Recently, we investigated the decreased growth of LPS treated 4T1 and discovered that the effect of LPS was dependent upon Myd88. Surprisingly, we also found that simply decreasing Myd88 expression was sufficient to slow the growth of the tumor cells in vitro and in vivo. We obtained similar data by inhibiting Myd88 function using a Myd88 inhibitory peptide. Treatment of four different murine mammary carcinomas with the Myd88 inhibitor led to a decrease in the in vitro growth rate of the tumor cells. These data suggest that signaling through Myd88 is important for tumor growth and may provide a novel way to target breast cancer. The hypothesis of this study is that Myd88 contributes to the growth of breast cancer. To address this hypothesis, there are three specific aims: 1) determine the extent to which Myd88 regulates cell cycle progression and/or inhibition of apoptosis in 4T1 tumor cells, 2) determine the therapeutic potential of targeting Myd88 in growing tumors, and 3) determine whether HMGB1 and HSP60 mediate Myd88 dependent signaling in 4T1 tumor cells. Specific aim 1 will be accomplished by treating 4T1 cells with a Myd88 inhibitor and siRNA specific for Myd88 and determining whether inhibition of growth is due to inhibition of cell cycle progression or apoptosis. For specific aim 2, we will deliver the Myd88 inhibitor in vivo. Tumor growth will be monitored in conjunction with immunogenicity and metastatic potential of the Myd88 targeted tumors in an attempt to correlate changes in growth with alterations in anti-tumor immunity and disease progression. These studies will be conducted with the 4T1 model as well as rat neu transgenic mice in order to determine the therapeutic potential of targeting Myd88 in a transplantable and spontaneous tumor model. Specific aim 3 will be accomplished by inhibiting expression of HMGB1 and HSP60 in 4T1 cells using RNA interference to determine (1) if inhibiting expression of these DAMPs impacts tumor cell growth, cell cycle progression, and/or apoptosis and (2) if inhibiting HMGB1 or HSP60 recapitulates the effects of inhibiting Myd88. Defining the role of Myd88 in tumors may lead to the development of more effective treatment strategies for patients with cancer. Although Myd88 antagonists would need to be evaluated, this is already an active field of research. Investigators are exploring variants of Myd88, have generated mimics which inhibit Myd88 function, and recently crystallized the Myd88-IRAK4-IRAK2 death domain complex, all of which may assist in developing novel strategies to manipulate Myd88 function. Therefore, if Myd88 antagonists can help control tumor progression, clinicians may be able to utilize this knowledge to help treat patients with cancer. Methods to block Mydd8 function may also be relevant where gain-of-function mutations have been shown to contribute to cancer. Finally, results from this project may lead to a better understanding of how DAMPs and PAMPs impact tumor growth and assist in generating therapies aimed at increasing anti-tumor immunity. PUBLIC HEALTH RELEVANCE: Because Myd88 expression has been correlated with tumor progression, there is great interest in not only defining the role of Myd88 in tumor growth, but also in understanding how to control the function of this protein. As a result, current investigations of natural variants of Myd88 and the generation of synthetic mimics which inhibit Myd88 function may assist in developing novel strategies to manipulate Myd88 function and as a consequence control tumor growth. The results from this project therefore may open the door to new strategies for the treatment of patients with cancer.