Abstract 4053: Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth.
Authors: McIntyre Alan , Hulikova Alzbeta , Ledaki Ioanna , Seden Peter , Turley Helen , Jones Dylan , Russell Angela , Swietach Pawel , Harris Adrian L. 
Published: August 2014

Background: Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia. CAIX extracellularly hydrates CO2 to HCO3− and H+, and we previously showed CAIX regulates pH in 3D spheroids. CAIX expression correlates with poor prognosis in most tumour types. We recently showed CAIX regulates tumour growth, necrosis and that knockdown enhances Bevacizumab treatment, highlighting the role of pH regulation in the hypoxic tumour milieu. We hypothesised that transport of HCO3−, which had been produced by CAIX extracellularly could facilitate quenching of intracellular H+. We identified increased expression of SLC4A4 (which encodes a bicarbonate transporter), in response to Bevacizumab, a treatment which increases the hypoxic fraction of cells, in vivo.

Method: We investigated a panel of cells lines (4 colon cancers, 2 glioblastomas, 1 breast cancer and 1 head and neck cancer) for changes in RNA expression of bicarbonate transporters in response to hypoxia (0.1% O2, 72 hours), and the role of HIF1α and HIF2α in key identified changes. We used a specific Na+ dependent bicarbonate transport inhibitor (SO859) to examine the effect of inhibition on pH regulation and growth in spheroids. Further to this we used shRNA knockdown of SLC4A4 and SLC4A9 in cell lines with increased hypoxic expression to further investigate their role.

Results: Hypoxia increased the RNA expression of one or more bicarbonate transporter in 6/8 cancer cell lines. The largest increases in expression change were for SLC4A4 in Ls174T and SCC25 (79.4 fold, p&lt;0.01 n=3: 2.4 fold, p&lt;0.05, n=3) and SLC4A9 in U87 and Ls174T (10.5 fold, p&lt;0.01 n=3; 19 fold, p&lt;0.01 n=3). SLC4A4 expression was regulated by HIF1α in Ls174T and SCC25 (p&lt;0.05, n=3). HIF1α bound directly to the HRE in the promoter of SLC4A4 in SCC25. SLC4A9 expression increase was regulated by HIF2α (80%) and HIF1α (29%) in Ls174T. SO859 treatment acidified the intracellular pH of cells in U87 spheroids. The most significant differences in pH were seen at the spheroid core (p&lt;0.001, n=20) and periphery (p&lt;0.001, n=20). U87 spheroids have HIF1α stabilisation and CAIX expression throughout, as detected by immunohistochemistry. Longer SO859 treatment of 24 hours resulted in a reduced effect on pH, suggesting a compensation mechanism. SO859 treatment reduced the spheroid growth rate of 4/4 cell lines tested, including MDA-MB-468 a cell line that showed no hypoxic increase in bicarbonate transport expression. In U87 spheroids, CAIX knockdown further reduced the reduction in growth rate seen with SO859. SLC4A4 and SLC4A9 knockdown reduced growth rate in Ls174T spheroids and SLC4A9 knockdown reduced spheroid growth rate in U87.

Conclusion: This work highlights the value of developing small molecules or antibodies, which inhibit bicarbonate transport to clinically deregulate pH regulation in the hypoxic microenvironment of tumours.

Citation Format: Alan McIntyre, Alzbeta Hulikova, Ioanna Ledaki, Peter Seden, Helen Turley, Dylan Jones, Angela Russell, Pawel Swietach, Adrian L. Harris. Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2013-4053