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

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

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

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

Correlation between clustering by expression arrays, genetic alterations and clinicopathological characteristics in CCC.

<p>Sensitive*: Complete response or Partial response.</p><p>Resistant**: Stable disease or Progressive disease.</p><p>Correlation between clustering by expression arrays, genetic alterations and clinicopathological characteristics in CCC.</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

Inhibition of cell proliferation by DS-7423 and rapamycin.

<p>(A) Cell viability for each cell line was analyzed using the methyl thiazolyl tetrazolium (MTT) assay 72 h after treatment with DS-7423 or rapamycin at the doses indicated. The data were normalized relative to the value of the control cells. In all nine cell lines, DS-7423 suppressed cell proliferation more robustly than rapamycin when both were used at higher doses. (B) IC₅₀ values for DS-7423 in seven ovarian serous adenocarcinoma (OSA) cell lines (left) were compared with those of nine OCCA cells (right). Four of seven OSA cells had IC₅₀ values >100 nM, which is higher than that of any OCCA cells.</p

DS-7423–mediated induction of apoptosis in ovarian clear cell adenocarcinoma cell lines.

<p>(A) All nine OCCA cells were treated with DS-7423 at 156 or 2,560 nM for 48 h, and apoptotic cell proportion was evaluated using annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining, followed by analysis using flow cytometry. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± standard deviation (SD). (B) The apoptotic cells were calculated using flow cytometry by counting the cell population in the right boxes. The example shown (OVISE cells) is representative of the results obtained for all the cell lines tested. (C) The proportion of cells rendered apoptotic by exposure to DS-7423 at 156 nM and 2,560 nM was significantly higher in OCCA cells without mutations in TP53 than in OCCA cells that carry mutations in TP53. (D) Cleaved poly(ADP-ribose) polymerase (PARP) induction was evaluated by immunoblotting in OVISE cells. OVISE cells were treated with DS-7423 at 156 nM for the times indicated (left) or for 4 h at the doses indicated (right).</p

Phosphorylation and mutational status of genes that encode components of the RTK/Ras/PI3K pathway.

<p>Nine ovarian clear cell adenocarcinoma (OCCA) and a control (Cntl) cell line (immortalized epithelial cells from ovarian endometrioma) were lysed in cell lysis buffer and analyzed by western blotting. In general, most of the OCCA cell lines displayed higher levels of phosphorylation of Akt (Thr308) and its downstream targets (GSK3β, FOXO 1/3a, 4EBP1 and S6) than the respective levels of phosphorylation in the control line. The abundances and levels of phosphorylation of c-MET (Tyr1234/1235), HER2 (Tyr1221/1222), and HER3 (Tyr1289) were also evaluated. The mutational status of <i>PIK3CA</i>, <i>PTEN</i>, and <i>K-Ras</i> is shown for each cell line.</p

Flow cytometric analysis of the cell cycle in cancer cells treated with DS-7423, and <i>in vivo</i> demonstration of the anti-tumor effect of DS-7423 in nude mice.

<p>(A) Cells (5×10⁵) were seeded in the presence of 10% serum and treated with DS-7423 for 48 h at doses of 9.8 nM, 256 nM, or 2,500 nM. DS-7423 blocked OCCA cell cycle progression into the S phase in a dose-dependent manner. The relative size of the sub-G1 population was increased in six of the cell lines (left) but was not affected in the remaining three cell lines (right). (B) Subcutaneous xenograft tumors in athymic BALB/c mice were established following the injection of OCCA cells of either the TOV-21G (left) or RMG-I (right) cell lines. Mice were treated daily (5–7 days per week) at the indicated doses of DS-7423 (1.5, 3, or 6 mg/kg, 8–10 days). Each treatment group contained five mice. Estimated tumor volumes (upper graphs) and body weight losses (BWL) (lower graphs) were shown in the two OCCA cells. Tumor volumes were calculated by the formula {(major axis)*(minor axis)²/2} mm³. Groups were compared at the end of treatment. Points, mean; bars, standard deviation (SD); *p<0.05.</p