Na+/H+ exchanger NHE1 regulation modulates metastatic potential and epithelial-mesenchymal transition of triple-negative breast cancer cells

Abstract

In triple-negative breast cancer (TNBC), the high recurrence rate, increased invasion and aggressive metastatic formation dictate patient survival. We previously demonstrated a critical role for the Na+/H+ exchanger isoform 1 (NHE1) in controlling metastasis of triple-negative cells. Here, we investigated the effect of changes to three regulatory loci of NHE1. Two via the Ras/Raf/ERK/p90RSK pathway: p90RSK/14-3-3 (S703A) and ERK1/2 (S766,770,771A, SSSA) and a third via a calmodulin-binding domain (K641,R643,645,647E, 1K3R4E). MDA-MB-231 cells with a mutation at the p90RSK site (S703A-NHE1) changed from a wild-type mesenchymal morphology to a smaller epithelial-like phenotype with a loss of expression of mesenchymal marker vimentin. S703A cells also had reduced metastatic potential and markedly decreased rates of migration, invasion, spheroid growth, anchorage-dependent and soft agar colony formation. Similarly, BI-D1870, a specific inhibitor of p90RSK, significantly inhibited the metastatic potential of highly invasive MDA-MB-231 and moderately invasive MDA-MB-468 TNBC cells, but was minimally effective in non-invasive Hs578T TNBC cells. In contrast, invasion and spheroid growth were unaffected in cells containing NHE1 with mutations interfering with its activation by ERK1/2 (SSSA), though rates of migration and colony formation were reduced. Cells with a constitutive activation of NHE1 via the 1K3R4E mutation exhibited higher rates of migration, invasion, and spheroid growth. Taken together, our data demonstrate the critical role of NHE1 in metastasis, and suggest a novel link between NHE1 and the expression and cytosolic organization of vimentin, a key factor in epithelial-mesenchymal transition, that is dependent on p90RSK/14-3-3-mediated activation of the exchanger.

Na + /H + exchanger-mediated hydrogen ion extrusion as a carcinogenic signal in triple-negative breast cancer etiopathogenesis and prospects for its inhibition in therapeutics

“…This change was accompanied by a dramatic decrease in metastatic potential, as demonstrated by many parameters including: low rates of migration, low invasion through extracellular matrix, poor anchorage-dependent clonogenic growth, poor anchorage-independent colony growth in softagar, and reduced 3D-spheroid growth in extracellular matrix. In fact, S703A-NHE1 cells behaved much like 231-NHE1ko cells, though the same morphological changes were not observed in the NHE1-knockout cells (79). We noted that the observed changes in morphology in the S703A-NHE1 cells correlated with a marked downregulation in both the mRNA and protein expression of vimentin intermediate filaments.…”

KR-33028, a potent inhibitor of the Na+/H+ exchanger NHE1, suppresses metastatic potential of triple-negative breast cancer cells

“…Thus, the exact reason for this discrepancy is not currently known, but previous work in our laboratory suggests that proliferation in these TNBC cells may not be mediated by the activity of NHE1 [9,10]. [26].…”

“…However, it could be presumed that expression of NHE1 protein alone cannot be used as a measure of metastatic potential of these cells. Indeed, our recent work suggests that it is the underlying regulatory mechanisms controlling the activity of the exchanger that may be a key determinant of metastatic potential [27]. Further experiments are necessary to confirm this hypothesis.…”

“…Anchorage-independent colony growth was assessed by colony formation in soft agar as previously described [10,20,21]. Briefly, 6-well plates were coated with 0.5 mL of 0.5% DNAgrade agar (Thermo Scientific) dissolved in complete growth medium and allowed to solidify at room temperature.…”

“…Indeed, when NHE1 is knocked out in highly metastatic triple-negative MDA-MB-231 breast cancer cells, xenograft tumor growth in athymic nude mice is almost abolished [9]. Furthermore, mutations to regulatory sites on the NHE1 C-terminal tail significantly modulate the behavior of metastatic triple-negative breast cancer cells [10]. Consequently, there is a growing body of evidence supporting the potential for pharmacological inhibition of NHE1 as a novel chemotherapeutic strategy [6,11].…”