Wookyeom Yang

Accumulation of cytoplasmic Cdk1 is associated with cancer growth and survival rate in epithelial ovarian cancer

Cyclin dependent kinase 1 (Cdk1) have previously reported correlation with cancer growth and a key regulator for cell cycle. Mostly, Cdk1′s function of nucleus for cell cycle is well known to be associated with cancer, but cytoplasmic Cdk1′s traits are not clearly identified, yet. We revealed that tissue microarray blocks of epithelial ovarian cancer (n = 249) showed increased level of cytoplasmic Cdk1 (p < 0.001), but not in nucleus (p = 0.192) of histologic cell type independently. On survival analysis, Cdk1 overexpression conferred a significantly worse prognosis in 5-year overall survival (Log-rank p = 0.028, Hazard ratio = 2.016, 95% CI = 1.097 to 4.635). Also, the expression of Cdk1 was increased in ovarian cancer cell lines and Gene Expression Omnibus datasets. When the expression and activity of Cdk1 were inhibited by si-Cdk1 or RO-3306 which is a potent Cdk1 inhibitor, the growth of ovarian cancer was diminished. Moreover, combined treatment with RO-3306 and cisplatin in ovarian cancer significantly elevated anti-cancer effects than single-agent treatment. In conclusion, cytoplasmic Cdk1 expression which was elevated in ovarian cancer predicts a poor overall survival. The inhibition of Cdk1 expression and activity reduced ovarian cancer growth.

Comparison of Clinical Features and Outcomes in Epithelial Ovarian Cancer according to Tumorigenicity in Patient-Derived Xenograft Models

Purpose Although the use of xenograft models is increasing, few studies have compared the clinical features or outcomes of epithelial ovarian cancer (EOC) patients according to the tumorigenicity of engrafted specimens. The purpose of this study was to evaluate whether tumorigenicity was associated with the clinical features and outcomes of EOC patients. Materials and Methods Eighty-eight EOC patients who underwent primary or interval debulking surgery from June 2014 to December 2015 were included. Fresh tumor specimens were implanted subcutaneously on each flank of immunodeficient mice. Patient characteristics, progression-free survival (PFS), and germline mutation spectra were compared according to tumorigenicity. Results Xenografts were established successfully from 49 of 88 specimens. Tumorigenicity was associated with lymphovascular invasion and there was a propensity to engraft successfully with high-grade tumors. Tumors from patientswho underwent non-optimal (residual disease ≥ 1 cm) primary orinterval debulking surgery had a significantly greater propensity to achieve tumorigenicity than those who received optimal surgery. In addition, patients whose tumors became engrafted seemed to have a shorter PFS and more frequent germline mutations than patients whose tumors failed to engraft. Tumorigenicity was a significant factor for predicting PFS with advanced International Federation of Gynecology and Obstetrics stage and high-grade cancers. Conclusion sTumorigenicity in a xenograft model was a strong prognostic factor and was associated with more aggressive tumors in EOC patients. Xenograft models can be useful as a preclinical tool to predict prognosis and could be applied to further pharmacologic and genomic studies on personalized treatments.

Abstract 2456: Overexpression of TOM40 (translocase in the outer mitochondrial membrane 40) inhibits the cell proliferation, invasion and migration abilities in ovarian cancer cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Mitochondria are essential for energy conversation and cellular metabolism in eukaryotic cell. Mitochondria contain mitochondrial proteins that are encoded in the nucleus and synthesized in cytosol. Synthesized mitochondrial proteins are specially sorted by sub-mitochondrial compartment and then transported to their functional location. If mitochondrial proteins are not transported to their specific regions, it is reported that Mitochondria has a tendency to be implicated in various diseases including cancer. Mitochondria consist of mitochondrial outer membrane and inner membrane, especially, TOM complex are integrated in mitochondrial outer membrane. TOM40 is a central component of TOM complex has General import pore (GIP) to pass mitochondrial pre-protein. The goal of this study is to investigate the function of TOM40 in ovarian cancer. Methods: At first, we performed microarray to identify new target gene in ovarian cancer for nine human ovarian cancer cell line and seven human ovarian surface epithelial cell lines (HOSEs). We investigated target gene expression level in ovarian cancer cell line by RT-PCR and Western blot. To evaluate the association of target gene expression level and clinicopatholigical parameter in ovarian cancer, we carried out immunohistochemistry by using normal and ovarian cancer tissue. Second, we constructed stable DOV-13 and RMUG-S cell lines for overexpression and knock down of TOM40 protein using lentiviral system. We carried out proliferation, invasion and migration assay of each individual stable cell line. Results: We identified TOM40 as a new target gene for ovarian cancer by microarray data that was overexpressed in all nine human ovarian cancer cell lines than HOSEs. TOM40 protein level in borderline and cancer tissue was higher than normal tissue and benign tissue. TOM40 immuno-staining score of normal ovarian tissues is 1.23 (95% CI, 0.44-2.01) that compare with TOM40 immuno-staining score of benign, borderline, and epithelial ovarian tumors is 1.23 (95% CI, 0.44-2.01), 1.41 (95% CI, 0.45-2.37), 3.42 (95% CI, 2.65-4.20), and 5.04 (95% CI, 4.82-5.27), respectively. We understood TOM40 expression level of DOV-13 cells and RMUG-S cells is lower and higher than other ovarian cancer cell line. TOM40 overexpression in DOV-13 cell line inhibited the cell proliferation, invasion and migration than that of control cell line. TOM40 knock down in RMUG-S cell line induced cell proliferation, invasion and migration abilities than in control RMUG-S cell line. Conclusion: It is important to transport pathways of mitochondrial proteins, actually, the paper discussing directly association of TOM complex involving mitochondrial proteins transport and various diseases including cancer is not found. Our results suggest that TOM40 can be a role as putative tumor suppressor for ovarian cancer. Citation Format: Sol Kim, Hanbyoul Cho, Wookyeom Yang, Hyunja Kwon, Ha yeon Shin, Eunju Lee, Eun-Suk Kang, Jae-hoon Kim. Overexpression of TOM40 (translocase in the outer mitochondrial membrane 40) inhibits the cell proliferation, invasion and migration abilities in ovarian cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2014-2456

Establishment of five immortalized human ovarian surface epithelial cell lines via SV40 T antigen or HPV E6/E7 expression

Background Human ovarian surface epithelial (HOSE) cells are a critical cell source for ovarian cancer research; however, they are difficult to obtain and maintain under standard laboratory conditions in large quantities. The aim of this study was to generate immortalized HOSE (IHOSE) cells with maintained properties to the original cell source, thereby guaranteeing a sufficiently large cell quantity for ovarian cancer research. Methods HOSE cells isolated from four non-cancer patients and five IHOSE cell lines were established by induction of HPV-E6/E7 expression or SV40 large T antigen using a lenti-viral system. Each of IHOSE cells was confirmed to be distinct by STR profiling. RNA-sequencing was used to compare gene expression profiles in HOSE, IHOSE and ovarian cancer cells. Results RNA-sequencing results revealed a stronger linear correlation in gene expression between IHOSE and HOSE cells (R2 = 0.9288) than between IHOSE or HOSE cells and ovarian cancer cells (R2 = 0.8562 and R2 = 0.7982, respectively). The gene expression pattern of 319 differentially expressed genes revealed minimal differences between HOSE and IHOSE cells, while a strong difference between ovarian cancer cells and HOSE or IHOSE cells was observed. Furthermore, the five IHOSE cell lines displayed morphological characteristics typical of epithelial cells but showed a lower level of EpCAM, CD133 and E-cadherin, as cancer stem marker, than ovarian cancer cells. Moreover, unlike cancer cells, IHOSE cells could not form colonies in the anchorage-independent soft agar growth assay. Conclusion These findings demonstrate that five newly established IHOSE cell lines have characteristics of progenitor HOSE cells while exhibiting continuous growth, and thus, should be highly useful as control cells for ovarian cancer research.

Prognostic implication of programmed cell death 1 protein and its ligand expressions in endometrial cancer

OBJECTIVE Monoclonal antibodies targeting programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1) demonstrated promising clinical response. The predictive/prognostic value of PD-1/PD-L1 immunohistochemistry (IHC) has been evaluated in many cancer types. However, the prognostic value of PD-1/PD-L1 IHC has not been evaluated in endometrial cancer. METHODS We conducted a retrospective study to quantify the IHC CD8, PD-1, and PD-L1 expressions in immune cells at center of tumor (CT), invasive margin (IM), and/or tumor cell in 183 primary endometrial cancer samples from a single cohort, followed by their reciprocal combinations, including compartmental differences, and correlated them with overall survival (OS) and progression-free survival (PFS). RESULTS In repeated Cox multivariable models adjusted by clinicoimmunopathologic factors, high CT-PD-L1 was an independent adverse prognostic factor for PFS in all patients and in the microsatellite-stable subgroup. Immune marker ratios revealed independently shorter PFS for high CT-PD-L1/CT-CD8 and CT-PD-L1/CT-PD-1 ratios. Classification of endometrial cancer into four groups based on CT-CD8 and CT-PD-L1 revealed significantly different survival among groups. CONCLUSIONS The high PD-L1/CD8 ratio and the high expression of PD-L1 on immune cells were independent poor prognostic factors for PFS in endometrial cancer, providing insights into the tumor microenvironment.

DNA Mismatch Repair Protein Immunohistochemistry and MLH1 Promotor Methylation Testing for Practical Molecular Classification and the Prediction of Prognosis in Endometrial Cancer

The incidence of endometrial cancer is rapidly increasing worldwide, and its molecular classification has gained importance for new therapeutic approaches. This study sought to examine the clinicopathologic features and immune markers associated with the DNA mismatch repair (MMR) status and MLH1 promoter methylation status of endometrial cancer patients. A total of 173 patients with primary endometrial cancer who had received a hysterectomy were evaluated for four MMR proteins (MLH1, MSH2, MSH6, and PMS2), immune markers (CD8, programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1)) and p53 by immunohistochemistry (IHC), followed by an MLH1 methylation test. Patients were classified into MMR deficiency or proficiency, sporadic cancer, or probable Lynch syndrome (PLS), and the clinicopathologic features (including the expression of peritumoral immune markers) and prognosis of each group were compared. Patients with MMR deficiency or PLS showed an increase in immune markers compared those with MMR proficiency or sporadic cancer, respectively, and PLS demonstrated higher immune marker expression than MLH1 promoter methylation. Regarding prognosis, patients with MMR deficiency showed significant adverse overall survival (OS) when in stages I and II. Practical molecular classifications based on p53 staining results, in addition to MMR or PLS status, revealed an increased predictive ability for OS compared with the European Society of Medical Oncologists (ESMO) risk groups. The results of this study suggest that PLS may be a better candidate for an immune checkpoint inhibitor than MMR deficiency. The practical molecular classification contributes not only to the screening of Lynch syndrome, but also assists in predicting the prognosis in endometrial cancer.

Abstract B82: Application of modulated electro-hyperthermia for the cellular target therapy in ovarian cancer cells.

Background: Oncothermia as modulated electro-hyperthermia is an anti-cancer therapy that was approved by TUV-SUD in Germany, Health Canada in Canada, and Therapeutic Goods Administration in Australian. Oncothermia induces apoptosis via destroying the plasma membrane of cancer cells and leads to accumulation of heat-induced damages mostly in cells located in the tumor tissue while providing minimal damages to the surrounding normal tissue. Thus, oncothermia has recently become a potentially effective therapeutic tool for cancer. Methods: LabEHY-100, an oncothermia device from Oncotherm (Germany & Hungary), was used for all in vitro studies. Ovarian cells were exposed to thermal damage induced by LabEHY-100, and subsequently, the cancer cells were analyzed in terms of viability, levels of apoptosis, autophagic markers and cell cycle status. In addition, xenograft tumors which generated by OVCAR-3 and patients derived tumor, were exposed to thermal damage induced by LabEHY-100 after which the tumor progression was monitored. The efficacy of combination therapy using both oncothermia and 3-methyladenine (3-MA), an autophagy inhibitor, was assessed using crystal violet assay. Results: Our studies showed that oncothermia resulted in growth inhibition in ovarian cancer cell tested. Apoptotic markers such as cleaved caspase-3 and PARP were upregulated in cells exposed to oncothermia compared to the control cells exposed to hyperthermia. Oncothermia also led to reduction in the mass and the volume of the tumors in the mouse transplanted with xenografts derived from cancer cells and ovarian cancer patient tumors. FACS analysis showed that cell cycles of these cells were not disrupted by oncothermia. Still there was a significant increase in the number of sub-G1 population, which consisted of dying cells including apoptotic cells. However, the xenograft cells recovered from cellular damage even after inducing autophagy via oncothermia. Combined treatment with oncothermia and 3-MA, a autophagy inhibitor, was more effective in inhibiting cancer cell growth than treatment with each alone. Conclusion: Oncothermia can potentially be an effective therapy for ovarian cancer cells. Conflict of interest : We obtained funding and in vitro device for electro-hyperthermia from HOSPICARE. Co., Ltd. (Seoul, Republic of Korea). Citation Format: Wookyeom Yang, Doo Byung Chay, Hanbyoul Cho, Sunghoon Kim, Jinkyoung Kong, Geumseon Son, Jihui Choi, Kim Jae-hoon. Application of modulated electro-hyperthermia for the cellular target therapy in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B82.

Abstract C155: Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer

Purpose: Ovarian cancer shows heterogeneous characteristics which cause chemoresistance and recurrence. The goal of this study was to elucidate characteristics common to various ovarian cancers in order to evaluate their potential for targeted therapy. Methods: Microarray results for cell lines of serous, mucinous, and brenner types of ovarian cancers were reanalyzed and validated via ovarian cancer cell lines, GEO datasets and TMA blocks. RO-3306 which is an inhibitor of Cdk1 and si-cdk1 was used to measure the growth rate of ovarian cancer cell lines via FACS analysis. In addition, combination treatment with RO-3306 and cisplatin was administered in vitro and in in vivo xenograft mouse model. Results: Microarray reanalysis revealed that expression of Cdk1 and cyclinB1 was higher in 5 types of ovarian cancer cell lines, 4 GEO datasets and ovarian cancer TMA blocks than in HOSE cells. Ovarian cancer TMA particularly showed increased level of cytoplasmic Cdk1 protein, but not in nucleus. When Cdk1 was inhibited by siRNA and a potent Cdk1 inhibitor (RO-3306), growth of ovarian cancer cells decreased, while G2/M arrest or apoptosis increased. In addition, tumor growth was significantly lesser in a xenograft mouse model treated with a combination of RO-3306 and cisplatin than in mice treated with each drug alone. Conclusions: Cdk1 is a promising gene for targeted anticancer therapy. It is expected that combined treatment with Cdk1 inhibitor and chemotherapeutic agents would maximize the effects of ovarian cancer treatment. Citation Format: Hanbyoul Cho, Wookyeom Yang, Ha-Yeon Shin, Eun-ju Lee, Doo-Byung Chay, Jae-Hoon Kim. Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C155.

Abstract POSTER-BIOL-1351: Molecular characterization of cyclin-dependent kinase 1 pathway in newly established epithelial ovarian cancer cell lines

Background: Epithelial ovarian cancer (EOC) is a heterogeneous gynecologic tumor. To better understand the biological pathways involved in the development of EOC, we performed mRNA expression profiling on newly established EOC cell lines. Methods: Five newly established EOC cell lines were characterized by transcript profiling. Microarray analyses were validated using quantitative real-time PCR (RT-PCT) and immunoblotting. Furthermore, biological networks of the differentially expressed genes were predicted using the STRING database and literature review. Results: A total of 239 genes represented by 24,957 probes were identified from microarray analysis, of which 98 and 141 genes are up- (> 2 fold, p Conclusions: These data reveal that the CDK1 pathway is important to EOC development and progression, providing new insight into the biology of EOC. Citation Format: Wookyeom Yang, Hanbyoul Cho, Kim Sol, Jae-Hoon Kim. Molecular characterization of cyclin-dependent kinase 1 pathway in newly established epithelial ovarian cancer cell lines [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1351.

Abstract 1864: Reptin as a tumor-associated antigen of epithelial ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Reptin is also known as RuvBl2, Tip48, TIP49b, ECP51 and CGI-46, is a member of AAA+ (ATPases associated with diverse cellular activities) superfamily. Reptin is associated with cell viability and development of S. cerevisiae, D. melanogaster, X. laevis, or D. rerio has a lot of functions including transcriptional regulation, DNA damage repair and telomerase activity. Self-reactive IgG autoantibodies are present on human sera.Each self-reactive IgG autoantibody derived from pre- and post-treatment of ovarian cancer patients is competitively combined with cancer tissue gotten during the operation, self-reactive IgG autoantibodies bound to tumor antigens. As a result, self-reactive IgG autoantibodies may be decreased after treatment. In this study, we have identified and investigated the function of Reptin as an available therapeutic biomarker for ovarian cancer using difference of self-reactive IgG autoantibody amounts. Method: We carried out the two-dimensional differential gel electrophoresis analysis of immunoprecipitated tumor antigens (2D-DITA) to compare with expression level of autoantibodies in pre- and post-treatment sera of 14 ovarian cancer patients. Autoantibody differentiated between pre- and post-treatment sera was identified by MALDI-TOF. We performed qRT-PCR with 8 human ovarian surface epitheliums (HOSEs) and 14 ovarian cancer cell lines for validation studies. Reptin was also detected in other ovarian cancer tissues by immunohistochemistry (IHC). We constructed SKOV-3 cell line for overexpression Reptin using lentiviral system. For investigating function of Reptin, we performed proliferation, wound healing, and invasion assay. Results: We identified Reptin as a tumor-associated antigen of epithelial ovarian cancer. Relative mRNA level of Reptin showed higher levels in ovarian cancer cell lines than in HOSEs. Reptin protein levels were significantly upregulated in ovarian cancer tissues than normal, benign, and borderline ovarian tumors. In SKOV-3 cells, overexpression of Reptin induced inhibition of cell proliferation (P<0.0001), migration and invasion (P<0.0001). Conclusion: Our results demonstrate that Reptin is a tumor -associated antigen of ovarian cancer, i.e. tumor suppressor. Citation Format: Eun-ju Lee, Hanbyoul Cho, Ha-Yeon Shin, Wookyeom Yang, Hyunja Kwon, Sol Kim, Jae-Hoon Kim. Reptin as a tumor-associated antigen of epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2014-1864