NaV1.5 sodium channels allosterically regulate the NHE-1 exchanger and promote breast cancer cell invadopodial activity

Abstract

SummaryThe degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. Na V 1.5 (also known as SCN5A) Na + channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that Na V 1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that Na V 1.5 colocalises with Na + /H + exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, Na V 1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of Na V 1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, Na V 1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that Na V 1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Suppression of PPARβ, and DHA treatment, inhibit NaV1.5 and NHE-1 pro-invasive activities

“…Cell growth was assessed with the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as described previously [4,42]. MDA-MB-231 cells were transduced with lentiviral particles coding for a short hairpin shRNA designed to knock down PPARβ expression (shPPARβ), or shCTL, and cells permanently expressing shPPARβ, or shCTL, were selected with puromycin dihydrochloride as described previously [10].…”

“…Thirdly, we have shown previously that nNa V 1.5 is functional in highly invasive human breast cancer cells, such as MDA-MB-231 cells, but not in non-cancer or in weakly invasive cancer cells [35]. Its activity increases cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix (ECM) by cysteine cathepsins [10,11,19]. Furthermore, Na V 1.5 interacts with and potentiates the ubiquitous Na + /H + exchanger type 1 (NHE-1, the major regulator of H + efflux in MDA-MB-231 cells) activity in plasma membrane rafts in focal ECM degradation sites corresponding to caveolin-1-containing invadopodia [10,11].…”

“…Its activity increases cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix (ECM) by cysteine cathepsins [10,11,19]. Furthermore, Na V 1.5 interacts with and potentiates the ubiquitous Na + /H + exchanger type 1 (NHE-1, the major regulator of H + efflux in MDA-MB-231 cells) activity in plasma membrane rafts in focal ECM degradation sites corresponding to caveolin-1-containing invadopodia [10,11]. Small interfering RNA (siRNA) targeting Na V 1.5 also reduces NHE-1 activity and cancer cell invasiveness [10].…”

“…Furthermore, Na V 1.5 interacts with and potentiates the ubiquitous Na + /H + exchanger type 1 (NHE-1, the major regulator of H + efflux in MDA-MB-231 cells) activity in plasma membrane rafts in focal ECM degradation sites corresponding to caveolin-1-containing invadopodia [10,11]. Small interfering RNA (siRNA) targeting Na V 1.5 also reduces NHE-1 activity and cancer cell invasiveness [10]. Finally, Isbilen et al (2006) reported that a 48 h treatment with 0.5 μM DHA inhibits Na V 1.5 messenger RNA (mRNA) and protein expression as well as MDA-MB-231 cell migration [24], but the potential effect of DHA or PPARβ on Na V 1.5 current and NHE-1 activity in breast cancer cells have not been investigated so far.…”

Invadopodia and basement membrane invasion in vivo

“…The advantages in imaging, biochemical approaches, and genetic and pharmacological perturbations in 2D conditions have led to remarkable advances in our understanding of invadopodia. 22,[39][40][41][42] These findings include the identification of different components of invadopodia, [43][44][45] elucidation of the stages of invadopodia formation, [46][47][48][49] identification of genes associated with cancer metastasis that regulate invadopodia formation, 19,21,[50][51][52] understanding of the trafficking of proteases to invadopodia, [53][54][55][56][57] and examination of invadopodia membrane dynamics. 58 Key insights have also been gained into the regulation of their formation by growth factors, integrin activation, and the microenvironment, including hypoxia, matrix stiffness, Over 20 years ago, protrusive, F-actin-based membrane structures, termed invadopodia, were identified in highly metastatic cancer cell lines.…”