Lucie Brisson

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.

pH regulators in invadosomal functioning: Proton delivery for matrix tasting

Invadosomes are actin-rich finger-like cellular structures sensing and interacting with the surrounding extracellular matrix (ECM) and involved in its proteolytic remodeling. Invadosomes are structures distinct from other adhesion complexes, and have been identified in normal cells that have to cross tissue barriers to fulfill their function such as leukocytes, osteoclasts and endothelial cells. They also represent features of highly aggressive cancer cells, allowing them to escape from the primary tumor, to invade surrounding tissues and to reach systemic circulation. They are localized to the ventral membrane of cells grown under 2-dimensional conditions and are supposed to be present all around cells grown in 3-dimensional matrices. Indeed invadosomes are key structures in physiological processes such as inflammation and the immune response, bone remodeling, tissue repair, but also in pathological conditions such as osteopetrosis and the development of metastases. Invadosomes are subdivided into podosomes, found in normal cells, and into invadopodia specific for cancer cells. While these two structures exhibit differences in organization, size, number and half-life, they share similarities in molecular composition, participation in cell-matrix adhesion and promoting matrix degradation. A key determinant in invadosomal function is the recruitment and release of proteases, such as matrix metalloproteinases (MMPs), serine proteases and cysteine cathepsins, together with their activation in a tightly controlled and highly acidic microenvironment. Therefore numerous pH regulators such as V-ATPases and Na(+)/H(+) exchangers, are found in invadosomes and are directly involved in their constitution as well as their functioning. This review focuses on the participation of pH regulators in invadosome function in physiological and pathological conditions, with a particular emphasis on ECM remodeling by osteoclasts during bone resorption and by cancer cells.

Carcinoma-specific expression of P2Y 11 receptor and its contribution in ATP-induced purinergic signalling and cell migration in human hepatocellular carcinoma cells

Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.

Metabolic reprogramming in cancer cells, consequences on pH and tumour progression: Integrated therapeutic perspectives with dietary lipids as adjuvant to anticancer treatment

While tumours arise from acquired mutations in oncogenes or tumour-suppressor genes, it is clearly established that cancers are metabolic diseases characterized by metabolic alterations in tumour cells, and also non-tumour cells of the host organism resulting in tumour cachexia and patient weakness. In this review, we aimed at delineating details by which metabolic alterations in cancer cells, characterized by mitochondrial bioenergetics deregulations and the preference for aerobic glycolysis, are critical parameters controlling the aggressive progression of tumours. In particular, metabolic alteration in cancer cells are coupled to the modulation of intracellular and extracellular pH, epithelial-to-mesenchymal transition and associated increased invasiveness, autophagy, and the development of anticancer treatment resistance. Finally, based on mechanistic, pre-clinical and clinical studies, we proposed the adjuvant supplementation of dietary n-3 polyunsaturated fatty acids for a complementary holistic treatment of the cancer disease.

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].