The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

Abstract

The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAF(V600E)). MEK inhibitor U0126 or RNA interference (RNAi) for BRAF(V600E) decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor alpha by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAF(V600E) RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAF(V600E) and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system.

Figure 1.

Decreased production of immunosuppressive soluble factors IL-6, IL-10, and VEGF from melanoma cell lines with constitutively active MAPK through the BRAF^V600E mutation by inhibition of the MAPK signaling with a MEK inhibitor, U0126. (a) Inhibition of phosphorylation of ERK1/2 was detected by Western blot analysis of the A375mel melanoma cell line with the BRAF^V600E mutation before and 2, 4, 6, and 8 h after treatment with MEK inhibitor U0126 at a concentration of 25 μM. (b) Inhibition of mRNA expression for immunosuppressive soluble factors IL-6, IL-10, and VEGF in A375 cells. The mRNAs for various soluble factors, including IL-6, IL-10, and VEGF, were measured by quantitative RT-PCR before and 2, 4, 6, and 8 h after the treatment with U0126. The control treatment was performed with a DMSO solution. The mRNA levels at each time point were normalized to GAPDH mRNA and indicated as the relative value to that of 0 h. (c) Decreased production of IL-6, IL-10, and VEGF proteins from A375 cells after an 18-h treatment with U0126 at a concentration of 25 μM. The expression was detected by ELISA with the culture supernatants. The ratio of viability of U0126-treated cells DMSO-treated cells at the harvest was 77%. The cytokine production was normalized to the value of control DMSO-treated cells based on the cell count. Western blot showed strong inhibition of ERK phosphorylation, but not of STAT3 protein, or its phosphorylation at Ser⁷²⁷ and Tyr⁷⁰⁵. (d) Decreased production of IL-10 and VEGF from three melanoma cell lines with the BRAF^V600E mutation, 624mel, 888mel, and 928mel after an 18-h treatment with U0126 at a concentration of 25 μM, which was detected by ELISA with the culture supernatants. The ratios of viability of U0126-treated cells DMSO-treated cells at the harvest were 95% for 624mel, 103% for 888mel, and 100% for 928mel. The cytokine production was normalized to the value of control DMSO-treated cells based on the cell count. Western blot showed strong inhibition of ERK phosphorylation, but not of STAT3 protein. Slight decrease of Ser727 phosphorylation of STAT3 was observed in 888mel and 928mel cells, but not in 624mel cells. These results are representative of three or four independent experiments with similar results.

Figure 2.

Decreased production of immunosuppressive factors IL-6, IL-10, and VEGF from three melanoma cell lines with the BRAF^V600E mutation by BRAF^V600E-specific RNAi. The three melanoma cell lines with the BRAF^V600E mutation, A375mel, 888mel, and 624mel, were infected with the lentivirus vectors encoding short hairpin RNA for either firefly luciferase mRNA (GL3B; as control) or BRAF^V600E mRNA (BRAF#1′) at 50 or 100 multiplicity of infection. At 5 or 6 d after the infection, proteins were extracted and subjected to Western blot analysis. Profound decrease of phosphorylation of ERK1/2 with decrease of the BRAF protein was observed by the BRAF^V600E-specific RNAi. No significant difference in STAT3 protein and phosphorylation of STAT3 at Ser⁷²⁷ or Tyr⁷⁰⁵ was observed. A slight decrease of phosphorylation at Ser⁷²⁷ was observed in 888mel and 624mel cells after BRAF RNAi. 5 or 6 d after the lentivirus infection, the equal number of the melanoma cells was dispensed at a density of 1–2 × 10⁶ cells/2 ml, and the culture supernatants after 18 h were subjected to ELISA for IL-6, IL-10, or VEGF. Profound decrease of IL-6, IL-10, and VEGF was observed. One representative result of two or three independent experiments with similar results is shown.

Figure 3.

Inhibition of IL-6, IL-10, and VEGF production in A375 cells through the RNAi for BRAF^V600E alone, STAT3 alone, or both, without significant changes in the DNA-binding and promoter activity of STAT3. (a) Profound inhibition of IL-6, IL-10, and VEGF production in A375 through the RNAi for BRAF^V600E alone, STAT3 alone, or both. 5 or 6 d after the infection, the equal number of the cells was dispensed at a density of 10⁶ cells/2 ml, and the culture supernatant after 18 h was subjected to ELISA for IL-6, VEGF, and IL-10. Decrease of BRAF and phosphorylated ERK1/2 by the BRAF^V600E-specific RNAi and decrease of STAT3 by the STAT3-specific RNAi were confirmed by Western blot analysis. One representative result of six independent experiments with similar results is shown. (b) No significant change of the STAT3 DNA-binding activity by the inhibition of MAPK signaling with BRAF RNAi in melanoma cell lines. STAT3 DNA-binding activity was examined by EMSA of the nuclear extracts from melanoma cell lines with either control GL3B or BRAF#1′ shRNA vector treatment. Specific STAT3 bands indicated by arrows disappeared with cold wild-type STAT3 DNA probe (wt), but not with mutant STAT3 DNA probe (mt). Only a slight decrease of STAT3 DNA-binding activity was observed in 888mel cells. (c) STAT3 reporter assays in A375, 624mel, and 888mel cells. 2–4 × 10⁵ cells with either control or BRAF#1′ shRNA vector infection were transfected with 0.4 μg pSTAT3-TA-Luc and 0.4 μg pRL-SV40 with Effectene Transfection Reagent. 24 h after the transfection, the cells were harvested and analyzed for firefly and renilla luciferase activity. Each firefly luciferase activity was normalized with the renilla luciferase activity. Shaded bars and error bars indicate the mean and standard deviation of the triplicate assays, respectively. One representative experiment of three or four independent experiments is shown. STAT3-driven transcription was not changed after the BRAF RNAi in A375 and 624mel, but it slightly decreased in 888mel.

Figure 4.

Decreased suppressive activity of the A375 melanoma culture supernatants by pretreatment with RNAi for BRAF^V600E alone, STAT3 alone, or both on LPS-induced IL-12 and TNF-α production from DCs. (a) Neutralization of IL-6, IL-10, or VEGF in the culture supernatants of A375 melanoma cells restored the inhibition of IL-12 production from LPS-stimulated human DCs. Human MoDCs were cultured with the A375 supernatants in the presence or absence of the monoclonal antibody for IL-6, IL-10, VEGF, or an isotype control antibody at 1 μg/ml. IL-12 production in the culture supernatants was determined by ELISA 24 h after the LPS stimulation at 100 ng/ml. (b) CD14⁺ monocytes were isolated from PBMCs by using MACS and cultured in the media containing RPMI 1640, 10% (vol/vol) FBS, 100 ng/ml GM-CSF, and 50 ng/ml IL-4 with or without 20% (vol/vol) of the culture supernatant of the A375mel cells prepared in Fig. 3. (a) Half of the media, cytokines, and the culture supernatant was changed every 2 d. On day 5 of the culture, LPS was added at 100 ng/ml. After 15 h, the culture supernatants were collected and subjected to ELISA of IL-12 and TNF-α.