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Carroll, Christopher (2020) The role of sodium driven bicarbonate transporters in triple negative breast cancer metastasis. PhD thesis, University of Nottingham.

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Abstract

Triple negative breast cancer is the most difficult breast cancer subtype to treat due to; hormonal therapy resistance, regions of hypoxia, propensity to metastasize, and a lack of targeted therapy options. Metastasis is the cause of over 90% of breast tumour deaths. Hypoxic regions are areas of low oxygen and arise in tumours due to a high metabolic and proliferative rate, and aberrant vasculature. Hypoxia induces resistance to chemotherapy and radiotherapy. Hypoxic tumour regions are also generally more acidic, both hypoxia and acidosis induce metastasis. The sodium driven bicarbonate transporter (NDBT) family regulate intracellular pH. Preliminary data has shown that under hypoxic conditions (0.5% O2) NDBT mRNAs are induced in triple negative breast cancers and knockdown of one of these (SLC4A9) or NDBT inhibition reduces migration and invasion in-vitro. The aim of this PhD was to investigate targeting NDBTs as a novel mechanism for inhibiting triple negative breast cancer metastasis.

To determine a functional role in breast cancer, generation of validated gene knockdowns of NDBTs was undertaken and these clones were compared to the effects of small molecule inhibitor. NDBT knockdown and inhibition was shown to reduce intracellular pH in normoxia and hypoxia. NDBT knockdown and inhibition reduced migration and invasion in vitro and metastasis in vivo. The mechanism of action for functional changes by NDBT knockdown were investigated with regards to intracellular signalling. In preliminary research this was primarily undertaken with regards to HIF-1α. This study expanded on the preliminary research by determining activation status in an array of phospho-tyrosine signalling proteins. NDBT knockdown or inhibition reduced the activation of mTOR, LCK, LYN and many other tyrosine kinases. The reduced mTOR activation in turn resulted in an ablation of HIF-1/2α stabilization and downstream signalling in hypoxia. Inhibitors against mTOR, LCK, and LYN were used to recapitulate the effects seen by NDBT knockdown but did not recapitulate the effects to the same extent.

Further to this, Epithelial to mesenchymal transition (EMT) status was assessed under hypoxic and acidic conditions to investigate intracellular pH control and EMT signalling. It was shown that knockdown of NDBT perturbed transcription of multiple EMT genes. This perturbation altered EMT protein expression culminating in an increased expression of E-cadherin and lower expression of vimentin by NDBT knockdown or inhibition.

NDBT knockdown or inhibition altered metabolism. Total ATP levels and extracellular lactate levels were reduced by NDBT knockdown or inhibition in normoxia and hypoxia. Furthermore, expression of PKM2 mRNA and protein expression was reduced by NDBT knockdown. Conversely, total mitochondrial mass was increased, but mitochondrial polarity was perturbed resulting in altered mitochondrial morphology by NDBT knockdown in normoxia and hypoxia.

This study presents evidence regarding the role of pH regulatory proteins in tumour metastasis. Accumulating evidence highlights the regulation of signal transduction, EMT, and cellular metabolism in response by acidosis and pH regulatory mechanisms.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	McIntyre, Alan Grabowska, Anna
Keywords:	Triple negative breast cancer; Metastasis; Sodium driven bicarbonate transporter(NDBT); pH regulatory proteins
Subjects:	W Medicine and related subjects (NLM Classification) > WP Gynecology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	60141
Depositing User:	Carroll, Christopher
Date Deposited:	17 Jul 2020 04:40
Last Modified:	17 Jul 2020 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/60141

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