Abstract: Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAFV600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway . In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN . Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAFV600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.
Keywords: melanoma brain metastasis; BRAF; PTEN; PI3K (phosphoinositide 3-kinase); MAPK (mitogen-activated protein kinase); mTOR; temsirolimus; vemurafenib
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Daphu, I.; Horn, S.; Stieber, D.; Varughese, J.K.; Spriet, E.; Dale, H.A.; Skaftnesmo, K.O.; Bjerkvig, R.; Thorsen, F. In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways. Int. J. Mol. Sci. 2014, 15, 8773-8794.
Daphu I, Horn S, Stieber D, Varughese JK, Spriet E, Dale HA, Skaftnesmo KO, Bjerkvig R, Thorsen F. In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways. International Journal of Molecular Sciences. 2014; 15(5):8773-8794.
Daphu, Inderjit; Horn, Sindre; Stieber, Daniel; Varughese, Jobin K.; Spriet, Endy; Dale, Hege A.; Skaftnesmo, Kai O.; Bjerkvig, Rolf; Thorsen, Frits. 2014. "In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways." Int. J. Mol. Sci. 15, no. 5: 8773-8794.