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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.

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

[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