Limits...
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.

Show MeSH

Related in: MedlinePlus

MITF expression is lost in B-RAF–transformed melanocytes. (A) Western blot analysis of melan-a cells, a neoR control line, WTB-RAF–expressing clones B2 and B9, and V600EB-RAF–expressing clone VE16 probed for myc-tagged B-RAF, total B-RAF, and ERK2. (B) Bright field image of melan-a cells, neoR controls, clones B2, B9, and VE16, and G12VRAS- or MEKEE-transformed melan-a cells under growing conditions. (C) Western blot analysis of MITF, phosphorylated ERK (ppERK), and ERK2 in melan-a cells, neoR controls, WTB-RAF–expressing clones (B2 and B9), V600EB-RAF–expressing clones (VE11, VE14, and VE16) and G12VRAS- or MEKEE-expressing cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171350&req=5

fig1: MITF expression is lost in B-RAF–transformed melanocytes. (A) Western blot analysis of melan-a cells, a neoR control line, WTB-RAF–expressing clones B2 and B9, and V600EB-RAF–expressing clone VE16 probed for myc-tagged B-RAF, total B-RAF, and ERK2. (B) Bright field image of melan-a cells, neoR controls, clones B2, B9, and VE16, and G12VRAS- or MEKEE-transformed melan-a cells under growing conditions. (C) Western blot analysis of MITF, phosphorylated ERK (ppERK), and ERK2 in melan-a cells, neoR controls, WTB-RAF–expressing clones (B2 and B9), V600EB-RAF–expressing clones (VE11, VE14, and VE16) and G12VRAS- or MEKEE-expressing cells.

Mentions: We previously described the generation of mouse melanocyte lines expressing myc-tagged versions of WTB-RAF (melan-a–B-RAF) or V600EB-RAF (melan-a–V600E [VE]; Wellbrock et al., 2004b). We demonstrated that melanocytes expressing V600EB-RAF show constitutive ERK signaling and proliferate in a factor-independent manner (Wellbrock et al., 2004b). Importantly, cells expressing high or low levels of WTB-RAF do not have elevated ERK activity or grow in a factor-independent manner, demonstrating that even high levels of WTB-RAF expression are not transforming. Melanocytes expressing V600EB-RAF (clone VE16; Fig. 1 A) display dramatically reduced dendricity and pigmentation, which is similar to the morphology that is observed in melanocytes expressing oncogenic RAS (G12VRAS) or constitutively active MAPK and ERK kinase (MEK; MEKEE; Fig. 1 B). In contrast, clones expressing low or high levels of WTB-RAF (clones B2 and B9) have a parental phenotype (Fig. 1 B). The reduction in pigmentation and dentricity that is induced by oncogenic B-RAF prompted us to examine known regulators of melanocyte differentiation. Importantly, we find that MITF is consistently down-regulated in cell lines expressing V600EB-RAF, G12VRAS, and MEKEE, and this loss correlates with constitutive ERK activation (Fig. 1 C).


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

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

MITF expression is lost in B-RAF–transformed melanocytes. (A) Western blot analysis of melan-a cells, a neoR control line, WTB-RAF–expressing clones B2 and B9, and V600EB-RAF–expressing clone VE16 probed for myc-tagged B-RAF, total B-RAF, and ERK2. (B) Bright field image of melan-a cells, neoR controls, clones B2, B9, and VE16, and G12VRAS- or MEKEE-transformed melan-a cells under growing conditions. (C) Western blot analysis of MITF, phosphorylated ERK (ppERK), and ERK2 in melan-a cells, neoR controls, WTB-RAF–expressing clones (B2 and B9), V600EB-RAF–expressing clones (VE11, VE14, and VE16) and G12VRAS- or MEKEE-expressing cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171350&req=5

fig1: MITF expression is lost in B-RAF–transformed melanocytes. (A) Western blot analysis of melan-a cells, a neoR control line, WTB-RAF–expressing clones B2 and B9, and V600EB-RAF–expressing clone VE16 probed for myc-tagged B-RAF, total B-RAF, and ERK2. (B) Bright field image of melan-a cells, neoR controls, clones B2, B9, and VE16, and G12VRAS- or MEKEE-transformed melan-a cells under growing conditions. (C) Western blot analysis of MITF, phosphorylated ERK (ppERK), and ERK2 in melan-a cells, neoR controls, WTB-RAF–expressing clones (B2 and B9), V600EB-RAF–expressing clones (VE11, VE14, and VE16) and G12VRAS- or MEKEE-expressing cells.
Mentions: We previously described the generation of mouse melanocyte lines expressing myc-tagged versions of WTB-RAF (melan-a–B-RAF) or V600EB-RAF (melan-a–V600E [VE]; Wellbrock et al., 2004b). We demonstrated that melanocytes expressing V600EB-RAF show constitutive ERK signaling and proliferate in a factor-independent manner (Wellbrock et al., 2004b). Importantly, cells expressing high or low levels of WTB-RAF do not have elevated ERK activity or grow in a factor-independent manner, demonstrating that even high levels of WTB-RAF expression are not transforming. Melanocytes expressing V600EB-RAF (clone VE16; Fig. 1 A) display dramatically reduced dendricity and pigmentation, which is similar to the morphology that is observed in melanocytes expressing oncogenic RAS (G12VRAS) or constitutively active MAPK and ERK kinase (MEK; MEKEE; Fig. 1 B). In contrast, clones expressing low or high levels of WTB-RAF (clones B2 and B9) have a parental phenotype (Fig. 1 B). The reduction in pigmentation and dentricity that is induced by oncogenic B-RAF prompted us to examine known regulators of melanocyte differentiation. Importantly, we find that MITF is consistently down-regulated in cell lines expressing V600EB-RAF, G12VRAS, and MEKEE, and this loss correlates with constitutive ERK activation (Fig. 1 C).

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