Investigating novel targets for topical immunoprevention of keratinocytic skin cancer - Abstract Non-melanoma skin cancer (NMSC) represents a major public health burden. Cutaneous exposure to solar ultraviolet (sUV) radiation is a causative factor in NMSC, and inflammatory dysregulation is a key mechanism underlying detrimental effects of acute and chronic UV exposure. The identification of relevant and targetable immune checkpoints such as PD1/PD-L1 is changing clinical outcomes of cancer patients. However, leveraging immune checkpoint modulation for cancer prevention remains mostly unexplored. Recent data from our laboratory has shown that PD-L1 is upregulated in epidermal keratinocytes after acute and chronic UV stimulation in human skin, and may therefore serve as a target for skin cancer immunoprevention (i.e., strategies harnessing tumorigenesis-directed innate or adaptive mechanisms of immune surveillance). However, the possibility that other stress-induced immune checkpoint proteins are also upregulated in the skin due to UV exposure and are therefore viable targets for immunoprevention of NMSC has not been explored. In this CIP- Net UG3/UH3 project we will pursue the novel hypothesis that overexpression of select UV-responsive immune checkpoint proteins early in the progression from normal skin to NMSC can promote an immune suppressive microenvironment and can be targeted to prevent photocarcinogenesis. In order to test this hypothesis, the UG3 phase (Aim 1) will use matched human samples of sun-protected skin, sun-damaged skin, actinic keratoses (AK), cutaneous squamous cell carcinomas (cSCC) and basal cell carcinomas (BCC) for transcriptomic profiling and immunohistochemistry analysis in order to identify novel immune checkpoint proteins that are upregulated early in the skin carcinogenesis process. The UH3 phase (Aim 2) of this study will confirm that these human targets are also upregulated early in mouse skin tumorigenic progression, can affect cellular responses to UV stress in keratinocytes in culture, and can influence cutaneous responses to UV and photocarcinogensis in a transgenic mouse model. Multiplex IHC will also be utilized to verify the top candidates using a translationally- relevant platform. The milestones associated with the UG3 phase are 1) Define 2-3 immune checkpoint proteins that are upregulated in keratinocytes of sun-damaged skin or AK as well as overexpressed in cSCC tumor cells (keratinocytic origin), and 2) Confirm these targets at the protein level using immunohistochemistry. The milestones associated with the UH3 phase are 1) validating immune checkpoint protein candidates to be UV responsive in mouse skin, 2) determining UV-inducible signaling/phenotypic responses to keratinocytic knockout (genetic deletion) of these immune checkpoint proteins in vitro and in vivo, 3) screen compound libraries to discover and characterize small molecules targeting these immune checkpoints, and 4) validating our previous IHC findings using clinically-relevant multiplex staining of the human samples. The proposed research will serve to identify novel molecular immune biomarkers for NMSC that will allow us to intervene at earlier stages of skin carcinogenesis to reduce the morbidity and mortality of cSCC.
