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Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer

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Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer Published OnlineFirst January 26, 2010; DOI: 10.1158/0008-5472.CAN-09-3215 Published Online First on January 26, 2010 as 10.1158/0008-5472.CAN-09-3215 Therapeutics, Targets, and Chemical Biology Cancer Research Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer Xuedong Yan1, Jie Yin1, Huiyu Yao2, Ning Mao2, Yili Yang3, and Lingya Pan1 Abstract Resistance to platinum drugs has emerged as a major obstacle in the treatment of ovarian cancers. Through proteomic analysis, we have found that the expression of annexin A3, a member of the Ca2+ and phospholipid- binding annexin family, is significantly increased in platinum-resistant ovarian cell lines. Anti–annexin A3 immunostaining indicated that cancers from platinum-resistant patients also possess higher levels of annex- in A3 than those from platinum-sensitive patients. Although expression of annexin A3 made susceptible ovarian cancer cells more resistant to platinum, expression of antisense annexin A3 downregulated its ex- pression and rendered the resistant cells more sensitive to platinum. In athymic mice, the growth of tumors from inoculated SKOV3 cells was inhibited by the administration of platinum, whereas tumors from annexin A3–expressing SKOV3/Ann were resistant to platinum treatment. Interestingly, the intracellular platinum concentration and platinum-DNA binding are significantly lower in annexin A3–overexpressing cells than those in parental cells. The lower cisplatin concentration was also accompanied by reduced induction of p53, which could be restored by downregulation of annexin A3. These results indicate that increased ex- pression of annexin A3 is a mechanism of platinum resistance in ovarian cancer. It seems to act by pre- venting uptake or accumulation of platinum in cells. Therefore, it is conceivable that annexin A3 could be a target for therapeutic intervention and may also serve as a biomarker for drug resistance in ovarian cancer patients. Cancer Res; 70(4); OF1–9. ©2010 AACR. Introduction decreased drug accumulation, enhanced detoxification capa- bility, aberrant apoptosis pathway, and increased repair of Ovarian cancer is the most common cause of cancer death drug-induced DNA damage (1–8). It has been reported that from gynecologic tumors in the world. Cytoreductive surgery metallothioneins (9), glutathione S-transferase π (10), p53 followed by a platinum-based chemotherapy is the main treat- (11), ERCC1 (12), copper transporters (13), and XIAP (14) ment strategy at present. However, initial responses to the can be responsible for these changes in cells. However, it is still drugs are often followed by the eventual evolution of fatal drug not fully understood how these genes render cells resistant to resistance. There are also no biomarkers that have achieved platinum, and there are conflicting data regarding their roles the necessary specificity to predict the development of plati- in platinum resistance in patients (15–18). num resistance in ovarian cancer patients. This has prompted Annexins belong to the protein family that includes five extensive studies to investigate the cellular and molecular me- groups (A–E); among which, those in group A are the human chanisms of platinum resistance in various cancer cells. Devel- and vertebrate orthologues and are identified numerically opment of platinum resistance has been attributed to (19). As Ca2+-dependent phospholipid-binding proteins, an- nexins often function as organizers of membrane domains and membrane-recruitment platforms for proteins in re- sponse to Ca2+ flux in cells (20). Therefore, they have diver- Authors' Affiliations: 1Department of Obstetrics and Gynecology, Peking sified activities that include mediating the anti-inflammation Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 2Department of Cell Biology, Institute action of glucocorticoids, participating in the regulation of of Basic Medical Sciences, Beijing, People's Republic of China; and blood coagulation, modulating ion channels, and participat- 3Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, Maryland ing in membrane fusion and exocytosis (19). The ability of annexin A5 to bind phosphatidylserine also makes it a valu- Note: Present address for X. Yan: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University School able tool to detect apoptosis (21). of Oncology, Beijing Cancer Hospital and Institute, 52 Fu-cheng Road, We have found that annexin A3 is one of the proteins up- Beijing 100142, China. regulated in platinum-resistant ovarian cancer cell lines (22). Corresponding Author: Lingya Pan, Department of Obstetrics and Gy- In present study, the role of annexin A3 in platinum resis- necology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Shuai Fu Yuan, tance is investigated through examining primary tumors Wang Fu Jing Street, Beijing 100730, China. Phone: 86-10-65296218; and their gene overexpression and downregulation in culture Fax: 86-10-65124875; E-mail: [email protected]. and in athymic mice. Our data indicate that increased ex- doi: 10.1158/0008-5472.CAN-09-3215 pression of annexin A3 is a specific mechanism for platinum ©2010 American Association for Cancer Research. resistance in ovarian cancers. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 26, 2010; DOI: 10.1158/0008-5472.CAN-09-3215 Yan et al. Materials and Methods Immunohistochemistry. Immunostaining of tissue sec- tions was performed using Dako LASB system (DakoCytoma- Cell lines and culture conditions. Human epithelial tion) according to the manufacturer's instruction. The ovarian cancer cell lines SKOV3, SKOV3/Cis, SKOV3/Car, sections of normal hepatic tissue were used as positive con- A2780, A2780/Cis, and A2780/Car were described previously trols for annexin A3. As a negative control, the sections were (16, 23). They were maintained in DMEM supplemented incubated with rabbit IgG at the same dilution. The tissue with 10% FCS, 100 units/mL penicillin, and 150 μg/mL sections were evaluated under a light microscope (400×) streptomycin. and scored as follows: 1, no detectable immunostaining; 2, Western blot analysis. The level of annexin A3 and p53 immunostaining <25%; 3, immunostaining between 25% were analyzed using standard Western blot procedures de- and 50%; 4, immunostaining between 50% and 75%; 5, immu- scribed previously (23) with the rabbit anti-human annexin nostaining >75%. The immunostaining intensity was evaluat- A3 primary antibody (24) and mouse anti-human p53 anti- ed by two independent observers who were blinded to body (Santa Cruz Biotechnology), respectively. clinical and annexin A3 data. Fluorescence microscopy. Thecellswerefixedwith4% Construction of sense and antisense annexin A3– paraformaldehyde for 20 min and made permeable with expressing plasmids and gene transfection. Annexin A3 0.2% Triton X-100. After blocking with 2% bovine serum al- cDNA (Genbank accession number gi: 4826642) was cloned bumin in PBS for 60 min, they were incubated at 4°C over- through reverse transcriptase-PCR from SKOV3 cells and night with anti–annexin A3 antibody. Following incubation inserted into the EcoRI site of pcDNA3.1(+) (Invitrogen). with goat anti-rabbit IgG antibodies conjugated with fluores- The sequence and orientation of the constructs were cein (Santa Cruz Biotechnology), cell nuclei were stained confirmed by sequencing. Lipofectamine 2000 (Invitrogen) with propidium iodide for 20 min. The specimens were ex- was used to transfect the cells according to manufacturer's amined under a Radiance 2100 confocal laser-scanning instruction. Stable transfectants were obtained by selecting microscope (400×; Bio-Rad). with medium containing 800 μg/mL G418. Patients, tissue specimens, and pathologic data. Tumor Drug sensitivity assay. Single-cell suspensions were pre- tissues were collected from 42 patients with epithelial ovar- pared and dispersed in 96-well plates. After incubation for ian cancer from 2000 to 2005 at the Department of Obstetrics 72 h with the platinum compounds, the MTT (Sigma) solu- and Gynecology, Peking Union Medical College Hospital, tion (0.5 mg/mL) was added. Following incubation for 4 h, Beijing, China. The patients gave informed consent according 100 μL of extraction buffer were added to each well. After to the criteria used at Peking Union Medical College Hospi- an overnight incubation, absorbance at 540 nm was mea- tal. Relevant clinical information was gathered, and tissue sured using an Immunoskan 340 (Labsystems). samples and clinical data were anonymized. The tissue speci- Intracellular content of cisplatin. Cells were seeded in mens were taken intraoperatively, immediately frozen in triplicate at a density of 2 × 106/mL in complete medium liquid nitrogen, and stored at −80°C. and incubated with 10 μmol/Lofcisplatinfor24h.After The 42 patients were consecutively selected as follows: (a) washingthreetimeswithPBS,cellswerescrapedfreein patients who suffered from primary epithelial ovarian cancer; 1 mL of PBS. Following centrifugation at 4°C, the pellets (b) patients who obtained the primary treatment in our hos- were digested in 70% nitric acid at 65°C for 2 h and diluted pital; (c) patients who nearly harbored no residual tumors to 5% nitric acid by adding an appropriate
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