a, Parental MM3MG cells, a cell line derived from mammary epithelial cells of wild-type BALB/c mice, do not express ERα but express low levels of HER2 and PGR-B compared to 4T1 and TUBO cells (TUBO cells were grown at low density; see Fig. 3 and Extended Data Figs 4, 5). b, Immunoblot confirming successful transduction of the MM3MG cell line with Her2 and Pgr-B. Note that transduction of PGR-B increases HER2 levels. c, d, Overexpression of Pgr-B in MM3MG cells (MM3MG-Pgr-B) reduces migration, whereas Her2 overexpression (MM3MG–Her2) increases migration of cells. Addition of progesterone does not alter migration of Pgr-B-overexpressing cells (MM3MG–Pgr-B). e, Overexpression of Pgr-B in MM3MG cells reduces sphere formation, whereas Her2 overexpression increases sphere formation. c–e, Migrating/sphere forming cells are not from the PGR+, but the PGR− population, which are responsive to PIPS. f–j, To investigate which PGR− cells were the target population of progesterone signalling, we exposed parental MM3MG cells and Her2-transduced cells to progesterone, PIPS or mixed the cells with PGR+ cells (only for migration experiments). Progesterone, WNT4 and RANKL, and co-culture with MM3MG–Pgr-B induced sphere formation and migration of MM3MG cells, but decreased these responses in MM3MG–Her2 cells. k, Overexpression of Her2 increases proliferation of MM3MG cells (MM3MG–Her2). WNT4 and RANKL (WR) further increase proliferation of MM3MG–Her2 cells, but decreases proliferation of the parental (MM3MG) cells. Therefore based on expression of HER2, cells either migrate (HER2low/−) or proliferate (HER2high). l, WNT4 and RANKL treatment induces proliferation of primary cultured cells derived from primary tumours, but reduces it in cells derived from early lesions. m, Reduction of HER2 signalling by lapatinib overrides the inhibitory effect of WNT4 and RANKL, and increases migration in MM3MG–Her2 cells. However, strong inhibition of HER2 signalling reduces migration. n, Lapatinib inhibits HER2 signalling by preventing phosphorylation. o, Cells that migrated through the pores of the migration chamber insert were stained for HER2 (FITC, green) and PGR or ERα (Cy3, red). In 1:1 co-culture of MM3MG–Pgr-B and MM3MG–Her2 (top) only HER2-expressing cells migrate. Migrated primary cells derived from early lesions (middle) do not express PGR and display faint HER2 staining (brightness of HER2 and PGR staining increased by 50% for better visibility). HER2 and PGR double-positive T47D cells fixed onto the filters of migration chambers served as positive control of staining. m–o, Cells with low/intermediate signalling of HER2 show the highest response in migration and sphere formation induction by PIPS. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS, not significant; (F-test of the slope (k, l) or Student’s t-test (other panels)); data are mean ± s.d. For gel source data, see Supplementary Fig. 1.