title: Inhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin.
abstract: Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Lahiru
authors.initials: L
authors.lastname: Gangoda
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Shivakumar
authors.initials: S
authors.lastname: Keerthikumar
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Pamali
authors.initials: P
authors.lastname: Fonseka
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Edgington
authors.affiliation: Bio21 Institute, University of Melbourne, Victoria, Australia.
authors.forename: Ching-Seng
authors.initials: CS
authors.lastname: Ang
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Cemil
authors.initials: C
authors.lastname: Ozcitti
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University, Stanford, California, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Belinda S
authors.initials: BS
authors.lastname: Parker
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: Suresh
authors.initials: S
authors.lastname: Mathivanan
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002465:Cell Movement; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D011480:Protease Inhibitors / Q000494:pharmacology; D040901:Proteomics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D014407:Tumor Cells, Cultured; D014945:Wound Healing
chemical_list: D000903:Antibiotics, Antineoplastic; D011480:Protease Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D004317:Doxorubicin; D002403:Cathepsins
keywords: N-Myc amplification; mass spectrometry; neuroblastoma; proteomics; secretome