AbstractAlthough trastuzumab is an effective treatment in early stage HER2⁺ breast cancer the majority of advanced HER2⁺ breast cancers develop trastuzumab resistance, especially in the 40% of breast cancers with loss of PTEN. However, HER2⁺ breast cancer patients continue to receive trastuzumab regardless PTEN status and the consequence of therapy in these patients is unknown. We demonstrate that continued use of trastuzumab in HER2⁺ cells with loss of PTEN induces the epithelial-mesenchymal transition (EMT) and transform HER2⁺ to a triple negative breast cancer. These transformed cells exhibited mesenchymal morphology and gene expression markers, while parent HER2⁺ cells showed epithelial morphology and markers. The transformed cells exhibited loss of dependence on ERBB family signaling (such as HER2, HER3, HER4, BTC, HRG, EGF) and reduced estrogen and progesterone receptors. Continued use of trastuzumab in HER2⁺ PTEN⁻ cells increased the frequency of cancer stem cells (CSCs) and metastasis potential. Strikingly, parental HER2⁺ cells and transformed resistant cells respond to treatment differently. Transformed resistant cells were sensitive to chemical probe (sulforaphane) through inhibition of IL-6/STAT3/NF-κB positive feedback loop whereas parental HER2⁺ cells did not respond. This data suggests that trastuzumab resistance in HER2⁺ PTEN₋ breast cancer induces EMT and subtype switching, which requires unique treatment options.