Garima Singh

NFAT-Induced Histone Acetylation Relay Switch Promotes c-Myc-Dependent Growth in Pancreatic Cancer Cells

BACKGROUND & AIMSnInduction 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.nnnMETHODSnExpression 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.nnnRESULTSnSerum 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.nnnCONCLUSIONSnOur 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.

Sequential Activation of NFAT and c-Myc Transcription Factors Mediates the TGF-β Switch from a Suppressor to a Promoter of Cancer Cell Proliferation

Transforming growth factor β (TGF-β) 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-β switch from a cell cycle inhibitor to a growth promoter pathway in cancer cells. Mechanistically, TGF-β 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-β, activated NFAT factors bind to and displace Smad3 repressor complexes from the previously identified TGF-β 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-β-induced cell cycle arrest and growth suppression. Taken together, this study provides the first evidence for a Smad-independent master regulatory pathway in TGF-β-promoted cell growth that is defined by sequential transcriptional activation of NFAT and c-Myc factors.

Restricted Heterochromatin Formation Links NFATc2 Repressor Activity With Growth Promotion in Pancreatic Cancer

BACKGROUND & AIMSnTranscriptional 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.nnnMETHODSnImmunohistochemistry, 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.nnnRESULTSnOur 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.nnnCONCLUSIONSnHere 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.

Inflammation-Induced NFATc1–STAT3 Transcription Complex Promotes Pancreatic Cancer Initiation by KrasG12D

UNLABELLEDnCancer-associated inflammation is a molecular key feature in pancreatic ductal adenocarcinoma. Oncogenic KRAS in conjunction with persistent inflammation is known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood. Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in pancreatic epithelial cells to promote Kras(G12D)-driven carcinogenesis. NFATc1 activation is induced by inflammation and itself accelerates inflammation-induced carcinogenesis in Kras(G12D) mice, whereas genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 complexes with STAT3 for enhancer-promoter communications at jointly regulated genes involved in oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1-STAT3 cooperativity is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer. Together, these studies unravel new mechanisms of inflammatory-driven pancreatic carcinogenesis and suggest beneficial effects of chemopreventive strategies using drugs that are currently available for targeting these factors in clinical trials.nnnSIGNIFICANCEnOur study points to the existence of an oncogenic NFATc1-STAT3 cooperativity that mechanistically links inflammation with pancreatic cancer initiation and progression. Because NFATc1-STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of inflammation and cancer, our study has significant relevance for potentially managing pancreatic cancer and other inflammatory-driven malignancies.

Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial–mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene‐induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation‐induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell‐like state through Sox2‐dependent transcription of EMT and stemness factors. Intriguingly, NFATc1–Sox2 complex‐mediated PDAC dedifferentiation and progression is opposed by antithetical p53‐miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell‐like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.

NFATc1 Links EGFR Signaling to Induction of Sox9 Transcription and Acinar-Ductal Transdifferentiation in the Pancreas.

BACKGROUND & AIMSnOncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of thexa0SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling withxa0activation of SOX9 during acinar-ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis.nnnMETHODSnWe analyzed pancreatic tissues from Kras(G12D);pdx1-Cre and Kras(G12D);NFATc1(Δ/Δ);pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue.nnnRESULTSnEGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras(G12D) mice. EGFR signaling also promoted formation of a complex between NFATc1 andxa0C-JUN in dedifferentiating mouse acinar cells, leading toxa0activation of Sox9 transcription and induction of acinar-ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar-ductal transdifferentiation and pancreatic cancer initiation in mice.nnnCONCLUSIONSnEGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.

Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid

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-3β-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-3β 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-3β 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-3β-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.