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.