Flowcytometric analysis of cell cycle in cancer cells treated with either NVP-BEZ235 or RAD001.

<p>(A–D) Cells (5×10⁵) were seeded in the presence of 10% serum and treated with NVP-BEZ235 or RAD001 for 48 h at a dose of 10 nM or 100 nM, respectively. A higher dose of NVP-BEZ235 (100 nM) significantly augmented the percentage of cells in the G0-G1phase of the cell cycle, compared with that of RAD001 (100 nM). (A)–(B); The data from two group A cells. (C)–(D); the data from two group B cells.</p

in vivo anti-tumor effect of NVP-BEZ235 and RAD001 in nude mice.

<p>Subcutaneous xenograft tumors in athymic BALB/c mice were established in the injection of endometrial carcinoma cells. Mice were treated with a daily dose of 40 mg/kg (NVP-BEZ235) or with a daily dose of 2.5 mg/kg RAD001 or vehicle alone (control). Each treatment group contained 6 mice; (A) HEC-59 and (B) AN3CA. Tumor volumes were calculated by the formula {(major axis)*(minor axis)2/2}mm3 twice a week. Groups were compared at the end of treatment. Points, mean; bars, SD, *;p<0.05. (C) Appearance of subcutaneous tumors in HEC-59 xenografts. (D) Western blot of total lysates from the HEC-59 xenografts. p-Akt, p-FOXO1/3a, p-GSK3beta, p-S6 were assessed 1 and 24 h after the last drug administration. Total Akt and beta-actin were shown as loading controls.</p

Copy number gain at the locus of

<p><b><i>K-Ras</i></b><b>(12p12.1) in the two group D cell lines.</b> (A) SNP array ‘karyograms’ (250K) of HEC-50B cells. The graph shows the total copy number through chromosome 9p-X. The locus of <i>K-Ras</i> is amplified as indicated. (B) Array CGH of KLE cells. The graphs show total copy number throughout the entire genome (Left) and chromosome 12 (Right). The locus of <i>K-Ras</i> is amplified as indicated.</p

Inhibition of cell proliferation by NVP-BEZ235 and RAD001.

<p>(A)–(D) WST-8 assay showing the sensitivity of endometrial cancer cells to NVP-BEZ235 and RAD001 at increasing concentrations of drug (nmol/L) for 72 h. The data was normalized to the value of control cells. (A) Four group A cell lines with double mutations of <i>PIK3CA</i> and <i>PTEN</i>, (B) Five group B cell lines with <i>PTEN</i> mutations, (C) Two group C cell lines with coexistent mutations of <i>K-Ras</i> and <i>PIK3CA</i>, and (D) Two group D cell lines with chromosomal copy number amplification at the locus of <i>K-Ras</i>. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D.</p

Inhibition of cell proliferation and augmentation of G1 arrest by combination of a MEK inhibitor and NVP-BEZ235 (or RAD001) in cells with alterations in

<p><b><i>K-Ras</i></b><b> (mutation or amplification).</b> (A)–(B) WST-8 assay was performed in HEC-1B (group C) and HEC-50B (group D) cell lines. All the experiments were repeated three times and each value is shown as the mean of three experiments +/− S.D. The combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 (or RAD001) significantly augmented anti-proliferative effect in these cell lines, compared with either inhibition alone (p<0.05 by Student's t-test). (C)–(D) Flowcytometric analysis of cell cycle in HEC-1B and HEC-50B cells. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D. Combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 significantly augmented the percentage of cells in the G0-G1phase of the cell cycle in these cells. *: p<0.05 by Student's t-test.</p

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

<div><p>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 <i>PIK3CA</i>. 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 <i>TP53</i> mutations than in the three cell lines with <i>TP53</i> 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 <i>TP53</i>, with induction of genes that mediate TP53-dependent apoptosis, including <i>p53AIP1</i> and <i>PUMA</i> 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 <i>TP53</i> wild-type OCCAs.</p></div

Integrated analyses reveal a poor-prognostic clear cell signature associated with high chromosomal instability.

<p>Comparison of clear cell carcinoma (CCC) clusters CCC-1 and CCC-2 by gene expression profiling. (A) <i>UGT1A6</i> and <i>UGT1A10</i> were significantly upregulated in CCC-1 compared with CCC-2. (B) The cluster of genes upregulated in CCC-1 compared with CCC-2. The cluster includes <i>STAT3</i> and <i>HIF2A</i>.</p

SNP profiling discriminates histology-related subgroups based on chromosomal instability status.

<p>Chromosomal instability status (CIN) according to the number of allele-specific copy number alterations (CNAs) and copy number neutral loss of heterozygosity (CNN LOH), using a human mapping 250K single nucleotide polymorphism (SNP) array with paired tumor DNA and normal DNA. CNAs were divided into three subgroups: CIN-high (≥9 arms with CNAs), CIN-low (1–8 arms with CNAs), and CIN-negative (0 CNAs). (A) Details of number of chromosomal arms with CNAs in each tumor of three histological subtypes (serous carcinomas, SC; clear cell carcinomas, CCC; endometrioid carcinomas, EC). Stage I/II and stage III/IV are colored differently. (B) Correlation between CIN status and histological subtypes. (C) Overview of CNAs by running SNP arrays with 57 ovarian cancer samples. Hierarchical clustering based on the Euclidean distance for dissimilarities is shown. The type A cluster includes tumors with a broad range and low frequency of CNAs, whereas the type B cluster includes tumors with a focal range and high frequency of CNAs. C, E, and S indicate clear cell carcinoma, endometrioid carcinoma, and serous carcinoma, respectively.</p

Ratio of whole arm CNAs among all the CNAs in serous and clear cell carcinomas.

<p>Ratio of whole arm CNAs among all the CNAs in serous and clear cell carcinomas.</p

Clustering by expression arrays and clinicopathological characteristics.

<p>Sensitive*: Complete response or Partial response.</p><p>Resistant**: Stable disease or Progressive disease.</p><p>p-value (by Fisher's exact test).</p><p>Clustering by expression arrays and clinicopathological characteristics.</p