Martin E. Fernandez-Zapico

Stromal Elements Act to Restrain, Rather Than Support, Pancreatic Ductal Adenocarcinoma

Sonic hedgehog (Shh), a soluble ligand overexpressed by neoplastic cells in pancreatic ductal adenocarcinoma (PDAC), drives formation of a fibroblast-rich desmoplastic stroma. To better understand its role in malignant progression, we deleted Shh in a well-defined mouse model of PDAC. As predicted, Shh-deficient tumors had reduced stromal content. Surprisingly, such tumors were more aggressive and exhibited undifferentiated histology, increased vascularity, and heightened proliferation--features that were fully recapitulated in control mice treated with a Smoothened inhibitor. Furthermore, administration of VEGFR blocking antibody selectively improved survival of Shh-deficient tumors, indicating that Hedgehog-driven stroma suppresses tumor growth in part by restraining tumor angiogenesis. Together, these data demonstrate that some components of the tumor stroma can act to restrain tumor growth.

Glycogen Synthase Kinase-3β Participates in Nuclear Factor κB–Mediated Gene Transcription and Cell Survival in Pancreatic Cancer Cells

Recent studies using glycogen synthase kinase-3beta (GSK-3beta)-deficient mouse embryonic fibroblasts suggest that GSK-3beta positively regulates nuclear factor kappaB (NFkappaB)-mediated gene transcription. Because NFkappaB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3beta in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3beta and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3beta by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3beta influences NFkappaB-mediated gene transcription at a point distal to the Ikappa kinase complex, as only ectopic expression of the NFkappaB subunits p65/p50, but not an Ikappa kinase beta constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3beta inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3beta in cancer cell survival and proliferation and suggest GSK-3beta as a potential therapeutic target in the treatment of pancreatic cancer.

Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB.

MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir‐29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir‐29b suppression is unknown. Here, we examined mir‐29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir‐29b downregulation. Using 5′ rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E‐box (Myc‐binding) sites, a Gli‐binding site, and four NF‐κB‐binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c‐Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir‐29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir‐29 promoter. Finally, activation of NF‐κB signaling, via ligation of Toll‐like receptors, also repressed mir‐29b expression and promoter function. Of note, activation of hedgehog, Toll‐like receptor, and c‐Myc signaling protected cholangiocytes from TRAIL‐induced apoptosis. Thus, in addition to c‐Myc, mir‐29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies. J. Cell. Biochem. 110: 1155–1164, 2010. Published 2010 Wiley‐Liss, Inc.

Aberrant Nuclear Accumulation of Glycogen Synthase Kinase-3β in Human Pancreatic Cancer: Association with Kinase Activity and Tumor Dedifferentiation

Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) β regulates nuclear factor-κB (NF-κB)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3β in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3β affects NF-κB activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3β in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Results: We found nuclear accumulation of GSK-3β in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3β nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3β can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-κB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts. Conclusions: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3β nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3β regulates NF-κB activity in pancreatic cancer.

Structural and Energetic Mechanisms of Cooperative Autoinhibition and Activation of Vav1

Vav proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases. They control processes including T cell activation, phagocytosis, and migration of normal and transformed cells. We report the structure and biophysical and cellular analyses of the five-domain autoinhibitory element of Vav1. The catalytic Dbl homology (DH) domain of Vav1 is controlled by two energetically coupled processes. The DH active site is directly, but weakly, inhibited by a helix from the adjacent Acidic domain. This core interaction is strengthened 10-fold by contacts of the calponin homology (CH) domain with the Acidic, pleckstrin homology, and DH domains. This construction enables efficient, stepwise relief of autoinhibition: initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation of the inhibitory helix and consequent GEF activation. Our findings illustrate how the opposing requirements of strong suppression of activity and rapid kinetics of activation can be achieved in multidomain systems.

An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation.

KLF11 is a biochemical paradigm for a subset of proteins that repress transcription via a Mad1‐like mSin3A interacting domain (SID). The biological role of these proteins and the significance of their biochemical activity, however, remain to be established. We report that KLF11 is downregulated in human cancers, inhibits cell growth in vitro and in vivo, and suppresses neoplastic transformation. Transgenic mice for KLF11 display a downregulation of genes encoding the oxidative stress scavengers SOD2 and Catalase1. Chromatin immunoprecipitation assays confirm that, indeed, these genes are bonafide targets of KLF11. KLF11 expression renders cells more sensitive to oxidative drugs, an effect that is rescued by infection with recombinant adenoviruses expressing SOD2 and Catalase1. KLF11‐regulated functions require the Mad1‐like SID, indicating that these target genes involved in these phenomena are regulated via this corepressor system. These results demonstrate that SID‐containing KLF repressor proteins can inhibit cell growth and neoplastic transformation, and outline for the first time cellular and molecular mechanisms by which these functions may be achieved.

NFκB p50-CCAAT/enhancer-binding protein β (C/EBPβ)-mediated transcriptional repression of microRNA let-7i following microbial infection.

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(−) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFκB p50-C/EBPβ silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.

Viral Factors Induce Hedgehog Pathway Activation in Humans with Viral Hepatitis, Cirrhosis, and Hepatocellular Carcinoma

Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.

MODY7 Gene, KLF11, Is a Novel p300-dependent Regulator of Pdx-1 (MODY4) Transcription in Pancreatic Islet β Cells

Pdx-1 (pancreatic-duodenal homeobox-1), a MODY4 homeodomain transcription factor, serves as a master regulator in the pancreas because of its importance during organogenesis and in adult islet insulin-producing β cell activity. Here, we show that KLF11, an SP/Krüppel-like (SP/KLF) transcription factor, mutated in French maturity onset diabetes of the young patients (MODY7), regulates Pdx-1 transcription in β cells through two evolutionarily conserved GC-rich motifs in conserved Area II, a control region essential to islet β cell-enriched expression. These regulatory elements, termed GC1 (human base pair −2061/−2055) and GC2 (−2036/−2027), are also nearly identical to the consensus KLF11 binding sequence defined here by random oligonucleotide binding analysis. KLF11 specifically associates with Area II in chromatin immunoprecipitation assays, while preventing binding to GC1- and/or GC2-compromised Pdx1-driven reporter activity in β cell lines. Mechanistically, we find that KLF11 interacts with the coactivator p300 via its zinc finger domain in vivo to mediate Pdx-1 activation. Together, our data identified a hierarchical regulatory cascade for these two MODY genes, suggesting that gene regulation in MODY is more complex than anticipated previously. Furthermore, because KLF11 like most MODY-associated transcription factors uses p300, these data further support a role for this coactivator as a critical chromatin link in forms of type 2 diabetes.

The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3–dependent Wnt activation

Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6‐O‐desulfatase activity on HSPGs, is up‐regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up‐regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/β‐catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2‐negative Hep3B HCC cells transfected with SULF2 and in SULF2‐expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. β‐catenin–dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized β‐catenin, and activated T cell factor transcription factor activity and expression of the Wnt/β‐catenin target gene cyclin D1. Opposite effects were observed in SULF2‐knockdown models. In vivo, nude mouse xenografts established from SULF2‐transfected Hep3B cells showed enhanced GPC3, Wnt3a, and β‐catenin levels. Conclusion: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3‐mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis. (HEPATOLOGY 2010;)