Fig 2: MTSS1 and SCAMP1 expression promotes cell–cell adhesions via promoting the expression of active RAC1.a, b Immunofluorescence images showing expression of F-actin, MTSS1-GFP and/or SCAMP1-HA in SkBr3 and MDA-MB-453 cells expressing MTSS1-GFP, SCAMP1-HA or both constructs and using Alexa Fluor® 568 Phalloidin, anti-GFP and anti-HA antibodies. Scale bar = 10 μm. c Immunoblotting from GST-PAK1-PBD pull down of RAC1 and from transfected SkBr3 and MDA-MB-453 whole-cell extracts using RAC1− antibody. d, e Graphs representing measurements from cell adhesion assays performed on SkBr3 and MDA-MB-453 expressing the empty vector, MTSS1-GFP, SCAMP1-HA or both constructs (48 h post transfection). ANOVA ****P < 0.0001) and ANOVA ****P < 0.0001

Fig 3: Kaplan–Meier models of survival and metastasis development for MTSS1 (a, b), SCAMP1 (c, d) and SERPIN B13 (e, f) loss, respectively. Less favourable prognosis is seen in patients with ‘pure’ HER2+ tumours compared to other patients showing loss of these biomarkers. Scale bar = 100 μm

Fig 4: MTSS1 and SCAMP1 expression prevents cell migration and invasion.a, b Wound-healing assay (Scratch assay) using SkBr3 and MDA-MB-453 expressing MTSS1-GFP, SCAMP1-HA or both constructs and the corresponding data quantifying gap closure at time points 0, 24 and 48 h following scratching. ANOVA ****P = < 0.0001 and ***P = 0.0001. Scale bar = 200 μm. c Wound-healing assay (Scratch assay) using BT-474 cells expressing empty vector, MTSS1-shRNA, SCAMP1 siRNA or both and the corresponding data quantifying gap closure at time points 24 h following scratching. ANOVA ***P < 0.0001. d, e Well-cell invasion assay using SkBr3 and MDA-MB-453 cells expressing MTSS1-GFP, SCAMP1-HA or both constructs and the data relating to the number of invading cells after 48 h of culture. ANOVA ***P = 0.0001 and ANOVA **P = 0.0084. Scale bar = 10 μm. f, g Graphs representing measurements of cell proliferation of SkBr3 and MDA-MB-453 expressing MTSS1-GFP, SCAMP1-HA or both constructs (24 and 48 h post transfection). Two-way ANOVA P(ns) = 0.1878) and two-way ANOVA P(ns) = 0.8040

Fig 5: Cytoplasmic staining was detected for (a) MTSS1, (b) SCAMP1 and (c) Serpin B13 protein in the malignant epithelium of breast cancer. Alternatively, the majority of ‘pure’ HER2+ (HER2+/ER−/PR−) tumours showed loss or in (d) MTSS1, (e) SCAMP1 and (f) SERPIN B13 staining. g Distribution of MTSS1, SCAMP1 and SERPIN B13 in the ‘pure’ HER2 patient class versus other tumour classes