Abstract
Worldwide breast cancer is considered to be the most common type of cancer among women and the third most common cancer in New Zealand. The principal cause of cancer-related mortality involves metastasis in which cells can escape the primary tumor, invade into neighboring blood vessels, migrate through the stroma, and disseminate to neighboring sites to formulate a secondary tumor thus allowing cancer progression. The epithelial-mesenchymal plasticity (EMP) underpins metastasis and allows epithelial cells to gain mesenchymal phenotypes, reflecting more migratory, invasive and proliferative characteristics.
Recent studies have identified ion channels as an attractive target that would aid EMP and subsequently promote metastasis. In particular, the epithelial sodium channel (ENaC), has been shown to have anti-proliferative and decreased migratory effects suggesting a potential protective tool against breast cancer progression. Further, aiding cellular migration the α subunit of ENaC expression has been shown to induce cell protrusions reflective of invadopodia in cancer cells. Hence, this project was designed to explore ENaC regulation of breast cancer migration. The aim of this research was to determine the presence of invadopodia and correlate this to the degree of migration in the case of breast cancer. With this aim, the project hypothesized that the upregulation of αENaC expression will decrease levels of invadopodia markers correlating to a decrease in cell migration.
Western blot analysis was able to confirm the overexpression of αENaC in the αENaC overexpressing cell line in comparison with the control. Scratch assay analysis revealed a decrease in migratory rate of the αENaC overexpressing cell line in comparison with the control. The use of phalloidin was able to provide variation in the distribution of actin between the two different cell lines. The αENaC overexpressing cell line highlighted a decrease in mean intensity of green fluorescence compared with the control cell line. A decreased actin presence may imply less cell stiffness and thus an increased cell motility. However, the decreased cell motility levels observed in this cell line could suggest that the overexpression of αENaC may alter the localization or perhaps cause the incomplete formation of migratory structures such as invadopodia. Thus, ICC methods were used to identify the cortactin protein, which is necessary for the formation of the core invadopodia precursor. Results showed no variation in the mean fluorescent green intensity with some cells showcasing the potential altered localization of invadopodia. Thus, these results were not able to fully satisfy the hypothesis providing to understand the correlation between αENaC overexpression and migratory structures such as invadopodia.
Collectively these results were able to partially support the hypothesis and highlight the important role which αENaC plays in aiding cancer cell motility. The findings were able to demonstrate that αENaC overexpression has the capacity to influence breast cancer cell motility. Further experiments into understanding the means by which ENaC can regulate the molecular mechanisms of the tumour microenvironment and other potential factors that influence both migration and features of metastasis may provide a viable avenue for therapeutical intervention in breast cancer.