Abstract
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.
Highlights
From the ‡Turku Centre of Biotechnology, University of Turku and Åbo Akademi, Tykistokatu 6, Turku 20520, Finland; §Department of Pathology, Medicity Research Unit, University of Turku and Turku University Hospital, Tykistokatu 6, Turku 20520, Finland; ¶Department of Pharmaceutical Sciences, University of Maryland, 20 North Pine Street, Room N707, Maryland 21201; ʈDepartment 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
Hyper-phosphorylation of SQSTM1 is Increased in the Platinum Resistant Cell Lines—Our analysis revealed that both SQSTM1 mRNA and protein expression, and protein phosphorylation were significantly elevated in platinum resistant
Patient derived cisplatin-sensitive M019i and generated cisplatin-resistant M019iCis spheroidal high-grade serous ovarian carcinoma (HGSOC) cells were used to model in vivo disease, especially in metastatic spread in ascites
Summary
Overview of M019i HGSOC Cellular Proteome and Phosphorylated Proteome—From the whole proteome and TiO2 enriched peptide samples of sensitive M019i (IC50 ϭ 0.6 mol/L) and resistant M019iCis (IC50 ϭ 7.0mol/L) cell lines (Fig. 1A–1B), 2790 and 1361 quantifiable proteins were identified in both the absence and presence of cisplatin, respectively. Proteome of M019i HGSOC Cell Lines in Absence and Presence of Cisplatin—In the Absence of Cisplatin—A total of 312 differentially expressed proteins were identified between sensitive M019i and resistant M019iCis HGSOC cell lines in the absence of cisplatin in the whole proteome samples.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
