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Mol Cell Proteomics. 2017 Jul;16(7):1377-1392. doi: 10.1074/mcp.M116.058321. Epub 2017 Apr 28.

Hyper-phosphorylation of Sequestosome-1 Distinguishes Resistance to Cisplatin in Patient Derived High Grade Serous Ovarian Cancer Cells.

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From the ‡Turku Centre of Biotechnology, University of Turku and Åbo Akademi, Tykistökatu 6, Turku 20520, Finland.
§Department of Pathology, Medicity Research Unit, University of Turku and Turku University Hospital, Tykistökatu 6, Turku 20520, Finland.
¶Department of Pharmaceutical Sciences, University of Maryland, 20 North Pine Street, Room N707, Maryland 21201.
‖Department of Pathology, University of Helsinki and HUSLAB, Helsinki University Hospital, Haartmaninkatu 3, 00290 Helsinki, Finland.
**Research Programs Unit, Genome-Scale Biology, Medicum and Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, POB 63, Helsinki, 00014 Finland.
‡‡Department of Obstetrics and Gynecology, University of Turku and Turku University Hospital, Kiinamyllynkatu 4-8, Turku 20521, Finland.
From the ‡Turku Centre of Biotechnology, University of Turku and Åbo Akademi, Tykistökatu 6, Turku 20520, Finland;


Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.

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