Ester Antelmi

NaV1.5 Na+ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia

Summary The 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. NaV1.5 (also known as SCN5A) Na+ channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 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, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 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, NaV1.5 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 NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe.

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1‑dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

ß1 integrin binding phosphorylates ezrin at T567 to activate a lipid raft signalsome driving invadopodia activity and invasion

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na+/H+ exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.

NHERF1 acts as a molecular switch to program metastatic behavior and organotropism via its PDZ domains

Tumor metastasis is the primary cause of death in cancer patients, but the molecular mechanisms driving the evolution of the phenotype toward a specific organ is one of its less understood aspects. The scaffolding protein NHERF1 reprograms the metastatic phenotype and organotropism via the differential function of its PDZ domains.

NaV1.5 sodium channels increase breast cancer cell invadopodial activity by both controlling Src kinase-dependent F-actin polymerization and promoting NHE-1-dependent proton efflux and extracellular matrix digestion

Background and aim. The degradation of the extracellular matrix (ECM) by cancer cells is a critical essential step in metastatic progression. NaV1.5 sodium channels are overexpressed in breast tumours and associated with metastatic occurrence. NaV1.5 activity in breast cancer cells promotes ECM degradation through the perimembrane acidification and the subsequent activation of cysteine cathepsins [1,2]. The aim of this study was to identify cellular pathways involved in the NaV1.5-dependent H+ efflux and invasiveness of highly aggressive human MDA-MB-231 breast cancer cells. Methods. Highly aggressive MDA-MB-231 breast cancer cells express functional NaV1.5 channels. By using cell fractionation and microscopy analyses, we studied the presence of NaV1.5 channels in invadopodia which are key cellular structures for cancer cells invasiveness. In MDA-MB-231-derived cell lines expressing or not NaV1.5 channels (shRNA) we examined the ability of cancer cells to form invadopodia, to invade through an ECM composed of Matrigel™, and to regulate the efflux of protons. Results. We showed that NaV1.5 was co-localised with NHE-1, and caveolin-1 in MDA-MB-231 breast cancer cells invadopodia, at sites of ECM remodelling. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicated a close association between these proteins. The expression of NaV1.5 was responsible for the allosteric modulation of NHE-1 rendering it more active at intracellular pH range 6.4 to 7, thus potentially extruding more protons in the extracellular space. Furthermore, NaV1.5 increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, controlled F-actin polymerization and the acquisition of an invasive morphology. Conclusions. This study suggests that NaV1.5 is an important regulator of invadopodia formation (F-actin polymerization) and degradative activity (NHE-1 over-activation resulting in extracellular acidic proteases activation) in breast cancer cells [3].