Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF

Abstract

The Microphthalmia-associated transcription factor (MITF) is an important regulator of cell-type specific functions in melanocytic cells. MITF is essential for the survival of pigmented cells, but whereas high levels of MITF drive melanocyte differentiation, lower levels are required to permit proliferation and survival of melanoma cells. MITF is phosphorylated by ERK, and this stimulates its activation, but also targets it for degradation through the ubiquitin-proteosome pathway, coupling MITF degradation to its activation. We have previously shown that because ERK is hyper-activated in melanoma cells in which BRAF is mutated, the MITF protein is constitutively down-regulated. Here we describe another intriguing aspect of MITF regulation by oncogenic BRAF in melanoma cells. We show oncogenic BRAF up-regulates MITF transcription through ERK and the transcription factor BRN2 (N-Oct3). In contrast, we show that in melanocytes this pathway does not exist because BRN2 is not expressed, demonstrating that MITF regulation is a newly acquired function of oncogenic BRAF that is not performed by the wild-type protein. Critically, in melanoma cells MITF is required downstream of oncogenic BRAF because it regulates expression of key cell cycle regulatory proteins such as CDK2 and CDK4. Wild-type BRAF does not regulate this pathway in melanocytes. Thus, we show that oncogenic BRAF exerts exquisite control over MITF on two levels. It downregulates the protein by stimulating its degradation, but then counteracts this by increasing transcription through BRN2. Our data suggest that oncogenic BRAF plays a critical role in regulating MITF expression to ensure that its protein levels are compatible with proliferation and survival of melanoma cells. We propose that its ability to appropriate the regulation of this critical factor explains in part why BRAF is such a potent oncogene in melanoma.

Figure 1. MITF expression requires BRAF in melanoma cells.

A) Western blot for MITF, phosphorylated ERK (ppERK) and total ERK2 in untreated (-) melanoma cells or in cells treated with DMSO (D) or U0126 (U0; 10 µM) for 48 h. B) Western blot for BRAF, MITF, ppERK and total ERK2 in melanoma cells 48 h after transfection with control (SC) or BRAF (B1, B2) siRNAs. C) Immunohistochemistry analysis of WM266-4 cells transfected with Alexa-Fluor-647-labelled control (SC) of BRAF siRNA probes (blue). Cells were stained for BRAF or MITF (red) after 48 h and DNA was stained using SYTOX®-green (green). D) Quantitative RT-PCR analysis of MITF in A375 and WM266-4 cells 24 and 48 h after transfection with control (SC) or BRAF (B1, B2) siRNA. MITF expression levels are expressed relative to the GAPDH control.

Figure 2. BRAF regulates the MITF promoter through BRN2.

A) MITF (−2293 to +120) promoter activity in A375 cells transfected with control (SC) or BRAF (B1, B2) siRNA. Extracts were prepared after 48 h and analysed for BRAF and ERK2 (loading control) by Western blotting and for luciferase activity [RLU]. B) MITF (−2293 to +120) promoter activity in A375 cells transfected with the indicated amounts (50–500 ng) of myc-tagged ^V600EBRAF, myc-tagged ^WTBRAF or an empty vector control (vec). Extracts, prepared 48 h after transfection were analysed for expression of myc-tagged BRAF (*: non-specific band), ERK2 (loading control) by Western blot and for luciferase activity [RLU]. C) MITF promoter activity induced by ^V600EBRAF. Cells were transfected with the indicated promoter fragments and the fold induction stimulated by ^V600EBRAF is indicated. Binding sites previously identified in the MITF promoter are indicated . D) The sequence of the MITF promoter from −53 to −27, with the putative BRN2 binding site indicated. The binding site mutations (mut 1 and mut 2) are also shown. E) Chromatin immunoprecipitation (ChIP) assays from A375 cells using BRN2 antibodies, non-specific antibodies (IgG) or no antibody (no Ab). The −170 to +120 region of the MITF promoter was amplified, and as a control the cyclin D1 promoter was also analysed. F) The activity of the −333 luciferase reporter, stimulated by ^V600EBRAF or BRN2 in A375 cells is shown. The effects of the BRN2 binding site mutations (mut 1, mut 2) are shown, relative (% Activity) to the activity of the non-mutated promoter. G) The activity of the −333 luciferase promoter in A375 cells transfected with control (SC) or BRN2 siRNA is shown. The cells were transfected with ^V600EBRAF and the reporter construct 24 h after the siRNA had been introduced. Luciferase activity was measured after a further 48 h.

Figure 3. BRN2 is required for MITF expression in melanoma cells.

A) Western blot for BRN2, ppERK and tubulin (loading control) in WM266-4 cells treated with control (SC), BRAF or ERK2 siRNA. B) Western blot for MITF, BRN2 and total ERK2 (loading control) in the indicated melanoma cells treated with control (SC) or BRN2 siRNA. C) Quantitative real time PCR of MITF mRNA expression in the indicated melanoma cells treated with control (SC) or BRN2 siRNA. MITF expression was normalised to GAPDH expression and is shown as fold expression in reference to SC transfected cells. Results are for one experiment assayed in triplicate. Similar results were obtained in three independent experiments. D) Western blot for BRN2, MITF and total ERK2 (loading control) in human melanoma samples expressing wild-type BRAF (WT) or ^V600EBRAF (VE). The expression of BRN2 and MITF relative to ERK2 expression is indicated below the blots.

Figure 4. MITF acts downstream of BRAF in melanoma cells.

A) DNA synthesis ([³H]-thymidine incorporation) in SKMel28, A375 and WM266-4 cells 48–60 h after transfection with control (SC) or MITF (MI, MI1, MI3) siRNA. B) Western blot for BRAF, MITF, ppERK, CDK4, CDK2, p21^Cip (p21) and total ERK2 (loading control) in WM266-4 and A375 cells 24 h and 48 h after transfection with control (SC), BRAF (B1) or MITF (MI, MI1, MI3) siRNA. C) Expression of CDK2 in A375 cells transfected with Alexa-Fluor-647-labelled control (SC) or BRAF siRNA (blue). The cells were microinjected with expression constructs for HA-tagged 4OHT-binding domain of the estrogen receptor (ER) or HA-tagged ER∶MITF after 24 hours (green). The cells were treated with 200 nM 4OHT and fixed and stained for CDK2 (red) after a further 40 h. D) Western blot for BRAF, MITF, CDK4, CDK2, ppERK and ERK in A375 cells transfected with control (SC) or BRAF (B1) siRNAs, together with an empty vector (vec) or MITF expression construct (rescue). E) DNA synthesis ([³H]-thymidine incorporation) in A375 cells 40 h after transfection with control (SC) or BRAF (B1) siRNA, together with either an empty vector (vec) or MITF expression construct (rescue). The levels of BRAF expressions are shown on a Western blot with ERK2 serving as the loading control. F) Colony formation assay of SKMel28 cells infected with empty vector (pBabe) or MITF expression construct, and treated with 1 µM PD184352 for 4 weeks. Whole plate and high-magnification images are shown. The inhibitory effect of PD184352 on ERK phosphorylation is shown on a Western blot and actin was used as the loading control.

Figure 5. ^V600EBRAF can regulate MITF expression in melanocytes.

A) Thymidine incorporation in normal melanocytes 40 h after transfection with control (SC), BRAF (B1) or MITF siRNA. B) Western blot for BRAF, MITF and ERK2 (loading control) in normal human melanocytes 48 h after transfection with control (SC) or BRAF (B1) siRNA. C) Western blot for MITF, ppERK and ERK2 (loading control) in normal human melanocytes 24 h after treatment with DMSO (D) or U0126 (U0; 10 µM). D) Western blot for CDK4, CDK2, p21^Cip (p21) and ERK2 (loading control) in human melanocytes 48 h after transfection with control (SC), BRAF (B1) or MITF (MI) siRNA. E) Western blot for BRN2 and total ERK2 (loading control) in Hela cells (negative control), human melanocytes and the indicated melanoma cell lines carrying mutant BRAF. F) Western blot for BRN2, ppERK and total ERK2 (loading control) in melanocytes transfected with empty vector (control), ^WTBRAF or ^V600EBRAF. G) MITF (−2293 to +120) promoter-reporter (luciferase) activity in melanocytes transfected with an empty vector (vec), or with a BRN2 or ^V600EBRAF expression construct.

Figure 6. A model of MITF regulation by oncogenic BRAF in melanoma cells.

MITF protein levels are critical in melanoma cells. High levels of MITF stimulate differentiation, whereas if the levels are too low, the cells die by apoptosis. Therefore to stimulate proliferation in melanoma cells, MITF protein levels must be constrained to within a narrow range. We propose that ^V600EBRAF stimulates MITF activation through ERK phosphorylation, but this targets MITF for degradation. This would reduce MITF protein to levels that are below those required for survival and proliferation, so to counter this, ^V600EBRAF stimulates MITF transcription through up-regulation of BRN2. Thus although MITF is constantly destroyed by proteasome-mediated degradation, its expression persists at a level that is sufficient to maintain expression of cell cycle components such as CDK4 and CDK2 and survival factors such as BCL2, thereby favouring proliferation and survival over differentiation or apoptosis.