Targeting hypoxia regulated sodium driven bicarbonate transporters reduces breast cancer metastasis


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Christopher Carroll1,Alan Mcintyre1
1University of Nottingham

Abstract

Background

Metastasis is the cause of 90% of breast cancer patient deaths. Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple receptor negative subtype (TNBC). Regions of tumour hypoxia tend to be more acidic, and hypoxia and acidosis increase tumour metastasis. The metastatic process is dependent on pH regulatory mechanisms increased in hypoxia. We and others previously identified increased expression of Na+-driven bicarbonate transporters (NDBT) in hypoxia as a major mechanism of pH regulation. We hypothesised that NDBT inhibition or knockdown would reduce tumour cell migration and invasion.

Method

Hypoxic induction of NDBT was investigated by QRT-PCR and western blot. An NBDT inhibitor (S0859) and shRNA knockdown of hypoxia increased NDBT was utilised in in vitro studies to investigate the impact on breast cancer migration (wound-healing) and invasion (modified-Boyden chamber). To identify the mechanisms underlying our results we investigated the impact of NDBT knockdown or inhibition upon phospho-receptor tyrosine kinase (RTK) signalling, epithelial-to-mesenchymal transition (EMT) protein expression, and ROS production.

Results

Hypoxia (0.5% O2) induced the expression of NDBTs; most frequently SLC4A4 (5/7) and SLC4A5 (6/7). S0859 treatment and shRNA knockdown supressed migration (40% reduction) and invasion (70% reduction) in vitro. Hypoxia and acidosis increased phosphorylation of a number of signalling molecules including JNK 1/2/3, STAT2 and FAK, which were downregulated in response to NDBT knockdown and inhibition. E-cadherin expression was increased and vimentin expression ablated by NDBT knockdown in hypoxia, suggesting they are key regulators of epithelial-to-mesenchymal transition (EMT).

Conclusion

These results identify that targeting hypoxia induced NDBT reduces migration and invasion via downregulation of phospho-signalling and EMT. Future work will focus on investigating these effects in vivo and further investigations of the mechanisms by which NDBT/pH-regulation enable breast cancer metastasis.