The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells.
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 AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 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, AS703026 synergized with small interfering RNA-mediated downregulation of BRAF to produce results similar to those of combined treatment with PLX4032 and AS703026.


CONCLUSIONS
Our results suggest that combined treatment with AS703026 and a BRAF inhibitor overcomes the resistance to BRAF inhibitors in malignant melanoma cells harboring a mutant form of BRAF.
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.
Seong Joon
SJ
Park
Seung-Woo
SW
Hong
Jai-Hee
JH
Moon
Dong-Hoon
DH
Jin
Jin-Sun
JS
Kim
Chang-Kyu
CK
Lee
Kyu-pyo
KP
Kim
Yong Sang
YS
Hong
Eun Kyung
EK
Choi
Jung Shin
JS
Lee
Jae-Lyun
JL
Lee
Tae Won
TW
Kim
D000970:Antineoplastic Agents / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D055785:Gene Knockdown Techniques; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D000077484:Vemurafenib
D000970:Antineoplastic Agents; D007211:Indoles; C000715470:MAS1 protein, human; C550600:N-(2,3-dihydroxypropyl)-1-((2-fluoro-4-iodophenyl)amino)isonicotinamide; D047428:Protein Kinase Inhibitors; D000090063:Proto-Oncogene Mas; D034741:RNA, Small Interfering; D013449:Sulfonamides; D000077484:Vemurafenib; D009536:Niacinamide; C482128:MAP2K2 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human