Novel breast cancer theranostics: targeting sodium to suppress tumour progression

我们将研究低氧和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，我们已经确定了几个钠转运基因作为假定的HIF-1靶点，包括NHE3、NCX1和一些钠依赖的溶质转运体；到目前为止，这些基因还没有在肿瘤进展的背景下进行研究。我们已经将这些与其他与癌细胞有关的钠转运蛋白结合在一起，编制了一份18个候选转运蛋白的清单，用于在该项目中进行评估。我们将在常氧和低氧条件下对两种乳腺癌细胞系中的18个候选钠转运体进行有针对性的CRISPR耗竭筛查。将使用一个定制排列的文库，每个目标至少包含5个引导RNA，阳性对照(例如基本生存基因)和阴性对照(安全港基因和非靶向序列)将被使用。在低氧和常氧中生长5-15天后耗尽的引导RNA将使用聚合酶链式反应和下一代测序进行鉴定。我们还将进行一项平行的化学筛选，尽可能使用相同通道的抑制剂。在筛选中确定的多达三个单独的钠转运体将在两个细胞系中沉默，以用于后续的生理特征。转运蛋白基因敲除将通过定量聚合酶链式反应、蛋白质印迹和全细胞膜片钳记录来确认。我们将评估这些转运蛋白基因敲除对细胞内钠浓度(SBFI-AM成像)和生长/存活的影响。我们还将评估放置在急性缺氧条件下的细胞，以测试敲除是否抑制我们先前报道的缺氧诱导的细胞内钠的增加。将使用现有的药理学工具分离和测量感兴趣的钠转运体/通道电流。最后，通过上述分析确定的最有希望的目标钠转运体基因敲除也将使用临床相关的椭球模型进行评估。

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.