Novel breast cancer theranostics: targeting sodium to suppress tumour progression

We will study the transcriptional and physiological mechanisms by which hypoxia and HIF-1 activation regulate sodium transport. By mining publicly available ChIP-seq datasets in breast cancer cell lines, we have identified several sodium transport genes as putative HIF-1 targets, including NHE3, NCX1, and a number of sodium-dependent solute transporters; until now these have not been studied in the context of tumour progression. We have combined these, together with other sodium transporters which have been implicated in cancer cells, to compile a list of 18 candidate transporters for evaluation in this project.

We will perform a targeted CRISPR depletion screen of the 18 candidate sodium transporters in two breast cancer cell lines under normoxic vs. hypoxic conditions. A custom arrayed library comprising at least 5 guide RNAs per target, positive controls (e.g. essential survival genes) and negative controls (safe harbour loci and non-targeting sequences) will be used. Guide RNAs depleted after 5-15 days' growth in hypoxia vs. normoxia will be identified using PCR and next generation sequencing. We will also undertake a parallel chemical screen using inhibitors of the same channels, where available. Up to three individual sodium transporters of interest identified in the screens will then be silenced in both cell lines for subsequent physiological characterization. Transporter knockout will be confirmed by qPCR, western blot, and whole-cell patch clamp recording. We will evaluate the effects of these transporter knockouts on intracellular sodium concentration (SBFI-AM imaging) and growth/survival. We will also evaluate cells placed under acute hypoxic conditions to test whether the knockout inhibits hypoxia-induced increase in intracellular sodium that we have reported previously. Sodium transporter/channel currents of interest will be isolated and measured using established pharmacological tools. Finally, the most promising target sodium transporter knockout identified through the above analyses will also be evaluated using clinically relevant spheroid models.