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PI3K pathway signaling has received attention as a molecular target in clear cell ovarian carcinoma (CCOC). MDM2 is one of the AKT effectors in the PI3K pathway, which binds to and degrades p53. In this study, we aimed to clarify the prognostic significance of PIK3CA and MDM2 expression, and potential therapeutic effect of a dual inhibition of the PI3K pathway and MDM2.cDNA expression was evaluated by using microarray data using 75 samples of CCOC. DS-7423 (dual inhibitor of pan-PI3K and mTOR) and RG7112 (MDM2 inhibitor) were used on CCOC cell lines to evaluate cell proliferation, expression level of MDM2 related proteins, and apoptosis by MTT assay, western blotting, and flow cytometry. DS-7423 (3 mg/kg) and/or RG7112 (50 mg/kg) were orally administrated every day for three weeks, and the anti-tumor effect was evaluated using tumor xenografts, along with immunohistochemistry.Tumors with high expression of both PIK3CA and MDM2 showed significantly worse prognosis in expression array of 71 CCOCs (P = 0.013). Dual inhibition of the PI3K pathway by DS-7423 and MDM2 by RG7112 showed synergistic anti-proliferative effect in 4 CCOC cell lines without TP53 mutations. The combination therapy more robustly induced pro-apoptotic proteins (PUMA and cleaved PARP) with increase of sub G1 population and apoptotic cells, compared with either single agent alone. The combination therapy significantly reduced tumor volume in mice (P 0.001 in OVISE, and P = 0.038 in RMG-I) without severe body weight loss. Immunohistochemistry from the xenograft tumors showed that the combination treatment significantly reduced vascularity and cell proliferation, with an increase of apoptotic cell death.A combination therapy targeting the PI3K pathway and MDM2 might be a promising therapeutic strategy in CCOC.

// Chinami Makii 1 , Katsutoshi Oda 1 , Yuji Ikeda 1 , Kenbun Sone 1 , Kosei Hasegawa 2 , Yuriko Uehara 1, 3 , Akira Nishijima 1, 3 , Kayo Asada 1, 3 , Takahiro Koso 1, 3 , Tomohiko Fukuda 1 , Kanako Inaba 1 , Shinya Oki 1 , Hidenori Machino 1 , Machiko Kojima 1 , Tomoko Kashiyama 1 , Mayuyo Mori-Uchino 1 , Takahide Arimoto 1 , Osamu Wada-Hiraike 1 , Kei Kawana 1 , Tetsu Yano 4 , Keiichi Fujiwara 2 , Hiroyuki Aburatani 3 , Yutaka Osuga 1 , Tomoyuki Fujii 1 1 Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Department of Gynecologic Oncology, Saitama Medical University International Medical Center, Saitama, Japan 3 Division of Genome Science, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan 4 Department of Obstetrics and Gynecology, National Center for Global Health and Medicine, Tokyo, Japan Correspondence to: Katsutoshi Oda, email: katsutoshi-tky@umin.ac.jp Keywords: MDM2, TP53, prognosis, molecular-targeted therapy, ovarian clear cell carcinoma Received: May 14, 2016 Accepted: September 02, 2016 Published: September 21, 2016 ABSTRACT MDM2, a ubiquitin ligase, suppresses wild type TP53 via proteasome-mediated degradation. We evaluated the prognostic and therapeutic value of MDM2 in ovarian clear cell carcinoma. MDM2 expression in ovarian cancer tissues was analyzed by microarray and real-time PCR, and its relationship with prognosis was evaluated by Kaplan-Meier method and log-rank test. The anti-tumor activities of MDM2 siRNA and the MDM2 inhibitor RG7112 were assessed by cell viability assay, western blotting, and flow cytometry. The anti-tumor effects of RG7112 in vivo were examined in a mouse xenograft model. MDM2 expression was significantly higher in clear cell carcinoma than in ovarian high-grade serous carcinoma ( P = 0.0092) and normal tissues ( P = 0.035). High MDM2 expression determined by microarray was significantly associated with poor progression-free survival and poor overall survival ( P = 0.0002, and P = 0.0008, respectively). Notably, RG7112 significantly suppressed cell viability in clear cell carcinoma cell lines with wild type TP53 . RG7112 also strongly induced apoptosis, increased TP53 phosphorylation, and stimulated expression of the proapoptotic protein PUMA. Similarly, siRNA knockdown of MDM2 induced apoptosis. Finally, RG7112 significantly reduced the tumor volume of xenografted RMG-I clear cell carcinoma cells ( P = 0.033), and the density of microvessels ( P = 0.011). Our results highlight the prognostic value of MDM2 expression in clear cell carcinoma. Thus, MDM2 inhibitors such as RG7112 may constitute a class of potential therapeutics.

金沢大学医薬保健研究域医学系, Correction to: PLoS ONE https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128066

DS-7423, a novel, small-molecule dual inhibitor of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), is currently in phase I clinical trials for solid tumors. Although DS-7423 potently inhibits PI3Kα (IC50 = 15.6 nM) and mTOR (IC50 = 34.9 nM), it also inhibits other isoforms of class I PI3K (IC50 values: PI3Kβ = 1,143 nM; PI3Kγ = 249 nM; PI3Kδ = 262 nM). The PI3K/mTOR pathway is frequently activated in ovarian clear cell adenocarcinomas (OCCA) through various mutations that activate PI3K-AKT signaling. Here, we describe the anti-tumor effect of DS-7423 on a panel of nine OCCA cell lines. IC50 values for DS-7423 were

Background PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. Results The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer.

Aims Mucinous borderline tumours of the ovary are subclassified as intestinal-type (IMBT) and endocervical-like (EMBT), which differ in their clinicopathological features. In this study, we attempted to elucidate characteristics of the mucinous epithelium in each subtype. Methods and results The expression of claudin-18, a marker of gastric differentiation, MUCs, CDX2, CK7, CK20, oestrogen receptor (ER), progesterone receptor (PgR), CA-125 and vimentin in IMBTs (n = 54), EMBTs (n = 25) and serous borderline tumours (SBTs) (n = 22) were compared by immunohistochemistry. Claudin-18 positivity was identified in 98% of the IMBTs, whereas only 4% of the EMBTs were claudin-18-positive. Expression of intestinal markers such as CDX2 and MUC2 was relatively infrequent in IMBTs (48% and 33%, respectively). Mullerian-lineage markers such as ER, PgR and vimentin were expressed rarely in IMBTs, while most EMBTs and SBTs were positive for these markers. Hierarchial clustering revealed a close association between EMBTs and SBTs, while IMBTs were clearly separate. Conclusions Claudin-18 positivity is a specific phenotype that is characteristic of IMBTs. Frequent and diffuse expression of gastric markers, along with less frequent and usually focal expression of intestinal markers, suggests that IMBTs are essentially composed of gastrointestinal-type mucinous epithelium (gastric-type epithelium with a variable degree of intestinal differentiation).

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

Introduction: Wnt/beta-catenin/cyclin D1 pathway has been suggested to be involved in cell proliferation in endometrioid endometrial carcinomas (EEC), in addition to the activation of the PI3K pathway via PIK3CA and/or PTEN mutations. We previously reported that the mutations of cyclin D1 (CCND1 gene) promotes cell proliferation in EEC. However, the role of beta-catenin and its relationship with cyclin D1 is not yet well known. In this study, we focused on alterations of beta-catenin and cyclin D1 and their clinical impact in EEC. Methods: This study was conducted under approval of ethics committee of the institute and informed consent from each patient. Expressions of cyclin D1 and beta-catenin were examined by tissue microarray in formalin fixed 120 samples of EEC. The expression levels were evaluated by two pathologists, and the staining was classified as overexpressed when >50% of tumor cells showed strong staining in cytoplasm (for beta-catenin) and nucleus (for cyclin D1). Genomic DNA was isolated in 91 primary surgical specimens. Mutations of beta-catenin (exon 3 of CTNNB1 gene) and Cyclin D1 (exon 5 of CCND1 gene) were examined by PCR-direct sequencing. We constructed expression plasmid of various types of mutant beta-catenin and performed colony formation assay using these plasmids. Result: In 120 samples, beta catenin and cyclin D1 were overexpressed in 5 (4%) and 7 (6%) cases, respectively. Median follow-up period of these patients is 85 months. Interestingly, over-expression of these two proteins was not overlapped each other. In these 12 samples, 7 of 12 cases (58%) were stage III or IV. However, recurrence was only observed in one patient (5-year progression free survival rate is 92%). Mutations of CCND1 and CTNNB1 were detected in 2 (2%) and 9 (10%) cases, respectively. We confirmed that all these mutations are somatic. Mutations of CCND1 and CTNNB1 were also mutually exclusive. Although the two cases with CCND1 mutations died of disease, none of the nine cases with CTNNB1 mutations got recurrent (p=0.018 by Fisher's exact test). In colony formation assay, mutant beta-catenin at codon 32 and codon 37 did not increase the colony number, compared with wild-type beta-catenin (control), although mutants at codon 34 significantly augmented the colony formation. Conclusion: Aberrant expression of both beta-catenin and cyclin D1 are present in EEC, through mutations and/or overexpression, and alterations of beta-catenin might be associated with favorable prognosis. Our data suggest that function of mutant beta-catenin might be distinct among each mutation type. Further study is warranted to clarify the role of wnt/beta-catenin/cyclin D1 pathway in EEC. Citation Format: Yuji Ikeda, Katsutoshi Oda, Takahiro Koso, Daichi Maeda, Osamu Wada Hiraike, Tomoko Kashiyama, Aki Miyasaka, Kei Kawana, Tetsu Yano, Yoshihiro Kikuchi, Masashi Fukayama, Yutaka Osuga, Tomoyuki Fujii. Aberrant expression of beta-catenin and cyclin D1 in endometrial 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 1314. doi:10.1158/1538-7445.AM2014-1314

Background: Ovarian clear cell carcinoma (OCCC) is a distinct histopathological subtype in epithelial ovarian cancer, and its pathological features and gene expression profiles are similar to those of clear cell renal cell carcinoma (ccRCC), including HIF pathway activation. Methods: We performed whole exome sequencing and/or SNP typing array in 45 OCCC clinical samples, and obtained the data of ccRCC from TCGA. Results: We identified several genetic mutations in SWI/SNF complex and PI3K/mTOR pathway, which had been reported in ccRCC, but not in OCCC. Mutations of ARID1A (BAF250a) were predominant in OCCC (55% vs 4%), whereas PBRM1 (BAF180) mutations were detected only in ccRCC at 35%. Mutations of PIK3CA (60% vs 3.0%) and the genetic alterations in the PI3K/mTOR pathway (65% vs 30%, p Citation Format: Kayo Asada, Takahiro Koso, Katsutoshi Oda, Yuriko Uehara, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Reiko Kurikawa, Yuji Ikeda, Osamu Hiraike, Kei Kawana, Tetsu Yano, Yutaka Osuga, Tomoyuki Fujii, Hiroyuki Aburatani. Comparison of genomic alteration patterns between ovarian clear cell carcinoma and renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A19.

Introduction. Cyclin D1 is an important regulator of cell cycle progression. Phosphorylation of cyclin D1 at Thr-286 by GSK3beta triggers nuclear export and its cytoplasmic proteolysis via the 26S proteasome. Cyclin D1 overexpression is a common event in various types of human cancers; however,mutations of CCND1 (encoding cyclin D1) have been only reported in endometrial cancer and esophageal cancer. We previously reported T286I mutations of CCND1 in 2 endometrial carcinomas (2.3%). The objective of this study was to identify the functions of T286I mutant of cyclin D1 in endometrial cancer. Materials and methods. T286I mutant cyclin D1 (CD1-T286I) plasmid was generated using wild-type cyclin D1 (CD1-WT) plasmid via site-directed mutagenesis technique. pcDNA3 empty plasmid was used as a control (CD1-Ct). Using these plasmids, the functions of mutant CD1-T286I were analyzed by luciferase assays, immunofluorescence, western blotting and colony formation assays in HEK293T (kidney) and HEC-50B (endometrial cancer) cells, both of which do not possess CCND1 mutations. Statistical analysis was performed by student-T test and p Citation Format: Yuji Ikeda, Katsutoshi Oda, Takahiro Koso, Aki Miyasaka, Tomoko Kashiyama, Osamu Hiraike-Wada, Daichi Maeda, Kei Kawana, Shunsuke Nakagawa, Osamu Tetsu, Yoshihiro Kikuchi, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Cyclin D1 harboring T286I mutation promotes oncogenic activation in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2013-1748

Background:Epithelial ovarian cancer is a leading cause of death from gynecological malignancies.Ovarian clear cell adenocarcinoma (OCCA) is the second most common cause of death from ovarian cancer with a higher incidence in Asia, especially Japan (>25%). Due to low response rates to conventional platinum-based chemotherapy, the clinical outcome is generally poor. PIK3CA mutations are frequent (>40%) in OCCA compared with other histology types and overexpression of receptor tyrosine kinases (RTKs), including HER2 and cMET, is also frequently observed in OCCA. Thus, the RTK-PI3K (phosphatidylinositol 3-kinase)/mTOR (mammalian target of rapamycin) signaling axis is broadly activated in OCCA. This study evaluated the anti-tumor efficacy of DS-7423, a novel PI3K/mTOR dual inhibitor, in a panel of OCCA cell lines. Material and methods: We evaluated the phosphorylation status of RTK-PI3K/mTOR signaling pathway in 9 OCCA cell lines. Four of those cell lines possessed PIK3CA mutations; 3 of the 5 remaining cell lines overexpressed HER2, HER3 and/or cMET. We determined the effect of DS-7423 by MTT assay, FACS analysis, and Western blotting. Apoptosis induction was detected by annexin-V and propidium iodide (PI) staining. Induction of p53 target genes, including p53AIP1, was evaluated by RT-PCR. Results: IC50 values for DS-7423 were Citation Format: Tomoko Kashiyama, Katsutoshi Oda, Yuji Ikeda, Yoshinobu Shiose, Yasuhide Hirora, Aki Miyasaka, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Keiko Shoji, Michihiro Tanikawa, Kazunori Nagasaka, Osamu Hiraike-Wada, Kei Kawana, Hiroyuki Aburatani, Shunsuke Nakagawa, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 525. doi:10.1158/1538-7445.AM2013-525

[Introduction] Synchronous cancers involving ovaries as well as uterine corpus are well-known events in gynecologic malignancies. These tumors may be independently derived, non-metastatic tumors (Dual Primary tumors; DP) or a tumor from one organ with metastasis to the other (Single Primary tumor with Metastasis; SPM). In the majority of these synchronous endometrial and ovarian cancers, both tumors are histologically endometrioid adenocarcinomas, which may cause a diagnostic difficulty to distinguish DP and SPM. By genome-wide genotyping, single nucleotide polymorphism (SNP) arrays, we can obtain chromosomal copy number alterations (CNA) throughout the genome in a single assay. We hypothesized that the copy number profiles by SNP arrays might be highly informative for genetic diagnosis in the synchronous endometrioid adenocarcinomas. [Material and Methods] We genetically diagnosed ten tumors from five patients with synchronous endometrial and ovarian carcinomas, using 250K SNP typing arrays and mutational analysis of PIK3CA, PTEN, K-Ras and CTNNB1 (beta-catenin). We evaluated whether conventional pathological diagnosis is compatible with the genetic diagnosis. [Results] Three of the five patients show identical copy number alterations (CNA), including type, loci and degree of each alteration, between the endometrial and the ovarian carcinomas. The other two show CNA only in either endometrial or ovarian carcinoma. All the five tumors possess one or more genetic mutations in the genes examined. One patient showed mutations both in PIK3CA and PTEN at discordant sites between endometrial and ovarian carcinomas, whereas the other four showed concordant mutations. Together, four of the five were genetically diagnosed as SPM and the remaining one was as DP. The pathological diagnosis was not in agreement with the molecular diagnosis in four of the five cases. [Conclusion] Our data suggest that most of the copy number alterations might occur before metastases and that genome-wide genotyping may represent a useful approach to distinguish between SPM and DP in synchronous endometrial and ovarian carcinomas. As chromosomal instability is commonly observed in various types of tumors, SNP array genotyping might be applicable to synchronous tumors in other organs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1702. doi:1538-7445.AM2012-1702