Novel breast cancer theranostics: targeting sodium to suppress tumour progression

新型乳腺癌治疗学：靶向钠来抑制肿瘤进展

• 批准号: 2752703
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2752703
• 关键词: Novel; breast; cancer; theranostics; targeting

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.

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。通过在乳腺癌细胞系中挖掘公开可用的ChIP-seq数据集，我们已经确定了几个钠转运基因作为推定的HIF-1靶点，包括NHE 3，NCX 1和一些钠依赖性溶质转运蛋白;到目前为止，这些尚未在肿瘤进展的背景下进行研究。我们将这些结合起来，再加上其他与癌细胞有关的钠转运蛋白，编制了一份18种候选转运蛋白的清单，用于本项目的评估。我们将在常氧与低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耗竭筛选。将使用包含每个靶至少5个向导RNA、阳性对照（例如必需存活基因）和阴性对照（安全港基因座和非靶向序列）的定制阵列文库。将使用PCR和下一代测序来鉴定在缺氧与常氧中生长5-15天后耗尽的向导RNA。我们还将在可能的情况下使用相同通道的抑制剂进行平行的化学筛选。在筛选中鉴定的多达三种感兴趣的单个钠转运蛋白随后将在两种细胞系中沉默，用于随后的生理表征。将通过qPCR、蛋白质印迹和全细胞膜片钳记录确认转运蛋白敲除。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/存活的影响。我们还将评估置于急性缺氧条件下的细胞，以测试敲除是否抑制我们之前报道的缺氧诱导的细胞内钠增加。将使用已建立的药理学工具分离和测量感兴趣的钠转运蛋白/通道电流。最后，还将使用临床相关球体模型评价通过上述分析鉴定的最有希望的靶钠转运蛋白敲除。