MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v01/MSV000082208/primaryOK2000000ProteomicsJennifer Zhang, Ph.D.Q ExactiveHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=997fff3b0cfc4979ae57db54343a31b1jennifer.zhang@duke.eduDuke UniversityMassIVE17UNIMOD:21 - "Phosphorylation."UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.<b>Significance:</b> These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. <i>Cancer Res; 78(22); 6462-72. ©2018 AACR</i>.UBE2N Promotes Melanoma Growth via MEK/FRA1/SOX10 Signaling.Dikshit Anushka A, Jin Yingai J YJ, Degan Simone S, Hwang Jihwan J, Foster Matthew W MW, Li Chuan-Yuan CY, Zhang Jennifer Y JYKinase Kinases, HEL-S-71, MEKs, MAPKK7, DSORT, WS2E, dsor1, MAP-ERK Kinase, MAPK-ERK Kinase, postnatal development, UbcH-ben, D-MEK/Dsor, MEK1/2, growth and development, PRKMK7, Malignant, D-mek, 1500026J17Rik, PCWH, melanoma (disease), Dom, DOM, melanoma, Dmek, Su(Raf)34B, UBC13, Kinase, JNKK2, CG15793, MAP, Map, WS4C, BB101821, MAPK ERK Kinase, MAPKKs, BLU, MAPK-ERK Kinases, MKK7, Naevocarcinoma, MEK 7, D-sor, DSor1, DSOR1, Map Kinase, Map Kinase Kinase, Kinase Kinase, SOR, dSor1, Sor, D-Mek, D-MEK, FRAGILE FIBER 1, single organism signaling., Sox21, SAPKK4, Mitogen-Activated Protein Kinase Kinase, sor/MEK1, MAP-ERK, D-sor-1, malignant, DSor, AL022654, Malignant Melanomas, sor, MCA23_16, DRODSOR1, MK, development, MEK/Dsor1, Melanomas, Melanoma, MAPK ERK Kinases, fra-1, MAPK, Malignant Melanoma, MAP Kinase Kinases, MEK, Mek, MAP Kinase, DMEK-1, Mitogen Activated Protein Kinase Kinase, dSor, D-SOR, D-Sor, EK1-1, DmelCG15793, MAPKK, MAP ERK Kinase, growth pattern, Mitogen Activated Protein Kinase Kinases, UbcH13, Kinases, non-developmental growth, postnatal growth, mek, SAPKK-4, MCA23.16, WS4, dMEK, signalling process, MAPK-ERK, AW538199, Dsor, malignant melanoma, FRA, UBCHBEN - UBC13, growth, MAPK Kinase, FRA1, Fra1, MAPK Kinasesliquid chromatography tandem mass spectroscopy, LC-MS2, LCMSMS, A375 cell, grupo, ensemble, number, LC-MS/MS, Rest, presence, free, Cell, LC-MS-MS, set, count in organism, LC/MS/MS, Phosphopeptide, grupos, label, liquid chromatography-tandem mass spectroscopy, A-375, LC-MSMS, A375, liquid chromatography tandem mass spectrometry, group., groupe, Gruppemelanoma (disease), malignant melanoma, Cell., Melanomas, Melanoma, Malignant Melanomas, melanoma, Malignant, malignant, Malignant Melanoma, NaevocarcinomaKinase Kinases, N Cadherin, P Cadherins, DmErk, extracellular signal-regulated kinase activity, dp53, pp44mapk, DSORT, Materials, p19<ARF>, Tyro5, croc-1, Liver Cell Adhesion Molecules, EDPF1, MEK1/2, growth and development, 0610011J09Rik, E-Cadherin, TRANSPL HETEROL, Tumor, 1500026J17Rik, PCWH, Carboxypeptidase G2, Carboxypeptidase G1, LeMPK3, p44mpk, bbl, symptoms, Dp38, 1110021H13Rik, EDAF-1, ARF-INK4a, Kinase, Analysis, DmelCG9881, SAPK2, p16(INK4a), SEM, Sem, MAP-k, treatment, BB101821, P16-ARC, molecules, Pen16, Analyses, MAPK-ERK Kinases, pp42, BCC7, DCAD2, DCad2, EK5, beta-Tub6D, dmp53, Dsor2, T, 1422/04, metabolic process resulting in cell growth, Molekuel, DSor1, DSOR1, sem, DECad, CG3722, Map Kinase, Immune Disease, DE Cad, Map Kinase Kinase, p16K, e, Xenotransplantations, Dp53, cellular growth, xenotransplant, CG17117, DEcad, p50/tubulin, Sox21, single organism signaling, Tumors, ERK-A, DE, Epithelial cadherin, screening, CG10873, dpERK, dpErk, metabolism resulting in cell growth, ERK-2, Shg, DmMAPK, Folate Conjugase, Malignant Melanomas, PMK-2, dp-ERK, PMK-1, CEP52, Spectrum Analysis, E Cadherin, signal transduction by trans-phosphorylation, Sytip, PMK-3, Folyl Poly-gamma-Glutamate Carboxypeptidase, Benign, ECadh, fra-1, Cadherin, MAP Kinase Kinases, CG17251, pMAPK, pMapK, Cellular, DMEK-1, bfy, UEV1A, Cadherins, C81524, Ubiq, HTH, Hth, EK1-1, MSH-R, Neural Cadherins, DmERKA, Xenotransplantation, D E-cad, rl/MAPK, cir1, Epithelial Cadherin, E-cadherin, N Cadherins, Spectrometry, CG3401, signs, Benign Neoplasms, beta3Tub, beta3TUB, l(2)41Ac, INSDC_feature:gene, Immunologic, WS4, gp150, N-Cadherins, Malignant Neoplasms, C78273, GH, DTB3, Epithelial Cadherins, CT34260, dpERk, signaling pathway, MAPK-ERK, AW538199, Material, Proliferation, ARC-1, SAPK, Immune System Disorder, bhy, FRA, 9230106F14Rik, other neoplasm, CT12481, INK4A, CG31325, NSC697923, 5730524P06Rik, DCad, ECad2, CDKN2, MAPK Kinases, 9030612K14Rik, CDHE, beta[[3]]-Tub, MEKs, DmelCG17117, ABCB5alpha, DE Cadh, UEV2, UEV1, INK4, Neoplasms, MAPK-ERK Kinase, UbcH-ben, 12559, ATP Dependent Proteolysis Factor 1, 143391_i_at, beta3 TU, Epithelial-Cadherins, CIR1, LFS1, l(3)05745, EK2-1, Human, l(2)k03401, melanoma (disease), Dom, DOM, Tob, AA407128, Prkm1, Number Growth, anon-EST:Liang-2.13, uev1, MAP, Map, 2018., ERK, Erk, CadE, PRKM1, Ecad, Folylpolyglutamate Hydrolase, P-Cadherin, PRKM2, BLU, cadh, Naevocarcinoma, MAPK signaling, PCBC, MEK 7, erk, Neoplasias, ECAD, ECad, ube2v, Cyp2b, 40S ribosomal protein S27a, Mshra, P-Cadherins, D-cad, Kinase Kinase, ARPC5, Immune Disorders, p19ARF, rll, beta[[3]]-tubulin, STK26, SOR, Sor, p38-2, protein tagging activity, myelin basic protein kinase activity, CADH, Heterologous, Cancer, AI256840, inhibitors, TP16, Dmbeta3, DRT, findings, Malignant Neoplasm, 6820402M05, MAP-ERK, Immune Diseases, prac, stress-activated kinase activity, beta3t, MAP-2 kinase activity, AL022654, sor, Folyl Conjugate Synthetase, CROC-1, p19-lt-ARF-gt-, Cell, Cadh, p16INK4a, MK, p16-ARC, E-Cadherins, Ubiquitin A-52 residue ribosomal protein fusion product 1, N-cad, MS, MT, XENOTRANSPL, Epithelial-Cadherin, MAPK ERK Kinases, Ubiquitin-related 2, P14ARF, Malignant Melanoma, stress-activated protein kinase activity, p53/tubulin, chemical analysis, mapk2, mapk1, Neoplasm, MEK, Mek, MAP Kinase, signalling cascade, Placental Cadherins, Mass Spectrum Analyses, P16INK4A, covalent modifier, CG33336, primary cancer, CG12559, Mitogen Activated Protein Kinase Kinases, underdeveloped, P42MAPK, UVO, dpERK1, postnatal growth, D-p53, mek, Dm-P53, Cancers, beta3, CROC1, AI848315, malignant tumor, E-cad, dtl, INK4a-ARF, Tub, AU018647, signalling process, 60S ribosomal protein L40, dpMAPK, betatub60D, Ubiquitin-related, Neoplasia, SAP kinase activity, FRA1, Fra1, E-CAD, E-Cad, l(2)k10220, MTS1, WS2E, dsor1, MAP-ERK Kinase, determination, gamma Glutamyl Hydrolase, Heterograft, ERK/MAPK cascade, Liver Cell Adhesion Molecule, ABCB5beta, Mpk2, postnatal development, Immunologic Diseases, D-MEK/Dsor, p42mapk, uev1a, Pctr1, neutral molecular compounds, PRKMK7, betaTub3, Cell Growth in Number, Tp53, SR2-1, DMP53, melanoma, Cell Number Growth, 1300007E16Rik, Su(Raf)34B, Arf, mitogen-activated protein kinase activity, Dmp53, ARF, mpk1, molecule, molecula, Mass Spectrum Analysis, signal transduction by protein phosphorylation, WS4C, 1323/07, Growth, Erk/Map kinase, High Mobility Protein 20, Diseases of Immune System, non-developmental growth of a unicellular organism, DERK-A, hypoplasia, Ubiquitin-related 1, MTS-1, DmP53, E(sina)7, Rl, APF-1, B3t, MP kinase activity, DmelCG3401, DERK, Immune, DE-CAD2, dEcad, malignant neoplasm, MAPKKK cascade during sporulation, Pteroyl Polyglutamate Hydrolase, disease management, MBP kinase II activity, Therapies, P14, Malignancies, P16, dERK, DECadh, Neural Cadherin, dSor1, P19, CD324, P16-INK4A, Therapy, MAP kinase 2 activity, SAPKK4, sor/MEK1, D-sor-1, Mapk, MLM, DmelCG34449, DSor, Erk1, signal transduction by conformational transition, ERK1, Immunological Disease, p16, ERK2, mapk1a, Erk2, DE-Cad2, MCA23_16, EDPF-1, CG12136, DRODSOR1, DE-cadh, Spectroscopy, 3t, Conjugase, signaling cascade, Melanoma, pen16, p41mapk, DE-Cadherin, mapk1b, MapK, MAPK, non-developmental cell growth, DE-cadherin, anon-Pen16, Genetic Materials, Cyp2b20, Immune System Disorders, regulator, Mitogen Activated Protein Kinase Kinase, erk2, Heterologous Transplantations, D7Bwg1382e, dSor, mapk, Genetic Material, Tub60D, Immune System Disease, Ubiquitin, SHEP2, Xenograft, shg/DE-Cad, ERKa, beta-tub, MAP ERK Kinase, BcDNA:RE08694, growth pattern, Transplantation, Cadherin-2, l(2R)EMS45-39, non-developmental growth, Cadherin-1, Folate Hydrolyzing Enzyme, Cadherin-3, CAPB, DmelCG33336, Human Ubiquitin, DmelCG12559, p38, Xenografts, MCA23.16, Treatments, Folacin Conjugase, DpErk, DpERK, dMEK, ErkA, ERKA, hth1, hth2, DmelCG3722, CG9881, p41, MAPK signalling, Dmel_CG32705, p40, P53, Cell Number, p44, Dsor, Cistron, UBCHBEN - UBC13, com5, DDVit-1, MAPK Kinase, pERK, betaTub, Uvomorulin, PSP2, HEL-S-71, Immunologic Disease, MAPKK7, GroupII, p50, p53, Benign Neoplasm, Immune System, Gene, Ubiquitin carboxyl extension protein 80, Spectrum Analyses, Transplantations, Malignant, 3.4.19.9, D-mek, heterologous transplantation, l(2)10469, UEV-1, UEV-2, Hek5, reduced, Dmek, EST422562, Mass, HMG-20, UBC13, p42-MAPK, tiny, JNKK2, CG15793, Mass Spectroscopy, DDVIT1, Neural, Nestin, MAPK ERK Kinase, Genetic, clone 2.13, MAPKKs, Malignancy, growth of cell, D.m.BETA-60D, MKK7, xp42, Cadherin 1, Cadherin 3, Cadherin 2, inhibiteur, Heterografts, Immunological Diseases, D-sor, p42 mitogen-activated protein kinase activity, CG18732, ATP:protein phosphotransferase (MAPKK-activated) activity, cell proliferation, signalling pathway, Trp53, Ink4a|Arf, inhibidor, E-Cad|CTF3, Disorder, CMM5, ubiquitin, E-Cad|CTF2, Carboxypeptidase G, E-Cad|CTF1, betaTub60C, UBE2V, CMM2, signal transduction by cis-phosphorylation, DmERK-A, beta3-tubulin, TRP53, D-Mek, D-MEK, FRAGILE FIBER 1, DE[cyto], small, beta-Tub60D, Multiplication, Disease, ert1, Cellular Proliferation, Dm-HTH, Placental, P16INK4, Mitogen-Activated Protein Kinase Kinase, malignant, BETA 60D, DE-cad, prkm2, beta3-Tub, prkm1, CT39192, N-Cadherin, inhibitor, CG32705, Cistrons, Xp53, development, MEK/Dsor1, p38delta, cell expansion, Melanomas, croc1, Cad, Protein, MBP kinase I activity, DE-CAD, DE-Cad, Ink4a/Arf, ATP-Dependent Proteolysis Factor 1, beta[[3]] tubulin, Gamma-Glu-X carboxypeptidase, D-SOR, D-Sor, Xp42, DmelCG15793, Mass Spectrum, cad, MAPKK, Folyl Polyglutamate Cleavage Enzyme, UbcH13, Kinases, mitogen activated kinase activity, Nestin Protein, Su(Raf)2B, CDK4I, beta60C, SAPKK-4, l(3)86Ca, HETEROL TRANSPL, EY2-2, MAPK2, 4632410D19Rik, Immunological, P Cadherin, DEC, Meis1, CAM 120|80, D-ERK, Therapeutic, ERT1, P19ARF, EPHT3, Immune Disorder, Cell Multiplication, CG15372, malignant melanoma, Treatment, assay, growth, MMS2, e-cad, E Cadherins, MAP kinase 1 activity0falsePhosphoproteome of shUBE2N in melanoma cellsQuantitative label-free LC-MS/MS was performed on TiO2-enriched phosphopeptides from A375 cells expressing shUBE2N versus shcontrol (n=3 per group).Sun Mar 25 15:18:00 BST 2018MSV00008220830224375