title: A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors.
abstract: Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.
authors.affiliation: a Department of Anatomy and Cell Biology ; University of Florida ; Gainesville , FL USA.
authors.forename: Debra
authors.initials: D
authors.lastname: Akin
authors.forename: S Keisin
authors.initials: SK
authors.lastname: Wang
authors.forename: Pouran
authors.initials: P
authors.lastname: Habibzadegah-Tari
authors.forename: Brian
authors.initials: B
authors.lastname: Law
authors.forename: David
authors.initials: D
authors.lastname: Ostrov
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yin
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: Kim
authors.forename: Nicole
authors.initials: N
authors.lastname: Horenstein
authors.forename: William A
authors.initials: WA
authors.lastname: Dunn
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D000818:Animals; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003198:Computer Simulation; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D012516:Osteosarcoma / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D000071183:Autophagy-Related Proteins; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D049452:Green Fluorescent Proteins; D008055:Lipids; D019869:Phosphatidylinositol 3-Kinases; D058570:TOR Serine-Threonine Kinases; C000601128:NSC185058; C546842:MTOR protein, human; C507524:ATG4B protein, human
keywords: 3MA, 3-methyladenine; ACTB, actin, beta; ATG, autophagy-related; ATG4B; ATG4B, autophagy-related 4B, cysteine protease; AV, autophagic vacuole; BECN1, beclin 1, autophagy related; CMPase, cytidine monophosphatase; DMEM, Dulbecco's modified Eagle medium; ECL, enhanced chemiluminescence; FYVE, zinc-finger domain named after 4 cysteine-rich proteins: FAB1, YOTB, VAC1, and EEA1; GABARAPL2, GABA(A) receptor-associated protein-like 2; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; IC50, half maximal inhibitory concentration; IP, intraperitoneal; LC3B; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3beta; MP, melting point; MTOR, mechanistic target of rapamycin; NCI, National Cancer Institute; NMR, nuclear magnetic resonance; PLA2, phospholipase A2; PVDF, polyvinylidene difluoride; PtdIns3K, phosphatidylinositol 3-kinase class III; PtdIns3P, phosphatidylinositol 3-phosphate; RFP, red fluorescent protein; RLU, relative luciferase units; RPS6, ribosomal protein S6; RPS6KB1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SEM, standard error of the mean; ULK1/2, unc-51-like autophagy activating kinase 1/2; and xenografts; antiautophagy compounds; dNGLUC, Gaussia luciferase; in silico docking; osteosarcoma