The Brn-2 transcription factor links activated BRAF to melanoma proliferation

Abstract

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

FIG. 1.

Brn-2 is expressed at high levels in melanoma cell lines. (A) Reverse transcription-PCR showing relative levels of Brn-2 mRNA in the indicated cell lines compared to a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control. 501 mel and HMB2 are human melanoma cell lines, melan-c is a mouse melanocyte cell line, and K1735 is an undifferentiated mouse melanoma cell line. Cos7 cells were used as a negative control. (B and C) Western blots with an anti-Brn-2 monoclonal antibody, showing relative levels of Brn-2 protein expressed in the indicated cell lines compared to the expression of α-tubulin. Melan-c is a mouse melanocyte cell line, melb is a mouse melanoblast line, 1° melanocytes are primary mouse melanocytes, and B16 and K1735 represent relatively differentiated and undifferentiated mouse melanomas, respectively. Cos7 cells were used as a negative control. The remaining cell lines were derived from human melanomas, VUP representing a uveal melanoma.

FIG. 2.

Brn-2 overexpression results in increased [³H]thymidine incorporation into melanocytes. (A) Western blot with anti-Brn-2 antibody of melan-a cells, melan-a cells infected with an empty retrovirus (SV5), or three independent clones of melan-a cells infected with a retrovirus expressing SV5 epitope-tagged Brn-2. The A375 melanoma cell line was used a positive control. The same blot was also probed for α-tubulin as a loading control. (B) Phase contrast image of melan-a cells and the indicated clones of melan-a cells stably overexpressing Brn-2 from the pBabePuro retrovirus. (C) [³H]thymidine incorporation into the indicated cell lines.

FIG. 3.

Robust activation of Brn-2 expression in response to a melanoma-associated RTK. (A) Schematic showing the MAP kinase signaling pathway downstream from the HER-mrk chimeric receptor in which the ligand binding domain of the EGFR is fused to the intracellular signaling domain of the Xmrk RTK. U0126 is a specific inhibitor of the MAP kinase kinase MEK. (B) Western blot showing expression of ERK in cells expressing the HER-mrk fusion protein (melan-a Hm) (36) grown in the presence of EGF (50 ng/ml). The smaller panel indicates that EGF-mediated stimulation of MAP kinase was inhibited by treatment with the MEK inhibitor U0126. (C) Western blot with anti-Brn-2 antibody, showing that Brn-2 expression is induced by treatment of cells expressing the HER-mrk fusion protein (melan-a Hm) with EGF (50 ng/ml) for 24 h. The location of Brn-2 is indicated, and the asterisk indicates a nonspecific band that cross-reacted with the antibody. This band was apparent when bovine serum albumin rather then skim milk was used as a blocking agent for the Western blot. Parental melan-a cells and melan-a Hm cells grown in the absence of EGF are shown as controls. The blot was reprobed with anti-ERK2 polyclonal antibody as a loading control. No expression of Brn-2 was observed in the absence of treatment. ERK2 was used as a loading control. (D) Western blot with anti-Brn-2 monoclonal antibody of melan-a Hm cells grown in either the absence or presence of EGF (50 ng/ml) for 24 or 48 h, as indicated. The blot was probed with anti-Brn-2 monoclonal or anti-ERK2 polyclonal antibodies. (E) Induction of Brn-2 expression by Xmrk requires MAP kinase signaling. Melan-a Hm cells were stimulated with EGF for 48 h in the presence or absence of 20 μM U0126. Cell extracts were Western blotted with anti-Brn-2 or anti-ERK2 antibody, as indicated.

FIG. 4.

Ras- and MAP kinase-induced expression of the Brn-2 promoter. (A) Western blot with anti-Brn-2 or anti-α-tubulin antibodies and either melan-a cells or Ras-transformed melan-a cells, as indicated. (B) Western blot of melan-a cells and a melan-a cell line stably expressing constitutively activated MEK (MEK.EE). MEK.EE expression was determined with an anti-His tag antibody. Antibodies against Brn-2, Rsk, phospho-Rsk, and α-tubulin were used where indicated. (C and D) Regulation of the Brn-2 promoter by Ras. The wild-type Brn-2 promoter fused to a luciferase reporter extending to −2.3 kb or to −266 (ΔNhe) was transfected into the melan-c melanocyte cell line together with either an empty expression vector or a vector expressing activated Ras (RasN12), as indicated. The results presented are the averages of four experiments.

FIG. 5.

Brn-2 is a target for BRAF signaling. (A) Western blot of human melanoma cell lines A375M and WM266.4, which express constitutively active forms of BRAF (11), with either an anti-Brn-2 antibody or anti-ERK2 antibody. Where indicated, cells were treated for 24 h with the MEK inhibitor U0126 at a concentration of 10 μM. (B) BRAF activates the Brn-2 promoter. B16 melanoma cells were transfected with the Brn-2 promoter (−266)-luciferase reporter (500 ng) and either an empty vector or a vector expressing wild-type BRAF or the indicated BRAF mutants (200 ng). (C) BRAF and β-catenin can cooperate for activation of the Brn-2 promoter. B16 melanoma cells were transfected with the Brn-2 promoter (−266)-luciferase reporter (100 ng) and either an empty vector or a vector expressing wild-type BRAF, β-catenin, or both, as indicated. Luciferase activity was determined 48 h posttransfection. (D) siRNA-mediated inhibition of BRAF leads to loss of MAP kinase activity and Brn-2 expression. Shown is a Western blot of WM266.4 cells either untreated or transfected for 60 h with BRAF-specific siRNA or a scrambled oligonucleotide control with either anti-Brn2 monoclonal, anti-ERK2, or anti-phospho-ERK2 antibodies, where indicated.

FIG. 6.

siRNA-mediated downregulation of Brn-2 leads to decreased [³H]thymidine incorporation in melanoma cells. (A) Western blot with anti-Brn-2 antibody of WM266.4 cell cells transfected with a Brn-2-specific siRNA or a control nonsilencing siRNA for the indicated times. Untransfected cells were used as an additional control. (B) Phase contrast image of WM266.4 cells transfected with a Brn-2-specific siRNA or a control nonsilencing siRNA, as indicated. (C) [³H]thymidine incorporation into untransfected WM266.4 cells or cells transfected with a Brn-2-specific siRNA or a control nonsilencing siRNA, as indicated, either 72 or 96 h posttransfection.