CRISPR Interference Screening and Pharmacological Targeting of Sodium Channels in Triple-Negative Breast Cancer Cells

Hypoxia is linked with more invasive forms of breast cancer, particularly triple negative breast cancer (TNBC). Elevated intratumoural sodium (Na+) level in patients correlates with disease severity and Na+ reduction predicts treatment response. Our in vivo data, using 23Na+ MRI, suggest that the majority of this Na+ is intracellular, implicating specific Na+ channels and transporters in its accumulation and regulation in the tumour microenvironment. While hypoxia has been shown to upregulate various Na+ channels, their combined involvement in hypoxia-driven breast cancer progression remains unclear.

Here, we performed the first systematic screen to identify Na⁺ channels which support TNBC proliferation under hypoxia. Using a targeted CRISPR interference (CRISPRi) screen with a dCas9-KRAB transcriptional repression system, we repressed 181 Na+ channel and transporter genes in two TNBC cell lines, MDA-MB-231 and MDA-MB-468, cultured under normoxic or hypoxic conditions for 7 days. The screen identified hypoxia-specific depletion of guide RNAs (gRNAs) targeting SCN5A (Nav1.5), SLC10A5, SLC38A5, and SLC34A2 in MDA-MB-231 cells, and GRIN3A, SLC22A12, and SLC34A3 in MDA-MB-468 cells. FXYD5 was also exclusively depleted under hypoxia in both cell lines.

To evaluate the therapeutic potential, we tested the Nav1.5 channel as a target in TNBC cells using propafenone, a clinically approved antiarrhythmic drug not previously studied in the cancer context. Electrophysiological analysis confirmed that propafenone (100 µM) significantly inhibited Na+ currents in MDA-MB-231 cells.

Our findings highlight multiple Na+ transporters involved in TNBC proliferation under hypoxia, with SCN5A/Nav1.5 as a promising therapeutic target in hypoxic TNBC. These results support the potential repurposing of existing Na+ channel-targeting drugs to inhibit hypoxic TNBC progression.