Antitumor activity of a combination of dual PI3K/mTOR inhibitor SAR245409 and selective MEK1/2 inhibitor pimasertib in endometrial carcinomas
Kanako Inaba a, Katsutoshi Oda a, , Yuji Ikeda a, Kenbun Sone a, Aki Miyasaka a, Tomoko Kashiyama a, Tomohiko Fukuda a, Yuriko Uehara a, Takahide Arimoto a, Hiroyuki Kuramoto b, Osamu Wada-Hiraike a, Kei Kawana a, Tetsu Yano c, Yutaka Osuga a, Tomoyuki Fujii a
a Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, Japan
b Kanagawa Health Service Association, Kanagawa, Japan
c Department of Obstetrics and Gynecology, National Center for Global Health and Medicine, Tokyo, Japan
HIGHLIGHTS
• MEK and PI3K/mTOR inhibitors synergistically suppress endometrial cancer cell growth.
• The MEK inhibitor (pimasertib) dose for the synergistic effect was much lower than its IC50.
• mTOR-independent AKT effectors might be essential for the synergistic effect.
a r t i c l e i n f o
Article history:
Received 1 March 2015
Received in revised form 24 May 2015 Accepted 26 May 2015 Available online 29 May 2015
Keywords:
Endometrial carcinoma
MEK
MAPK
PI3K/mTOR
Molecular target therapy
a b s t r a c t
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 N20 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.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
Endometrial cancer is the most common gynecological malignancy in developed countries [1]. Although conventional therapies, including surgery, radiation, and chemotherapy, yield favorable prognoses for
Corresponding author at: Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan.
E-mail address: [email protected] (K. Oda).
http://dx.doi.org/10.1016/j.ygyno.2015.05.031 0090-8258/© 2015 Elsevier Inc. All rights reserved.
early-stage endometrial cancers, therapeutic options for advanced and recurrent tumors are limited. Especially, no targeted therapy has been approved for the treatment of endometrial cancer.
The phosphatidylinositol-3 kinase (PI3K) pathway is frequently acti-vated in various types of cancers [2,3], and the coexistence of multiple alterations in the PI3K pathway is common in endometrial cancer [4–6], e.g., PTEN, PIK3CA, PIK3R1, PIK3R2, and AKT1 mutations are reported at 55–64%, 36–53%, 20–33%, 5%, and 2%, respectively [4,7–9]. Upon acti-vation, PI3K phosphorylates AKT, which subsequently phosphorylates
324 K. Inaba et al. / Gynecologic Oncology 138 (2015) 323–331
various types of effectors including mammalian target of rapamycin (mTOR) [10,11]. Therefore, the PI3K/mTOR pathway is considered a promising target for endometrial cancer therapy. In preclinical studies, the antitumor effect of PI3K pathway inhibitors has been demonstrated in a panel of endometrial cancer cell lines [12,13]. mTOR inhibitors in-cluding everolimus, temsirolimus, and a combination of bevacizumab and temsirolimus have been evaluated in endometrial cancer patients [14–18]. However, the response rate of mTOR inhibition alone was b10%, and no mTOR inhibitor has been approved for the treatment of endometrial cancer. Additionally, several phase II clinical trials evaluating PI3K pathway inhibitors (SAR245408, BEZ-235, PF-04691502, PF-05212384) in endometrial cancer have been conducted (ClinicalTrials.gov Identifier: ctgov ID NCT01013324, NCT01290406, NCT01420081), but none of these monotherapies have demonstrated clinical benefit in this patient population and progressed to phase III clinical trials.
The resistance to PI3K pathway inhibition can be ascribed to the con-comitant activation of the mitogen-activated protein kinase (MAPK) pathway [19,20]. This pathway is activated by alterations in BRAF, KRAS, and neurofibromin-1 (NF1) in various types of cancers [21–23]. In endometrial cancer, KRAS mutations and copy number loss of NF1 has been reported in 20–30% and 13% of patients, respectively [6,8,22]. There are only a few reports on the impact of inhibition of the MAPK pathway in endometrial cancer [12,24].
The rationale to inhibit both the RAS/MAPK and PI3K/mTOR pathways is that RAS mutations are associated with the activation of both PI3K and MAPK pathways [25–27]. We therefore aimed to clarify whether combination therapy with a dual PI3K/mTOR inhibitor and a
MEK inhibitor synergistically induces antitumor effects in endometrial cancer cell lines using various dose settings. We also explored whether the inhibition of mTOR might be associated with a synergistic effect in a combination therapy with a MEK inhibitor. Furthermore, we looked for biomarkers to predict sensitivity to the combined inhibition of PI3K and MAPK pathways.
2. Materials and methods
2.1. Cell lines and inhibitors
We used 12 endometrial cancer cell lines, including the Ishikawa3-H-12 cell line (hereafter referred to as Ishikawa; a generous gift from Dr. Masato Nishida, National Hospital Organization, Kasumigaura Medical Center, Japan), 8 human endometrial cancer (HEC) cell lines established by Hiroyuki Kuramoto [28], and the AN3CA, KLE, and HHUA cell lines that were purchased from the American Type Culture Collection (Manassas, VA, USA). The culture conditions have been described earlier [5]. HHUA and KLE were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, whereas the other 10 cell lines were maintained in Eagle’s minimum essential medium (MEM) with 10% FBS. Histologically, all these cell lines were endometrioid adenocarcinomas. SAR245409 (voxtalisib, XL-765), a PI3K/mTOR inhibitor, and pimasertib, a MEK inhibitor, were provided by Sanofi (Paris, France) and Merck Serono (Darmstadt, Germany), respectively (Fig. 1A). SAR245409, a pyridopyrimidinone-derivative, is a highly selec-tive and potent ATP-competitive inhibitor of pan-class I PI3Ks and mTORC1/mTORC2. Pimasertib is a highly potent ATP-noncompetitive
A
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Fig. 1. Inhibition of cell proliferation by SAR245409 and pimasertib. (A) Scheme of the PI3K/mTOR and MAPK pathways with downstream molecules, and various types of inhibitors targeting these pathways. (B and C) IC50 values were calculated according to the data of the MTT assay. (B) and (C) show the IC50 values of SAR245409 and pimasertib, respectively. Of the 12 cell lines, 6 with IC50 values of b5 μM for pimasertib were classified into the pimasertib-sensitive group. *The IC50-concentration of HEC-59 and HHUA is N25 μM. The results are shown as the mean ± SE of 3 independent experiments.
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second-generation inhibitor of MEK1 and MEK2. The preclinical pharma-cological characterization of SAR245409 and pimasertib (molecular structure, biochemical, cellular, and in vivo data) has been described previously [29,30]. SAR245409 inhibits mTOR signaling in both PI3K-dependent and PI3K-independent manner, which results in suppression of mTOR signaling at lower doses than PI3K/AKT signaling [29] as reported for other dual PI3K/mTOR inhibitors [12,31]. Both agents are orally bioavailable small-molecule inhibitors and have been under phase I/II clinical trials for some malignant tumors. Additionally, an initial evaluation of SAR245409 and preliminary toxicity data for pimasertib in cancer patients has been reported [32,33]. Rapamycin, an mTOR inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA).
2.2. Proliferation assays
Cell proliferation was evaluated by the methyl thiazolyl tetrazolium (MTT) assay with the Cell Counting Kit-8 using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8; Dojindo, Tokyo, Japan) [12]. Cells were seeded into 96-well plates at a rate of 2 × 103 cells/well, and exposed to each drug for 72 h. Cell proliferation was quantified by monitoring changes in the absorbance at 450 nm, normalized relative to the absorbance of cell cultures treated with DMSO alone, and a dose–response curve was composed for each drug. The half-maximal inhibitory concentra-tion (IC50) values were determined from the dose–response curves as
the concentration at which 50% inhibition was obtained. Each experi-ment was repeated at least three times.
2.3. Immunoblotting
Cells in 6-well plates were exposed to SAR245409 and/or pimasertib for the indicated times and concentrations. The cells were then lysed as described previously [5]. Primary antibodies against AKT, phosphorylated AKT (p-AKT) (Ser473), S6, p-S6 (Ser235/236), p-MDM2 (Ser166), ERK, p-ERK (ERK1/2-Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA), MDM2 (Santa Cruz Biotechnology, Dallas, TX, USA), and beta-actin (Sigma-Aldrich, St. Louis, MO, USA) were used for immuno-blotting. Signals were detected using BioRad immunoblotting systems (BioRad, Hercules, CA, USA) with ECL Select detection agents (GE Healthcare, Piscataway, NJ, USA). Immunoblotting results were quanti-fied using ImageJ software [34].
2.4. Cell cycle analysis
Cells (5 × 105) were seeded in 60-mm dishes and treated with SAR245409 and/or pimasertib for 72 h. Both floating and adherent cells were collected after trypsinization, and cell cycle distribution was analyzed by flow cytometry (BD FACSCalibur HG, Franklin Lakes, NJ, USA) and CELL Quest pro ver. 3.1. (Beckman Coulter Epics XL, Brea, CA, USA) [12]. All experiments were repeated three times.
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Fig. 2. Inhibition of PI3K/mTOR signaling by SAR245409 and inhibition of MAPK signaling by pimasertib. (A) Western blot of total lysates of AN3CA (pimasertib-sensitive) and Ishikawa (pimasertib-resistant) cells, treated with SAR245409 (0–3000 nM) or pimasertib (0–1000 nM). p-AKT, p-S6, and p-ERK levels were evaluated to assess the suppression of PI3K, mTOR, and MAPK pathways, respectively. (B) p-AKT levels and p-S6 in response to SAR245409, as well as p-ERK levels in response to pimasertib, were quantified using ImageJ. The ratio of phosphorylated over total protein was compared using 4 dose settings. The results are shown as the mean ± SE of 3 independent experiments.
326 K. Inaba et al. / Gynecologic Oncology 138 (2015) 323–331
2.5. Small interfering RNA (siRNA) transfection
siRNAs were used to inhibit the expression of MAPK3 and MAPK1, encoding ERK1 and ERK2 protein, respectively. Three siRNAs specific
to MAPK3 (HSS108538, 108539, 108540) and MAPK1 (HSS183535, 183536, 183537) were purchased from Invitrogen (Carlsbad, CA, USA). Non-silencing siRNA (siCT) was used as a control (Stealth RNAi siRNA Negative Control Kit; Invitrogen). AN3CA cells were seeded 24 h before
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Table 1
IC50 values to SAR245409 under absence or presence of pimasertib, and Combination Index (CI) of SAR245409 and pimasertib in each cell line.
SAR245409 SAR245409 + SAR245409 + SAR245409 + Rapamycin +
Pimasertib 30 nM Pimasertib 100 nM Pimasertib 10 nM Pimasertib 30 nM
IC50 (uM) IC50 (uM) CI CI CI CI
HEC-116 5 0.2 0.07 0.14 1.01 1.1
HEC-1B 6 0.1 0.22 0.68 1.07 0.28
HEC-88 4 1 0.25 0.52 0.25 2
HEC-50B 3.5 0.1 0.32 1.03 0.53 1.3
AN3CA 0.7 0.2 0.36 0.53 1.03 2.1
HEC-151 6 1 0.46 1.67 0.27 1.8
HEC-6 4 4 1
HEC-108 7 7 1
HEC-59 2.2 3 1.4
Ishikawa 0.5 0.7 1.4
HHUA 3 7 2.3
KLE 3 9 3
transfection at approximately 30% confluence in 100-mm plates and transfected with 20 nmol/L siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen).
2.6. Statistical analysis
Means ± standard errors (SE) of parameters from 3 independent experiments were determined. The significance of differences was analyzed by Student’s t-test, and p b 0.05 was considered statistically sig-nificant. For combination experiments with SAR245309 and pimasertib, or rapamycin and pimasertib, combination indexes (CIs) were calculated according to the Chou–Talalay method using the IC50 values under the monotherapy and the combination therapy [35]. Synergism, additive effects, and antagonism were defined as CI b 1, CI = 1, and CI N 1, respectively.
3. Results
3.1. Antiproliferative effects of SAR245409 and pimasertib in a panel of endometrial cancer cell lines
The IC50 values of SAR245409 varied from 0.5 μM to 7 μM, whereas those of pimasertib varied from 0.1 μM to N20 μM (Fig. 1B and C). Of the 12 cell lines, 6 (50%) showed a high sensitivity to pimasertib (IC50 b 5 μM) (sensitive group), while the remaining 6 cell lines (resistant group) show a lower sensitivity to pimasertib (IC50 N 5 μM).
Mutations (or chromosomal gains) in PTEN, PIK3CA, and KRAS were found in 9, 5, and 4 of the 12 cell lines, respectively (Table S1). Two cell lines (Ishikawa and AN3CA) with PTEN mutations and wild-type PIK3CA showed IC50 values of b1 μM in response to SAR245409. However, neither PIK3CA status nor PTEN status was associated with SAR245409 IC50 values. Additionally, sensitivity to pimasertib was found to be unrelated with the mutational status of KRAS and TP53 (Table S1). Only 2 (HEC-1B and HEC-50B) of the 4 cell lines with KRAS alterations were included in the pimasertib-sensitive group. Overall, there was no correlation between sensitivity to SAR245409 and pimasertib and molecular alteration status.
The effects of SAR245409 and pimasertib on signaling pathways were evaluated by immunoblotting. SAR245409 suppressed phosphorylation
of AKT (p-AKT) and MDM2 (p-MDM2) at 0.1–1 μM or higher and that of p-S6 at 0.1–0.3 μM or higher in all 5 cell lines tested (Figs. 2A and S1). Pimasertib suppressed phosphorylation of ERK at ≥10–30 nM (Figs. 2A and S1). We quantified the phosphorylation levels of the target proteins using ImageJ. Inhibition of the PI3K and MAPK pathways was more important in AN3CA than in Ishikawa cells with both inhibitors (Fig. 2B). The phosphorylation levels of all target proteins were sup-pressed at lower inhibitor concentrations in AN3CA than in Ishikawa cells (Fig. 2B).
3.2. SAR245409 plus low-dose pimasertib has a synergistic effect in sensi-tive cell lines
SAR245409 and pimasertib were concurrently added to each cell line according to 4 series of concentration ratios of the drugs (Fig. 3A). MTT assays demonstrated that the combination suppressed cell proliferation more robustly than either agent alone in AN3CA cells (Fig. 3A). Cell proliferation was significantly suppressed by the combination in all 6 pimasertib-sensitive cell lines, but not in the 6 pimasertib-resistant cell lines (data not shown).
Next, we examined whether the effect of the combination was syn-ergistic. We fixed pimasertib concentrations at 100 nM, 30 nM, or 10 nM and added SAR245409 at 8 different doses to each cell line. The effect of pimasertib at 30 nM was comparable with that at 100 nM in the pimasertib-sensitive cell lines AN3CA, HEC-1B, and HEC-50b (Fig. 3B). However, 10 nM pimasertib did not show a significant additive effect in combination with SAR245409 in any of these 3 pimasertib-sensitive cells (Fig. 3B). No additive effect was observed at any dose in the 6 pimasertib-resistant cell lines including Ishikawa cells (Fig. 3B). The CI was ≤ 0.46 in all 6 pimasertib-sensitive cell lines at 30 nM of SAR245409 but was N1 in 3 of the 6 cell lines with SAR245409 at 10 nM (Table 1). Thus, the synergistic effect was high at doses of 1 μM SAR245409 and 30 nM pimasertib.
We also fixed the concentration of SAR245409 at 300 nM or 1 μM and added pimasertib at 8 different doses. SAR245409 at 300 nM did not cause an additive effect with pimasertib. However, the combination of SAR245409 at 1.0 μM with pimasertib more robustly suppressed cell proliferation in pimasertib-sensitive cells (HEC-1B, HEC-50B, and AN3CA) compared with SAR245409 alone (Fig. 3C).
Fig. 3. Enhanced antitumor effect of a combination of SAR245409 and pimasertib in pimasertib-sensitive cell lines. (A) Combination treatment (SAR245409 plus pimasertib) in AN3CA (pimasertib-sensitive) cells was compared with treatment with each agent alone by the MTT assay. Four drug concentration settings (a, b, c, d) were tested as indicated in the table below the graphs. The horizontal axis in the upper graphs represents the concentration of SAR245409, and the effect of the combination was compared with SAR245409 alone. The hor-izontal axis in the lower graphs represents the concentration of pimasertib, and the effect of the combination was compared with that of pimasertib alone. The results are shown as the mean ± SE of 3 independent experiments. (B) MTT assays of the combination of SAR245409 and pimasertib in 4 cell lines. AN3CA, HEC-1B, and HEC-50B were pimasertib-sensitive, where-as Ishikawa cells were pimasertib-resistant in the pimasertib-only experiment. The concentration of pimasertib was fixed at 100 nM, 30 nM, or 10 nM, and the data were compared with those of SAR245409 alone. The results are shown as the mean ± SE of 3 independent experiments. (C) MTT assays of the combination therapy in the 4 cell lines examined in (B). The concentration of SAR245409 was fixed at 300 nM or 1000 nM, and the data were compared with those of pimasertib alone. The results are shown as the mean ± SE of 3 independent experiments.
328 K. Inaba et al. / Gynecologic Oncology 138 (2015) 323–331
3.3. SAR245409 plus low-dose pimasertib induces G1 arrest
We examined the inhibition of phosphorylation of target proteins by using the combination of 1 μM SAR245409 and 30 nM pimasertib. As shown in Fig. 2A, phosphorylation of AKT, MDM2, and S6 was sup-pressed by exposure to SAR245409, and phosphorylation of ERK was inhibited by exposure to pimasertib. The combination of SAR245409 and pimasertib suppressed phosphorylation of all these proteins (Fig. 4A). The combination of 1 μM SAR245409 and 30 nM pimasertib markedly decreased the S-phase population and increased the G1
population in the sensitive cells (Fig. 4B). The sub-G1 population was not increased by either of the compounds, singly or in combination, suggesting that the synergistic effect was mainly cytostatic (Fig. 4B).
3.4. Knockdown of ERK1/2 enhances sensitivity to SAR245409
To confirm the more robust antitumor effect of the combination therapy of SAR245409 and inhibition of the MAPK pathway, we knocked down ERK1/2 in AN3CA cells before treatment with various concentrations of SAR245409. ERK1/2 expression was suppressed by
A
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Fig. 4. Inhibition of phosphorylation of target proteins and induction of G1 cell cycle arrest by the combination of SAR245409 and pimasertib. (A) The phosphorylation levels of AKT and MDM2 (in the PI3K pathway), S6 (in the mTOR pathway), and ERK (in the MAPK pathway) were evaluated by Western blotting after treatment with 1 μM SAR245409, 30 nM pimasertib, or a combination thereof. (B) Flow cytometric analysis of the cell cycle in HEC-116 (left), HEC-1B (middle), and HEC-50B (right) cells treated with 1 μM SAR245409, 30 nM pimasertib, or a combination thereof. (C) Suppression of ERK1/2 expression by siRNAs was confirmed by immunoblotting. siRNAs targeting MAP2K1and MAP2K2 (at 20 nmol/L each) were transfected into AN3CA cells. (i) siERK1/2-1, (ii) siERK1/2-2, and (iii) siERK1/2-3 correspond to mixtures of (i) HSS108538 and HSS183535, (ii) HSS108539 and HSS183536, and (iii) HSS108540 and HSS183537, respectively. Non-silencing siRNA (siCT) was used as a control. (D) MTT assays were performed under exposure to SAR245409 (25–100,000 nM) with siRNAs to ERK1/2 in AN3CA cells. The results are shown as the mean ± SE of 3 independent experiments.
K. Inaba et al. / Gynecologic Oncology 138 (2015) 323–331 329
N80% with the three siRNAs as indicated by immunoblotting (Fig. 4C). Knockdown of ERK1/2 significantly enhanced the antiproliferative effect of SAR245409 compared with the control (combination of siCT and SAR245409) (Fig. 4D). These data supported the synergistic effect of SAR245409 and the inhibition of the MAPK pathway in AN3CA cells.
3.5. Combination of rapamycin and pimasertib does not effectively induce synergy
Finally, we assessed whether the synergistic effect is also observed when using the mTOR inhibitor rapamycin. When rapamycin (2.5–2500 nM) was combined with pimasertib at 30 nM in an MTT assay, the synergistic effect was observed in only 1 (HEC-1B) of the 6 pimasertib-sensitive cell lines (Fig. 5A and B). We confirmed that rapamycin at 2.4 nM or higher doses clearly suppressed the level of p-S6 in the endometrial cancer cells (Fig. S2). The CI was N1.0 in 5 of the 6 pimasertib-sensitive cell lines (Fig. 5B and Table 1). No synergy was observed in the pimasertib-resistant KLE cells (CI = 1.1) (Fig. 5B).
4. Discussion
We herein focused on combination therapy using the PI3K/mTOR in-hibitor SAR245409 and the MEK inhibitor pimasertib in endometrial cancer cell lines, and found that the combination of SAR245409 with low-dose pimasertib synergistically inhibited cell growth, particularly in cells with high sensitivity to the MEK inhibitor.
First, we clarified the synergistic effects of the combination of SAR245409 and pimasertib in certain endometrial cancer cell lines. The synergistic effect was observed only in cells sensitive to pimasertib. Simultaneous inhibition of PI3K and MAPK pathways confers synergistic
antitumor effects on several types of tumors, including colorectal, ovarian, and lung cancer cells and rhabdomyosarcoma cells [36–38]. Furthermore, RAS/MEK activation is linked with the synergistic effect of the combination of PI3K and MAPK pathway inhibitors [39–41]. How-ever, in this study, the mutational status of KRAS was not associated with sensitivity to the MEK inhibitor or the synergistic effect of the com-bination therapy. One possibility is that hyperactivation of the PI3K pathway contributes to the resistance to the MEK inhibitor. KRAS muta-tions frequently coexist with PIK3CA mutations in endometrial cancer [5], e.g., the resistant cell line HHUA possesses triple mutations in KRAS, PIK3CA, and PTEN. Another possibility is that other pathways are associated with the resistance. The Wnt/beta-catenin/cyclin D1 path-way is frequently activated by mutations in beta-catenin (CTNNB1) and cyclin D1 (CCND1) [9,42], which might cause variable responses to this combination therapy. Further studies are warranted to elucidate how sensitivity to MEK inhibitors is controlled and why the sensitivity to pimasertib itself might be a predictive biomarker for the synergistic effect of this combination therapy.
Second, the 30-nM dose of pimasertib, which was required for the synergistic effect in combination with SAR245409, was much lower than the IC50 of pimasertib alone. The optimal combination dose (30 nM of pimasertib and 1 μM of SAR245409) suggests the importance of pharmacodynamic markers for each inhibitor. Immunoblot analysis indicated that the phosphorylation levels of the target proteins in the PI3K/mTOR pathway (AKT and S6) and the MAPK pathway (ERK) were significantly downregulated by ≥1 μM SAR245409 and ≥30 nM pimasertib, respectively. The higher IC50 values (μM range) of pimasertib than those of the combination suggested that activation of the PI3K pathway might contribute to the resistance to selective inhibition of MEK alone. Our data also implied that PI3K/mTOR pathway inhibition
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1000 0 10 1000
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100
Rapamycin+Pimas
ertib 30nM
50
0 Rapamycin (nM)
0.001 0.1 10 1000
HEC-50B
100
viability(%) 50
Cell 0
0 10 1000
KLE B
100 index 2
50 combination 1
0 0
0 10 1000
Rapamycin (nM)
Sensitive to pimasertib
Fig. 5. Non-synergistic effect of the combination of the mTOR inhibitor rapamycin and pimasertib in all cell lines except one. (A) MTT assays were performed under exposure to the combination of rapamycin (2.5–2,500 nM) and pimasertib (30 nM) in AN3CA, HEC-1B, HEC-50B, and KLE cells. The results are shown as the mean ± SE of 3 independent experiments.
(B) The combination indexes were calculated by Chou–Talalay method. Seven cell lines (6 pimasertib-sensitive and 1 pimasertib-resistant) were treated with a combination of rapamycin at 2.5–2500 nM and pimasertib at 30 nM. The results are shown as the mean ± SE of 3 independent experiments.
330 K. Inaba et al. / Gynecologic Oncology 138 (2015) 323–331
alone is not as efficient as the combination with MEK inhibition, because the IC50 values for SAR245409 were N2 μM in all the pimasertib-sensitive cells, except for AN3CA. Cell cycle analysis suggested that the synergistic effect was mainly cytostatic and supported that a low dose of pimasertib (30 nM) was sufficient to enhance the cytostatic effect. However, we should carefully interpret the dose dependency of each drug. For example, the pERK level in the Ishikawa cells was decreased by 30 nM of pimasertib (Figs. 2A and 4A); however, the pERK/ERK ratio was more robustly decreased by 300 nM than by 30 nM pimasertib (Fig. 2B). In addition, we cannot exclude the possibility that pERK-independent mechanisms affect the sensitivity to pimasertib and its combination with SAR245409. Clinical trials using both SAR245409 and pimasertib are underway, including for ovarian cancer (clinicaltrials.gov/ NCT01936363). Optimization of the concentration of each drug, especial-ly of pimasertib, is essential in these clinical trials because an overdose can be toxic and expensive, while underdose treatment can result in poor response.
Third, we confirmed that the combination of an mTOR inhibitor and pimasertib did not show a synergistic effect in the endometrial cancer cells, regardless of their sensitivity to pimasertib. In the past, we have shown that the dual PI3K/mTOR inhibitor NVP-BEZ235 provides more robust growth suppression than the mTOR inhibitor everolimus in en-dometrial cancer cell lines [12,18]. Moreover, low-dose rapamycin has been found to be sufficient to suppress phosphorylation of S6 [43]. The present data suggest that mTOR-independent effectors might be crucial and that inhibiting both mTOR and PI3K are essential to induce the synergistic effect with a MEK inhibitor in endometrial cancer.
This study has several limitations. First, no predictive biomarkers for either pimasertib or the combination of pimasertib and SAR245409 were identified. Second, it remains to be determined whether a low dose of a MEK inhibitor with an adequate dose of a PI3K/mTOR inhibitor induces synergistic effect in other types of tumors. Third, in vivo exper-iments using animal models such as mice inoculated with endometrial cancer cells and treated with SAR245409 and pimasertib are important to verify our results. Finally, although several clinical trials for ovarian and other types of tumors are underway, trials to evaluate the synergis-tic effect and tolerability of this combination in endometrial carcinomas are warranted.
In conclusion, we demonstrated that the combination of a MEK inhibitor and a PI3K/mTOR inhibitor synergistically suppressed cell growth in certain endometrial cancer cells. The dose of the MEK inhibi-tor in this combination might be reduced in patients with endometrial cancer. We also highlight that mTOR-independent AKT effectors might be required for induction of the synergy. Therefore, targeting both PI3K and mTOR would be essential for the combination therapy with a MEK inhibitor in endometrial cancer.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgments
We thank Coumaran Egile and Vincent Loic for their insightful comments on the data. We thank Merck KGaA and Sanofi for pimasertib and SAR245409, respectively. We also thank Masato Nishida for the generous gift of the Ishikawa cell line. We would like to thank Editage (www.editage.com) for English language editing.
This work was financially supported by Grants-in-Aid for Scientific Research (C), 23592437 and 24592505, and Grants-in-Aid for Young Scientific Research (B), 25893229, 25861473, and 25861471 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This study was also performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ygyno.2015.05.031.
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