Novel breast cancer theranostics: targeting sodium to suppress tumour progression

我们将研究缺氧和HIF-1激活调节钠转运的转录和生理机制。通过挖掘乳腺癌细胞系中公开可用的ChIP-seq数据集，我们已经确定了几种钠转运基因作为HIF-1的假定靶点，包括NHE3、NCX1和一些钠依赖性溶质转运蛋白；到目前为止，这些还没有在肿瘤进展的背景下进行研究。我们将这些与其他与癌细胞有关的钠转运蛋白结合起来，编制了一份18个候选转运蛋白的清单，用于本项目的评估。我们将在常氧和缺氧条件下对两种乳腺癌细胞系中的18种候选钠转运蛋白进行靶向CRISPR耗尽筛选。将使用自定义阵列文库，包括每个靶标至少5个引导rna，阳性对照（例如必需生存基因）和阴性对照（安全港位点和非靶向序列）。在低氧和常氧条件下生长5-15天后缺失的引导rna将通过PCR和下一代测序进行鉴定。如有可能，我们还将使用相同通道的抑制剂进行平行化学筛选。在筛选中鉴定出的最多三种感兴趣的钠转运蛋白将在两种细胞系中沉默，以进行后续的生理表征。转运蛋白敲除将通过qPCR、western blot和全细胞膜片钳记录来证实。我们将评估这些转运蛋白敲除对细胞内钠浓度（SBFI-AM成像）和生长/生存的影响。我们还将评估处于急性缺氧条件下的细胞，以测试敲除是否抑制缺氧诱导的细胞内钠的增加，我们之前报道过。感兴趣的钠转运体/通道电流将使用既定的药理学工具进行分离和测量。最后，通过上述分析确定的最有希望的靶钠转运蛋白敲除也将使用临床相关的球体模型进行评估。

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.