Triple-negative breast cancer (TNBC) does not express hormone receptors, making it more aggressive and challenging to treat compared to estrogen receptor-positive breast cancer (Hartkopf et al., 2020). In TNBC, the Na_v1.5 isoform of voltage-gated sodium channels (VGSCs), encoded by SCN5A, is up-regulated and promotes invasion and metastasis (Nelson et al., 2015). TNBCs often develop intratumoral hypoxic microenvironments. The hypoxic response is regulated transcriptionally by hypoxia-inducible factors (HIFs), most critically the HIF-1α subunit. HIFs bind to hypoxia-response elements (HREs) and activate target genes that are critical for tumour progression (Semenza, 2016). Given that hypoxia plays a key role in up-regulating Na_v1.5 activity in the ischaemic heart (Plant et al., 2020) and HIF-1α significantly enhances breast cancer metastasis (Liao et al., 2007), we sought to investigate whether HIF-1 might regulate Na_v1.5 in TNBC.

To identify whether hypoxia/HIF-1 regulates VGSC expression in TNBC, an in silico analysis was performed to identify HRE consensus motifs (ACGTG) in key VGSC genes expressed in breast cancer. The mRNA expression levels of SCN5A and SCN1B (encoding the VGSC β1 subunit also expressed in TNBC) were quantified using rt-qPCR in MDA-MB-231 cells treated with dimethyloxalylglycine (DMOG; 1 mM), a chemical stabiliser of HIF-1α, for 0, 24, 48, and 72 hours. Na_v1.5 channel activity was studied using the whole-cell patch clamp technique. Cell proliferation and viability were measured using the RealTime-Glo™ MT cell viability assay and trypan blue exclusion assay, respectively. 

In silico HIF binding site prediction showed 4 putative HREs in the SCN5A promoter and 4 HREs in the 5 kb upstream of its transcription start site (TSS). 1 HRE was found in the 5 kb upstream of the SCN1B TSS. SCN1B was upregulated in DMOG-treated cells in a time-dependent manner (2.54±0.46 fold at 72 hours; p<0.001) while SCN5A expression was not affected by DMOG (1.08 ± 0.26 fold at 72 hours; p>0.05, one-way ANOVA, n=4, mean±S.D.). Transient and persistent Na⁺ currents were not significantly different between control and DMOG-treated cells after 24 or 48 hours of treatment (p>0.05, t-test, n=4-11). The RealTime-Glo™ results showed significant decreases in cell proliferation in DMOG-treated cells at all time points (24, 48, and 72 hours); this reduction is likely unrelated to VGSC signalling since the combination of DMOG (1 mM) and tetrodotoxin (TTX; 30 µM), a VGSC blocker, did not alter cell proliferation when compared to cells treated with DMOG alone. The trypan blue exclusion assay confirmed that the decrease in cell proliferation after 72 hours of DMOG treatment was not due to cell death. Cell viability was not statistically different between control (99.12±0.74%) and DMOG-treated cells (98.23±1.78%) (p>0.05, t-test, n=3). Overall, this study suggests that HIF stabilisation using DMOG in MDA-MB-231 cells increases SCN1B expression but does not alter SCN5A expression or Na_v1.5 activity. Further work is required to establish whether hypoxia may affect Na_v1.5 activity independent of HIF-1 stabilisation in TNBC.