PROJECT SUMMARY / ABSTRACT: Cancer stem cells are a rare and yet critically important subpopulation of cells in tumors associated with treatment failure and metastatic recurrence. Though it is becoming increasingly clear that targeting this subpopulation could lead to therapies with better odds of a cure it is still unknown how these cells originate and what are the biochemical processes that promote “stemness” in cancer. Our laboratory found that alterations in the metabolism of mitochondrial reactive oxygen species (ROS) promote aberrant activation of hypoxia-induced factor 2α (HIF2α). The activation of HIF2α is a well-established mechanism of stemness that has also been implicated in metastatic recurrence as well as treatment failure in women with breast cancer. We found that a posttranslational modification (i.e. acetylation) of a primary enzyme involved in the metabolism of mitochondrial ROS, manganese superoxide dismutase (MnSOD) breaks the tetrameric structure that has antioxidant function turning the enzyme into a monomer that promotes ROS formation and activates HIF2α. We also found that MnSOD-K68Ac accumulates prominently in breast cancers expressing low estrogen receptor levels. Hence, it is proposed that MnSOD has a dichotomous behavior functioning as a suppressor of tumor initiation (antioxidant function) and yet promotes cancer stem cell reprogramming later on in established tumors. This proposition is supported by the finding of strong associations between a MnSOD/HIF2α signature present in metastatic lesions compared to primary tumor samples from the same breast cancer patients. Hence, this application aims at determining: (1) if MnSOD-Ac reprograms tumor cells to stem-like phenotypes associated with chemoresistance. (2) if the biochemical and/or genetic targeting of MnSOD-Ac or HIF2α in established tumors of mice with the MnSODhigh/HIF2α signature (MMTV.PyVT) suppresses chemoresistance and/or metastasis. (3) if there is an association between subsets of women with breast cancer that exhibit a MnSODhigh or MnSOD-Ac, or MnSOD-ROS-HIF2α molecular axis signature and develop chemoresistance or have increased risk of metastatic recurrence.