PROJECT ABSTRACTTLR4 is a member of the Toll-like receptor family which act as sentinels for activation of the innate immuneresponse. TLR4 is typically stimulated by ligand binding of pathogenic or endogenous stimuli leading toactivation of inflammatory transcription factors such as NF-B AP-1 and IRF3. Dysregulation of inflammatoryresponses is a hallmark of many cancers including UV-induced non-melanoma skin cancer (NMSC). Whilemany studies focus upon the reaction of immune cells after UV stimulation of the skin the keratinocytes thatgive rise to NMSC also respond to this environmental stress by activating inflammatory genes and proteins.We have recently shown that TLR4 is overexpressed in human NMSC compared to normal skin andcontributes to UV-induced inflammatory/stress responses in cultured keratinocytes. In addition inhibition ofTLR4 using the specific pharmacological antagonist resatorvid (TAK-242) blocks UV-induced signaling inkeratinocytes and in mouse epidermis. Remarkably long-term topical resatorvid application also significantlyinhibits UV-induced skin tumorigenesis in SKH-1 mice. Profiling of protein/phosphoprotein expression intumors from resatorvid treated mice compared to those from control mice have revealed some interestingresults. While we do see the expected inhibition of p38 MAPK and Akt phosphorylation in resatorvid-treatedtumors phosphorylation of the TLR-regulated kinase IRAK4 is increased by resatorvid treatment. In additionchronic treatment with UV causes strong upregulation of TLR4 protein expression in mouse epidermiscompared to untreated skin which is maintained in each of the skin tumor treatment groups. Our datasuggests that there are likely compensatory mechanisms activated in resatorvid-treated skin tumors that allowfor partial escape from the influence of this drug. This proposal aims to use our currently banked mouse skinand tumor samples to generate whole transcriptome gene expression data for use in confirming the patterns ofTLR4-linked signaling inhibition that we have noted previously with resatorvid treatment and in querying whatcompensatory mechanisms might be in play. We also plan to utilize wildtype and TLR4 knockout mouseembryonic fibroblasts (MEFs) to confirm the specificity of resatorvid as a TLR4 inhibitor. Little is known aboutthe regulation of TLR4 expression in the skin especially in the context of UV exposure. We plan to use theexpression data and a systems biology approach to define which signaling pathways are significantly differentin the resatorvid treated tumors chronically treated skins and MEFs compared to controls in order to evaluatetargets for future combinatorial approaches for prevention and treatment of NMSC.