p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins
Introduction
Ovarian cancer, the deadliest gynaecological malignancy,1 is typically treated in its advanced stages with cytoreductive surgery and platinum-based chemotherapy. Cisplatin (cis-diamminedichloroplatinum) is widely used as a chemotherapeutic agent for ovarian cancers. However, cisplatin resistance limits its use in cancer patients;2, 3 some cancers develop cisplatin resistance over time, while others are intrinsically resistant. Several mechanisms of cisplatin that confer drug resistance have been proposed.4, 5 Recent studies have shown that cisplatin-induced apoptotic signalling can occur through endoplasmic reticulum (ER) stress, suggesting that the ER is a cytosolic target of cisplatin. This indicates that stress tolerance in the ER is possibly involved in the development of cisplatin resistance.6, 7 Physiological and pathological conditions, including anticancer therapies, viral infections, hypoxia and oxidative injury, may interfere with protein folding, leading to accumulation of misfolded proteins in the ER lumen, a condition called ‘ER stress’. Parts of soluble misfolded proteins are mainly degraded by ubiquitin-proteasomes,8, 9 while most misfolded proteins that include polymers and aggregates are degraded by autophagy-lysosomes.10, 11 Roles of production and degradation of misfolded proteins in cisplatin resistance are unclear.
The ubiquitin-binding protein, p62/SQSTM1 (sequestosome 1) is multifunctional; it promotes survival–critical signals, including proliferation, differentiation and induction of anti-apoptotic genes.12 The protein p62 has unique features: an N-terminal Phox, and a Bem1p domain (capable of self-oligomerisation), and a C-terminal ubiquitin-associated domain, which interacts with ubiquitinated proteins.13, 14, 15 These properties imply p62’s involvement in aggregate formation. In autophagy-impaired mice and flies, additional p62 loss is linked to reduced formation of ubiquitin inclusions.16, 17 p62 is selectively degraded by autophagy. It can act as a receptor or adaptor for ubiquitinated substrate autophagy,18, 19, 20 which is mediated through microtubule-associated protein 1 light chain 3 (LC3). The LC3 is drawn to phagophore/isolation membranes and remains coupled to completed autophagosomes.15 Bjorkoy et al. showed that p62 may affect ubiquitinated proteins’ linkage to autophages through LC3, thus maintaining cell homeostasis and survival.18
We found p62 to be associated with cisplatin resistance in human ovarian cancer cells (HOCCs), and that cisplatin-resistant SKOV3/DDP cells express much higher levels of p62 than do cisplatin-sensitive SKOV3 cells. The p62 binds and targets ubiquitinated proteins for degradation through autophagy, thus maintaining cell homeostasis and causing cisplatin resistance in HOCCs.
Section snippets
Cell lines
Cisplatin-sensitive HOCCs SKOV3 and their cisplatin-resistant clones SKOV3/DDP were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College. Both cells were cultured in Roswell Park Memorial Institute – 1640 culture (RPMI-1640, GIBCO, Carlsbad, CA), supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C, 5% CO2 with high humidity. According to the recommendation from the Chinese Academy of Medical Sciences and Peking Union Medical College,
Cisplatin inhibits growth and induces apoptosis in ovarian cancer cells
We treated cisplatin-sensitive SKOV3 cells and cisplatin-resistant SKOV3/DDP cells with increasing doses of cisplatin for 24 h and 48 h, and then examined growth inhibition using MTT assays. While cisplatin inhibited growth of both cell lines, SKOV3 cells were more sensitive to cisplatin than SKOV3/DDP cells (Fig. 1A).
Based on these MTT results and previous studies,21 we treated both cell lines with 6 μg/ml cisplatin, and examined apoptotic chromatin condensation with Hoechst 33258 staining by
Discussion
Cisplatin is widely used to treat ovarian cancer, as a monotherapy or in combination with other anticancer agents.28, 29 While about 70% of ovarian cancer patients respond to cisplatin initially, most relapse as resistance to cisplatin develops.2, 3 Better understanding of the mechanism of cisplatin resistance is, therefore, critical. Mechanisms of cisplatin resistance in ovarian cancer involve alteration of the gene and also some anti-apoptotic pathway.30, 31 Recently, the ER was reported to
Conflict of interest statement
None declared.
Acknowledgements
This work was funded by the National Natural Science Foundation of China (No. 30801354 and No. 30970791) and ‘211 project’ of Jilin University.
References (40)
- et al.
Autophagy in the pathogenesis of disease
Cell
(2008) - et al.
Signal integration and diversification through the p62 scaffold protein
Trends Biochem Sci
(2007) - et al.
Structural and functional link between the mitochondrial network and the endoplasmic reticulum
Int J Biochem Cell Biol
(2009) - et al.
Physiological significance of selective degradation of p62 by autophagy
FEBS Letters
(2010) - et al.
Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice
Cell
(2007) - et al.
P62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy
J Biol Chem
(2007) - et al.
Structural basis for sorting mechanism of p62 in selective autophagy
J Biol Chem
(2008) - et al.
Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling
J Biol Chem
(2003) The glucose-regulated proteins: stress induction and clinical applications
Trends Biochem Sci
(2001)- et al.
P62 serves as a shuttling factor for TrkA interaction with the proteasome
Biochem Biophys Res Commun
(2008)
Phase I trial of sirolimus combined with radiation and cisplatin in non-small cell lung cancer
J Thorac Oncol
Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells
Kidney Int
Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation
Blood
Global cancer statistics, 2002
CA Cancer J Clin
Ovarian cancer: strategies for overcoming resistance to chemotherapy
Nat Rev Cancer
Recurrent ovarian cancer
Clin Adv Hematol Oncol
BCL-2 family protein expression and platinum drug resistance in ovarian carcinoma
Br J Cancer
Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function
Int J Cancer
Glucose-regulated protein 78 antagonizes cisplatin and adriamycin in human melanoma cells
Carcinogenesis
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These authors contributed equally to this study.