Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells

Abstract

Voltage-gated sodium channels (Na V ) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na V 1.5 and associated auxiliary ␤-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (ϳ65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na V function. Inhibiting Na V has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na V activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na v enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.Breast cancer is the most common female cancer and the primary cause of death in women by cancer worldwide (1).Deaths occur primarily after the development of metastases. The invasive potential of malignant cells is mainly linked to their capacity to degrade basement membranes and extracellular matrices by various proteases. Studies have mostly focused on metalloproteases, including matrix metalloproteinases and the closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs) (2), that are key factors in growth, invasion, and angiogenesis, and to a lesser extent on aspartyl and serine proteases. Pharmaceutical inhibitors of matrix metalloproteinases have been developed, but the results from clinical trials with these drugs have so far been disappointing (3,4

P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness

“…They are known to be extracellularly active, even if the events leading to their extracellular secretion or activation are not well understood. Importantly, we confirm in this study that high amounts of immature forms of cathepsins are tonically released by cancer cells at much higher contents than active forms, which as already been reported (Roshy et al, 2003;Gillet et al, 2009). These cathepsins might be constitutively extruded from cellular compartments different from lysosomes, from which it is expected to only find active forms.…”

“…These channels have also been demonstrated to enhance the cysteine cathepsin-dependent invasiveness of breast cancer cells through the sodium influx (Gillet et al, 2009). P2X 7 R being non-specific cation-permeant channels, this raises the question of a possible involvement of internal sodium in the regulation of cathepsins activity/release and in the invasiveness of cancer cells.…”

“…To assess the cell survival, 2 Â 10 5 cells were seeded per well in six-well plates in a control culture medium or in presence of different concentrations of ATP/inhibitors, and were grown for 24 h. Cell numbers were assayed by the tetrazolium salt assay as previously described (Gillet et al, 2009). Cell survival was expressed as formazan 492 nm absorbance and expressed as a ratio by comparison with the control condition, in the absence of drug or ATP (on the same day of the experiment).…”

“…In the last decade, studies mainly focused on the involvement of matrix metalloproteinases in the neoplastic progression (Egeblad and Werb, 2002), but recently, it appeared that cysteine cathepsins are highly upregulated in a wide variety of cancers, including breast cancer (Mohamed and Sloane, 2006). Indeed elevated cathepsin B expression correlates with poor prognosis in breast cancer as in other cancers (Jedeszko and Sloane, 2004) and its activity has been shown to enhance breast cancer cells invasiveness in vitro (Gillet et al, 2009). Cathepsins classical localization within intracellular lysosomes is found to be modified in tumours.…”

NaV1.5 enhances breast cancer cell invasiveness by increasing NHE1-dependent H+ efflux in caveolae

“…We and others have shown that voltage-gated sodium channels (Na V ) are abnormally expressed in cancer cells of epithelial origin and associated with cancer progression (Laniado et al, 1997;Roger et al, 2003Roger et al, , 2006Roger et al, , 2007Fraser et al, 2005;Diaz et al, 2007). In the highly invasive MDA-MB-231 breast cancer cell line, Na V 1.5 activity enhances extracellular matrix invasion by increasing the activity of acidic cysteine cathepsins B and S through an acidification of the pericellular microenvironment (Gillet et al, 2009). In this study, we investigated the potential pH regulators involved in the Na V -dependent invasiveness of MDA-MB-231 breast cancer cells.…”