LAUSR

The E3 ubiquitin ligase Constitutive Photomorphogenesis (COP)-1 inhibits UVB-induced light activation in plants by interaction with UV Receptor (UVR)8 and effects on hypocotyl elongation (HY)5-induced growth. UVB is recognized as a causative factor in human melanomas, and this is the first identification of the role of COP1 in inhibition of melanoma growth and the correlation between effects in UVB-stimulated growth in plants and human melanomas. Following tail-vein injection of 200, 000 B16F10 cells in 14-week-old female C57BL/6J mice, intravenous treatment with rhARSB (2.0 mg/ml) or saline occurred on days 2 and 7 and mice were euthanized on day 14. Marked reduction in number and size of pulmonary melanomas was evident in the ARSB-treated mice. To determine the mechanisms underlying the inhibition of melanoma growth, assays were performed in the melanoma tissue, in the human A375 melanoma cell line, and in normal human melanocytes. Experiments included: activated caspase-3 by live cell imaging of A375 cells following ARSB and PBMC; ELISA assays of activated caspase-3/7, cytosolic cytochrome c, BCL2, nuclear ETS1, and phospho(Tyr)-ROR1; Western blots of phospho(Ser473)-AKT1; disaccharide analysis of A375 cells following ARSB silencing and exogenous ARSB; chromatin immunoprecipitation of nuclear FOXO3 and COP1 promoter; mRNA expression of COP1; siRNA inhibition of COP1, ARSB, Insulin Growth Factor 2 Receptor, carbohydrate sulfotransferase (CHST)15; treatment by ROR1 inhibitor cirmtuzumab; and treatment by other inhibitors of signaling pathways. Enhanced apoptosis was evident by increase in activated caspase-3. Live imaging of human A375 melanoma cells treated with rhARSB with and without PBMC showed increase in activated caspase 3/7. The apoptotic mechanism was determined to follow declines in nuclear ETS1 and in ETS1-mediated expression of BCL2, with increase in cytosolic cytochrome c and activated caspase-3. COP1 silencing blocked these effects. Expression of COP1 increased following rhARSB-initiated declines in phospho(Tyr)-ROR1 and phospho(Ser473)-AKT1, leading to increased nuclear FOXO3 and COP1 expression, demonstrated by chromatin immunoprecipitation assay of FOXO3 and COP1 promoter sequence. Effect of exogenous ARSB required insulin-like growth factor 2 receptor (IGF2R), a mannose-6-phosphate receptor. Exogenous ARSB reduced carbohydrate sulfotransferase (CHST)15 expression, shown by disaccharide analysis of chondroitin 4, 6-sulfate disaccharides. Decline in CHST15, by rhARSB or CHST15 siRNA reduced phospho(Tyr)-ROR1 and phospho(Ser473)-AKT1, leading to increased nuclear FOXO3 and COP1. This series of signaling and transcriptional events in A375 cells and normal human melanocytes provides new insight into how a conserved, inhibitory response to light-stimulated growth can impact on inhibition of human melanoma growth. Joanne Kramer Tobacman, Insug O-Sullivan, Jiyuan Yang, Fuming Zhang, Sumit Bhattacharyya. Constitutive photomorphogenesis (COP)-1 leads to apoptosis in B16F10 metastatic melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1395.