Mucinous adenocarcinoma of the ovary (MAC) is the third most common type of epithelial ovarian cancer (EOC), comprising 10% to 12% of EOC.1–3 Mucinous adenocarcinoma of the ovary seems to have a clinical behavior distinct from other EOC. Several studies have shown that MAC often is diagnosed at an early stage, and therefore, confers a relatively good prognosis. However, advanced MAC has a poorer prognosis than other histopathologic subgroups.4–6 Poor response (13% to 42%) to conventional platinum- or taxane-based chemotherapy is associated with poor prognosis because chemosensitivity is one of the main prognostic factors for patients with advanced EOC.4–9 Thus, novel treatment strategies (eg, incorporation of molecular-targeted agents) for advanced MAC are needed.
The phosphatidylinositol 3′-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a key role in cellular metabolism, proliferation, survival, and motility.10 This pathway is hyperactivated by several mechanisms, such as deletion or decreased function of phosphatase and tensin homolog (PTEN), activating mutations of PI3K or Akt, and activating receptor tyrosine kinases (eg, epidermal growth factor receptor, human epidermal growth factor receptor 2).10,11 Activating this cascade is indicated to relate drug resistance and poor prognosis in many cancers including EOC.10,12 The frequent mutation of PIK3CA has been reported in clear cell carcinoma (33%) and endometrioid adenocarcinoma (20%) of the ovary, and this cascade is thought to be a potential therapeutic target.13,14 Several studies have shown that inhibiting the Akt/mTOR signaling pathway is one of these promising therapeutic targets in clear cell carcinoma of the ovary.15,16 Therefore, we focused on this cascade to treat MAC. In MAC, overexpressed proteins of the epidermal growth factor receptor family and activated downstream signaling of Akt/mTOR have been found in 48% of tumors and were associated with poor patient outcomes.17,18 This pathway could be an attractive target for therapy for MAC.
Recently, a novel imidazoquinoline derivative, NVP-BEZ235 (BEZ235), was developed and has entered clinical trials.19–21 NVP-BEZ235 is an orally bioavailable agent that targets the ATP-binding clefts of the class 1 PI3K and mTOR kinases, thereby inhibiting the activity of PI3K, mTOR complex (mTORC) 1, and mTORC2. Several studies have shown potential antitumor activities by BEZ235 in vitro and in vivo in a variety of cancers, including EOC.19,22–25 However, the effects of a dual inhibitor of PI3K and mTOR have not been evaluated systematically in MAC. We therefore conducted the present study to assess the possibility of molecular-targeted therapy with BEZ235 to treat MAC.
MATERIALS AND METHODS
Cell Lines and Culture Conditions
The 7 human MAC cell lines used in this study were obtained as follows: JHOM-1 and JHOM-2B from Riken BioResource Center cell bank (Tsukuba City, Ibaraki, Japan); MCAS from Health Science Research Resources Bank (Chiyoda-ku, Tokyo, Japan); OMC-1 from Dr Tsuyoshi Saito (Sapporo Medical University, Sapporo, Japan); and RMUG-L and RMUG-S from Dr Daisuke Aoki (Keio University, Tokyo, Japan). TU-OM-1 was established by our department. These cell lines were maintained in DMEM/Ham’s F-12 medium (Wako Pure Chemical Industries, Ltd) with 10% fetal bovine serum, 100 IU/mL penicillin, and 50 µg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37°C.
The sensitivity of the cell lines to BEZ235 (LC Laboratories) and temsirolimus (LC Laboratories) was determined by a cytotoxicity assay using Cell Counting Kit-8 (Dojindo Laboratories). Briefly, cells were diluted with culture medium to a seeding density of 3 to 5 × 104/mL, plated on 96-well tissue culture plates at 180 μL per well, and incubated at 37°C for 24 hours. The next day, the cells were treated continuously with 20 μL of various concentrations of the agents to obtain a dose-response curve for each agent. The concentration for each agent was 1 to 10,000 nmol/L. After being incubated for 72 hours, 20 μL of Cell Counting Kit-8 solution was added to each well, and the plates were incubated for another 1 to 2 hours. Absorbance was measured at 450 nm with a microplate reader (iMark Microplate Absorbance Reader; Bio-Rad). Inhibition of cell growth was calculated as the percentage of viable cells compared with the percentage in untreated cultures.
NVP-BEZ235 was combined with each of the different anticancer agents at a fixed ratio that spanned the individual half-maximal inhibitory concentrations (IC50) of each drug. The IC50 was determined from dose-effect curves created by a cytotoxicity assay. Median effect plot analyses and calculated combination indices (CI) were analyzed by the method of Chou and Talalay.26 CalcuSyn software (Biosoft) was used to analyze data from the cytotoxicity assays in which cells were exposed to agents alone or combined with the anticancer drugs and BEZ235. CalcuSyn provided a measure of the combined agents in an additive or synergistic manner. We used the Chou and Talalay26 definition of CI as synergistic (CI < 0.9), additive (0.9 < CI < 1.1), or antagonistic (CI > 1.1).
Mutation screening for PIK3CA was performed at exons 9 and 20 and K-Ras at exons 2 and 3, covering the mutational hot spots in human cancers. These exons of PIK3CA and K-Ras were amplified using polymerase chain reaction (PCR) for genomic DNA. The primers for PCR and sequencing were prepared according to previous reports.27,28
The PCR conditions were as follows: 1 cycle at 94°C for 5 minutes, 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, followed by 1 cycle at 72°C for 7 minutes. The PCR products subsequently were subjected to direct sequencing PCR with BigDye terminator v3.1 cycle sequencing reagents (Applied Biosystems). The samples were finally analyzed on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) with Sequence Scanner Software v1.0 (Applied Biosystems).
Western Blot Analyses
Cells were washed twice with PBS and then lysed in lysis buffer [50 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% glycerol, 1% Nonidet P-40, 2 mmol/L EDTA, 50 mmol/L NaF, 2 mmol/L Na3VO4, and protease inhibitors (complete protease inhibitor cocktail tablets [Roche Diagnostics])]. Protein concentrations were measured against a standardized control using a protein assay kit (Bio-Rad Laboratories). A total of 50 μg protein was separated by electrophoresis on a 5% to 20% or 15% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). All the antibodies that were used came from Cell Signaling Technology, except for mouse antiactin (Sigma)—rabbit anti-PTEN (1:500), rabbit anti-Akt (1:1000), rabbit anti–phosphorylated (p-) Akt (1:500), rabbit anti-mTOR (1:500), rabbit anti–p-mTOR (1:500), rabbit anti–4E-BP1 (1:1000), rabbit anti–p-4E-BP1 (threonine 37/46, 1:1000), rabbit anti–p70 s6 kinase (1:1000), rabbit anti–p-p70 s6 kinase (1:1000), rabbit anticleaved caspase 9 (1:500), rabbit anticleaved PARP (1:1000), and mouse antiactin (1:1000). These were visualized with secondary antimouse or antirabbit immunoglobulin G antibody coupled with horseradish peroxidase using enhanced chemiluminescence according to the manufacturer’s recommendation.
Flow Cytometric Analysis
To analyze cell cycle distribution, the cells (2 × 106/L) were trypsinized, collected by centrifugation, fixed in 70% ethanol at 4°C for 1 hour, and resuspended in PBS containing 50 μg/mL propidium iodide and 0.1 mg/mL RNase. After 30 minutes at 37°C, the cells were analyzed with a flow cytometer (EPICS Altra HyperSort; Beckman Coulter, Inc).
Annexin V Staining
The annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BioVision) was used to assess apoptosis as the externalization of phosphatidylserine residues according to the specifications of the manufacturer. Briefly, cells were suspended in 500 μL of 1 × binding buffer. The cells then were stained with 5 μL annexin V-FITC for 5 minutes in the dark at room temperature. Finally, the cells were analyzed with a flow cytometer (EPICS Altra HyperSort; Beckman Coulter, Inc).
Ovarian Cancer Xenograft Model
This study was carried out at the Laboratory Animal Research Center under the control of the animal research committee in accordance with the Guidelines for Animal Experimentation in the Faculty of Medicine of Tottori University in Yonago, Japan. For these experiments, OMC-1 or RMUG-S cells in log-phase growth were trypsinized, washed twice with PBS, and centrifuged at 250 × g. Viable cells were counted, then 5 × 106 viable cells (in 0.3 mL PBS) were inoculated subcutaneously under aseptic conditions into the left flank of female nude mice. The mice were assigned randomly to 1 of 3 groups (7 mice per group), and treatment was started 7 days later, which are as follows: group 1, oral administration of vehicle (PBS); and groups 2 and 3, oral administration of 25 or 50 mg/kg per day BEZ235 for 3 weeks, respectively (5 days per week). Tumor size was measured with a caliper twice weekly, and tumor volume was calculated according to the following equation: tumor volume (mm3) = π/6 × L × W 2, where L and W are the long and short dimensions of the tumor, respectively. Two mice of each group were killed on day 25, and tumors were collected and fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries) and embedded in paraffin for immunohistochemical analysis. Paraffin blocks were sliced in 4-μm sections and deparaffinized. The expression of p-Akt, p-mTOR, p-4E-BP1, and p-p70S6K proteins on the tumor tissue sections was detected using the Histofine Simple Stain PO kit (Nichirei Corporation). Slides were counterstained with hematoxylin. The primary antibodies used were anti–p-Akt (dilution, 1:100; Cell Signaling Technology), anti–p-mTOR (dilution, 1:100; Cell Signaling Technology), anti–p-4E-BP1 (dilution, 1:100; Cell Signaling Technology), and anti–p-p70S6K (dilution, 1:100; Cell Signaling Technology).
Statistical analyses were performed using the GraphPad Prism program Version 5 (GraphPad Software, Inc). Data are presented as mean (1 SD). The means for all data were compared by 1-way analysis of variance with post hoc testing. A P value of less than 0.05 was considered statistically significant.
Sensitivity to Targeted Agents and the PI3K/Akt/mTOR Pathway in MAC Cell Lines
The effects of BEZ235 or temsirolimus on the proliferation of 7 MAC cell lines are shown in Figure 1A. NVP-BEZ235 decreased the cell viability rate in a dose-dependent manner in all MAC cell lines tested. Contrarily, reduced cell viability by temsirolimus was significantly less compared with BEZ235 in all lines.
We next determined in each MAC cell line the relationship between sensitivity to BEZ235 or temsirolimus and expression of protein in the PI3K/Akt/mTOR signaling pathway, or the presence of activating mutations of PIK3CA and K-Ras genes. The IC50 of these cell lines to BEZ235 ranged from 18 to 328 nmol/L and more than 10,000 nmol/L for temsirolimus, except for TU-OM-1 cells (530 nmol/L). The protein expression of Akt, mTOR, p70S6K, 4E-BP1, and these phosphorylation forms were confirmed in all cell lines (Fig. 1B). Although the expression levels were not related to the sensitivity to BEZ235 or temsirolimus, 2 cell lines, MCAS and OMC-1, with activating mutations of H1047R and E545K in the PIK3CA gene, respectively, exhibited low IC50 to BEZ235 (Fig. 1C). Mutations of G12A in the K-Ras gene also were observed in MCAS cells.
NVP-BEZ235 Inhibits the PI3K/Akt/mTOR Pathway
We examined the effect of BEZ235 and temsirolimus on the PI3K/Akt/mTOR signaling pathway in MAC cells. Treating OMC-1 and RMUG-S cells with BEZ235 suppressed the protein expression levels of p-Akt, p-p70S6K, and p-4E-BP1 in a dose-dependent manner (Fig. 2A). In contrast, the mTOR inhibitor used alone for temsirolimus treatment slightly down-regulated p-p70S6K and p-4E-BP1, whereas p-Akt protein expression levels were up-regulated in both cell lines (Fig. 2B). Similar results were obtained in the other 5 cell lines (data not shown).
NVP-BEZ235 Increased Gap (G)0/G1-Phase Fraction and Up-regulates the Apoptotic Pathway
We then assessed the cell cycle distribution by flow cytometry to confirm whether treatment with BEZ235 influenced cell cycle progression. After treatment with BEZ235, the proportion of the cells in G0/G1 phase increased markedly and in the S phase was decreased in OMC-1 and RMUG-S cells (Fig. 3A–C). Moreover, 72 hours after treatment with BEZ235, the subG1 population was significantly increased compared with untreated controls.
To confirm whether the apoptotic pathway was activated by BEZ235, we assessed the protein expression levels of cleaved PARP and caspase 9 and the proportion of annexin V-positive cells among MAC cells. Forty-eight hours after treatment with BEZ235 at higher doses, the expression of cleaved PARP and caspase 9 were up-regulated in OMC-1 and RMUG-S cells, and the proportion of annexin V-positive cells increased in a dose-dependent manner (Fig. 4A and B). Similar results were obtained in the other 5 cell lines (data not shown).
NVP-BEZ235 Suppressed Tumor Growth in MAC Xenograft Models
After confirming that BEZ235 reduced cell viability and enhanced apoptosis in vitro, we examined the effect of treatment of BEZ235 in xenograft models of MAC. Female nude mice were given subcutaneous injections of OMC-1 or RMUG-S cells and then treated with daily oral PBS or BEZ235 (25 or 50 mg/kg per day). There were no signs of overt toxicity (weight loss or gross clinical signs) in any group (Fig. 5A).
To confirm the inhibition of PI3K/Akt/mTOR pathway by BEZ235 in vivo, we performed immunohistochemical analysis of tumor tissues (Fig. 5B). As expected, p-Akt, p-mTOR, p-4E-BP1, and p-p70S6K proteins were down-regulated in tumors from mice treated with BEZ235.
In nude mice bearing OMC-1 or RMUG-S, the mean volume of subcutaneous tumors in the group treated with BEZ235 25 or 50 mg/kg per day doses was significantly smaller than what was in the group treated with PBS (P < 0.05; Fig. 5C). These findings indicated that BEZ235 suppressed growth of subcutaneous tumors in nude mice bearing OMC-1 or RMUG-S cells.
Combination Effects of BEZ235 and Anticancer Agents
We analyzed the synergistic activities of combining BEZ235 with each anticancer agent by calculating CI values by using the method of Chou and Talalay.26 Data representative of BEZ235 combined with paclitaxel or cisplatin in OMC-1 cells are shown in Figure 6A. The CI value at an effective dose of 50 (effective dose being the percentage inhibition of cell growth using the drug combinations in the actual experiment) was less than 0.9 (synergism) for 6 cell lines treated with paclitaxel, 5 cell lines with cisplatin and SN-38, representing an active metabolite of irinotecan, 7 cell lines with etoposide, and 3 lines with gemcitabine (Fig. 6B).
In this exploration of the effects of BEZ235, we found that dual inhibition of PI3K and mTOR significantly inhibited proliferation activity in a panel of MAC cells. We also showed that BEZ235 induced cell cycle arrest at the G0/G1 phase and activated apoptotic pathways. The effectiveness of this agent was confirmed in xenograft models of MAC. NVP-BEZ235 markedly suppressed tumor growth in these mice compared with control animals. To our knowledge, this is the first study to show that the novel dual PI3K/mTOR inhibitor BEZ235 is effective against MAC, both in vitro and in vivo.
Numerous molecular-targeted agents have been developed and have already entered clinical practice.29,30 The PI3K/Akt/mTOR pathway has been a focus for attractive treatment options for cancers.20,21 The activation of PI3K caused phosphorylation and increased the activity of Akt, and thus activated mTORC1.10 Rapamycin and its derivatives, such as temsirolimus and everolimus, bind to FK506-binding protein 12, a member of the immunophilin protein family, and this complex inhibits kinase activity of mTORC1 allosterically by binding directly to mTOR. When the downstream signaling of mTOR such as 4E-BP1 and p70S6K1 is blocked, it leads to arrest in the G1-phase cell cycle and cell death.21 However, several studies have reported that mTOR inhibition alone up-regulated the expression of p-Akt in some cells because a feedback loop that depends on mTORC1 is induced that limits activation of PI3K and/or continued activation of Akt mediated by mTORC2. Activated Akt may attenuate the antitumor effect of rapalogues.24,31–33 In addition, rapalogues may cause feedback activation of the PI3K/Akt pathway mediated by insulinlike growth factor 1 receptor signaling.34 Indeed, we found that p-Akt increased in 6 of 7 cell lines after being treated with temsirolimus and that those 6 cells resisted cell cycle arrest induced by temsirolimus. These results suggested that the effect of the mTOR inhibitor alone might be limited to treat patients with MAC.
In contrast to the mTOR inhibitor alone, the dual PI3K/mTOR inhibitor, BEZ235, down-regulated p-Akt expression and downstream signaling of mTOR (p-4E-BP1 and p-p70S6K) and suppressed proliferation activity of MAC cells. Moreover, BEZ235 induced cell death by up-regulating the apoptotic pathway in MAC cells. Consistent with our findings, other studies have reported that BEZ235 had more pronounced effects compared with an mTOR inhibitor alone on cell growth in breast cancer and EOC.22,24 This convergence of results suggested that dual inhibition of PI3K and mTOR may be required for therapeutic efficacy in patients with MAC.
Several studies have reported that activating mutations of the PIK3CA gene may predict enhanced sensitivity to treatment with inhibitors of the PI3K/Akt/mTOR pathways in patients with advanced cancers, including EOC.24,35 –37 PIK3CA is located on chromosome 3q26.32 and encodes the p110α catalytic subunit of the class IA PI3K. Most PIK3CA mutations are at E542K and E545K in the helical domain (exon 9) and H1047R in the kinase domain (exon 20), resulting in kinase activation of p110α, which then activates Akt.38 Indeed, 2 cell lines with PIK3CA mutations at an E545K or H1047R showed low IC50 to BEZ235. However, we observed that BEZ235 was effective (IC50 < 100 nmol/L) in 4 of the other 5 MAC cell lines with wild-type PIK3CA. Similar effectiveness of BEZ235 on some cancer cells without mutations in this gene has been reported.24 ,39 Our findings suggested that BEZ235 may have clinical benefit not only in patients with MAC with activating mutation in PIK3CA but also for those with wild-type PIK3CA.
Mutational activation of K-Ras has been reported to predict poor response to PI3K/Akt inhibitors,37 and K-Ras is thought to mutate frequently (average, 43%) in MAC.40 In this study, however, only 1 cell line had alterations in K-Ras and PIK3CA genes, and it was sensitive to BEZ235. Consistent with our results, Santiskulvong et al24 showed that BEZ235 was effective to established ovarian tumor disease using transgenic and immunocompetent LSL-K-Ras G12D/+ Pten loxP/loxP mice. However, further studies are needed to elucidate the effect of BEZ235 in MAC with K-Ras mutation.
Further, we confirmed the effect of BEZ235 in vivo on MAC in murine xenograft models. NVP-BEZ235 decreased the expression of p-Akt and p-mTOR in the tumors of these mice compared with those treated with PBS alone. The present study provided clear evidence that BEZ235 down-regulated PI3K/Akt/mTOR signaling and suppressed proliferation of MAC cells both in vitro and in vivo.
Although MAC is known to resist platinum- and taxane-based chemotherapy, patients with MAC are usually treated with the standard chemotherapy regimen as used for EOC. Interestingly, a synergistic effect was observed from combining BEZ235 at even lower doses with paclitaxel (6 cell lines) or cisplatin (5 cell lines) on cell proliferation. Several researchers also have reported that BEZ235 could sensitize the cytotoxic effects of paclitaxel or cisplatin in EOC and breast cancer cells, suggesting that dually inhibiting PI3K/mTOR might overcome resistance to anticancer agents.24,25 ,41 In contrast, we previously found antagonistic effects when the mTOR inhibitor rapamycin was combined with paclitaxel or cisplatin on more than 4 of 6 ovarian serous adenocarcinoma cell lines.42 However, combining the PI3K inhibitor LY294002 with paclitaxel or cisplatin had an additive inhibitory effect on cell growth in serous adenocarcinoma cells.43,44 These observations suggested that BEZ235 could be incorporated into first-line chemotherapy for MAC.
In summary, our study showed that the dual PI3K/mTOR inhibitor BEZ235 has growth inhibition and antitumor effects, and it enhances the cytotoxic effect of chemotherapeutic agents in MAC cells. We also found that BEZ235 is therapeutically effective in both PIK3CA mutant and PIK3CA wild-type MAC cells. Therefore, we concluded that BEZ235 is worth exploring in a therapeutic strategy for MAC. We hope that such therapies will improve the survival of patients with advanced MAC.
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