Spleen tyrosine kinase functions as a tumor suppressor in melanoma cells by inducing senescence-like growth arrest

Abstract

Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation-mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated beta-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity.

Figure 1. Reintroduction of Syk in melanoma cells abrogates colony formation, growth in 3D culture and migration

(A) The indicated melanoma cell lines were transfected with a puromycin resistance vector and pEF-V5-wt-Syk expression vector or control empty vector (pEF). Stable puromycin-resistant colonies were stained and counted. Colony growth of cells transfected with control vector is normalized to 100% for each cell lines. Columns, mean of triplicate of a representative experiment; bars, SD. Representative plates are shown. A fraction of transfected cells was lysed and analyzed by immunoblotting for V5-tagged Syk expression. (B) Spheroids from WM793 cells infected with Syk-expressing (Ad-Syk) or empty vector adenoviruses were formed in liquid overlay cultures with agarose coat preventing cell adhesion to culture dishes. After 3 days, spheroids were viewed using a phase-contrast microscope and the diameter (μm) of at least 13 separate spheroids was determined. *, P < 0.001, Student’s t test. Representative examples of spheroids are shown. Immunoblots show expression of Syk in spheroids transduced with control or with Syk-expressing adenoviruses. (C) Transwell migration and invasion assays of A375 infected with Syk-expressing or empty vector adenoviruses. The values are the mean ± SD of three independent experiments. *, P < 0.01, Student’s t test.

Figure 1. Reintroduction of Syk in melanoma cells abrogates colony formation, growth in 3D culture and migration

(A) The indicated melanoma cell lines were transfected with a puromycin resistance vector and pEF-V5-wt-Syk expression vector or control empty vector (pEF). Stable puromycin-resistant colonies were stained and counted. Colony growth of cells transfected with control vector is normalized to 100% for each cell lines. Columns, mean of triplicate of a representative experiment; bars, SD. Representative plates are shown. A fraction of transfected cells was lysed and analyzed by immunoblotting for V5-tagged Syk expression. (B) Spheroids from WM793 cells infected with Syk-expressing (Ad-Syk) or empty vector adenoviruses were formed in liquid overlay cultures with agarose coat preventing cell adhesion to culture dishes. After 3 days, spheroids were viewed using a phase-contrast microscope and the diameter (μm) of at least 13 separate spheroids was determined. *, P < 0.001, Student’s t test. Representative examples of spheroids are shown. Immunoblots show expression of Syk in spheroids transduced with control or with Syk-expressing adenoviruses. (C) Transwell migration and invasion assays of A375 infected with Syk-expressing or empty vector adenoviruses. The values are the mean ± SD of three independent experiments. *, P < 0.01, Student’s t test.

Figure 2. Syk suppresses colony growth independently of its two tandem SH2 domains

(A) Functional domains of Syk and schematic of the mutants. (B) Control immunoblot shows expression of the V5-tagged Syk wt and mutants in transiently transfected A375 cells. (C) Cells were transfected with empty vector (pEF), Syk wt, the SH2 deleted (ΔSH2), the SH2 mutated (SH2D) or the Kinase Dead (KD) Syk expression vector. Colony formation was analyzed as described in the legend of Fig. 1. Representative plates are shown. *, P < 0.001, Student’s t tests.

Figure 3. Syk reduces the growth of melanoma cells and induces a G0/G1 cell cycle arrest

(A) Proliferation curves of control and Syk-expressing cells. A375 cells were infected with control adenovirus or with adenoviruses expressing Syk for the indicated times. Result is from two independent experiments performed in triplicate, shown with SD. (B) Immunoblots of cellular lysates corresponding to cells transduced with control or with Syk-expressing adenoviruses. (C) Cell-cycle distribution of Syk-expressing and control populations as determined by BrdU incorporation and DNA-content flow cytometry analysis. Four days post-infection, cells were pulsed with BrdU and stained with anti-BrdU-APC to detect BrdU incorporation (vertical axis) and 7-AAD to detect total DNA (horizontal axis). The upper box indicates cells in S phase, the lower-left box identifies G0/G1 cells, and the lower-right box displays cells in G2/M. The percentage of cells in the different phases of the cell cycle is indicated. (D) Expression of Syk decreases S phase population. Percentage of BrdU positive cells in A375 transduced with control or Syk expressing adenoviruses. Data are mean ± SD of three separate experiments.

Figure 4. Syk triggers senescence-like phenotypes

(A) Phase-contrast images of A375 cells infected for 6 days with empty vector or with adenovirus encoding Syk. Syk-transduced cells show a large and flat morphology with increased cytoplasmic granularity. The figure shown is a typical representation of the experiment that was performed at least five times (B) Induction of SA-β-gal activity in A375 cells by Syk. Cells were fixed and stained for SA-β-gal (pH 6.0) after 6 days of infection. Upper panels are low-magnification images and lower panels are high-magnification images. (C) Percentage of SA-β-gal-positive cells 7 days post-infection. At least 200 cells were scored from multiple randomly selected fields. Values represent the mean ± SD of three independent measurements. (D) Syk induces senescence-associated heterochromatin foci (SAHF). A375 cells were stained by immunofluorescence with anti-Syk (red) and anti-HP1β (green) 6 days post-infection and viewed under a fluorescence microscope. The percentage of SAHF positive cells is indicated in insert. Numbers given are representative of three independent experiments.

Figure 5. Syk regulates expression and activity of senescence-associated markers

(A) Lysates from A375 infected for 4 days with empty vector or with adenovirus encoding Syk were subjected to immunoblotting with the antibodies indicated on the left. (B) Immunofluorescent staining of vector- and Syk-expressing A375 cells. After 4 days of infection, cells were stained with anti-p53 antibody (green), anti-Syk antibody (red) or DAPI (blue). A treatment for 4 hr with bleomycin was used as positive control. Boxed regions correspond to larger magnifications of representative cells. The percentage of Syk-positive cells showing nuclear labeling of p53 is indicated in insert and is representative of three independent experiments.

Figure 6. Induction of the cdk inhibitor p21 by Syk expression is p53 dependent

(A) A375 cells infected with empty vector or with adenovirus encoding Syk for 2 days were incubated with pifithrin α (PFTα) (20 μg/ml) or DMSO as a vehicle control for additional 2 days. Cell lysates were tested for Syk, p53, Phospho (Ser15) p53, p21 and ERK2 by immunoblotting. (B) A375 cells were transfected with p53-siRNA or luciferase-siRNA (control). 6 hr later, cells were infected with empty vector or with adenovirus encoding Syk for 4 days. A treatment with bleomycin was used as positive control. Total cell lysates were subjected to immunoblotting analysis as in A). (C) MeWo (p53 defective) human melanoma cells were infected with empty vector or with adenovirus encoding Syk for 4 days. Cell lysates were tested for Syk, p53, p21 and ERK2 by immunoblotting.