-mesenchymal plasticity (EMP). During EMP, epithelial cells with apical-basal polarity and cell-cell junctions, gradually transition into mesenchymal-like cells with enhanced invasiveness and migratory abilities, which are features necessary for metastasis. Recent studies have reported that ion channels regulate EMP-associated cancer, including epithelial sodium channel (ENaC) that functions in sodium reabsorption and blood pressure regulation. The α-ENaC subunit has been suggested to play an anti-proliferative role in breast cancer, possibly via EMP. However, the mechanism through which α-ENaC influences EMP and breast cancer progression has not been thoroughly investigated.
Therefore, this project aimed to study the potential role of α-ENaC in regulating EMP during breast cancer progression by investigating the mRNA and protein expression of EMP markers following overexpression of α-ENaC or inhibition of α-ENaC’s activity with amiloride. Experiments were conducted in the mesenchymal-like BT-549 and epithelial-like T-47D breast cancer cells using RT-qPCR and western blot. It was hypothesised that α-ENaC overexpression will promote epithelial phenotypes by causing an increase in E-cadherin with a decrease in Vimentin and N-cadherin in both cell lines, whereas amiloride will promote mesenchymal phenotypes by reversing the changes expected for the overexpression experiments. Additionally, the mRNA expression of α-ENaC in both cell lines were also compared with RT-qPCR, while immunohistochemistry was conducted to study the ex vivo expression patterns of α-ENaC and E-cadherin within human breast cancer tissues.
Here, I showed that epithelial-like T-47D cells have significantly higher α-ENaC expression compared to mesenchymal-like BT-549 cells. In support of the hypothesis, overexpression of α-ENaC in BT-549 and T-47D cells significantly increased E-cadherin mRNA levels, while amiloride significantly decreased E-cadherin in the BT-549 cells. Interestingly, Vimentin mRNA levels were also increased in the BT-549 cells but were not altered in the T-47D cells following α-ENaC overexpression. On the other hand, inhibiting α-ENaC’s activity with amiloride did not alter the mRNA levels of E-cadherin and Vimentin in the T-47D cells. Similarly, no significant changes were observed in N-cadherin mRNA levels in both cell lines with amiloride or increased α-ENaC. Despite changes in the EMP markers are observed at the transcriptional level, western blot was unable to detect E-cadherin proteins in the BT-549 cells and overexpressing α-ENaC in T-47D cells did not alter the protein levels of E-cadherin.
Taken together, these results partially supported the hypothesis and showed that α-ENaC enhances E-cadherin gene expression, which suggest that α-ENaC may promote epithelial phenotypes in breast cancer cells. Furthermore, these findings also demonstrated that both α-ENaC’s expression and activity influences EMP. Further investigation into the role of α-ENaC and the mechanisms by which α-ENaC regulates EMP during breast cancer progression may potentially offer a novel therapeutic target for the treatment of breast cancer.