SOX2 (Sex-determining region Y (SRY)-Box2) has important functions during embryonic development and is involved in cancer stem cell (CSC) maintenance, in which it impairs cell growth and tumorigenicity. However, the function of SOX2 in pancreatic cancer cells is unclear. The objective of this study was to analyze SOX2 expression in human pancreatic tumors and determine the role of SOX2 in pancreatic cancer cells regulating CSC properties. In this report, we show that SOX2 is not expressed in normal pancreatic acinar or ductal cells. However, ectopic expression of SOX2 is observed in 19.3% of human pancreatic tumors. SOX2 knockdown in pancreatic cancer cells results in cell growth inhibition via cell cycle arrest associated with p21Cip1 and p27Kip1 induction, whereas SOX2 overexpression promotes S-phase entry and cell proliferation associated with cyclin D3 induction. SOX2 expression is associated with increased levels of the pancreatic CSC markers ALDH1, ESA and CD44. Importantly, we show that SOX2 is enriched in the ESA+/CD44+ CSC population from two different patient samples. Moreover, we show that SOX2 directly binds to the Snail, Slug and Twist promoters, leading to a loss of E-Cadherin and ZO-1 expression. Taken together, our findings show that SOX2 is aberrantly expressed in pancreatic cancer and contributes to cell proliferation and stemness/dedifferentiation through the regulation of a set of genes controlling G1/S transition and epithelial-to-mesenchymal transition (EMT) phenotype, suggesting that targeting SOX2-positive cancer cells could be a promising therapeutic strategy.

WASH regulates endosomal sorting, but its roles are ill defined. WASH-knockout MEFs display enlarged yet ordered endosomes without aberrant tubulation and a collapsed lysosomal network. Without WASH, EGFR is basally degraded, whereas TfnR is not, which supports discrete receptor trafficking via WASH-dependent and WASH-independent mechanisms.

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein B-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with B1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of B-catenin, which leads to an increasing expression of the B-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.A utophagy is an evolutionarily conserved catabolic process in eukaryotes that involves lysosomal degradation of cellular components, including long-lived proteins and organelles. There are four main forms of autophagy: macroautophagy (referred to here as autophagy), selective autophagy, microautophagy, and chaperone-mediated autophagy (1-4). Autophagy serves as an adaptive response to protect cells or organisms during periods of cellular stress, such as nutrient deprivation. In addition, autophagy can participate in several cellular and developmental processes, including homeostasis, clearance of intracellular pathogens, and immunity (1). Due to its fundamental importance for cellular survival, autophagy regulation has been implicated in several human diseases, such as cancer and neurodegenerative disorders (2, 5).Autophagy initiation involves the de novo synthesis of a double-membrane structure known as the phagophore, which ultimately elongates and closes to sequester cytoplasmic proteins and organelles, forming the autophagosome. The autophagosome subsequently undergoes a stepwise maturation process that culminates in its fusion with acidified endosomal/lysosomal vesicles, resulting in the degradation of its contents into useful biomolecules (2). A screen of yeast mutants unable to survive under nitrogen deprivation characterized a network of autophagy-related (ATG) genes (6). Mammalian homologues of these ATGs were later identified and shown to participate during distinct steps of autophagy. For example, microtubule-associated protein light chain 3 (LC3B) undergoes lipidation and is recruited to the pha...

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 were examined by RT-PCR and immunoblotting, and their implications in cell cycle progression and growth were determined by flow cytometry and [3H] thymidine incorporation. Intracellular Ca2+ concentrations, calcineurin activity and cellular NFAT distribution were analyzed. Transcription factor complex formations and promoter regulation were examined by immunoprecipitations, reporter gene assays and chromatin immunoprecipitation (ChIP). Using a combination of RNAi 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 Ca2+ 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 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. Conclusion: 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.

Background & Aims Oncogenic Kras mutation is a defining genetic alteration in pancreatic ductal adenocarcinoma (PDAC), but is not sufficient to promote cancer formation on ist own. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. In this study we sought to identify the underlying mechanisms which link EGFR signaling to Sox9 gene induction during acinar–ductal metaplasia (ADM), a transdifferentiation process that precedes pancreatic carcinogenesis. Methods We analyzed pancreatic tissues from KrasG12D;pdx1-Cre and KrasG12D;NFATc1Δ/Δ;pdx1-Cre mice after intraperitoneal administration of caerulein or dimethyl suloxide (controls). Pharmacological inhibition of NFATc1 activation was achieved by application of cyclosporin A. Induction of EGFR signaling and its effects on expression of NFATc1 or SOX9 were investigated by quantitative reverse transcription PCR, 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. Results EGFR activation induced NFATc1 expression in metaplastic tissues from patients with chronic pancreatitis and in pancreatic tissues from KrasG12D mice and promoted complex-formation with c-Jun in dedifferentiating acinar cells, thereby stimulating the transcription of ductal gene signatures to provoke ADM. This process involved NFATc1:c-Jun-mediated activation of Sox9 transcription in converting acinar cells. Pharmacological 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. Conclusion Our findings identify an EGFR-NFATc1-Sox9 signaling cascade as a critical mediator of inflammation-induced PDAC initiation and suggest that disruption of this pathway may offer a novel chemopreventive target for high-risk pancreatitis patients.

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 complexmediated 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.

Glycogen synthase kinase-3 beta (GSK-3β) is overexpressed in a number of human malignancies and has been shown to contribute to tumor cell proliferation and survival. Although regulation of GSK-3β activity has been extensively studied, the mechanisms governing GSK-3β gene expression are still unknown. Using pancreatic cancer as a model, we find that constitutively active Ras signaling increases GSK-3β 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-3β gene transcription in pancreatic cancer cells. Lastly, we show that ETS2 cooperates with p300 histone acetyltransferase to remodel chromatin and promote GSK-3β expression. Taken together, these results provide a general mechanism for increased expression of GSK-3β in pancreatic cancer and perhaps other cancers, where Ras signaling is deregulated.