Purpose: Accumulating evidence suggests that a tumor is consisting of heterogeneous cell populations with a different fate under radiotherapy.The cancer stem cells (CSCs) being less than 1% of total tumor cell population are able to self-renew and may survive the genotoxic conditions of anticancer chemo and radiation therapy.In clinic, overexpression of HER-2, a member of ErbB receptor family, is linked with increased tumor recurrence and metastatic lesions.This work is to identify the signaling pathways associated with the radiation-resistant phenotype of cancer stem cells.Elucidation of the role of CSCs in radiation-mediated tumor adaptive response and cell repopulation will provide critical insights for re-sensitization of resistant fractions.Materials and methods: Establishment of radioresistant breast cancer cell lines by fractionated ionizing radiation; proteomics with 2-DIGE; Western blot and IHC detecting HER2/CD44/CD24 expression; FAC sorting cells with cancer stem cell markers.Results: The HER2/Neu is found to be causally related with the radioresistant breast cancer stem cells (CSCs) expressing CD44+ and CD24-/low.About half of the breast CSCs showed elevated HER2 expression, and these CSCs were associated with more aggressive growth and higher aldehyde dehydrogenase 1 AL(ALDH1) than CSCs with no HER2 expression.Proteomics with 2-DIGE generate a large profile of proteins differentially expressed in HER2+/CD44+/CD24-/low versus HER2-/CD44+/CD24-/low cells, including factors in DNA repair, cell migration and invasion.HER2+/CD44+/CD24-/low cells showed a higher potential in tumorigenesis than HER2-/CD44+/CD24-/low cells in mouse xenograft.The co-expression of HER2+ and CD44+ was more frequently detected in recurrent versus primary breast tumors.Conclusions: These results suggest that HER2+/CD44+/CD24-/low CSCs represent a novel radioresistant subpopulation within breast CSCs and a potential therapeutic target to treat radioresistant recurrent and metastatic cancer cells.