Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA114

Poster Communications

Effects of knockout versus pharmacological inhibition of Na+/H+exchanger NHE1 on growth and chemosensitivity of cancer cell spheroids

L. O. Elingaard-Larsen1, M. G. Rolver2, A. Poder Andersen2, S. F. Pedersen1

1. Department of Biology, University of Copenhagen, Copenhagen NV, Denmark. 2. University of Copenhagen, Copenhagen, Denmark.

Rapid proliferation of cancer cells is accompanied by an increased metabolic acid production, in turn increasing reliance on net-acid-extruding transporters. The Na+/H+ exchanger NHE1 (SLC9A1) is upregulated in a variety of cancers including breast cancers, is a key player in intracellular pH homeostasis, and contributes to tumor growth. NHE1 inhibition has been found to synergize with cisplatin chemotherapy to induce cancer cell death, yet the importance of NHE1 for specific breast cancer subtypes is essentially unknown (1). The aim of this project is to investigate the dependence on NHE1 for cell growth and survival in Luminal-A and triple-negative (TNBC) breast cancer subtypes. MCF-7 (Luminal-A) and MDA-MB-231 (TNBC) human breast cancer cellswere grown as 3-dimensional spheroids. Under these conditions, NHE1 knockdown- or knockout (KO) delays spheroid growth (2). Spheroids were treated with NHE1 inhibitors EIPA or cariporide, alone or in combination with chemotherapy (Tamoxifen or a CAF regimen, respectively), every 2-3 days. Spheroid size was monitored by imaging and a final assessment of cell viability was performed on day 9. Cell viability was not affected by cariporide (10 µM) but was drastically reduced by EIPA (10 µM) in both cell lines. No synergy between NHE1 inhibitors and chemotherapy was observed. To investigate the dramatic effect of EIPA, an EIPA dose-response (0-10 µM) spheroid growth experiment was performed. EIPA potently reduced MCF-7 cell viability, with an EC50 of app. 3.2 µM for WT, whereas MDA-MB-231 cell spheroids were only significantly affected at the highest dose (n= 3, One-way ANOVA, p>0.05). To assess the NHE1-dependence of the response, this assay was next applied to two NHE1 CRISPR-KO cell lines for each cell type. Surprisingly, the response was identical between WT and NHE1 KO cell lines, consistent with a potent, NHE1-independent cytotoxic effect of EIPA. Propidium iodide staining of MDA-MB-231 and MCF-7 spheroids suggested that EIPA acted via a combination of antiproliferative effects and cell death in the spheroid core. This was confirmed by immunoblotting of MCF-7 spheroids lysed at day 2 of inhibitor treatment: EIPA (10 µM) essentially abolished pRb phosphorylation and increased cleavage of PARP and caspase-7 consistent with cell cycle arrest and apoptosis induction. Collectively, these results show that KO or cariporide-mediated inhibition of NHE1 in Luminal-A and TNBC breast cancer cells grown in spheroid culture has no effect on chemotherapy sensitivity. In contrast, the NHE1 inhibitor EIPA has potent growth-inhibitory, NHE1-independent effects involving increased apoptosis and decreased proliferation. This warrants reevaluation of previous work based on this compound. Future studies should assess whether EIPA may be of therapeutic interest despite its lack of specificity.

Where applicable, experiments conform with Society ethical requirements