The incidence of melanoma has risen rapidly in the past

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1 BASIC INVESTIGATION The MEK1/2 Inhibitor AS Circumvents Resistance to the BRAF Inhibitor PLX4032 in Human Malignant Melanoma Cells Seong Joon Park, MD, Seung-Woo Hong, PhD, Jai-Hee Moon, PhD, Dong-Hoon Jin, PhD, Jin-Sun Kim, PhD, Chang-Kyu Lee, MS, Kyu-pyo Kim, MD, PhD, Yong Sang Hong, MD, PhD, Eun Kyung Choi, MD, PhD, Jung Shin Lee, MD, PhD, Jae-Lyun Lee, MD, PhD and Tae Won Kim, MD, PhD Abstract: Background: Although inhibitors of the proto-oncogene BRAF have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired drug resistance, a process in which reactivation of MAP kinase (MEK) is known to play an important role. In this study, we evaluated the efficacy of AS703026, a new MEK inhibitor, in BRAF inhibitor resistant melanoma cell lines. Methods: Two melanoma cells lines, RPMI-7951 and SK-MEL5, harboring an activating mutation of BRAF (V600E) were treated with the BRAF inhibitor PLX4032 to select a BRAF inhibitor resistant cell line for further study. Cell viability assay was determined with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] assay and trypan blue exclusion method; apoptosis assay was performed by annexin-v staining. Knockdown of BRAF was investigated by small interfering RNA. Results: RPMI-7951 cells exhibited an increased sensitivity to combined treatment with PLX4032 and AS compared to either drug alone. Consistent with this, the combination of PLX4032 and AS significantly induced apoptosis, whereas each drug used alone did not, as demonstrated by a flow cytometric analysis of annexin-v/propidium iodide stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblot analyses also showed a depletion of phosphorylated-erk with combined drug treatment. In addition, AS synergized with small interfering RNA mediated downregulation of BRAF to produce results similar to those of combined treatment with PLX4032 and AS Conclusions: Our results suggest that combined treatment with AS and a BRAF inhibitor overcomes the resistance to BRAF inhibitors in malignant melanoma cells harboring a mutant form of BRAF. Key Indexing Terms: AS703026; MEK1/2 inhibitor; PLX4032; BRAF inhibitor; Melanoma. [Am J Med Sci 2013;346(6): ] From Institute for Innovative Cancer Research, Asan Medical Center (SJP, S-WH, J-HM, D-HJ, J-SK, C-KL, K-PK, YSH, EKC, JSL, J-LL, TWK), Departments of Oncology (SJP, S-WH, J-HM, D-HJ, J-SK, C-KL, K-PK, YSH, JSL, J-LL, TWK), and Radiation Oncology (EKC), College of Medicine, University of Ulsan, Asan Medical Center, Songpa-gu, Seoul, South Korea. Submitted November 28, 2012; accepted in revised form April 1, S.J. Park and S.-W. Hong contributed equally to this project. The authors have no financial or other conflicts of interest to disclose. Supported by grants from the Korea Health 21 R&D Project, Ministry of Health and Welfare and Family Affairs (A102059), and Covering Research Center Program, Ministry of Education Science and Technology (2011K000874), South Korea. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal s website ( Correspondence: Tae Won Kim, MD, PhD, Department of Oncology, College of Medicine, University of Ulsan, Asan Medical Center, 86 Asanbyeongwon-gil, Songpa-gu, Seoul, , South Korea ( twkimmd@amc.seoul.kr). The incidence of melanoma has risen rapidly in the past 3 decades and has become a significant health risk worldwide. 1 Surgical therapy is effective in patients with localized or early melanoma. However, melanoma that has spread to locoregional lymph nodes or distant sites is largely refractory to currently available systemic therapies. 2 Constitutive activation of the BRAF proto-oncogene and subsequent activation of the RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is critical for the proliferation and survival of melanoma cells. 3 The most common activating somatic mutation in BRAF is a substitution of glutamic acid for valine at position 600 (V600E), which is found in 50% to 60% of melanomas. Several BRAF and MEK inhibitors have been developed and are currently undergoing preclinical and clinical investigation. Although BRAF inhibitors have shown efficacy in melanoma cell lines harboring an activating mutation of BRAF, acquired drug resistance frequently develops after the initial response. 4 Recent studies have reported that the resistance to BRAF inhibitors is associated with the recovery of MAPK signaling through other types of RAF isoforms, such as ARAF and CRAF. 5,6 Another study identified MAP3K8 (COT) as a key element in a BRAF inhibitor resistance mechanism that activates ERK through a MEK-dependent pathway, which does not require RAF signaling. 7 Although other mechanisms may exist, reactivation of the MAP kinase pathway has taken on added significance in BRAF inhibitor resistance, as demonstrated by these studies. In this context, inhibition of MEK1/2 may be critical for circumventing the resistance to BRAF inhibitors. AS (N-[(2S)-2,3-dihydroxypropyl]-3-[(2-fluoro-4- iodophenyl)amino]isonicotinamide hydrochloride), a highly selective and potent, ATP-noncompetitive allosteric inhibitor of MEK1/2 has shown efficacy in myeloma and colorectal cancer cell lines. 8,9 However, it has not yet been tested in melanoma cell lines. Accordingly, we evaluated the efficacy of AS in BRAF inhibitor resistant melanoma cell lines harboring a BRAF V600E activating mutation. MATERIALS AND METHODS Cell Culture and Reagents Human melanoma cell lines, RPMI-7951 and SK-MEL5, expressing the BRAF V600E activating mutation were purchased from American Type Culture Collection (ATCC, Manassas, VA). RPMI-7951 and SK-MEL5 cells were maintained at 37 C in a humidified atmosphere at 5% CO 2 in Modified Eagle s Medium (GIBCO BRL, Grand Island, NY) or RPMI-1640 medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (Thermo Scientific Hyclone, Barrington, IL), of penicillin (100 IU/mL), and streptomycin (100 mg/ml). 494 The American Journal of the Medical Sciences Volume 346, Number 6, December 2013

2 AS Enhances Sensitivity to BRAF Inhibitor PLX4032 and AS were purchased from Selleck Chemicals (Houston, TX). Cellular Viability Assay Approximately cells/well were seeded into 96-well plates. One day later, cells were treated with PLX4032 and/or AS at 0 to 10 mm concentrations for 72 hours. Cell viability was determined using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] assay kit (Promega Corporation, Madison, WI) according to the manufacturer s instructions. Cell Cycle Analysis and Apoptosis Assay RPMI-7951 cells in 60-mm dishes were cultured in a medium supplemented with 10% fetal bovine serum for 24 hours. The cells were treated with PLX4032 (10 mm), AS (10 mm), and PLX4032 (10 mm) together with AS (10 mm), or vehicle control (dimethyl sulfoxide) for 24 hours. After harvesting by trypsinization, cells were resuspended in 1 ml of phosphate-buffered saline, stained with propidium iodide (PI) and annexin-v solution, and analyzed by flow cytometry using a FACS Calibur fluorescence-activated cell sorter (Becton Dickinson, San Jose, CA). Immunoblot Analysis Immunoblot analyses were performed by preparing cell lysates using RIPA lysis buffer (50 mm HEPES [ph7.4], 150 mm NaCl, 1 mm EDTA, 2.5 mm ethylene glycol tetraacetic acid (EGTA), 1 mm Dithiothreitol (DTT), and 0.1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Sigma). Protein concentrations were determined using the Bradford assay. Total cellular proteins (15 mg/sample) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to an Immobilon-PVDF membrane (Millipore Corporation, Billerica, MA). Membranes were blocked using 5% nonfat dry milk in TBS-T (20 mm Tris-HCl [ph 7.4], 150 mm NaCl, and 0.1% Tween 20) and probed with anti-braf, anti-g-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-craf, anti-phospho-erk1/ 2, anti-erk1/2, anti-phospho-mek1/2, anti-mek1/2, or anticleaved caspase-3 (Cell Signaling Technology, Beverly, MA) primary antibodies. Primary antibodies were detected using horseradish peroxidase conjugated goat antimouse or donkey antirabbit secondary antibodies and enhanced chemiluminescence detection reagents (Amersham, Buckinghamshire, United Kingdom). RNA Interference RPMI-7951 cells were transiently transfected with small interfering RNA (sirna) (150 pmol/60-mm dish) targeting mutant BRAF (59-AAG UGG CAU GGU GAU GUG GCA-39) or MEK1/2 (MEK1; 59-AAG CAA CTC ATG GTT CAT GCT-39 and MEK2; 59-AAG AAG GAG AGC CTC ACA GCA-39)using Lipofect-AMINE 2000 (Invitrogen, Carlsbad, CA). Scrambled sirna (mutant BRAF; 59-GCG CAU UCC AGC UUA CGU A-39 and MEK1/2; 59-AGG UCG AAC UAC GGG UCA AUC-39) was used as a control. All sirnas were synthesized by Genolution Pharmaceuticals Inc (Seoul, South Korea). Statistical Analysis Figures 1 and 2 were analyzed by 1-way analysis of variance using GraphPad Prism Ver 5.01 (Systat Software, San Jose, CA) and followed to Tukey post hoc test. A 2-sided t test was used for Figure 3. P, 0.05 was considered statistically significant. RESULTS AS Enhances Sensitivity to the BRAF Inhibitor PLX4032 in the Human Malignant Melanoma Cell Line RPMI-7951 It was recently reported that resistance to BRAF inhibitors is correlated with MEK1/2 activity. 10 AS703026, a selective MEK1/2 inhibitor originally developed by MERCK, Ltd, was reported to exhibit an inhibitory effect on myeloma and KRAS-mutated colon cancer that were resistant to epidermal growth factor receptor monoclonal antibody therapy. 9 On the basis of these reports, we analyzed the effect of AS on sensitivity to the highly selective BRAF inhibitor, PLX4032. For these experiments, we used the RPMI-7951 cell line, which shows de novo resistance to BRAF inhibitor treatment. 7 These cells are known to harbor the BRAF V600E activating mutation and exhibit elevated COT protein expression and MAP kinase pathway activation. 7 In preliminary treatments, we tested PLX4032 resistance in RPMI-7951 cells and the SK-MEL5 cell line, which also harbors the BRAF V600E mutant. These experiments, which measured the concentration-dependent sensitivity of both cell lines to PLX4032, showed that cell viability was lower in SK-MEL-5 cells than in RPMI-7951 cells (Figure 1A), indicating that RPMI-7951 cells are indeed resistant to PLX4032. Next, we examined the effect of AS in PLX4032- resistant RPMI-7951 cells. As shown in Figure 1A, AS alone (like PLX4032) also did not decrease the viability of RPMI-7951 cells (Figure 1B). Notably, however, combined treatment with AS and PLX4032 decreased the viability of RPMI-7951 cells (Figure 1C), implying that AS may overcome resistance to PLX4032 in human melanoma cells. Taken together, these data suggest that treatment with AS increases the sensitivity to PLX4032. Combined Treatment With AS and PLX4032 Induces Apoptotic Cell Death Through the Suppression of ERK Phosphorylation On the basis of the above results, we further examined the effect of PLX4032 and AS in RPMI-7951 cells. Cell death in RPMI-7951 cells significantly increased after combined exposure to PLX4032 and AS703026, whereas only a small fraction of RPMI-7951 cells were killed by single treatment with PLX4032 (10 mm) or AS (5 and 10 mm) (Figure 2). To investigate the effects of PLX4032 and AS alone and in combination on RAF downstream effectors in RPMI-7951 cells, we analyzed changes in the MAPK signaling pathway using Western blotting. We also examined CRAF, which is known to be involved in the resistance mechanism to BRAF inhibitors. Treatment with PLX4032 alone did not affect the levels of phosphorylated ERK1/2 (p-erk1/2), an established downstream effector, whereas AS induced a small change in p-erk1/2 levels. In contrast, PLX4032 treatment in combination with 5 mm AS induced a significant decrease in p-erk1/ 2 levels and combined treatment with PLX4032 and 10 mm AS completely inhibited ERK1/2 phosphorylation. These results suggest that combined treatment with AS and PLX4032 induces cell death through the suppression of ERK phosphorylation. In addition, CRAF expression was slightly increased after combined drug treatment compared with baseline. To determine whether the decrease in cell viability induced by combined treatment with AS and PLX4032 was attributable to an increase in apoptosis, we treated PLX4032- resistant RPMI-7951 cells with PLX4032 and AS703026, alone and in combination, and then quantified apoptosis by analyzing Ó 2013 Lippincott Williams & Wilkins 495

3 Park et al FIGURE 1. The MEK inhibitor AS synergizes with the BRAF inhibitor PLX4032 in PLX4032-resistant RPMI-7951 cells. (A) SK-MEL- 5 or RPMI-7951 cells were seeded in 96-wells and then treated with the indicated concentrations of PLX4032. After 72 hours, MTS assays were performed. The results represent means 6 standard deviations of 3 independent experiments (**P, 0.001). (B) RPMI-7951 cells were treated with the indicated concentrations of the MEK inhibitor AS for 72 hours. Cell viability was determined by MTS assay (**P, 0.001). (C) RPMI-7951 cells were treated with PLX4032 with or without AS for 72 hours (*P, 0.01; **P, 0.001). annexin-v stained and PI-stained cells using flow cytometry. As shown in Figure 3, although treatment with PLX4032 alone did not increase annexin-v or PI staining of RPMI-7951 cells, a small portion of cells (5.75%) treated with AS alone was stained with annexin-v. Notably, treatment with both drugs together induced a much more substantial and significant increase in annexin-v positive cells (27.8%) in RPMI-7951 cells. Consistent with this, immunoblotting revealed that combined treatment with PLX4032 and AS induced an increase in the cleaved form of caspase-3, a molecular marker of apoptosis. Collectively, these results suggest that treatment of PLX4032- resistant RPMI-7951 cells with AS in addition to PLX4032 induces apoptosis through the suppression of ERK phosphorylation. Knockdown of BRAF Enhances Sensitivity to AS To further confirm the beneficial effect of combined BRAF inactivation and treatment with AS703026, we analyzed the effect of AS on RPMI-7951 cells in which BRAF was silenced by transfection with sirna. Notably, cell death in BRAF-siRNA transfected cells was significantly increased after exposure to AS Also, as shown in Figure 4, AS treatment induced the cleavage of caspase-3 in cells transfected with BRAF-siRNA, whereas cells transfected with BRAF-siRNA only and control (scrambled) sirna-transfected cells treated with AS alone showed no increase in the cleaved form of caspase-3 (Figure 4). These results suggest that AS also increases the sensitivity to knockdown BRAF, an effect similar to that of AS on PLX4032 efficacy. DISCUSSION For many years, disseminated melanoma was assumed to be resistant to all forms of therapeutic intervention. However, a recent phase II study showed that vemurafenib (PLX4032), a potent inhibitor of mutated BRAF, exhibited marked antitumor effects against malignant melanoma, producing approximately a 50% response rate and an 80% disease control rate. 11 Although very encouraging, the clinical responses to vemurafenib observed to date are relatively short lived, with an initial period of response followed by relapse and resistance. 4,11,12 Thus, acquired drug resistance has become an important issue, and many studies are currently underway to elucidate the 496 Volume 346, Number 6, December 2013

AS703026 Enhances Sensitivity to BRAF Inhibitor FIGURE 2. Cotreatment of PLX4032-resistant RPMI-7951 cells with AS703026 sensitizes cells to PLX4032-induced death.

Values are presented as means 6 standard deviations from 3 separate experiments performed in triplicate (*P, 0.05; **P, 0.005).

4 AS Enhances Sensitivity to BRAF Inhibitor FIGURE 2. Cotreatment of PLX4032-resistant RPMI-7951 cells with AS sensitizes cells to PLX4032-induced death. RPMI cells were treated with the indicated concentrations of PLX4032 and/or AS for 24 hours and then cell death was determined using the trypan blue exclusion method. Values are presented as means 6 standard deviations from 3 separate experiments performed in triplicate (*P, 0.05; **P, 0.005). underlying resistance mechanism and develop strategies to overcome the resistance. Reactivation of the MAP kinase pathway through CRAF or MAP3K8 (COT) and compensatory reactivation of alternative signaling pathways, such as PI3K/AKT, have arisen as the mechanisms responsible for the resistance to BRAF inhibitors. 5 7 These findings suggest that MEK1/2 kinase is an important component in the BRAF inhibitor resistance mechanism. However, in the current study, the MEK inhibitor AS703026, when used alone did not show efficacy, whereas combined treatment with AS and PLX4032 did. This supports earlier findings that dual BRAF/MEK inhibition may help overcome resistance to BRAF inhibitors. 6,7,13 Villanueva et al 6 reported that acquired resistance to BRAF inhibitors was mediated by reactivation of the MAPK pathway through flexible switching among the 3 RAF isoforms. However, resistance was not overcome by MEK inhibition, which resulted in cell cycle arrest only. These results suggest the involvement of various and complicated pathways in the resistance mechanism. In addition, the use of BRAF and MEK inhibitors in combination has been shown to override resistance to single agents in experimental models. 13 Johannessen et al 7 reported that COT activates the ERK pathway through a MEK-dependent mechanism and also suggested the existence of a possible MEK-independent mechanism because the RPMI-7951 cell line was not sensitive to MEK inhibitors. Interestingly, combined RAF/MEK inhibition circumvented COT-driven resistance in this latter study, indicating that the MEK-dependent pathway is the primary mechanism, although a MEK-independent pathway may contribute to the underlying resistance mechanism. The MEK1/2 inhibitor, AS703026, is a novel, orally bioavailable, small molecule inhibitor that binds to an allosteric site in close proximity to the ATP-binding site of MEK1/2. Binding of AS to this allosteric site prevents the activation of MEK1/2. AS has favorable pharmacological characteristics and has been shown to completely and specifically block MEK1/2 activity without affecting the activity of 217 other kinases tested. 8 One study 8 with AS showed both inhibition of growth and apoptosis of multiple myeloma cells in vitro and in vivo, and another recent study 9 showed that AS was also effective in KRAS-mutated colorectal cancer cells both in vitro and in vivo. Because of such favorable preclinical test results, this new drug is currently undergoing evaluation in clinical trials. However, it has never been tested against human melanoma cells. In the current study, we showed that AS in combination with PLX4032 was effective against the RPMI-7951 human melanoma cell line, which harbors the BRAF V600E activating mutation and is resistant to BRAF inhibitors. Consistently, rate of cell death was clearly increased in cells that express MEK1/2-siRNA after treatment with PLX4032 but not in cells that express scramble sirna, as a control (see Figure, Supplemental Digital Content 1, By extension, cotreatment with AS might be expected to be efficacious against human melanomas harboring BRAF-activating mutations, although this needs further study. CONCLUSIONS We showed that combined treatment with AS and the BRAF inhibitor PLX4032 overcame BRAF inhibitor FIGURE 3. The combination of PLX4032 and AS synergistically induces apoptosis in RPMI-7951 cells. RPMI-7951 cells were treated with 10 um PLX4032 and/or 5 um AS for 24 hours and then analyzed for apoptosis by annexin-v/pi double staining and flow cytometry. Ó 2013 Lippincott Williams & Wilkins 497

Park et al FIGURE 4. The MEK inhibitor AS703026 synergizes with sirnamediated knockdown of BRAF to induce cell death.

5 Park et al FIGURE 4. The MEK inhibitor AS synergizes with sirnamediated knockdown of BRAF to induce cell death. RPMI-7951 cells were transfected with a BRAF V600E -specific sirna, and then 48 hours later, were treated with AS for an additional 24 hours. Cell death was determined using the trypan blue exclusion method. Values represent means 6 standard deviations of 3 independent experiments (**P, 0.01). resistance in a malignant melanoma cell line harboring an activating mutation of BRAF. Our findings indicate that the new MEK inhibitor, AS703026, warrants further study in research areas in which resistance to BRAF inhibition is emerging as an important issue. REFERENCES 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61: Flaherty KT. Chemotherapy and targeted therapy combinations in advanced melanoma. Clin Cancer Res 2006;12:2366s 70s. 3. Dhomen N, Marais R. BRAF signaling and targeted therapies in melanoma. Hematol Oncol Clin North Am 2009;23:529 45, ix. 4. Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363: Montagut C, Sharma SV, Shioda T, et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res 2008;68: Villanueva J, Vultur A, Lee JT, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010;18: Johannessen CM, Boehm JS, Kim SY, et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 2010;468: Kim K, Kong SY, Fulciniti M, et al. Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo. Br J Haematol 2010;149: Yoon J, Koo KH, Choi KY. MEK1/2 inhibitors AS and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy. Cancer Res 2011;71: Wagle N, Emery C, Berger MF, et al. Dissecting therapeutic resistance RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol 2011;29: Hersey P, Smalley KS, Weeraratna A, et al. Meeting report from the 7th International Melanoma Congress, Sydney, November, Pigment Cell Melanoma Res 2011;24:e Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364: Emery CM, Vijayendran KG, Zipser MC, et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc Natl Acad Sci U S A 2009;106: Volume 346, Number 6, December 2013

HCC1937 is the HCC1937-pcDNA3 cell line, which was derived from a breast cancer with a mutation

SUPPLEMENTARY INFORMATION Materials and Methods Human cell lines and culture conditions HCC1937 is the HCC1937-pcDNA3 cell line, which was derived from a breast cancer with a mutation in exon 20 of BRCA1