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The Role of Glycogen Synthase Kinase 3B in the Transformation of Epidermal Cells

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The Role of Glycogen Synthase Kinase 3B in the Transformation of Epidermal Cells Research Article The Role of Glycogen Synthase Kinase 3B in the Transformation of Epidermal Cells Cuiling Ma,1,3 Jian Wang,4 Ying Gao,3 Tian-Wen Gao,3 Gang Chen,1 Kimberly A. Bower,1 Mohammed Odetallah,1 Min Ding,1,2 Zunji Ke,5 and Jia Luo1,5 1Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center; 2National Institute for Occupational Safety and Health, Morgantown, West Virginia; Departments of 3Dermatology and 4Obstetrics and Gynecology, Xijing Hospital, Xian, P.R. China; and 5Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China Abstract number of external signals (3). GSK3h activity is regulated by site- B B specific phosphorylation. Full activity of GSK3h generally requires Glycogen synthase kinase 3 (GSK3 ) is a multifunctional 216 serine/threonine kinase. We showed that the expression of phosphorylation on Tyr , and conversely, phosphorylation at 9 h h GSK3B was drastically down-regulated in human cutaneous Ser inhibits GSK3 activity. GSK3 is a negative regulator of Wnt/ h squamous cell carcinomas and basal cell carcinomas. Due -catenin signaling (1, 4). Because some oncogenic transcription to its negative regulation of many oncogenic proteins, we factors [e.g., activator protein-1 (AP-1)] and proto-oncoproteins h h hypothesized that GSK3B may function as a tumor suppressor (i.e., -catenin) are putative GSK3 substrates for phosphorylation- h during the neoplastic transformation of epidermal cells. We dependent inactivation (4), it has been hypothesized that GSK3 tested this hypothesis using an in vitro model system, JB6 may interfere with cellular neoplastic transformation and tumor mouse epidermal cells. In response to epidermal growth factor development. However, there are only limited studies examining h (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the the involvement of GSK3 in tumor development; the findings h promotion-sensitive JB6 P+ cells initiate neoplastic transfor- are sometimes contradictory (5–8). The role of GSK3 during mation, whereas the promotion-resistant JB6 PÀ cells do not. tumorigenesis remains unclear. Carcinogenesis is a complex JB6 PÀ cells expressed much higher levels of GSK3B than JB6 process that can be divided experimentally into three stages, P+ cells; JB7 cells, the transformed derivatives of JB6, had the namely, initiation, promotion, and progression. Initiation is least amount of GSK3B. The activity of GSK3B is negatively associated with irreversible, carcinogen-mediated DNA mutation. regulated by its phosphorylation at Ser9. EGF and TPA induced In contrast, promotion is a reversible process in which there are strong Ser9 phoshorylation in JB6 P+ cells, but phosphoryla- increases in the rate of cell replication and/or alterations in gene tion was seen at a much lesser extent in JB6 PÀ cells. EGF expression. Progression represents the final genetic changes and TPA-stimulated Ser9 phosphorylation was mediated by associated with the conversion of benign tumors into fully phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C malignant cells. Skin cancer is the most common cancer worldwide (PKC) pathways. Inhibition of GSK3B activation significantly (9). Our understanding of the mechanisms underlying the stimulated activator protein-1 (AP-1) activity. Overexpression development and progression of skin tumors is still fragmentary. of wild-type (WT) and S9A mutant GSK3B in JB6 P+ cells JB6 P+ mouse epidermal cells (Cl 41), originally derived from suppressed EGF and TPA-mediated anchorage-independent primary mouse epidermal cells, offer an excellent model to growth in soft agar and tumorigenicity in nude mice. Over- investigate the molecular events that are associated with tumor expression of a kinase-deficient (K85R) GSK3B, in contrast, promotion. These cells undergo a response analogous to second- potentiated anchorage-independent growth and drastically stage tumor promotion in mouse skin when treated with various enhanced in vivo tumorigenicity. Together, these results tumor promoters. For example, exposure of JB6 P+ cells to indicate that GSK3B plays an important role in skin epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13- tumorigenesis. [Cancer Res 2007;67(16):7756–64] acetate (TPA) induces the phenotype of anchorage-independent growth and tumorigenicity in vivo (10–12). In contrast, JB6 PÀ cells Introduction are promotion resistant; EGF and TPA fail to initiate neoplastic transformation in these cells (10, 13). JB6 cells and their derivatives h h Glycogen synthase kinase 3 (GSK3 ) is a serine/threonine have been extensively used as an in vitro model for the promotion kinase that was first identified as a critical mediator in glycogen h of neoplastic transformation (10, 12–15). It has been shown that metabolism and insulin signaling. It is now known that GSK3 is three signaling pathways are involved in the transformation of JB6 a multifunctional kinase; more than 40 proteins are substrates of P+ cells, namely, phosphoinositide-3-kinase (PI3K)/Akt, PKC and GSK3h, including transcription factors, cell cycle/survival regu- mitogen-activated protein (MAP)/extracellular signal-regulated lators and oncogenic/proto-oncogenic proteins (1, 2). Unlike most kinase (ERK) kinase 1 (MEK1)/Erk (13–16). Activation of these protein kinases, GSK3h is constitutively active in resting cells signaling pathways by EGF or TPA results in AP-1 transactivation, and undergoes a rapid and transient inhibition in response to a which is essential for the transformation of JB6 P+ cells (13, 14, 16). In this study, we compared the expression of GSK3h in human nonmelanoma skin cancers (cutaneous squamous cell carcinomas Requests for reprints: Jia Luo, West Virginia University School of Medicine, P.O. and basal cell carcinomas) to normal skin tissues. Using the JB6 Box 9177, Morgantown, WV 26506. Phone: 304-293-7208; Fax: 304-293-7823; E-mail: cell system, we have investigated the role of GSK3h in neoplastic [email protected]. I2007 American Association for Cancer Research. transformation and delineated the signal pathways that regulate doi:10.1158/0008-5472.CAN-06-4665 GSK3h activity. Cancer Res 2007; 67: (16). August 15, 2007 7756 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. GSK3b Regulates the Transformation of Epidermal Cells j Materials and Methods 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 Materials and cell cultures. 2 All antibodies except antiactin antibody containing eight or more cells were counted in four 0.5-cm areas were obtained from Cell Signaling Technology, Inc.. Antiactin antibody was randomly chosen with respect to distance from the center of the well, and purchased from Santa Cruz Biotechnology. PKC inhibitors (GF10203X or the count was multiplied by the appropriate factor to give the colony h bisindolylmaleimide I and Go6976), PKA inhibitor (H89), GSK3 inhibitor number per well. (TDZD-8), and MEK1 inhibitor (PD98059) were purchased from Calbio- Tumorigenicity in nude mice. To evaluate in vivo tumorigenicity, h chem. PI3K inhibitors (LY294002 and wortmannin), GSK3 inhibitor 5-week-old male nude mice (BALB/c nu/nu, f25 g; Charles River (SB216763), LiCl, and lactacystin were purchased from Sigma Chemical Laboratories) were used. JB6 P+ cells and stable transfectants expressing Co.. c-Jun-NH2-kinase (JNK) inhibitor (D-JNKI) was purchased from Alexis various GSK3h constructs were treated with EGF or TPA (0 or 10 ng/mL) for Biochemicals. 6 days and then dissociated from monolayer cultures by trypsinization. À JB6 P+ mouse epidermal cell line (Cl 41), JB6 P and transformed JB7 Cells were counted and centrifuged at 1,500 rpm for 5 min and resuspended cells were grown in EMEM containing 10% fetal bovine serum (FBS), in PBS. An aliquot of cells (5 Â 106 in 100 AL of PBS) that were treated with A j 2 mmol/L L-glutamine, and 25 g/mL gentamicin at 37 C with 5% CO2. JB7 EGF or TPA (0 or 10 ng/mL) was directly injected to both flanks of the cells were derived from soft agar colonies of JB6 P+ treated with TPA for animals. One injection per flank was done for each mouse. Eight animals 3 weeks. These cells form colonies in soft agar and display tumorigenicity were used for each treatment group. Mice were maintained in a pathogen- in vivo . The stable transfectants of JB6 P+ cells expressing AP-1-luciferase free environment; food and water were given ad libitum. Seven weeks after reporter (Cl 41 AP-1) have been previously described (17, 18). the initial injections, the length (L) and width (W) of the s.c. tumor mass B Human skin samples and immunohistochemical study of GSK3 . were measured by calipers, and the tumor volume (TV) was calculated as Human skin tissues were obtained from surgical specimens at the described by Yaguchi et al. (20): TV = 0.5 Â L Â W2. At the end of the Department of Dermatology, Xijing Hospital (Xi’an, China). The protocol experiments, mice were sacrificed using a CO2 chamber. Animal housing for collecting human tissues was approved by the Ethical Committee of and all procedures followed the NIH Guide for the Care and Use of Xijing Hospital. The specimens were formalin fixed and paraffin embedded. Laboratory Animals and were approved by the West Virginia University The samples comprised of 31 primary cutaneous squamous cell carcinomas Animal Care and Use Committee. Every effort was made to reduce the and 12 basal cell carcinomas. The median patient age was 61 years. Tumor number of animals and their suffering. diagnoses were established through pathologic evaluation of paraffin- Measurement of AP-1 activity. AP-1transactivationinJB6P+ embedded tissues stained with H&E. None of the patients received radiation epidermal cells was determined by assaying the activity of the luciferase or chemotherapy before the operation.
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