Yuriko Uehara

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.

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.

PI3K/mTOR pathway inhibition overcomes radioresistance via suppression of the HIF1-α/VEGF pathway in endometrial cancer

Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, biomarkers that predict radiosensitivity and drugs to enhance this sensitivity have not yet been established. We aimed to investigate the roles of TP53 and MAPK/PI3K pathways in endometrial carcinomas and to identify appropriate radiosensitizing therapeutics. D10 values (the irradiating dose required to reduce a cell population by 90%) were determined in eight endometrial cancer cell lines with known mutational statuses for TP53, PIK3CA, and KRAS. Cells were exposed to ionizing radiation (2-6Gy) and either a dual PI3K/mTOR inhibitor (NVP-BEZ235) or a MEK inhibitor (UO126), and their radiosensitizing effects were evaluated using clonogenic assays. The effects of silencing hypoxia-inducible factor-1 α (HIF-1α) expression with small interfering RNAs (siRNAs) were evaluated following exposure to ionizing radiation (2-3Gy). D10 values ranged from 2.0 to 3.1Gy in three cell lines expressing wild-type TP53 or from 3.3 to more than 6.0Gy in five cell lines expressing mutant TP53. NVP-BEZ235, but not UO126, significantly improved radiosensitivity through the suppression of HIF-1α/vascular endothelial growth factor-A expression. HIF-1α silencing significantly increased the induction of the sub-G1 population by ionizing radiation. Our study data suggest that TP53 mutation and PI3K pathway activation enhances radioresistance in endometrial carcinomas and that targeting the PI3K/mTOR or HIF-1α pathways could improve radiosensitivity.

Antitumor activity of a combination of dual PI3K/mTOR inhibitor SAR245409 and selective MEK1/2 inhibitor pimasertib in endometrial carcinomas

OBJECTIVE We aimed to clarify whether dual inhibition of PI3K/MAPK and MAPK pathways synergistically suppresses cell growth in endometrial cancer cells. METHODS We exposed a panel of 12 endometrial cancer cell lines to a PI3K/mTOR inhibitor (voxtalisib, SAR245409) and/or a MEK inhibitor (pimasertib). The effect of each drug singly or in combination was evaluated by MTT assay, flow cytometry, and immunoblotting. Combination indexes (CIs) were calculated using the Chou-Talalay method to evaluate the synergy. RESULTS The IC50 values for SAR245409 and pimasertib varied from 0.5 μM to 7 μM and from 0.1 μM to >20 μM, respectively. A combination of both compounds (1 μM SAR245409 and 30 nM pimasertib) caused a synergistic antitumor effect in 6 out of 12 endometrial cell lines (CI, 0.07-0.46). The synergistic effect was exclusively observed in 6 pimasertib-sensitive cell lines (IC50 of pimasertib, ≤5 μM). We found that 30 nM pimasertib, a concentration much lower than the IC50 for each cell line, was sufficient to cause a synergistic effect with SAR245409. Flow cytometric analysis showed that this combination significantly increased the population of G1 cells. However, a combination of rapamycin (an mTOR inhibitor) and pimasertib did not induce a synergistic effect in endometrial cancer cells, except for HEC-1B cells. CONCLUSIONS The combination of a PI3K/mTOR inhibitor and a MEK inhibitor induced a synergistic antitumor effect in certain endometrial cancer cells. This study underscores the importance of using optimized doses of antitumor agents, singly or in combination, in treating endometrial cancer.

Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer

BackgroundPrevious studies have shown that the cell polarity protein partitioning defective 3 (Par3) plays an essential role in the formation of tight junctions and definition of apical-basal polarity. Aberrant function of this protein has been reported to be involved in epithelial–mesenchymal transition (EMT) and cancer invasion. The aim of this study was to examine the functional mechanism of Par3 in ovarian cancer.MethodsFirst, we investigated the association between Par3 expression level and survival of 50 ovarian cancer patients. Next, we conducted an in vitro analysis of ovarian cancer cell lines, focusing on the cell line JHOC5, to investigate Par3 function. To investigate the function of Par3 in invasion, the IL-6/STAT3 pathway was analyzed upon Par3 knockdown with siRNA. The effect of siRNA treatment was assessed by qPCR, ELISA, and western blotting. Invasiveness and cell proliferation following treatment with siRNA against Par3 were investigated using Matrigel chamber, wound healing, and cell proliferation assays.ResultsExpression array data for ovarian cancer patient samples revealed low Par3 expression was significantly associated with good prognosis. Univariate analysis of clinicopathological factors revealed significant association between high Par3 levels and peritoneal dissemination at the time of diagnosis. Knockdown of Par3 in JHOC5 cells suppressed cell invasiveness, migration, and cell proliferation with deregulation of IL-6/STAT3 activity.ConclusionTaken together, these results suggest that Par3 expression is likely involved in ovarian cancer progression, especially in peritoneal metastasis. The underlying mechanism may be that Par3 modulates IL-6 /STAT3 signaling. Here, we propose that the expression of Par3 in ovarian cancer may control disease outcome.

MDM2 is a potential therapeutic target and prognostic factor for ovarian clear cell carcinomas with wild type TP53

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.

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 4917: Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives: Circulating cell-free DNA (cfDNA) is expected to be useful for diagnosis of genetic alterations in cancer. However, it has been still difficult to comprehensively analyze somatic mutations using cfDNA, because of the low quantity in blood. We examined the feasibility of whole exome sequencing using cfDNA and assessed the ratio of mutation reads in cfDNA for somatic mutations comparing with the corresponding tumor DNA in ovarian cancer specimens. In addition, we assessed whether cell free DNA might reflect the tumor heterogeneity in ovarian cancer. Methods: The pair of fresh frozen tumor samples and blood samples was obtained from two cases of ovarian serous adenocarcinoma under informed consent and approval of institutional review board. The tumor DNA and cfDNA were extracted from the specimens and whole-exome sequencing was performed using 1ug of the tumor DNA or 25ng of cfDNA. The list of the mutated genes was compared between cell free DNA and the corresponding tumor DNA. Validation of the mutations and calculation of the tumor content ratio in cfDNA was assessed using target sequencing. Result: We successfully detected the somatic mutations from cfDNA. The somatic mutations detected in cfDNA covered from 21% (49/233) to 55% (141/258) of the mutations detected in the corresponding tumor DNA. TP53 was included in the mutated genes, and the base substitution (chr17:7578257 C>A) was identified in 5 of 121 reads (4%) in cfDNA. We validated the tumor content ratio of cfDNA is 8% by target sequencing. One of the two cases showed the correlation between the mutant allele frequency in the tumor DNA and the detection ratio of the mutations in cfDNA (p = 0.004, logistic regression analysis). However, the correlation was not observed in another case (p = 0.3923, logistic regression analysis). Of note, we detected various types of mutations in cfDNA alone. One of such genes is RAPGEF2. The base substitution (ch4:160253031 C>T) was detected only in cfDNA (11/53, 21%), but not in tumor DNA (0/120, 0%). Conclusion: Our data suggest that whole exome sequencing of cfDNA is feasible. The mutations detected only in cfDNA might reflect the tumor heterogeneity, and cfDNA might provide us novel biomarkers for diagnosis of recurrence and/or metastasis. Further study is warranted to elucidate the method to utilize the genetic information from cfDNA. Note: This abstract was not presented at the meeting. Citation Format: Kayo Asada, Katsutoshi Oda, Kengo Gotoh, Reiko Kurikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Yuji Ikeda, Yuriko Uehara, Kenbun Sone, Osamu Hiraike-Wada, Kei Kawana, Tetsu Yano, Yutaka Osuga, Tomoyuki Fujii, Hiroyuki Aburatani. Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4917. doi:10.1158/1538-7445.AM2015-4917

Abstract 2402: Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The loss of PTEN is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in the nucleus. Poly (ADP-ribose) polymerase (PARP) plays a key role 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 a panel of 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 introduction on the sensitivity of cells 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. Results The SF50 values were 100 nM or less in six of the 16 (38%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in five of the 16 (31%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 995 ± 1,096 nM; Wild-type [n = 4]: 144 ± 91 nM, p = 0.17 by Studentst 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. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. The inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. Citation Format: Aki Miyasaka, Katsutoshi Oda, Yuji Ikeda, Tomoko Kashiyama, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Chinami Makii, Kayo Asada, Reiko Kurikawa, Osamu Wada Hiraike, Kenbun Sone, Yuriko Uehara, Yoko Matsumoto, Takahide Arimoto, Hiroyuki Kuramoto, Tetsu Yano, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii. Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma 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 2402. doi:10.1158/1538-7445.AM2014-2402

Abstract A19: Comparison of genomic alteration patterns between ovarian clear cell carcinoma and renal cell carcinoma

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<0.05 by Fishers exact test) were more common in OCCC. Distinct from ccRCC, we did not detect any mutations in VHL and copy number losses at chr 3p in OCCC. Conclusions: Genetic alteration patterns in the SWI/SNF complex and the PI3K/mTOR pathway are distinct between OCCC and ccRCC. Our data suggest that targeting mTOR pathway might be a promising therapeutic strategy in OCCC, as well as in ccRCC. 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.