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mTOR inhibitor NVP-BEZ235 on human melanoma
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Cancer Letters xxx (2017) 1e11
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Original Article
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Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma
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R. Calero a, E. Morchon b, I. Martinez-Argudo c, R. Serrano a, * a
Biochemistry Section, Faculty of Biochemistry and Environmental Sciences, University of Castilla-La Mancha, Toledo, Spain Albacete University Hospital, Albacete, Spain c Genetics Section, Faculty of Biochemistry and Environmental Sciences, University of Castilla-La Mancha, Toledo, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2017 Received in revised form 7 June 2017 Accepted 22 July 2017
Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/ AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. © 2017 Elsevier B.V. All rights reserved.
Keywords: HSP90 Melanoma PI3K/mTOR RTK
Introduction Malignant melanoma represents one of the most aggressive malignancies with a 5-year survival rate of less than 10% at the metastatic setting of the disease [1]. Despite the advances in the last decade to understand the biology of this tumor, it remains as one of the most refractory neoplasia to systemic drug therapy. The RAS/RAF/MAPK and PI3K/AKT/mTOR pathways are two major signal transduction cascades that are often hyper-activated in various human cancers, including melanoma [2,3]. These pathways can be constitutively activated in melanoma cells due to oncogenic mutations of their components (e.g.V600EBRAF, Q61R NRAS, Q79KAKT1) or by hyper-activation of Tyrosine Kinase Receptors (RTKs) (e.g. HER4, IGF-1R, PDGFRb) [4e7]. Furthermore, there are cross-interactions between both pathways showing reciprocal regulation through positive or negative feedbacks, and finally converging in the activation of common downstream proteins involved in cell survival and proliferation [8,9]. Because of the interplay between these pathways, patients treated with
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* Corresponding author. Castilla La Mancha University, Toledo, 45071 Spain. E-mail address: [email protected] (R. Serrano).
monotherapy directed to a single target gave only partial positive response and developed acquired resistance [10]. Inhibition of constitutively mutated V600EBRAF with tyrosine kinase inhibitors have shown promising results in recent years [11,12]. Unfortunately, most of the responses observed are transient, with relapse and resistance in most cases [13,14]. This acquired resistance is associated with MAPK pathway reactivation and PI3K/AKT/mTOR pathway activation by CRAF, NRAS activating mutations or RTKs hyper-activation [15e17]. It has been found that in 10e20% of tumors with resistance to MAPK inhibition there is a loss of PTEN or mutations in PI3K or AKT. Furthermore, dual inhibition of BRAF and PI3K/AKT pathway in PTEN melanoma cells induces apoptosis in BRAF inhibitors resistant cells [13]. Heat shock protein 90 (HSP90) is a chaperone involved in the folding and function of several proteins and is essential for cell survival. Many proteins related with melanoma initiation and progression, such as PDGFRb, COT, IGF-1R, CRAF, ARAF, S6, cyclin D1, and AKT, are client proteins of this chaperone [18e20]. Oncogenic mutations in such proteins cause them instability and demand higher expression of HSP90 to keep these proteins in a functional state [21]. HSP90 inhibitors have shown limited antitumor activity alone but more promising results have been obtained when they are combined with other agents [22,23]. This has
http://dx.doi.org/10.1016/j.canlet.2017.07.021 0304-3835/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 1. Tyrosine kinase receptors are expressed and correlate with MAPK and PI3K/AKT pathways in melanoma tumors and cell lines. (A) Volcano plot representation of the analyses of Oncomine datasets showing high expression of RTKs in human melanoma tumors compared with healthy skin tissue. Red dots represent more than 1.5-fold significantly overexpressed RTKs in melanoma tumors (p 0.05). (B) R2 platform (r2.amc.nl) was used to check for correlation between the expression of RTKs and the components of PI3K/AKT/
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 2. Activation of tyrosine kinase receptors in human melanoma cell lines and tumors. (A) Whole-cell lysates (500 mg of protein) from cell lines and patient samples were incubated on phospho-RTK antibody arrays. Each phospho-RTK antibody is spotted in duplicate; positive controls are included in corners. RTKs phosphorylation were analyzed in three melanoma cell lines, eight tumors (arrays from patient 1, 2, 5 and 8 are shown) and nontumoral sample from patient 5 (M5N). Each experiment was performed in triplicate. (B) Heatmap shows the RTKs activation (Fold-change compared to negative controls included in the array) of cell lines, tumors and healthy skin patient's samples.
made HSP90 an attractive target for melanoma sensitization to other therapies [18]. On this basis, we have studied the expression of 42 membrane tyrosine kinase receptors in melanoma cell lines. We also found a
positive correlation of the expression of these receptors with both PI3K/AKT/mTOR and RAS/RAF/MAPK pathways. We evaluated the phosphorylation profile of these receptors and the activation status of downstream kinases in human melanoma tumors and cell lines.
mTOR and RAS/RAF/MAPK pathways from KEGG Pathway database in 41 melanoma cell lines and 470 melanoma tumors (p 3 106). (C) mRNA expression level of RTKs in 41 melanoma cell lines ordered by BRAF and NRAS mutation status (green-BRAF wt; red-BRAF mutated; blue-NRAS wt; yellow-NRAS mutated). Gene expression analyses was performed using R2 Genomics Analysis and Visualization Platform (r2.amc.nl). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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We tested a panel of tyrosine kinase inhibitors in melanoma cell lines showing higher sensitivity to the dual PI3K/mTOR inhibitor NVP-BEZ235. Finally, we investigated the effect on these cells of the dual inhibition of HSP90 and PI3K/mTOR pathway with 17AAG and NVP-BEZ235. Our results highlight the importance of attacking multiple targets simultaneously to inhibit efficiently the growth of melanoma tumors.
Materials and methods Gene expression analysis Data analyses of gene expression (genesets correlation, KaplaneMeier survival curves and mRNA expression analyses) on publicly available data sets were performed using the R2 microarray analysis and visualization platform (http://r2.amc. nl) and Oncomine™ (Compendia Bioscience, Ann Arbor, MI). IC50 values for melanoma cell lines drug sensitivity analyses were obtain from Sanger Institute (http:// www.cancerrxgene.org/).
Fig. 3. PI3K/AKT/mTOR and RAS/RAF/MAPK pathways are upregulated in melanoma cell lines and tissues. (A) The AKT and MAPK activation status were determined in melanoma cell lines (EGF 100 ng/mL 300 vs. control cells) by immunoblotting for p-AKTSer473 and p-ERK1/2Thr202/Tyr204 levels. Total AKT and ERK1/2 levels were analyzed as loading controls (50 mg of protein). (B) Melanoma tissues derived from patients were assayed for phospho-ERK1/2 and phospho-AKT by immunohistochemistry in paraffin-embedded samples. Healthy skin from the same patients were assayed as negative controls. Magnification 10x; higher magnification is shown in some samples (squares inside). See Table 1 for tumor histology data and staining intensity quantification (0-none, 1-weak, 2-moderate, 3-strong). Table 1 Tumor localization, subtype, thickness and Clarck level of patient samples used in the study. P-ERK1/2 and p-AKT staining intensity quantification of immunohistochemistry experiments. Histology and Staining data of Malignant Melanomas Melanoma cases
Tumor site
Subtype
Thickness
Clark level
P-ERK1/2
p-Akt
M1 M2 M3 M4 M5 M6 M7 M8
Head Head Trunk Trunk Extremities Extremities Head Head
Lentigo maligna Superficial spreading Superficial spreading Nodular Acral lentiginous Superficial spreading Nodular Nodular
3,50 mm 9 mm 1,85 mm 6 mm 0,60 mm 0,48 mm 4 mm 1,5 mm
III IV IV III IV III IV IV
2 2 1 3 3 1 2 2
0 2 1 2 1 1 0 2
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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R. Calero et al. / Cancer Letters xxx (2017) 1e11 Patient samples Melanoma samples from 8 patients were obtained from the biobank of the Albacete University General Hospital. Samples from healthy skin, from the margins of resected specimens, were also extracted from these patients. Malignant melanoma samples were microscopically analyzed for subtype, Breslow tumor thickness (in mm), and level of invasion (Clark level, I-V). The Ethical Committee of the Albacete University General Hospital approved the study. Written informed consent was obtained from patients. Cell culture and reagents Human melanoma cell lines SK-MEL-2, WM115 and A2058 were purchased from the American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco's Modified Eagle's Medium Ham's F-12 (DMEM F-12) with 10% of heatinactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 C in a humidified 5% CO2 atmosphere. Sorafenib was purchased for research purposes in the pharmacy of the Albacete University Hospital, 17-allylamino-17demethoxygeldanamycin (17-AAG) was from Sigma-Aldrich and NVP-BEZ235 was from Selleck Chemicals.
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using the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich). Colorimetric evaluation was performed using a SPECTROstar Omega (BMG LABTECH GmbH, Offenburg/Germany) spectrophotometer at 555 nm and 690 nm. Calculation of IC50 values was done from log (concentration) vs effect curves. To determine whether the combinations of drugs were synergistic, additive or antagonistic we used the Calcusyn v2.0 Software (Biosoft, Cambridge, UK). Western blotting Cells were seeded at 106 cells per flask and incubated overnight at 37 C followed by treatment with selected compounds. After treatment, cells were collected and lysed in RIPA buffer (Sigma) with protease and phosphatase inhibitor cocktails (Sigma). Protein concentrations were determined using BCA protein assay. Equal amounts of proteins were loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes and incubated with primary antibodies specific for: total-AKT, phosphoAKT (Ser473), total p42/44 MAPK (ERK1/2), phospho-p42/44 MAPK (Thr202/ Tyr204), p27 (Cell Signaling Technology), cyclin D1 (BD Pharmingen) and GAPDH (Fitzgerald Ind. Int.). The membranes were washed three times with PBS buffer and then incubated with HRP-conjugated secondary antibodies followed by detection with enhanced chemiluminescence.
Immunohistochemically assays Sections (3e3.5 mm thick) of formalin-fixed paraffin-embedded tissue were immunostained with phospho-AKT (Ser473) (D9E) XP®Rabbit monoclonal antibody (4060, Cell Signaling Technology) and phospho-p44/42 MAPK (ERK1/2) (Thr202/ Tyr204) (D13.14.4E) XP®Rabbit monoclonal antibody (4370, Cell Signaling Technology). Immunohistochemical analysis was performed by an external laboratory (LV tech XXI Biotechnology). In the absence of independent healthy tissue in some of the cases, healthy areas adjacent to the tumor in the same slide were considered as negative controls. The immunostains were evaluated and manually scored using light microscopy to determine intensity of immunostaining.
Tyrosine kinase receptors arrays To identify the relative levels of phosphorylation of RTKs, protein lysates were used with Human Phospho-RTK Array Kit (R&D Systems, Minneapolis, US) according to manufacturer instructions. Quantitation of RTKs phosphorylation in the array was performed using the Multigauge V3.0 software (Fujifilm). Corresponding levels of tyrosine phosphorylation were calculated by subtracting the background from the values of RTK spots and normalized with the positive controls values present in each membrane. Cell cycle analyses
Cell viability assay Cells were seeded in 48-well plates overnight and treated with increasing concentrations of drugs or DMSO (vehicle) for 72 h. Cell viability was determined by
To evaluate cell cycle changes cells seeded in 6-well plates were treated with inhibitors. After 48-h incubation, cells were trypsinized, washed with ice-cold phosphate-buffered saline (PBS), and fixed in 70% ethanol at 20 C. Cells were
Fig. 4. PI3K/AKT/mTOR signaling pathway and HSP90 are overexpressed and correlates with poor survival in melanoma tumors. Analyses of Oncomine datasets showing high expression of (A) AKT (B) 4EBP1 and (C) HSP90 mRNA in human melanoma compared to normal skin tissue. KaplaneMeier survival estimates of high/low (D) AKT (E) 4EBP1 and (F) HSP90 expression in melanoma. The Kaplan scanning tool in the R2 platform (r2.amc.nl) was used to check for mRNA expression in a dataset of 44 metastatic melanoma tumors. Expression data were scanned to find the most optimal cut-off between high and low gene expression and the log-rank test that gave the lowest P-value were calculated to search for significant differences between tumor samples expressing high and low AKT, 4EBP1 or HSP90 mRNA levels. P-values were corrected for multiple testing (one-way ANOVA).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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R. Calero et al. / Cancer Letters xxx (2017) 1e11 washed with PBS and incubated with RNase 10 mg/mL and propidium iodide (PI) 30 mg/mL (Sigma-Aldrich, Inc.). Cell cycle analysis was performed using a FACSCanto II Flow Cytometer (BD Biosciences) and the data were analyzed using FACSDiva v 6.1.3. Software (BD Biosciences). Apoptosis assay Cells were seeded in 6-well plates overnight and treated with inhibitors for 48h. Apoptosis was determined by Annexin-V/PI method using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Apoptosis analysis was performed using a FACSCanto II Flow Cytometer and the data were analyzed using FACSDiva v 6.1.3. Software (BD Biosciences). Statistical analysis All experiments were performed in triplicate unless otherwise noted. Data are presented as the mean ± SD. Statistical significance between groups was performed using a two-sided Student's t-test. Multiple groups were compared with one-way ANOVA. Differences were considered to be statistically significant when P values were less than 0.05. All statistical analyses were performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
Results Expression and activation of tyrosine kinase receptors in human melanoma cell lines and tumors RTKs activation is a well-known acquired resistance mechanism of melanoma. In order to evaluate the expression of RTKs in human melanoma we compared the mRNA levels between normal skin and malignant melanoma tumors from published gene profiling studies available in the Oncomine platform (https://www. oncomine.org) (Fig. 1A). We found significant overexpression of several RTKs including ErbB2, ErbB3, PDGFRA and IGF1R. As constitutive signaling throw some of these receptors has been associated with BRAF inhibitors resistance, we compared the mRNA expression level of a panel of these receptors in 41 melanoma cell lines (30 BRAF mutated, 8 NRAS mutated and 3 BRAF/NRAS nonmutated) from Sanger Cell Line Cancer Panel dataset and in 36 melanoma cell lines (27 BRAF mutated, 5 NRAS mutated and 4 BRAF/NRAS nonmutated) from Cancer Cell Line Encyclopedia (CCLE), using the R2 microarray analysis and visualization platform (http://r2.amc.nl) (Fig. 1C and Supplemental Fig. 1B). We did not found a differential pattern of RTKs gene expression between BRAF mutated, NRAS mutated or WT cell lines. PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways can be activated by RTKs and are frequently upregulated in melanoma. We analyzed the Sanger Cell Line Cancer Panel, CCLE and The Cancer Genome Atlas (TCGA) mRNA expression datasets with R2 platform and we found significant correlation between RTKs and both PI3K/ AKT/mTOR and RAS/RAF/MAPK signaling pathways in melanoma cell lines and tumors (Fig. 1B and Supplemental Fig. 1A). Several RTKs, AKT and MAPK are constitutively phosphorylated in melanoma cell lines and tumors As tyrosine phosphorylation is indicative of the signaling activity of RTKs, we used human phospho-RTK arrays to analyze the activation of RTKs in melanoma cell lines and tumors (Fig. 2). Activated receptors included EGFR, FGFR, IGF1R, Axl, MSPR, PDGFR, RET, Tie, VEGFR and EphR families. EGFR family receptors can be
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seen activated in all tumor samples as in melanoma cell lines. PDGFRB was phosphorylated in WM115 cell line and two tumor samples (M4T and M8T). IGF1R phosphorylation was only detected in SK-MEL-2 cell line. None of the nontumoral samples showed RTK activity. Moreover, to assess if PI3K/AKT/mTOR and RAS/RAF/MAPK downstream signaling pathways are activated, cell lysates were analyzed by immunoblotting with the indicated antibodies (Fig. 3A). AKT and MAPK (ERK1/2) proteins were phosphorylated at standard culture conditions and no differences were detected after epidermal growth factor (EGF) stimulation. We next evaluated by immunohistochemistry the phosphorylated status of ERK 1/2 and AKT in melanoma tumors (Fig. 3B). ERK 1/2 appeared phosphorylated in Thr202/Tyr204 residues in all tumors with different signal intensities from weak staining in M3 and M6 samples to strong staining in M4 and M5 samples (Table 1). AKT (Ser473), was phosphorylated in 6/8 of the tumors with weak and moderate intensities. The staining was negative for M1 and M7 tumors. All the nontumoral samples were negative for p-ERK 1/2 and p-AKT staining. Our findings show that activation of MAPK was present in all the analyzed melanomas independently of thickness and level of invasion and that AKT activation is a very common feature of metastatic melanoma.
PI3K/AKT/mTOR pathway genes and HSP90 are overexpressed in melanoma and correlates with poor survival We used Oncomine platform to evaluate the mRNA expression of the PI3K/AKT/mTOR pathway components, AKT and 4EBP1, and the HSP90 chaperone in human melanoma tumors (Fig. 4AeC). We analyzed mRNA expression of AKT and 4EBP1 as downstream effectors of PI3K and mTOR protein complexes. We found these genes are highly expressed (15 and 3-fold respectively) in tumors compared to healthy skin tissue. HSP90 is also highly expressed (10.6 fold) in tumors compared to healthy skin tissue. Moreover, Kaplan Meier analysis revealed that high expression of AKT, 4EBP1 and HSP90 correlates with poor survival in human metastatic melanoma (Fig. 4DeF).
Melanoma cell lines show high sensitivity to PI3K/AKT/mTOR pathway inhibition by NVP-BEZ235 We also screened different tyrosine kinase inhibitors in a panel of 44 melanoma cell lines from Sanger Cell Line Cancer Panel (http://www.cancerrxgene.org/) (Fig. 5A). We tested Dabrafenib, SB590885 and PLX4720 (BRAF inhibitors); Sorafenib (BRAF/RTKs inhibitor); Sunitinib and Lapatinib (multi-RTKs inhibitors) and the dual PI3K/mTOR inhibitor NVP-BEZ235. Cells showed significantly higher sensitivity (lower IC50) to NVP-BEZ235 (64.04-fold; p 0.03). To validate these results, we performed MTT assays with A2058 and SK-MEL-2 cell lines treated with increasing concentrations of Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235 for 72 h (Fig. 5B). We observed similar response with higher sensitivity of these cells to NVP-BEZ235 than to other tyrosine kinase inhibitors (6e20 fold lower IC50).
Fig. 5. HSP90 inhibitor 17AAG show a synergistic effect with NVP-BEZ235 in melanoma cell lines. (A) Analyses of melanoma cell lines sensitivity to panel of tyrosine kinase inhibitors (Dabrafenib, SB590885, PLX4720, Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235) using Sanger Institute Drug Sensitivity Platform (http://www.cancerrxgene.org). Melanoma cell lines show high sensitivity (lower IC50) to the dual PI3K/mTOR inhibitor NVP-BEZ235 (p < 0.0001). (B) MTT assays of A2058 and SK-MEL-2 cell lines treated with increasing concentrations of Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235 (72 h; mean ± SD, n ¼ 4). (C) MTT assays of melanoma cell lines in response to NVP-BEZ235,17AAG or the combination of both drugs in a fixed ratio (2,5:1) (72 h; mean ± SD, n ¼ 4 experiments). (D) Combination index (CI) showing the synergistic effect of NVP-BEZ235 and 17AAG on SK-MEL-2 and A2058 cell lines. Used concentrations are specified in the table where strength of synergy was quantified from – (strong antagonism) to þþþþ (strong synergism) (E) Western blots showing AKT and ERK1/2 phosphorylation from cells treated with Sorafenib, 17AAG, NVP-BEZ235 or combinations at the indicated doses for 24 h. Representative western blots (top) and summaries (bottom) (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. Control; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 6. Effect of NVP-BEZ235 and 17AAG on melanoma cell lines apoptosis and cell cycle. (A) (left) Representative flow cytometry dot plots for Annexin V and PI staining in SKMEL-2 and A2058 cell lines treated with NVP-BEZ235, 17AAG or combination of both at the indicated concentrations for 48 h. (Right) Quantification of annexin-V positive cells (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. DMSO; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone). (B) Quantification of cell cycle phases in melanoma
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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HSP90 inhibition combined with NVP-BEZ235 has a synergistic effect on melanoma cell lines As PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways components and some RTKs we found overexpressed and/or overactivated in melanoma cells are client proteins of HSP90, we investigated the effect on cell viability of the HSP90 inhibitor 17AAG alone or combined with NVP-BEZ235. MTT assays showed that the combination of the two drugs was markedly more effective in the inhibition of cell viability than 17AAG or NVP-BEZ235 alone (Fig. 5C). Combination index (CI) values showed synergism between both drugs for almost all the concentrations tested (Fig. 5D). We also combined the RTKs/RAF inhibitor, Sorafenib, with 17AAG (Supplemental Fig. 1AeB). MTTs results showed that this combination has also a synergistic effect on the viability of melanoma cell lines. We next determined the phosphorylation status of AKT and ERK1/2 proteins treated with NVP-BEZ235, 17AAG and Sorafenib alone or in combination (Fig. 5E). Treatment with NVP-BEZ235, produced a moderate to no effect on AKT phosphorylation and a significant increase in ERK1/2 phosphorylation in SK-MEL-2 and A2058 cell lines. These cells treated with Sorafenib showed a decrease in ERK1/2 phosphorylation. This effect was accompanied by an increase in AKT phosphorylation level in both cell lines. 17AAG 1 mM treatment produced a significant decrease in both AKT and ERK1/2 phosphorylation levels in SK-MEL-2 cells but no effect on A2058 p-AKT level. SK-MEL-2 cells treated with NVPBEZ235 þ Sorafenib showed an increase in p-AKT level compared to control and a moderate decrease in ERK1/2 phosphorylation. The same drug combination showed no effect on A2058 cell line compared to untreated cells. When Sorafenib and 17AAG were combined both pathways were inhibited in SK-MEL-2 cells but no effect on p-AKT was observed in A2058 cells. Finally, combination of NVP-BEZ235 and 17AAG reduced AKT and ERK1/2 phosphorylation significantly in both cell lines. This combination was markedly more effective not only in cell viability reduction, but also in the inhibition of PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways.
Dual inhibition of HSP90 and PI3K/AKT/mTOR pathway induces apoptosis and reduces the proliferation of melanoma cells Next, we examined whether these drugs have a pro-apoptotic effect in melanoma cells by measuring the percentage of Annexin-V positive cells using flow cytometry analyses (Fig. 6A). NVP-BEZ235 and 17AAG combination increased the percentage of apoptotic population in SK-MEL-2 and A2058 cell lines with respect to the control (DMSO) or both drugs alone (p < 0.05). We also analyzed the effect of 17AAG, NVP-BEZ235 or the combination of both on the cell cycle (Fig. 6B). 17AAG treatment produce a moderate G2 cell cycle arrest (increase respect to control: 9% SK-MEL-2; 7% A2058; p < 0,05). Treatment of melanoma cells with 17AAG and NVP-BEZ235 simultaneously for 48 h led to an increase in the Sub G0 population of around 13% in SK-MEL-2 and A2058 cells (p < 0,05) and proliferation inhibition (decrease in S phase: 7,6% and 5,4% respectively for SK-MEL-2 and A2058 cells; p < 0,05) versus control cells. These cells cycle changes are concomitant with a slight G1 phase arrest. Western blot shows that SK-MEL-2 cell line treated with NVP-BEZ235 and 17AAG showed a significant decrease in Cyclin D1 protein and up-regulation of p27 expression (p 0,05)
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compared to single treatment with NVP-BEZ235, 17AAG, Sorafenib or 17AAG þ Sorafenib (Fig. 6C). A2058 cell line treated with NVPBEZ235 þ 17AAG showed a significant decrease in Cyclin D1 protein compared to single treatment with NVP-BEZ235 and Sorafenib but differences between NVP-BEZ235 þ 17AAG and 17AAG or 17AAG þ Sorafenib were not significant. P27 expression was significantly up-regulated when A2058 cells are treated with NVPBEZ235 þ 17AAG (p 0,0001). These results are consistent with the anti-proliferative effect for this combination of drugs.
Discussion Drug resistance is the major factor that limits the long-term efficacy of targeted therapies in melanoma patients. BRAF inhibition has not reached the expected results in the clinical practice with temporary responses and the development of resistance to these inhibitors. This resistance is the result of diverse intrinsic and acquired mechanisms like the recovery of MAPK signaling or activation of alternative signaling pathways, such as PI3K/AKT/mTOR by upregulation of RTKs like IGF-1R, EPHA2 or PDGFRb [12,16,24,25]. Currently, the combination of BRAF with MEK inhibitors represents the gold standard of targeted therapy in BRAF mutated melanoma. Unfortunately, the efficacy of MEK inhibitors is also limited by the activation of the PI3K/AKT/mTOR pathway that drives resistance development [26,27]. In this work, we studied the expression and activation status of RTKs in both melanoma cell lines and tumors and we show its correlation with the expression and phosphorylation of PI3K/AKT/ mTOR and RAS/RAF/MAPK pathways components. It is known that membrane tyrosine kinase receptors can activate both, PI3K/AKT and MAPK pathways and hyper-activation of these receptors, deregulated expression of its ligands and aberrant expression of integrins and cytokines, contribute to tumor cell proliferation, survival and resistance to cell death [28e30]. Activation of several RTKs has been detected previously in melanoma cell lines and short hairpin RNA knockdown of some of these receptors inhibited the proliferation of melanoma cells by AKT or STAT3 signaling pathways modulation [31]. There are evidences that support the role of PI3K/AKT pathway in melanoma initiation [32]. The transformation of normal nevi to melanoma in situ, and from melanoma in situ to metastasis, is drived by p-AKT progressive accumulation [33]. Moreover, co-expression of the PI3K p110 subunit and mTOR have been demonstrated in melanoma tumors and mTOR inhibition with Rapamycin enhances the activity of PI3K/AKT [34]. Here we show that HSP90 and PI3K/AKT/mTOR pathway components are highly expressed in melanoma tumors and this expression correlates with poor survival in melanoma patients representing potential therapeutic targets. HSP90 inhibition has been demonstrated to be a highly effective strategy to overcome BRAF and MEK inhibitors resistance in vitro and in vivo [23,35e37]. Nevertheless, no objective response has been observed in phase II clinical trials with 17AAG [38]. There are other ongoing clinical trials combining HSP90 inhibitors with BRAF and MEK inhibitors but no phase II data has been published yet. Interestingly, it has been demonstrated that HSP90 inhibition induces HSP70 expression as a compensatory mechanism that contributes to drug resistance and lower sensitivity to targeted HSP90 inhibition of melanoma [39]. This mechanism seems to be regulated by mTOR-dependent translational activity supporting the rationale of using NVP-BEZ235 in combination with HSP90 targeted therapy in melanoma [40]. Our data
cell lines treated with 17AAG, NVP-BEZ235 or both for 48 h. Histograms show the mean percentages of cells in SubG0, G0/G1, S and G2/M phases (n ¼ 3). (C) Representative western blots (top) and summaries (bottom) showing cell cycle-related proteins in SK-MEL-2 and A2058 cells after 24 h treatment with the indicated concentrations of NVP-BEZ235, 17AAG and Sorafenib. GAPDH was used as loading control (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. Control; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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shows that treatment with 17AAG reduces AKT and ERK1/2 phosphorylation in SK-MEL-2 cells but in A2058 cells AKT phosphorylation is still present. On the other hand, previous studies showed that NVP-BEZ235 effectively reduced the viability and clonogenicity of 23 melanoma cell lines, regardless of BRAF mutation status. This drug also produced the downregulation of p-AKT and pP70S6K [34,41]. In the present study, we show that melanoma cell lines are highly sensible to NVP-BEZ235 in comparison to other tyrosine kinase inhibitors including RTKs and BRAF inhibitors. We observed that treatment of melanoma cell lines with NVP-BEZ235 slightly reduces the phosphorylation of AKT but increases the level of phosphorylated ERK1/2. Furthermore, treatment with the RTKs/RAF inhibitor, Sorafenib, produced an inverse effect with an increase in AKT phosphorylation and a reduction in phosphorylated ERK1/2. There are numerous evidences in melanoma and other tumors of MAPK and PI3K pathways cross-interaction by reciprocal regulation through positive or negative feedbacks and how inhibitors of one of these pathways induce a compensatory mechanism that over-activates the other pathway to maintain survival and proliferation signaling [9,42,43]. This effect has been described previously, with NVP-BEZ235 in particular, in melanoma and other tumors [43]. Treatment with the combination of 17AAG and NVPBEZ235 highly abolished this effect allowing a strong suppression of p-ERK and p-AKT. Combined treatment also shows a synergistic effect on cell viability and a significant induction of apoptosis. Interestingly, little effect of single treatment with NVP-BEZ235 on apoptosis due to incomplete growth inhibition in melanoma has been reported [44]. NVP-BEZ235 and 17AAG combined treatment has also an antiproliferative effect on melanoma cells and results in Cyclin D1 decrease and p27 up-regulation when compared with each drug alone. In conclusion, our findings suggest that both signaling pathways, RAS/RAF/MAPK and PI3K/AKT/mTOR, must be strongly inhibited to achieve a notably effect on proliferation and apoptosis. In this line, combinations of drugs targeting these two pathways have been proposed in the last years for melanoma treatment, especially for those unresponsive to BRAF inhibitors [45e47]. The combination of PI3K/AKT/mTOR and HSP90 inhibition appears to be very effective because it abrogates the activation of AKT and ERK that is promoted by the increase of RTK activity, RAF and RAS signaling in metastatic melanoma. On one side, NVP-BEZ235 inhibits the PI3K/AKT/mTOR pathway that functions as survival mechanism in melanoma cells and on the other the use of an HSP90 inhibitor, like 17AAG, potentiates NVP-BEZ235 effect and provides additional inhibitory activity that target multiple oncogenic kinases that cooperate to drive tumor growth, survival, and resistance to therapy. Therefore, our study provides a basis for the development of new specific drugs that target these pathways. Animal model studies and clinical trials will give valuable information about this mechanisms. The identification of the subgroup of melanoma patients that are most likely to respond to this treatment remains as a major challenge. Acknowledgements This study was partially supported by the scientific foundation of Spanish Association against Cancer (AECC), the Castilla La Mancha University, the Castilla la Mancha Foundation for Health Research and Castilla la Mancha Health Service. The authors are ~ o for her technical assistance in cultures grateful to Laura Avendan procedures and Syong H Nam-Cha for the immunostains evaluation. We also greatly thank the Biobank of the Albacete University Hospital, integrated into the National Biobank Network of Carlos III Institute (RETICS BIOBANK, RD09/0076/00047), for kindly supplying human samples.
Conflict of interest The authors state no conflict of interest.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2017.07.021.
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Original Article
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Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma
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R. Calero a, E. Morchon b, I. Martinez-Argudo c, R. Serrano a, * a
Biochemistry Section, Faculty of Biochemistry and Environmental Sciences, University of Castilla-La Mancha, Toledo, Spain Albacete University Hospital, Albacete, Spain c Genetics Section, Faculty of Biochemistry and Environmental Sciences, University of Castilla-La Mancha, Toledo, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2017 Received in revised form 7 June 2017 Accepted 22 July 2017
Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/ AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. © 2017 Elsevier B.V. All rights reserved.
Keywords: HSP90 Melanoma PI3K/mTOR RTK
Introduction Malignant melanoma represents one of the most aggressive malignancies with a 5-year survival rate of less than 10% at the metastatic setting of the disease [1]. Despite the advances in the last decade to understand the biology of this tumor, it remains as one of the most refractory neoplasia to systemic drug therapy. The RAS/RAF/MAPK and PI3K/AKT/mTOR pathways are two major signal transduction cascades that are often hyper-activated in various human cancers, including melanoma [2,3]. These pathways can be constitutively activated in melanoma cells due to oncogenic mutations of their components (e.g.V600EBRAF, Q61R NRAS, Q79KAKT1) or by hyper-activation of Tyrosine Kinase Receptors (RTKs) (e.g. HER4, IGF-1R, PDGFRb) [4e7]. Furthermore, there are cross-interactions between both pathways showing reciprocal regulation through positive or negative feedbacks, and finally converging in the activation of common downstream proteins involved in cell survival and proliferation [8,9]. Because of the interplay between these pathways, patients treated with
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* Corresponding author. Castilla La Mancha University, Toledo, 45071 Spain. E-mail address: [email protected] (R. Serrano).
monotherapy directed to a single target gave only partial positive response and developed acquired resistance [10]. Inhibition of constitutively mutated V600EBRAF with tyrosine kinase inhibitors have shown promising results in recent years [11,12]. Unfortunately, most of the responses observed are transient, with relapse and resistance in most cases [13,14]. This acquired resistance is associated with MAPK pathway reactivation and PI3K/AKT/mTOR pathway activation by CRAF, NRAS activating mutations or RTKs hyper-activation [15e17]. It has been found that in 10e20% of tumors with resistance to MAPK inhibition there is a loss of PTEN or mutations in PI3K or AKT. Furthermore, dual inhibition of BRAF and PI3K/AKT pathway in PTEN melanoma cells induces apoptosis in BRAF inhibitors resistant cells [13]. Heat shock protein 90 (HSP90) is a chaperone involved in the folding and function of several proteins and is essential for cell survival. Many proteins related with melanoma initiation and progression, such as PDGFRb, COT, IGF-1R, CRAF, ARAF, S6, cyclin D1, and AKT, are client proteins of this chaperone [18e20]. Oncogenic mutations in such proteins cause them instability and demand higher expression of HSP90 to keep these proteins in a functional state [21]. HSP90 inhibitors have shown limited antitumor activity alone but more promising results have been obtained when they are combined with other agents [22,23]. This has
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Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 1. Tyrosine kinase receptors are expressed and correlate with MAPK and PI3K/AKT pathways in melanoma tumors and cell lines. (A) Volcano plot representation of the analyses of Oncomine datasets showing high expression of RTKs in human melanoma tumors compared with healthy skin tissue. Red dots represent more than 1.5-fold significantly overexpressed RTKs in melanoma tumors (p 0.05). (B) R2 platform (r2.amc.nl) was used to check for correlation between the expression of RTKs and the components of PI3K/AKT/
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 2. Activation of tyrosine kinase receptors in human melanoma cell lines and tumors. (A) Whole-cell lysates (500 mg of protein) from cell lines and patient samples were incubated on phospho-RTK antibody arrays. Each phospho-RTK antibody is spotted in duplicate; positive controls are included in corners. RTKs phosphorylation were analyzed in three melanoma cell lines, eight tumors (arrays from patient 1, 2, 5 and 8 are shown) and nontumoral sample from patient 5 (M5N). Each experiment was performed in triplicate. (B) Heatmap shows the RTKs activation (Fold-change compared to negative controls included in the array) of cell lines, tumors and healthy skin patient's samples.
made HSP90 an attractive target for melanoma sensitization to other therapies [18]. On this basis, we have studied the expression of 42 membrane tyrosine kinase receptors in melanoma cell lines. We also found a
positive correlation of the expression of these receptors with both PI3K/AKT/mTOR and RAS/RAF/MAPK pathways. We evaluated the phosphorylation profile of these receptors and the activation status of downstream kinases in human melanoma tumors and cell lines.
mTOR and RAS/RAF/MAPK pathways from KEGG Pathway database in 41 melanoma cell lines and 470 melanoma tumors (p 3 106). (C) mRNA expression level of RTKs in 41 melanoma cell lines ordered by BRAF and NRAS mutation status (green-BRAF wt; red-BRAF mutated; blue-NRAS wt; yellow-NRAS mutated). Gene expression analyses was performed using R2 Genomics Analysis and Visualization Platform (r2.amc.nl). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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We tested a panel of tyrosine kinase inhibitors in melanoma cell lines showing higher sensitivity to the dual PI3K/mTOR inhibitor NVP-BEZ235. Finally, we investigated the effect on these cells of the dual inhibition of HSP90 and PI3K/mTOR pathway with 17AAG and NVP-BEZ235. Our results highlight the importance of attacking multiple targets simultaneously to inhibit efficiently the growth of melanoma tumors.
Materials and methods Gene expression analysis Data analyses of gene expression (genesets correlation, KaplaneMeier survival curves and mRNA expression analyses) on publicly available data sets were performed using the R2 microarray analysis and visualization platform (http://r2.amc. nl) and Oncomine™ (Compendia Bioscience, Ann Arbor, MI). IC50 values for melanoma cell lines drug sensitivity analyses were obtain from Sanger Institute (http:// www.cancerrxgene.org/).
Fig. 3. PI3K/AKT/mTOR and RAS/RAF/MAPK pathways are upregulated in melanoma cell lines and tissues. (A) The AKT and MAPK activation status were determined in melanoma cell lines (EGF 100 ng/mL 300 vs. control cells) by immunoblotting for p-AKTSer473 and p-ERK1/2Thr202/Tyr204 levels. Total AKT and ERK1/2 levels were analyzed as loading controls (50 mg of protein). (B) Melanoma tissues derived from patients were assayed for phospho-ERK1/2 and phospho-AKT by immunohistochemistry in paraffin-embedded samples. Healthy skin from the same patients were assayed as negative controls. Magnification 10x; higher magnification is shown in some samples (squares inside). See Table 1 for tumor histology data and staining intensity quantification (0-none, 1-weak, 2-moderate, 3-strong). Table 1 Tumor localization, subtype, thickness and Clarck level of patient samples used in the study. P-ERK1/2 and p-AKT staining intensity quantification of immunohistochemistry experiments. Histology and Staining data of Malignant Melanomas Melanoma cases
Tumor site
Subtype
Thickness
Clark level
P-ERK1/2
p-Akt
M1 M2 M3 M4 M5 M6 M7 M8
Head Head Trunk Trunk Extremities Extremities Head Head
Lentigo maligna Superficial spreading Superficial spreading Nodular Acral lentiginous Superficial spreading Nodular Nodular
3,50 mm 9 mm 1,85 mm 6 mm 0,60 mm 0,48 mm 4 mm 1,5 mm
III IV IV III IV III IV IV
2 2 1 3 3 1 2 2
0 2 1 2 1 1 0 2
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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R. Calero et al. / Cancer Letters xxx (2017) 1e11 Patient samples Melanoma samples from 8 patients were obtained from the biobank of the Albacete University General Hospital. Samples from healthy skin, from the margins of resected specimens, were also extracted from these patients. Malignant melanoma samples were microscopically analyzed for subtype, Breslow tumor thickness (in mm), and level of invasion (Clark level, I-V). The Ethical Committee of the Albacete University General Hospital approved the study. Written informed consent was obtained from patients. Cell culture and reagents Human melanoma cell lines SK-MEL-2, WM115 and A2058 were purchased from the American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco's Modified Eagle's Medium Ham's F-12 (DMEM F-12) with 10% of heatinactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 C in a humidified 5% CO2 atmosphere. Sorafenib was purchased for research purposes in the pharmacy of the Albacete University Hospital, 17-allylamino-17demethoxygeldanamycin (17-AAG) was from Sigma-Aldrich and NVP-BEZ235 was from Selleck Chemicals.
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using the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich). Colorimetric evaluation was performed using a SPECTROstar Omega (BMG LABTECH GmbH, Offenburg/Germany) spectrophotometer at 555 nm and 690 nm. Calculation of IC50 values was done from log (concentration) vs effect curves. To determine whether the combinations of drugs were synergistic, additive or antagonistic we used the Calcusyn v2.0 Software (Biosoft, Cambridge, UK). Western blotting Cells were seeded at 106 cells per flask and incubated overnight at 37 C followed by treatment with selected compounds. After treatment, cells were collected and lysed in RIPA buffer (Sigma) with protease and phosphatase inhibitor cocktails (Sigma). Protein concentrations were determined using BCA protein assay. Equal amounts of proteins were loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes and incubated with primary antibodies specific for: total-AKT, phosphoAKT (Ser473), total p42/44 MAPK (ERK1/2), phospho-p42/44 MAPK (Thr202/ Tyr204), p27 (Cell Signaling Technology), cyclin D1 (BD Pharmingen) and GAPDH (Fitzgerald Ind. Int.). The membranes were washed three times with PBS buffer and then incubated with HRP-conjugated secondary antibodies followed by detection with enhanced chemiluminescence.
Immunohistochemically assays Sections (3e3.5 mm thick) of formalin-fixed paraffin-embedded tissue were immunostained with phospho-AKT (Ser473) (D9E) XP®Rabbit monoclonal antibody (4060, Cell Signaling Technology) and phospho-p44/42 MAPK (ERK1/2) (Thr202/ Tyr204) (D13.14.4E) XP®Rabbit monoclonal antibody (4370, Cell Signaling Technology). Immunohistochemical analysis was performed by an external laboratory (LV tech XXI Biotechnology). In the absence of independent healthy tissue in some of the cases, healthy areas adjacent to the tumor in the same slide were considered as negative controls. The immunostains were evaluated and manually scored using light microscopy to determine intensity of immunostaining.
Tyrosine kinase receptors arrays To identify the relative levels of phosphorylation of RTKs, protein lysates were used with Human Phospho-RTK Array Kit (R&D Systems, Minneapolis, US) according to manufacturer instructions. Quantitation of RTKs phosphorylation in the array was performed using the Multigauge V3.0 software (Fujifilm). Corresponding levels of tyrosine phosphorylation were calculated by subtracting the background from the values of RTK spots and normalized with the positive controls values present in each membrane. Cell cycle analyses
Cell viability assay Cells were seeded in 48-well plates overnight and treated with increasing concentrations of drugs or DMSO (vehicle) for 72 h. Cell viability was determined by
To evaluate cell cycle changes cells seeded in 6-well plates were treated with inhibitors. After 48-h incubation, cells were trypsinized, washed with ice-cold phosphate-buffered saline (PBS), and fixed in 70% ethanol at 20 C. Cells were
Fig. 4. PI3K/AKT/mTOR signaling pathway and HSP90 are overexpressed and correlates with poor survival in melanoma tumors. Analyses of Oncomine datasets showing high expression of (A) AKT (B) 4EBP1 and (C) HSP90 mRNA in human melanoma compared to normal skin tissue. KaplaneMeier survival estimates of high/low (D) AKT (E) 4EBP1 and (F) HSP90 expression in melanoma. The Kaplan scanning tool in the R2 platform (r2.amc.nl) was used to check for mRNA expression in a dataset of 44 metastatic melanoma tumors. Expression data were scanned to find the most optimal cut-off between high and low gene expression and the log-rank test that gave the lowest P-value were calculated to search for significant differences between tumor samples expressing high and low AKT, 4EBP1 or HSP90 mRNA levels. P-values were corrected for multiple testing (one-way ANOVA).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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R. Calero et al. / Cancer Letters xxx (2017) 1e11 washed with PBS and incubated with RNase 10 mg/mL and propidium iodide (PI) 30 mg/mL (Sigma-Aldrich, Inc.). Cell cycle analysis was performed using a FACSCanto II Flow Cytometer (BD Biosciences) and the data were analyzed using FACSDiva v 6.1.3. Software (BD Biosciences). Apoptosis assay Cells were seeded in 6-well plates overnight and treated with inhibitors for 48h. Apoptosis was determined by Annexin-V/PI method using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Apoptosis analysis was performed using a FACSCanto II Flow Cytometer and the data were analyzed using FACSDiva v 6.1.3. Software (BD Biosciences). Statistical analysis All experiments were performed in triplicate unless otherwise noted. Data are presented as the mean ± SD. Statistical significance between groups was performed using a two-sided Student's t-test. Multiple groups were compared with one-way ANOVA. Differences were considered to be statistically significant when P values were less than 0.05. All statistical analyses were performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
Results Expression and activation of tyrosine kinase receptors in human melanoma cell lines and tumors RTKs activation is a well-known acquired resistance mechanism of melanoma. In order to evaluate the expression of RTKs in human melanoma we compared the mRNA levels between normal skin and malignant melanoma tumors from published gene profiling studies available in the Oncomine platform (https://www. oncomine.org) (Fig. 1A). We found significant overexpression of several RTKs including ErbB2, ErbB3, PDGFRA and IGF1R. As constitutive signaling throw some of these receptors has been associated with BRAF inhibitors resistance, we compared the mRNA expression level of a panel of these receptors in 41 melanoma cell lines (30 BRAF mutated, 8 NRAS mutated and 3 BRAF/NRAS nonmutated) from Sanger Cell Line Cancer Panel dataset and in 36 melanoma cell lines (27 BRAF mutated, 5 NRAS mutated and 4 BRAF/NRAS nonmutated) from Cancer Cell Line Encyclopedia (CCLE), using the R2 microarray analysis and visualization platform (http://r2.amc.nl) (Fig. 1C and Supplemental Fig. 1B). We did not found a differential pattern of RTKs gene expression between BRAF mutated, NRAS mutated or WT cell lines. PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways can be activated by RTKs and are frequently upregulated in melanoma. We analyzed the Sanger Cell Line Cancer Panel, CCLE and The Cancer Genome Atlas (TCGA) mRNA expression datasets with R2 platform and we found significant correlation between RTKs and both PI3K/ AKT/mTOR and RAS/RAF/MAPK signaling pathways in melanoma cell lines and tumors (Fig. 1B and Supplemental Fig. 1A). Several RTKs, AKT and MAPK are constitutively phosphorylated in melanoma cell lines and tumors As tyrosine phosphorylation is indicative of the signaling activity of RTKs, we used human phospho-RTK arrays to analyze the activation of RTKs in melanoma cell lines and tumors (Fig. 2). Activated receptors included EGFR, FGFR, IGF1R, Axl, MSPR, PDGFR, RET, Tie, VEGFR and EphR families. EGFR family receptors can be
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seen activated in all tumor samples as in melanoma cell lines. PDGFRB was phosphorylated in WM115 cell line and two tumor samples (M4T and M8T). IGF1R phosphorylation was only detected in SK-MEL-2 cell line. None of the nontumoral samples showed RTK activity. Moreover, to assess if PI3K/AKT/mTOR and RAS/RAF/MAPK downstream signaling pathways are activated, cell lysates were analyzed by immunoblotting with the indicated antibodies (Fig. 3A). AKT and MAPK (ERK1/2) proteins were phosphorylated at standard culture conditions and no differences were detected after epidermal growth factor (EGF) stimulation. We next evaluated by immunohistochemistry the phosphorylated status of ERK 1/2 and AKT in melanoma tumors (Fig. 3B). ERK 1/2 appeared phosphorylated in Thr202/Tyr204 residues in all tumors with different signal intensities from weak staining in M3 and M6 samples to strong staining in M4 and M5 samples (Table 1). AKT (Ser473), was phosphorylated in 6/8 of the tumors with weak and moderate intensities. The staining was negative for M1 and M7 tumors. All the nontumoral samples were negative for p-ERK 1/2 and p-AKT staining. Our findings show that activation of MAPK was present in all the analyzed melanomas independently of thickness and level of invasion and that AKT activation is a very common feature of metastatic melanoma.
PI3K/AKT/mTOR pathway genes and HSP90 are overexpressed in melanoma and correlates with poor survival We used Oncomine platform to evaluate the mRNA expression of the PI3K/AKT/mTOR pathway components, AKT and 4EBP1, and the HSP90 chaperone in human melanoma tumors (Fig. 4AeC). We analyzed mRNA expression of AKT and 4EBP1 as downstream effectors of PI3K and mTOR protein complexes. We found these genes are highly expressed (15 and 3-fold respectively) in tumors compared to healthy skin tissue. HSP90 is also highly expressed (10.6 fold) in tumors compared to healthy skin tissue. Moreover, Kaplan Meier analysis revealed that high expression of AKT, 4EBP1 and HSP90 correlates with poor survival in human metastatic melanoma (Fig. 4DeF).
Melanoma cell lines show high sensitivity to PI3K/AKT/mTOR pathway inhibition by NVP-BEZ235 We also screened different tyrosine kinase inhibitors in a panel of 44 melanoma cell lines from Sanger Cell Line Cancer Panel (http://www.cancerrxgene.org/) (Fig. 5A). We tested Dabrafenib, SB590885 and PLX4720 (BRAF inhibitors); Sorafenib (BRAF/RTKs inhibitor); Sunitinib and Lapatinib (multi-RTKs inhibitors) and the dual PI3K/mTOR inhibitor NVP-BEZ235. Cells showed significantly higher sensitivity (lower IC50) to NVP-BEZ235 (64.04-fold; p 0.03). To validate these results, we performed MTT assays with A2058 and SK-MEL-2 cell lines treated with increasing concentrations of Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235 for 72 h (Fig. 5B). We observed similar response with higher sensitivity of these cells to NVP-BEZ235 than to other tyrosine kinase inhibitors (6e20 fold lower IC50).
Fig. 5. HSP90 inhibitor 17AAG show a synergistic effect with NVP-BEZ235 in melanoma cell lines. (A) Analyses of melanoma cell lines sensitivity to panel of tyrosine kinase inhibitors (Dabrafenib, SB590885, PLX4720, Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235) using Sanger Institute Drug Sensitivity Platform (http://www.cancerrxgene.org). Melanoma cell lines show high sensitivity (lower IC50) to the dual PI3K/mTOR inhibitor NVP-BEZ235 (p < 0.0001). (B) MTT assays of A2058 and SK-MEL-2 cell lines treated with increasing concentrations of Sorafenib, Sunitinib, Lapatinib and NVP-BEZ235 (72 h; mean ± SD, n ¼ 4). (C) MTT assays of melanoma cell lines in response to NVP-BEZ235,17AAG or the combination of both drugs in a fixed ratio (2,5:1) (72 h; mean ± SD, n ¼ 4 experiments). (D) Combination index (CI) showing the synergistic effect of NVP-BEZ235 and 17AAG on SK-MEL-2 and A2058 cell lines. Used concentrations are specified in the table where strength of synergy was quantified from – (strong antagonism) to þþþþ (strong synergism) (E) Western blots showing AKT and ERK1/2 phosphorylation from cells treated with Sorafenib, 17AAG, NVP-BEZ235 or combinations at the indicated doses for 24 h. Representative western blots (top) and summaries (bottom) (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. Control; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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Fig. 6. Effect of NVP-BEZ235 and 17AAG on melanoma cell lines apoptosis and cell cycle. (A) (left) Representative flow cytometry dot plots for Annexin V and PI staining in SKMEL-2 and A2058 cell lines treated with NVP-BEZ235, 17AAG or combination of both at the indicated concentrations for 48 h. (Right) Quantification of annexin-V positive cells (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. DMSO; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone). (B) Quantification of cell cycle phases in melanoma
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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HSP90 inhibition combined with NVP-BEZ235 has a synergistic effect on melanoma cell lines As PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways components and some RTKs we found overexpressed and/or overactivated in melanoma cells are client proteins of HSP90, we investigated the effect on cell viability of the HSP90 inhibitor 17AAG alone or combined with NVP-BEZ235. MTT assays showed that the combination of the two drugs was markedly more effective in the inhibition of cell viability than 17AAG or NVP-BEZ235 alone (Fig. 5C). Combination index (CI) values showed synergism between both drugs for almost all the concentrations tested (Fig. 5D). We also combined the RTKs/RAF inhibitor, Sorafenib, with 17AAG (Supplemental Fig. 1AeB). MTTs results showed that this combination has also a synergistic effect on the viability of melanoma cell lines. We next determined the phosphorylation status of AKT and ERK1/2 proteins treated with NVP-BEZ235, 17AAG and Sorafenib alone or in combination (Fig. 5E). Treatment with NVP-BEZ235, produced a moderate to no effect on AKT phosphorylation and a significant increase in ERK1/2 phosphorylation in SK-MEL-2 and A2058 cell lines. These cells treated with Sorafenib showed a decrease in ERK1/2 phosphorylation. This effect was accompanied by an increase in AKT phosphorylation level in both cell lines. 17AAG 1 mM treatment produced a significant decrease in both AKT and ERK1/2 phosphorylation levels in SK-MEL-2 cells but no effect on A2058 p-AKT level. SK-MEL-2 cells treated with NVPBEZ235 þ Sorafenib showed an increase in p-AKT level compared to control and a moderate decrease in ERK1/2 phosphorylation. The same drug combination showed no effect on A2058 cell line compared to untreated cells. When Sorafenib and 17AAG were combined both pathways were inhibited in SK-MEL-2 cells but no effect on p-AKT was observed in A2058 cells. Finally, combination of NVP-BEZ235 and 17AAG reduced AKT and ERK1/2 phosphorylation significantly in both cell lines. This combination was markedly more effective not only in cell viability reduction, but also in the inhibition of PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways.
Dual inhibition of HSP90 and PI3K/AKT/mTOR pathway induces apoptosis and reduces the proliferation of melanoma cells Next, we examined whether these drugs have a pro-apoptotic effect in melanoma cells by measuring the percentage of Annexin-V positive cells using flow cytometry analyses (Fig. 6A). NVP-BEZ235 and 17AAG combination increased the percentage of apoptotic population in SK-MEL-2 and A2058 cell lines with respect to the control (DMSO) or both drugs alone (p < 0.05). We also analyzed the effect of 17AAG, NVP-BEZ235 or the combination of both on the cell cycle (Fig. 6B). 17AAG treatment produce a moderate G2 cell cycle arrest (increase respect to control: 9% SK-MEL-2; 7% A2058; p < 0,05). Treatment of melanoma cells with 17AAG and NVP-BEZ235 simultaneously for 48 h led to an increase in the Sub G0 population of around 13% in SK-MEL-2 and A2058 cells (p < 0,05) and proliferation inhibition (decrease in S phase: 7,6% and 5,4% respectively for SK-MEL-2 and A2058 cells; p < 0,05) versus control cells. These cells cycle changes are concomitant with a slight G1 phase arrest. Western blot shows that SK-MEL-2 cell line treated with NVP-BEZ235 and 17AAG showed a significant decrease in Cyclin D1 protein and up-regulation of p27 expression (p 0,05)
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compared to single treatment with NVP-BEZ235, 17AAG, Sorafenib or 17AAG þ Sorafenib (Fig. 6C). A2058 cell line treated with NVPBEZ235 þ 17AAG showed a significant decrease in Cyclin D1 protein compared to single treatment with NVP-BEZ235 and Sorafenib but differences between NVP-BEZ235 þ 17AAG and 17AAG or 17AAG þ Sorafenib were not significant. P27 expression was significantly up-regulated when A2058 cells are treated with NVPBEZ235 þ 17AAG (p 0,0001). These results are consistent with the anti-proliferative effect for this combination of drugs.
Discussion Drug resistance is the major factor that limits the long-term efficacy of targeted therapies in melanoma patients. BRAF inhibition has not reached the expected results in the clinical practice with temporary responses and the development of resistance to these inhibitors. This resistance is the result of diverse intrinsic and acquired mechanisms like the recovery of MAPK signaling or activation of alternative signaling pathways, such as PI3K/AKT/mTOR by upregulation of RTKs like IGF-1R, EPHA2 or PDGFRb [12,16,24,25]. Currently, the combination of BRAF with MEK inhibitors represents the gold standard of targeted therapy in BRAF mutated melanoma. Unfortunately, the efficacy of MEK inhibitors is also limited by the activation of the PI3K/AKT/mTOR pathway that drives resistance development [26,27]. In this work, we studied the expression and activation status of RTKs in both melanoma cell lines and tumors and we show its correlation with the expression and phosphorylation of PI3K/AKT/ mTOR and RAS/RAF/MAPK pathways components. It is known that membrane tyrosine kinase receptors can activate both, PI3K/AKT and MAPK pathways and hyper-activation of these receptors, deregulated expression of its ligands and aberrant expression of integrins and cytokines, contribute to tumor cell proliferation, survival and resistance to cell death [28e30]. Activation of several RTKs has been detected previously in melanoma cell lines and short hairpin RNA knockdown of some of these receptors inhibited the proliferation of melanoma cells by AKT or STAT3 signaling pathways modulation [31]. There are evidences that support the role of PI3K/AKT pathway in melanoma initiation [32]. The transformation of normal nevi to melanoma in situ, and from melanoma in situ to metastasis, is drived by p-AKT progressive accumulation [33]. Moreover, co-expression of the PI3K p110 subunit and mTOR have been demonstrated in melanoma tumors and mTOR inhibition with Rapamycin enhances the activity of PI3K/AKT [34]. Here we show that HSP90 and PI3K/AKT/mTOR pathway components are highly expressed in melanoma tumors and this expression correlates with poor survival in melanoma patients representing potential therapeutic targets. HSP90 inhibition has been demonstrated to be a highly effective strategy to overcome BRAF and MEK inhibitors resistance in vitro and in vivo [23,35e37]. Nevertheless, no objective response has been observed in phase II clinical trials with 17AAG [38]. There are other ongoing clinical trials combining HSP90 inhibitors with BRAF and MEK inhibitors but no phase II data has been published yet. Interestingly, it has been demonstrated that HSP90 inhibition induces HSP70 expression as a compensatory mechanism that contributes to drug resistance and lower sensitivity to targeted HSP90 inhibition of melanoma [39]. This mechanism seems to be regulated by mTOR-dependent translational activity supporting the rationale of using NVP-BEZ235 in combination with HSP90 targeted therapy in melanoma [40]. Our data
cell lines treated with 17AAG, NVP-BEZ235 or both for 48 h. Histograms show the mean percentages of cells in SubG0, G0/G1, S and G2/M phases (n ¼ 3). (C) Representative western blots (top) and summaries (bottom) showing cell cycle-related proteins in SK-MEL-2 and A2058 cells after 24 h treatment with the indicated concentrations of NVP-BEZ235, 17AAG and Sorafenib. GAPDH was used as loading control (mean ± SD, n ¼ 3 experiments; *p < 0.05, **p < 0.001 vs. Control; a p < 0.05 vs. NVP-BEZ235 alone; b p < 0.05 vs. 17AAG alone).
Please cite this article in press as: R. Calero, et al., Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.07.021
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shows that treatment with 17AAG reduces AKT and ERK1/2 phosphorylation in SK-MEL-2 cells but in A2058 cells AKT phosphorylation is still present. On the other hand, previous studies showed that NVP-BEZ235 effectively reduced the viability and clonogenicity of 23 melanoma cell lines, regardless of BRAF mutation status. This drug also produced the downregulation of p-AKT and pP70S6K [34,41]. In the present study, we show that melanoma cell lines are highly sensible to NVP-BEZ235 in comparison to other tyrosine kinase inhibitors including RTKs and BRAF inhibitors. We observed that treatment of melanoma cell lines with NVP-BEZ235 slightly reduces the phosphorylation of AKT but increases the level of phosphorylated ERK1/2. Furthermore, treatment with the RTKs/RAF inhibitor, Sorafenib, produced an inverse effect with an increase in AKT phosphorylation and a reduction in phosphorylated ERK1/2. There are numerous evidences in melanoma and other tumors of MAPK and PI3K pathways cross-interaction by reciprocal regulation through positive or negative feedbacks and how inhibitors of one of these pathways induce a compensatory mechanism that over-activates the other pathway to maintain survival and proliferation signaling [9,42,43]. This effect has been described previously, with NVP-BEZ235 in particular, in melanoma and other tumors [43]. Treatment with the combination of 17AAG and NVPBEZ235 highly abolished this effect allowing a strong suppression of p-ERK and p-AKT. Combined treatment also shows a synergistic effect on cell viability and a significant induction of apoptosis. Interestingly, little effect of single treatment with NVP-BEZ235 on apoptosis due to incomplete growth inhibition in melanoma has been reported [44]. NVP-BEZ235 and 17AAG combined treatment has also an antiproliferative effect on melanoma cells and results in Cyclin D1 decrease and p27 up-regulation when compared with each drug alone. In conclusion, our findings suggest that both signaling pathways, RAS/RAF/MAPK and PI3K/AKT/mTOR, must be strongly inhibited to achieve a notably effect on proliferation and apoptosis. In this line, combinations of drugs targeting these two pathways have been proposed in the last years for melanoma treatment, especially for those unresponsive to BRAF inhibitors [45e47]. The combination of PI3K/AKT/mTOR and HSP90 inhibition appears to be very effective because it abrogates the activation of AKT and ERK that is promoted by the increase of RTK activity, RAF and RAS signaling in metastatic melanoma. On one side, NVP-BEZ235 inhibits the PI3K/AKT/mTOR pathway that functions as survival mechanism in melanoma cells and on the other the use of an HSP90 inhibitor, like 17AAG, potentiates NVP-BEZ235 effect and provides additional inhibitory activity that target multiple oncogenic kinases that cooperate to drive tumor growth, survival, and resistance to therapy. Therefore, our study provides a basis for the development of new specific drugs that target these pathways. Animal model studies and clinical trials will give valuable information about this mechanisms. The identification of the subgroup of melanoma patients that are most likely to respond to this treatment remains as a major challenge. Acknowledgements This study was partially supported by the scientific foundation of Spanish Association against Cancer (AECC), the Castilla La Mancha University, the Castilla la Mancha Foundation for Health Research and Castilla la Mancha Health Service. The authors are ~ o for her technical assistance in cultures grateful to Laura Avendan procedures and Syong H Nam-Cha for the immunostains evaluation. We also greatly thank the Biobank of the Albacete University Hospital, integrated into the National Biobank Network of Carlos III Institute (RETICS BIOBANK, RD09/0076/00047), for kindly supplying human samples.
Conflict of interest The authors state no conflict of interest.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2017.07.021.
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