ABSTRACT
Glycogen synthase kinase-3 beta (GSK-3ß) is overexpressed in a number of human malignancies and has been shown to contribute to tumor cell proliferation and survival. Although regulation of GSK-3ß activity has been extensively studied, the mechanisms governing GSK-3ß gene expression are still unknown. Using pancreatic cancer as a model, we find that constitutively active Ras signaling increases GSK-3ß gene expression via the canonical mitogen-activated protein kinase signaling pathway. Analysis of the mechanism revealed that K-Ras regulates the expression of this kinase through two highly conserved E-twenty six (ETS) binding elements within the proximal region. Furthermore, we demonstrate that mutant K-Ras enhances ETS2 loading onto the promoter, and ETS requires its transcriptional activity to increase GSK-3ß gene transcription in pancreatic cancer cells. Lastly, we show that ETS2 cooperates with p300 histone acetyltransferase to remodel chromatin and promote GSK-3ß expression. Taken together, these results provide a general mechanism for increased expression of GSK-3ß in pancreatic cancer and perhaps other cancers, where Ras signaling is deregulated.
Subject(s)
Genes, ras , Glycogen Synthase Kinase 3/genetics , Mutation , Pancreatic Neoplasms/genetics , RNA-Binding Protein EWS/genetics , p300-CBP Transcription Factors/genetics , Animals , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Immunohistochemistry , MAP Kinase Signaling System , Mice , Mice, Transgenic , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms.
Subject(s)
Apoptosis/drug effects , Hedgehog Proteins/antagonists & inhibitors , Kruppel-Like Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Apoptosis/genetics , Cell Line, Tumor , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/genetics , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/pathology , Drug Resistance, Neoplasm , Hedgehog Proteins/metabolism , Humans , Immunoblotting , Kruppel-Like Transcription Factors/genetics , Nerve Tissue Proteins/genetics , RNA, Small Interfering/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Up-Regulation , Zinc Finger Protein Gli3ABSTRACT
Transforming growth factor-beta (TGFbeta) plays a critical role in pancreatic development and cell proliferation. Binding of TGFbeta to its membrane receptor kinases activates the Smad signaling proteins, allowing them to translocate to the nucleus and participate in the transcriptional control of TGFbeta target genes. In addition, there is an increasing number of cellular mechanisms affecting the final response of a cell to TGFbeta. This includes crosstalk with other signaling pathways and the induction of TGFbeta early response genes, such as the TGFbeta-inducible early response gene (TIEG) family of transcription factors. Like the Smads, TIEGs behave as downstream effector proteins in TGFbeta-mediated pancreatic growth control. The discovery of the Smads and TIEGs has provided new insights into TGFbeta-regulated functions. Their significance in pancreatic development and cancer is discussed in this review.
ABSTRACT
The effect of dietary quercetin (Q) was evaluated in rats treated with nitrosomethylurea (NMU). Pancreatic nodules and focal acinar cell hyperplasias were observed in groups treated with NMU (87%) and Q-NMU (100%). Although rats with dysplastic foci (27%) were found in the NMU-treated group, Q-NMU treatment resulted in a significantly higher number of rats with dysplastic foci (73%). Furthermore, carcinomas in situ (12%) and one microcarcinoma (4%) were found in these animals. Mitosis was significantly increased and apoptosis was diminished in focal acinar cell hyperplasias of the Q-NMU group. Our present results support a promoting and progressing effect of quercetin in the NMU model of rat pancreatic carcinogenesis.
