Inhibition of the Na+/K+-ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

Mia A Shandell,Alina L Capatina,Samantha M Lawrence,William J Brackenbury,Dimitris Lagos

doi:10.1016/j.jbc.2022.101707

Mia A Shandell, Alina L Capatina + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101707

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Abstract

Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2′,3′-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.

Highlights

Understanding and treating cancer is a fundamental struggle of modern medicine
An initial screen of MDA-MB-231 cells resulted in decreased kynurenine upon treatment with 50 μM ouabain: 3.8 ± 1.3 μM, n = 3, vs. 1% DMSO control: 27.5 ± 4.1 μM, n = 6 (p < 0.0001, one-way ANOVA Tukey’s post-hoc) (Figure 1C)
An initial screen of A549 cells resulted in decreased kynurenine upon treatment with 50 μM ouabain: 1.5 ± 1.6 μM, n = 3, vs. 1% DMSO control: 15.0 ± 2.9 μM, n = 6 (p < 0.001, one-way ANOVA Tukey’s post-hoc) (Figure 1D)

Summary

Introduction

Understanding and treating cancer is a fundamental struggle of modern medicine. Breast cancer alone is responsible for 30% of newly diagnosed cancers in women in the United States over the last year, with more than 240,000 new cases registered only in 2019 [1]. A key feature of highly metastatic breast tumors is represented by ionic imbalances characterized primarily by elevated intracellular Na+, and a slightly depolarized cell membrane [2]. Growing attention has focused on the role of ion transport in cancer progression [3, 4]. Changes in ion transport drive a number of cellular phenotypes associated with cancer [5]. Cytoskeletal remodelling, and cell motility underpinning cell migration [6, 7], growth and cell cycle progression [8,9,10], and gene expression [11, 12] can all be impacted by altered ion flux. Increased expression of a range of ion channels is associated with metastasis [4, 17,18,19]

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