Exposure to the ultraviolet (UV) rays of sunlight is the primary cause of skin cancer, which kills greater than 20,000 U.S. individuals each year. Veterans are at increased risk for skin cancer development due to increased sun exposure during training and deployment. UV exposure also causes photoaging that results in cells acquiring a post-mitotic phenotype called senescence. In addition to sun exposure, age and treatment with immunosuppressants are the most significant risk factors for skin cancer development. In this proposal, we will examine our novel 2 component cancer field model of cutaneous carcinogenesis that links photoaging and immunosuppression to skin cancer development. Historically, the cancer field refers to multifocal areas of otherwise normal epithelium that are at increased risk for cancer development. This epithelial cancer field is characterized by the stepwise accumulation of mutations that eventually lead to malignant conversion. We have shown that UV induces a multifocal dermal senescent field that is characterized by inflammatory angiogenesis that can be visualized and measured by imaging the dermal hemoglobin (Hb) content. This dermal field persists & expands following cessation of UV treatments and predicts sites of overlying epidermal clonal expansion & tumor formation. In this proposal, we propose that a dermal field also exists and that the key characteristic of this dermal field is the presence of senescent cells. These senescent cells produce and secrete specific pro-inflammatory, mitogenic and immunomodulatory mediators that we propose drive the continued development of the overlying epithelial mutant field. However, the immune system can recognize and clear senescent cells (senescence surveillance). We predict that this immune-mediated clearance acts to suppress the effects of the senescent dermal field on tumor formation. We expect that treatment with immunosuppressive drugs would promote tumorigenesis by blocking immune-mediated clearance of dermal senescent cells. Transforming growth factor beta (TGF-β) is a cytokine that is necessary for UV-induced senescence and is produced by senescent cells. This TGF-β production can in turn result in the induction of senescence in nearby cells (bystander senescence). We propose that bystander senescence, in an immunosuppressed setting, provides a self-perpetuating mechanism that drives senescent field persistence AND expansion. Finally, not all immunosuppressive agents have the same capacity to promote skin cancer development. Cyclosporine A promotes skin cancer development, TGF-β production, and induces senescence. In contrast, sirolimus has a limited effect on skin cancer risk, is known to suppresses TGF-β production, and suppresses senescent cell formation and secretory activity. In this proposal, we will determine whether the UV- induced senescent field is necessary for skin cancer development, determine whether immunosuppressive therapy promotes senescent field development and persistence, and determine whether TGF-β signaling & bystander senescence play central roles in this process. This will be done in three aims: Aim 1: Determine whether senolytic therapy inhibits photocarcinogenesis and dermal field persistence. Examine whether senolytic therapy and loss of TGF-β signaling block the ability of cyclosporine A to enhance, and sirolimus to suppress, UV-induced tumor formation. Aim 2. Use single cell RNA sequencing (scRNAseq) and dermal whole transcriptome sequencing and differential expression analysis to characterize the differences between hyperemic and non-hyperemic foci. Aim 3. Determine whether cyclosporine A suppresses senescence surveillance and promotes bystander senescence in wildtype while sirolimus acts to suppress bystander senescence. The role of TGF-β on bystander senescence will also be assessed.

The primary risk factors for skin cancer development are sun exposure, age and an immunocompromised status. We propose that these risk factors are linked by their ability to promote the development, persistence and expansion of a senescent field that promotes overlying tumorigenesis. We will also determine whether transforming growth factor β production plays a central role in senescent field development, persistence and expansion. If successful, these studies will validate a novel two-field hypothesis for skin cancer development and provide evidence that anti-aging therapies may prove highly useful in suppressing skin cancer risk in individuals, particularly the immunosuppressed.