Expression,localization, and activity of glycogen synthase kinase 3beta during mouse skin tumorigenesis

Glycogen synthase kinase 3 (GSK-3) is a protein kinase that plays essential roles in the control of several developmental, metabolic, and apoptotic processes. Owing to its negative actions on several oncogenic insults, it has been considered a putative functional tumor suppressor. We studied the expression, activity, and localization of GSK-3beta during the process of chemically induced two-stage mouse skin carcinogenesis and also in the tumors generated upon subcutaneous injection of Akt-transformed keratinocytes. We found that GSK-3 activity was downregulated at the later stages of promotion by tyrosine 216 dephosphorylation and serine 9 phosphorylation. The data obtained with Akt-transformed keratinocytes clearly suggested the involvement of Akt in serine 9 phosphorylation of GSK-3beta. Finally, besides functional inactivation, significant basal activity of GSK-3beta was detected in all cases, indicating that this enzyme provides essential functions to malignant keratinocytes.     

Adiponectin modulates the glycogen synthase kinase-3beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice

Adiponectin is an adipokine that has pleiotropic beneficial roles in systemic insulin resistance and inflammation. Several recent clinical studies suggest that low serum levels of adiponectin are associated with increased risks of breast cancer. Here, we investigated the direct effects of adiponectin on breast cancer development in vitro and in vivo. Our results showed that adiponectin significantly attenuated the proliferations of two typical human breast cancer cells, MDA-MB-231 and T47D, in a cell type-specific manner. Further analysis revealed that adiponectin could induce apoptosis and arrest the cell cycle progression at G(0)-G(1) phase in MDA-MB-231 cells. Prolonged treatment with adiponectin in this cell line blocked serum-induced phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), suppressed intracellular accumulation of beta-catenin and its nuclear activities, and consequently reduced expression of cyclin D1. Adiponectin-mediated suppression of cyclin D1 expression and attenuation of cell proliferation was abrogated by the GSK-3beta inhibitor lithium chloride. These results suggest that the inhibitory role of adiponectin on MDA-MB-231 cell growth might be attributed to its suppressive effects on the GSK-3beta/beta-catenin signaling pathway. Furthermore, our in vivo study showed that both supplementation of recombinant adiponectin and adenovirus-mediated overexpression of this adipokine substantially reduced the mammary tumorigenesis of MDA-MB-231 cells in female nude mice. Taken together, these data support the role of adiponectin as a negative regulator of breast cancer development and also suggest that adiponectin might represent a novel therapeutic target for this disease.     

Glycogen synthase kinase 3beta (GSK3beta) in tumorigenesis and cancer chemotherapy

This study aimed to analyse (i) the metastatic behaviour of human melanoma FEMX-1 cells in scid mice after surgical excision of the PT and (ii) to evaluate the feasibility of magnetic resonance imaging (MRI) for the detection of melanoma metastases. Histology proved both high specificity (95%), and high sensitivity of MRI detection of melanoma metastasis. CEACAM1, L1, and HPA-binding site expression, all markers predicting metastasis in clinical studies, were preserved in the metastatic nodules. Thus, our xenograft model closely resembles the clinical situation of post-operative development of distant organ metastasis and demonstrates that MRI is a sensitive and highly qualified technology for intra-vital monitoring of melanoma progression.     

Oncogene-mediated inhibition of glycogen synthase kinase 3 beta impairs degradation of prolactin receptor

Prolactin receptors (PRLr) expressed in a majority of breast cancer are activated by prolactin and growth hormone. The PRLr is commonly stabilized in human breast cancer due to decreased phosphorylation of residue Ser(349), which, when phosphorylated, recruits the beta Trcp E3 ubiquitin ligase and facilitates PRLr degradation. Here, we show that constitutive oncogenic signaling downstream of ErbB2 and Ras stabilizes PRLr via inhibitory phosphorylation of glycogen synthase kinase-3beta (GSK3 beta) on Ser(9). Importantly, inactivation of GSK3 beta correlates with elevated levels of PRLr protein in clinical human breast cancer specimens. Additional studies using pharmacologic, biochemical, and genetic approaches reveal that GSK3 beta is a bona fide PRLr kinase that phosphorylates PRLr on Ser(349) and is required for the recognition of PRLr by beta Trcp, as well as for PRLr ubiquitination and degradation.     

Role of glycogen synthase kinase 3beta in rapamycin-mediated cell cycle regulation and chemosensitivity

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.     

The role of glycogen synthase kinase 3beta in the transformation of epidermal cells

Glycogen synthase kinase 3beta (GSK3beta) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3beta 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 GSK3beta 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 GSK3beta than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3beta. The activity of GSK3beta 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 GSK3beta activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3beta 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) GSK3beta, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3beta plays an important role in skin tumorigenesis.     

The glycogen synthase kinase (GSK) 3beta represses RNA polymerase I transcription

Several oncogenic proteins and tumour suppressors target the RNA polymerase I and interfere with rRNA synthesis. Here, we show that the glycogen synthase kinase (GSK) 3beta, which phosphorylates the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), is selectively enriched in nucleoli of RAS-transformed cells. Immunoprecipitation and chromatin immunoprecipitation assays performed on epithelial and endothelial cells transformed with oncogenic RAS show that GSK3beta and PTEN are part of the same complex and associate with promoter and coding region of the rDNA. An active GSK3beta mutant abolished nucleolar BrUTP incorporation and associated with the member of the selectivity factor 1 complex TAF(I)110. Finally, GSK3beta inhibition upregulated 45S, 18S and 28S rRNA synthesis in RAS-transformed epithelial cells as revealed by semiquantitative real-time PCR and promoted cellular proliferation. Our results underscore a repressive function for GSK3beta in rRNA biogenesis supporting its role as a tumour supressor.     

Inhibition of glycogen synthase kinase 3 beta attenuates neurocognitive dysfunction resulting from cranial irradiation

There are now more than 10 million cancer survivors in the United States. With these numbers, chronic sequelae that result from cancer therapy have become a major health care problem. Although radiation therapy of the brain has improved cancer cure rates, learning disorders and memory deficits are a common consequence of this therapy. Here we show that glycogen synthase kinase 3beta (GSK-3beta) is required for radiation-induced hippocampal neuronal apoptosis and subsequent neurocognitive decline. Inhibition of GSK-3beta either by small molecules (SB216763 or SB415286) or by ectopic expression of kinase-inactive GSK-3beta before irradiation significantly attenuated radiation-induced apoptosis in hippocampal neurons. GSK-3beta inhibition with SB216763 or SB415286 also decreased apoptosis in the subgranular zone of the hippocampus in irradiated mice, leading to improved cognitive function in irradiated animals. Studies of the molecular mechanisms of the cytoprotective effect showed that GSK-3beta activity in hippocampal neurons was not significantly altered by radiation, pointing to the indirect involvement of this enzyme in radiation-induced apoptosis. At the same time, radiation led to increased accumulation of p53, whereas inhibition of the basal level of GSK-3beta activity before radiation prevented p53 accumulation, suggesting a possible mechanism of cytoprotection by GSK-3beta inhibitors. These findings identify GSK-3beta signaling as a key regulator of radiation-induced damage in hippocampal neurons and suggest that GSK-3beta inhibitors may have a therapeutic role in protecting both pediatric and adult cancer patients and may help to improve quality of life in cancer survivors.     

BP75, bromodomain-containing M(r) 75,000 protein,binds dishevelled-1 and enhances Wnt signaling by inactivating glycogen synthase kinase-3 beta

To identify novel regulators of Wnt signaling, we performed yeast two-hybrid analyses with Dvl-1 and identified BP75 as a candidate. Here, we demonstrated that BP75 directly interacts with Dvl-1 in mammalian cells and enhances TCF-dependent gene expression induced by Dvl-1. In support of these results, BP75 in cooperation with Dvl-1 was found to facilitate dephosphorylation at Tyr216 of glycogen synthase kinase-3beta and consequently inhibit its kinase activity. Furthermore, the nuclear translocation and formation of vesicular structures of beta-catenin were induced by BP75 and Dvl-1 in a synergistic manner. Collectively, these results provided us a novel mechanism in Wnt signaling where BP75 plays important regulatory roles between glycogen synthase kinase-3beta and Dvl.     

Wnt signaling and gastrointestinal tumorigenesis in mouse models

The canonical Wnt signaling plays important roles in embryonic development and tumorigenesis. For the latter, induced mutations in mice have greatly contributed to our understanding of the molecular mechanisms of cancer initiation and progression. Here, I will review recent reports on gastrointestinal cancer model mice, with an emphasis on the roles of the Wnt signal pathway. They include: mouse models for familial adenomatous polyposis; modifying factors that affect mouse intestinal polyposis, including the genes that help cancer progression; Wnt target genes that affect mouse intestinal polyposis; and a mouse model of gastric cancer that mimics Helicobacter pyroli infection.     

Detection of active fraction of glycogen synthase kinase 3beta in cancer cells by nonradioisotopic in vitro kinase assay

Glycogen synthase kinase 3beta (GSK3beta) is a well-known marker and potential therapeutic target in non-insulin-dependent diabetes mellitus and Alzheimer's disease. Our recent demonstration that GSK3beta has a previously unrecognized role in colorectal cancer facilitates the development of a nonradioisotopic in vitro kinase assay (NRIKA) for detecting GSK3beta activity in gastrointestinal cancer cells. The NRIKA uses a sequential combination of immunoprecipitations to isolate GSK3beta in sample cells' lysates, and an in vitro kinase reaction that uses recombinant beta-catenin protein (substrate) and nonradioisotopic ATP, followed by immunoblotting to detect beta-catenin phosphorylated in serine 33, 37 and/or threonine 41 residues. The NRIKA detected higher expression of active GSK3beta in stomach, colon, pancreas and liver cancer cell lines than in human embryonic kidney cells (HEK293) considered nonneoplastic. Inhibition of cancer cell-derived GSK3beta activity by GSK3beta inhibitors (SB-216763, AR-A014418) was detected by the NRIKA. GSK3beta inhibition attenuated survival and proliferation and induced apoptosis in all types of cancer cells but not in HEK293. These findings supported the idea that the pathologic roles of GSK3beta are definite and common in various types of cancer. The NRIKA provides a basis for evolving a high-throughput tool for testing substances for GSK3beta inhibition, and for screening and identifying novel GSK3beta inhibitors with a view to discovering drugs for treatment of cancer as well as non-insulin-dependent diabetes mellitus and Alzheimer's disease.     

Protein kinase B/Akt-dependent phosphorylation of glycogen synthase kinase-3beta in irradiated vascular endothelium

The vascular endothelium plays a critical role in the response of cancer to ionizing radiation. Activation of the phosphoinositide-3-kinase/Akt pathway is one initial signaling event in irradiated endothelial cells. Specifically, a low dose of ionizing radiation (3 Gy) induces phosphorylation of Akt at Ser473 in the vascular endothelium within minutes of irradiation. However, signaling events that are downstream of Akt have not been well defined. Here, we show that phosphorylation of the Akt downstream target glycogen synthase kinase-3beta (GSK-3beta) at Ser9 also occurred within minutes of exposure to ionizing radiation. In addition, ionizing radiation caused the dissociation of GSK-3beta from the cell membrane, consistent with the inactivation of GSK-3beta enzyme activity. Overexpression of the dominant negative mutant Akt attenuated GSK-3beta phosphorylation at Ser9 and enhanced radiation-induced apoptosis. X-irradiated endothelial cells formed capillaries in both in vitro and in vivo models, whereas overexpression of the dominant negative mutant Akt inhibited capillary tubule formation. Studies using GSK-3beta antagonists showed that GSK-3beta activity was required for apoptosis in endothelial cells treated simultaneously with Akt antagonists and radiation. In mouse vascular models, radiation-induced microvascular destruction in response to Akt antagonists also required GSK-3beta function. These data indicate that on exposure of vascular endothelium to ionizing radiation, activation of Akt signaling contributes to GSK-3beta inhibition, which in turn promotes endothelial cell survival and capillary formation. Thus, pharmacologic regulation of Akt/GSK-3beta signaling may present a new approach to the radiation response in the tumor microvasculature.     

LRP5/6 in Wnt signaling and tumorigenesis

The canonical Wnt signaling pathway is frequently overactivated in several types of human cancer. Defects in different components of the Wnt signaling pathway promote tumorigenesis and tumor progression. Accordingly, the pathway has been intensely studied to understand its importance in cancer biology and as therapeutic target. Recent studies have found that the low-density lipoprotein receptor-related protein (LRP)5 and 6 are essential Wnt coreceptors and interact with several key components of the Wnt signaling pathway. Furthermore, it has been demonstrated that LRP5 and 6 are potential oncogenic proteins. Thus, blockade of LRP56 function may lead to new strategies in cancer treatment.     

Cripto-1 induces phosphatidylinositol 3'-kinase-dependent phosphorylation of AKT and glycogen synthase kinase 3beta in human cervical carcinoma cells.

Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3beta (GSK-3beta)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3beta. Phosphorylation of AKT and GSK-3beta by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3beta.     

Transforming acidic coiled coil 1 promotes transformation and mammary tumorigenesis

Transforming acidic coiled coil 1 (TACC1) is a putative oncogene located within a breast cancer amplicon found on human chromosome 8p11. Although TACC1 has been reported to transform fibroblasts, it is also down-regulated in a subset of mammary tumors treated with anthracyclin. Here, we show that ectopic TACC1 overexpression can cooperate with Ras to induce focus formation in murine fibroblast cultures and prevent death caused by overexpression of Pten or a dominant-negative form of protein kinase B (PKB)/Akt. In transgenic mice carrying TACC1 under the control of the mouse mammary tumor virus promoter, TACC1 expression reduced apoptosis during mammary gland involution, increased the penetrance of mammary tumors in a pten+/- background, and decreased the average age of mammary tumor onset in a mouse model based on a phosphatidylinositol 3'-kinase (PI3K)-decoupled mutant of polyoma middle T. Elevated levels of both phospho-PKB and phospho-extracellular signal-regulated kinase were found in mammary tissue containing the TACC1 transgene. Thus, TACC1 positively regulates the Ras and PI3K pathways, promotes Ras-mediated transformation, and prevents apoptosis induced by PI3K pathway inhibition. TACC1 also cooperates with tumorigenic mutations in the PI3K pathway and thereby plays an oncogenic role in tumor formation in the murine mammary gland.