In vitro treatment of melanoma brain metastasis by simultaneously targeting the MAPK and PI3K signaling pathways

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 [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. 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.

Figure 1.

Cell proliferation and survival of H1_DL2 and H3 melanoma brain metastasis cells grown as monolayer cultures, after treatment with vemurafenib and temsirolimus. (A,B) Treatment of H1_DL2 melanoma cells, harboring the BRAF^V600E mutation. (A) H1_DL2 cells treated with increasing drug concentrations for 72 h (n = 6, mean ± SD); (B) A more detailed analysis of the cell survival shown in (A), after drug treatment with 0, 0.05, 5 or 10 μM of vemurafenib, temsirolimus or a combination therapy. Scatter dot plot (n = 6, mean ± SD). *** p < 0.001; **** p < 0.0001; (C,D) Treatment of H3 melanoma cells, harboring wild-type (WT) BRAF. (C) H3 cells treated with increasing drug concentrations for 72 h (n = 6, mean ± SD); and (D) A more detailed analysis of the cell survival shown in (C), after drug treatment with 0, 0.05, 5 or 10 μM vemurafenib, temsirolimus or combined therapy. Scatter dot plot (n = 6, mean ± SD). n.s.: not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 2.

Wound healing assay of H1_DL2 melanoma brain metastasis cells after treatment with vemurafenib and temsirolimus. (A) Untreated H1_DL2 cells at 0 h; (B) Untreated cells 60 h after initiating a scratch wound; (C) Representative picture 60 h after initiating a scratch wound and starting the treatment with 5 μM of temsirolimus; (D) Representative picture 60 h after initiating a scratch wound and starting the treatment with 5 μM of vemurafenib; (E) Representative picture 48 h after initiating a scratch wound and starting combined treatment (5 μM temsirolimus and 5 μM vemurafenib); and (F) Representative picture 60 h after initiating a scratch wound and starting combined treatment (5 μM temsirolimus and 5 μM vemurafenib). Scale bar = 200 μm.

Figure 3.

Multicellular spheroid growth of H1_DL2 cells after treatment with vemurafenib and temsirolimus. (A, left) Spheroid diameters measured zero and eight days after treatment with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib) (n = 6, mean ± SD). n.s.: not significant; * p < 0.05, **** p < 0.0001. (A, right) The percentage of dead cells within the spheroids after treatment for three days with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib); (B) Maximum intensity projection images of the red channel (dead cells) of confocal image stacks of spheroids treated with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib); (C) The surface volume of the green channel (live cells) of the same spheroids as in (B); and (D) An overlay of the red and green volume surfaces of the same spheroids as in (B). Scale bar = 100 μm. neg. control = negative control.

Figure 4.

Western blot of H1_DL2 cells after drug treatment with vemurafenib and temsirolimus. Western blot of phosphorylated ERK1 and phosphorylated ERK2 (pERK1/2), phosphorylated mTOR (p-mTOR) and phosphorylated AKT (pAKT), in H1_DL2 cells treated with 15 μM of vemurafenib (Vem), 25 μM of temsirolimus (Tems), or the combined treatment (Combi; 10 μM vemurafenib + 10 μM temsirolimus). The K16 cell line (A) served as positive control for pMAPK; and the 293T cell line (B) was a positive control for pAKT and p-mTOR. GAPDH protein levels were assessed as a loading control.

Figure 5.

Principal component analysis (PCA) of H1_DL2 cells after drug treatment with vemurafenib and temsirolimus. PCA analysis of all 20 samples resulted in four distinct clusters for each of the respective treatments: Untreated samples (blue), vemurafenib-treated cells (15 μM; green), temsirolimus-treated cells (25 μM; purple) and the combination treatment (5 μM each; red) (n = 5 for all treatments).

Figure 6.

Graphical representation of two of the top score networks identified by Ingenuity Pathway Analysis (IPA). Canonical pathways from Ingenuity for combination treatment (5 μM vemurafenib and 5 μM temsirolimus) compared to untreated cells. Molecular relationships between genes downregulated (green) or upregulated (red) after treatment are shown. (A) The canonical pathway “Cell cycle: G1/S checkpoint regulation”; and (B) The canonical pathway “Cell cycle: G2/M DNA damage checkpoint regulation”.

Figure 7.

Ingenuity function analysis. (A) Bar plot of the activation z-scores of selected downstream functions in combination treated samples (5 μM vemurafenib and 5 μM temsirolimus) compared to untreated cells. Functions with z-scores greater than two are predicted as being “activated” in this comparison, while those with scores less than −2 are “inhibited”. All columns were statistically significant; p < 0.005. Column numbers correspond to the list of downstream functions shown in Table 3; and (B) A detailed presentation of bar plots of activation z-scores, showing the most activated downstream functions for each of the three treatments; 25 μM temsirolimus vs. untreated cells, 15 μM vemurafenib vs. untreated cells or 5 μM vemurafenib + 5 μM temsirolimus vs. untreated cells. All columns were statistically significant; p < 0.005.