Abstract 276: Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer

Abstract

Abstract Changes in membrane potential dramatically impact the progression of cancer, yet little is known as to how ion transport contributes to disease progression. Voltage-gated sodium channels (VGSCs) and Na,K-ATPases are particularly attractive targets as both are upregulated in many cancers and have been correlated specifically with invasiveness in vitro and metastasis and in vivo. However, their precise upstream regulators and downstream effectors remain unclear. Additionally, it has not been determined whether these transporters are co-regulated in cancer, although they display overlap in their downstream effectors and have complementary activity on sodium ions. Such a system has, however, been described in neuronal models of pain, wherein inflammatory injury leads to an upregulation in VGSC expression and a subsequent proportional increase in Na,K-ATPase. We sought to describe a similar phenomenon in cancer cells, which could indicate that this neuronal response to injury may be hijacked as a driver of metastatic development. To elucidate the impact of inflammatory mediators on these ion transporters in cancer cells, we assessed the effects of TNFα and PGE2 on VGSCs and Na,K-ATPases in the human breast epithelial or cancer cells MCF-10a, MCF-7, and MDA-MB-231. RT-qPCR revealed increases in RNA expression for the clinically relevant VGSC subunit Nav1.5. Similar RNA increases were observed in the α1 and α3 subunits of Na,K-ATPase, although in all cases the magnitude and time point of maximal effect differed by cell line. Then a 1hr challenge of TNFα on MDA-MB-231 cells was used to examine whether the targets were independently regulated by inflammatory mediators, co-regulated via the transport of sodium ions, or co-regulated by the expression of one protein influencing the expression of the other protein. Transfection with siRNA against either the α1 or α3 subunit of Na,K-ATPase did not inhibit the inflammatory-mediated increase in RNA for Nav1.5. However, transfection with anti-Nav1.5 siRNA inhibited the upregulation of both Na,K-ATPase subunits. TNFα challenge following pre-incubation of MDA-MB-231 cells in a sodium-free medium completely inhibited the inflammatory-mediated upregulation of Nav1.5, Na,K-ATPase α1 or α3 RNA. Co-incubation of TNFα with the VGSC blocking drug protoxin I inhibited the inflammatory-mediated increase in Na,K-ATPase α1 or α3 RNA, but co-incubation with the Na,K-ATPase blocking drug ouabain did not inhibit the increase in Nav1.5 RNA and indeed may have potentiated the increase. Together these results are evidence that VGSCs and Na,K-ATPase are co-regulated by inflammatory mediators in metastatic breast cancer, that sodium is essential to this mechanism, and that the presence and ion-transporting function of VGSCs is necessary for the inflammatory-mediated effect on Na,K-ATPase but not vice versa. Citation Format: Steven D. Scahill, Harry J. Gould, Dennis Paul. Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 276.