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

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/p90(RSK) pathway: p90(RSK)/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 p90(RSK) 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 p90(RSK), 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 p90(RSK)/14-3-3-mediated activation of the exchanger.