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Activation of the AKT and mammalian target of rapamycin pathways and the inhibitory effects of rapamycin on those pathways in canine malignant melanoma cell lines

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  • 1 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 2 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 3 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.

Abstract

Objective—To investigate the activation of the AKT and mammalian target of rapamycin (mTOR) pathways and assess the inhibitory effects of rapamycin on those pathways in canine malignant melanoma cells.

Sample Population—3 established primary canine melanoma cell lines generated from naturally occurring tumors.

Procedures—Expressions of total and phosphorylated AKT, mTOR, and p70 ribosomal S6 kinase 1 (p70S6K) in canine melanoma cells that were or were not exposed to 10nM rapamycin were assessed via western blot analysis. Clonogenic assays were performed to determine the surviving fraction of melanoma cells after exposure to 0.1, 1, 10, or 100nM rapamycin.

Results—Expressions of total and phosphorylated AKT, mTOR, and p70S6K proteins were detected (ie, the AKT and mTOR pathways were activated) in all 3 cell lines. Rapamycin treatment resulted in decreases in phosphorylated mTOR expression and phosphorylated p70S6K expression but no change in phosphorylated AKT expression. Expression of total AKT, mTOR, and p70S6K persisted after rapamycin treatment. There was a significant dose-dependent decrease in surviving tumor cell fraction for each cell line following treatment with rapamycin.

Conclusions and Clinical Relevance—These data indicated that AKT and mTOR, as well as their downstream product p70S6K, are present and active in canine melanoma cells. Activation of the mTOR pathway can be inhibited by rapamycin; treatment of melanoma cells with rapamycin decreased the surviving tumor cell fraction. Use of mTOR inhibitors as antineoplastic treatments in dogs with melanoma warrants investigation. Furthermore, these data support the use of canine melanoma cells as a molecular model for melanoma in humans.

Contributor Notes

Supported in part by the UCD Cancer Center Core Support Grant NIH CA093373.

The authors thank John Doval for assistance with preparation of the figures.

Address correspondence to Dr. Kent.