p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins

https://doi.org/10.1016/j.ejca.2011.01.019Get rights and content

Abstract

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we show a critical role for the ubiquitin-binding protein p62/SQSTM1 in cisplatin resistance in human ovarian cancer cells (HOCCs). Specifically, we found that cisplatin-resistant SKOV3/DDP cells express much higher levels of p62 than do cisplatin-sensitive SKOV3 cells. The protein p62 binds ubiquitinated proteins for transport to autophagic degradation, reducing apoptosis induced by endoplasmic reticulum (ER) stress in SKOV3/DDP cells. Knockdown of p62 or inhibition of autophagy using 3-methyladenine resensitises SKOV3/DDP cells to cisplatin. Collectively, our data indicate that p62 acts as a receptor or adaptor for autophagic degradation of ubiquitinated proteins, and plays an important role in preventing ER stress-induced apoptosis, leading to cisplatin resistance in HOCCs.

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)

  • J.N. Sarkaria et al.

    Phase I trial of sirolimus combined with radiation and cisplatin in non-small cell lung cancer

    J Thorac Oncol

    (2007)
  • S. Periyasamy-Thandavan et al.

    Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells

    Kidney Int

    (2008)
  • Y. Hiruma et al.

    Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

    Blood

    (2009)
  • D.M. Parin et al.

    Global cancer statistics, 2002

    CA Cancer J Clin

    (2005)
  • R. Agarwal et al.

    Ovarian cancer: strategies for overcoming resistance to chemotherapy

    Nat Rev Cancer

    (2003)
  • M.K. Tummala et al.

    Recurrent ovarian cancer

    Clin Adv Hematol Oncol

    (2005)
  • P.J. Beale et al.

    BCL-2 family protein expression and platinum drug resistance in ovarian carcinoma

    Br J Cancer

    (2000)
  • M. Fraser et al.

    Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

    Int J Cancer

    (2008)
  • Lin Y, Wang Z, Liu L, Chen L. Akt is the downstream target of GRP78 in mediating cisplatin resistance in ER...
  • C.C. Jiang et al.

    Glucose-regulated protein 78 antagonizes cisplatin and adriamycin in human melanoma cells

    Carcinogenesis

    (2009)
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