Prolactin Signaling Stimulates Invasion via Na+/H+ Exchanger NHE1 in T47D Human Breast Cancer Cells

Abstract

Prolactin (PRL) and its receptor (PRLR) are implicated in breast cancer invasiveness, although their exact roles remain controversial. The Na(+)/H(+) exchanger (NHE1) plays essential roles in cancer cell motility and invasiveness, but the PRLR and NHE1 have not previously been linked. Here we show that in T47D human breast cancer cells, which express high levels of PRLR and NHE1, exposure to PRL led to the activation of Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5), Akt, and ERK1/2 signaling and the rapid formation of peripheral membrane ruffles, known to be associated with cell motility. NHE1 was present in small ruffles prior to PRL treatment and was further recruited to the larger, more dynamic ruffles induced by PRL exposure. In PRL-induced ruffles, NHE1 colocalized with activated Akt, ERK1/2, and the ERK effector p90Ribosomal S kinase (p90RSK), known regulators of NHE1 activity. Stimulation of T47D cells with PRL augmented p90RSK activation, Ser703-phosphorylation of NHE1, NHE1-dependent intracellular pH recovery, pericellular acidification, and NHE1-dependent invasiveness. NHE1 activity and localization to ruffles were attenuated by the inhibition of Akt and/or ERK1/2. In contrast, noncancerous MCF10A breast epithelial cells expressed NHE1 and PRLR at lower levels than T47D cells, and their stimulation with PRL induced neither NHE1 activation nor NHE1-dependent invasiveness. In conclusion, we show for the first time that PRLR activation stimulates breast cancer cell invasiveness via the activation of NHE1. We propose that PRL-induced NHE1 activation and the resulting NHE1-dependent invasiveness may contribute to the metastatic behavior of human breast cancer cells.

The Na+/H+ exchanger NHE1 localizes as clusters to cryptic lamellipodia and accelerates collective epithelial cell migration

“…To allow visual inspection of NHE1 localization and direct comparison between individual experiments, a cell line stably expressing NHE1 with a C‐terminal GFP tag (referred to as NHE1‐MDCK) was used (Pedraz‐Cuesta et al . ) (Fig. A ).…”

“…Also consistent with a dynamic interaction of NHE1 with cytoskeletal elements, NHE1 localized to membrane ruffles in T47D cells but did not colocalize with focal adhesions in these cells (Pedraz‐Cuesta et al . ). In migrating melanoma cells, NHE1 localization was increased at focal adhesions in the leading edge but not in the trailing part (Ludwig et al .…”

“…; Pedraz‐Cuesta et al . ). Cells were loaded with 1.6 μ m BCECF‐AM (B1150; Thermo Fisher Scientific, Wlatham, MA, USA) by directly adding from a 1:1000 DMSO stock into the cell culture medium and incubated for 30 min.…”

“…Measurements of pH i were carried out essentially as described previously (Lauritzen et al 2010;Pedraz-Cuesta et al 2016). Cells were loaded with 1.6 μM BCECF-AM (B1150; Thermo Fisher Scientific, Wlatham, MA, USA) by directly adding from a 1:1000 DMSO stock into the cell culture medium and incubated for 30 min.…”

“…This is important for understanding how NHE1 contributes to tissue homeostasis and development, as well as disease states where NHE1 is overexpressed, such as early cancer development. NHE1 expression and have been described previously (Pedraz-Cuesta et al 2016). Because NHE1-GFP was overexpressed at low levels, which could not be obtained for green fluorescent protein (GFP) or enhanced GFP (EGFP), and because EGFP overexpression in MDCK did not appear to affect cellular polarity and migration (data not shown), WT MDCK cells were employed as the control cell line.…”