Published OnlineFirst May 20, 2016; DOI: 10.1158/1535-7163.MCT-16-0032 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Cyclin-Dependent Kinase 11 (CDK11) Is Required for Ovarian Cancer Cell Growth In Vitro and In Vivo, and Its Inhibition Causes Apoptosis and Sensitizes Cells to Paclitaxel Xianzhe Liu1,2, Yan Gao1, Jacson Shen1, Wen Yang1,3, Edwin Choy1, Henry Mankin1, Francis J. Hornicek1, and Zhenfeng Duan1 Abstract Ovarian cancer is currently the most lethal gynecologic compared with the matched, original primary tumors. RNAi- malignancy with limited treatment options. Improved tar- mediated CDK11 silencing by synthetic siRNA or lentiviral geted therapies are needed to combat ovarian cancer. Here, shRNA decreased cell proliferation and induced apoptosis in we report the identification of cyclin-dependent kinase 11 ovarian cancer cells. Moreover, CDK11 knockdown enhances (CDK11) as a mediator of tumor cell growth and proliferation the cytotoxic effect of paclitaxel to inhibit cell growth in in ovarian cancer cells. Although CDK11 has not been impli- ovarian cancer cells. Systemic in vivo administration of CDK11 cated previously in this disease, we have found that its expres- siRNA reduced the tumor growth in an ovarian cancer xeno- sion is upregulated in human ovarian cancer tissues and graft model. Our findings suggest that CDK11 may be a associated with malignant progression. Metastatic and recur- promising therapeutic target for the treatment of ovarian rent tumors have significantly higher CDK11 expression when cancer patients. Mol Cancer Ther; 15(7); 1691–701. Ó2016 AACR. Introduction stagnant at approximately 45% (6). Therefore, there is an urgent need to develop novel targeted therapies to improve the treatment Ovarian cancer is currently the most lethal gynecologic malig- of ovarian cancer. nancy and the leading cause of death from gynecologic cancers in Cyclin-dependent kinases (CDK) are members of the serine/ the United States, which is predominantly a disease of elderly, threonine protein kinase family and play crucial roles in tumor postmenopausal women over 50 years of age (1). There were more cell proliferation and growth by controlling cell-cycle, tran- than 239,000 newly diagnosed cases of ovarian cancer worldwide scription, and RNA splicing (7). Recently, studies have shown in 2012, including about 20,800 cases in the United States and that overexpression and activation of CDKs are common fea- 65,600 in Europe (2, 3). The current standard treatment for tures for most human cancers, and targeting CDKs in tumor ovarian cancer patients consists of radical surgery with paclitaxel cells has become a promising therapeutic strategy (7–10). More and carboplatin-based chemotherapy (4). Although the surgical recently, the FDA has approved the CDK4/6 inhibitor palbo- and chemotherapeutic strategies lead to small improvements in ciclib for treating metastatic breast cancer (11–13). Palbociclib clinical outcome, most ovarian cancer survivors are eventually has also shown promising preclinical activity in glioblastoma, diagnosed with recurrent disease that will develop resistance to renal, and ovarian cancer models that may provide direction for several types of chemotherapeutic reagents (5, 6). Ultimately, the their future clinical development. Other CDK inhibitors, progression of residual disease leads to significant morbidity and including abemaciclib and ribociclib, have demonstrated very finally mortality with current 5-year overall survival rates being promising clinical activity in breast cancer, melanoma, lipo- sarcoma, and mantel cell lymphoma (8, 14). Among other members of the CDK family, more recent studies have shown 1Sarcoma Biology Laboratory, Center for Sarcoma and Connective that CDK11 (formerly known as PITSLREA or CDC2L1) also Tissue Oncology, Massachusetts General Hospital and Harvard Med- plays critical roles in cancer cell growth and proliferation (15). 2 ical School, Boston, Massachusetts. Department of Orthopaedic CDK11 is usually overexpressed and/or overactivated in human Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 3Department of Anesthe- malignancies, and this overexpression is highly associated with siology, Union Hospital, Tongji Medical College, Huazhong University poor outcomes in cancer patients (13, 16, 17). Inhibition of of Science and Technology, Wuhan, China. CDK11 has been shown to lead to cancer cell death and Corresponding Author: Zhenfeng Duan, Massachusetts General Hospital apoptosis in difference tumor cells (18–21). Significant evi- and Harvard Medical School, 55 Fruit Street, Jackson Building Room 1115, Boston, dence suggests that CDK11 may be a novel and promising MA 02114. Phone: 617-724-3144; Fax: 617-726-3883; E-mail: therapeutic target for the treatment of cancers. [email protected] High expression levels and activities of different CDKs, as well doi: 10.1158/1535-7163.MCT-16-0032 as aberrant expression of the CDK inhibitors (such as p16, p21, Ó2016 American Association for Cancer Research. and p27) have been frequently characterized among women with www.aacrjournals.org 1691 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 20, 2016; DOI: 10.1158/1535-7163.MCT-16-0032 Liu et al. ovarian cancer (8, 22–25). Although previous studies have found lines were cultured in RPMI1640 from Invitrogen supplemented that knockdown of CDK11 decreased cell viability and increased with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin apoptosis in many type of cancers, the significance of CDK11 (Invitrogen). Paclitaxel was obtained as unused residual clinical expression in ovarian cancer is unknown. Therefore, in this study, material at the Massachusetts General Hospital. Cells were main- we first comparatively examined the expression of CDK11 in an tained in a humidified incubator in an atmosphere of 5% CO2 ovarian cancer tissue microarray (TMA) by immunohistochem- –95% air at 37C. Light microscopic images were documented ical analysis. Subsequently, we investigated the functional role of using Zeiss microscope from Carl Zeiss, Inc. with an attached CDK11 in the growth and proliferation of ovarian cancer in in vitro Nikon D40 digital camera from Nikon Corp. cultures and in mouse xenografts model of ovarian cancer in vivo. Our study indicates that CDK11 represents a novel promising Synthetic CDK11 siRNA and transfection drug target for further study in ovarian cancer. CDK11 knockdown in ovarian cells was performed by trans- fection of synthetic human CDK11 siRNA (ID: s2734), which was purchased from Ambion at Applied Biosystems. The siRNA Materials and Methods sequence targeting CDK11 corresponded to coding regions (50- Ovarian cancer TMA and IHC AGAUCUACAUCGUGAUGAAtt-30, antisense 50-UUCAUCAC- The TMA used in our study was generated from samples of GAUGUAGAUCUtg-30) of the CDK11 gene. Nonspecific siRNA ovarian cancer patients with long-term follow-up as reported oligonucleotides from Applied Biosystems were used as negative previously (26, 27). The unique features of this ovarian cancer control. The siRNA oligonucleotides were dissolved in nuclease- TMA are that tissues are collected from matched primary, meta- free water at a concentration of 100 mmol/L and kept at À20C static, and recurrent tumor tissues each from 26 individual ovar- until the following transfection experiment. Ovarian cancer ian cancer patients at the time of surgery. In total, the TMA slide SKOV-3 or OVCAR-8 cells were either plated on 96-well plates contained 78 epithelial ovarian cancer tissue samples. For IHC of for cell proliferation assays or plated on dishes for Western blot CDK11, the slide was baked at 62 C for 1 hour, deparaffinizedin protein isolation. Transfections were performed with Lipofecta- xylene for 15 minutes, washed with 100% ethanol for 12 minutes, mine RNAiMAX (Invitrogen) according to the manufacturer's and then rehydrated with 95%, 70%, and 50% ethanol, respec- instructions. Media were replaced with RPMI1640 supplemen- tively. Antigen retrieval was processed with Target Retrieval Solu- ted with 10% FBS, 100 U/mL penicillin, and 100 mg/mL strep- tion from Vector Laboratories following the manufacturer's tomycin 24 hours after transfection. Effects of CDK11 siRNA on instructions. After antigen retrieval, the slide was washed with cellular growth and proliferation were assessed in vitro using the PBS three times for 5 minutes and covered with peroxidase Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay blocking reagent for 5 minutes at room temperature. The slide (Sigma-Aldrich) as described previously (29, 30). Experiments was then incubated with serum-blocking reagent, avidin-blocking were performed in triplicate. All data were processed with the reagent, and then biotin-blocking reagent for 15 minutes. Primary use of GraphPad Prism 4 software from GraphPad Software, Inc. CDK11 antibody from Santa Cruz Biotechnology, Inc. (1:50 dilu- Total protein was isolated with RIPA Lysis Buffer (Upstate tion) was applied at 4 C overnight in a humidified chamber. The Biotechnology) 48 hours after siRNA transfection. next day, the biotinylated secondary antibody (1:200 dilution) diluted with 1% BSA was added to the slide and incubated for Immunofluorescence 1 hour. The slide was then incubated with HSS-HRP for 30 For the immunofluorescence assay, 4 Â 103 SKOV-3 or minutes. Bound antibody was detected and visualized with the OVCAR-8