TLR4 is a member of the Toll-like receptor family, which act as sentinels for activation of the innate immune response. TLR4 is typically stimulated by ligand binding of pathogenic or endogenous stimuli, leading to activation of inflammatory transcription factors such as NF-κB, AP-1 and IRF3. Dysregulation of inflammatory responses is a hallmark of many cancers, including UV-induced non-melanoma skin cancer (NMSC). While many studies focus upon the reaction of immune cells after UV stimulation of the skin, the keratinocytes that give 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, and contributes to UV-induced inflammatory/stress responses in cultured keratinocytes. In addition, inhibition of TLR4 using the specific pharmacological antagonist resatorvid (TAK-242) blocks UV-induced signaling in keratinocytes and in mouse epidermis. Remarkably, long-term topical resatorvid application also significantly inhibits UV-induced skin tumorigenesis in SKH-1 mice. Profiling of protein/phosphoprotein expression in tumors from resatorvid treated mice compared to those from control mice have revealed some interesting results. While we do see the expected inhibition of p38 MAPK and Akt phosphorylation in resatorvid-treated tumors, phosphorylation of the TLR-regulated kinase IRAK4 is increased by resatorvid treatment. In addition, chronic treatment with UV causes strong upregulation of TLR4 protein expression in mouse epidermis compared to untreated skin, which is maintained in each of the skin tumor treatment groups. Our data suggests that there are likely compensatory mechanisms activated in resatorvid-treated skin tumors that allow for partial escape from the influence of this drug. This proposal aims to use our currently banked mouse skin and tumor samples to generate whole transcriptome gene expression data for use in confirming the patterns of TLR4-linked signaling inhibition that we have noted previously with resatorvid treatment, and in querying what compensatory mechanisms might be in play. We also plan to utilize wildtype and TLR4 knockout mouse embryonic fibroblasts (MEFs) to confirm the specificity of resatorvid as a TLR4 inhibitor. Little is known about the regulation of TLR4 expression in the skin, especially in the context of UV exposure. We plan to use the expression data and a systems biology approach to define which signaling pathways are significantly different in the resatorvid treated tumors, chronically treated skins and MEFs compared to controls, in order to evaluate targets for future combinatorial approaches for prevention and treatment of NMSC.