A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

For (C) and (D), viability was assessed using the trypan blue assay method described previously (1).Data are mean ± SEM. *P < 0.05, ns, not significant.

Supplementary Figure 3 .Supplementary Figure 4 .CSupplementary Figure 5 .Supplementary Figure 6 .
Transcriptional response to SCN5A downregulation in xenograft tumours.(A) Principal component analysis (PCA) biplot of tumour sample coloured by cell type (shControl vs shRNA SCN5A).(B) PCA biplot of tumour sample coloured by animal cage.(C) Gene set enrichment analysis (GSEA) of invasion-Enrichment of GO terms in differentially expressed genes in SCN5A knock-down tumours for (A) Biological process, (B) Molecular function, and (C) Cellular compartment.positive thymic T cell selection response to interferon−alpha cytokine production involved in immune response antigen processing and presentation of exogenous peptide antigen response to interferon−gamma production of molecular mediator of immune response antigen processing and presentation of peptide or polysaccharide antigen via MHC class II antigen processing and presentation of peptide antigen via MHC class II antigen processing and presentation of exogenous peptide antigen via MHC class II response to type I interferon cellular response to type I interferon type I interferon signaling pathway defense response to virus regulation of actin filament−based process positive regulation of cell activation Log2 fold change < 0, Padj < 0.05 antigen processing and presentation of exogenous peptide antigen via MHC class II antigen processing and presentation of peptide antigen via MHC class II antigen processing and presentation of peptide or polysaccharide antigen via MHC class II A B Effect of 3-bromopyruvate on [Na + ]i of MDA-MB-231 cells.(A) SBFI-AM fluorescence ratios of MDA-MB-231 cells after 2 h treatment with 3bromopyruvate at the indicated concentrations.Data are normalised to the ratio in vehicle-treated cells (0.6% DMSO).*P < 0.05; ns, not significant (n = 3 experimental repeats; one sample t tests).Effect of glycolytic and mitochondrial respiration on viability of MDA-MB-231 and MCF7 cells.(A) Viability of MDA-MB-231 cells after 6 h treatment with ouabain (300 nM), or 2 h treatment with sodium iodoacetate (2 mM) or oligomycin (1 µM).Data are normalised to the ratio in vehicle-treated cells (n = 3 experimental repeats; one sample t tests).(B) Viability of MCF7 cells after 6 h treatment with ouabain (300 nM) or 2 h treatment with sodium iodoacetate (2 mM) or oligomycin (1 µM).Data are normalised to the ratio in vehicle-treated cells (n = 3 experimental repeats; one sample t tests).(C) Viability of MDA-MB-231 cells after 90 min treatment in Na + -free physiological saline solution (PSS) with veratridine (100 µM) or DMSO vehicle (n = 3 experimental repeats; t tests).(D) Viability of MCF7 cells after 90 min treatment in Na +free PSS with veratridine (100 µM) or DMSO vehicle (n = 3 experimental repeats; t tests).

Table 2 .
Receptor status of patient tumour tissue slices

Table 3 .
Characteristics of primary cell samples

Table 4 .
Calculation of buffering capacity of medium used in Seahorse analyzer experiments.