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

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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.