Christina Wilkens Olesen:
Numerous cellular processes are highly pH-dependent in the physiological range, and regulation of intracellular pH (pHi) is therefore fundamental for maintaining normal cell function. The electroneutral Na+;HCO3- co-transporter NBCn1 (SLC4A7) plays essential roles in pHi homeostasis in a wide range of cell types. It contributes importantly to the normal physiological function of many human organs, and its dysfunction has been linked to several pathologies including cardiovascular diseases and breast cancer. Expression of a truncated constitutive active HER2 receptor, p95HER2, is associated with poor clinical prognosis due to resistance to treatments with monoclonal antibodies and chemotherapy. We previously showed that p95HER2 expression increased NBCn1 protein expression in MCF-7 breast cancer cells. The purpose of this PhD project was (i) to gain further insight into the regulation of HCO3- transporters in cancer, and to increase the understanding of the regulation and roles of NBCn1 in normal and cancerous epithelial cells, (ii) by determining the mechanisms through which expression of p95HER2 and NCBn1 impacts on cisplatin chemotherapy-induced cell death and (iii) by elucidating the kinetics and mechanisms of NBCn1 trafficking, membrane retention and degradation in MCF-7 cells and other epithelial cell types.
In Paper-I we review existing evidence regarding the possible roles of the SLC4 and SLC26 families of HCO3 - transporters in breast, colon and lung cancer. Bioinformatic analysis of publicly available datasets revealed that several HCO3 - transporters are dysregulated in various cancers. In particular, we found that expression of the SLC4A3, SLC4A7 and SLC26A6 transporters was upregulated in lung cancer, whereas the expression of the SLC4A1 and SLC4A4 transporters was decreased in all of the three examined cancer tissues.
In Paper-II, the roles of p95HER2 and NBCn1 during cisplatin-induced breast cancer cell death were investigated. Expression of p95HER2 slightly sensitized the cells to cisplatin-induced cell death, as did inhibition of NHE1 (confirming our previous work), whereas inhibition of NBCn1 had no effect. This may in part be explained by a loss of the transporter from the membrane, as NBCn1 yet not NHE1, was lost from the membrane and appeared in a perinuclear compartment upon treatment with cisplatin.
In Paper-III we undertook for the first time a thorough analysis of the sorting and regulation of NBCn1 in breast and kidney epithelial cells. We found that NBCn1 protein turnover was very slow and that the transporter did not undergo constitutive endocytosis over a 24 h period, suggesting that the protein is unusually stable in the epithelial cell plasma membrane under normal conditions. Furthermore, we found that the small scaffolding protein RACK1 co-localizes with NBCn1 in membrane protrusions and is important for NBCn1 membrane stability.
Collectively, the work in this thesis has contributed with new understanding of the mechanisms involved in cisplatin-induced cell death in p95HER2 positive breast cancer cells and suggested a reason for why NBCn1 inhibition does not exacerbate cisplatin-induced MCF-7 cell death, despite its major importance for pHi regulation in these cells. Moreover, this work comprises the first detailed analysis of the kinetics and mechanisms of NBCn1 sorting, targeting, membrane retention, and degradation, providing a broader understanding of how NBCn1 is regulated in epithelial cells. These findings may guide future investigations into the regulation and roles of NBCn1 in normal and diseased epithelial tissues.