Investigating hypoxia induced pH regulatory mechanisms and their regulation of protein stability and metabolic adaptation in breast cancer

缺氧（低氧）在50%的乳腺肿瘤中发现。缺氧是由肿瘤的高增殖率和代谢率引起的，这导致肿瘤生长超过其血液供应所能维持的范围。缺氧会推动乳腺癌的进展，通过调节癌症主要标志的基因促进适应。临床上，缺氧与化疗和放疗抵抗、转移增加和患者生存率降低相关。低分化肿瘤区域也是酸性的，对肿瘤pH值的评估已经确定许多肿瘤是酸性的，pH值低至pH6.4。我们确定了增加表达的Na+驱动的碳酸氢盐转运蛋白（NDBT）在缺氧的pH调节的主要机制。我们表明，NDBT敲低或抑制酸化细胞的细胞内pH值。NDBT抑制或敲低降低了肿瘤缺氧核心中的细胞存活，以及体外和体内的肿瘤生长。此外，我们最近发现，NDBT抑制或敲低抑制肿瘤细胞迁移，侵袭和转移。我们已经表明，通过NDBT敲低或抑制来调节细胞的细胞内pH显著下调许多磷酸化信号传导途径。此外，NDBT敲除或抑制调节细胞代谢，抑制代谢适应缺氧，减少糖酵解和增加氧化磷酸化。本博士学位的重点是进一步研究NDBT抑制或敲除引起的细胞内酸化反应中发生的表型，代谢和信号转导变化，并确定发生变化的机制。这将通过三个主要目标实现。1)使用3D OrbiSIM确定NDBT抑制对代谢的影响，以在3D肿瘤球体中描绘缺氧/酸性核心、常氧外围和缺氧/常氧相间的代谢物。3D肿瘤球体精确地模拟肿瘤中发现的氧气，pH值和代谢梯度。2)使用iTRAQ高级蛋白质组学定量相对和绝对蛋白质水平，研究常氧/缺氧/酸中毒中NDBT抑制反应的蛋白质稳定性变化。3）研究我们先前确定的磷酸信号传导变化与NDBT抑制反应中发生的表型和代谢变化之间的关系。我们将使用各种抑制剂，针对NDBT和激酶，以及CRISPR CAS9基因敲除。

Hypoxia (low oxygen) is found in 50% of breast tumours. Hypoxia is caused by the high proliferative and metabolic rates of tumours that leads to them growing beyond what their blood supply can sustain. Hypoxia drives breast cancer progression, promoting adaptation through genes regulating the major hallmarks of cancer. Clinically, hypoxia is associated with chemotherapy and radiotherapy resistance, increased metastasis and worse patient survival. Hypoxic tumour regions are also acidic, and assessment of the pH of tumours has identified that many tumours are acidic with pH as low as pH6.4. We identified increased expression of Na+ driven bicarbonate transporters (NDBT) in hypoxia as a major mechanism of pH regulation. We showed that NDBT knockdown or inhibition acidifies the intracellular pH of cells. NDBT inhibition or knockdown reduces cell survival in the hypoxic core of tumours, and tumour growth in vitro and in vivo. Furthermore we recently identified that NDBT inhibition or knockdown inhibits tumour cell migration, invasion and metastasis. We have shown that modulating the intracellular pH of cells by NDBT knockdown or inhibition significantly downregulates many phospho-signalling pathways. Further to this NDBT knockdown or inhibition modulates cell metabolism, inhibiting the metabolic adaptation to hypoxia, reducing glycolysis and increasing oxidative phosphorylation.The focus of this PhD is to further investigate the phenotypic, metabolic and signalling changes that occur in response to intracellular acidification by NDBT inhibition or knockdown and identify the mechanisms underpinning the changes that occur.This will be achieved via three main goals. 1) Identify the impact of NDBT inhibition on metabolism using 3D OrbiSIMs to profile metabolites in the hypoxic/acidic core, normoxic periphery and the hypoxic/normoxic interphase in 3D tumour spheroids. 3D tumour spheroids accurately model the oxygen, pH and metabolic gradients found in tumours. 2) Investigate changes in protein stability in response to NDBT inhibition in normoxia/hypoxia/acidosis using iTRAQ advanced proteomics to quantify the relative and absolute protein levels.3) Investigate the relationship between the changes in phospho-signalling, that we have previously identified, and the phenotypic and metabolic changes that occur in response to NDBT inhibition. We will use a variety of inhibitors, against NDBT and kinases, and CRISPR CAS9 gene knockouts.