Knockout MEFs Research Articles

Abstract 4880: CASZ1, a neuroblastoma tumor suppressor gene, is epigenetically silenced by EZH2

Abstract Casz1 is a C2H2 Zn-finger transcriptional factor that localizes to a region of LOH on Chr1p36.22 in tumors of Neuroblastoma patients who have a poor prognosis. Moreover, high CASZ1 expression is found in tumors of patients with a good prognosis while tumors from patients with a poor prognosis express low levels of CASZ1 (58 patients, p= 2.07×10-6). Studies indicate that CASZ1 functions as a tumor suppressor gene. Our finding that treatment of NB cells with HDAC inhibitors results in a 10-fold increase in CASZ1 expression suggests that epigenetic silencing contributes to the functional loss of CASZ1 expression from the remaining Chr1p36.22 allele. In studying the mechanism of CASZ1 epigenetic silencing, we found a bivalent chromatin modification region around the CASZ1 Transcription Start Site (TSS) that contains both a H3K27 methylation mark as well as an H3K4 methylation mark. The H3K27 methylation is thought to be a gene repressive marker, which is catalyzed by Polycomb repressor complex 2 (PRC2) proteins. PRC2 contains three core components, EZH2, EED and SUZ12, in which EZH2 (enhancer of zeste 2) is a methyltransferase catalyzing H3K27 methylation. Using Chromatin Immunoprecipitation (ChIP) we identified a 5-fold decrease in binding of EZH2, EED and SUZ12 to the bivalent region after HDAC inhibitor treatment. Consistent with decreased PRC2 binding is the finding of decreased tri-methylation of H3K27 and an increase in H3K4me3, a histone mark associated with transcriptionally active genes. The changes in histone modifications are associated with the activation of the CASZ1 gene. RNA silencing of EZH2 also leads to increases in CASZ1 expression. Consistent with this, CASZ1 expression is also up-regulated (3- fold) in EZH2 knockout MEFs compared to EZH2 wild type MEFs. This indicates that CASZ1 expression on the remaining allele is silenced by epigenetic modifications mediated by EZH2. High expression of EZH2 and EED are also found in tumor samples of neuroblastoma patients with a poor prognosis (58 patients, p=2.6×10-3 for EED and p=5.87×10-4 for EZH2). This is consistent with a model in which aberrant epigenetic gene silencing of the tumor suppressor gene CASZ1 contributes to neuroblastoma tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4880.

Abstract 1262: Bif-1 is required for keeping mitochondrial and lysosomal positive feedback loops in check to prevent apoptosis induced by death receptors in type II cells

Abstract Bif-1, also known as endophilin B1 and SH3GLB1, is involved in the regulation of apoptosis, mitochondrial morphogenesis, and autophagy. We have previously shown that increasing the levels of Bif-1 accelerates Bax conformational change, caspase-3 activation, and apoptotic cell death in response to a variety of intrinsic apoptotic stimuli, whereas the loss of Bif-1 delays all these processes. Here, we report the role of Bif-1 as a negative regulator in the extrinsic pathway of apoptosis by controlling mitochondrial and lysosomal amplification loops in type II cells. There are two different types of cells distinguished by their distinct extrinsic apoptotic signaling pathways. In type I cells, the activation of caspase-8 by death receptor ligation is sufficient to activate downstream effector caspases (e.g. caspaspe-3) for cell killing, whereas in type II cells, caspase cascade amplification through mitochondria and/or lysosome is required. We found that depletion of Bif-1 dramatically enhances not only caspase-3 but also caspase-8 activation and cell death in type II cell lines including MEF, HeLa, and Jurkat, but not in type I H9 cells, following treatment by the extrinsic apoptotic stimuli TNFα/cycloheximide, TRAIL or Fas ligand. Overexpression of Bcl-XL or XIAP in Bif-1 knockout MEFs significantly diminishes the enhanced caspase activation, indicating that a mitochondrial feedback loop is elevated by the loss of Bif-1. Moreover, while treatment with cathepsin inhibitors does not suppress TNFα/cycloheximide-induced caspase-3 activation in Bif-1 knockout MEFs, it completely blocks the activation of caspase-3 in Bif-1 knockout MEFs overexpressing Bcl-XL or XIAP, implying that lysosomal pathway is also involved. Furthermore, inhibition of caspase-8 is able to completely block the activation of caspase-3 induced by TNFα/cycloheximide, indicating that caspase-8 initiates the mitochondrial and lysosomal amplification loops observed in Bif-1 deficient type II cells. Taken together, these studies suggest that Bif-1 plays an important role in the regulation of caspase-8-initiated mitochondrial and lysosomal positive feedback loops for killing type II cells by death receptors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1262.

Abstract 4836: RB/E2F1 in the crossroad of autophagy and apoptosis

Abstract Autophagy is a protective mechanism that renders cells viable in stressful conditions. Mounting evidence suggests that this cellular process is also a tumor suppressor pathway. We hypothesized that retinoblastoma protein (RB), a key tumor suppressor, induces autophagy. To test this hypothesis, we first transduced RB into RB-defective human cancer cells: sarcoma osteogenic (Saos-2) cells, hepatoma (Hep3B) cells, and brain tumor stem cells (MDNSC23). The ectopic RB induced autophagy as demonstrated by significantly increased percentage of cells with acidic vesicular organelles (from less than 10% to more than 30%, P < 0.05), and the lipidation of LC3-I leading to the formation of autophagosomes/autolysosomes that were visualized by EGFP-LC3 punctation (from less than 20% to more than 80%, P = 0.003). These double-membraned vesicles were clearly shown by ultrastructural study via transmission electron microscopy. However, when Beclin 1 was silenced with siRNA in Saos-2 cells, the RB-mediated autophagy was blocked. In addition, study of autophagy flux with Saos-2 cells expressing double-tagged mRFP-EGFP-LC3 fusion protein revealed that RB stimulated the formation and maturation of autophagosomes. Consistently, RB activators p16INK4a and p27/kip1 caused autophagy in an RB-dependent manner. Importantly, we found RB mutants Delta-22 and R661W deficient for binding E2F failed to induce autophagy. Moreover, E2F1 overexpression antagonized RB-mediated autophagy, leading to apoptosis in Saos-2 cells. In agreement with the above observations, silencing E2F1 with siRNA resulted in autophagy in U-87 MG cells; and autophagy levels increased from 4% in wild-type MEFs to 40% in E2F1 knockout MEFs (P = 0.002). Collectively, our data reveal that RB/E2F1 plays a key role in the decision of a cell to undergo autophagy or apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4836.

Redundancy and Specificity of the Metalloprotease System Mediating Oncogenic NOTCH1 Activation in T-ALL.

Abstract 988Poster Board I-10Aberrant NOTCH signaling plays a critical role in the development of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL) harboring mutations of NOTCH1. The NOTCH1 receptor functions as a ligand activated transcription factor which requires a double proteolytic processing for activation. NOTCH1 is first processed by an ADAM metalloprotease in the so called S2 site located in the extracellular portion of the receptor, and subsequently by the presenilin γ-secretase complex, which cleaves at the S3 site located in the transmembrane domain. Notably, small molecule inhibitors of the γ-secretase complex effectively block NOTCH1 S3 processing and are under development as anti-NOTCH1 therapies in T-ALL. Moreover, selective inhibitors of ADAM10 and ADAM17, two ADAM proteases implicated in the S2 processing of NOTCH1, have been developed as inhibitors of ERBB signaling for the treatment of solid tumors; suggesting that inhibition of NOTCH1 S2 processing could be exploited as an anti-NOTCH1 therapy in T-ALL. However, activating mutations in NOTCH1 found in T-ALL frequently alter the structure of the HD-LNR domains that normally control the access of ADAM proteases to the S2 site, raising the possibility that S2 cleavage may be processed by alternative proteases in the context of T-ALL. To address the role of ADAM proteases in the shedding and activation of oncogenic forms of NOTCH1, we tested the effects of metalloprotease inhibitors in NOTCH1 processing and activation using a luciferase reporter assay. In these experiments we transiently transfected HeLa cells with expression vectors encoding the a NOTCH1 HD class I mutant (NOTCH1 L1601P), a NOTCH1 HD class II allele (P12 ICHIKAWA) and a NOTCH1 JME construct (Jurkat JME17), which are characterized by increased and aberrant NOTCH1 S2 processing, to test the effect of Timp1, Timp2 and Timp3 in NOTCH1 signaling. Notably Timp3, a metalloprotease inhibitor that blocks soluble, membrane and ADAM metalloproteases, decreased the activity of all three NOTCH1 mutant alleles. In contrast, inhibition of soluble metalloproteases with Timp1 had no effect on NOTCH1 activation and blocking soluble and membrane metalloproteases with Timp2 showed only a mild inhibitory activity towards the L1601P mutant allele. These results suggest that, as in the case of wild type NOTCH1, ADAM proteases are responsible for the processing of oncogenic NOTCH1 at the S2 site. Consistent with these results, inhibition of Adam10 or Adam17 in mouse embryonic fibroblasts resulted in marked decrease in the activity of all three oncogenic forms of NOTCH1. Moreover, knocking down Adam17 in Adam10 deficient cells and vice versa, knocking down Adam10 in Adam17 knockout MEFs, showed that simultaneous inactivation of Adam10 and Adam17 resulted in further decreases in the activity of oncogenic NOTCH1. These results suggest that both Adam10 and Adam17 can be competent in NOTCH1 processing. However, recent data from Adam10 conditional knockout mice has shown that Adam10 may play a more prominent role in the processing and activation of Notch1 signaling during thymocyte development. To address the specific requirement of ADAM10 for NOTCH1 processing and activation in T-ALL we performed shRNA knock down of this protease in T-ALL cell lines harboring a NOTCH1 HD class I mutation (KOPTK1), a NOTCH1 HD class II mutation (P12 ICHIKAWA) and a NOTCH1 JME mutation (Jurkat). These experiments showed that ADAM10 inactivation results in a marked decrease in the levels of activated intracellular NOTCH1 (ICN1) protein detected by Western blot and diminished expression of the NOTCH1 target Deltex1. Moreover, inhibition of ADAM10 by shRNA knock down in KOPTK1 cells, a T-ALL cell line dependent on NOTCH1 signaling for growth and proliferation, caused growth inhibition and sensitized these cells to low concentrations (10nM) of Compound E, a highly active γ-secretase inhibitor. Overall these results provide further insight on the mechanisms that control the activation of oncogenic NOTCH1 mutants and identify ADAM10 as a potential therapeutic target for the inhibition of oncogenic NOTCH1 in T-ALL. Disclosures:Ferrando:Merck, Pfizer: Research Funding.

Smif (Smad4 Interacting Factor) Is a Negative Regulator of T Cell Activation and Autoimmunity In Vivo.

Abstract 712Transforming growth factor β (TGFβ) plays a critical role in regulating cellular processes like proliferation, extracellular matrix production, vasculogenesis and angiogenesis as well as immunomodulation. TGFβ is a pluripotent cytokine with a pronounced immunosuppressive effect by controlling proliferation, differentiation and activation of immune cells. TGFβ binding to its receptor leads to the phosphorylation of R-Smads. R-Smads again form a heteromeric complex with the cytosolic common Smad4. This Smad complex, together with additional cofactors, translocate into the nucleus, where they control the transcription of TGFβ target genes. Smif was originally identified in our lab as an interaction partner of Smad4. Functional analysis revealed a stimulatory effect in regulating TGFβ-dependent genes like the early target gene JunB. After TGFβ stimulation, Smif tranlocates, together with Smad4, into the nucleus, where Smif acts as a coactivator. To investigate the role of Smif in mammals, we generated a Smif knockout mouse. To this end exon 2 of Smif was replaced by GFP and an inverted neomycin selection cassette. Smif-deficient mice were viable but exhibit a shortened life span. On the average, these mice die at 12 month of age due to multifocal inflammatory disease. Overall pathological analysis of diseased mice revealed extensive lymphocytic infiltrates in multiple organs. Moreover, Smif-deficiency caused immune complex induced glomerulonephritis associated with proteinuria. In line with these findings, autoantibodies could be detected in the serum of Smif knockout mice. Interestingly, we identified T cells and not B-cells as the important target in Smif-deficient mice. T cells lacking Smif were spontaneously activated. In addition, TGFβ was not able to block T cell proliferation of CD4+ cells in vitro, whereas B cells isolated from Smif knockout spleens behave as wildtype. Transcription of TGFβ responsive reporter constructs was greatly reduced in Smif knockout Mefs and could be rescued by the reexpression of functional Smif. Taken all together, the observed autoimmune phenotype found in Smif-deficient mice is at least partially caused by overactivated T cells due to downregulation of the inhibitory TGFβ pathway. Disclosures:No relevant conflicts of interest to declare.

Differential Involvement of Atg16L1 in Crohn Disease and Canonical Autophagy

A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.

Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF(+/-)) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF(+/-), may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

Presenilin‐2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake

We have previously shown that familial Alzheimer’s disease mutants of presenilin-2 (PS2) and, to a lesser extent, of presenilin-1 (PS1) lower the Ca2+ concentration of intracellular stores. We here examined the mechanism by which wild-type and mutant PS2 affect store Ca2+ handling. By using HeLa, SH-SY5Y and MEFs as model cells, and recombinant aequorins as Ca2+ probes, we show evidence that transient expression of either wild-type or mutant PS2 increases the passive Ca2+ leakage: both ryanodine- and IP3-receptors contribute to Ca2+ exit out of the ER, whereas the ribosome translocon complex is not involved. In SH-SY5Y cells and MEFs, wild-type and mutant PS2 potently reduce the uptake of Ca2+ inside the stores, an effect that can be counteracted by over-expression of SERCA-2B. On this line, in wild-type MEFs, lowering the endogenous level of PS2 by RNA interference, increases the Ca2+-loading capability of intracellular stores. Furthermore, we show that in PS double knockout MEFs, reduction of Ca2+ stores is mimicked by the expression of PS2-D366A, a loss-of-function mutant, uncleaved because also devoid of presenilinase activity but not by co-expression of the two catalytic active fragments of PS2. In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca2+ uptake by intracellular stores. To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

Loss of Stearoyl‐CoA Desaturase activity increases ER stress and PKCζ mediated inhibition of Akt in response to saturated fatty acids in breast cancer cells.

Increased de novo lipogenesis is a hallmark of invasive tumors and is negatively correlated with survival. However, the end product is the saturated fatty acid (SFA) palmitate that is lipotoxic unless desaturated. Stearoyl‐CoA desaturase (SCD) converts SFA into monounsaturated fatty acids and is the rate limiting step in the storage of triglycerides and the generation of phospholipids. We demonstrate that SCD increases proliferation in HeLa cells in the presence of Cerulenin or exogenous SFA and that stearate decreased cell viability in a dose dependent fashion. While the product of SCD (oleate) can activate Akt, substrate (stearate or palmitate) treatment of cells exhibited a dose dependent increase in PARP cleavage and PKC? expression. We further probed the role of SCD‐1 in WT and SCD‐1 knockout MEFs and established that SFA prevents Akt translocation to lipid rafts. In the presence of a small molecule SCD‐1 inhibitor, palmitate treatment induced ER stress as evidenced by Xbp‐1 splicing. Density gradient analysis and IP experiments of palmitate treated cells showed a decrease in both PKC? and Akt lipid raft localization and an increase in TLR4/IKKβ complex formation. Finally, in transgenic MMTV‐PyMT mice, SCD‐1 deficiency reduced invasiveness of mammary gland tumors. Therefore, we conclude that SCD regulates MUFA mediated proliferation, prevents ER stress, and reduces tumor invasion.

Phosphorylation by CDK1 and JNK1 regulate the DNA binding activity of transcription factor Sp1 during mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated at threonine 739 by JNK and CDK1 and not co‐localized with the chromatin at the beginning of mitotic stage. EMSA, DNA affinity precipitation assay, and ChIP assay were performed and revealed that during mitosis the DNA‐binding ability of phosphorylated Sp1was decrease as well as dephosphorylation of Sp1 by phosphatase restored the DNA binding affinity. In addition, the phosphorylation of Sp1 during mitotic stage also suppressed Sp1 to interact with p300, Brg1, and RNA polII and reduced the transcriptional activity of Sp1 target genes. Moreover, in late mitosis, the Pin1 interacted with the high phosphorylation of Sp1 and recruited the PP2A to dephosphorylate Sp1. In Pin1 knockout MEF cells, there was a delay in the relocalization of Sp1 to the nucleus during telophase. Taken together, our results indicated that Sp1 interacts with JNK and CDK1, followed by phosphorylation of Sp1 at early mitosis. Sp1, then, is disassociated from DNA and loses interaction with co‐activator. In late mitosis, Pin1 and PP2A are recruited by phosphorylation of Sp1, leading to the enhancement of the DNA binding‐ability and protein interactional function of Sp1.

Activation Mechanism of Protein Kinase B by DNA-dependent Protein Kinase Involved in the DNA Repair System.

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be 2nd upstream kinase for protein kinase B (PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells (MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK (M059J) and a wild-type of DNA-PK (M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

Regulatory Effects of Mammalian Target of Rapamycin-activated Pathways in Type I and II Interferon Signaling

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNalpha or IFNgamma results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNalpha- or IFNgamma-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1(-/-)) knockout MEFs, as compared with wild-type 4E-BP1(+/+) MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2(-/-)) or hamartin (TSC1(-/-)) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.

Suppressing Wnt Signaling by the Hedgehog Pathway through sFRP-1

The hedgehog (Hh) signaling pathway is essential for embryonic development and carcinogenesis. Activation of Hh signaling has been identified in several types of gastrointestinal cancers, including esophageal, gastric, pancreatic, and liver cancers. Several recent studies suggest that Hh signaling activation can inhibit Wnt signaling. However, the molecular basis underlying this inhibition remains unclear. As transcription factors in the Hh signaling pathway, Gli molecules transform cells in culture, and their expression are associated with cancer development. Here we report that expression of a secreted frizzled-related protein-sFRP-1 in mouse embryonic fibroblasts is dependent on Gli1 and Gli2. In human gastric cancer cells, inhibition of Hh signaling reduces the level of sFRP-1 transcript, whereas ectopic expression of Gli1 increases the level of sFRP-1 transcript. Results from chromatin immunoprecipitation indicate that Gli1 is involved in transcriptional regulation of sFRP-1. In 293 cells with Gli1 expression, Wnt-1-mediated beta-catenin accumulation in the cytosol and DKK1 expression are all abrogated, which can be reversed by inhibiting sFRP-1 expression. Furthermore, while SIIA cells do not respond to Wnt-1-conditioned medium, inhibition of Hh signaling by smoothened (SMO) antagonist KAAD-cyclopamine (keto-N-aminoethylaminocaproyldihydrocinnamoylcyclopamine) leads to Wnt1-mediated beta-catenin accumulation in the cytosol. These data indicate that sFRP-1, a target gene of the hedgehog pathway, is involved in cross-talk between the hedgehog pathway and the Wnt pathway.

Control of cellular senescence by CPEB

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16(INK4A); however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.

IKK-i/IKKϵ Controls Constitutive, Cancer Cell-associated NF-κB Activity via Regulation of Ser-536 p65/RelA Phosphorylation

Nuclear factor kappaB (NF-kappaB) has been studied extensively as an inducible transcriptional regulator of the immune and inflammatory response. NF-kappaB activation downstream of lipopolysaccharide or cytokine stimulation is controlled by the IkappaB kinase complex, which contains IKKalpha and IKKbeta. Significantly, the constitutive activity of NF-kappaB has been implicated as an important aspect of many cancer cells, but mechanisms associated with this activity are poorly understood. An inducible kinase, IKK-i/IKKepsilon, related to the catalytic forms of the IkappaB kinase, has been studied as an anti-viral, innate immune regulator through its ability to control the activity of the transcription factors IRF-3 and IRF-7. Here, we demonstrate that IKK-i/IKKepsilon is expressed in a number of cancer cells and is involved in regulating NF-kappaB activity through its ability to control basal/constitutive, but not cytokine-induced, p65/RelA phosphorylation at Ser-536, a modification proposed to contribute to the transactivation function of NF-kappaB. Knockdown of IKK-i/IKKepsilon or expression of a S536A mutant form of p65 suppresses HeLa cell proliferation. The data indicate a role for IKK-i/IKKepsilon in controlling proliferation of certain cancer cells through regulation of constitutive NF-kappaB activity.

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

A Nuclear Function of β-Arrestin1 in GPCR Signaling: Regulation of Histone Acetylation and Gene Transcription

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. Here, we show that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of beta-arrestin 1 (betaarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, betaarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. Our results reveal a novel function of betaarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification.

Triptolide-Induced Apoptosis Is Dependent on Caspase Activation and NFκB Signaling and Mediated by XIAP and Survivin Downregulation through the Mitochondrial Pathway in Leukemic Cells.

Triptolide, an immunosuppressor isolated from the Chinese herb, Tripterygium wilfordii Hook. F, has recently shown anti-tumor activities in a broad range of solid tumors. We examined its effects on leukemic cells and investigated mechanisms of apoptosis. Triptolide, at less than 100 nM, arrested cell growth and potently induced cell death in myeloid and lymphoid leukemic cells tested, including OCI-AML3, U937, Jurkat, KBM5, and K562 cells. In OCI-AML3 cells, triptolide induced caspase 3 activation, PARP cleavage and annexin V positivity with an IC50 of about 30 nM, at 24 hrs, all of which were inhibited by a general caspase inhibitor suggesting caspase dependent cell death. However, Triptolide-induced cell growth arrest was not affected by caspase inhibition. Treatment of OCI-AML3 cells with triptolide decreased XIAP and survivin expression, but did not affect Bcl2 and BclXL levels. Forced overexpression of XIAP attenuated Triptolide-induced cell death. Triptolide induced Bid cleavage, but Jurkat cells deficient in caspase 8 were only slightly less sensitive to triptolide than the wild-type counterpart indicating that Triptolide-induced cell death is caspase 8 independent. Jurkat cells deficient in receptor interacting protein (RIP) and therefore deficient in NFκB activation were resistant to Triptolide demonstrating that NFκB signaling is essential for Triptolide-induced cell death. Triptolide treatment induced cytosolic release of cytochrome C and loss of mitochondrial membrane potential, overexpression of Bcl2 effectively suppressed apoptosis induced by Triptolide, and caspase 9 knockout MEF cells were resistant to Triptolide suggesting criticality of the mitochondrial pathway. The antioxidants GSH (5 mM) and vitamin C (150 μM) did not protect from apoptotic cell death induced by Triptolide. In addition, Triptolide-induced apoptosis of blast crisis CML KBM5 cells was independent of their sensitivity or resistance to Imatinib: Triptolide killed Imatinib resistant KBMSTI cells as effectively as Imatinib sensitive KBM5 cells. Ex vivo studies showed that Triptolide also induced cell death in primary AML blasts. Collectively, our studies demonstrate that Triptolide potently induces caspase-dependent apoptosis and arrests cell growth in leukemic cells. Triptolide-induced cell death is dependent on NFκB signaling, and mediated by downregulation of XIAP and survivin through the mitochondrial pathway. The potent anti-leukemic activity of Triptolide in vitro warrants further investigation of this compound for the treatment of leukemia and other malignancies. This drug may also be potentially useful in overcoming Imatinib resistance in CML and Philadelphia chromosome positive ALL.