Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma

Summary Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N‐RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathw...

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Published in	Pigment cell and melanoma research Vol. 29; no. 6; pp. 643 - 655
Main Authors	Wei, Bih-Rong, Michael, Helen T., Halsey, Charles H.C., Peer, Cody J., Adhikari, Amit, Dwyer, Jennifer E., Hoover, Shelley B., El Meskini, Rajaa, Kozlov, Serguei, Weaver Ohler, Zoe, Figg, William. D., Merlino, Glenn, Simpson, R. Mark
Format	Journal Article
Language	English
Published	England Blackwell Publishing Ltd 01.11.2016 Wiley Subscription Services, Inc John Wiley and Sons Inc
Subjects	Animals Apoptosis - drug effects BRAF and NRAS wild type Cell Proliferation - drug effects dactolisib dog Dogs Drug Synergism Female Gene Expression Regulation, Neoplastic - drug effects Humans MAP Kinase Kinase Kinases - antagonists & inhibitors MAP Kinase Kinase Kinases - metabolism MAP Kinase Signaling System - drug effects melanoma Melanoma - drug therapy Melanoma - metabolism Melanoma - pathology Mice Mice, Nude Mucous Membrane - drug effects Mucous Membrane - metabolism Mucous Membrane - pathology Original Phosphatidylinositol 3-Kinases - antagonists & inhibitors Phosphatidylinositol 3-Kinases - metabolism preclinical model Protein Kinase Inhibitors - pharmacology Proto-Oncogene Proteins c-akt - antagonists & inhibitors Proto-Oncogene Proteins c-akt - metabolism Signal Transduction TOR Serine-Threonine Kinases - antagonists & inhibitors TOR Serine-Threonine Kinases - metabolism trametinib Tumor Cells, Cultured Xenograft Model Antitumor Assays trametinib BRAF and NRAS wild type melanoma preclinical model dactolisib dog
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Summary:	Summary Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N‐RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP‐BEZ235. The two‐drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl‐2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p‐ERK, p‐AKT, p‐S6, and 4E‐BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.
Bibliography:	ArticleID:PCMR12512 ark:/67375/WNG-QC9CS25H-B Figure S1. Western blot analysis of additional patient tumors indicates that canine melanomas exhibit a diverse range of ERK1/2 and AKT activities. Tumor lysates were prepared from snap-frozen melanoma specimens. Lane numbers indicate unique canine patient primary tumor samples.Figure S2. Inhibition of MEK by GSK1120212 (GSK, trametinib) and PI3K/mTOR by NVP-BEZ235 (NVP, dactolisib) in MM was time- and dose-dependent. Five canine MM cell lines were treated with GSK or NVP at 31, 125, 500, and 2000 nM concentrations in vitro for 4 and 24 h, and analyzed by Western blot with anti-p-ERK1/2 or anti-p-AKT. (D, DMSO Control).Figure S3. Densitometric assessment of p-AKT(serine 473) in Western blots, normalized to (A) beta actin or (B) native AKT as internal controls, using Image J, revealed variable levels of inhibition in MM cell lines due to exposure to NVP-BEZ235 (N, dactolisib) separately, or in combination (C) with GSK1120212 (G, trametinib) for 48 h. Results expressed as a ratio to control DMSO-treated cells. p-AKT blot images are from Figure .Figure S4. Cell cycle analysis of canine MM cell lines (A) M1, (B) M2, and (C) M5 and (D) Jones. Cells were treated with GSK1120212 (G), NVP-BEZ235 (N), a combination of the two agents (C), or DMSO control (D) for 48 h. Cells were fixed and labeled with propidium iodide after 48 h treatment to analyze the distribution of cells in various phases of cell cycle. Ratios of cycle phases varied by treatment and cell line and typically included relative increased population in G0/G1 (N), and sub-G fraction for (G) and (C).Figure S5. Body weights of M5 MM xenograft bearing mice treated with GSK1120212, NVP-BEZ235, a combination of the two agents, or vehicle control. Mice were treated for up to 39 days after tumors were established. Intramural Research Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland Animal Cancer Foundation istex:B9E86CE01808222C0F788D9700D5546D7C09A857 Canine Comparative Oncology and Genomics Consortium Biorepository ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1755-1471 1755-148X
DOI:	10.1111/pcmr.12512