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CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer
Gynecologic Oncology xxx (xxxx) xxx
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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer Jihye Kim a, 1, Young-Jae Cho b, 1, Ji-Yoon Ryu b, Ilseon Hwang c, h, Hee Dong Han d, Hyung Jun Ahn e, Woo Young Kim f, Hanbyoul Cho g, Joon-Yong Chung c, Stephen M. Hewitt c, Jae-Hoon Kim g, Byoung-Gie Kim b, Duk-Soo Bae b, Chel Hun Choi b, **, Jeong-Won Lee b, * a
Department of Obstetrics and Gynecology, Dankook University Hospital, Cheonan, Republic of Korea Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea c Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA d Department of Immunology, School of Medicine, Konkuk University, Chungju, Republic of Korea e Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 136-791, South Korea f Department of Obstetrics and Gynecology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea g Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea h Department of Pathology, Keimyung University School of Medicine, Dongsan Medical Center, Daegu, Republic of Korea b
h i g h l i g h t s CDK7 CDK7 CDK7 CDK7
inhibition has an anti-cancer effect on platinum-sensitive EOC. inhibition could induce responsiveness to platinum chemotherapy in platinum-resistant EOC. overexpression had independent negative prognostic value for disease recurrence of EOC. might play a critical role in EOC tumorigenesis, and it also serves as a possible therapeutic target in EOC.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2019 Received in revised form 2 November 2019 Accepted 4 November 2019 Available online xxx
Objective: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). Methods: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. Results: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P ¼ 0.035 and P ¼ 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P ¼ 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis.
Keywords: Cyclin-dependent kinase 7 Epithelial ovarian cancer Prognosis THZ1 Therapeutic target
* Corresponding author.Department of Obstetrics and Gynecology, Samsung Seoul Hospital, Sungkyunkwan University College of Medicine, 81 Irwon-ro, Gangnamgu, Seoul, 06351, South Korea. ** Corresponding author. Department of Obstetrics and Gynecology, Samsung Seoul Hospital, Sungkyunkwan University College of Medicine, 81 Irwon-ro, Gangnamgu, Seoul, South Korea. E-mail addresses: [email protected] (C.H. Choi), garden.lee@samsung. com (J.-W. Lee). 1 The first two authors contributed equally to this paper. https://doi.org/10.1016/j.ygyno.2019.11.004 0090-8258/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Conclusion: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant. © 2019 Elsevier Inc. All rights reserved.
1. Introduction Epithelial ovarian cancer (EOC) is the most life-threatening gynecologic malignancy, with 238,700 cases newly diagnosed worldwide in 2012 [1,2]. Despite multimodal treatments, including aggressive cytoreductive surgery, platinum-based chemotherapy, and molecular targetebased therapies, about 75% of patients experience relapse, and the five year survival rate is only 39 to 45% [3,4]. EOC is a heterogeneous disease with varying histology, grade, and physiologic behavior. The heterogeneity of EOC is consistently observed at both the genetic and molecular levels. In addition, EOC is specifically characterized by a low prevalence of actionable genetic mutations [5,6]. Accordingly, despite novel molecular targetebased therapies such as bevacizumab and poly(ADP-ribose) polymerase inhibitors, the anticipated therapeutic effect is observed in patients with less than expected or only in patients with certain genetic mutations [7]. Therefore, therapeutic approaches involving tumor-specific genetic mutations may not be an attractive option for EOC. Meanwhile, discovering novel targets for EOC requires other molecular approaches, including intercellular or intracellular ubiquitous molecules which are generally involved in tumorigenesis [8]. In general, most cancer cells exhibit defects in cell cycle regulation and gene transcription, which promotes uncontrolled cell proliferation leading to tumorigenesis [9]. Cyclin-dependent kinases (CDKs) are members of the serine/threonine protein kinase family and are involved in cell cycle control and transcription regulation, which play central roles in tumorigenesis [10,11]. Among the CDKs, cyclin dependent kinase 7 (CDK7) is a component of transcription factor IIH (TFIIH), which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII), and also acts as a CDK activating kinase (CAK) which activates other CDKs involved in cell-cycle control [10,12]. In tumorigenesis, CDK7 encourages tumor growth by directly linking transcription regulation to cell cycle control. Based on these molecular traits, CDK7 has emerged as an attractive target for novel anti-neoplastic treatments in various cancers, including EOC [8,9]. THZ1, a covalent CDK7 inhibitor, was first described as an antineoplastic agent in T-cell acute lymphoblastic leukemia [13]. THZ1, a phenylaminopyrimidine bearing a potentially cysteinereactive acrylamide moiety, downregulates gene transcription by completely inhibiting phosphorylation of established intracellular CDK7 substrate RNAPII CTD Serine 5 and Serine 7 residues. In addition, it can also inhibit CDK12 kinase activity at slightly higher concentrations [13]. Interestingly, THZ1 is involved in transcription addiction by reducing the expression of several transcription factors of “super-enhancer”, thereby substantially reducing the associated target gene expression [14]. Its anti-neoplastic effect also has been demonstrated in a variety of tumors, including triple negative breast cancer [15], glioblastoma [16], pancreatic cancer [17], small cell lung carcinoma [18], colorectal cancer [19], and MYCdependent ovarian cancer [20] in preclinical studies. However, the clinical significance of CDK7 expression in EOC remains elusive. Here, we evaluated the clinical implications and prognostic significance of CDK7 expression in a relatively large EOC cohort and
validated our findings with publicly available datasets. We also investigated the anti-neoplastic effects of CDK7 inhibition by using short interfere RNAs (siRNAs) and THZ1 in preclinical studies and found that CDK7 is a potential target for EOC treatment. 2. Materials and methods 2.1. Patients and tissue specimens A total of 436 tissue samples (62 normal ovarian epithelial tissues, 69 benign tumors, 57 borderline tumors, 197 EOCs, and 51 metastatic tumors) were collected from patients who underwent surgery at Gangnam Severance Hospital (Seoul, South Korea) between 1996 and 2010, and some tissue samples were obtained from the Korea Gynecologic Cancer Bank (NRF-2017M3A9B8069610). The tissue samples and patients’ clinical information were obtained with the informed consent of all patients and the approval of the Institutional Review Board of Samsung Medical Center (approval no. 2015e07e122; Seoul, South Korea). All procedures were conducted in accordance with the guidelines of the Declaration of Helsinki. The patients’ clinical information was obtained from medical records, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, histology based on the World Health Organization grading system, the response to platinumbased chemotherapy, residual disease after debulking surgery (optimal: residual disease < 1 cm, suboptimal: residual disease 1 cm), and levels of cancer antigen 125 (CA-125). All patients were treated with maximal debulking surgery, followed by combination treatment with paclitaxel/carboplatin. Progression-free survival (PFS) was evaluated from the date of surgery to the period of recurrence/progression or the time of the last follow-up visit. Date of recurrence/progression was defined as disease progression according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1), based on either computed tomography (CT), positron emission tomography (PET)-CT, or CA-125 levels [21]. Overall survival (OS) was assessed from the date of surgery to patient death or the date of last contact for living patients. We also analyzed CDK7 gene expression profiles and clinical information about EOC from publicly available databases, including The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) and the Genomics of Drug Sensitivity in Cancer (https://www. cancerrxgene.org), to verify the clinical significance of CDK7 expression. 2.2. Immunohistochemistry and its quantitative evaluation Tissue cylinders of 1.0 mm in diameter were extracted from the most representative areas of donor blocks and transplanted into recipient blocks using a tissue arrayer (Beecher Instruments, Inc., Silver Spring, MD, USA). Depending on the block, 2e3 punches from each patient were included in the tissue microarray (TMA), and the final expression values were averaged. The TMA sections were cut to 5 mm thickness, deparaffinized with xylene, and dehydrated through a graded ethanol series. Antigen retrieval was performed in heat-activated antigen retrieval buffer (Dako, Carpinteria, CA, USA) at pH 6.0. The endogenous peroxidase activity was blocked
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
J. Kim et al. / Gynecologic Oncology xxx (xxxx) xxx
with 3% H2O2 for 10 min. The sections were incubated with antiCDK7 mouse monoclonal antibody (clone M01; Cell Signaling, Danvers, MA, USA) at a 1:1000 dilution for 60 min at room temperature. The antigeneantibody reaction was detected with an EnVision þ Dual Link System-HRP and visualized with DABþ (3, 30 diaminobenzidine; Dako). Negative controls were performed by omitting the primary antibody and rabbit immunoglobulin (IgG). The results of the immunohistochemistry (IHC) staining were evaluated by staining intensity (0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong) (Supplementary Fig. 1) and proportion (0 10%, 1 > 10% and 30%, 2 > 30%, and 60%, 3 > 60%). The final score was calculated by multiplying the intensity and positivity scores (overall score range, 0e9), and in the analyses for survival and clinicopathologic characteristics, the expression values of CDK7 were dichotomized into high (overall score 3) and low (overall score < 3) using the most statistically significant value as a cut-off. 2.3. Cell lines and treatments A2780-CP20 and HeyA8 (RRID: CVCL_8878) cells were a gift from Dr. Anil K. Sood, Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA, and RMG-1(RRID: CVCL_1662) cells were purchased from the Japanese Collection of Research Bioresources cell bank (Osaka, Japan). The A2780 (RRID: CVCL_0134) cells were obtained from the European Collection of Cell Cultures (Cat NO.93112520). All ovarian EOC cells were maintained in complete medium (RPMI 1640 for A2780, A2780-CP20, and HeyA8 and Ham’s F12 for RMG-1) supplemented with 10% fetal bovine serum in 5% CO2 at 37 C. A covalent CDK7 inhibitor, THZ1, was obtained from ApexBio (#A8882), and cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). All EOC cell lines in this research have been authenticated using STR profiling, and all experiments were performed with mycoplasma and virus free cells. 2.4. siRNA transfection siRNA of CDK7 and negative control siRNA were obtained from Bioneer (Daejeon, Korea). The siRNA targeting CDK7 sequences were as follows: 50 -GAGCAAUCAAAUCCAGCUU-30 (siRNA#1), 50 CUGAAACCAAACAACUUGU-30 (siRNA#2) and 50 - GAGAUUCAGACCUUGAUCA-30 (siRNA#3). A nonspecific, scrambled siRNA with a sequence of 50 -UUCUCCGAACGUGUCA CGU-30 was used as a negative control. Western blot analyses were performed to identify the inhibition effects of each CDK7-siRNA, including the negative control. All EOC cells were transfected with the CDK7-siRNA selected in the Western blot analysis using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.5. Cell proliferation assay Cells were seeded at 3 103 cells/well in a 96-well microplate in culture medium. After 24 h, cells were transfected with the siRNA for 48 h and then treated with THZ1 for 48 and 72 h 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays were performed as previously described [22]. Each sample was assayed in triplicate. 2.6. Apoptosis assay Cell apoptosis was measured 48 h after treatment with THZ1 by measuring the activated caspase-3 with ELISA (Invitrogen, #KH01091) according to the manufacturer’s protocol. Each sample was measured in triplicate.
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2.7. Wound-healing and migration assay A2780 and RMG-1 cells were used for the wound healing and migration assays. For the wound healing assay, cells were seeded in 24-well plates. Confluent monolayers of cells were “wounded” using the end of a sterilized tip. Photographs were taken 24, 32, and 48 h after treatment with THZ1 at 100X magnification, and the analysis was carried out using T-Scratch software (ETH Zurich). In addition, the migration assay was conducted using a Cytoselect 24well cell migration kit according to the manufacturer’s protocol (Cell Biolabs, CA, USA). It was prepared as cell suspension containing 1 x 106 cells/mL in serum free media. 40 nM THZ1 was added directly to the cell suspension. It was added 500 mL of media containing 10% fetal bovine serum to the lower well of the migration plate and added 300 mL of the cell suspension solution to the inside of each insert. Chamber were incubated for 24 h in a cell culture incubator. After removal of non-migratory cells, migratory cells were stained and quantified. 2.8. Cell cycle analysis A2780 cells were treated with various doses of THZ1 or dimethyl sulfoxide (DMSO, control) for 48 h. Then cells were trypsinized for dissociation into a single cell in potassium-buffered saline (PBS), pH 7.5. Cell suspensions were rinsed twice with ice-cold PBS and added dropwise to ice-cold 70% ethanol for 12 h at 4 C. After removing the ethanol, cells were treated with RNaseA (1 mg/mL) followed by the addition of propidium iodide (Sigma; 0.5 mg/mL) for 30 min. The prepared cells were immediately analyzed using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, USA). 2.9. Western blot analysis Cells were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined with a Bradford assay kit (BIO-RAD, Hercules, USA). Cell lysates (50 mg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with mouse monoclonal anti-CDK7 (1:1000, Santa Cruz, #sc-365075), rabbit monoclonal anti-CDK4 (1:1000, Cell signaling, #12790), mouse monoclonal anti-cyclin D1 (1:1000, Santa Cruz, #sc-450), rabbit monoclonal anti-cyclin H (1:1000, Abcam, #EPR3929), and mouse monoclonal anti-b-actin antibody (1:3000,Santa Cruz, #sc47778) and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences) according to the manufacturer’s protocol. 2.10. Animal care and development of in vivo models using established cell lines and patient-derived xenografts This study was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute (Seoul, Korea; protocol No. H-A9-003), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International and abides by the guidelines of the Institute of Laboratory Animal Resources. Female BALB/c nude mice were purchased from Orient Bio (Seongnam, Korea). The mice used in these experiments were 6- to 8-weeks old. To establish orthotopic models, A2780 (1 106 cells/0.2 mL HBSS) and HeyA8 (2.5 105 cells/0.2 mL HBSS) cells were injected into the peritoneal cavities of mice. For the patient-derived xenograft (PDX) model of
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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EOC, a patient tumor specimen retrieved from an operating room was cut into small pieces (less than 2e3 mm), implanted into the subrenal capsule of the left kidney, and propagated by serial transplantation. The tumor of PDX was derived from one patient with ovarian high-grade serous adenocarcinoma at FIGO stage IIIC (OV-71). She was treated with three lines of neo-adjuvant chemotherapy with a paclitaxel-carboplatin combination regimen prior to cytoreductive surgery, and followed by additional six cycles of adjuvant paclitaxel-carboplatin combination chemotherapy for the primary treatment. After completion of adjuvant chemotherapy, there were no residual tumors. To date, she has not experienced relapse for 54 months follow up. After 7 days of cell injections for the cell line models and 4 weeks for the PDX model, THZ1 (10 mg/ kg) or PBS was i.p. injected twice daily in the A2780 and PDX models. In the HeyA8 model, Poly(d,l-lactide-co-glycolide) (PLGA)CDK7-siRNA (7.5 mg) or PLGA-control siRNA was i.v. injected twice weekly [23]. Two groups of xenograft mice for each cell line (n ¼ 10 per group) and two groups for PDX (n ¼ 10 per group) were monitored daily for tumor development and side effects, and they were sacrificed on day 28e35 after the injection of cancer cells or when they appeared moribund. We recorded their body weight, tumor weight, and number of tumor nodules. Tumors were fixed in formalin and embedded in paraffin or snap-frozen in OCT compound (Sakura Finetek Japan, Tokyo, Japan) in liquid nitrogen. Using tumor tissues harvested from the in vivo experiments, IHC staining was done for Ki-67 (NOVUS, NB 600e1252), and TUNEL assays were performed using the ApopTag Peroxidase in situ Apoptosis kit as described previously [24]. 2.11. Data analysis Statistical analyses of the CDK7 expression data from the TMA and publicly available datasets were performed using R software, version 3.1.3 (R Foundation, Vienna, Austria; http://www.R-project. org). Student’s t-tests or ANOVA tests were performed to determine whether CDK7 expression was associated with clinicopathological characteristics in EOC. Kaplan-Meier survival curves were plotted to compare survival between groups. A Cox proportional hazards model was created to identify independent predictors of survival. For the rest of the experiments, statistical analyses were performed using two-tailed Student’s t-tests with GraphPad Prism 3.0. The data are presented as the mean ± SEM for the indicated number of separate experiments. P < 0.05 was considered to be statistically significant. 3. Results 3.1. High CDK 7 expression correlates with aggressive oncogenic behavior in EOC To evaluate the clinical implications of CDK7 expression in EOC, we performed IHC testing in 436 ovarian samples ranging from normal to metastatic and examined the association between CDK7 expression and various clinicopathological characteristics. Immunoreactivity was observed in the nucleus of malignant cells, as shown in the representative photomicrographs of CDK7 immunostaining in Fig. 1A. In detail, the CDK7 expression pattern of normal fallopian tube epithelium was similar to the normal ovarian epithelium whereas high CDK7 expression was observed in clear cell adenocarcinoma (Supplementary Fig. 2). Patient incidence of high CDK7 expression (CDK7High) and the CDK7 expression scores increased gradually from normal tissue to metastatic ovarian tumors (P < 0.001) (Supplementary Table 1), although there was a strikingly high expression score in borderline ovarian tumor tissue (P < 0.001) (Fig. 1B). In addition, we found no correlation between CDK7 expression and cell type, chemo-sensitivity, or preoperative
CA-125 levels, but CDK7High was significantly associated with advanced FIGO stage (III or IV) (P ¼ 0.035) and high-grade histology (P ¼ 0.011) (Supplementary Table 1). In particular, high-grade histology had significantly higher CDK7 expression than low-grade histology (P ¼ 0.035) (Fig. 1B). We also compared CDK7 expression with sensitivity to a CDK7 inhibitor (THZ2) in ovarian cancer using a variety of cancer cell lines in an in silico analysis with the Genomics of Drug Sensitivity in Cancer dataset (https://www.cancerrxgene.org). As shown in Supplementary Fig. 3, CDK7 expression and IC50 levels to THZ2 in ovarian cancer cell lines did not differ when compared with other cancer cell lines (Z-score (interquartile range) ¼ 0.5 (‒ 0.57e1.22), ‒ 1.28 (‒ 2.49e0.40)), and we found no association between CDK7 expression and IC50 levels to THZ2 in ovarian cancer cell lines (Supplementary Fig. 4). 3.2. CDK7 expression is an independent prognostic factor for disease recurrence in EOC We next examined the association between CDK7 expression and patient survival outcomes in 192 EOC patients with available survival data. Kaplan-Meier plots demonstrated that patients with CDK7High was significantly associated with disease recurrence (P ¼ 0.003), whereas no correlation was observed with OS (P ¼ 0.079) (Fig. 1C). As shown in Table 1, the univariate analysis for PFS revealed that CDK7High (P ¼ 0.003), older age at diagnosis (P ¼ 0.023), advanced FIGO stage, non-serous, and high-grade histology (all, P < 0.001) were all associated with poor PFS. Furthermore, the multivariate analysis also demonstrated that CDK7High (P ¼ 0.034), advanced FIGO stage (P < 0.001), and highgrade histology (P ¼ 0.011) independently predicted poor PFS. However, CDK7High had no prognostic value for OS in EOC patients. To validate these findings, we also performed an in silico analysis using the TCGA ovarian cancer dataset. KaplaneMeier survival analysis demonstrated that higher CDK7 mRNA expression correlated significantly with decreased PFS when the upper and lower 50% of CDK7 mRNA expression was used as the cut-off point for stratification (P ¼ 0.029). This result became more pronounced when the cut-off point was set to the upper and lower 25% (P ¼ 0.012) (Supplementary Fig. 4). 3.3. THZ1, a potent CDK7 inhibitor, reduces neoplastic characteristics in EOC To investigate whether CDK7 inhibition interferes with the cell growth of EOC, we conducted in vitro and in vivo experiments with EOC cell lines and PDX. We first evaluated endogenous expression of CDK7 in diverse EOC cell lines using Western blotting. CDK7 was expressed variously in most EOC cell lines (Fig. 2A). We chose HeyA8, RMG-1, A2780, and A2780-CP20 because they have relatively regular, high expression of CDK7 and different histology and platinum sensitivity. As shown in Fig. 2B, THZ1, a covalent CDK7 inhibitor, significantly reduced the proliferation of A2780, HeyA8, and RMG-1 cells in a dose-dependent manner over both 48 and 72 h, whereas it did not reduce the proliferation of A2780-CP20, a cisplatin-resistant EOC cell line. Furthermore, this anti-proliferative effect of THZ1 was observed more significantly at 72 h exposure compared to 48 h when treated at concentrations of 40 nM or higher in A2780, HeyA8 and RMG-1 cells. This confirmed that THZ1 has a time dependent inhibition effect suggesting covalent bonds. Nevertheless, cell proliferation of A2780-CP20 was reduced by sequential treatment of cisplatin after 72 h of treatment with THZ1 (Fig. 2C), and that combination effect became more pronounced at higher doses of THZ1 (Fig. 2C). To determine the roles of CDK7 in the EOC cell lines, we silenced CDK7 in HeyA8 cells using three kinds of siRNAs: siCDK7 #1, #2 or #3 (Supplementary Fig. 5). siCDK7 #3
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 1. CDK7 overexpression in epithelial ovarian cancer and its clinicopathologic significance. (A) Representative images of immunohistochemical staining of CDK7 in ovarian tissues from normal, benign, borderline, and high-grade serous carcinoma patients. High magnification images are shown in inset. N, normal; Be, benign; Bo, borderline; EOC, epithelial ovarian cancer. Scale bar shown is 50 mm. (B) Boxplot of CDK7 expression according to pathological characteristics. N, normal (n ¼ 62); Be, benign (n ¼ 69); Bo, borderline (n ¼ 57); EOC, epithelial ovarian cancer (n ¼ 197); Mets, metastasis (n ¼ 51), Gr1/2; grade 1/2, Gr3; grade 3. (C) Kaplan-Meier plots of progression-free survival and overall survival according to CDK7 expression. of CDK7High, high CDK7 expression; CDK7Low, Low CDK7 expression.
Table 1 Univariate and multivariate analyses of progression-free survival and overall survival according to prognostic variables in ovarian cancer patients (n ¼ 192). Univariate
Progression free survival CDK7 High Age (>50) FIGO stage (III) Cell type (Serous vs. Others) Grade (High) Suboptimal Overall survival CDK7 High Age (>50) FIGO stage (III) Cell type (Serous vs. Others) Grade (High) Suboptimal
Multivariate
Hazard ratio [95%CI]
P value
Hazard ratio [95%CI]
P value
1.8 [1.22e2.65] 1.59 [1.07e2.36] 6.3 [3.16e12.54] 0.38 [0.23e0.62] 2.01 [1.32e3.07] 2.3 [1.51e3.48]
0.003* 0.023* <0.001* <0.001* 0.001* <0.001*
1.58 [1.03e2.41] 1.25 [0.81e1.94] 4.18 [2.07e8.45] 0.58 [0.33e1.03] 1.76 [1.14e2.72] 1.7 [1.28e4.39]
0.034* 0.310 <0.001* 0.062 0.011* 0.006*
1.67 [0.94e2.99] 2.3 [1.23e4.32] 4.07 [1.61e10.35] 0.3 [0.13e0.7] 2.05 [1.1e3.82] 2.37 [1.28e4.39]
0.082 0.009* 0.003* 0.006* 0.023* 0.006*
1.33 2.04 2.63 0.55 2.04 2.38
0.366 0.039* 0.047* 0.177 0.031* 0.013*
[0.72e2.45] [1.04e4.01] [1.01e6.82] [0.23e1.31] [1.07e3.92] [1.2e4.72]
Abbreviations: FIGO, International Federation of Gynecology and Obstetrics; CDK7, cyclin dependent kinase 7. *Significant at the level of P < 0.05.
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 2. Effect of THZ1 (CDK7 inhibitor) on cell proliferation of EOC cell lines. (A) Western blot analysis of CDK7 in various epithelial ovarian cancer cell lines. (B) Effect of THZ1, a covalent CDK7 inhibitor, on EOC cell proliferation in A2780, HeyA8, RMG-1, and A2780-CP20 cells after 48 and 72 h. Each line represents an individual cell viability curve (square:
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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represented the most inhibitory effect, so we selected siCDK7 #3 for further study. The expression of CDK7 was effectively silenced in A2780, A2780-CP20, and RMG-1 cells by siCDK7 #3 (Supplementary Fig. 5B). Cell proliferation was significantly reduced in siCDK7 #3etransfected cells compared with the nontargeted controls in A2780, HeyA8, and RMG-1 cells treated with 40 nM or more siCDK7 #3 (Supplementary Fig. 5C). We measured apoptosis induction with active caspase-3 in ELISA after 48 h of treatment with THZ1, and our results were similar to those from the proliferation assay. THZ1 induced significantly greater apoptosis in A2780, HeyA8, and RMG-1 cells in a dose dependent manner, while it did not have an apoptotic effect in A2780-CP20 cells (Fig. 3A). We evaluated the migration assay in two different ways using A2780 and RMG-1 cells. In the wound healing assay, the cell migration rate decreased dramatically in both A2780 and RMG-1 cells compared with the control (Fig. 3B and C), and migration capability also decreased significantly after treatment with 40 nM THZ1 in both cell lines (Supplementary Fig. 6A and B). 3.4. Inhibition of CDK7 interferes with cell cycle control in EOC cell lines We performed flow cytometry analyses and immunoblotting in A2780 and HeyA8 cells after treated with THZ1. The flow cytometry analysis revealed that THZ1 markedly caused G0/G1 cell cycle arrest in A2780 cells (Fig. 4A and B). As shown in Fig. 4C, the expression of CDK4, cyclin D1, and cyclin H, which all participate in cell cycle control, decreased in A2780 and HeyA8 cells treated with THZ1 in a dose dependent manner. 3.5. CDK7 inhibition significantly decreased tumor growth in in vivo EOC models We performed in vivo experiments to confirm the anti-cancer effects of CDK7 inhibition in EOC in tumor xenograft models. As shown in Fig. 5, THZ1 significantly inhibited tumor growth in the A2780 xenograft model, compared with the control (Fig. 5A), and PLGAeCDK7-siRNA also inhibited tumor growth in the HeyA8 model (Supplementary Fig. 7A). In IHC staining against Ki-67 and the TUNEL assay, we found that tumor tissues isolated from THZ1 and PLGA-CDK7-siRNA treated mice showed proliferation inhibition and apoptosis induction (Fig. 5B and Supplementary Fig. 7B and C). We also developed a PDX model with high-grade serous ovarian adenocarcinoma (OV-71). The tumor weight in the THZ1-treated mice was significantly lower than in the control (P ¼ 0.009) (Fig. 5C and D). Furthermore, IHC staining against Ki-67 and the TUNEL assay showed similar results to those obtained from the cell line xenograft models (Fig. 5E). Based on these results, we demonstrated that CDK 7 inhibition has anti-neoplastic effects in in vivo xenograft models of EOC. 4. Discussion In this study, we have demonstrated that CDK7High is positively associated with aggressive clinicopathologic characteristics and has independent prognostic value in disease recurrence of EOC through our sample cohort analyses. We verified this prognostic significance of CDK7High using in silico analyses of the TCGA ovarian cancer dataset. These results are consistent with those of previous
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studies, which have also reported that CDK7 overexpression is oncogenic and has prognostic significance in various cancers [15e17,25e28]. Despite the prognostic significance of CDK7 in EOC, CDK7 expression and drug sensitivity to the CDK7 inhibitor THZ2 (an analogue of THZ1) were not dramatic in ovarian cancer cell lines compared with other cancer cell lines in in silico analysis of the Genomics of Drug Sensitivity in Cancer dataset. These results could reflect that CDK7 is commonly observed in a broad spectrum of human cancers as a ubiquitous kinase which is associated with cell cycle control and transcription regulation. In addition, we found no association between CDK7 expression and IC50 of THZ2 in EOC cell lines. This result does not correspond to a previous finding in gastric cancer [29]. In summary, our findings indicate that CDK7 expression in post-operative tissue samples has prognostic value for the recurrence of EOC, and it could provide valuable information for treatment decisions to reduce recurrence and for follow-up policies for early recurrence detection. The anti-cancer effects of CDK7 inhibition in EOC has been demonstrated in both in vitro and in vivo experiments using A2780, HeyA8, and RMG-1 cells and the PDX model with a genetic targeting approach (siRNA) and a pharmacological inhibitor (THZ1). These finding were consistent with previous report by Zhang et al. [26]. Even though the anti-neoplastic effect of THZ1 was not observed in A2780-CP20 cells (platinum-resistant EOC cells), a synergistic effect of THZ1 was observed with cisplatin in A2780CP20 when the two agents were used sequentially. As a chemotherapeutic agent, platinum compounds (e.g, cisplatin) adhere to DNA strands and cause single or doublestranded DNA breaks which induce cell apoptosis. Platinum resistance of EOC is a complex and multifactorial process, and nucleotide excision repair (NER) of damaged DNA is one of the main mechanisms of platinum resistance of EOC by eliminating platinum-DNA adducts [30,31]. NER has two DNA repair pathways; the global genomic NER (GG-NER) and the transcription-coupled NER (TC-NER) [32]. Of these, TC-NER was reported in previous studies to be related to the cytotoxic activity of cisplatin in testicular cancer and EOC cells [30,33]. Given that TC-NER is initiated via RNAPII anchoring in DNA breaks and CDK7 phosphorylates RNAPII to initiate transcription [12,32,33], it can be presumed that CDK7 inhibition may help overcome platinum resistant in EOC cells by disrupting the TC-NER process. There also have been several studies supporting the above that CDK7High is associated with the underlying resistance of cisplatin in lung adenocarcinoma and endometrial cancer [30,34]. Besides, this synergistic effect of CDK7 inhibition with other chemotherapeutic agents was also observed in previous studies of endometrial carcinoma with cisplatin [34], colorectal carcinoma with 5-fluorouracil [35], and TNBC with doxorubicin [15]. As the anti-cancer mechanism of CDK7 inhibition, G0/G1 cell cycle arrest was observed after THZ1 treatment in the flow cytometry analysis with A2780 cells. Furthermore, CDK4, cyclin D1, and cyclin H were dose dependently reduced by THZ1 in A2780 and HeyA8 cells, as shown by Western blotting. Given that the CDK7/ cyclin H complex acts as a CAK and the complex of CDK4/cyclin D1 is involved in the Rb/E2F pathway and promotes the G1-S cell cycle transition [10,36], THZ1 apparently interferes with cell cycle control by disrupting CDK7 action as a CAK and thereby causing G0/G1 cell cycle arrest. Prior to present study, Zhang et al. focused on transcriptional dysregulation as an effective therapeutic approach for EOC and conducted an unbiased high-throughput drug screening to discover
48 h; triangle: 72 h). THZ1 significantly reduced the proliferation of A2780, HeyA8 and RMG-1 cells in a dose-dependent manner in both 48 and 72 h, and this anti-proliferative effect of THZ1 was observed more significantly at 72 h exposure in A2780, HeyA8 and RMG-1 (40 nM: 79.0% vs. 69.0% (P ¼ 0.012); 60 nM: 67.0% vs. 53.0% (P < 0.001)) cells compared with 48 h. Representative images of cell proliferation from the MTT assay after THZ1 treatment (40 nM, 48 h) are also shown above. (C) Synergistic effect of THZ1 with cisplatin on EOC cell proliferation in A2780-CP20 cells. Cell proliferation was significantly decreased by treatment with 40 nM THZ1 and cisplatin. *P < 0.05, **P < 0.01, ***P < 0.001.
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 3. Effect of THZ1 (CDK7 inhibitor) on apoptosis and migration of EOC cell lines. (A) Apoptosis increased dose dependently after treatment with THZ1 in A2780, HeyA8, and RMG-1 cells but not in A2780-CP20 cells. (B, C) Wound healing assay after treatment with THZ1 (40 nM) in A2780 and RMG-1 cells. Inhibition of CDK7 interfered with the migratory ability of EOC cells. All photographs were taken at 100X magnification. *P < 0.05, **P < 0.01, ***P < 0.001.
novel transcriptional target compound. THZ1 was identified as a novel transcriptional target compound for EOC. As with our research, THZ1 showed antineoplastic effects in in vitro and in vivo experiments across various ovarian cancer cell lines with CDK7 expression. In mechanistic analysis, they demonstrated that THZ1 is involved in cell cycle control and transcription regulation which led to antineoplastic effects. These findings were also observed consistently in our mechanistic experiments. Furthermore, they showed for the first time the super-enhancer landscape of EOC and found that many super-enhancers were enriched at THZ1-targeted transcripts [26].
Although preclinical research on CDK7 inhibition has already been conducted in EOC as above, our study adds value to those results. First, our IHC data included more samples than previous study, and clinicopathologic information about tissues ranging from normal epithelial ovaries to metastatic tumors. By virtue of our relatively large dataset, we were able to identify that CDK7 overexpression correlates with aggressive clinicopathologic traits in EOC and has prognostic significance for EOC recurrence. Second, our study successfully demonstrates the in vivo therapeutic efficacy of CDK7 inhibition in EOC cell lines, and we also observed the therapeutic efficacy of THZ1 in an in vivo PDX model. Lastly, the
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 4. Effect of THZ1 in cell cycle control of EOC cell lines. (A) Flow cytometry analysis in A2780 cells treated with THZ1. (B) THZ1 induces G0/G1 cell cycle arrest in A2780 cells. (C) Western blot analysis of CDKs (CDK4 and CDK7) and cyclins (cyclin D1 and cyclin H) in A2780 and HeyA8 cells treated with THZ1. CDKs and cyclins decreased after THZ1 treatment in a dose-dependent manner. *P < 0.05, **P < 0.01.
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Fig. 5. Effect of CDK7 inhibition from THZ1 on tumor weight, cell proliferation, and apoptosis in in vivo experiments with EOC cell line xenograft and patient-derived xenograft models. (A) THZ1 significantly decreased tumor weight, compared with control. (B) In the THZ1-treated group of A2780 cells, the harvested tumor tissues showed significantly reduced cellular proliferation and increased apoptosis. (C) THZ1 significantly reduced tumor weight compared with the DMSO-injected control in the PDX model. (D) In each image, a normal kidney (left) and a developed PDX (right) are shown. (E) In the PDX model, harvested tumor tissues showed significantly decreased the Ki-67 proliferative indices and increased apoptosis in the TUNEL assay. *P < 0.05, **P < 0.01, ***P < 0.001.
in vitro experiments of our research proposed that CDK7 inhibition might be a potential therapeutic target to overcome platinum resistant of EOC. In conclusion, CDK7 might play a critical role in EOC tumorigenesis, and it also serves as an independent molecular prognostic marker for disease recurrence in EOC. CDK7 inhibition via genetics
(siRNA) or pharmacology (CDK7 inhibitor such as THZ1) has potential anti-neoplastic effects in platinum sensitive EOC. Furthermore, the results of in vitro experiment with A2780-CP20 may provide novel ideas and a theoretical basis to discover overcoming platinum resistance in EOC. Although further clinical researches are needed to elucidate the detailed functional mechanism of CDK7
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inhibition in EOC tumorigenesis, CDK7 could be a potential therapeutic target for the treatment of EOC whether platinum sensitive or resistant. Author contributions J-HK, CHC and J-WL conceived of the study and devised the experimental design. CHC, YJC, J-YR, ISH, HDH, HJA, HBC, and J-YC performed the experiments. J-HK, CHC, J-WL, J-HK, B-GK, D-SB, and SMH performed data analysis for the experiments and the clinical records. J-HK, YJC, J-YR, J-YC, CHC, and J-WL drafted the final version of the manuscript and figure legends. WYK, SMH, J-WL revised the figures, added critical content to the discussion, and was responsible for revising all submitted portions of the manuscript. All of the authors read and approved the final manuscript. Data availability statements All data generated or analyzed during this study are available on request from the corresponding authors. Declaration of competing interest All authors declare that no conflict of interest exists. Acknowledgements This research was supported by grants from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (1520100). And, further funds and experimental supporting have been provided via the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), the Ministry of Health & Welfare, Republic of Korea (HI18C1953), National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2016R1A2B3006644), and the National Research Foundation of Korea (NRF) grant funded by the Korean Government(MSIP) (2016R1A5A2945889). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ygyno.2019.11.004. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J Clin 67 (2017) 7e30, 2017. [2] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, CA Cancer J Clin 65 (2012) 87e108, 2015. [3] P. Jessmon, T. Boulanger, W. Zhou, P. Patwardhan, Epidemiology and treatment patterns of epithelial ovarian cancer, Expert Rev. Anticancer Ther. 17 (2017) 427e437. [4] P.M. Webb, S.J. Jordan, Epidemiology of epithelial ovarian cancer, Best Pract. Res. Clin. Obstet. Gynaecol. 41 (2017) 3e14. [5] N. Cancer Genome Atlas Research, Integrated genomic analyses of ovarian carcinoma, Nature 474 (2011) 609e615. [6] S. Vaughan, J.I. Coward, R.C. Bast Jr., A. Berchuck, J.S. Berek, J.D. Brenton, et al., Rethinking ovarian cancer: recommendations for improving outcomes, Nat. Rev. Cancer 11 (2011) 719e725. [7] J. Liu, U.A. Matulonis, New strategies in ovarian cancer: translating the molecular complexity of ovarian cancer into treatment advances, Clin. Cancer Res. 20 (2014) 5150e5156. [8] Q. Zhou, Targeting cyclin-dependent kinases in ovarian cancer, Canc. Invest. 35 (2017) 367e376.
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Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer Jihye Kim a, 1, Young-Jae Cho b, 1, Ji-Yoon Ryu b, Ilseon Hwang c, h, Hee Dong Han d, Hyung Jun Ahn e, Woo Young Kim f, Hanbyoul Cho g, Joon-Yong Chung c, Stephen M. Hewitt c, Jae-Hoon Kim g, Byoung-Gie Kim b, Duk-Soo Bae b, Chel Hun Choi b, **, Jeong-Won Lee b, * a
Department of Obstetrics and Gynecology, Dankook University Hospital, Cheonan, Republic of Korea Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea c Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA d Department of Immunology, School of Medicine, Konkuk University, Chungju, Republic of Korea e Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 136-791, South Korea f Department of Obstetrics and Gynecology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea g Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea h Department of Pathology, Keimyung University School of Medicine, Dongsan Medical Center, Daegu, Republic of Korea b
h i g h l i g h t s CDK7 CDK7 CDK7 CDK7
inhibition has an anti-cancer effect on platinum-sensitive EOC. inhibition could induce responsiveness to platinum chemotherapy in platinum-resistant EOC. overexpression had independent negative prognostic value for disease recurrence of EOC. might play a critical role in EOC tumorigenesis, and it also serves as a possible therapeutic target in EOC.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2019 Received in revised form 2 November 2019 Accepted 4 November 2019 Available online xxx
Objective: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). Methods: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. Results: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P ¼ 0.035 and P ¼ 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P ¼ 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis.
Keywords: Cyclin-dependent kinase 7 Epithelial ovarian cancer Prognosis THZ1 Therapeutic target
* Corresponding author.Department of Obstetrics and Gynecology, Samsung Seoul Hospital, Sungkyunkwan University College of Medicine, 81 Irwon-ro, Gangnamgu, Seoul, 06351, South Korea. ** Corresponding author. Department of Obstetrics and Gynecology, Samsung Seoul Hospital, Sungkyunkwan University College of Medicine, 81 Irwon-ro, Gangnamgu, Seoul, South Korea. E-mail addresses: [email protected] (C.H. Choi), garden.lee@samsung. com (J.-W. Lee). 1 The first two authors contributed equally to this paper. https://doi.org/10.1016/j.ygyno.2019.11.004 0090-8258/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Conclusion: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant. © 2019 Elsevier Inc. All rights reserved.
1. Introduction Epithelial ovarian cancer (EOC) is the most life-threatening gynecologic malignancy, with 238,700 cases newly diagnosed worldwide in 2012 [1,2]. Despite multimodal treatments, including aggressive cytoreductive surgery, platinum-based chemotherapy, and molecular targetebased therapies, about 75% of patients experience relapse, and the five year survival rate is only 39 to 45% [3,4]. EOC is a heterogeneous disease with varying histology, grade, and physiologic behavior. The heterogeneity of EOC is consistently observed at both the genetic and molecular levels. In addition, EOC is specifically characterized by a low prevalence of actionable genetic mutations [5,6]. Accordingly, despite novel molecular targetebased therapies such as bevacizumab and poly(ADP-ribose) polymerase inhibitors, the anticipated therapeutic effect is observed in patients with less than expected or only in patients with certain genetic mutations [7]. Therefore, therapeutic approaches involving tumor-specific genetic mutations may not be an attractive option for EOC. Meanwhile, discovering novel targets for EOC requires other molecular approaches, including intercellular or intracellular ubiquitous molecules which are generally involved in tumorigenesis [8]. In general, most cancer cells exhibit defects in cell cycle regulation and gene transcription, which promotes uncontrolled cell proliferation leading to tumorigenesis [9]. Cyclin-dependent kinases (CDKs) are members of the serine/threonine protein kinase family and are involved in cell cycle control and transcription regulation, which play central roles in tumorigenesis [10,11]. Among the CDKs, cyclin dependent kinase 7 (CDK7) is a component of transcription factor IIH (TFIIH), which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII), and also acts as a CDK activating kinase (CAK) which activates other CDKs involved in cell-cycle control [10,12]. In tumorigenesis, CDK7 encourages tumor growth by directly linking transcription regulation to cell cycle control. Based on these molecular traits, CDK7 has emerged as an attractive target for novel anti-neoplastic treatments in various cancers, including EOC [8,9]. THZ1, a covalent CDK7 inhibitor, was first described as an antineoplastic agent in T-cell acute lymphoblastic leukemia [13]. THZ1, a phenylaminopyrimidine bearing a potentially cysteinereactive acrylamide moiety, downregulates gene transcription by completely inhibiting phosphorylation of established intracellular CDK7 substrate RNAPII CTD Serine 5 and Serine 7 residues. In addition, it can also inhibit CDK12 kinase activity at slightly higher concentrations [13]. Interestingly, THZ1 is involved in transcription addiction by reducing the expression of several transcription factors of “super-enhancer”, thereby substantially reducing the associated target gene expression [14]. Its anti-neoplastic effect also has been demonstrated in a variety of tumors, including triple negative breast cancer [15], glioblastoma [16], pancreatic cancer [17], small cell lung carcinoma [18], colorectal cancer [19], and MYCdependent ovarian cancer [20] in preclinical studies. However, the clinical significance of CDK7 expression in EOC remains elusive. Here, we evaluated the clinical implications and prognostic significance of CDK7 expression in a relatively large EOC cohort and
validated our findings with publicly available datasets. We also investigated the anti-neoplastic effects of CDK7 inhibition by using short interfere RNAs (siRNAs) and THZ1 in preclinical studies and found that CDK7 is a potential target for EOC treatment. 2. Materials and methods 2.1. Patients and tissue specimens A total of 436 tissue samples (62 normal ovarian epithelial tissues, 69 benign tumors, 57 borderline tumors, 197 EOCs, and 51 metastatic tumors) were collected from patients who underwent surgery at Gangnam Severance Hospital (Seoul, South Korea) between 1996 and 2010, and some tissue samples were obtained from the Korea Gynecologic Cancer Bank (NRF-2017M3A9B8069610). The tissue samples and patients’ clinical information were obtained with the informed consent of all patients and the approval of the Institutional Review Board of Samsung Medical Center (approval no. 2015e07e122; Seoul, South Korea). All procedures were conducted in accordance with the guidelines of the Declaration of Helsinki. The patients’ clinical information was obtained from medical records, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, histology based on the World Health Organization grading system, the response to platinumbased chemotherapy, residual disease after debulking surgery (optimal: residual disease < 1 cm, suboptimal: residual disease 1 cm), and levels of cancer antigen 125 (CA-125). All patients were treated with maximal debulking surgery, followed by combination treatment with paclitaxel/carboplatin. Progression-free survival (PFS) was evaluated from the date of surgery to the period of recurrence/progression or the time of the last follow-up visit. Date of recurrence/progression was defined as disease progression according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1), based on either computed tomography (CT), positron emission tomography (PET)-CT, or CA-125 levels [21]. Overall survival (OS) was assessed from the date of surgery to patient death or the date of last contact for living patients. We also analyzed CDK7 gene expression profiles and clinical information about EOC from publicly available databases, including The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) and the Genomics of Drug Sensitivity in Cancer (https://www. cancerrxgene.org), to verify the clinical significance of CDK7 expression. 2.2. Immunohistochemistry and its quantitative evaluation Tissue cylinders of 1.0 mm in diameter were extracted from the most representative areas of donor blocks and transplanted into recipient blocks using a tissue arrayer (Beecher Instruments, Inc., Silver Spring, MD, USA). Depending on the block, 2e3 punches from each patient were included in the tissue microarray (TMA), and the final expression values were averaged. The TMA sections were cut to 5 mm thickness, deparaffinized with xylene, and dehydrated through a graded ethanol series. Antigen retrieval was performed in heat-activated antigen retrieval buffer (Dako, Carpinteria, CA, USA) at pH 6.0. The endogenous peroxidase activity was blocked
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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with 3% H2O2 for 10 min. The sections were incubated with antiCDK7 mouse monoclonal antibody (clone M01; Cell Signaling, Danvers, MA, USA) at a 1:1000 dilution for 60 min at room temperature. The antigeneantibody reaction was detected with an EnVision þ Dual Link System-HRP and visualized with DABþ (3, 30 diaminobenzidine; Dako). Negative controls were performed by omitting the primary antibody and rabbit immunoglobulin (IgG). The results of the immunohistochemistry (IHC) staining were evaluated by staining intensity (0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong) (Supplementary Fig. 1) and proportion (0 10%, 1 > 10% and 30%, 2 > 30%, and 60%, 3 > 60%). The final score was calculated by multiplying the intensity and positivity scores (overall score range, 0e9), and in the analyses for survival and clinicopathologic characteristics, the expression values of CDK7 were dichotomized into high (overall score 3) and low (overall score < 3) using the most statistically significant value as a cut-off. 2.3. Cell lines and treatments A2780-CP20 and HeyA8 (RRID: CVCL_8878) cells were a gift from Dr. Anil K. Sood, Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA, and RMG-1(RRID: CVCL_1662) cells were purchased from the Japanese Collection of Research Bioresources cell bank (Osaka, Japan). The A2780 (RRID: CVCL_0134) cells were obtained from the European Collection of Cell Cultures (Cat NO.93112520). All ovarian EOC cells were maintained in complete medium (RPMI 1640 for A2780, A2780-CP20, and HeyA8 and Ham’s F12 for RMG-1) supplemented with 10% fetal bovine serum in 5% CO2 at 37 C. A covalent CDK7 inhibitor, THZ1, was obtained from ApexBio (#A8882), and cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). All EOC cell lines in this research have been authenticated using STR profiling, and all experiments were performed with mycoplasma and virus free cells. 2.4. siRNA transfection siRNA of CDK7 and negative control siRNA were obtained from Bioneer (Daejeon, Korea). The siRNA targeting CDK7 sequences were as follows: 50 -GAGCAAUCAAAUCCAGCUU-30 (siRNA#1), 50 CUGAAACCAAACAACUUGU-30 (siRNA#2) and 50 - GAGAUUCAGACCUUGAUCA-30 (siRNA#3). A nonspecific, scrambled siRNA with a sequence of 50 -UUCUCCGAACGUGUCA CGU-30 was used as a negative control. Western blot analyses were performed to identify the inhibition effects of each CDK7-siRNA, including the negative control. All EOC cells were transfected with the CDK7-siRNA selected in the Western blot analysis using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.5. Cell proliferation assay Cells were seeded at 3 103 cells/well in a 96-well microplate in culture medium. After 24 h, cells were transfected with the siRNA for 48 h and then treated with THZ1 for 48 and 72 h 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays were performed as previously described [22]. Each sample was assayed in triplicate. 2.6. Apoptosis assay Cell apoptosis was measured 48 h after treatment with THZ1 by measuring the activated caspase-3 with ELISA (Invitrogen, #KH01091) according to the manufacturer’s protocol. Each sample was measured in triplicate.
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2.7. Wound-healing and migration assay A2780 and RMG-1 cells were used for the wound healing and migration assays. For the wound healing assay, cells were seeded in 24-well plates. Confluent monolayers of cells were “wounded” using the end of a sterilized tip. Photographs were taken 24, 32, and 48 h after treatment with THZ1 at 100X magnification, and the analysis was carried out using T-Scratch software (ETH Zurich). In addition, the migration assay was conducted using a Cytoselect 24well cell migration kit according to the manufacturer’s protocol (Cell Biolabs, CA, USA). It was prepared as cell suspension containing 1 x 106 cells/mL in serum free media. 40 nM THZ1 was added directly to the cell suspension. It was added 500 mL of media containing 10% fetal bovine serum to the lower well of the migration plate and added 300 mL of the cell suspension solution to the inside of each insert. Chamber were incubated for 24 h in a cell culture incubator. After removal of non-migratory cells, migratory cells were stained and quantified. 2.8. Cell cycle analysis A2780 cells were treated with various doses of THZ1 or dimethyl sulfoxide (DMSO, control) for 48 h. Then cells were trypsinized for dissociation into a single cell in potassium-buffered saline (PBS), pH 7.5. Cell suspensions were rinsed twice with ice-cold PBS and added dropwise to ice-cold 70% ethanol for 12 h at 4 C. After removing the ethanol, cells were treated with RNaseA (1 mg/mL) followed by the addition of propidium iodide (Sigma; 0.5 mg/mL) for 30 min. The prepared cells were immediately analyzed using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, USA). 2.9. Western blot analysis Cells were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined with a Bradford assay kit (BIO-RAD, Hercules, USA). Cell lysates (50 mg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with mouse monoclonal anti-CDK7 (1:1000, Santa Cruz, #sc-365075), rabbit monoclonal anti-CDK4 (1:1000, Cell signaling, #12790), mouse monoclonal anti-cyclin D1 (1:1000, Santa Cruz, #sc-450), rabbit monoclonal anti-cyclin H (1:1000, Abcam, #EPR3929), and mouse monoclonal anti-b-actin antibody (1:3000,Santa Cruz, #sc47778) and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences) according to the manufacturer’s protocol. 2.10. Animal care and development of in vivo models using established cell lines and patient-derived xenografts This study was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute (Seoul, Korea; protocol No. H-A9-003), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International and abides by the guidelines of the Institute of Laboratory Animal Resources. Female BALB/c nude mice were purchased from Orient Bio (Seongnam, Korea). The mice used in these experiments were 6- to 8-weeks old. To establish orthotopic models, A2780 (1 106 cells/0.2 mL HBSS) and HeyA8 (2.5 105 cells/0.2 mL HBSS) cells were injected into the peritoneal cavities of mice. For the patient-derived xenograft (PDX) model of
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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EOC, a patient tumor specimen retrieved from an operating room was cut into small pieces (less than 2e3 mm), implanted into the subrenal capsule of the left kidney, and propagated by serial transplantation. The tumor of PDX was derived from one patient with ovarian high-grade serous adenocarcinoma at FIGO stage IIIC (OV-71). She was treated with three lines of neo-adjuvant chemotherapy with a paclitaxel-carboplatin combination regimen prior to cytoreductive surgery, and followed by additional six cycles of adjuvant paclitaxel-carboplatin combination chemotherapy for the primary treatment. After completion of adjuvant chemotherapy, there were no residual tumors. To date, she has not experienced relapse for 54 months follow up. After 7 days of cell injections for the cell line models and 4 weeks for the PDX model, THZ1 (10 mg/ kg) or PBS was i.p. injected twice daily in the A2780 and PDX models. In the HeyA8 model, Poly(d,l-lactide-co-glycolide) (PLGA)CDK7-siRNA (7.5 mg) or PLGA-control siRNA was i.v. injected twice weekly [23]. Two groups of xenograft mice for each cell line (n ¼ 10 per group) and two groups for PDX (n ¼ 10 per group) were monitored daily for tumor development and side effects, and they were sacrificed on day 28e35 after the injection of cancer cells or when they appeared moribund. We recorded their body weight, tumor weight, and number of tumor nodules. Tumors were fixed in formalin and embedded in paraffin or snap-frozen in OCT compound (Sakura Finetek Japan, Tokyo, Japan) in liquid nitrogen. Using tumor tissues harvested from the in vivo experiments, IHC staining was done for Ki-67 (NOVUS, NB 600e1252), and TUNEL assays were performed using the ApopTag Peroxidase in situ Apoptosis kit as described previously [24]. 2.11. Data analysis Statistical analyses of the CDK7 expression data from the TMA and publicly available datasets were performed using R software, version 3.1.3 (R Foundation, Vienna, Austria; http://www.R-project. org). Student’s t-tests or ANOVA tests were performed to determine whether CDK7 expression was associated with clinicopathological characteristics in EOC. Kaplan-Meier survival curves were plotted to compare survival between groups. A Cox proportional hazards model was created to identify independent predictors of survival. For the rest of the experiments, statistical analyses were performed using two-tailed Student’s t-tests with GraphPad Prism 3.0. The data are presented as the mean ± SEM for the indicated number of separate experiments. P < 0.05 was considered to be statistically significant. 3. Results 3.1. High CDK 7 expression correlates with aggressive oncogenic behavior in EOC To evaluate the clinical implications of CDK7 expression in EOC, we performed IHC testing in 436 ovarian samples ranging from normal to metastatic and examined the association between CDK7 expression and various clinicopathological characteristics. Immunoreactivity was observed in the nucleus of malignant cells, as shown in the representative photomicrographs of CDK7 immunostaining in Fig. 1A. In detail, the CDK7 expression pattern of normal fallopian tube epithelium was similar to the normal ovarian epithelium whereas high CDK7 expression was observed in clear cell adenocarcinoma (Supplementary Fig. 2). Patient incidence of high CDK7 expression (CDK7High) and the CDK7 expression scores increased gradually from normal tissue to metastatic ovarian tumors (P < 0.001) (Supplementary Table 1), although there was a strikingly high expression score in borderline ovarian tumor tissue (P < 0.001) (Fig. 1B). In addition, we found no correlation between CDK7 expression and cell type, chemo-sensitivity, or preoperative
CA-125 levels, but CDK7High was significantly associated with advanced FIGO stage (III or IV) (P ¼ 0.035) and high-grade histology (P ¼ 0.011) (Supplementary Table 1). In particular, high-grade histology had significantly higher CDK7 expression than low-grade histology (P ¼ 0.035) (Fig. 1B). We also compared CDK7 expression with sensitivity to a CDK7 inhibitor (THZ2) in ovarian cancer using a variety of cancer cell lines in an in silico analysis with the Genomics of Drug Sensitivity in Cancer dataset (https://www.cancerrxgene.org). As shown in Supplementary Fig. 3, CDK7 expression and IC50 levels to THZ2 in ovarian cancer cell lines did not differ when compared with other cancer cell lines (Z-score (interquartile range) ¼ 0.5 (‒ 0.57e1.22), ‒ 1.28 (‒ 2.49e0.40)), and we found no association between CDK7 expression and IC50 levels to THZ2 in ovarian cancer cell lines (Supplementary Fig. 4). 3.2. CDK7 expression is an independent prognostic factor for disease recurrence in EOC We next examined the association between CDK7 expression and patient survival outcomes in 192 EOC patients with available survival data. Kaplan-Meier plots demonstrated that patients with CDK7High was significantly associated with disease recurrence (P ¼ 0.003), whereas no correlation was observed with OS (P ¼ 0.079) (Fig. 1C). As shown in Table 1, the univariate analysis for PFS revealed that CDK7High (P ¼ 0.003), older age at diagnosis (P ¼ 0.023), advanced FIGO stage, non-serous, and high-grade histology (all, P < 0.001) were all associated with poor PFS. Furthermore, the multivariate analysis also demonstrated that CDK7High (P ¼ 0.034), advanced FIGO stage (P < 0.001), and highgrade histology (P ¼ 0.011) independently predicted poor PFS. However, CDK7High had no prognostic value for OS in EOC patients. To validate these findings, we also performed an in silico analysis using the TCGA ovarian cancer dataset. KaplaneMeier survival analysis demonstrated that higher CDK7 mRNA expression correlated significantly with decreased PFS when the upper and lower 50% of CDK7 mRNA expression was used as the cut-off point for stratification (P ¼ 0.029). This result became more pronounced when the cut-off point was set to the upper and lower 25% (P ¼ 0.012) (Supplementary Fig. 4). 3.3. THZ1, a potent CDK7 inhibitor, reduces neoplastic characteristics in EOC To investigate whether CDK7 inhibition interferes with the cell growth of EOC, we conducted in vitro and in vivo experiments with EOC cell lines and PDX. We first evaluated endogenous expression of CDK7 in diverse EOC cell lines using Western blotting. CDK7 was expressed variously in most EOC cell lines (Fig. 2A). We chose HeyA8, RMG-1, A2780, and A2780-CP20 because they have relatively regular, high expression of CDK7 and different histology and platinum sensitivity. As shown in Fig. 2B, THZ1, a covalent CDK7 inhibitor, significantly reduced the proliferation of A2780, HeyA8, and RMG-1 cells in a dose-dependent manner over both 48 and 72 h, whereas it did not reduce the proliferation of A2780-CP20, a cisplatin-resistant EOC cell line. Furthermore, this anti-proliferative effect of THZ1 was observed more significantly at 72 h exposure compared to 48 h when treated at concentrations of 40 nM or higher in A2780, HeyA8 and RMG-1 cells. This confirmed that THZ1 has a time dependent inhibition effect suggesting covalent bonds. Nevertheless, cell proliferation of A2780-CP20 was reduced by sequential treatment of cisplatin after 72 h of treatment with THZ1 (Fig. 2C), and that combination effect became more pronounced at higher doses of THZ1 (Fig. 2C). To determine the roles of CDK7 in the EOC cell lines, we silenced CDK7 in HeyA8 cells using three kinds of siRNAs: siCDK7 #1, #2 or #3 (Supplementary Fig. 5). siCDK7 #3
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 1. CDK7 overexpression in epithelial ovarian cancer and its clinicopathologic significance. (A) Representative images of immunohistochemical staining of CDK7 in ovarian tissues from normal, benign, borderline, and high-grade serous carcinoma patients. High magnification images are shown in inset. N, normal; Be, benign; Bo, borderline; EOC, epithelial ovarian cancer. Scale bar shown is 50 mm. (B) Boxplot of CDK7 expression according to pathological characteristics. N, normal (n ¼ 62); Be, benign (n ¼ 69); Bo, borderline (n ¼ 57); EOC, epithelial ovarian cancer (n ¼ 197); Mets, metastasis (n ¼ 51), Gr1/2; grade 1/2, Gr3; grade 3. (C) Kaplan-Meier plots of progression-free survival and overall survival according to CDK7 expression. of CDK7High, high CDK7 expression; CDK7Low, Low CDK7 expression.
Table 1 Univariate and multivariate analyses of progression-free survival and overall survival according to prognostic variables in ovarian cancer patients (n ¼ 192). Univariate
Progression free survival CDK7 High Age (>50) FIGO stage (III) Cell type (Serous vs. Others) Grade (High) Suboptimal Overall survival CDK7 High Age (>50) FIGO stage (III) Cell type (Serous vs. Others) Grade (High) Suboptimal
Multivariate
Hazard ratio [95%CI]
P value
Hazard ratio [95%CI]
P value
1.8 [1.22e2.65] 1.59 [1.07e2.36] 6.3 [3.16e12.54] 0.38 [0.23e0.62] 2.01 [1.32e3.07] 2.3 [1.51e3.48]
0.003* 0.023* <0.001* <0.001* 0.001* <0.001*
1.58 [1.03e2.41] 1.25 [0.81e1.94] 4.18 [2.07e8.45] 0.58 [0.33e1.03] 1.76 [1.14e2.72] 1.7 [1.28e4.39]
0.034* 0.310 <0.001* 0.062 0.011* 0.006*
1.67 [0.94e2.99] 2.3 [1.23e4.32] 4.07 [1.61e10.35] 0.3 [0.13e0.7] 2.05 [1.1e3.82] 2.37 [1.28e4.39]
0.082 0.009* 0.003* 0.006* 0.023* 0.006*
1.33 2.04 2.63 0.55 2.04 2.38
0.366 0.039* 0.047* 0.177 0.031* 0.013*
[0.72e2.45] [1.04e4.01] [1.01e6.82] [0.23e1.31] [1.07e3.92] [1.2e4.72]
Abbreviations: FIGO, International Federation of Gynecology and Obstetrics; CDK7, cyclin dependent kinase 7. *Significant at the level of P < 0.05.
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Fig. 2. Effect of THZ1 (CDK7 inhibitor) on cell proliferation of EOC cell lines. (A) Western blot analysis of CDK7 in various epithelial ovarian cancer cell lines. (B) Effect of THZ1, a covalent CDK7 inhibitor, on EOC cell proliferation in A2780, HeyA8, RMG-1, and A2780-CP20 cells after 48 and 72 h. Each line represents an individual cell viability curve (square:
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represented the most inhibitory effect, so we selected siCDK7 #3 for further study. The expression of CDK7 was effectively silenced in A2780, A2780-CP20, and RMG-1 cells by siCDK7 #3 (Supplementary Fig. 5B). Cell proliferation was significantly reduced in siCDK7 #3etransfected cells compared with the nontargeted controls in A2780, HeyA8, and RMG-1 cells treated with 40 nM or more siCDK7 #3 (Supplementary Fig. 5C). We measured apoptosis induction with active caspase-3 in ELISA after 48 h of treatment with THZ1, and our results were similar to those from the proliferation assay. THZ1 induced significantly greater apoptosis in A2780, HeyA8, and RMG-1 cells in a dose dependent manner, while it did not have an apoptotic effect in A2780-CP20 cells (Fig. 3A). We evaluated the migration assay in two different ways using A2780 and RMG-1 cells. In the wound healing assay, the cell migration rate decreased dramatically in both A2780 and RMG-1 cells compared with the control (Fig. 3B and C), and migration capability also decreased significantly after treatment with 40 nM THZ1 in both cell lines (Supplementary Fig. 6A and B). 3.4. Inhibition of CDK7 interferes with cell cycle control in EOC cell lines We performed flow cytometry analyses and immunoblotting in A2780 and HeyA8 cells after treated with THZ1. The flow cytometry analysis revealed that THZ1 markedly caused G0/G1 cell cycle arrest in A2780 cells (Fig. 4A and B). As shown in Fig. 4C, the expression of CDK4, cyclin D1, and cyclin H, which all participate in cell cycle control, decreased in A2780 and HeyA8 cells treated with THZ1 in a dose dependent manner. 3.5. CDK7 inhibition significantly decreased tumor growth in in vivo EOC models We performed in vivo experiments to confirm the anti-cancer effects of CDK7 inhibition in EOC in tumor xenograft models. As shown in Fig. 5, THZ1 significantly inhibited tumor growth in the A2780 xenograft model, compared with the control (Fig. 5A), and PLGAeCDK7-siRNA also inhibited tumor growth in the HeyA8 model (Supplementary Fig. 7A). In IHC staining against Ki-67 and the TUNEL assay, we found that tumor tissues isolated from THZ1 and PLGA-CDK7-siRNA treated mice showed proliferation inhibition and apoptosis induction (Fig. 5B and Supplementary Fig. 7B and C). We also developed a PDX model with high-grade serous ovarian adenocarcinoma (OV-71). The tumor weight in the THZ1-treated mice was significantly lower than in the control (P ¼ 0.009) (Fig. 5C and D). Furthermore, IHC staining against Ki-67 and the TUNEL assay showed similar results to those obtained from the cell line xenograft models (Fig. 5E). Based on these results, we demonstrated that CDK 7 inhibition has anti-neoplastic effects in in vivo xenograft models of EOC. 4. Discussion In this study, we have demonstrated that CDK7High is positively associated with aggressive clinicopathologic characteristics and has independent prognostic value in disease recurrence of EOC through our sample cohort analyses. We verified this prognostic significance of CDK7High using in silico analyses of the TCGA ovarian cancer dataset. These results are consistent with those of previous
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studies, which have also reported that CDK7 overexpression is oncogenic and has prognostic significance in various cancers [15e17,25e28]. Despite the prognostic significance of CDK7 in EOC, CDK7 expression and drug sensitivity to the CDK7 inhibitor THZ2 (an analogue of THZ1) were not dramatic in ovarian cancer cell lines compared with other cancer cell lines in in silico analysis of the Genomics of Drug Sensitivity in Cancer dataset. These results could reflect that CDK7 is commonly observed in a broad spectrum of human cancers as a ubiquitous kinase which is associated with cell cycle control and transcription regulation. In addition, we found no association between CDK7 expression and IC50 of THZ2 in EOC cell lines. This result does not correspond to a previous finding in gastric cancer [29]. In summary, our findings indicate that CDK7 expression in post-operative tissue samples has prognostic value for the recurrence of EOC, and it could provide valuable information for treatment decisions to reduce recurrence and for follow-up policies for early recurrence detection. The anti-cancer effects of CDK7 inhibition in EOC has been demonstrated in both in vitro and in vivo experiments using A2780, HeyA8, and RMG-1 cells and the PDX model with a genetic targeting approach (siRNA) and a pharmacological inhibitor (THZ1). These finding were consistent with previous report by Zhang et al. [26]. Even though the anti-neoplastic effect of THZ1 was not observed in A2780-CP20 cells (platinum-resistant EOC cells), a synergistic effect of THZ1 was observed with cisplatin in A2780CP20 when the two agents were used sequentially. As a chemotherapeutic agent, platinum compounds (e.g, cisplatin) adhere to DNA strands and cause single or doublestranded DNA breaks which induce cell apoptosis. Platinum resistance of EOC is a complex and multifactorial process, and nucleotide excision repair (NER) of damaged DNA is one of the main mechanisms of platinum resistance of EOC by eliminating platinum-DNA adducts [30,31]. NER has two DNA repair pathways; the global genomic NER (GG-NER) and the transcription-coupled NER (TC-NER) [32]. Of these, TC-NER was reported in previous studies to be related to the cytotoxic activity of cisplatin in testicular cancer and EOC cells [30,33]. Given that TC-NER is initiated via RNAPII anchoring in DNA breaks and CDK7 phosphorylates RNAPII to initiate transcription [12,32,33], it can be presumed that CDK7 inhibition may help overcome platinum resistant in EOC cells by disrupting the TC-NER process. There also have been several studies supporting the above that CDK7High is associated with the underlying resistance of cisplatin in lung adenocarcinoma and endometrial cancer [30,34]. Besides, this synergistic effect of CDK7 inhibition with other chemotherapeutic agents was also observed in previous studies of endometrial carcinoma with cisplatin [34], colorectal carcinoma with 5-fluorouracil [35], and TNBC with doxorubicin [15]. As the anti-cancer mechanism of CDK7 inhibition, G0/G1 cell cycle arrest was observed after THZ1 treatment in the flow cytometry analysis with A2780 cells. Furthermore, CDK4, cyclin D1, and cyclin H were dose dependently reduced by THZ1 in A2780 and HeyA8 cells, as shown by Western blotting. Given that the CDK7/ cyclin H complex acts as a CAK and the complex of CDK4/cyclin D1 is involved in the Rb/E2F pathway and promotes the G1-S cell cycle transition [10,36], THZ1 apparently interferes with cell cycle control by disrupting CDK7 action as a CAK and thereby causing G0/G1 cell cycle arrest. Prior to present study, Zhang et al. focused on transcriptional dysregulation as an effective therapeutic approach for EOC and conducted an unbiased high-throughput drug screening to discover
48 h; triangle: 72 h). THZ1 significantly reduced the proliferation of A2780, HeyA8 and RMG-1 cells in a dose-dependent manner in both 48 and 72 h, and this anti-proliferative effect of THZ1 was observed more significantly at 72 h exposure in A2780, HeyA8 and RMG-1 (40 nM: 79.0% vs. 69.0% (P ¼ 0.012); 60 nM: 67.0% vs. 53.0% (P < 0.001)) cells compared with 48 h. Representative images of cell proliferation from the MTT assay after THZ1 treatment (40 nM, 48 h) are also shown above. (C) Synergistic effect of THZ1 with cisplatin on EOC cell proliferation in A2780-CP20 cells. Cell proliferation was significantly decreased by treatment with 40 nM THZ1 and cisplatin. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 3. Effect of THZ1 (CDK7 inhibitor) on apoptosis and migration of EOC cell lines. (A) Apoptosis increased dose dependently after treatment with THZ1 in A2780, HeyA8, and RMG-1 cells but not in A2780-CP20 cells. (B, C) Wound healing assay after treatment with THZ1 (40 nM) in A2780 and RMG-1 cells. Inhibition of CDK7 interfered with the migratory ability of EOC cells. All photographs were taken at 100X magnification. *P < 0.05, **P < 0.01, ***P < 0.001.
novel transcriptional target compound. THZ1 was identified as a novel transcriptional target compound for EOC. As with our research, THZ1 showed antineoplastic effects in in vitro and in vivo experiments across various ovarian cancer cell lines with CDK7 expression. In mechanistic analysis, they demonstrated that THZ1 is involved in cell cycle control and transcription regulation which led to antineoplastic effects. These findings were also observed consistently in our mechanistic experiments. Furthermore, they showed for the first time the super-enhancer landscape of EOC and found that many super-enhancers were enriched at THZ1-targeted transcripts [26].
Although preclinical research on CDK7 inhibition has already been conducted in EOC as above, our study adds value to those results. First, our IHC data included more samples than previous study, and clinicopathologic information about tissues ranging from normal epithelial ovaries to metastatic tumors. By virtue of our relatively large dataset, we were able to identify that CDK7 overexpression correlates with aggressive clinicopathologic traits in EOC and has prognostic significance for EOC recurrence. Second, our study successfully demonstrates the in vivo therapeutic efficacy of CDK7 inhibition in EOC cell lines, and we also observed the therapeutic efficacy of THZ1 in an in vivo PDX model. Lastly, the
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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Fig. 4. Effect of THZ1 in cell cycle control of EOC cell lines. (A) Flow cytometry analysis in A2780 cells treated with THZ1. (B) THZ1 induces G0/G1 cell cycle arrest in A2780 cells. (C) Western blot analysis of CDKs (CDK4 and CDK7) and cyclins (cyclin D1 and cyclin H) in A2780 and HeyA8 cells treated with THZ1. CDKs and cyclins decreased after THZ1 treatment in a dose-dependent manner. *P < 0.05, **P < 0.01.
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Fig. 5. Effect of CDK7 inhibition from THZ1 on tumor weight, cell proliferation, and apoptosis in in vivo experiments with EOC cell line xenograft and patient-derived xenograft models. (A) THZ1 significantly decreased tumor weight, compared with control. (B) In the THZ1-treated group of A2780 cells, the harvested tumor tissues showed significantly reduced cellular proliferation and increased apoptosis. (C) THZ1 significantly reduced tumor weight compared with the DMSO-injected control in the PDX model. (D) In each image, a normal kidney (left) and a developed PDX (right) are shown. (E) In the PDX model, harvested tumor tissues showed significantly decreased the Ki-67 proliferative indices and increased apoptosis in the TUNEL assay. *P < 0.05, **P < 0.01, ***P < 0.001.
in vitro experiments of our research proposed that CDK7 inhibition might be a potential therapeutic target to overcome platinum resistant of EOC. In conclusion, CDK7 might play a critical role in EOC tumorigenesis, and it also serves as an independent molecular prognostic marker for disease recurrence in EOC. CDK7 inhibition via genetics
(siRNA) or pharmacology (CDK7 inhibitor such as THZ1) has potential anti-neoplastic effects in platinum sensitive EOC. Furthermore, the results of in vitro experiment with A2780-CP20 may provide novel ideas and a theoretical basis to discover overcoming platinum resistance in EOC. Although further clinical researches are needed to elucidate the detailed functional mechanism of CDK7
Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004
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inhibition in EOC tumorigenesis, CDK7 could be a potential therapeutic target for the treatment of EOC whether platinum sensitive or resistant. Author contributions J-HK, CHC and J-WL conceived of the study and devised the experimental design. CHC, YJC, J-YR, ISH, HDH, HJA, HBC, and J-YC performed the experiments. J-HK, CHC, J-WL, J-HK, B-GK, D-SB, and SMH performed data analysis for the experiments and the clinical records. J-HK, YJC, J-YR, J-YC, CHC, and J-WL drafted the final version of the manuscript and figure legends. WYK, SMH, J-WL revised the figures, added critical content to the discussion, and was responsible for revising all submitted portions of the manuscript. All of the authors read and approved the final manuscript. Data availability statements All data generated or analyzed during this study are available on request from the corresponding authors. Declaration of competing interest All authors declare that no conflict of interest exists. Acknowledgements This research was supported by grants from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (1520100). And, further funds and experimental supporting have been provided via the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), the Ministry of Health & Welfare, Republic of Korea (HI18C1953), National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2016R1A2B3006644), and the National Research Foundation of Korea (NRF) grant funded by the Korean Government(MSIP) (2016R1A5A2945889). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ygyno.2019.11.004. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J Clin 67 (2017) 7e30, 2017. [2] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, CA Cancer J Clin 65 (2012) 87e108, 2015. [3] P. Jessmon, T. Boulanger, W. Zhou, P. Patwardhan, Epidemiology and treatment patterns of epithelial ovarian cancer, Expert Rev. Anticancer Ther. 17 (2017) 427e437. [4] P.M. Webb, S.J. Jordan, Epidemiology of epithelial ovarian cancer, Best Pract. Res. Clin. Obstet. Gynaecol. 41 (2017) 3e14. [5] N. Cancer Genome Atlas Research, Integrated genomic analyses of ovarian carcinoma, Nature 474 (2011) 609e615. [6] S. Vaughan, J.I. Coward, R.C. Bast Jr., A. Berchuck, J.S. Berek, J.D. Brenton, et al., Rethinking ovarian cancer: recommendations for improving outcomes, Nat. Rev. Cancer 11 (2011) 719e725. [7] J. Liu, U.A. Matulonis, New strategies in ovarian cancer: translating the molecular complexity of ovarian cancer into treatment advances, Clin. Cancer Res. 20 (2014) 5150e5156. [8] Q. Zhou, Targeting cyclin-dependent kinases in ovarian cancer, Canc. Invest. 35 (2017) 367e376.
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Please cite this article as: J. Kim et al., CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer, Gynecologic Oncology, https://doi.org/10.1016/j.ygyno.2019.11.004