Crosstalk in Skin: Loss of Desmoglein 1 in Keratinocytes Inhibits BRAFV600E-induced Cellular Senescence in Human Melanocytes

Melanoma arises from transformation of melanocytes in the basal layer of the epidermis where they are surrounded by keratinocytes, with which they interact through cell contact and paracrine communication. Although research focuses on how the accumulation of oncogene and tumor suppressor gene mutations in melanocytes drive melanoma development, how alterations in keratinocytes serve as extrinsic drivers of melanoma initiation and progression is poorly understood. We recently identified keratinocyte desmoglein 1 (Dsg1) as an important mediator of keratinocyte:melanoma crosstalk. Here we address the extent to which Dsg1 loss, which occurs in response to acute environmental stress such as ultraviolet radiation, affects early steps in melanomagenesis. RNA-Seq analysis revealed that paracrine signals from Dsg1-deficient keratinocytes mediate a transcriptional switch from a differentiated to undifferentiated cell state in melanocytes expressing BRAFV600E. Of 221 differentially expressed genes in BRAFV600E cells treated with Dsg1-deficient conditioned media (CM), the laminin superfamily member NTN4/Netrin-4, which inhibits senescence in endothelial cells, stood out. Indeed, while BRAFV600E melanocytes treated with Dsg1-deficient CM showed signs of senescence bypass, knockdown of NTN4 reversed these effects. These results suggest that Dsg1 loss in keratinocytes provides an extrinsic signal to push melanocytes towards oncogenic transformation once an initial mutation has been introduced. Overall design: Keratinocytes and melanocytes were isolated from neonatal foreskin provided by the Northwestern University Skin Biology and Diseases Research-Based Center as previously described (Roth-Carter et al., 2022). Keratinocytes were grown in M154 medium (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (Thermo Fisher Scientific), 1,000 x gentamycin/amphotericin B solution (ThermoFisher Scientific), and 0.07mM CaCl2. Melanocytes were cultured in OptiMEM (Thermo Fisher Scientific) containing 5% fetal bovine serum (MilliporeSigma), 10 ng/ml bFGF (ConnStem), 1 ng/ml heparin (MilliporeSigma), 0.1 mM N6, 2'-O-dibutyryladenosine 3:5-cyclic monophosphate (MilliporeSigma), 0.1 mM 3-isobutyl-1-methyl xanthine (MilliporeSigma), and 1% penicillin/streptomycin (Corning). All cells were maintained at 37 °C with 5% CO2. Conditioned media (CM) from keratinocytes were prepared by following previously published procedure (Arnette et al., 2020). Melanocytes were transduced with wild type BRAF or mutant BRAFV600E, then treated with CM from keratinocytes expressing shCTL or shDsg1 for 3 days. Total RNA was extracted from cells using the Quick-RNA miniprep kit (Zymo Research) following the manufacturer's protocol. Novogene Co., LTD conducted preparation of the mRNA library and transcriptome sequencing.