998 EMT-like phenotype in normal keratinocytes driven by TLR3 activation

Abstract

Toll-Like-Receptor 3 (TLR3), a pattern recognition receptor that recognizes dsRNA, activation results in NFkB or interferon-regulatory factor 3 (IRF3) mediated gene transcription of inflammatory cytokines (e.g. IL-6) and type I and III interferons (e.g. IFN-λ). TLR3 also contributes to induced-pluripotent stem cell (IPS) programming, wound healing and skin regeneration through Wnt, SHH, and epithelial-mesenchymal transition (EMT) pathways. Our hypothesis is that TLR3 signaling within normal keratinocytes contributes to non-melanoma skin cancer (NMSC) through activation of EMT. In vitro, normal neonatal human embryonic keratinocytes (NHEKs) treated with a TLR3 agonist, polyinosinic:polycytidylic acid (poly IC), exhibit sustained EMT morphology changes that correlate with elevated levels of the intermediate filament Vimentin (∼2 fold), and transcription factors, ZEB1 (∼3-fold), SNAI1 (∼2-fold), and TWIST1 (∼2-fold), all key EMT markers. Poly IC treatment significantly increased levels of IL-6 (∼8-fold) and IFN-λ1 (∼200-fold) in normal keratinocytes. However, treatment of NHEKs with recombinant IL-6 or IFN-λ1 proteins do not induce an EMT-like morphology or increase in EMT gene expression. UVB radiation, the major player in NMSC development, also activates TLR3 signaling in keratinocytes. In vitro, NHEKs exposed to UVB (10mJ/cm2) exhibit similar EMT-like morphology and gene expression changes as poly IC treated cells. Chemical inhibition of TLR3 (CU CPT 4a) prior to poly IC or UVB exposure prevents EMT gene expression. We performed RNA-sequencing to investigate common pathways activated by both Poly IC and UVB to uncover additional downstream players responsible for driving the TLR3 induced EMT-phenotype. Taken together, TLR3 activation and the resulting EMT-like phenotype is a result of multiple players, and not likely driven by a single downstream mediator of TLR3 signaling.