Fig 2: The loss of SCN4B/ß4 expression promotes human cancer cell migration and invasiveness in two and three dimensions.(a) Cancer cell invasiveness was assessed using Matrigel-invasion chambers from shCTL or shSCN4B MDA-MB-231 cells, in the absence (-) or presence of the protease inhibitors GM6001 (10 µM), leupeptin (200 µM) or E64 (100 µM). Results from three to seven independent experiments are presented and were expressed relative to shCTL cells in the absence of inhibitors. Results are expressed as mean values±s.e.m. *** denotes a statistical difference from the shCTL at P<0.001, and # indicates a statistical difference from shSCN4B at P<0.05 (ANOVA). (b) Cancer cell migration of shCTL and shSCN4B cells measured by time-lapse microscopy to track the movement of cells over 180 min, 1 frame per min (n=20 representative cells in each condition). Distances are indicated in µm. (c) The speed of migration (in µm min-1) was analysed in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at P<0.001 (MW). (d) The track length of cell migration (in µm) was analysed over 180 min in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at P<0.001 (MW). (e) Three-dimension (3D) invasiveness of shCTL and shSCN4B cells, embedded inside Matrigel, was measured by time-lapse microscopy to track the movement of cells over 48 h (1 frame per 30 min) in the absence (CTL) or presence of the MMP inhibitor GM6001 (10 µM) (n=13 representative cells in each condition). Distances are indicated in µm. (f) The track length of 3D cell invasiveness (in µm) was analysed over 48 h in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 30 cells in each condition. ** and *** denote statistical difference from the shCTL, CTL condition at P<0.01 and P<0.001, respectively. ## denotes a statistical difference from the shSCN4B, CTL condition at P=0.002. § denotes a statistical difference from the shCTL, GM6001 condition at P=0.038 (Dunn's test). (c,d and f) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Fig 3: NaVß4 downregulation induces morphological changes in non-cancer mammary cells. (a) The expression level of the SCN4B gene, coding for NaVß4, was analysed from datasets from The Cancer Genome Atlas (http://cancergenome.nih.gov, accessed on 19 December 2020), from the US National Cancer Institute, in the non-tumoral adjacent tissue (n = 178), and in the different stages of primary breast tumours: I (n = 125), IIA (n = 243), IIB (n = 115), IIIA (n = 85), IIIB (n = 10), IIIC (n = 31), IV (n = 4). For each array, data were log2-transformed and centred to the median. ***, statistically different with p < 0.001 (Mann–Whitney rank sum test) when comparing with adjacent non-tumoral tissue; *, p < 0.05 when comparing Stage I with stage IIA. (b) NaVß4 protein expression level was assessed by western blotting in non-cancer MCF10A human mammary epithelial cells and in human breast cancer MDA-MB-231 cells. The upper section shows a WB representative of 5 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of NaVß4 protein expression in the two cell lines expressed relatively to that of MCF10A. *, statistically different with p < 0.05 (Mann–Whitney rank sum test). (c) NaVß4 protein expression level was assessed by western blotting in control MCF10A cells and in cells stably knocked down for the expression of SCN4B gene (MCF10A Crß4). The upper section shows a WB representative of 8 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of NaVß4 protein expression in the two cell lines expressed relatively to that of MCF10A CTL (n = 8). *, statistically different with p < 0.05 (Mann–Whitney rank sum test). (d) Representative images of MCF10A CTL and MCF10A Crß4 cells in phase contrast microscopy. Scale bar, 50 µm. (e) Maximal cell length (n = 31 MCF10A CTL and n = 20 MCF10A Crß4) and, in (f), number of intercellular contacts per cell (n = 60 MCF10A CTL and n = 57 MCF10A Crß4), assessed from images taken as in (d). Cells were randomly selected from pictures, and the number of joint cells was manually counted. ***, statistically different with p < 0.001 (Student’s t-test). (g) MCF10A CTL and MCF10A Crß4 cells were stained for the identification of nuclei (DAPI, blue staining) and F-actin (phalloidin-594, red staining). Scale bar, 125 µm. (h) Mean cell area (n = 40 MCF10A CTL and n = 40 MCF10A Crß4) and, in (i), F-actin fluorescence intensity per cell surface (n = 100 MCF10A CTL and n = 100 MCF10A Crß4) were calculated from images taken as in (g). ***, statistically different with p < 0.001 (Student’s t-test).

Fig 4: SCN4B/ß4 protein is expressed in normal epithelial cells of human breast tissues and is downregulated in cancer cells.(a,b) ß4 protein (expression of the SCN4B gene) was analysed by immunohistochemistry on human breast tissue samples. (a) The expression of ß4 protein was strong in epithelial cells of mammary acini (some examples are indicated by the black arrows), and not in non-epithelial cells of normal breast tissues. (b) In breast cancer tissue, the expression of ß4 protein was strong in normal epithelial cells of mammary acini (black arrows), but significantly reduced in cancer cells (tumour area indicated by the red arrow, ‘T'). Scale bars, 50 µm.

Fig 5: Loss of SCN4B/ß4 expression promotes human cancer cell invasiveness independently of the pore-forming NaV subunit.(a) Cancer cell invasiveness was assessed, using Matrigel-invasion chambers, from MDA-MB-231-Luc cells stably transfected with null-target shRNA (shCTL), SCN5A-targeting shRNA (shSCN5A) or SCN4B-targeting shRNA (shSCN4B), in the absence (-) or presence (+) of 30 µM TTX. The results from 8 to 16 independent experiments were expressed relative to control cells transfected with shCTL in the absence of TTX. ***, statistically different from shCTL at P<0.001 and #, statistically different from shSCN4B in the absence of TTX at P<0.05. (b) Cancer cell invasiveness was likewise assessed in shCTL or shSCN4B cells, transiently transfected with null-target siRNA (siCTL) or SCN5A-targeting siRNA (siSCN5A). The results from 12 independent experiments were expressed relative to shCTL cells transfected with siCTL. ***, statistically different from the shCTL/siCTL condition at P<0.001 and #, statistically different from shSCN4B/siCTL at P<0.05. (c) Cancer cell invasiveness was assessed in MDA-MB-231-Luc cells stably expressing the SCN5A-targeting shRNA (shSCN5A), not expressing the NaV1.5 protein, and transiently transfected with null-target siRNA (siCTL) or SCN4B-targeting siRNA (siSCN4B). This effect was assessed in the absence (-) or presence (+) of two TTX concentrations (3 or 30 µM), or 30 nM of the NaV1.8 inhibitor A803467. The results from six independent experiments were expressed relative to shSCN5A cells transfected with siCTL, in the absence of any NaV inhibitor. NS stands for no statistical difference and *** denotes a statistical difference from shSCN5A/siCTL at P<0.001. (d) Cancer cell invasiveness was assessed using Matrigel-invasion chambers for MDA-MB-468 breast, H460 and A549 non-small-cell lung, and PC3 prostate cancer cells transfected with null-target siRNA (siCTL, black bar) or SCN4B-targeting siRNA (siSCN4B, red bars). Cancer cell lines known to express or not functional NaV channels are indicated as NaV+ and NaV-, respectively. The results from 3 to 12 independent experiments were presented and are expressed relative to the results obtained with the same cells transfected with siCTL. *, different from siCTL at P<0.05 and *** at P<0.001. Statistics presented in this figure were performed using ANOVA for multiple group comparison (a–c) or Student's t-test (d). All results presented in this figure are mean values±s.e.m.