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Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.

Wellbrock C, Marais R - J. Cell Biol. (2005)

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

ABSTRACT
The protein kinase B-RAF is a human oncogene that is mutated in approximately 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

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V600EB-RAF activates ERK and suppresses MITF expression in human melanocytes. (A) Western blot analysis of ppERK and ERK2 in primary human melanocytes (NHM) that were untreated or treated with 10 μM U0126 for 24 h or with DMSO control (second lane). (B) Thymidine incorporation into NHM treated with 10 μM U0126 for 24 h, DMSO, or no treatment control in the presence of melanocyte growth factor supplement. (C) Western blot analysis of A-RAF, B-RAF, C-RAF, ppERK, and ERK2 in NHM transfected with either scrambled control siRNA (sc) or siRNAs specific for A-RAF, B-RAF, or C-RAF. (D) Thymidine incorporation into NHM in the presence of melanocyte growth factor supplement 72 h after transfection with the indicated siRNAs. Error bars represent SD. (E) Western blot analysis for myc-tagged B-RAF, ppERK, and ERK2 in NHM transiently expressing either myc-V600EB-RAF or myc-WTB-RAF. B-RAF was revealed using the antibody 9E10. (F) Immunofluorescence analysis of transiently expressed myc-V600EB-RAF and myc-WTB-RAF and of endogenous MITF in NHM transfected with myc-V600EB-RAF or myc-WTB-RAF. B-RAF proteins are revealed with anti-myc, and MITF is revealed with C5. Nuclei are counterstained with DAPI. Arrows indicate nuclei of transfected cells expressing either wild-type B-RAF or mutant V600EB-RAF. (G) Quantification of immunofluorescence data. Means of three experiments are shown (100 cells were counted in each experiment). Vector-transfected cells served as a control.
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fig4: V600EB-RAF activates ERK and suppresses MITF expression in human melanocytes. (A) Western blot analysis of ppERK and ERK2 in primary human melanocytes (NHM) that were untreated or treated with 10 μM U0126 for 24 h or with DMSO control (second lane). (B) Thymidine incorporation into NHM treated with 10 μM U0126 for 24 h, DMSO, or no treatment control in the presence of melanocyte growth factor supplement. (C) Western blot analysis of A-RAF, B-RAF, C-RAF, ppERK, and ERK2 in NHM transfected with either scrambled control siRNA (sc) or siRNAs specific for A-RAF, B-RAF, or C-RAF. (D) Thymidine incorporation into NHM in the presence of melanocyte growth factor supplement 72 h after transfection with the indicated siRNAs. Error bars represent SD. (E) Western blot analysis for myc-tagged B-RAF, ppERK, and ERK2 in NHM transiently expressing either myc-V600EB-RAF or myc-WTB-RAF. B-RAF was revealed using the antibody 9E10. (F) Immunofluorescence analysis of transiently expressed myc-V600EB-RAF and myc-WTB-RAF and of endogenous MITF in NHM transfected with myc-V600EB-RAF or myc-WTB-RAF. B-RAF proteins are revealed with anti-myc, and MITF is revealed with C5. Nuclei are counterstained with DAPI. Arrows indicate nuclei of transfected cells expressing either wild-type B-RAF or mutant V600EB-RAF. (G) Quantification of immunofluorescence data. Means of three experiments are shown (100 cells were counted in each experiment). Vector-transfected cells served as a control.

Mentions: Because V600EB-RAF mutations occur in 50–70% of human melanomas (Davies et al., 2002), we examined whether our mouse cell studies were relevant to human melanocytes. First, we analyzed the RAF–MEK–ERK pathway in primary normal human melanocytes (NHMs). ERK inhibition by U0126 (Fig. 4 A) blocks DNA synthesis (Fig. 4 B), demonstrating that ERK signaling is essential for NHM proliferation, so we examined the contribution of individual RAF isoforms by RNA interference (RNAi). Depletion of A-RAF from these cells did not affect basal ERK activity (Fig. 4 C) or DNA synthesis (Fig. 4 D), whereas depletion of B-RAF or C-RAF suppresses ERK activity (Fig. 4 C) and significantly inhibits DNA synthesis (Fig. 4 D). Thus, A-RAF is not required for ERK-dependent melanocyte proliferation, whereas B- and C-RAF are both required. This contrasts with observations in melanoma cells harboring V600EB-RAF in which only B-RAF is required for ERK activation but all three RAF kinases are required for proliferation (Karasarides et al., 2004). The observation that B- and C-RAF both contribute to ERK signaling and proliferation in NHM can be explained by the fact that in melanocytes, these isoforms stimulate nonredundant growth signals (Wellbrock et al., 2004a).


Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.

Wellbrock C, Marais R - J. Cell Biol. (2005)

V600EB-RAF activates ERK and suppresses MITF expression in human melanocytes. (A) Western blot analysis of ppERK and ERK2 in primary human melanocytes (NHM) that were untreated or treated with 10 μM U0126 for 24 h or with DMSO control (second lane). (B) Thymidine incorporation into NHM treated with 10 μM U0126 for 24 h, DMSO, or no treatment control in the presence of melanocyte growth factor supplement. (C) Western blot analysis of A-RAF, B-RAF, C-RAF, ppERK, and ERK2 in NHM transfected with either scrambled control siRNA (sc) or siRNAs specific for A-RAF, B-RAF, or C-RAF. (D) Thymidine incorporation into NHM in the presence of melanocyte growth factor supplement 72 h after transfection with the indicated siRNAs. Error bars represent SD. (E) Western blot analysis for myc-tagged B-RAF, ppERK, and ERK2 in NHM transiently expressing either myc-V600EB-RAF or myc-WTB-RAF. B-RAF was revealed using the antibody 9E10. (F) Immunofluorescence analysis of transiently expressed myc-V600EB-RAF and myc-WTB-RAF and of endogenous MITF in NHM transfected with myc-V600EB-RAF or myc-WTB-RAF. B-RAF proteins are revealed with anti-myc, and MITF is revealed with C5. Nuclei are counterstained with DAPI. Arrows indicate nuclei of transfected cells expressing either wild-type B-RAF or mutant V600EB-RAF. (G) Quantification of immunofluorescence data. Means of three experiments are shown (100 cells were counted in each experiment). Vector-transfected cells served as a control.
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fig4: V600EB-RAF activates ERK and suppresses MITF expression in human melanocytes. (A) Western blot analysis of ppERK and ERK2 in primary human melanocytes (NHM) that were untreated or treated with 10 μM U0126 for 24 h or with DMSO control (second lane). (B) Thymidine incorporation into NHM treated with 10 μM U0126 for 24 h, DMSO, or no treatment control in the presence of melanocyte growth factor supplement. (C) Western blot analysis of A-RAF, B-RAF, C-RAF, ppERK, and ERK2 in NHM transfected with either scrambled control siRNA (sc) or siRNAs specific for A-RAF, B-RAF, or C-RAF. (D) Thymidine incorporation into NHM in the presence of melanocyte growth factor supplement 72 h after transfection with the indicated siRNAs. Error bars represent SD. (E) Western blot analysis for myc-tagged B-RAF, ppERK, and ERK2 in NHM transiently expressing either myc-V600EB-RAF or myc-WTB-RAF. B-RAF was revealed using the antibody 9E10. (F) Immunofluorescence analysis of transiently expressed myc-V600EB-RAF and myc-WTB-RAF and of endogenous MITF in NHM transfected with myc-V600EB-RAF or myc-WTB-RAF. B-RAF proteins are revealed with anti-myc, and MITF is revealed with C5. Nuclei are counterstained with DAPI. Arrows indicate nuclei of transfected cells expressing either wild-type B-RAF or mutant V600EB-RAF. (G) Quantification of immunofluorescence data. Means of three experiments are shown (100 cells were counted in each experiment). Vector-transfected cells served as a control.
Mentions: Because V600EB-RAF mutations occur in 50–70% of human melanomas (Davies et al., 2002), we examined whether our mouse cell studies were relevant to human melanocytes. First, we analyzed the RAF–MEK–ERK pathway in primary normal human melanocytes (NHMs). ERK inhibition by U0126 (Fig. 4 A) blocks DNA synthesis (Fig. 4 B), demonstrating that ERK signaling is essential for NHM proliferation, so we examined the contribution of individual RAF isoforms by RNA interference (RNAi). Depletion of A-RAF from these cells did not affect basal ERK activity (Fig. 4 C) or DNA synthesis (Fig. 4 D), whereas depletion of B-RAF or C-RAF suppresses ERK activity (Fig. 4 C) and significantly inhibits DNA synthesis (Fig. 4 D). Thus, A-RAF is not required for ERK-dependent melanocyte proliferation, whereas B- and C-RAF are both required. This contrasts with observations in melanoma cells harboring V600EB-RAF in which only B-RAF is required for ERK activation but all three RAF kinases are required for proliferation (Karasarides et al., 2004). The observation that B- and C-RAF both contribute to ERK signaling and proliferation in NHM can be explained by the fact that in melanocytes, these isoforms stimulate nonredundant growth signals (Wellbrock et al., 2004a).

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

ABSTRACT
The protein kinase B-RAF is a human oncogene that is mutated in approximately 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

Show MeSH
Related in: MedlinePlus