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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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Enhanced MITF expression contributes to cAMP-induced growth inhibition of melanoma cells. (A) Western blot analysis of endogenous MITF, ppERK, and ERK2 in melanoma cell lines expressing oncogenic RAS or B-RAF compared with NHM. MITF was revealed using anti-MITF (D5). (B) Western blot of endogenous MITF (using antibody D5) and ERK2 as a loading control in NHM treated with 20 μM forskolin or DMSO for the indicated times in the absence of melanocyte growth factor supplement. (C) Thymidine incorporation into NHM in the absence of melanocyte growth factor supplement after 20 μM forskolin treatment for 24 h or DMSO treatment. (D) Western blot analysis of MITF (D5) and ERK2 in untreated or 20 μM forskolin-treated (16 h) Colo826 or WM266-4 cells in the presence or absence of either MITF-specific siRNA or scrambled control (sc). (E) Thymidine incorporation in parallel samples from D. Thymidine incorporation in DMSO-treated cells was set at 100%. Results are from triplicate assays with error bars to represent SD from the mean.
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fig5: Enhanced MITF expression contributes to cAMP-induced growth inhibition of melanoma cells. (A) Western blot analysis of endogenous MITF, ppERK, and ERK2 in melanoma cell lines expressing oncogenic RAS or B-RAF compared with NHM. MITF was revealed using anti-MITF (D5). (B) Western blot of endogenous MITF (using antibody D5) and ERK2 as a loading control in NHM treated with 20 μM forskolin or DMSO for the indicated times in the absence of melanocyte growth factor supplement. (C) Thymidine incorporation into NHM in the absence of melanocyte growth factor supplement after 20 μM forskolin treatment for 24 h or DMSO treatment. (D) Western blot analysis of MITF (D5) and ERK2 in untreated or 20 μM forskolin-treated (16 h) Colo826 or WM266-4 cells in the presence or absence of either MITF-specific siRNA or scrambled control (sc). (E) Thymidine incorporation in parallel samples from D. Thymidine incorporation in DMSO-treated cells was set at 100%. Results are from triplicate assays with error bars to represent SD from the mean.

Mentions: Thus, MITF protein levels are significantly reduced in melanocytes in which B-RAF–ERK signaling is elevated. However, MITF is present in most melanoma cell lines expressing oncogenic B-RAF or RAS, albeit generally at reduced levels compared with NHM (Fig. 5 A). Our data suggests that MITF is antiproliferative and that one function of oncogenic B-RAF is to suppress its expression to overcome its growth-inhibitory activity. This model is supported by our observation that MITF expression is reduced in NHM expressing V600EB-RAF (Fig. 4, F and G) and the finding that forskolin, which up-regulates MITF (Fig. 5 B), also inhibits DNA synthesis in these cells (Fig. 5 C). Forskolin also up-regulates MITF expression in Colo829 and WM266-4 cells (Fig. 5 D, lanes 1, 2, 5, and 6) and in two melanoma cell lines that express oncogenic B-RAF (Davies et al., 2002), and this is accompanied by reduced proliferation (Fig. 5 E). Importantly, when RNAi is used to prevent MITF up-regulation (Fig. 5 D, lanes 4 and 8), forskolin does not inhibit proliferation of Colo829 and WM266-4 cells (Fig. 5 E), clearly demonstrating that elevated MITF protein levels are growth inhibitory to melanoma cells.


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

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

Enhanced MITF expression contributes to cAMP-induced growth inhibition of melanoma cells. (A) Western blot analysis of endogenous MITF, ppERK, and ERK2 in melanoma cell lines expressing oncogenic RAS or B-RAF compared with NHM. MITF was revealed using anti-MITF (D5). (B) Western blot of endogenous MITF (using antibody D5) and ERK2 as a loading control in NHM treated with 20 μM forskolin or DMSO for the indicated times in the absence of melanocyte growth factor supplement. (C) Thymidine incorporation into NHM in the absence of melanocyte growth factor supplement after 20 μM forskolin treatment for 24 h or DMSO treatment. (D) Western blot analysis of MITF (D5) and ERK2 in untreated or 20 μM forskolin-treated (16 h) Colo826 or WM266-4 cells in the presence or absence of either MITF-specific siRNA or scrambled control (sc). (E) Thymidine incorporation in parallel samples from D. Thymidine incorporation in DMSO-treated cells was set at 100%. Results are from triplicate assays with error bars to represent SD from the mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171350&req=5

fig5: Enhanced MITF expression contributes to cAMP-induced growth inhibition of melanoma cells. (A) Western blot analysis of endogenous MITF, ppERK, and ERK2 in melanoma cell lines expressing oncogenic RAS or B-RAF compared with NHM. MITF was revealed using anti-MITF (D5). (B) Western blot of endogenous MITF (using antibody D5) and ERK2 as a loading control in NHM treated with 20 μM forskolin or DMSO for the indicated times in the absence of melanocyte growth factor supplement. (C) Thymidine incorporation into NHM in the absence of melanocyte growth factor supplement after 20 μM forskolin treatment for 24 h or DMSO treatment. (D) Western blot analysis of MITF (D5) and ERK2 in untreated or 20 μM forskolin-treated (16 h) Colo826 or WM266-4 cells in the presence or absence of either MITF-specific siRNA or scrambled control (sc). (E) Thymidine incorporation in parallel samples from D. Thymidine incorporation in DMSO-treated cells was set at 100%. Results are from triplicate assays with error bars to represent SD from the mean.
Mentions: Thus, MITF protein levels are significantly reduced in melanocytes in which B-RAF–ERK signaling is elevated. However, MITF is present in most melanoma cell lines expressing oncogenic B-RAF or RAS, albeit generally at reduced levels compared with NHM (Fig. 5 A). Our data suggests that MITF is antiproliferative and that one function of oncogenic B-RAF is to suppress its expression to overcome its growth-inhibitory activity. This model is supported by our observation that MITF expression is reduced in NHM expressing V600EB-RAF (Fig. 4, F and G) and the finding that forskolin, which up-regulates MITF (Fig. 5 B), also inhibits DNA synthesis in these cells (Fig. 5 C). Forskolin also up-regulates MITF expression in Colo829 and WM266-4 cells (Fig. 5 D, lanes 1, 2, 5, and 6) and in two melanoma cell lines that express oncogenic B-RAF (Davies et al., 2002), and this is accompanied by reduced proliferation (Fig. 5 E). Importantly, when RNAi is used to prevent MITF up-regulation (Fig. 5 D, lanes 4 and 8), forskolin does not inhibit proliferation of Colo829 and WM266-4 cells (Fig. 5 E), clearly demonstrating that elevated MITF protein levels are growth inhibitory to melanoma cells.

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