Clonal variation drives heterogeneous necrotic and apoptotic cell death responses to UVB radiation in human keratinocytes

Summary: Abstract Body: Ultraviolet B radiation (UVB) is the primary etiological contributor to skin cancer. UVB-induced apoptosis is a non-inflammatory process that suppresses tumor development. However, UVB induces pro-carcinogenic inflammation which may be triggered by necrosis. Necrosis encompasses multiple regulated cell death (RCD) pathways, including necroptosis, pyroptosis, secondary necrosis and PANoptosis. UVB-induced necrotic RCD in keratinocytes is not well understood. We investigated RCD cell fates in HaCaT keratinocytes exposed to 30 mJ/cm2 UVB after 12 hours by immunofluorescence (IF) staining for phospho-MLKL (necroptosis), cleaved Gasdermin D (pyroptosis), ASC (pyroptosis), and nuclear condensation (apoptosis). Our results showed that individual cells died through different RCD fates including apoptosis, pyroptosis, necroptosis, or PANoptosis. To understand this heterogeneity, twelve HaCaT clonal populations were analyzed by Annexin V/PI flow cytometry and IF, with one population being early apoptotic, two populations necrotic, and nine populations double positive, suggesting either primary or secondary necrosis, similar to the parent HaCaTs. IF staining revealed clones undergoing predominately apoptosis, necroptosis, pyroptosis, or PANoptosis, indicating clonal variation in the parental HaCaT cells. Normal human epidermal keratinocytes (NHEKs) exposed to 30 mJ/cm2 UVB were also analyzed by IF and found to undergo pyroptosis or apoptosis. The caspase inhibitor zVAD inhibited UVB-induced cleaved Gasdermin D but not ASC clustering, while the RIPK3 inhibitor HS-1371 strongly upregulated pyroptotic markers, indicative of a compensatory RCD cell fate switch. The clonal variation and cell death compensation supports the hypothesis that human keratinocytes exhibit a heterogenic necrotic and apoptotic cell death response upon UVB radiation which is not a stochastic process but regulated by intrinsic cellular properties. Madeline Z. Tara¹, Mitchell Denning¹ 1. Cancer Biology, Loyola University Chicago Graduate School, Chicago, IL, United States. UV Biology/Injury and Non-melanoma Cancers