Kenbun Sone

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.

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: An implicative role of SIRT1 in the ovary

BackgroundResveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary.MethodsThe physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining.ResultsSIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol.ConclusionsThese results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.

Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

The presence of phosphorylated histone H2AX (γ-H2AX) is associated with the local activation of DNA-damage repair pathways. Although γ-H2AX deregulation in cancer has previously been reported, the molecular mechanism involved and its relationship with other histone modifications remain largely unknown. Here we find that the histone methyltransferase SUV39H2 methylates histone H2AX on lysine 134. When H2AX was mutated to abolish K134 methylation, the level of γ-H2AX became significantly reduced. We also found lower γ-H2AX activity following the introduction of double-strand breaks in Suv39h2 knockout cells or on SUV39H2 knockdown. Tissue microarray analyses of clinical lung and bladder tissues also revealed a positive correlation between H2AX K134 methylation and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemosensitivity of cancer cells. Overall, our results suggest that H2AX methylation plays a role in the regulation of γ-H2AX abundance in cancer.

Repression of estrogen receptor β function by putative tumor suppressor DBC1

It has been well established that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor (ER) alpha appears to promote the proliferation of cancer tissues, while ERbeta can protect against the mitogenic effect of estrogen in breast tissue. The expression status of ERalpha and ERbeta may greatly influence on the development, treatment, and prognosis of breast cancer. Previous studies have indicated that the deleted in breast cancer 1 (DBC1/KIAA1967) gene product has roles in regulating functions of nuclear receptors. The gene encoding DBC1 is a candidate for tumor suppressor identified by genetic search for breast cancer. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of the endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. In addition, DBC1 modulates ERalpha expression and promotes breast cancer cell survival by binding to ERalpha. Here we report an ERbeta-specific repressive function of DBC1. Immunoprecipitation and immunofluorescence studies show that ERbeta and DBC1 interact in a ligand-independent manner similar to ERalpha. In vitro pull-down assays revealed a direct interaction between DBC1 amino-terminus and activation function-1/2 domain of ERbeta. Although DBC1 shows no influence on the ligand-dependent transcriptional activation function of ERalpha, the expression of DBC1 negatively regulates the ligand-dependent transcriptional activation function of ERbetain vivo, and RNA interference-mediated depletion of DBC1 stimulates the transactivation function of ERbeta. These results implicate the principal role of DBC1 in regulating ERbeta-dependent gene expressions.

Loss of Hugl-1 expression associates with lymph node metastasis in endometrial cancer

Mutation of neoplastic tumor suppressor genes, scribble, discs large, and lethal giant larvae (lgl), causes disruption of cell polarity and overproliferation of Drosophila epithelial cells and neuroblasts. Reduced expression of human homologue of lgl, Hugl-1, has been reported to be involved in development and progression of human colon cancer and malignant melanoma. To explore the association between Hugl-1 expression and clinical character in endometrial cancer, we examined the expression of Hugl-1 in primary endometrial cancer tissues. The expression of Hugl-1 mRNA in 86 primary endometrial cancer tissues was examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). All samples were categorized into two groups: Hugl-1 positive and Hugl-1 negative. Clinical data of each group were analyzed by Fishers exact probability test and survival rates of each group were compared by Kaplan-Meier method and Log-rank test. Loss of Hugl-1 expression had correlation with the higher incidence of lymph node metastasis, but not to the patients age at onset, distant metastasis, clinical stage, lymph or venous vessel invasion, or histopathological grade of differentiation. The Hugl-1-positive group had poorer prognosis compared with the Hugl-1-negative group. These results indicate that loss of Hugl-1 expression in endometrial cancer may contribute to lymph node metastasis and it can be a factor of poor prognosis.

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.

The anti-malarial chloroquine suppresses proliferation and overcomes cisplatin resistance of endometrial cancer cells via autophagy inhibition

OBJECTIVE The anti-malarial drug chloroquine (CQ) is also known as an autophagy inhibitor. Autophagy can promote tumor growth by fueling the necessary energy metabolism and inducing resistance to chemotherapy and/or irradiation in various human cancers. However, the role of autophagy in endometrial cancer has not yet been established. We investigated the anti-tumor effects and autophagy inhibition caused by CQ in endometrial cancer cells. METHODS Cell proliferation and cell cycle were assessed in response to CQ in six endometrial cancer cell lines by using an MTT assay and/or flow cytometry. To assess the level of autophagy, western blotting and an immunofluorescence assay were used to measure LC3 expression. The effects of knockdown of either ATG5 or ATG7, both of which are indispensable for induction of autophagy, were assessed via an MTT assay. Sensitivity to CQ was compared between parental and cisplatin-resistant (CP-r) Ishikawa endometrial cancer cells. RESULTS CQ suppressed proliferation in all six endometrial cancer cell lines in a dose-dependent manner, whereas it increased the population of apoptotic cells. Inhibition of autophagy via knockdown of either ATG5 or ATG7 decreased the sensitivity to CQ. Additionally, sensitivity to cisplatin was improved by knocking down ATG5 or ATG7. Finally, CP-r Ishikawa cells, with a high basal level of autophagy, were more sensitive to CQ than parental Ishikawa cells. CONCLUSIONS Our data suggest that autophagy is involved in endometrial tumor growth and cisplatin resistance. Furthermore, our data support a therapeutic role for CQ in endometrial cancer cells with upregulation of autophagy.

Preoperative diagnosis of colouterine fistula secondary to diverticulitis by sonohysterography with contrast medium

Colouterine fistulae secondary to sigmoid diverticulitis are unusual. Methods for diagnosis remain to be established. We report a case with a colouterine fistula in which sonohysterography detected the flow of ultrasound contrast medium between the uterine cavity and the sigmoid colon through the posterior uterine wall, thus confirming the diagnosis. The diagnosis was further substantiated by a charcoal challenge test. The patient underwent en bloc resection of the uterus, Fallopian tubes, ovaries and sigmoid colon, the organs involved with diverticulitis. This is the first report to describe a colouterine fistula successfully diagnosed by sonohysterography using ultrasound contrast medium. Copyright