Abstract
More than 666,000 females died worldwide from breast cancer in 2022, mostly resulting from metastasis, which accounts for over 90% of all cancer deaths. Our lab previously observed that higher levels of the alpha subunit of the Epithelial Sodium Channel (αENaC) exert tumour-suppressive and anti-metastatic effects in breast cancer cells, by inhibiting proliferation, migration and invasion. This study aims to further characterise αENaC’s role and elucidate the molecular underpinnings of its effects in repressing breast cancer progression.
Bioinformatic analyses were performed on publicly available datasets from breast cancer patients hosted by repositories including the Gene Expression Omnibus and The Cancer Genome Atlas, and followed by western blotting on breast cancer cell lines to further characterise αENaC’s prognostic significance. MDA-MB-231 breast cancer cells were generated to stably overexpress αENaC or a control construct for molecular investigations. RNA-sequencing and transcriptomic analysis were performed on the αENaC-overexpressing cells and the control. Cell cycle assays, comprising cell cycle-synchronization with 2mM thymidine treatment or serum-starvation, followed by western blotting and live fluorescent imaging of the αENaC-overexpressing and control cells were performed.
The bioinformatic analyses showed significantly reduced αENaC mRNA levels in metastatic breast cancer tissues compared to normal breast (Dunn's P = 3.56x10-5) and primary breast cancer (Dunn's P = 1.5x10-5) tissues. Repression of αENaC expression was associated with significantly reduced metastasis-free (Cox-Mantel log rank P = 3.8x10-4) and relapse-free (Cox-Mantel log rank P = 4.3x10-10) survival. Western blotting showed lower αENaC protein in the highly proliferative and metastatic breast cancer cell lines (MDA-MB-231 and BT-549). The transcriptomic analysis uncovered 385 genes which were differentially expressed (Benjamini-Hochberg adjusted P < 0.05) when αENaC was overexpressed. These genes were core to processes involved in signal transduction, cellular migration, epithelial-mesenchymal transition and immune interactions. The cell cycle assays revealed altered Cyclin E,Cyclin A and Cyclin B protein profiles, and an ~2-hour delay in cell division completion by the αENaC-overexpressing cells.
These findings suggest αENaC may reduce breast cancer progression towards metastasis and relapse by altering the expression of key genes and delaying cell division.