Abstract The overall cure rate of primary tumors has been significantly improved in the last three decades; however, metastatic and recurrent tumors with aggressive growth and therapy-resistance remain as the major factors shortening breast cancer patients’ survival. MKP1, a mitogen-activated protein kinase (MAPK) phosphatase, was shown to be linked with chemoresistance in breast cancer cells and was over-expressed in radio-resistant breast cancer cells that survived long-term fractionated irradiation. Herein, we showed that MKP1 is expressed exclusively in HER2 positive clinical breast tumors compared to HER2 negative tumors. Comparison of normal and tumor breast tissue samples from same patients revealed that MKP1 expression is induced in breast tumors along with HER2 expression in patients, who showed low/no expression of MKP1 and HER2 in their normal breast tissue. Most importantly, we have shown that HER2 positive breast cancer cells rely heavily on MKP1 for survival as knocking down MKP1 resulted in a massive cell death. MKP1 was found to translocate into the mitochondria in breast cancer cells upon radiation and mitochondrial MKP1 levels were significantly enhanced in HER2 over-expressing and radio-resistant breast cancer cells, suggesting a survival mechanism where increased MKP1 translocation into the mitochondria functions to attenuate pro-apoptotic signals initiated from mitochondria-localized MAPK, c-Jun N-terminal kinase (JNK). Consistent with this, increased mitochondrial MKP1 levels were correlated with the reduced levels of phosphorylated active JNK in the mitochondria of irradiated cells. Such mitochondrial MKP1-mediated dephosphorylation of JNK was predominantly enhanced in the radio-resistant breast cancer cells that can be sensitized by siRNA-mediated inhibition of MKP1. The identification of cancer stem cells (CSCs) in all human tumors and their involvement in tumor radioresistance encouraged the studies investigating whether MKP1-mediated survival mechanism is present in radio-resistant breast cancer stem cells. Here, we showed that MKP1 is overexpressed in irradiated HER2-positive breast CSCs (HER2+/CD44+/CD24-/low) and knocking down MKP1 in these cells significantly reduced their survival, which was further reduced with radiation treatment. These data provide critical insights on the aggressive phenotype of breast tumors and offer MKP1 as a novel diagnostic and therapeutic target to re-sensitize resistant breast cancer stem cells. Identification of the mechanism of MKP1-mediated survival in these cells may provide additional druggable targets to control breast cancer and its recurrence. Citation Format: Demet Candas, Chung-Ling Lu, Ming Fan, Frank Chuang, Colleen Sweeney, Alexander Borowsky, Jian Jian Li. MKP1-mediated survival of HER2 positive breast cancer stem cells. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2014-3029