1. RAD6B Loss Disrupts Expression of Melanoma Phenotype in Part by Inhibiting WNT/β-Catenin Signaling
- Author
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Ambikai Gajan, Katherine Gurdziel, Malathy P.V. Shekhar, Pratima Nangia-Makker, Seongho Kim, Guangzhao Mao, and Ashapurna Sarma
- Subjects
0301 basic medicine ,Mice, Nude ,Melanocyte ,Biology ,Cell Line ,Pathology and Forensic Medicine ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Melanoma ,Wnt Signaling Pathway ,Transcription factor ,Cell growth ,Wnt signaling pathway ,Regular Article ,medicine.disease ,Microphthalmia-associated transcription factor ,Protein ubiquitination ,Cell biology ,Gene Expression Regulation, Neoplastic ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Ubiquitin-Conjugating Enzymes ,Heterografts ,Stem cell - Abstract
Canonical Wnt signaling is critical for melanocyte lineage commitment and melanoma development. RAD6B, a ubiquitin-conjugating enzyme critical for translesion DNA synthesis, potentiates β-catenin stability/activity by inducing proteasome-insensitive polyubiquitination. RAD6B expression is induced by β-catenin, triggering a positive feedback loop between the two proteins. RAD6B function in melanoma development/progression was investigated by targeting RAD6B using CrispR/Cas9 or an RAD6-selective small-molecule inhibitor #9 (SMI#9). SMI#9 treatment inhibited melanoma cell proliferation but not normal melanocytes. RAD6B knockout or inhibition in metastatic melanoma cells downregulated β-catenin, β-catenin–regulated microphthalmia-associated transcription factor (MITF), sex-determining region Y-box 10, vimentin proteins, and MITF-regulated melan A. RAD6B knockout or inhibition decreased migration/invasion, tumor growth, and lung metastasis. RNA-sequencing and stem cell pathway real-time RT-PCR analysis revealed profound reductions in WNT1 expressions in RAD6B knockout M14 cells compared with control. Expression levels of β-catenin–regulated genes VIM, MITF-M, melan A, and TYRP1 (a tyrosinase family member critical for melanin biosynthesis) were reduced in RAD6B knockout cells. Pathway analysis identified gene networks regulating stem cell pluripotency, Wnt signaling, melanocyte development, pigmentation signaling, and protein ubiquitination, besides DNA damage response signaling, as being impacted by RAD6B gene disruption. These data reveal an important and early role for RAD6B in melanoma development besides its bonafide translesion DNA synthesis function, and suggest that targeting RAD6B may provide a novel strategy to treat melanomas with dysregulated canonical Wnt signaling.
- Published
- 2021