KR-33028, a potent inhibitor of the Na + /H + exchanger NHE1, suppresses metastatic potential of triple-negative breast cancer cells Schammim Ray Amith 1 , Jodi Marie Wilkinson, Larry Fliegel ⇑ Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada article info Article history: Received 29 April 2016 Accepted 9 August 2016 Available online 10 August 2016 Keywords: NHE1 Triple-negative breast cancer Metastasis KR-33028 abstract Hyper-activation of the Na + /H + exchanger NHE1 occurs at the onset of oncogenic transformation and plays a critical role in breast cancer carcinogenesis. Dysregulation of NHE1 activity results in intracellular alkalinization and the acidification of the extracellular tumor microenvironment that promotes metasta- sis. Hence, the use of chemical inhibitors of NHE1 as chemotherapeutic agents is an alluring prospect. We previously demonstrated that two structurally different NHE1 inhibitors, EMD87580 [(2-methyl-4,5-di- (methylsulfonyl)-benzoyl)-guanidine], and HMA [5-(N,N-hexamethylene)-amiloride], were effective as co-adjuvants to potentiate paclitaxel-mediated cytotoxic chemotherapy in triple-negative breast cancer (TNBC) cells. Both these drugs, however, had reduced or minimal anti-cancer effects when used alone. Here, we tested KR-33028 (4-cyano (benzo[b]thiophene-2-carbonyl)guanidine), a potent and selective inhibitor of NHE1, to determine its efficacy in inhibition of metastatic potential of TNBC cells. In highly invasive MDA-MB-231, moderately invasive MDA-MB-468, and lowly invasive Hs578T TNBC cells, KR- 33028 considerably reduced rates of cell migration and anchorage-independent colony growth. Invasion of MDA-MB-231 and MDA-MB-468 cells through extracellular matrix was also dramatically decreased in response to KR-33028. We further tested the effect of KR-33028 on MDA-MB-231 cells lack- ing NHE1 expression (231koNHE1); no differences were observed between untreated control and KR- 33028-treated 231koNHE1 cells. Taken together, our results highlight the in vitro efficacy of KR-33028- mediated NHE1 inhibition on limiting cellular functions that are predictive of metastasis in vivo. We sug- gest that targeting NHE1 in the development of novel chemotherapeutics could be highly effective in combatting triple-negative breast cancer and that KR-33028 is potentially useful in prevention of metastasis. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction At the onset of oncogenic transformation, several cellular pro- cesses are dysregulated. The dysregulation of pH homeostasis, in particular, is key in the establishment of the acidic extracellular tumor microenvironment that maintains the transformed pheno- type and facilitates metastasis. This is chiefly due to the aberrant behavior of the major pH regulatory protein, the Na + /H + exchanger NHE1 [1,2]. NHE1 is an integral membrane protein 815 amino acids in length that is ubiquitously expressed in mammalian cells [3]. It comprises an N-terminal trans-membrane domain through which ion exchange occurs, and a cytosolic C-terminal domain that regulates exchanger activity via phosphorylation by kinases and association with lipid and protein binding partners [4,5]. NHE1 plays a critical role in cancer cells, becoming hyperactive in cells undergoing neoplastic transformation. This hyper-activation results in a reversal of the proton gradient and intracellular alkalin- ization, and causes acidification of extracellular interstitial spaces between tumor cells. The hypoxic and serum-depleted tumor microenvironment further promotes NHE1 hyper-activation [6]. In metastatic cancer cells, NHE1 localizes to leading edges of cells, accumulating in invasive structures like lamellipodia and invadopodia, where high proton extrusion provides the optimal acidic pH for extracellular matrix digestion by proteolytic enzymes [7,8]. Indeed, when NHE1 is knocked out in highly metastatic triple-negative MDA-MB-231 breast cancer cells, xenograft tumor http://dx.doi.org/10.1016/j.bcp.2016.08.010 0006-2952/Ó 2016 Elsevier Inc. All rights reserved. Abbreviations: HMA, [5-(N,N-hexamethylene)-amiloride]; NHE1, Na + /H + exchanger isoform 1; pH i , intracellular pH; TNBC, triple negative breast cancer. ⇑ Corresponding author at: Dept. of Biochemistry, 345 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. E-mail addresses: amith@ualberta.ca (S.R. Amith), jmwilkin@ualberta.ca (J.M. Wilkinson), lfliegel@ualberta.ca (L. Fliegel). 1 Present address: School of Life Sciences, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom. Biochemical Pharmacology 118 (2016) 31–39 Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm