Gitte Lauritzen:
Novel roles of the Na+/H+ exchanger NHE1 and the Na+,HCO3 - cotransporter NBCn1 in cell survival, proliferation and motility

Date: 09-01-2014    Supervisor: Stine Falsig Pedersen

Due to a shift towards glycolytic metabolism requiring increased capacity for acid extrusion, tumor cells upregulate acid extruding transport proteins. Dysregulation of pH regulatory ion transporters has been assigned important roles in tumor growth, cell survival/death balance, proliferation and cell motility. The molecular mechanisms contributing to altered pHi regulation in cancer cells are incomplete understood. Overexpression of ErbB2 is common in breast cancer and the expression of an N-terminally truncated, constitutively active ErbB2 receptor (ΔNErbB2) is associated with increased glycolysis, malignancy and poor prognosis. This thesis focuses on the regulation and roles of two pH regulatory transporters, the Na+/H+ exchanger NHE1 (SLC9A1) and the Na+,HCO3 - cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells expressing ΔNErbB2. Specifically, how these transporters contribute to an aberrant pH profile, chemotherapy resistance, cell cycle arrest after chemotherapy-induced DNA damage and motility.

In agreement with the fact that malignancy in cancer is associated with increased capacity for acid extrusion, we show in Paper I that NBCn1 expression, yet not NHE1, is upregulated upon ΔNErbB2 expression, and that the total acid extrusion capacity is significantly increased by expression of ΔNErbB2. Experiments based on siRNA-knockdown and pharmacological inhibition show, that acid extrusion from the MCF-7 cells can be accounted for by the joint activities of NBCn1 and NHE1. Pharmacological inhibition of NHE1 enhances cisdiamminedichloroplatinum (II) (cisplatin) induced cell death, especially in ΔNErbB2 expressing cells.

In Paper III we show that upon cisplatin treatment, expression of ΔNErbB2 results in increased caspase-9 and -7 cleavage, which is further augmented by specific inhibition of NHE1. Moreover, NBCN1, yet not NHE1, is lost from the plasma membrane upon cisplatin treatment, and this may explain why inhibition of NHE1 sensitizes the cells to cisplatin-induced cell death.

In Paper II we show that in MCF-7 breast cancer cells, the expression levels of NBCn1 and NHE1 are not further changed in response to serum deprivation. However, both cell adhesion and 2D motility on collagen I were increased under serum deprivation of ΔNErbB2 expressing cells. NHE1 inhibition augmented both adhesion and 2D motility of ΔNErbB2 MCF-7 cells. Additionally, NHE1 and ERM localize to invadopodial rosettes, and these rosettes are increased in size upon NErbB2 expression.

In Paper IV, we identify a specific ATM phosphorylation site at Ser788 at the distal Cterminus of hNHE1, and show that NHE1 activity is increased in response to DNA damage induced by the topoisomerase II inhibitor etoposide. G2/M cell cycle arrest induced by etoposide is attenuated and cell death reduced, in cells expressing NHE1 lacking the ATM phosphorylation site, compared to cells expressing wild type NHE1.

Collectively, the results confirm a strong requirement for acid extrusion in cancer cells, and provide new knowledge on the regulation and roles of acid extruding transporters in cell survival, proliferation and motility. Finally, it is interesting from both a clinical and a basic research perspective that two functionally similar transporters, perhaps through different localization or interaction partners, have very different effects on these processes.