Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis.

Christopher Paul Carroll,Philip A Clarke,Adrian L Harris,Alan Mcintyre,Anna M Grabowska,Hannah Bolland,Alison A Ritchie,Pamela Collier,Eric Vancauwenberghe

doi:10.1016/j.neo.2022.01.003

Abstract

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

Highlights

More than half of breast tumours contain regions of low oxygen [1], arising from high metabolic and proliferative rates and aberrant tumour vascularisation [2]
As Na+ driven bicarbonate transporters (NDBTs) knockdown modulated metabolism we validated this result by immunoblot which identified that phosphorylation of mTOR was reduced by SLC4A4 (70%) and SLC4A5 (80%) knockdown in hypoxia (Fig. 5G)(HCC1806). mTOR regulates hypoxia inducible factor (HIF) stabilization in hypoxia [24] and we identified that NDBT knockdown ablated HIF-1α stabilisation and expression of HIF-1α target CA9 in hypoxia (Fig. 5H)(HCC1806)
We previously identified the role of hypoxia-regulated NDBT in regulation pHi and that NDBTs knockdown/inhibition reduced spheroid growth, increased apoptosis at the core of spheroids and reduced xenograft growth rates [16]

Summary

Introduction

More than half of breast tumours contain regions of low oxygen (hypoxia) [1], arising from high metabolic and proliferative rates and aberrant tumour vascularisation [2]. Normal breast median pO2 is 65 mmHg, compared with a median pO2 of 10 mmHg in breast tumours with regions

Methods

Results

Conclusion