Keiko Shoji

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

Background:The phosphatidylinositol 3′-kinase (PI3K)–AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation.Methods:We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing.Results:We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras.Interpretation:Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K–AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas.

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.

Identification of DBC1 as a transcriptional repressor for BRCA1

Background:DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression.Methods:A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied.Results:We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1.Conclusion:These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.

Genome-wide single-nucleotide polymorphism arrays in endometrial carcinomas associate extensive chromosomal instability with poor prognosis and unveil frequent chromosomal imbalances involved in the PI3-kinase pathway

Endometrial cancer is one of the tumor types in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur. It is known to possess mutations frequently in the Ras-PI3K (phosphatidylinositol 3′-kinase) pathway. We performed a comprehensive genomic survey in 31 endometrial carcinomas with paired DNA for chromosomal imbalances (25 by the 50K and 6 by the 250K single-nucleotide polymorphism (SNP) array), and screened 25 of the 31 samples for MSI status and mutational status in the Ras-PI3K pathway genes. We detected five or more copy number changes (classified as CIN-extensive) in 9 (29%), 1 to 4 changes (CIN-intermediate) in 17 (55%) and no changes (CIN-negative) in 5 (16%) tumors. Positive MSI was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%), and multivariate analysis showed that CIN-extensive is an independent poor prognostic factor. SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN. In addition to eight (26%) tumors with PTEN deletions, we detected chromosomal imbalances of NF1, K-Ras and PIK3CA in four (13%), four (13%) and six (19%) tumors, respectively. In all, 7 of the 9 CIN-extensive tumors harbor deletions in the loci of PTEN and/or NF1, whereas all the 10 MSI-positive tumors possess PTEN, PIK3CA and/or K-Ras mutations. Our results showed that genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability, and suggest that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas.

Antitumor Activity and Induction of TP53-Dependent Apoptosis toward Ovarian Clear Cell Adenocarcinoma by the Dual PI3K/mTOR Inhibitor DS-7423

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 <75 nM in all the lines, regardless of the mutational status of PIK3CA. In mouse xenograft models, DS-7423 suppressed the tumor growth of OCCA in a dose-dependent manner. Flow cytometry analysis revealed a decrease in S-phase cell populations in all the cell lines and an increase in sub-G1 cell populations following treatment with DS-7423 in six of the nine OCCA cell lines tested. DS-7423-mediated apoptosis was induced more effectively in the six cell lines without TP53 mutations than in the three cell lines with TP53 mutations. Concomitantly with the decreased phosphorylation level of MDM2 (mouse double minute 2 homolog), the level of phosphorylation of TP53 at Ser46 was increased by DS-7423 in the six cell lines with wild-type TP53, with induction of genes that mediate TP53-dependent apoptosis, including p53AIP1 and PUMA at 39 nM or higher doses. Our data suggest that the dual PI3K/mTOR inhibitor DS-7423 may constitute a promising molecular targeted therapy for OCCA, and that its antitumor effect might be partly obtained by induction of TP53-dependent apoptosis in TP53 wild-type OCCAs.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function

Background:The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams–Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date.Methods:A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated.Results:We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1.Conclusion:These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Reply: Somatic mutations are present in all members of the AKT family in endometrial carcinoma

Reply: Somatic mutations are present in all members of the AKT family in endometrial carcinoma

Abstract 525: Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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 <100 nM for all 9 OCCA cell lines. Efficacy did not significantly differ between PIK3CA mutant and wild type OCCA cells. AKT and its downstream targets (GSK3β, FOXO1/3a, MDM2, 4EBP1, and S6) were phosphorylated in OCCA cell lines regardless of PIK3CA mutational status. DS-7423 suppressed the phosphorylation levels of AKT and its target proteins in all these OCCA cell lines. FACS analysis revealed an increase in sub-G1 cell populations by treatment at ≥156 nM DS-7423 in 6 out of 9 OCCA cell lines. In these 6 cell lines, the number of apoptotic cells, as determined by annexin-V and PI staining, increased in a dose-dependent manner and DS-7423 induced apoptosis more effectively in TP53 wild type OCCA cell lines than in TP53 mutant OCCA cell lines. In accordance with decreased phospho-MDM2 levels, phosphorylation levels of p53 (Ser46) were increased and expression of p53AIP1 and PUMA, genes regulating p53-dependent apoptosis, were induced by DS-7423 in these p53 wild type cells. We also evaluated the expression status of bcl-2 family proteins in the OCCA cell line OVISE, and observed that DS-7423 treatment induced up-regulation of proapoptotic Bim and down-regulation of anti-apoptotic Mcl-1. Conclusions: Dual PI3K/mTOR inhibitors such as DS-7423 may constitute a promising molecular targeted therapy for OCCA, regardless of PIK3CA status. DS-7423 may induce apoptosis via activation of p53 and bcl-2 family members. As p53 mutations ar generally rare in OCCA, DS-7423 might induce p53-dependent apoptosis in this cancer type. 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

Abstract 1702: A novel diagnostic approach using genome-wide single nucleotide polymorphism arrays for ovarian metastases in endometrial cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL [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

Abstract 4494: In vivo antitumor activity of NVP-BEZ235, a dual PI3K/mTOR inhibitor, and RAD001 (everolimus), an mTOR inhibitor, in endometrial cancer

Purpose; PI3K (phosphatidylinositol-3-kinase) and its downstream mTOR (mammalian target of rapamycin) pathway play a critical role in diverse cellular functions, including proliferation, growth and cell survival. PI3K-mTOR pathway is frequently activated in endometrial cancer through various PI3K/AKT activating genetic alterations, such as mutations in PTEN, PIK3CA, and K-Ras. It has not been well analyzed whether mTOR inhibition alone is as effective as dual PI3K/mTOR inhibition to cancer cells with the activating PI3K pathway mutations, especially in vivo. In this study, we compared in vivo anti-tumor activity of a dual PI3K.inhibitor, NVP-BEZ235, with an mTOR inhibitor, RAD001, in endometrial cancer cell lines with the PI3K/AKT activating mutations.Experimental procedure; We screened 13 endometrial cancer cell lines, 11 of which possess one or more PI3K/AKT activating mutations. Among them, we selected two cell lines; AN3CA with a nonsense mutation in PTEN, and Hec-59 with double mutations in PIK3CA and PTEN. The IC50 values for cell proliferation by RAD001 were 14nM in AN3CA and 220nM in Hec-59, whereas the values by NVP-BEZ235 were 20nM in AN3CA and 24nM in Hec-59. We examined the effect of RAD001 (2.5mg/kg/day) and BEZ235 (40mg/kg/day) on tumor growth in vivo, using mice inoculated with endometrial cancer cells. We analyzed the phosphorylation levels of Akt, FOXO, GSK3beta and S6 in the tumors by immunoblotting. Results;Both BEZ and RAD001, compared with placebo, significantly suppressed the tumor growth in xenograft mice in the two cell lines. No significant adverse effects were observed with either compounds in any of the mice. Inconsistent with the in vitro data, the effect was comparable between BEZ235 and RAD001 even in Hec-59 cells, although the IC50 value was much higher with RAD001 than NVP-BEZ235. In Hec-59, NVP-BEZ235 clearly suppressed all the phosphorylation level of Akt, FOXO, GSK3beta and S6 in one hour from the treatment. However, all the phosphorylation levels were completely recovered to the base level within 24 hours. RAD001 clearly suppressed the p-S6 level in 1 hour and the effect partly remained even after 24 hours from the treatment. Conclusion; We demonstrated that both NVP-BEZ235 and RAD001 suppressed tumor growth in vivo in endometrial cancer cell lines with PIK3CA and/or PTEN mutations. The comparable effect of NVP-BEZ235 and RAD001 might be explained by the recovery of the phosphorylation levels of the target proteins, such as p-Akt, suggesting that sufficient suppression of the PI3K/mTOR pathway for full time-course might be required to robustly induce the anti-tumor effect by these inhibitors. In clinical trials, both pharmacokinetic and pharmacodinamic analyses would be important to appropriately assess the effect of these inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2011-4494