Putative tumor suppression function of SIRT6 in endometrial cancer

AbstractSIRT6, 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

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

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

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

Inhibition of PI3K/mTOR signaling by DS-7423 and rapamycin in ovarian clear cell adenocarcinoma cell lines.

<p>(A) Immunoblotting of total protein extracts from OCCA cells (OVISE and OVMANA) treated with DS-7423 at concentrations ranging from 0 to 2,500 nM. (B) Immunoblotting of total protein extracts from OVISE cells treated with rapamycin at concentrations ranging from 0 to 2,500 nM.</p

Induction of the phosphorylation of TP53 at Ser46 and the accumulation of transcripts of the genes targeted by TP53, which participate in TP53-mediated apoptosis.

<p>(A) Immunoblotting in OVMANA cells treated with DS-7423 at the indicated doses. Phosphorylation levels of MDM2 were inversely associated with p-TP53 at Ser46, but not with p-TP53 at Ser15. (B) Semi-quantitative RT-PCR in OVMANA and OVISE cells treated with DS-7423 at the indicated doses. Both <i>p53AIP1</i> and <i>PUMA</i> were induced by DS-7423. CT, untreated (negative) control; IR, irradiation at 14 Gy (positive control). GADD45 was induced in OVISE, but not in OVMANA cells. (C) Quantification of the semi-quantitative RT-PCR in (B). Each experiment was repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (D) Effect of <i>TP53</i> knockdown on apoptosis induction by DS-7423. TP53 was knocked down by two independent siRNAs specific to <i>TP53</i> (siRNA1 and 2) in OVISE cells, which do not carry any mutation in <i>TP53</i>. The apoptotic cell population was evaluated using annexin-V staining, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087220#pone-0087220-g005" target="_blank">Figure 5</a>. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (E) Suppression of TP53 expression by siRNAs was confirmed by immunoblotting. (F) Effect of <i>TP53</i> knockdown on cell proliferation by DS-7423 in MTT assay of OVISE cells. TP53 was knocked down by a siRNA1 specific to <i>TP53</i> and MTT assay was subsequently performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087220#pone-0087220-g002" target="_blank">Figure 2</a>. Knockdown of TP53 diminished the anti-proliferative effect caused by DS-7423 on OVISE cells. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (G) TP53 expression plasmid (0.1 µg/µL) was cotransfected with pp53 TA Luc (0.25 µg/mL) plasmid into ES-2 cells mutated in <i>TP53</i>. The addition of DS-7423 increased the relative luciferase activity of TP53 in a dose-dependent manner. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05.</p