Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development

Abstract

Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease.

Figure 1.

bcat^sta construct and characteristics. (A) Map of the Tyr∷β-cat-mut-nls-egfp (bcat^sta) transgene (see Materials and Methods for details). (B) Localization of bcat^sta in FO-1 melanoma cells. In cells transfected with the bcat^sta construct (bcat^sta), bcat^sta protein was detected by its autofluorescence (EGFP). Note that the cells were transfected when they were at high confluency. Endogenous and exogenous β-catenin was detected with an antibody specific for the protein. Nuclear DNA was detected by DAPI staining. (C) Transient transfection of FO-1 melanoma cells with TOP–FOP flash luciferase reporters and various expression vectors. Note that β-cat-mut-nls is similar to bcat^sta without egfp. (D) bcat^sta expression in melanocyte cultures from two independent transgenic bcat^sta lines.

Figure 2.

Activated β-catenin cooperates with N-Ras to produce melanoma. (A) Photomicrography of a melanoma on the hairy part of the back of a bcat^sta/°; Tyr∷N-Ras^Q61K/° mouse. (B) Kaplan-Meier graph of melanoma incidence in various mouse genotypes, as indicated. The age of mice was scored according to the appearance of a cutaneous melanoma and additional signs of morbidity (Serrano et al. 1996; Ackermann et al. 2005). (C) Histological section of a hair follicle of a 3-mo-old Tyr∷N-Ras^Q61K/°; bcat^sta/° mouse. (D) Histological section of a cutaneous melanoma showing intradermal invasion. Immunostaining for tyrosinase (E) and Tyrp-1 (F) in an amelanotic melanoma was revealed by the brown color.

Figure 3.

Activated β-catenin does not induce melanocyte proliferation. (A) Number of wild-type and bcat^sta melanocytes 10 d after the explantation of newborn skin. One-million cells isolated from the skin of wild-type and bcat^sta 3-d-old pups were grown in culture for 10 d. The number of melanocytes in each culture was estimated for five independent cultures of wild-type and bcat^sta pups. (B) The number of melanoblasts in wild-type and bcat^sta embryos, from E10.5 to E15.5. The number of melanoblasts, identified as X-Gal-positive cells, was determined by eye and with the help of Adobe Photoshop. The cell number indicated on the figure corresponds to the melanoblasts on one side of the embryo in the trunk region between the front and back limbs, from somites 13–25. The mean number of melanoblasts is indicated, and the standard deviation is shown for each day of development. The number of embryo sides studied is indicated in brackets. Note that the number of melanoblasts is identical in wild-type, Ink4a–Arf^+/−, and Ink4a^−/− embryos (see Supplementary Fig. S9). (C) Growth curves of two independent wild-type (blue) and two independent bcat^sta (red) melanocyte cell lines. Note that these growth curves were established in the presence of TPA with cells that were immortalized for at least 6 mo. Each point is derived from the mean hemocytometer count of cells from three replicate dishes from two independent experiments. On day 0, 150,000 wild-type and bcat^sta cells were seeded. Cell numbers were determined 1, 3, 4, and 6 d after plating.

Figure 4.

bcat^sta promotes melanocyte immortalization in vitro. (A) Direct (2, 6, and 8 wk) and phase-contrast (16 wk) photomicrographs of wild-type and bcat^sta melanocyte colonies after explantation. The circle indicates a growing melanocyte colony surrounded by senescent cells. Bar, 100 μm. (B) Melanocyte growth during the first 24 wk in culture. (C) Acidic β-galactosidase is produced in a minority of bcat^sta melanocytes (arrow), 8 wk after seeding. The relative number (rel nb) of blue wild-type cells may be underestimated due to the high melanin content of some melanocytes.

Figure 5.

bcat^sta melanocyte lines do not express p16^INK4a. (A) Primary wild-type and bcat^sta melanocytes were processed for immunofluorescence microscopy using anti-Mitf and anti-p16^INK4a antibodies and DAPI staining 2, 6, and 20 wk after explantation. Arrows indicate Mitf-positive melanocytes and asterisks indicate nonmelanocytic Mitf-negative cells, which express p16^INK4a. Bar, 50 μm. Note that the cells were not passaged during the experiments. Morevover, wild-type melanocytes that were not dividing (big, flat, and hyperpigmented) remained p16^INK4a positive (not shown). (B) Expression of bcat^sta in culture 2, 6, and 20 wk after explantation. DNase-treated total RNA was subjected to RT–PCR analysis using oligonucleotides hybridizing to sequences around the intron sequence in the construct. Wild-type L14 and bcat^sta L10 melanocyte lines were used as controls. (C) Expression of p16^INK4a and p19^ARF in two wild-type (L9 and L14) and three independent bcat^sta (L10, L13, and L17) immortal melanocyte lines. Experiments were performed 6 mo after immortalization. Immunoblot analyses (top panel) and RT–PCR (bottom panel) were performed on identical amounts of protein and RNA from wild-type and bcat^sta cells. Murine 3T3 fibroblasts and melan-a melanocytes were used as controls. (D) RT–PCR analysis of p16^INK4a, p19^ARF, and hprt in wild-type (L14) and bcat^sta (L10) melanocytes treated with (+) or without (−) TPA for 3 d. The morphology of bcat^sta melanocytes (and of wild-type melanocytes, not shown) is greatly affected in the absence (−) of TPA, with cells becoming larger and flatter. The repression of p16^INK4a is reversible in bcat^sta melanocyte. Note that when TPA is removed from untransformed wild-type and bcat^sta melanocyte cultures, cells slowly stop growing.

Figure 6.

β-Catenin inhibits p16^INK4a transcription. (A) β-catenin/Lef-binding sites in mouse and human p16^INK4a promoters aligned with similar sites in mouse and human Mitf-M and Brn-2 genes. The Lef-binding site present in the p16^INK4a human promoter is the same as that published (Saegusa et al. 2006). It is in the same orientation as the Lef-binding sites of MITF-M but opposite to Brn-2 (Saito et al. 2002; Goodall et al. 2004). Mutated β-catenin/Lef sites (mut1 and mut2) of the p16^INK4a promoter used in B and D are indicated. (B) EMSA using p16^INK4a probe with LEF-1 protein produced in bacteria. The arrow indicates the specific p16^INK4a–Lef1 complex. Competitor oligonucleotides were used at 10 and 50 ng. (C) ChIP assays of β-catenin binding to the p16^INK4a promoter in 501Mel melanoma cells. (Top panel) ChIP assays are performed using antibody against β-catenin and analyzed after 25-cycle PCR (exponential phase). Brn2 promoter is used as a positive control and HSP70 as a negative control. (Lane 1) Input represents 0.4% of the input used for the ChIP. For each promoter, two negative controls (no Ab and IgG) are included. (Bottom panel) qPCR analysis of ChIP on p16^INK4a promoter using specific primers encompassing the Lef site and an antibody against β-catenin were performed in triplicate. No Ab, IgG, and the E-cadherin promoters are used as negative controls. All the data shown are representative of a minimum of two independent assays. (D, top) Diagram of the mouse p16^INK4a promoter coupled to the luciferase reporter gene. (Bottom) Activities of wild-type and mut2 p16^INK4a–luciferase reporters with various amounts (0, 0.2, 0.4, and 0.8 μg) of bcat^sta coexpressed in FO-1 cells, showing means of three independent experiments performed in duplicate; errors bars represent standard deviations. (E) siRNA-mediated down-regulation of β-catenin results in increased p16^INK4a expression. (F) Increasing amounts of bcat^sta inhibit endogenous p16^INK4a mRNA expression in human melanoma cell lines. Note that the cells were transfected when they were at low confluency.