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1.
Gastroenterology ; 166(2): 298-312.e14, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37913894

ABSTRACT

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Gemcitabine , Cell Line, Tumor , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , Mitogen-Activated Protein Kinases/metabolism , Smad3 Protein/metabolism
2.
Gut ; 71(12): 2561-2573, 2022 12.
Article in English | MEDLINE | ID: mdl-35365570

ABSTRACT

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease. DESIGN: NFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a . and NFATc1Δ/Δ ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo. RESULTS: NFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration. CONCLUSION: NFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.


Subject(s)
Non-alcoholic Fatty Liver Disease , Mice , Animals , Non-alcoholic Fatty Liver Disease/metabolism , Liver/metabolism , Hepatocytes/metabolism , Transcription Factors/metabolism , Inflammation/metabolism , Mice, Transgenic , Disease Progression , Mice, Inbred C57BL , NFATC Transcription Factors/metabolism
3.
Cells ; 10(12)2021 12 08.
Article in English | MEDLINE | ID: mdl-34943970

ABSTRACT

BACKGROUND: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in PDAC progression. METHODS: NFATc1 and EZH2 mRNA and protein expression and complex formation were determined in transgenic PDAC models and human PDAC specimens. NFATc1 binding on the Ezh2 gene and the consequences of perturbed NFATc1 expression on Ezh2 transcription were explored by Chromatin Immunoprecipitation (ChIP) and upon transgenic or siRNA-mediated interference with NFATc1 expression, respectively. Integrative analyses of RNA- and ChIP-seq data was performed to explore NFATc1-/EZH2-dependent gene signatures. RESULTS: NFATc1 targets the Ezh2 gene for transcriptional activation and biochemically interacts with the methyltransferase in murine and human PDAC. Surprisingly, our genome-wide binding and expression analyses do not link the protein complex to joint gene regulation. In contrast, our findings provide evidence for chromatin-independent functions of the NFATc1:EZH2 complex and reveal posttranslational EZH2 phosphorylation at serine 21 as a prerequisite for robust complex formation. CONCLUSION: Our findings disclose a previously unknown NFATc1-EZH2 axis operational in the pancreas and provide mechanistic insights into the conditions fostering NFATc1:EZH2 complex formation in PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , NFATC Transcription Factors/genetics , Pancreatic Neoplasms/genetics , Animals , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation/genetics , Chromatin/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic/genetics , Homeodomain Proteins/genetics , Humans , Mice , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/pathology , Protein Processing, Post-Translational/genetics , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Small Interfering/genetics , Trans-Activators/genetics
4.
Gastroenterology ; 142(2): 388-98.e1-7, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22079596

ABSTRACT

BACKGROUND & AIMS: Transcriptional silencing of the p15(INK4b) tumor suppressor pathway overcomes cellular protection against unrestrained proliferation in cancer. Here we show a novel pathway involving the oncogenic transcription factor nuclear factor of activated T cells (NFAT) c2 targeting a p15(INK4b)-mediated failsafe mechanism to promote pancreatic cancer tumor growth. METHODS: Immunohistochemistry, real-time polymerase chain reaction, immunoblotting, and immunofluorescence microscopy were used for expression studies. Cancer growth was assessed in vitro by [(3)H]thymidine incorporation, colony formation assays, and in vivo using xenograft tumor models. Protein-protein interactions, promoter regulation, and local histone modifications were analyzed by immunoprecipitation, DNA pull-down, reporter, and chromatin immunoprecipitation assays. RESULTS: Our study uncovered induction of NFATc2 in late-stage pancreatic intraepithelial neoplasia lesions with increased expression in tumor cell nuclei of advanced cancers. In the nucleus, NFATc2 targets the p15(INK4b) promoter for inducible heterochromatin formation and silencing. NFATc2 binding to its cognate promoter site induces stepwise recruitment of the histone methyltransferase Suv39H1, causes local H3K9 trimethylation, and allows docking of heterochromatin protein HP1γ to the repressor complex. Conversely, inactivation of NFATc2 disrupts this repressor complex assembly and local heterochromatin formation, resulting in restoration of p15(INK4b) expression and inhibition of pancreatic cancer growth in vitro and in vivo. CONCLUSIONS: Here we describe a novel mechanism for NFATc2-mediated gene regulation and identify a functional link among its repressor activity, the silencing of the suppressor pathway p15(INK4b), and its pancreatic cancer growth regulatory functions. Thus, we provide evidence that inactivation of oncogenic NFATc2 might be an attractive strategy in treatment of pancreatic cancer.


Subject(s)
Adenocarcinoma/metabolism , Biomarkers, Tumor/metabolism , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Heterochromatin/metabolism , NFATC Transcription Factors/metabolism , Pancreatic Neoplasms/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Biomarkers, Tumor/genetics , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , Cyclin-Dependent Kinase Inhibitor p15/genetics , Female , Gene Silencing , Humans , Mice , Mice, Nude , NFATC Transcription Factors/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Random Allocation , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
5.
J Biol Chem ; 286(33): 28761-28771, 2011 Aug 19.
Article in English | MEDLINE | ID: mdl-21628454

ABSTRACT

The aminobisphosphonate zoledronic acid has elicited significant attention due to its remarkable anti-tumoral activity, although its detailed mechanism of action remains unclear. Here, we demonstrate the existence of a nuclear GSK-3ß-NFATc2 stabilization pathway that promotes breast and pancreatic cancer growth in vitro and in vivo and serves as a bona fide target of zoledronic acid. Specifically, the serine/threonine kinase GSK-3ß stabilizes nuclear NFATc2 through phosphorylation of the serine-rich SP2 domain, thus protecting the transcription factor from E3-ubiquitin ligase HDM2-mediated proteolysis. Zoledronic acid disrupts this NFATc2 stabilization pathway through two mechanisms, namely GSK-3ß inhibition and induction of HDM2 activity. Upon nuclear accumulation, HDM2 targets unphosphorylated NFATc2 for ubiquitination at acceptor lysine residues Lys-684/Lys-897 and hence labels the factor for subsequent proteasomal degradation. Conversely, mutagenesis-induced constitutive serine phosphorylation (Ser-215, Ser-219, and Ser-223) of the SP2 domain prevents NFATc2 from HDM2-mediated ubiquitination and degradation and consequently rescues cancer cells from growth suppression by zoledronic acid. In conclusion, this study demonstrates a critical role of the GSK-3ß-HDM2 signaling loop in the regulation of NFATc2 protein stability and growth promotion and suggests that double targeting of this pathway is responsible, at least to a significant part, for the potent and reliable anti-tumoral effects of zoledronic acid.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Nucleus/metabolism , Diphosphonates/pharmacology , Imidazoles/pharmacology , NFATC Transcription Factors/metabolism , Neoplasm Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Animals , Bone Density Conservation Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Nucleus/genetics , Female , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mice , Mice, Nude , NFATC Transcription Factors/genetics , Neoplasm Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Stability , Protein Structure, Tertiary , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Ubiquitination/drug effects , Ubiquitination/genetics , Zoledronic Acid
6.
J Biol Chem ; 285(35): 27241-27250, 2010 Aug 27.
Article in English | MEDLINE | ID: mdl-20516082

ABSTRACT

Transforming growth factor beta (TGF-beta) has a dual role in carcinogenesis, acting as a growth inhibitor in early tumor stages and a promoter of cell proliferation in advanced diseases. Although this cellular phenomenon is well established, the underlying molecular mechanisms remain elusive. Here, we report that sequential induction of NFAT and c-Myc transcription factors is sufficient and required for the TGF-beta switch from a cell cycle inhibitor to a growth promoter pathway in cancer cells. Mechanistically, TGF-beta induces in a calcineurin-dependent manner the expression and activation of NFAT factors, which then translocate into the nucleus to promote c-Myc expression. In response to TGF-beta, activated NFAT factors bind to and displace Smad3 repressor complexes from the previously identified TGF-beta inhibitory element (TIE) to transactivate the c-Myc promoter. c-Myc in turn stimulates cell cycle progression and growth through up-regulation of D-type cyclins. Most importantly, NFAT knockdown not only prevents c-Myc activation and cell proliferation, but also partially restores TGF-beta-induced cell cycle arrest and growth suppression. Taken together, this study provides the first evidence for a Smad-independent master regulatory pathway in TGF-beta-promoted cell growth that is defined by sequential transcriptional activation of NFAT and c-Myc factors.


Subject(s)
Cell Cycle , NFATC Transcription Factors/metabolism , Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription, Genetic , Transforming Growth Factor beta/metabolism , Animals , Calcineurin/genetics , Calcineurin/metabolism , Cell Line, Tumor , Humans , Mice , NFATC Transcription Factors/genetics , Neoplasms/genetics , Proto-Oncogene Proteins c-myc/genetics , Smad Proteins/genetics , Smad Proteins/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Transforming Growth Factor beta/pharmacology
7.
Gastroenterology ; 138(3): 1189-99.e1-2, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19900447

ABSTRACT

BACKGROUND & AIMS: Induction of immediate early transcription factors (ITF) represents the first transcriptional program controlling mitogen-stimulated cell cycle progression in cancer. Here, we examined the transcriptional mechanisms regulating the ITF protein c-Myc and its role in pancreatic cancer growth in vitro and in vivo. METHODS: Expression of ITF proteins was examined by reverse-transcription polymerase chain reaction and immunoblotting, and its implications in cell cycle progression and growth was determined by flow cytometry and [(3)H]-thymidine incorporation. Intracellular Ca(2+) concentrations, calcineurin activity, and cellular nuclear factor of activated T cells (NFAT) distribution were analyzed. Transcription factor complex formations and promoter regulation were examined by immunoprecipitations, reporter gene assays, and chromatin immunoprecipitation. Using a combination of RNA interference knockdown technology and xenograft models, we analyzed the significance for pancreatic cancer tumor growth. RESULTS: Serum promotes pancreatic cancer growth through induction of the proproliferative NFAT/c-Myc axis. Mechanistically, serum increases intracellular Ca(2+) concentrations and activates the calcineurin/NFAT pathway to induce c-Myc transcription. NFAT binds to a serum responsive element within the proximal promoter, initiates p300-dependent histone acetylation, and creates a local chromatin structure permissive for the inducible recruitment of Ets-like gene (ELK)-1, a protein required for maximal activation of the c-Myc promoter. The functional significance of this novel pathway was emphasized by impaired c-Myc expression, G1 arrest, and reduced tumor growth upon NFAT depletion in vitro and in vivo. CONCLUSIONS: Our study uncovers a novel mechanism regulating cell growth and identifies the NFAT/ELK complex as modulators of early stages of mitogen-stimulated proliferation in pancreatic cancer cells.


Subject(s)
Adenocarcinoma/metabolism , Cell Proliferation , Chromatin Assembly and Disassembly , Histones/metabolism , NFATC Transcription Factors/metabolism , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Acetylation , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Binding Sites , Blotting, Western , Calcineurin/metabolism , Calcium/metabolism , Cell Cycle , Cell Line, Tumor , Chromatin Immunoprecipitation , Flow Cytometry , Humans , Immunohistochemistry , Male , Mice , Mice, Nude , NFATC Transcription Factors/genetics , Neoplasm Transplantation , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Serum/metabolism , Serum Response Element , Signal Transduction , Time Factors , Transcription, Genetic , Transfection , ets-Domain Protein Elk-1/metabolism , p300-CBP Transcription Factors/metabolism
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