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

50%的乳腺肿瘤存在缺氧（低氧）。缺氧是由肿瘤的高增殖和代谢率引起的，这导致肿瘤生长超出其血液供应所能维持的范围。缺氧驱动乳腺癌的进展，通过调节癌症主要特征的基因促进适应。在临床上，缺氧与化疗和放疗抵抗、转移增加和患者生存差有关。缺氧肿瘤区域也是酸性的，对肿瘤pH值的评估发现，许多肿瘤呈酸性，pH值低至pH6.4。我们发现缺氧时Na+驱动的碳酸氢盐转运体（NDBT）的表达增加是pH调节的主要机制。我们发现NDBT的敲除或抑制会使细胞内pH值酸化。NDBT抑制或敲低会降低肿瘤缺氧核心的细胞存活，以及肿瘤在体外和体内的生长。此外，我们最近发现NDBT抑制或敲除可抑制肿瘤细胞的迁移、侵袭和转移。我们已经证明，通过敲低或抑制NDBT来调节细胞内pH值可显著下调许多磷酸化信号通路。此外，NDBT敲低或抑制调节细胞代谢，抑制对缺氧的代谢适应，减少糖酵解和增加氧化磷酸化。本博士的重点是进一步研究通过NDBT抑制或敲除细胞内酸化而发生的表型、代谢和信号变化，并确定发生这些变化的机制。这将通过三个主要目标来实现。1)利用3D OrbiSIMs分析三维肿瘤球体缺氧/酸性核心、常氧外周和缺氧/常氧间期代谢物，确定NDBT抑制对代谢的影响。三维肿瘤球体精确地模拟了肿瘤中发现的氧、pH和代谢梯度。2)利用iTRAQ高级蛋白质组学方法量化NDBT抑制在常氧/缺氧/酸中毒中蛋白稳定性的变化。3)研究我们之前发现的磷酸化信号的变化与NDBT抑制引起的表型和代谢变化之间的关系。我们将使用多种抑制剂，对抗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.