Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3β was drastically down-regulated in human cutaneous squamous cell carcinomas and basal cell carcinomas. Due to its negative regulation of many oncogenic proteins, we hypothesized that GSK3β may function as a tumor suppressor during the neoplastic transformation of epidermal cells. We tested this hypothesis using an in vitro model system, JB6 mouse epidermal cells. In response to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the promotion-sensitive JB6 P+ cells initiate neoplastic transformation, whereas the promotion-resistant JB6 P− cells do not. JB6 P− cells expressed much higher levels of GSK3β than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3β. The activity of GSK3β is negatively regulated by its phosphorylation at Ser9. EGF and TPA induced strong Ser9 phoshorylation in JB6 P+ cells, but phosphorylation was seen at a much lesser extent in JB6 P− cells. EGF and TPA-stimulated Ser9 phosphorylation was mediated by phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C (PKC) pathways. Inhibition of GSK3β activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3β in JB6 P+ cells suppressed EGF and TPA-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient (K85R) GSK3β, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3β plays an important role in skin tumorigenesis. [Cancer Res 2007;67(16):7756–64]

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase that was first identified as a critical mediator in glycogen metabolism and insulin signaling. It is now known that GSK3β is a multifunctional kinase; more than 40 proteins are substrates of GSK3β, including transcription factors, cell cycle/survival regulators and oncogenic/proto-oncogenic proteins (1, 2). Unlike most protein kinases, GSK3β is constitutively active in resting cells and undergoes a rapid and transient inhibition in response to a number of external signals (3). GSK3β activity is regulated by site-specific phosphorylation. Full activity of GSK3β generally requires phosphorylation on Tyr216, and conversely, phosphorylation at Ser9 inhibits GSK3β activity. GSK3β is a negative regulator of Wnt/β-catenin signaling (1, 4). Because some oncogenic transcription factors [e.g., activator protein-1 (AP-1)] and proto-oncoproteins (i.e., β-catenin) are putative GSK3β substrates for phosphorylation-dependent inactivation (4), it has been hypothesized that GSK3β may interfere with cellular neoplastic transformation and tumor development. However, there are only limited studies examining the involvement of GSK3β in tumor development; the findings are sometimes contradictory (58). The role of GSK3β during tumorigenesis remains unclear. Carcinogenesis is a complex process that can be divided experimentally into three stages, namely, initiation, promotion, and progression. Initiation is associated with irreversible, carcinogen-mediated DNA mutation. In contrast, promotion is a reversible process in which there are increases in the rate of cell replication and/or alterations in gene expression. Progression represents the final genetic changes associated with the conversion of benign tumors into fully malignant cells. Skin cancer is the most common cancer worldwide (9). Our understanding of the mechanisms underlying the development and progression of skin tumors is still fragmentary. JB6 P+ mouse epidermal cells (Cl 41), originally derived from primary mouse epidermal cells, offer an excellent model to investigate the molecular events that are associated with tumor promotion. These cells undergo a response analogous to second-stage tumor promotion in mouse skin when treated with various tumor promoters. For example, exposure of JB6 P+ cells to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) induces the phenotype of anchorage-independent growth and tumorigenicity in vivo (1012). In contrast, JB6 P− cells are promotion resistant; EGF and TPA fail to initiate neoplastic transformation in these cells (10, 13). JB6 cells and their derivatives have been extensively used as an in vitro model for the promotion of neoplastic transformation (10, 1215). It has been shown that three signaling pathways are involved in the transformation of JB6 P+ cells, namely, phosphoinositide-3-kinase (PI3K)/Akt, PKC and mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1)/Erk (1316). Activation of these signaling pathways by EGF or TPA results in AP-1 transactivation, which is essential for the transformation of JB6 P+ cells (13, 14, 16). In this study, we compared the expression of GSK3β in human nonmelanoma skin cancers (cutaneous squamous cell carcinomas and basal cell carcinomas) to normal skin tissues. Using the JB6 cell system, we have investigated the role of GSK3β in neoplastic transformation and delineated the signal pathways that regulate GSK3β activity.

Materials and cell cultures. All antibodies except antiactin antibody were obtained from Cell Signaling Technology, Inc.. Antiactin antibody was purchased from Santa Cruz Biotechnology. PKC inhibitors (GF10203X or bisindolylmaleimide I and Go6976), PKA inhibitor (H89), GSK3β inhibitor (TDZD-8), and MEK1 inhibitor (PD98059) were purchased from Calbiochem. PI3K inhibitors (LY294002 and wortmannin), GSK3β inhibitor (SB216763), LiCl, and lactacystin were purchased from Sigma Chemical Co.. c-Jun-NH2-kinase (JNK) inhibitor (D-JNKI) was purchased from Alexis Biochemicals.

JB6 P+ mouse epidermal cell line (Cl 41), JB6 P− and transformed JB7 cells were grown in EMEM containing 10% fetal bovine serum (FBS), 2 mmol/L l-glutamine, and 25 μg/mL gentamicin at 37°C with 5% CO2. JB7 cells were derived from soft agar colonies of JB6 P+ treated with TPA for 3 weeks. These cells form colonies in soft agar and display tumorigenicity in vivo. The stable transfectants of JB6 P+ cells expressing AP-1-luciferase reporter (Cl 41 AP-1) have been previously described (17, 18).

Human skin samples and immunohistochemical study of GSK3β. Human skin tissues were obtained from surgical specimens at the Department of Dermatology, Xijing Hospital (Xi'an, China). The protocol for collecting human tissues was approved by the Ethical Committee of Xijing Hospital. The specimens were formalin fixed and paraffin embedded. The samples comprised of 31 primary cutaneous squamous cell carcinomas and 12 basal cell carcinomas. The median patient age was 61 years. Tumor diagnoses were established through pathologic evaluation of paraffin-embedded tissues stained with H&E. None of the patients received radiation or chemotherapy before the operation. Eight samples of histologically normal adult skin tissues were collected as controls.

Immunohistochemistry for GSK3β was done by the avidin-biotin indirect immunoperoxidase method. Briefly, 4-μm-thick sections were dewaxed, rehydrated, and incubated with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Sections were microwave treated in 0.01 mol/L citrate buffer (pH, 6.0) at 700 W for 10 min and rinsed with 0.01 mol/L TBS. Sections were incubated with normal horse serum for 20 min and then with primary antibodies (dilution 1:100) overnight at 4°C. After rinsing in TBS, sections were incubated with biotinylated secondary antibodies at room temperature for 30 min, followed by an avidin-biotin-peroxidase complex (Fisher Scientific) for 30 min. The reaction was visualized with 3,3′-diaminobenzidine tetrahydrochloride (0.5 mg/mL, Sigma Chemical Co.) supplemented with 0.01% hydrogen peroxide. Sections were counterstained with Harris hematoxylin.

Establishing stable transfectants. Stable transfectants expressing various GSK3β constructs were established as previously described (19). V5-tagged GSK3β constructs (wild-type, S9A, and K85R) carried by vector pcDNA3 were generous gifts from Dr. Thilo Hagen (University Hospital Nottingham). Cell transfection was carried out with LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Stable cell clones expressing exogenous GSK3β were screened by the treatment of G418 (600 μg/mL) for 3 to 4 weeks. Positive clones were verified by the expression of V5 as well as the evidence of GSK3β overexpression. The clones expressing the highest levels of exogenous GSK3β were selected for subsequent experiments.

Immunoblotting. The immunoblotting procedure to detect phosphorylation and expression of signal proteins was done as previously described (17). To control for the loading, the blots were stripped and reprobed with an antiactin antibody (Santa Cruz Biotechnology). In some cases, the density of immunoblotting was quantified with the software of Quantity One (Bio-Rad Laboratories).

Anchorage-independent growth. Anchorage-independent growth of JB6 P+, JB7 cells, and stable transfectants expressing various GSK3β constructs was determined by a previously described method (17). The cell growth matrix consists of two layers of basal medium Eagle (BME) agar in six-well culture trays. The base layer (2 mL) contained 10% FBS and 0.5% BME agar. The top layer (0.5 mL) contained 10% FBS, 0.33% BME agar, and the suspension of cells (0.5 × 104). EGF (10 ng/mL) or TPA (10 ng/mL) were applied in both top and bottom layers. The cultures were maintained at 37°C with an atmosphere of 5% CO2 for 10 to 14 days, and the number of induced cell colonies was counted under a microscope. Colonies containing eight or more cells were counted in four 0.5-cm2 areas randomly chosen with respect to distance from the center of the well, and the count was multiplied by the appropriate factor to give the colony number per well.

Tumorigenicity in nude mice. To evaluate in vivo tumorigenicity, 5-week-old male nude mice (BALB/c nu/nu, ∼25 g; Charles River Laboratories) were used. JB6 P+ cells and stable transfectants expressing various GSK3β constructs were treated with EGF or TPA (0 or 10 ng/mL) for 6 days and then dissociated from monolayer cultures by trypsinization. Cells were counted and centrifuged at 1,500 rpm for 5 min and resuspended in PBS. An aliquot of cells (5 × 106 in 100 μL of PBS) that were treated with EGF or TPA (0 or 10 ng/mL) was directly injected to both flanks of the animals. One injection per flank was done for each mouse. Eight animals were used for each treatment group. Mice were maintained in a pathogen-free environment; food and water were given ad libitum. Seven weeks after the initial injections, the length (L) and width (W) of the s.c. tumor mass were measured by calipers, and the tumor volume (TV) was calculated as described by Yaguchi et al. (20): TV = 0.5 × L × W2. At the end of the experiments, mice were sacrificed using a CO2 chamber. Animal housing and all procedures followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the West Virginia University Animal Care and Use Committee. Every effort was made to reduce the number of animals and their suffering.

Measurement of AP-1 activity. AP-1 transactivation in JB6 P+ epidermal cells was determined by assaying the activity of the luciferase reporter (17, 18). The assay accurately measures AP-1 transactivation (13, 18). Briefly, JB6 P+ cells expressing AP-1-luciferase reporter (Cl 41 AP-1) were cultured in 96-well plates and grown in a medium containing 10% FBS. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Subconfluent cultures were maintained in a medium containing 0.1% FBS for 24 h and treated with or without various protein kinase inhibitors 30 min before exposure to EGF or TPA. After treatment, cellular protein was extracted with a 1× lysis buffer supplied in the luciferase assay kit (Promega), and luciferase activity was measured with a monolight luminometer (3010, Analytical Luminescence Laboratory). AP-1 activity (luciferase activity) was calculated and expressed relative to the untreated cultures.

Statistical analysis. Differences among treatment groups were tested using an ANOVA. Differences in which P was < 0.05 were considered statistically significant. In cases where significant differences were detected, specific post hoc comparisons between treatment groups were examined with Student-Newman-Keuls tests.

Expression of GSK3β in human skin tissues. We first examined the expression of GSK3β and its phsophorylated forms [pGSK3β (Ser9 and Tyr216)] in histologically normal skin specimens. In all eight normal samples examined, a strong expression of GSK3β and pGSK3β (Ser9) was observed in the keratinocytes (Fig. 1A); the immunostaining of pGSK3β (Tyr216) was either very weak or negative. GSK3β and pGSK3β (Ser9) were also expressed in keratinocytes of patients with cutaneous squamous cell carcinomas or basal cell carcinomas; however, the immunostaining of GSK3β and pGSK3β (Ser9) in patients with skin carcinomas was generally weaker than that of age- and sex-matched normal subjects (Fig. 1B). In all tumor specimens (31 cases of primary cutaneous squamous cell carcinomas and 12 cases of basal cell carcinomas), cutaneous squamous cell carcinomas and basal cell carcinomas expressed much less GSK3β and pGSK3β (Ser9) than adjacent nontumor-bearing keratinocytes (Fig. 1B). The expression of pGSK3β (Tyr216) in the skin specimens of cancer patients was consistently negative (data not shown).

Figure 1.

Immunohistochemical study of GSK3β expression in normal human skin tissues and tumor-bearing tissues. A, the expression of GSK3β and phosphorylated forms (Ser9 and Tyr216) was examined in paraffin-embedded normal human skin tissues as described in Materials and Methods. a, expression of GSK3β. b, expression of phosphorylated GSK3β (Ser9) [pGSK3β (Ser9)]. c, expression of pGSK3β (Tyr216). d, human colon cancer tissues were used as a positive control for immunostaining of pGSK3β (Tyr216). B, a representative microphotograph shows GSK3β and pGSK3β (Ser9) immunostaining in tumor-bearing skin tissues and histologically normal skin tissues obtained from age- and sex-matched subjects. SCC, squamous cell carcinomas; BCC, basal cell carcinomas. Arrows, normal keratinocytes; arrowheads, tumor tissues. Bar, 50 μm.

Figure 1.

Immunohistochemical study of GSK3β expression in normal human skin tissues and tumor-bearing tissues. A, the expression of GSK3β and phosphorylated forms (Ser9 and Tyr216) was examined in paraffin-embedded normal human skin tissues as described in Materials and Methods. a, expression of GSK3β. b, expression of phosphorylated GSK3β (Ser9) [pGSK3β (Ser9)]. c, expression of pGSK3β (Tyr216). d, human colon cancer tissues were used as a positive control for immunostaining of pGSK3β (Tyr216). B, a representative microphotograph shows GSK3β and pGSK3β (Ser9) immunostaining in tumor-bearing skin tissues and histologically normal skin tissues obtained from age- and sex-matched subjects. SCC, squamous cell carcinomas; BCC, basal cell carcinomas. Arrows, normal keratinocytes; arrowheads, tumor tissues. Bar, 50 μm.

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Expression of GSK3β in JB7, JB6 P+ and P− cells. JB6 cells and their derivatives offer an excellent system to study the role of GSK3β in the transformation of epidermal cells. Because GSK3β was apparently down-regulated in skin carcinomas, we sought to compare the expression of GSK3β among tumor promotion-resistant JB6 P− cells, promotion-sensitive JB6 P+ cells, and transformed JB7 cells. Among these cells, JB7 had the least amount of GSK3β, whereas JB6 P− cells expressed the highest levels of GSK3β; the levels of GSK3β in JB6 P+ were intermediate (Fig. 2A). Cyclin D1 is a substrate of GSK3β; GSK3β may regulate the expression of cyclin D1 by transcriptional activation or controlling its degradation (21). The expression of cyclin D1 in these cells was inversely correlated to the levels of GSK3β (Fig. 2A). Two potent tumor promoters for JB6 cells, EGF and TPA, induced pGSK3β (Ser9; Fig. 2B). EGF produced a rapid (5 min) induction of pGSK3β (Ser9). TPA-mediated pGSK3β (Ser9) was slower but more sustained. EGF and TPA had little effect on pGSK3β (Tyr216; data not shown). Although JB6 P− cells expressed high levels of GSK3β (Fig. 2A), they were less sensitive to the induction of EGF and TPA of pGSK3β (Ser9; Fig. 2B), indicating that they were more resistant to negative regulation.

Figure 2.

Expression of GSK3β in JB6 cells and their derivatives and establishment of cells expressing ectopic GSK3β mutants. A, The expression of GSK3β and cyclin D1 in transformation-sensitive JB6 P+ cells, transformation-resistant JB6 P− cells, and transformed JB7 cells was examined with immunoblotting. B, JB6 P+ and P− cells cultured in serum-free media were treated with EGF (10 ng/mL) and TPA (10 ng/mL) for a specified period. The expression of phosphorylated GSK3β (Ser9) was examined with immunoblotting. C, establishment of JB6 cells stably expressing wild-type, S9A, and K85R GSK3β was carried out as described in Materials and Methods. The expression of V5, GSK3β, and cyclin D1 in these cells was examined with immunoblotting. D, JB6 cells expressing various GSK3β mutants were treated with EGF (10 ng/mL) for a specified period. The expression of phosphorylated GSK3β (Ser9) and cyclin D1 was examined with immunoblotting. E, JB6 cells expressing various GSK3β mutants were treated with lactacystin (Lac, 0 or 10 μmol/L) for 1 h. The expression of cyclin D1 was examined with immunoblotting.

Figure 2.

Expression of GSK3β in JB6 cells and their derivatives and establishment of cells expressing ectopic GSK3β mutants. A, The expression of GSK3β and cyclin D1 in transformation-sensitive JB6 P+ cells, transformation-resistant JB6 P− cells, and transformed JB7 cells was examined with immunoblotting. B, JB6 P+ and P− cells cultured in serum-free media were treated with EGF (10 ng/mL) and TPA (10 ng/mL) for a specified period. The expression of phosphorylated GSK3β (Ser9) was examined with immunoblotting. C, establishment of JB6 cells stably expressing wild-type, S9A, and K85R GSK3β was carried out as described in Materials and Methods. The expression of V5, GSK3β, and cyclin D1 in these cells was examined with immunoblotting. D, JB6 cells expressing various GSK3β mutants were treated with EGF (10 ng/mL) for a specified period. The expression of phosphorylated GSK3β (Ser9) and cyclin D1 was examined with immunoblotting. E, JB6 cells expressing various GSK3β mutants were treated with lactacystin (Lac, 0 or 10 μmol/L) for 1 h. The expression of cyclin D1 was examined with immunoblotting.

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GSK3β mediates the transformation of JB6 P+ cells. To determine whether GSK3β is involved in the transformation of JB6 P+ cells, we have established JB6 cells stably expressing wild-type (WT), S9A, or K85R GSK3β. S9A mutant is unable to be phosphorylated at Ser9 and, therefore, resistant to inhibitory regulation; K85R mutant is kinase deficient and functions as a dominant negative protein (6, 22). Overexpression of these exogenous GSK3β proteins was verified by immunoblotting using either anti-GSK3β or V5 antibody (Fig. 2C). The levels of cyclin D1 were correlated with the status of GSK3β expression. JB6 P+ cells overexpressing S9A expressed the least amount of cyclin D1, whereas cells overexpressing K85R had the highest expression (Fig. 2C). Cells overexpressing S9A were less sensitive to EGF-induced pGSK3β (Ser9) and up-regulation of cyclin D1 (Fig. 2D), indicating that these cells were more resistant to the negative regulation of GSK3β. In contrast, cells overexpressing K85R were more sensitive to EGF-induced up-regulation of cyclin D1 (Fig. 2D), verifying a dominant negative role of K85R mutant. GSK3β may regulate the expression of cyclin D1 by transcriptional activation or controlling its degradation (21). Lactacystin, a proteasome inhibitor, failed to reverse S9A-induced down-regulation of cyclin D1, whereas it enhanced K85R-mediated up-regulation of cyclin D1 (Fig. 2E). The results suggested that GSK3β regulated cyclin D1 expression in JB6 cells primarily through transcriptional control.

The effect of GSK3β on the transformation of JB6 P+ cells was first determined by anchorage-independent growth in soft agar. As shown in Fig. 3A, EGF and TPA stimulated the formation of cell colonies in soft agar. EGF- and TPA-induced cell colonies were significantly suppressed by the overexpression of WT and S9A GSK3β. In contrast, EGF- and TPA-induced anchorage-independent growth was drastically enhanced by the overexpression of K85R mutant. It was noted that JB6 P+ cells expressing K85R formed some small cell colonies (contained less than eight cells) in the absence of EGF and TPA. These small cell colonies were not scored. The results suggested that GSK3β was a negative regulator of cell transformation. JB7 cells formed colonies in soft agar (Fig. 3B). Two inhibitors of GSK3β (LiCl and SB216763) significantly enhanced anchorage-independent growth of JB7 cells. In contrast, overexpression of GSK3β in JB7 cells suppressed anchorage-independent growth (Fig. 3B). To further assess the role of GSK3β in tumorigenicity, we injected EGF- or TPA-exposed JB6 P+ cells expressing WT, S9A, or K85R GSK3β to nude mice and evaluated the formation of s.c. tumors. Cells were injected to both flanks for each mouse, and each flank received one injection. There were eight animals for each injection group. The number and the size of s.c. tumors were measured 7 weeks following initial injection. EGF- or TPA-exposed JB6 P+ cells expressing control vectors formed s.c. tumors in nude mice (Fig. 3C). For example, eight animals received injections of EGF-exposed JB6 P+ cells (each animals received two injections; one in each flank), a total of 11 tumors formed, and the average volume of each tumor was 1157 ± 105 mm3 (Table 1). However, in the eight animals injected with EGF-exposed cells expressing WT or S9A GSK3β, only three and two tumors formed, respectively. In addition, the average volume of tumors was significantly smaller. The tumor volumes in animals injected with cells expressing WT or S9A GSK3β were 632 ± 79 and 478 ± 81 mm3, respectively. In contrast, EGF-exposed cells overexpressing K85R showed enhanced tumorigenicity in nude mice; a total of 14 tumors formed, and the average volume of each tumor was 3756 ± 279 mm3 (Table 1). Similar results were obtained with the treatment of TPA (Table 1). It was noted that three small tumors (368 ± 65 mm3) formed in the nude mice that received an injection of cells expressing K85R that was not exposed to EGF or TPA. Thus, the results obtained from the nude mice agreed with anchorage-independent growth.

Figure 3.

Role of GSK3β in the transformation of JB6 P+ cells in vitro and in vivo. A, anchorage-independent growth of JB6 P+ cells expressing various GSK3β mutants. JB6 P+ cells stably expressing wild-type, S9A, and K85R GSK3β, which were grown in a matrix of soft agar, were exposed to EGF (0 or 10 ng/mL) or TPA (0 or 10 ng/mL). Cell colonies were scored after 14 d of incubation at 37°C in an atmosphere of 5% CO2 as described in Materials and Methods. The number of soft agar colonies/104 cells in the untreated control was arbitrarily designated as 1. The numbers of colonies in experimental groups were expressed as an arbitrary unit relative to the untreated control group. The experiment was replicated four times. *, P < 0.05 denotes a statistically significant difference from untreated controls. #, P < 0.05 denotes a statistically significant difference from EGF- or TPA-treated JB6 cells expressing the control vector. B, role of GSK3β in anchorage-independent growth of JB7 cells. The effect of GSK3β inhibitors (LiCl, 20 mmol/L; and SB216763, 10 μmol/L) on anchorage-independent growth of JB7 cells or JB7 cells overexpressing wild-type GSK3β (JB7/GSK3) was evaluated as described above. *, P < 0.05, statistically significant difference from untreated JB6 P+ and JB7/GSK3 cells. #, P < 0.05, statistically significant difference from JB7 cells. C, influence of GSK3β on tumorigenesis in nude mice. Nude mice were s.c. inoculated with JB6 P+ cells and their derivatives stably expressing wild-type, S9A, and K85R GSK3β constructs. The cells were treated with EGF or TPA (0 or 10 ng/mL) for 6 d. For each experimental group, there were eight animals. Seven weeks following inoculation, the number of s.c. tumor masses in each animal was scored, and the volume of tumors was measured by calipers as described in Materials and Methods. A representative photo shows s.c. tumors induced by EGF treatment in nude mice. 1, JB6; 2, JB6 + EGF; 3, S9A; 4, S9A + EGF; 5, K85R; 6, K85R + EGF. TPA-induced tumors were not shown. Arrows, s.c. tumors.

Figure 3.

Role of GSK3β in the transformation of JB6 P+ cells in vitro and in vivo. A, anchorage-independent growth of JB6 P+ cells expressing various GSK3β mutants. JB6 P+ cells stably expressing wild-type, S9A, and K85R GSK3β, which were grown in a matrix of soft agar, were exposed to EGF (0 or 10 ng/mL) or TPA (0 or 10 ng/mL). Cell colonies were scored after 14 d of incubation at 37°C in an atmosphere of 5% CO2 as described in Materials and Methods. The number of soft agar colonies/104 cells in the untreated control was arbitrarily designated as 1. The numbers of colonies in experimental groups were expressed as an arbitrary unit relative to the untreated control group. The experiment was replicated four times. *, P < 0.05 denotes a statistically significant difference from untreated controls. #, P < 0.05 denotes a statistically significant difference from EGF- or TPA-treated JB6 cells expressing the control vector. B, role of GSK3β in anchorage-independent growth of JB7 cells. The effect of GSK3β inhibitors (LiCl, 20 mmol/L; and SB216763, 10 μmol/L) on anchorage-independent growth of JB7 cells or JB7 cells overexpressing wild-type GSK3β (JB7/GSK3) was evaluated as described above. *, P < 0.05, statistically significant difference from untreated JB6 P+ and JB7/GSK3 cells. #, P < 0.05, statistically significant difference from JB7 cells. C, influence of GSK3β on tumorigenesis in nude mice. Nude mice were s.c. inoculated with JB6 P+ cells and their derivatives stably expressing wild-type, S9A, and K85R GSK3β constructs. The cells were treated with EGF or TPA (0 or 10 ng/mL) for 6 d. For each experimental group, there were eight animals. Seven weeks following inoculation, the number of s.c. tumor masses in each animal was scored, and the volume of tumors was measured by calipers as described in Materials and Methods. A representative photo shows s.c. tumors induced by EGF treatment in nude mice. 1, JB6; 2, JB6 + EGF; 3, S9A; 4, S9A + EGF; 5, K85R; 6, K85R + EGF. TPA-induced tumors were not shown. Arrows, s.c. tumors.

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Table 1.

Name and volume of tumor (mm3) in nude mice

Control, n (volume, mm3)EGF, n (volume, mm3)TPA, n (volume, mm3)
JB6 0 (0) 11 (1,157 ± 105) 11 (902 ± 112) 
WT 0 (0) 3 (632 ± 79)* 4 (424 ±67)* 
S9A 0 (0) 2 (478 ± 81)* 2 (358 ± 56)* 
K85R 3 (368 ± 65)* 14 (3,756 ± 279)* 15 (2,371 ± 199)* 
Control, n (volume, mm3)EGF, n (volume, mm3)TPA, n (volume, mm3)
JB6 0 (0) 11 (1,157 ± 105) 11 (902 ± 112) 
WT 0 (0) 3 (632 ± 79)* 4 (424 ±67)* 
S9A 0 (0) 2 (478 ± 81)* 2 (358 ± 56)* 
K85R 3 (368 ± 65)* 14 (3,756 ± 279)* 15 (2,371 ± 199)* 

NOTE: The number and the mean volume of subcutaneous tumors were calculated.

*

P < 0.05, denotes a statistically significant difference from parental JB6 P+ cells treated with EGF or TPA.

PKC and PI3K/Akt mediate EGF- and TPA-induced inactivation of GSK3β. Previous studies show that EGF and TPA activated PI3K/Akt, PKC, and MEK1/Erk pathways in JB6 P+ cells (13, 14, 23). These pathways are potential upstream components of GSK3β. We sought to determine whether EGF- and TPA-induced inactivation of GSK3β was mediated by these signaling pathways. Both EGF and TPA induced phosphorylation of Akt and PKC (Fig. 4A). The profiles of EGF- and TPA-mediated phosphorylation, however, were different. EGF-induced Akt phosphorylation was rapid (5 min) and strong, whereas TPA-mediated Akt phosphorylation was modest and more persistent. As detected by an antibody recognizing phosphorylated panPKC, EGF-induced phosphorylation of panPKC was weaker than that induced by TPA. The profiles of EGF- and TPA-induced pGSK3β (Ser9) were also different. EGF induced a rapid and strong phosphorylation of GSK3β (Ser9), the maximal phosphorylation occurred between 5 and 15 min after EGF treatment. TPA-mediated phosphorylation of GSK3β (Ser9) was gradual; it became evident at 15 min, and maximal phosphorylation occurred at 3 to 6 h. Inhibitors of PI3K blocked EGF-stimulated pGSK3β (Ser9) in JB6 P+ cells; an inhibitor of PKC also decreased EGF-stimulated pGSK3β (Ser9), but to a lesser extent (Fig. 4B and C), suggesting that the PI3K/Akt pathway played a major role in EGF regulation of pGSK3β (Ser9). On the other hand, inhibitor of PKC was more effective than the PI3K inhibitor in down-regulating TPA-induced pGSK3β (Ser9). The inhibitors for MEK1, JNK, and PKA had little effect on EGF- and TPA-induced pGSK3β (Ser9; Fig. 4B and C). These results indicated that PI3K/Akt and PKC mediated EGF- and TPA-induced pGSK3β (Ser9), although the profiles of regulation were different.

Figure 4.

Signaling pathways that regulate EGF- and TPA-induced pGSK3β (Ser9). A, JB6 P+ cells, cultured in serum-free media, were treated with EGF (10 ng/mL) and TPA (10 ng/mL) for a specified period. The expression of phosphorylated Akt, PKC, and GSK3β (Ser9) was examined with immunoblotting. The phosphorylated Akt was detected with an antibody recognizing pAkt (Thr308); the phosphorylated PKC was detected with an antibody recognizing p-panPKC. B, JB6 P+ cells were pretreated with inhibitors for 30 min and exposed to EGF or TPA for a specified period. The expression of pGSK3β (Ser9) was examined with immunoblotting. PD, PD98059, 50 μmol/L, MEK1 inhibitor; JNKi, 1 μmol/L, inhibitor for JNK; H89, 10 μmol/L, inhibitor for PKA; Bis, bisindolylmaleimide I, 1 μmol/L, inhibitor for PKC; LY, LY294002, 10 μmol/L, inhibitor for PI3K. C, the relative amounts of pGSK3β (Ser9) were measured microdensitometrically. The experiment was replicated thrice. D, effect of EGF on the phosphorylation of ERK in JB6 P+ cells expressing various GSK3β mutants. JB6 P+ cells stably expressing wild-type, S9A, and K85R GSK3β, which were grown in serum-free media, were exposed to EGF (0 or 10 ng/mL) for a specified period. The expression of phosphorylated ERK was determined by immunoblotting.

Figure 4.

Signaling pathways that regulate EGF- and TPA-induced pGSK3β (Ser9). A, JB6 P+ cells, cultured in serum-free media, were treated with EGF (10 ng/mL) and TPA (10 ng/mL) for a specified period. The expression of phosphorylated Akt, PKC, and GSK3β (Ser9) was examined with immunoblotting. The phosphorylated Akt was detected with an antibody recognizing pAkt (Thr308); the phosphorylated PKC was detected with an antibody recognizing p-panPKC. B, JB6 P+ cells were pretreated with inhibitors for 30 min and exposed to EGF or TPA for a specified period. The expression of pGSK3β (Ser9) was examined with immunoblotting. PD, PD98059, 50 μmol/L, MEK1 inhibitor; JNKi, 1 μmol/L, inhibitor for JNK; H89, 10 μmol/L, inhibitor for PKA; Bis, bisindolylmaleimide I, 1 μmol/L, inhibitor for PKC; LY, LY294002, 10 μmol/L, inhibitor for PI3K. C, the relative amounts of pGSK3β (Ser9) were measured microdensitometrically. The experiment was replicated thrice. D, effect of EGF on the phosphorylation of ERK in JB6 P+ cells expressing various GSK3β mutants. JB6 P+ cells stably expressing wild-type, S9A, and K85R GSK3β, which were grown in serum-free media, were exposed to EGF (0 or 10 ng/mL) for a specified period. The expression of phosphorylated ERK was determined by immunoblotting.

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Like PKC and PI3K/Akt, ERKs are critical regulators of the transformation of JB6 cells (13). MEK1 inhibitor (PD98059) did not affect EGF- and TPA-mediated pGSK3β (Ser9; Fig. 4B and C), suggesting that ERK was not involved in GSK3β inactivation in JB6 cells. Reversely, GSK3β may inhibit ERK activation (24). We therefore sought to determine whether GSK3β affected ERK activation in JB6 P+ cells. As shown in Fig. 4D, EGF elicited similar phosphorylation of ERK in cells expressing WT, S9A, and K85R GSK3β; furthermore, two inhibitors of GSK3β (LiCl and TDZD8) failed to affect ERK phosphorylation (data not shown), indicating that GSK3β was not involved in ERK activation.

GSK3β is involved in the regulation of AP-1. It has been shown that AP-1 activity is essential for the transformation of JB6 P+ cells (12, 13, 23). We sought to determine whether GSK3β regulates the activation of AP-1. As shown in Fig. 5A, two inhibitors of GSK3β (LiCl and TDZD8) stimulated the basal as well as EGF- and TPA-induced activation of AP-1. In contrast, inhibitors for PI3K and PKC decreased EGF- and TPA-induced activation of AP-1. Furthermore, we showed that overexpression of wild-type and S9A GSK3β significantly inhibited AP-1 activity; in contrast, down-regulation of GSK3β by small interfering RNA (siRNA) enhanced AP-1 activity (Fig. 5B). Together, these results indicated that GSK3β was a negative regulator of AP-1.

Figure 5.

Role of GSK3β in AP-1 transactivation. A, JB6 P+ epidermal cells stably expressing AP-1 luciferase reporter were pretreated with two GSK3β inhibitors, LiCl (20 mmol/L) and TDZD8 (10 μmol/L) or PI3K inhibitor (LY, 10 μmol/L) and PKC inhibitor (Bis, 1 μmol/L) for 30 min and then exposed to EGF (10 ng/mL) or TPA (10 ng/mL) for 12 h. The activity of AP-1 was measured by a luciferase assay as described in Materials and Methods. The activity of AP-1 was expressed relative to untreated cultures. The experiment was replicated thrice. *, P < 0.05, statistically significant difference from untreated JB6 P+ cells. #, P < 0.05, statistically significant difference from paired EGF- or TPA-treated JB6 cells. B, JB6 P+ epidermal cells stably expressing AP-1 luciferase reporter were transfected with either wild-type, S9A-mutated GSK3β constructs or an siRNA for GSK3β for 48 h. The activity of AP-1 was measured as described above. The experiment was replicated thrice. *, P < 0.05, statistically significant difference from cells transfected with an empty vector.

Figure 5.

Role of GSK3β in AP-1 transactivation. A, JB6 P+ epidermal cells stably expressing AP-1 luciferase reporter were pretreated with two GSK3β inhibitors, LiCl (20 mmol/L) and TDZD8 (10 μmol/L) or PI3K inhibitor (LY, 10 μmol/L) and PKC inhibitor (Bis, 1 μmol/L) for 30 min and then exposed to EGF (10 ng/mL) or TPA (10 ng/mL) for 12 h. The activity of AP-1 was measured by a luciferase assay as described in Materials and Methods. The activity of AP-1 was expressed relative to untreated cultures. The experiment was replicated thrice. *, P < 0.05, statistically significant difference from untreated JB6 P+ cells. #, P < 0.05, statistically significant difference from paired EGF- or TPA-treated JB6 cells. B, JB6 P+ epidermal cells stably expressing AP-1 luciferase reporter were transfected with either wild-type, S9A-mutated GSK3β constructs or an siRNA for GSK3β for 48 h. The activity of AP-1 was measured as described above. The experiment was replicated thrice. *, P < 0.05, statistically significant difference from cells transfected with an empty vector.

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GSK3β is implicated in many biological processes, including embryonic development, cell differentiation, and cell survival/cell cycle control (4, 25). Because many oncogenic transcription factors (e.g., c-Jun, c-Myc) and proto-oncoproteins (i.e., β-catenin) are substrates of GSK3β for phosphorylation-dependent inactivation (4), GSK3β may play a role in oncogenesis. However, information regarding the involvement of GSK3β in tumorigenesis is limited, and the function of GSK3β during cell transformation and cancer progression remains unclear.

Here, we show a decreased expression of GSK3β and pGSK3β (Ser9) in human nonmelanoma skin cancers (cutaneous squamous cell carcinomas and basal cell carcinomas) compared with adjacent normal keratinocytes. In addition, the immunostaining for GSK3β and pGSK3β (Ser9) in keratinocytes of patients with cutaneous squamous cell carcinomas or basal cell carcinomas is generally weaker than keratinocytes of age- and sex-matched normal subjects. The decreased immunostaining for pGSK3β (Ser9) likely results from the down-regulation of total GSK3β expression. Mouse epidermal (JB6) cells have been extensively used as an in vitro model for studying the promotion of neoplastic transformation (10, 1215). Consistent with the observations in human skin tissues, the expression levels of GSK3β in JB6 cells are correlated to the stage or potential of cell transformation. JB7 cells, the transformed derivatives of JB6, have the least amount of GSK3β, whereas promotion-resistant JB6 P− cells express the highest levels of GSK3β; the levels of GSK3β in promotion-sensitive JB6 P+ cells are intermediate. In addition, tumor promoters EGF and TPA induce strong phosphorylation of GSK3β at Ser9 in JB6 P+ cells, indicating an inactivation of GSK3β. On the other hand, JB6 P− cells are much less responsive to the negative regulation of GSK3β. The involvement of GSK3β in skin tumorigenesis is further shown by the modulation of GSK3β activity in JB6 P+ cells. Overexpression of WT and S9A mutant GSK3β inhibits EGF- and TPA-mediated anchorage-independent growth in soft agar, as well as in vivo tumorigenicity in nude mice. In contrast, overexpression of a kinase-deficient K85R GSK3β drastically potentiates EGF- and TPA-mediated anchorage-independent growth and greatly enhances in vivo tumorigenicity in response to EGF. Overexpression of a kinase-deficient K85R GSK3β also has a modest promoting effect on anchorage-independent growth in soft agar and in vivo tumorigenicity of JB6 P+ cells in the absence of EGF. These results verify that GSK3β negatively regulates epidermal cell transformation, and modulating GSK3β activity is sufficient to affect cell transformation.

We further show that GSK3β is a negative regulator of AP-1 transactivation in JB6 P+ cells; the inhibitors of GSK3β increase basal as well as EGF- and TPA-mediated AP-1 transactivation. This result is consistent with previous findings using other cells, which show that GSK3β activation results in the inhibition of AP-1 activity (26, 27). AP-1 is a heterodimeric transcription factor complex composed of a Jun family member and a FOS family member that binds the TRE DNA sequence (5′-TGAGTCA-3′). It is involved in a variety of cellular processes, including growth, apoptosis, and differentiation (28). The mouse epidermal multistage carcinogenesis model provides a well-defined system for examining the transformation of squamous epithelial cells to benign squamous papillomas and their subsequent progression into squamous cell carcinomas (29). With this carcinogenesis model system, a number of studies show that AP-1 activity is required for skin tumorigenesis as well as malignant transformation (3033). These findings are confirmed by in vitro studies that show that AP-1 activity is essential for the transformation of JB6 P+ cells (12, 13, 23). Three signaling pathways, namely, PI3K/Akt, PKC, and MEK1/Erk, have been shown to regulate EGF- and TPA-induced AP-1 transactivation and transformation of JB6 P+ cells (13, 14, 23). We show here that PI3K/Akt and PKC are upstream of GSK3β, and both EGF- and TPA-stimulated pGSK3β (Ser9) is mediated by PI3K/Akt and PKC. Thus, suppressing GSK3β activity is one of the mechanisms for EGF- and TPA-induced AP-1 transactivation (Fig. 6). The interaction between Erk and GSK3β has been previously reported (24, 34); however, we fail to find such interaction in JB6 cells. Based on these findings, we propose a signal cascade in which GSK3β plays an important role in regulating AP-1 activity and cell transformation (Fig. 6): EGF and TPA activate PI3K/Akt, PKC, and MEK1/Erk pathways. Activation of PI3K/Akt and PKC results in the inhibition of GSK3β, which is a negative regulator of AP-1. In the meantime, EGF- and TPA-mediated ERK activation can also cause AP-1 transactivation. Hyperactivity of AP-1 initiates the transformation process. In addition to negatively regulating AP-1 activity, GSK3β is a well-known inhibitor for Wnt/β-catenin signaling (1). Hyperactivity of the Wnt/β-catenin signaling pathway is associated with the development of a number of tumors as well as malignancies (3539). In terms of skin tumors, Wnt signaling is mainly implicated in the development and progression of melanomas (40). From the present study, however, it is not clear whether Wnt/β-catenin signaling is also involved in GSK3β-induced suppression of skin tumorigenesis.

Figure 6.

Diagram of the role of GSK3β in skin tumorigenesis. EGF and TPA activate PI3K/Akt and PKC, which induce phosphorylation of GSK3β (Ser9) and inactivate GSK3β. Inactivation of GSK3β stimulates oncogenic transcription factors, such as AP-1. EGF and TPA can also activate AP-1 through the MEK1/Erk pathway. Hyperactivity of AP-1 promotes skin tumorigenesis.

Figure 6.

Diagram of the role of GSK3β in skin tumorigenesis. EGF and TPA activate PI3K/Akt and PKC, which induce phosphorylation of GSK3β (Ser9) and inactivate GSK3β. Inactivation of GSK3β stimulates oncogenic transcription factors, such as AP-1. EGF and TPA can also activate AP-1 through the MEK1/Erk pathway. Hyperactivity of AP-1 promotes skin tumorigenesis.

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Other studies also support a role of GSK3β as a “tumor suppressor.” For example, expression of a kinase-inactive GSK3β in adult mouse mammary glands promotes mammary tumorigenesis, indicating that antagonism of GSK3β activity is oncogenic for mammary epithelial cells (6). In a mouse epidermal multistage carcinogenesis model, Leis et al. (41) show a dramatic increase in pGSK3β (Ser9) in late papillomas and squamous cell carcinomas. Furthermore, a significant decrease in pGSK3β (Tyr216) is observed in squamous cell carcinoma samples (41), indicating an inactivation of GSK3β during mouse skin carcinogenesis. Together, these observations support the notion that GSK3β is a negative regulator of skin tumorigenesis; down-regulation or inactivation of GSK3β is oncogenic for epidermal cells.

The mechanisms underlying skin tumorigenesis are complex and involve interactions among multiple signal cascades and various transcription factors. Our study clearly shows that GSK3β is an important component in the cascades, and modulation of GSK3β expression/activity is sufficient to alter the transformation potential of epidermal cells. Thus, GSK3β is a target for developing prevention/intervention strategies. GSK3β has emerged as an attractive therapeutic target for the treatment of multiple neurologic diseases such as Alzheimer's and stroke. Lithium has been used as a mood stabilizer to treat bipolar mood disorder, and other inhibitors of GSK3β have entered clinical trials for diabetes. The potential role of these inhibitors in tumorigenesis should be considered.

Grant support: NIH (AA015407), the National Natural Science Foundation of China (30470544, 30471452, and 30570580), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars that is sponsored by the State Education Ministry. Dr. Z.-J. Ke was also supported by the One Hundred Talents Program of the Chinese Academy of Sciences.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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