Abstract
Breast cancer remains the leading cause of mortality in females both worldwide, and in New Zealand (NZ). In addition, like most diseases, ethnic disparities in NZ are also evident where the mortality rate for Māori women is 60% greater compared to non-Māori, displaying the unequal disease burden among ethnicities. Although advancements in therapeutics have been made over the decades, the burden of the disease still remains high, where a major proportion of deaths are attributed to metastasis, a process involved in tumour cell separation and migration from the primary tumour site to a secondary site. The initiation of metastasis requires cells to undergo morphological changes, which is often achieved through epithelial-mesenchymal transition (EMT). EMT is an incredible phenomenon which entails significant changes in cell fate and phenotype and thereby enables the transition of immobile epithelial cells to mobile mesenchymal cells, essentially resulting in the acquisition of migratory capabilities. Recently, the epithelial sodium channel, ENaC, has emerged as a novel player in the pathogenesis of breast cancer, possibly due to its EMT-opposing effects. In addition, uric acid (UA), the final product of purine metabolism in humans has been associated with increased risk of breast cancer. UA is known in the context of gout, where chronically elevated serum UA levels, termed hyperuricemia, causing the painful inflammation/arthritis of the joints. Interestingly, hyperuricemia also causes hypertension which is most likely based on the UA-mediated regulation of ENaC expression in the kidneys. Besides being the main antioxidant in the blood, the metabolic and inflammatory effects of UA have been shown to influence EMT in certain cancers. However, the effect of UA on ENaC and EMT in breast cancer remains elusive, hence its investigation is the aim of this project.
The epithelial-like breast cancer cell line, MCF-7, was used to determine the impact of hyperuricemia and inflammation on ENaC and ultimately EMT/metastasis of breast cancer cells. It was hypothesised that hyperuricemia will reduce the expression of ENaC in MCF-7 cells, promoting EMT. This will be established by altering the transcription and post-translational modifications (PTMs) of ENaC and the expression of key EMT and inflammatory markers.
MTT assays were conducted to determine changes in cell metabolic activity. A statistically significant decrease in metabolic activity reflecting a loss of cell viability was observed in MCF-7 cells when subjected to hyperuricemic conditions in the presence and absence of lipopolysaccharide (LPS), a known inducer of inflammation. ROS assays and RT-qPCR were conducted to determine changes in redox homeostasis and inflammation, and no significant changes in ROS levels and the mRNA levels of the inflammatory marker, NLRP3 were observed. Subsequent RT-qPCR and western blots were performed to determine changes in the expression of ENaC subunits and , and , respectively, revealing no significant changes in the expression of any ENaC subunits under the different treatment conditions. Follow up experiments included more western blots to determine changes in the expression of ENaC regulatory protein, Nedd4-2, and no significant changes were observed. To confirm the obtained findings, immunocytochemistry was performed and no visible changes in ENaC localisation/distribution were observed under the different conditions. In addition, a pull-down of -ENaC was performed to investigate potential changes in ENaC PTMs and no significant changes in ubiquitination or uratylation of -ENaC were observed. Finally, RT-qPCR was conducted to determine changes in gene expression of key EMT markers and no significant changes in the mRNA levels of epithelial and mesenchymal markers, E-cadherin and N-cadherin, were observed.
Taken together, these findings were unexpected and do not support the stated hypotheses. These findings reveal that UA does not alter the expression of any ENaC subunits and key EMT/inflammatory markers in MCF-7 cells. However, these results do indicate a potential role of UA in the modulation of breast cancer cell metabolism, independent of inflammation, and more importantly, these results reveal for the first time that ENaC is subjected to a novel PTM, uratylation, thereby opening new avenues for future research.