As breast tumours grow, oxygen availability becomes limited, resulting in upregulation of key survival genes via hypoxia-inducible factors (HIFs) binding to core hypoxia response elements (HREs) ¹. Voltage gated sodium channels (VGSCs) are upregulated in several solid tumours, including breast ². In triple negative breast cancer (TNBC) tumours and MDA-MB-231 cells, the neonatal splice variant of Na_v1.5 (nNa_v1.5) encoded by SCN5A is overexpressed, and drives invasiveness of these cancer cells in vivo and in vitro ^2,3. In hypoxic pulmonary arterial smooth muscle cells, the HIF-1α isoform increases sodium/proton exchanger 1 (NHE1) expression ⁴. HIF-1α also mediates expression of other membrane transporters, including MCT-4 and GLUT1 ⁵. Due to the evidence implicating HIF-1α in modulating ion and solute transport, we wanted to investigate if VGSCs and NHE1 are HIF-1α-targets in breast cancer cell lines.

To understand hypoxia-dependent dysregulation of sodium transporters, both ER +ve MCF-7 and TNBC MDA-MB-231 cells were cultured for 0, 4 or 24 hours in hypoxia (1% O2). To elucidate HIF-1α involvement, MCF-7 and MDA-MB-231 cells were treated with the chemical PHD inhibitor, dimethyloxalylglycine (DMOG; 1 mM) in normoxia for up to 72 hours (0, 2, 4, 8, 16, 24, 48 and 72 hour time course). Western blot was performed to identify HIF-1α stabilisation. RT-qPCR was used to assess changes in VGSCs (SCN5A, SCN8A and SCN9A) and NHE1 mRNA expression. Data were analysed using one-way ANOVA, with post-hoc Dunnett’s tests as appropriate. 

Hypoxic MCF-7 cells displayed significantly increased expression of SCN5A (6.03 ± 1.60 fold at 24 hours  p <0.0001; n = 6, mean ± SD) and SCN8A (87.04 ± 32.28 fold at 24 hours, p <0.05, n = 3; mean ± SD). However, SCN9A was not significantly altered (p = 0.48, n = 3). Preliminary data indicate that NHE1 transcription was also increased at 24 hours (n = 2). Hypoxic MDA-MB-231 cells showed significantly increased expression of SCN8A (4.79 ± 0.13 fold at 4 hours, p <0.05; 8.78 ± 1.92 fold at 24 hours, p <0.001, n = 3, mean ± SD) but not SCN5A (p = 0.30, n = 6), SCN9A (p = 0.32, n = 3), or NHE1 (p = 0.27, n = 3). DMOG-dependent stabilisation of HIF-1α peaked at 4 hours in both MCF-7 and MDA-MB-231 cells, with protein levels decreasing after 16 hours (western blot, n=3). SCN5A expression was increased in MCF-7 cells after DMOG treatment, reaching a significant fold change in transcript levels at 48 hours (3.74 ± 2.19 fold at 48 hours; p <0.05 n = 3, mean ± SD). However, no significant increase in SCN8A (p = 0.51, n = 3), SCN9A (p = 0.17, n = 3) or NHE1 (p = 0.25, n = 3) was observed within the 72 hour DMOG treatment time course. 

These findings suggest hypoxia enhances expression of VGSC and NHE1 transcripts in a breast cancer subtype specific manner. Further work is required to depict the involvement of HIF-1α in aberrant sodium transporter expression.