FOXD3 is a mutant B-RAF-regulated inhibitor of G(1)-S progression in melanoma cells

Abstract

The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Transformation of melanocytes gives rise to melanoma. In two thirds of melanomas, the serine/threonine kinase B-RAF is mutated to a constitutively active form. Here, we show that FOXD3 levels are upregulated following attenuation of B-RAF and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling in mutant B-RAF harboring human melanoma cells. This effect was selective because FOXD3 was not upregulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G(1) cell cycle arrest and was associated with p53-dependent upregulation of p21(Cip1). FOXD3-induced cell cycle arrest was prevented by p53 depletion and, to a lesser extent, p21(Cip1) depletion. These studies show that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator, and have implications for the repression of melanocytic lineage cells.

Figure 1

Inhibition of B-RAF up-regulates FOXD3 levels in melanoma cells. A, WM793 and WM115 cells were transfected with non-targeting control (Ctl) or B-RAF (duplex #1) siRNA. Seventy-two hours post-transfection, cells were lysed and analyzed by Western blotting for FOXD3, B-RAF, phospho-ERK1/2 (pERK1/2) and ERK1/2. B, WM793TR cells expressing either control (LacZ2.1) or B-RAF (hairpins #1, #3, and #7) shRNA were induced (+) with tetracycline or not induced (−) for 6 days. Cells were lysed and samples analyzed by Western blotting as in A. The right-hand panels show A375TR cells expressing either Ctl or B-RAF #1 shRNA −/+ doxycycline for 6 days. C, WM793TR cells expressing either control or B-RAF shRNA were induced (+) with doxycycline or not induced (−) for 5 days. FOXD3 and actin mRNA levels were analyzed by quantitative RT-PCR. Graphed is the average and standard deviation from three independent experiments. The asterisk indicates statistical significance comparing B-RAF shRNA + doxycycline to other conditions (p<0.05). D, WM793 cells were transfected with Ctl, B-RAF#1 or cyclin D1 siRNA. After 72 hours, cell lysates were analyzed by Western blotting for FOXD3, B-RAF, cyclin D1 and ERK1/2.

Figure 2

MEK signaling is required to suppress FOXD3 expression. A, WM793 and A375 cells were treated with 3.3 μM AZD6244 or 5 μM U0126 for 16 and 24 hours. Cell lysates were analyzed by Western blotting for the indicated proteins. B, Three independent melanocytes cultures (C322, C334 and C337) and WM793 (as a positive control) were treated −/+ AZD6244 for 24 hours. Cell lysates were analyzed by Western blot as indicated. C, Wild-type B-RAF WM3211 melanoma cells were treated with AZD6244 for 5 and 24 hours. WM793 represents a positive control. Cell lysates were analyzed by Western blot as indicated. D, Wild-type and mutant B-RAF thyroid cancer cell lines were analyzed, as in A–C.

Figure 3

Inducible expression of Foxd3 blocks melanoma growth in vitro. A, WM793TR cells expressing doxycycline-inducible β-galactosidase (LacZ) or murine Foxd3 were induced with 0.1 μg/ml doxycycline for 5 days. Lysates were immunoblotted for the respective transgenes, ERK1/2 and the C-terminal V5-epitope tag found on both transgenes. B, Induced WM793TR cells were stained with anti-V5-epitope (green) to detect LacZ or Foxd3, as well as Hoechst 33342 (blue) and anti-RhoGDI (red) to label nuclei and cytoplasm, respectively. C, WM793TR and WM115TR cells expressing LacZ or Foxd3 were treated −/+ doxycycline, trypsinized, counted and replated every 3 days for nine days. Average cell numbers and standard error bars are given for 3 independent experiments. * p<0.05. D, SK-MEL-28TR and A375TR cells expressing LacZ or Foxd3 were embedded in 0.3% agar and allowed to grow for approximately 2 weeks. SK-MEL-28TR colonies were photographed at 20X magnification and A375TR colonies were photographed at 10X magnification.

Figure 4

Foxd3 induces a G0/G1 arrest in melanoma. A, WM793TR cells were induced for 5 days. For the final 8 hours, 10 μM EdU was included. Cells were processed for flow cytometry and analyzed on a BD FacsCanto^™. The graph represents the average percent of EdU-positive cells and standard error of 4 independent experiments (* p<0.05). B, Lysates from WM793TR cells were immunoblotted for the V5-epitope, cyclin A, p21 and ERK1/2. C, WM793TR cells were induced for 0, 1, 2, 3, and 4 days. Lysates were immunoblotted for the V5-epitope, cyclin A, p21^Cip1 and ERK1/2. D, WM793TR/LacZ and WM793TR/Foxd3 cells were induced (+) with doxycycline or not induced (−) for 5 days. p21^Cip1, and cyclin A mRNA levels were analyzed by quantitative RT-PCR. Graphed is the average fold change relative to actin and standard deviation from three independent experiments (* p<0.05).

Figure 5

Expression of Foxd3 up-regulates p21^Cip1 in melanoma cell lines but not in a breast or a fibrosarcoma cell line. A, Foxd3-inducible WM115, SK-MEL-28, and A375 cells were treated with 0.1 μg/ml doxycycline for 5 days. Cell lysates were analyzed by Western blotting for the V5-epitope, cyclin A, p21^Cip1 and ERK1/2. B, Foxd3 and LacZ inducible forms of the human breast cancer line, MDA-MB-231, and human fibrosarcoma line, HT-1080, were generated. Cell lines were treated −/+ 0.1 μg/ml doxycycline for 5 days. Cell lysates were analyzed by Western blotting for the V5-epitope, p21^Cip1 and ERK1/2. C, WM793 expressing inducible LacZ, mouse Foxd3-V5, human FOXD3 and human FOXD3-V5 were treated −/+ doxycycline for 5 days. Lysates were analyzed by Western blotting for FOXD3, p21^Cip1, cyclin A, phospho-ERK1/2 (pERK1/2) and total ERK1/2.

Figure 6

p21^Cip1 and p53 are required for in FOXD3-induced cell cycle arrest. A, WM793TR/FOXD3-V5 were transfected with either control, p21^Cip1 or p53 siRNA. After 24 hours for recovery, cells were treated −/+ 0.1 μg/ml doxycycline for 3 days. Lysates were analyzed by Western blotting for V5, p21^Cip1, p53 and total ERK1/2. B, Cells were processed as above except that cells were treated for 5 days with doxycycline and for the final 8 hours, 10 μM EdU was included. Cells were processed for flow cytometry and analyzed on a BD FacsCanto^™. The graph represents the average percent of EdU-positive cells and standard error of 3 independent experiments. Asterisks indicate statistical significance (p<0.05). C, WM115TR/FOXD3-V5 were transfected with either control, p21^Cip1 or p53 siRNA. After 24 hours for recovery, cells were treated −/+ 0.1 μg/ml doxycycline for a further 5 days. Lysates were analyzed by Western blotting for cyclin A, V5, p21^Cip1, p53 and total ERK1/2.