Kanako Inaba

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.

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.

Putative tumor suppression function of SIRT6 in endometrial cancer.

SIRT6, a member of the sirtuin family, has been identified as a candidate tumor suppressor. To pursue the role of SIRT6 in endometrial cancer, we investigated the anti‐tumorigenic function of SIRT6. The expression of SIRT6 negatively affected the proliferation of AN3CA and KLE endometrial cancer cells. Increased expression of SIRT6 resulted in the induction of apoptosis by repressing the expression of the anti‐apoptotic protein survivin. Consistent with this result, a survivin inhibitor YM155 efficiently inhibited cellular proliferation and induced apoptosis. These results revealed that SIRT6 might function as a tumor suppressor of endometrial cancer cells.

Significance of survivin as a prognostic factor and a therapeutic target in endometrial cancer

INTRODUCTION Survivin is an anti-apoptotic protein encoded by the baculoviral inhibitor of apoptosis repeat-containing (BIRC5) gene and is upregulated in 83% of endometrial cancers. We aimed to elucidate the prognostic importance of BIRC5 expression, and evaluate survivin as a therapeutic target for endometrial cancer, by knock-down of BIRC5 and using the survivin inhibitor-YM155. METHODS RNA sequencing data in 234 patients with endometrial carcinoma was obtained from The Cancer Genome Atlas database, and analyzed using Kaplan-Meier method, log-rank test and Cox proportional hazard model. Expressions of survivin in 16 endometrial cancer cell lines were analyzed by western blotting. Knocking down effect on survivin expression was evaluated using a small interfering RNA (siRNA). The anti-proliferative and pro-apoptotic effects of YM155 were assessed with cell viability, flow cytometry, and annexin V/propidium iodide assays. RESULTS High expression of BIRC5 was associated with poor progression free survival (P=0.006), and shown to be an independent prognostic factor (HR=1.97, 95% CI=1.29-4.5, P=0.045). Survivin was upregulated in 14 of 16 (87.5%) endometrial cancer cell lines, compared with endometrial immortalized cells. Apoptosis was induced by knockdown of BIRC5 in all 3 cell lines examined. YM155 showed increased population of sub-G1 cells (P<0.001) in all 16 cell lines, and IC50 values to YM155 were <50nm in 15 cell lines. YM155 dose-dependently and significantly increased the apoptotic cell population in all 16 cell lines (P<0.001). CONCLUSIONS Present study indicated that survivin expression is a significant prognostic factor and that survivin is a promising therapeutic target for endometrial cancer.

Characterization of TP53 and PI3K signaling pathways as molecular targets in gynecologic malignancies

Recent developments in genomic analysis have unveiled the key signaling pathways in human cancer. However, only a limited number of molecular‐targeted drugs are applicable for clinical use in gynecologic malignancies. TP53 signaling and phosphatidylinositol 3 kinase pathways are frequently mutated in cancer, and have received much attention as molecular targets in human cancers. In this review, we mainly focus on the functions of these pathways, and discuss the molecular‐targeted drugs under clinical trials. The molecular‐targeted drugs described in this review include dual phosphatidylinositol 3 kinase/mTOR inhibitors (NVP‐BEZ235, DS‐7423, SAR245409), an mTOR inhibitor (everolimus), an MEK inhibitor (pimasertib), an autophagy inhibitor (chloroquine), a cyclin‐dependent kinases 4/6 inhibitor (PD0332991), and a poly (ADP‐ribose) polymerase inhibitor (olaparib).

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.

Interstitial pneumonitis induced by pegylated liposomal doxorubicin in a patient with recurrent ovarian cancer

Interstitial pneumonitis after treatment with pegylated liposomal doxorubicin (PLD) has been rarely reported. We describe herein a case of interstitial pneumonitis in a 49-year-old woman with relapsed ovarian carcinoma treated with PLD. Twenty-five days after the second administration of PLD, she presented with fever and dry cough, and chest CT scans revealed bilateral interstitial infiltrates and ground-glass opacities. She was diagnosed to have interstitial pneumonitis induced by PLD. Steroid therapy improved her symptoms.

Autophagy inhibition augments resveratrol‑induced apoptosis in Ishikawa endometrial cancer cells

Resveratrol (RSV), a polyphenolic compound derived from red wine, inhibits the proliferation of various types of cancer. RSV induces apoptosis in cancer cells, while enhancing autophagy. Autophagy promotes cancer cell growth by driving cellular metabolism, which may counteract the effect of RSV. The present study aimed to elucidate the correlation between RSV and autophagy and to examine whether autophagy inhibition may enhance the antitumor effect of RSV in endometrial cancer cells. Cell proliferation, cell cycle progression and apoptosis were examined, following RSV exposure, by performing MTT assays, flow cytometry and annexin V staining, respectively, in an Ishikawa endometrial cancer cell line. Autophagy was evaluated by measuring the expression levels of light chain 3, II (LC3-II; an autophagy marker) by western blotting and immunofluorescence. Chloroquine (CQ) and small interfering RNAs targeting autophagy related (ATG) gene 5 (ATG5) or 7 (ATG7) were used to inhibit autophagy, and the effects in combination with RSV were assessed using MTT assays. RSV treatment suppressed cell proliferation in a dose-dependent manner in Ishikawa cells. In addition, RSV exposure increased the abundance of the sub-G1 population and induced apoptosis. LC3-II accumulation was observed following RSV treatment, indicating that RSV induced autophagy. Combination treatment with CQ and RSV more robustly suppressed growth inhibition and apoptosis, compared with RSV treatment alone. Knocking down ATG5 or ATG7 expression significantly augmented RSV-induced apoptosis. The results of the present study indicated that RSV-induced autophagy may counteract the antitumor effect of RSV in Ishikawa cells. Combination treatment with RSV and an autophagy inhibitor, such as CQ, may be an attractive therapeutic option for treating certain endometrial cancer cells.

Prognostic importance of CDK4/6-specific activity as a predictive marker for recurrence in patients with endometrial cancer, with or without adjuvant chemotherapy.

Background:Pathologically low-risk endometrial cancer patients do not receive postoperative treatment; however, 10–15% of these patients show recurrence with poor prognosis. We evaluated the clinical importance of cyclin-dependent kinase 4/6 (CDK4/6) activity, and its significance as a novel biomarker for the prognosis and chemo-sensitivity of endometrioid endometrial carcinoma (EEC).Methods:Cyclin-dependent kinase 4/6 expression and enzyme activity in 109 tumour samples from patients with EEC were examined with a cell-cycle profiling (C2P) assay. CDK4/6-specific activity (CDK4/6SA) was determined, and its relationship with clinicopathological factors and expression of Ki-67 was analysed.Results:CDK4/6-specific activity was significantly correlated with Ki-67 (P=0.035), but not with any other clinicopathological characteristics. CDK4/6SA was significantly higher (P=0.002) in pathologically low-risk patients (not receiving adjuvant chemotherapy, n=74) than in intermediate- or high-risk patients (receiving adjuvant chemotherapy, n=35). In addition, patients with high CDK4/6SA (>3.0) showed significantly (P=0.024) shorter progression-free survival (PFS) than those with low CDK4/6SA (<3.0). Although Ki-67 expression itself was not a marker for prognosis, the combination of high CDK4/6SA and high Ki-67 expression (>15%) was robustly associated with shorter PFS (P=0.015), and this combination was an independent poor prognostic factor in the low-risk group. Inversely, in the intermediate-/high-risk group, patients with high CDK4/6SA had a tendency of a more favourable prognosis compared with patients with low CDK4/6SA (P=0.063).Conclusions:CDK4/6-specific activity can be used as a biomarker to predict prognosis and, possibly, chemo-sensitivity. The combination of Ki-67 expression might strengthen the clinical usefulness of CDK4/6SA as a biomarker.