Graph, percentage s.e.m. might occur during the early stages of tumour evolution3,8,9. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown8. Here we show that, Edicotinib in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelialCmesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal Edicotinib branching that generates eDCCs that are capable of forming metastasis after a dormancy phase. We investigated whether the loss Edicotinib of tumour-suppressive p38 signalling and gain of oncogenic Her2 upregulation, which induce resistance to anoikis (apoptosis induced by lack of correct cellCextracellular matrix attachment) in early cancer cells10, might activate a disseminating phenotype. eDCCs were defined as those originating at times when the MMTVCHer2 (Her2 under the control of mouse mammary tumour virus) mice had normal ductal architecture, hyperplasia and mammary intraepithelial neoplasia, as confirmed by histopathology11, but no detectable tumours8 (Extended Data Fig. 1aCc). Her2 causes E-cadherin (E-cad, also known as cadherin 1) downregulation12, whereas p38 can maintain E-cadherin expression13. We found that more than 85% of Her2+ cells were E-cadlo (Fig. 1a), and in Her2+ (wild-type gene) and Her2-T+ (mutant active gene) tissues of early lesions, E-cadhi early cancer cells were more frequently phosphorylated (70C75%) (p-) ATF2hi (Fig. 1b and Extended Data Fig. 1d, e). In each duct, 60C70% of all cells from early lesions were positive for membrane -catenin (the inactive form) (Extended Data Fig. 2a). However, when analysing Her2+ cells alone, Edicotinib only 30% of cells showed membrane localization for -catenin (Fig. 1c). Overall, these results suggest that Her2+ cells display a loss of E-cadherin- and -catenin-based junctions and are p-ATF2lo. Open in a separate window Figure 1 E-cadherin, Her2 and p-ATF2 levels and function in early lesion cellsa, Top, MMTVCHer2 early lesion tissue sections co-stained for the indicated antigens. Bottom, intra-ductal heterogeneity of Her2hiE-cadlo cells. Inset, magnified view of the boxed region. Arrowheads, Her2hiE-cadlo cells; arrows, Her2loE-cadhi cells. Right, percentage of Her2hi cells that were E-cadlow or E-cadhigh (=20 ducts; =2 mice). *** 0.0001. b, Representative images of E-cadhip-ATF2hi (top) and E-cadlop-ATF2lo (bottom) ducts in MMTVCHer2 early lesion tissues. Arrowhead, p-ATF2hiE-cadhi cell, arrow, p-ATF2loE-cadlo cell. Scale bars, 25 m and 10 m (inset) (a and b). c, MMTVCHer2 early lesion tissue sections stained for Her2 and -catenin. Arrow, Her2+ cells with low membrane-associated -catenin (-catMEM-lo); arrowhead, Her2?-catMEM-hi cells. Scale bar, 10 m. Graph, percentage of early lesion cells with -catMEM that were Her2? or Her2+ (Her2, *=0.035 and Her2-T, *=0.0008). MMTVCHer2 = 30 ducts per mouse, =3 mice; MMTVCHer2-T = 10 ducts per mouse, =2 mice. d, MMTVCHer2 early lesion organoids treated for 48 h with DMSO or 5 M SB203580. BF, bright field. Bottom imagees indicate magnified, boxed regions. Scale bars, 15 m (left), 40 m (right). e, MMTVCHer2 organoids stained for the indicated antigens. Arrowheads, invasive cells; arrow, intact laminin-V layer. Scale bars, 25 m (e), 10 m (insets 1 and 2). f, MMTVCHer2 early lesion sections co-stained for the indicated antigens. Arrows, Her2+E-cadlo invading cells. Top numbers, percentage of Her2+ E-cadlo invading cells in early lesion sections, = 58 cells per section, = 2 mice. Graph, percentage E-cadlo invading cells in MCF10A-HER2 (10A-Her2; see also Rabbit polyclonal to ACVRL1 Extended Data Fig. 3a) or MMTVCHer2 organoids. = 20 MCF10A-HER2 organoids. a, c, one-sided MannCWhitney (DCIS) lesions retained both high p-ATF2 expression levels and organized E-cadherin junctions, whereas HER2+ DCIS lesions showed low expression levels for p-ATF2 and E-cadherin (Extended Data Fig. 2e, f). The HER2+p-p38lop-ATF2lo profile was also present in larger human HER2+ breast carcinomas, whereas only HER2? tumours showed strong nuclear staining.