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s: Tuomela J. et al. Low Toll−Like Receptor 9 Expression Is Required for the Aggressive Behavior of Triple Negative Breast Cancer Cells in Hypoxia, PROGRAM #P2−11−10, San Antonio Breast Cancer Symposium, San Antonio, TX, December 2011 Degrees obtained that are supported by this award; Jouko Sandholm, PhD (pending) Funding applied for based on work supported by this award: Susan Komen grant (pending), RO1 (pending), DOD (pending) Grants received: from the UAB Comprehensive Cancer Center to study the prognostic role of TLR9 expression in African-American breast cancer patients. Employment or research opportunities applied for and/or received based on experience/training supported by this award: Dr. Johanna Tuomela, PhD, post-doctoral fellow, Jouko Sandholm, M.Sc., graduate student. CONCLUSION: We have determined that DNA, which is derived from chemotherapy-killed breast cancer cells has important biological effects on living breast cancer cells and these effects induced by the “Dead DNA” may determine the course of the disease in a dramatic way, especially in triple negative breast cancer. First, we demonstrate that such “dead DNA” is taken up into living breast cancer cells and that complexing with LL-37 increases this cellular up-take. Second, “dead DNA” induces in vitro invasion in various breast cancer cells, also in brain cancer cells and this invasion is TLR9mediated and due to TLR9-regulated activation of proteolytic enzymes. This effect, however, requires a lot of DNA and it is unclear whether or not such an invasion takes place in vivo. It does not seem to do so in the animal models that we have used, but this phenomenon may be important in other cancers where more DNA is released upon cell death. Our results suggest that the pathophysiological significance of the dead DNA-induced invasion should be studied in an animal model that better mimics cancers that are undergoing rapid cell death and where large amounts of “dead DNA” are available in the vicinity of the tumor. We also discovered that such “dead DNA” has pro-inflammatory effects in living cancer cells. Our animal experiments suggest that these effects are also TLR9-mediated, although we have to still characterize the findings further. These findings may partly explain why the low TLR9-triple negative subgroup of breast cancer patients, which we have first described (Tuomela et al. 2012) has such poor prognosis. We also demonstrated that TLR9 expression is hypoxiaregulated and in the absence of hypoxia-induced TLR9 expression, triple negative, but not ER+ breast cancer cells, become highly invasive. We are now seeking further funding to test the hypotheses derived from the current work. SO WHAT: We set out to test the hypothesis that DNA from dead cancer cells induces invasion in living cancer cells and that this “death-induced vicious cycle” contributes to tumor spread and metastasis in breast cancer. While we have shown that this indeed is the case in vitro, we have been unable to demonstrate that it is important for metastasis of small breast cancers in vivo. However, while doing these experiments, we ended up discovering a novel, poor-prognosis subgroup of breast cancer patients that have low tumor TLR9 expression + triple negative tumors. These patients may represent as many as 10 % of all breast cancer patients. Interestingly, however, the experiments proposed in the original grant may