Topical sirolimus prevents cutaneous squamous cell carcinomas by disrupting the balance between differentiation and proliferation

Summary: Abstract Body: Topical mTor inhibitors are increasingly being used for several skin disorders. We previously demonstrated in a double-blind study that applying topical sirolimus for 12 weeks effectively reduced the risk of keratinocyte cancers in solid organ transplant recipients, with a significant decrease in the occurrence of intraepidermal carcinomas 24 months post-treatment. To gain deeper insights into the mechanisms of this chemoprevention, patients' skin samples were collected after 12 weeks of treatment (n=10 treated forearm vs. n=10 placebo forearm from the same patient) for Spatial Transcriptomics (10X Visium). We found a significant downregulation of genes involved in the mitosis phase and the G2/M transition, with most keratinocytes arrested in the G1 phase, confirmed by a marked decrease in PCNA expression and reduced p63 synthesis, as confirmed by immunofluorescence. Despite these changes, cell viability remained unaffected, with no increase in apoptosis. Simultaneously and unexpectedly, the differentiation process was also altered, showing downregulation in pathways associated with keratinocyte differentiation. This translated into a significant upregulation of keratin 14 expression, E-cadherin signaling, and integrin regulation as well as an activation of the Hippo-YAP pathway into upper epidermal layers, leading to YAP phosphorylation and restricted cell proliferation. Additionally, NOTCH1 signaling and related reactome pathways were downregulated. To conclude, our findings emphasise the importance of changes in the balance between proliferation and differentiation in the early carcinogenesis process, and the effect of sirolimus in disrupting that balance by controlling excess proliferation despite reduced differentiation. Lea Dousset¹, Chenhao Zhou¹, Yung-Ching Kao¹, Sam Tan¹, Nicholas Muller¹, Laura Sormani¹, Charlotte Cox¹, H P. Soyer¹, Mitchell S. Stark¹, Ho Yi Wong¹, Edwige Roy¹, Kiarash Khosrotehrani¹ 1. Frazer Institute, Dermatology Research Centre, The University of Queensland, Brisbane, QLD, Australia. UV Biology/Injury and Non-melanoma Cancers