Functional interdependence of NHE1 and merlin in human melanoma cells

Abstract

Upregulation of the Na(+)/H(+) exchanger isoform 1 (NHE1) has been correlated with tumor malignancy. In contrast, moesin-radixin-ezrin-like protein (merlin) is a tumor suppressor that protects from cancerogenesis. Merlin is highly related to the members of the ezrin, radixin, and moesin (ERM) protein family that are directly attached to and functionally linked with NHE1. In addition, merlin inhibits the MAPK cascade and the Rho-GTPases known to activate NHE1 activity. The present study investigates whether NHE1 expression and activity affect merlin or, conversely, whether merlin has an impact on NHE1 in human melanoma (MV3) cells. Indeed, features of merlin-deficient MV3 cells point to a functional link: merlin-deficient cells showed a decreased NHE1 expression and, paradoxically, an increase in NHE1 activity as measured upon cytosolic acidification (NH4Cl prepulse method). Loss of merlin also led to an elevated cell motility that could be further increased by NHE1 overexpression, whereas NHE1 overexpression alone had no effect on migration. In contrast, neither NHE1 expression nor its activity had an impact on merlin expression. These results suggest a novel tumor suppressor function of merlin in melanoma cells: the inhibition of the proto-oncogenic NHE1 activity, possibly including its downstream signaling pathways.

MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

“…Human melanoma cells of the MV3 cell line [21], stably transfected with an empty pcDNA3 vector (control) or with the pcDNA3 vector carrying NHE1 (NHE1 overexpressing; [22]), were grown in bicarbonate buffered Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Taufkirchen, Germany) supplemented with 10% ( v/v ) fetal calf serum (FCS) at 37 °C in a humidified atmosphere of 5% CO 2 , 95% air. The culture medium contained 0.6 g l −1 geneticin (G-418-sulfate; PAA Laboratories, Pasching, Austria) in order to select the transfected cells.…”

Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells

“…To study possible effects of NHE1 on cell adhesion, we used two established cell clones that have been characterized previously: (i) NHE1-overexpressing MV3 cells 14 and (ii) an NHE1-deficient MV3 cell clone 15 with the respective controls. The degree of NHE1-overexpression corresponds to that induced by prolonged incubation in acidic (pH6.8) culture medium.…”

“…MV3 spheroids from cell aggregation assays were harvested, washed in PBS twice, lysed in radioimmunoprecipitation assay (RIPA) lysis buffer and further treated as described previously for confluent MV3 monolayer (20 μg protein/sample) 14 . A primary monoclonal rabbit anti-MCAM (CD146) antibody (dilution 1:2.000; Merck Millipore, Billerica, USA) and, as a loading control, a monoclonal mouse anti-β-actin antibody (dilution: 1:10.000, Sigma-Aldrich) were used.…”

“…It thereby contributes to an extracellular acidosis and was already described to be constitutively active in tumour cells 10 11 . Both, NHE1 activity and/or NHE1 expression may be increased in tumour cells among others because of dysregulation of its C-terminus 12 13 , because of mutations of tumour suppressors such as merlin or because of the local acidosis 14 . In migrating human melanoma cells, NHE1 is not homogeneously expressed but concentrates at the leading edge of the lamellipodium 15 16 .…”

Role of Merlin/NF2 inactivation in tumor biology

“…An additional molecular mechanism that contributes to increased melanoma cell motility observed with merlin loss was identified in merlin-deficient MV3 human melanoma cells. Despite decreased Na+/H+ exchanger 1 (NHE1) expression, NH 4 Cl pre-pulse experiments revealed increased NHE1 activity and over-expression of NHE1 further increased migration, suggesting a functional link between merlin and NHE1 activity ( 182 ). Importantly, treatment of the human melanoma cell line A375 (this cell line carries the BRAF gain-of-function V600E mutation) with the RAF inhibitor vemurafenib allowed screening for genes involved in development of drug resistance in surviving cells by negative selection with the new CRISPR-Cas9 technology.…”