Abstract
Breast cancer is the most common cancer diagnosed in women worldwide, leading to over 600 000 deaths annually. The vast majority of breast cancer-related deaths are attributed to metastasis from the primary site in the breast to visceral organs or the brain. Therefore, understanding how these cells achieve metastasis is vital. Ion channels, such as the heterotrimeric epithelial sodium channel (ENaC), are emerging as new targets for cancer research due to their role in regulating cell functions, such as the process that allows cancer cells to undergo phenotypic changes necessary for metastasis. Ion channel dysregulation may be crucial in cancer development due to the ion channels’ potential role in controlling cancer cell characteristics such as migration and proliferation. However, there is limited research investigating the role ENaC may have in breast cancer, particularly if it has a role in cell migration. Therefore, this research aimed to develop an understanding of the influence that ENaC has on breast cancer cells with a focus on cell migration.
The relationship between ENaC expression and breast cancer patient prognosis was examined using publicly available data sets, which showed higher expression of the alpha subunit of ENaC correlated with improved overall survival compared to low expression (p < 0.01) in the SCAN-B data set of 1705 breast cancer patients. In this data set, a higher expression of alpha-ENaC was also shown to be negatively correlated with migratory gene profile (Spearmon correlation= -0.298, p = 2.3x10^67), showing higher alpha-ENaC expression in breast cancer patients was linked with cells expressing less migratory gene mRNA. When examining the expression of ENaC in breast cancer cell lines using western blotting and RTqPCR, the more migratory mesenchymal cell lines (BT-549 and MDA-MB-231) trended towards a lower expression of alpha-ENaC than the two epithelial cell lines (T-47D and MCF- 7).
The impact of transient transfection of ENaC into BT-549 and MDA-MB-231 cells was examined using two cell migration assays: scratch and Boyden chamber. Overexpressing alpha- ENaC alone or a combination of alpha-, beta- and gamma-ENaC using transient transfection did not result in any changes in migration in two different breast cancer cell lines (BT-549 and MDA-MB-231) across two migration assays. However, due to inconsistent overexpression with this method, a stable cell line that stably overexpressed alpha-ENaC was produced to continue examining the relationship between migration and ENaC expression.
MDA-MB-231 breast cancer cells were engineered via a stable transfection to overexpress alpha-ENaC or to express an empty vector control. Several clones were isolated, expanded and characterised for alpha-ENaC mRNA level using RTqPCR. The alpha-ENaC protein overexpression was confirmed to be increased in three clones (n = 7, p < 0.01). Using an EdU assay, the alpha-ENaC overexpressing cells were shown to have a significant reduction in proliferation compared to the control cell line (n = 3, p < 0.01). In addition, the alpha-ENaC overexpressing cells showed reduced migratory ability when examined in two established migration assays. In scratch wound assays, the alpha-ENaC overexpressing cells significantly reduced cell migration at 24 hours post-scratch (n = 4, p < 0.01). In Boyden chamber assays, the number of alpha-ENaC-overexpressing cells that migrated through the membrane was significantly reduced compared to the control cell line (n = 4, p < 0.01).
Finally, the MDA-MB-231 alpha-ENaC overexpressing cells were used as part of a pilot study to determine if they were able to form tumours in a mice model, and to determine the most suitable number of cells needed to be injected into the mammary fat pad for tumour cell establishment. A study of 35 NU/J mice was undertaken with seven groups of five animals, and although a large number of animals had to be culled before the experimental endpoint, this study showed that these cells were able to form tumours and that the cells of the tumours formed maintained alpha-ENaC overexpression.
Altogether the results of this PhD project provide an insight into the link between ENaC expression and breast cancer cell functions, particularly cell migration. The work presented here highlights ENaC as a potential target for future breast cancer therapies, particularly in more metastatic breast cancers which have low expression of this sodium channel.