AbstractHER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2^stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2^labile cell line gave rise to HER2-negative tumors from which MamBo38HER2^loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2^labile cells induced the loss of HER2 expression. MamBo38HER2^loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2^loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2^loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss.