Apc/β-catenin genes in the Wntsignaling pathway are frequently mutated and active Notch signalingcontributes to tumorigenesis by keeping the epithelial cells in aproliferative state. These pathways are simultaneously active inproliferative adenoma cells and a crosstalk between them has previously beensuggested in normal development as well as in cancer.Notch and Wnt pathways are key regulators of intestinal homeostasis andalterations in these pathways may lead to the development of colorectalcancer (CRC). In CRC the in silico analysis of putative promotersinvolved in transcriptional regulation of genes coding for proteins in theNotch signaling pathway revealed several putative LEF-1/TCF sites aspotential targets for β-catenin and canonical Wnt signaling. Furtherresults from competitive electrophoretic mobility-shift assay (EMSA) studiessuggest binding of several putative sites in Notch pathway gene promoters toin vitro translated β-catenin/Lef-1. Wild type(wt)-Apc negatively regulates β-catenin. By induction of wt-Apc orβ-catenin silencing in HT29 cells, we observed that several genes in theNotch pathway, including Notch-2, were downregulated.Finally, active Notch signaling was verified in theMin/Apc+ mouse modelwhere Hes-1 mRNA levels were found significantlyupregulated in intestinal tumors compared to normal intestinal mucosa.Luciferase assays showed an increased activity for the core and proximalNotch-2 promoter upon co-transfection of HCT116 cellswith high expression recombinant Tcf-4, Lef-1 or β-catenin.In this study, Notch-2 as a novel target forβ-catenin-dependent Wnt signaling. Furthermore our data supports thenotion that additional genes in the Notch pathway might be transcriptionallyregulated by Wnt signaling in colorectal cancer.In this paper, we identified  The epithelium of the gastrointestinal tract is continually replaced with a turn-overrate of two to seven days. In order to maintain homeostasis of the intestinalepithelium, processes cellular proliferation, differentiation, migration and deathmust be strictly regulated Fringe gene family  Mastermind-likefamily (Maml) Hes -and Hey family of genes, which functionas transcriptional repressors of further downstream targets likeMath-1 (mouse homolog of human Hath-1) Kip1 and p57Kip2Math-1 mRNA and protein Notch-2 has also been proposed to have a tumor suppressiveeffect in CRC The maturation of intestinal stem cells is also regulated by the Notch signalingpathway representing another evolutionary conserved signaling system involved inmaintaining colon epithelium homeostasis Hath-1 expression is increased when the Wnt pathway isinhibited Hes-1 is a direct target of canonical Wnt signaling incolorectal adenomas and carcinomas Jagged-1gene is directly regulated by β-catenin/Tcf-4 In addition to the independent roles of Wnt and Notch signaling pathways intumorigenesis in the colon, the findings that tumor development in Apc-deficientmice is enhanced upon simultaneous activation of Notch and Wnt signals Apc mutations.In this study, we have investigated the potential of the Wnt signaling pathway fordirect regulation of genes involved in Notch signaling. This revealed that severalof the putative promoter regions in Notch pathway associated genes containLef-1/Tcf-binding sites. Some of these genes were downregulated upon expression of afunctional Apc gene in the HT29 CRC cell line, supporting the idea that abnormal Wntsignaling has a direct impact on the expression of genes encoding proteins involvedin Notch signaling. Thus, we propose that crucial components of the Notch signalingpathway are directly influenced by Wnt signaling with the implication that even agenetically normal Notch pathway can contribute to tumorigenesis due to the responseto β-catenin or www.ingenuity.com),together with extensive literature searches. Gene promoter sequences wereextracted using the Genomatix Gene2Promoter software and the average length ofthe putative core, proximal and parts of the distal promoters were adjusted toapproximately 2500 bp (details available on request). By means of theMatInspector software 5′-(A/T)(A/T)CAA(A/T)G-3′To identify potential targets for interactions between the Notch and Wntsignaling pathways, 65 genes, known to be important for Notch and Wnt signaling,were selected bioinformatically using the Ingenuity Pathways Analysis with a radioactively labeled consensus LEF-1/TCF strong bindingprobe  compared to normal intestinal mucosa , indicating overactivatedNotch signaling in the mouse adenomas. A trend towardsupregulation was observed for mNotch-2 in tumor tissue  compared to normal intestinal mucosa , while no statisticallysignificant differences could be detected for mNotch-1 ) vs. ) .Mutational inactivation of the ignaling , containal cells . The 19 nduction . As apovariants . To detevariants , using and Numbl , displayn/+mice  carryingmHes-1 is significantlyupregulated in intestinal tumors from Apc deficient mice. This further supportsa direct transcriptional crosstalk suggested by the in silicoand in vitro DNA-binding analyses.In conclusion, inactivation of the Wnt pathway through activation of wt-Apc inHT29 CRC cells downregulates several target genes in the Notch pathway whilemRNA-levels of the Notch target Notch genesand a potential regulation of Notch-2 through Wnt signalingcould be of importance in the development and/or progression of CRC andpotentially in other malignancies as well. Previously, it has been shown thatboth Hes-1 and Jagged-1 are direct targets ofcanonical Wnt signaling in CRC in silico,in vitro DNA-binding analyses and activation of wt-Apc,strongly suggest that there is a direct positive regulation ofNotch-2 through Wnt-signaling. Therefore we wanted tofurther elucidate the β-catenin/Lef-1/Tcfs regulating potential of theNotch-2 promoter in the CRC cell lines HT29 and HCT116.Notch-2 functions redundantly with Notch-1 in colon epithelial cells where bothgenes are important for keeping the cells in the crypt compartment in aproliferative and undifferentiated state in silico analyses were used to identify thehuman Notch-2 promoter and a potential transcriptional startsite (TSS) was found 256 bp upstream the translational start site, verifying theresult from Gene2Promoter analysis. Four putative LEF-1/TCF consensus sites wereidentified in the core and proximal promoter region at positions −2261,−869, −689 and −110 relative to the translational start site ,thereby covering all four putative LEF-1/TCF-sites and one covering putativeLEF-1/TCF-site −110. They were cloned into a firefly luciferase reportervector  containing a TATA-box todetect possible enhancer and repressor cis-acting elements containingS33Y-β-catenin (S33Y mutation), hTcf-4 or mLef-1 sequences and the relativeluciferase activity was calculated by normalizing Notch-2promoter activity (N2PR −2327/−99 or N2PR −110) vs.pGL3-TATA-activity. Western blots confirmed expression of His-tagged Tcf-4 andLef-1 (p = 0.03) (p = 0.02) and 2.8 (Lef-1)(p = 0.001), respectively. However, the increase inactivity was not as significant as that seen upon β-catenin co-transfection,indicating more specific effects of the DNA-binding proteins. Tcf-4 and Lef-1failed to significantly affect the activity of N2PR −110 and nodifferences could be detected between wild-type and mutated construct, where theputative LEF1/TCF site at position −110 was mutated into aSpeI restriction site. These results suggest that LEF1/TCFconsensus site −110 is not a direct target of canonical Wnt signaling inthis system through Tcf-4 or Lef-1  .The resor Lef-1 . Howeverin/Lef-1 , where 3nd Tcf-4 . Followind Lef-1 ,somewhapitation .Notch-2 can betranscriptionally regulated by canonical Wnt signaling, however the exactmechanism and interactions sites remain to be elucidated.Overall, these results suggest that Apc deficient mice results inearlier onset and more tumors in the small intestine, compared toApc deficient mice alone. However, overexpression of Notchsignaling does not by itself cause intestinal tumor formation in the mouse et al. et al. Apc and RBJ-κgenes were deleted simultaneously, the latter reflecting a model of more acute Apcloss. In the present study, in silico analysis of Notch and Wntpathway gene promoters resulted in the identification of several putativeβ-catenin regulated LEF-1/TCF-sites in Notch pathway gene promoters andRBP-Jκ-sites in Wnt pathway gene promoters . Speculatively, β-catenin may interact with N2ICD topotentiate the expression of Notch-2, but more studies are neededto outline this interaction. Contrasting this, EMSA assays suggested binding ofLef-1 to the −110 LEF-1/TCF-site was transcriptionally regulated by the NICD/Rbp-jκ complex, and therebyindicating a regulatory feed-back loop, through β-catenin. By γ-secretasetreatment all Notch signaling is effectively inhibited and in our experiments thewell-known Notch target gene Hes-1e.g. through upregulation of Sfrp1,Sfrp5, Dkk1, which are known to encodeinhibitors of the Wnt pathway cyclin D1 by DAPT treatment  . These rNotch-2 is a noveltarget, activated by Wnt signaling in colon cancer cells.In conclusion, in this paper we identify several potential target genes ofWnt/β-catenin signaling among genes traditionally classified as belonging to theNotch pathway. Analysis of the genetic networks directly involved in Notch and Wntsignaling suggests functional overlap and direct crosstalk via target geneactivation. More specifically, we suggest that http://www.genomatix.de,Genomatix Software GmbH, Munich, Germany) Notch-2 promoteridentified with Gene2Promoter.Identification of potential promoter regions and putative LEF-1/TCF-sites thereinwas performed with Genomatix software (The Matrix Family Library Version 7.0 (October 2007) was used and the selectedgroups were ALL vertebrates.lib where the standard (0.75) core similarity andthe optimized matrix similarity was used.Full length murine Lef-1 (mLef-1) and human Tcf-4 (hTcf-4) expressed in pCDNA6were kind gifts from professor B.O Williams while HA-S33Y-β-catenin,expressed in pCGN, was a kind gift from professor Avri Ben-Ze'ev (viaprofessor Anita Sjölander).in vitro translation experiments HA-S33Y-β-catenin wassubcloned into pCDNA3  using XbaI and BamHIrestriction enzymes.For Notch-2 luciferase promoter reporter construct (N2PR−2327/−99) ,covering all four LEF-1/TCF-sites in the Notch-2 promoter, wasconstructed using primers:A larger N2PR −2327/−99-forward: 5′-GGTACCTGGGGATTAATAGGCTGTGG-3′N2PR −2327/−99-reverse: 5′-CTCGAGCTCCTGCTTCAAAGGCTCAG-3′,as well as a smaller (N2PR −110) covering the most proximal LEF-1/TCF-siteusing primers:N2PR −110-forward: 5′-GGTACCGTTGCACACCCGAGAAAGTT-3′N2PR −110-reverse: 5′- CTCGAGATCTTCTCGGTCGCCTCCT-3′.Forward primers were designed with an additional KpnI restriction site andreverse primers with a XhoI restriction site. PCR-products were cloned into thepGEM-T vector (Promega) according to manufacturer's conditions and thenfurther cloned into pGL3 luciferase reporter containing a TATA-box  and the pGL3-specific primer5′-CTTTATGTTTTTGGCGTCTTCCA-3′, and the secondusing the pGL3-specific primer RVprimer3: 5′-CTAGCAAAATAGGCTGTCCC-3′ and N2PR−110 SpeI-reverse: 5′-CTCGAGGCTAGC ACTAGT GCTCAGGCCCTGGCGCTA-3′(containing the SpeI restriction sequence), with pGL3-TATA-N2PR−110 as a template. Amplicons were separately cloned into the pGEM-Tvector and sequentially transferred into pGL3-TATA using KpnI,XhoI and SpeI restriction enzymes therebymutating the −110 LEF1/TCF core sequence CTTTGA into ACTAGT.GLprimer2: In vitro translated proteins were prepared with theTNT® coupled reticulocyte lysate system (Promega). Forprotein-DNA binding interaction studies, in vitro translatedLef-1 and β-catenin were incubated with the following 32P-labeledduplex oligonucleotide probe CD1TOP (5′-CTCTGCCGGGCTTTGATCTTTGCTTAACAACA-3′). Thebinding reaction contained 50 fmol of 32P-labeled probe that wasincubated for 30 min with the in vitro translated proteins in10 mM Hepes/70 mM KCl/1 mM dithiotreitol/1 mM EDTA/7.5 mMMgCl2/4% (vol/vol) glycerol/8 µg/ml salmon sperm DNA.For competition experiments, 300 -or 900-fold excess unlabeled double-strandedoligonucleotides  and counted.HCT116 and HT29 colon cancer cells  were cultivated inMcCoy's 5A media (Gibco/Invitrogen) supplemented with 10% foetalbovine serum at 37°C in 5% COApc gene(HT29-APC) or the control gene β-galactosidase, regulated by a zinc inducible promoter (generousgift from professor B. Vogelstein) were cultivated as described above, withthe exception that cells were selectively grown by the addition of1.2% hygromycine B (Invitrogen). Twenty-four hours before harvest,the cells were split with 0.05% Trypsin-EDTA and counted. HT29-APCand HT29-β-gal. were seeded in MULTIWELL™ 6 well   and 100 µMZnCl2 was added to the medium in the interval of 24, 18, 12,6, 3 and 0 hours before cell harvest and RNA as well as proteinisolation.HT29 cells harbouring a vector carrying the wt-5/well), as described above. A pool of fourdifferent siRNA oligonucleotides targeting β-catenin mRNA  with the sequence:GAUCCUAGCUAUCGUUCUU,UAAUGAGGACCUAUACUUA,GCGUUUGGCUGAACCAUCA,GGUACGAGCUGCUAUGUUC,were transfected into HT29 with DharmaFECT4™ transfection reagent(Dharmacon) according to the manufacturer's recommendations. The finalsiRNA concentration was 100 nM. As control experiment, cells were inparallel mock transfected with siGLO® transfection indicator (Dharmacon)only or a non-specific siRNA pool .Cells for siRNA transfection were seeded in MULTIWELL™ 12 well (2.5×102O and1 mL Bradford Reagent. As standard curve 0/1/2/4/8 1 mg/mL BSA (Sigma) was mixedwith Milli-Q H2O to a final volume of 100 µL and then 1 mLBradford reagent was added. The samples were immediately vortexed and 200µL sample was added to an ELIZA plate and measured by THERMOmax microplatereader (Molecular Devices) with SOFTMAX software (Molecular Devices). Allmeasurements were performed in duplicates.RNA and protein was isolated with the PARIS™ Kit  according to the supplier's recommendations. The protein concentrationwas spectrophotometrically measured using Bradford Reagent . Briefly, 2 µL sample was mixed with 98 uL Milli-Q Hcyclin D1, was used as a positive control Gapdh was used. For quantitative assessments, the mRNAexpression was normalized against Gapdh. 1 µL of cDNAsolution was added to 19 µL of a standard master mix containing 0.5 unitsof ThermoWhite DNA Polymerase  and 1 µMof the appropriate primers  according to the supplier'srecommendation. To determine the expression of Apc or silencing ofβ-catenin, the well-established target gene of an active Wnt pathway,2O, 0.25% NuPAGE® Antioxidant (Invitrogen) was addedto the inner chamber. After the separation, the proteins were blotted to PVDFmembranes  at 30 V for 60 min in NuPAGE® TransferBuffer (20×) (Invitrogen) diluted 1∶20 in H2O with20% methanol and 0.1% NuPAGE® Antioxidant. The PVDF membraneswere blocked with 5% Blotto  dissolved in Tris-buffered saline with 0.1% Tween®- 20(TBS-T) (Sigma) for 60 min at room temperature. The primary antibodies added tothe PVDF membranes were diluted in TBS-T as follows: anti-APC 1∶500, anti-β-catenin 1∶1000 , anti-Gapdh 1∶2000 (Millipore), anti-Notch-2 1∶2000(Santa Cruz) and anti-His (6× His tag® antibody) 1∶1000 (Abcam).The antibodies were incubated for 60 min at room temperature or over night in4°C, washed in TBS-T, followed by incubation for 60 min at room temperaturewith appropriate secondary antibodies, goat-anti-mouse 1∶10000  and goat-anti-rabbit 1∶10000. The PVDF membranes were exposed to ECLWestern Blotting Substrate (Pierce) and photographed digitally. Beforereincubation of the membranes, the antibodies were stripped with Restore™Western Blot Stripping Buffer  for 15 minat room temperature and washed in TBS-T.Cell lysates containing equal amounts of total protein were denatured inNuPAGE® LDS Sample Buffer (Invitrogen) at 70°C for 10 min withNuPAGE® Sample Reducing Agent (Invitrogen). The proteins were separated at200 V for 35 min in NuPAGE® 4–12% Bis-tris gels (Invitrogen)held in NuPAGE® MES DS Running Buffer (20×) diluted 1∶20 withHMin/+ genotype  were used in the present study Animals were housed inventilated cages at 23±1°C with a 12-h light/dark cycle. Standarddiet  and water wereavailable ad libitum. DNA for genotyping was isolated from tailbiopsies with the Extract-N-Amp Tissue PCR Kit™ (Sigma), according tosupplier's recommendations. The Apc encoding gene weregenotyped as previously described C57BL/6 mice with the APCmNotch-1,mNotch-2 and mHes-1 was subsequentlydetermined with the 7500 Fast Real-Time PCR System , using predesigned primer/probe assays purchased from AppliedBiosystems (sequences available at request). Ctvalues were related to the endogenous control gene GAPDH(ΔCt), and relative expression(2Ct−Δ) was normalised to theaverage expression in non-tumor intestinal mucosa ofAPCMin/+mPGES-1+/+ mice(2−ΔΔCt).Tumors and non-tumor intestinal mucosa specimens were collected and handled aspreviously described 24 hours prior to luciferase reporter assays, HCT116 or HT29 cells were seededand co-transfected with 200 ng reporter vector (pGL3-TATA with and without N2PR−2327/−99 or −110 insert), 95 ng pSV-β-galactosidase and455 ng pCGN-HA-S33Y-β-catenin, pcDNA6-mLef-1 or pcDNA6-hTcf-4, usingLipofectamine LTX® according to supplier's recommendations. Controlswere co-transfected with empty pCDNA6 vector and 24 h post transfection bothluciferase and β-galactosidase activities were measured. As a backgroundcontrol, pGL3-TATA without insert was used and treated as described above. Theluciferase activity was determined by the ratio of luciferase toβ-galactosidase activity and the relative activity by the ratio of promoteractivity vs. background control.Notch-2 LEF-1/TCF-site −110, chromatinimmunoprecipitation (ChIP) assays were carried out using a ChIP Assay Kit(Millipore). Briefly, 2.5×105 HCT116 cells/well were seeded inMULTIWELL™ 6 well  and co-transfected with 200 ng pGL3-TATA-N2PR−2327/−99, 200 ng pGL3-TATA-N2PR −110 and either 2.1 µg6×histidine tagged pcDNA6-hTcf-4 or pcDNA6-mLef-1, respectively, usingLipofectamine LTX (Invitrogen). 48 h post transfection, cells were fixed with1% formaldehyde for 10 minutes and washed twice in ice-coldphosphate-buffered saline. Cells were lysed in sodium dodecyl sulfate lysisbuffer  containing protease inhibitors, and DNA in the cross-linked chromatinpreparations was sonicated to an average fragment size of 0.2–1.0 kb usinga Bioruptor™  and 14 pulses for 30 seconds sonicationfollowed by 30 seconds of rest. The insoluble material was removed bycentrifugation, and soluble chromatin samples were precleared with a 50%slurry of protein A-Sepharose-salmon sperm DNA. Each sample was incubatedovernight at 4°C with 5 µg of rabbit polyclonal antibodies 6×His tag® antibody - ChIP Grade (Abcam). As a negative isotype control,Normal rabbit IgG (Millipore) was used. Immune complexes were collected withprotein A-Sepharose and eluted. Input templates were purified from 5% ofthe original lysates in parallel with the eluted immunoprecipitated samples.Cross-linking was reversed by incubation at 65°C for 4 h in 200 mM NaCl.After phenol-chloroform extraction and ethanol precipitation, the recovered DNA(2 µl from immunoprecipitated chromatin DNA samples or 2 µl from theinput DNA control) was subjected to PCR amplification with the followingprimers:To investigate binding of LEF-1/TCF-family members Lef-1 and Tcf-4 toN2PR −110 ChIP forward: 5′- GGGGAGTCGAGGCATTTG -3′N2PR −110 ChIP reverse: 5′- AGGAGCCCCACTCTCTCCT -3′.As a negative control 170 bp of the NCAPD2  were amplified using primers:NCAPD2 ChIP forward: 5′-ATGGTTGCCACTGGGGATCT-3′NCAPD2 ChIP reverse: 5′-TGCCAAAGCCTAGGGGAAGA-3′.in vitro luciferase assays were performedusing Independent sample t-tests and error bars describe the standard error ofthe mean (SEM). Differences in mRNA levels between mouse adenomas and normalmurine intestinal tissue were analyzed with the Mann-WhitneyU-test  and error bars describethe interquartile range. Figure composition and adjustments in whole imagecontrast levels were performed in Adobe Photoshop CS4.All statistics and graphs are computed in GraphPad Prism 5  or SPSS v15.0 . Groupcomparisons for the Figure S1Competitive electro mobility-shift assay reveals binding ofin vitro translatedβ-catenin/Lef-1 to Notchpathway gene promoters. Duplex CD1TOP probes were “endlabeled” with [32P]dATP, incubated within vitro translated Lef-1/β-catenin and exposed tocompetition with abundance of cold duplex oligonucleotides . The protein-DNA complexes were separated byelectrophoresis and visualized by autoradiography. As a competition controlcold CD1TOP and cold mutated CD1FOP competed with radiolabeled CD1TOP and toconfirm that radioactive labeled CD1TOP binds Lef-1 specifically,plasmid-free reticulocyte lysate were subjected to in vitrotranslation and incubated with radiolabeled probe. Competition of cold and–cold mutated Jagged-1 −1933, −1635,−1083, −238, Maml-1 −746, −698,−1864 and Hes-1 −528, −568, −476,Positive competitor control, plasmid free reticulocyte lysate,Notch-2 −2261, −869, −689,−110, Rfng −2481, Numb−342, Lfng −2128, −1513, −1291,−1023 and Numbl −2587, −2480 and−1919. Numbering of putative LEF-1/TCF-sites is relative to eachgene's translational start site.(TIF)Click here for additional data file.Figure S2Notch pathway gene expression followingRNAi silencing ofβ-catenin in HT29 cells. Gene expression was normalizedagainst the negative control Gapdh. The expression ispresented 0–96 h post transfection. (A) The expression ofCyclin D1, Gapdh,Hes-1, Hes-7,Notch-2, Maml-1, Lfng,Rfng, Numb land Numb.(B) The protein expression of β-catenin (∼88 kDa) and loadingcontrol, Gapdh, 0–96 h post transfection ofanti-β-catenin-siRNA.(TIF)Click here for additional data file.Figure S3β-catenin and cyclin D1expression remain unaffected in DAPT treated HT29 cells. HT29cells were treated with 12.5 µM of the γ-secretase inhibitor DAPTfor 24 h thereby inhibiting Notch signaling. (A) Western blots with Hes-1and β-catenin antibodies on HT29 whole-cell lysate following 24 h DAPTtreatment. Gapdh was used as a loading control. (B) Theexpression of cyclin D1 was semi-quantitatively determinedin DAPT treated HT29-cells. Semi-quantitative RT-PCR was carried out on cDNAreversely transcribed from 200 ng total RNA. Bars describe the relativeexpression of cyclin D1 in HT29 normalized againstGapdh expression.(TIF)Click here for additional data file.Figure S4HT29 cells were co-transfected with pGL3-TATA carrying N2PR -110 (or emptypGL3-TATA) and HA-S33Y-β-catenin in pCGN as well aspSV-β-galactosidase control vector (n = 6).(TIF)Click here for additional data file.Table S1(XLS)Click here for additional data file.Supplemental Information S1(DOC)Click here for additional data file.


CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC) cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF)-1α expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1α bound to the hypoxia-response element  of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1α significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1α may regulate cancer cell migration through CX3CR1. The HIF-1α/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC. CX3CR1 is normally expressed by hematopoetic cells Despite diagnostic and therapeutic advances, PDAC still has a very poor prognosis. PDAC accounts for the fourth largest cause of cancer-related deaths in the United States, and its 5-year survival rate is only 5% It is well known that the expression of cytokines is usually regulated by specific transcription factors. Hung et al. In this study, we aimed to investigate (i) the mechanism of CX3CR1 regulation by hypoxia, (ii) the role of HIF/CX3CR1 in the chemotactic migration of PDAC, and (iii) the correlation between HIF and CX3CR1 in specimens of pancreatic cancer.2, using Dulbecco’s modified Eagle media (DMEM) with 10% fetal bovine serum. For hypoxic treatment, cells were placed in a modulator incubator  in an atmosphere consisting of 93.5% N2, 5.0%CO2 and 1.5% O2.MiaPaCa2, AsPc1 and CaPan1 human PDAC cells were from the American Type Culture Collection, Patu8988 cells Whole-cell extracts were prepared by lysing cells with SDS lysis buffer supplemented with proteinase inhibitors cocktail (Sigma). Protein concentrations were quantified using Pierce protein assay kit. Protein lysates (20 µg) were separated by SDS-PAGE, and target proteins were detected by Western-blotting with antibodies against HIF-1α, HIF-2α, CX3CR1 and β-actin . SpecifiTotal RNA was isolated from transfected cells by the TriPure Isolation Reagent (Roche) and used for first-strand cDNA synthesis through the First-Strand Synthesis System for reverse transcription-PCR. Then, 1 µg sample of the cDNA was quantified by real-time PCR using primer pairs with SYBR Green PCR Master mix . Each sample was done in triplicate. β-actin was used as loading control. PCR primers used are indicated in Cells treated with siHIF1α duplexes or pcDNA3.1-HIF1α overexpression plasmids were analyzed in an EPICS XL (Beckman Coulter) flow cytometer through FITC-labeled antibody against CX3CR1 (Biolegend).To assess HIF-1α and CX3CR1 distribution, MiaPaCa2 and Patu-8988 cells were seeded on glass slides pretreated with poly-L-lysine for different treatment. Then, cells were washed once with PBS and fixed with 4% PFA for 15 min at room temperature. After blocking with 2% bovine serum albumin and normal goat serum in PBS, cells were stained with anti-CX3CR1  antibodies, followed with a DAPI staining. Coverslips were mounted with 90% glycerol, and sections examined with a confocal microscopy.A chromatin immunoprecipitation assay was performed using a commercial kit (Upstate Biotechnology) according to the manufacturer’s instruction. Primers flanking the HRE of the VEGF promoter were used as a positive control Small interfering RNAs (siRNAs) against HIF-1α and HIF-2α and pEGFP-C1-CX3CR1 plasmids were designed and synthesized from Ribobio  . pcDNA3.Genomic DNA fragments of the human CX3CR1 gene 5 cells/well in 6-well plates containing serum-containing medium. When the cells were 80% confluent, siHIF1α duplexes or pcDNA3.1-HIF1α overexpression plasmids were transfected into cells using lipofectamine-2000 (Invitrogen) for 48 h. The cells were collected for migration analysis, Western-blotting, RT-PCR, flow cytometry and confocal microscopy.For transfection, cells were plated at a density of 5×106 cells/mL in 6-well plates with serum-containing medium and stimulated with tumor necrosis factor α (TNFα) (20 ng/mL) and IFNγ (500 units/mL) (Peprotech). After overnight incubation, medium was replaced with serum-free medium for 24 h. The conditioned supernatants were collected SK-N-BE neuroblastoma cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences . Cells were seeded at 105 cells was placed into the top of migration chambers with 8-µm filters , which were standing in wells containing 700 µl of media with recombinant CX3CL1 (200 ng/ml) or containing 700 µl of SK-N-BE conditioned supernatants. The cells were incubated at 37°C for 12 h, after which the chambers were removed from the wells. Cells that had migrated to the other side of the filter bottom were fixed with 2.5% glutaraldehyde for 15 min, rinsed thoroughly with PBS, and stained with trypan blue for 5 min. Cells were counted in 10 fields . Each experiment was performed in triplicate.For transfection, cells were incubated with siHIF1α duplexes or pcDNA3.1-HIF1α overexpression plasmids for 48 h. Then, cells were rinsed with PBS and suspended in DMEM. A total of 200 µl of serum-free media containing 2.5×10With an approval from local ethics committee, PDAC samples were obtained from 95 patients  undergoing surgical resection between July 1997 and April 2010. Histologic diagnosis of PDAC was confirmed at the Tianjin Cancer Institute & Hospital. H&E slides of all the 95 cases were reviewed to evaluate the degree of neural invasion, defined as the presence of cancer cells in the perineurium of nerve fascicles as previously described 2O2, sections were blocked with goat serum and incubated with anti-HIF-1α (1∶100 dilution), anti-CX3CR1 (1∶350 dilution) or anti-S-100 antibodies (overnight at 4°C)  . The sec5) were injected subcutaneously into the right flanks of female nude mice (eight mice per group). For delivery of cholesterol-conjugated siHIF-1α, 10 nmol RNA in 0.1 ml saline buffer was injected into tumor mass once every 3 days for 3 weeks All animal experiments were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of Tianjin Cancer Institute & Hospital. For preparation of subcutaneous model, MiaPaCa2 cells  or hypoxia (1.5% O2) for different times. The hypoxic condition was defined to mimic the average oxygen tension in tumors reported previously Since hypoxia is a common feature of the microenvironment of solid tumors, it is important to detect whether hypoxia promotes the expression of CX3CR1 in pancreatic cancer cells. CX3CR1 expression was determined by Western-blotting on MiaPaCa2 and Patu8988 cells cultured under normoxia . In contrast, repression of HIF-1α notably decreased the expression of CX3CR1 (P<0.01).To determine the roles of HIF-1α in the expression of CX3CR1, we up-regulated the expression of HIF-1α by tansfecting pcDNA3.1-HIF1α plasmids. Following the overexpression of HIF-1α, CX3CR1 protein and mRNA expression and CX3CR1 distribution were markedly increased  under hypoxia. However, CX3CR1 promoter was not found in samples pulled down by a control IgG antibody.Having shown that HIF-1α plays a critical role in CX3CR1 expression, we further investigated whether HIF-1α directly regulated CX3CR1 gene. Screening of the 5′-flanking region of the CX3CR1 gene revealed eight potential HIF-1-binding sites . To demoP<0.01) and Patu8988 cells  compared with control vectors (pcDNA3.1)  markedly increased the CX3CR1 promoter activity in MiaPaCa2  .P<0.01). Repression of HIF-1α decreased the chemotactic migration of MiaPaCa2 cells (P<0.01).To explore the roles of HIF-1α in migration of PDAC cells in vitro, we performed chemotactic migration assays as described previously P<0.01). Repression of HIF-1α decreased the chemotactic migration of MiaPaCa2 (P<0.01). Taken together, both loss-of-function and gain-of-function studies demonstrated that HIF-1α is involved in the chemotactic migration of PDAC cells in vitro.It has been reported that CX3CR1 mediated chemotactic migration toward CX3CL1 gradients. We further detected that overexpression of HIF-1α increased the chemotactic migration of MiaPaCa2 , left anMiaPaCa2 , right aMiaPaCa2 , right cP<0.01), suggesting that CX3CR1 is involved in the chemotactic migration mediated by HIF-1α.To verify the roles of CX3CR1 in HIF-1α mediated chemotactic migration, we co-transfected siHIF1α duplexes and pEGFP-C1-CX3CR1 plasmids into MiaPaCa2 cells. As shown in F = 7.81, P<0.01), lymph node metastasis , the pathological tumor node metastasis stage  and PNI  of PDAC samples (P<0.01) (P<0.05) (We assessed the expression of HIF-1α in PDAC samples by immunohistochemistry and found that HIF-1α was correlated with tumor diameter ( samples . PDAC pa(P<0.01) , left. F(P<0.05) , right.r = 0.58, P<0.01).CX3CR1 has been identified as an important factor for PNI Nerve terminations were surrounded by tumor cells with high expression of HIF-1α and CX3CR1 . Hence, In this study, we have confirmed that CX3CR1 was highly expressed in both clinical samples and cell lines of pancreatic cancer and that hypoxia induced the expression of CX3CR1. Furthermore, small interfering RNAs (siRNAs) analysis revealed that HIF-1α, but not HIF-2α, influenced the hypoxia-induced expression of CX3CR1 in vitro and in vivo. Meanwhile, overexpression of HIF-1α protein upregulated the expression of CX3CR1 in pancreatic cancer cells.As a transcription factor, HIF-1 exerts its biological function through activating target genes. By chromatin immunoprecipitation and luciferase analysis, we showed evidence that HIF-1α bound to the HREs region of CX3CR1 promoter and upregulated the transcription. It has been demonstrated that CX3CR1 could regulate migration and PNI of pancreatic cancer cells The CX3CR1 receptor has been shown to influence the prognosis of PDAC patients. In our study, clinicopathological and experimental data uncovered the co-expression of HIF and CX3CR1 around nerve tissues in PDAC. Although PNI is a key pathological feature of PDAC relating to recurrence, little is known about its mechanism. Previous investigations were limited to the cytokines and their receptors for the interaction between cancer cells and nerve cells. In our opinion, it is more significant to uncover the mechanism of chemotactic migration at transcriptional levels because the transcription factor represents the mutual pathways for multiple effectors. HIF-1 might be a key transcription factor to influence chemotactic migration and could be an effective target to prevent the recurrence of PDAC.Previous studies showed that HIF-1 was constitutively expressed in pancreatic cancer. In deed, intratumoral hypoxia and oncogene mutation (such as Akt or MAPK pathways) induce the expression of HIF-1α. HIF-1α expression in PDAC cells is heterogeneous. So, different PDAC cells have various extents of chemotactic migration. Recent evidence has verified that CX3CL1 upregulated the expression of HIF-1α in human aortic endothelial cells CX3CR1 was expressed by dendritic cells, natural killer cells, macrophages and monocytes and was associated with immune-related diseases. According to our findings, HIF-1 may also mediate the expression of CX3CR1 in immune cells and be involved in hypoxia-related inflamatory response. Nowadays, many chemical compounds are available as inhibitors of HIF-1 Figure S1MiaPaCa2 (left) and Patu-8988 (right) cells were treated with CX3CL1 (200ng/ml) for different times and then were lysed for western-blotting.(TIF)Click here for additional data file.Table S1The antibodies, siRNAs and primer sequences in the experiment.(DOC)Click here for additional data file.


Sacrococcygeal teratoma accounts for half of all fetal tumors, with a prevalence of 1 : 40,000 births. It is believed to originate from pluripotent cells in Hensen's nodule. Although most are benign, they are associated with high morbidity and mortality rates because the fetus develops congestive heart failure and hydrops. Factors leading to poor prognosis include solid components in the mass, and hydrops diagnosed before the 30th week. A case of prenatal sacrococcygeal teratoma diagnosed using B-mode and color Doppler two-dimensional ultrasonography (2DUS) is described, in which three-dimensional ultrasonography (3DUS) enabled characterization of the extent of fetal lesions and allowed the parents to understand the pathological condition better. A 20-year-old primigravida was referred with a solid mass diagnosed in the lumbosacral spine. Examinations performed at our institution revealed pregnancy of 23 weeks and 4 days, with a female fetus presenting a bulky solid mass with cystic components and calcifications, measuring 7.7 × 9.1 × 12.2 cm, starting from the sacral region, with internal flow seen on color Doppler. A new ultrasound confirmed fetal death at 25 weeks and 4 days. Postnatal findings confirmed the diagnosis of sacrococcygeal teratoma. 3DUS can be used in cases of sacrococcygeal teratoma to assess the development of tumor during the prenatal and to allow better understanding of this anomaly by the parents. Sacrococcygeal teratomas are the commonest retroperitoneal benign tumors. They are believed to originate from pluripotent cells in Hensen's nodule, which is located on the anterior surface of the sacrum or coccyx. Although they constitute more than half of all fetal tumors, their incidence is only 1 : 40,000 live births, with a female-to-male ratio of 4 : 1  describeAdvances in ultrasonography have enabled accurate early diagnosis of sacrococcygeal teratomas when they present in the form of cystic, solid, or mixed masses that form starting in the sacral region and protrude toward the perineum or buttocks . HoweverThree-dimensional ultrasonography (3DUS) has been used for diagnosing sacrococcygeal teratomas, and there have been reports in which these tumors have been diagnosed during the first trimester, by means of the surface mode , 5, 6, aWe present a case of solid-cystic sacrococcygeal teratoma that was diagnosed in the 23rd week of pregnancy by means of two-dimensional ultrasonography (2DUS) in association with color Doppler. We emphasize the main findings from 3DUS in rendering mode.A 20-year-old primigravida was referred to the Gynecology and Obstetrics Service of the University Hospital of the Federal University of Maranhão (UFMA) with a diagnosis of a solid mass in the lumbosacral spine. A 2DUS examination performed in our institution revealed pregnancy of 23 weeks and 4 days, with a female fetus presenting a bulky solid mass with cystic components and calcifications, measuring 7.7 × 9.1 × 12.2 cm, starting from the sacral region, with internal flow seen on color Doppler . 3DUS waDiagnosing sacrococcygeal teratoma prenatally is very important, because large tumors can lead to congestive heart failure, hydrops, and high rates of perinatal mortality. Differentiation between histological types and their potential for malignancy is an important factor in decision making, such as deciding to terminate the pregnancy in countries where this is possible. However, prenatal ultrasonography does not allow such differentiation .In our case, the diagnosis of sacrococcygeal teratoma was made in the 23rd week of pregnancy. With more widespread availability of prenatal screening programs for aneuploidy in the first trimester, it is now possible to make an early diagnosis of sacrococcygeal teratoma , 5. NoneIn our case, 2DUS showed the sacrococcygeal teratoma as a solid mass with cystic components. Highly vascularized tumors in which solid components predominate are generally associated with immature histology and a higher degree of malignant transformation . These t3DUS has been used in a manner that is complementary to 2DUS in diagnosing sacrococcygeal teratoma prenatally . TogetheIn our case, we used 3DUS in rendering mode to evaluate the morphology of the mass and its relationships with the fetal pelvis. 3DUS in rendering mode has already been used in diagnosing two cases of sacrococcygeal teratoma in the first trimester of pregnancy , 5. In tIn summary, we have presented a case of sacrococcygeal teratoma that was diagnosed by means of 2DUS in the 23rd week of pregnancy. 3DUS in rendering mode-enabled assessment of the morphology of the mass and its spatial relationships with the fetal and made it possible for the parents to have a better understanding of the pathological condition. We believe that 3DUS may be an important diagnostic adjuvant to 2DUS in diagnosing sacrococcygeal teratoma prenatally.


We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both  The canonical Wnt/β-catenin signaling is crucial for normal embryonic development, patterning, and its abnormal expression is associated with many forms of oncogenesis The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain is identified at a large number of human proteins, including transcription factors, ion channel proteins, oncogenes, and cytoskeletal proteins in vivo and in vitro. Moreover, KCTD1 downregulated the expression of β-catenin and Wnt-specific genes by the proteasome pathway, which was dependent on CK1/GSK-3β-mediated phosphorylation. Furthermore, KCTD1-mediated degradation of β-catenin was blocked by APC but enhanced by β-TrCP or p53. These results revealed that KCTD1 enhances β-catenin degradation linking CK1, GSK-3β, APC and p53.KCTD1 and KCTD15 are two closely related proteins in the KCTD family 4) Plasmids expressing pCMV-Myc-KCTD1, pCMV-HA-KCTD1 and pGEX-4T-2-KCTD1 were described previously 2. Cells were transfected with plasmid DNA or siRNA using Lipofectamine 2000  according to the manufacturer's instructions.All cell lines were obtained from American Tissue Culture Collection. HeLa and HEK293 cells were cultured in DMEM medium , SW480 cells were cultured in RPMI 1640 medium (Gibco). All of these cells were cultured with 10% fetal calf serum , 4 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% COHEK293 and SW480 cells were cultured in 12-well plates and transfected with reporter plasmids pTOPFLASH, pFOPFLASH or A2-Luc and the indicated plasmids using Lipofectamine 2000. The pCMV-LacZ vector was cotransfected as an internal control and β-galactosidase activity was used to normalize for different transfection efficiencies. The amount of DNA in each transfection was kept constant by the addition of empty expression vector. In certain experiments HEK293 cells were transfected for 24 h and then treated with 100 ng/ml of Wnt-3a for 36 h. Following the transfection, β-galactosidase and luciferase activity were measured using the luciferase assay system  in a TD-20/20 Luminometer . Each transfection was performed in triplicate wells and replicated in at least three independent experiments.HeLa cells in 10 cm dishes were grown to 80% confluence, and transfected with 5 µg of Myc-KCTD1. After 30 h, the cells were harvested and lysed as previously described Full-length and truncated GST-KCTD1 fusion proteins, GST and His-β-catenin were expressed and purified according to manufacturer's instructions (Amersham). 5 µg of the GST or GST fusion proteins were mixed with 40 µl of 50% suspension of Glutathione Sepharose 4B beads (Amersham) for 1 h in binding buffer . Then 5 µg of His fusion proteins were added followed by incubation for another 2 h. The pellets were washed extensively and boiled in SDS sample buffer. The bound proteins were resolved by 10% SDS-polyacrylamide gel and detected by western blot analysis with mouse monoclonal antibodies against His-tag (631212) (Clontech).Truncated His-β-catenin fusion proteins were expressed and purified according to manufacturer's instructions (Amersham). 5 µg of the His-β-catenin or His-β-catenin deletion fusion proteins were mixed with 40 µl of 50% suspension of Chelating Sepharose Fast Flow  charged with nickel ions for 1 h in the binding buffer. Then 5 µg of GST fusion proteins were added followed by incubation for another 1 h. The pellets were washed extensively and boiled. A negative control experiment was performed simultaneously with His-β-catenin fusion proteins and GST. The bound proteins were resolved by 12% SDS-PAGE gel and detected by western blot analysis with mouse monoclonal antibodies against GST (sc-138) (Santa Cruz Biotech).HeLa cells were grown to 70% confluence on glass coverslips and cotransfected with 3 µg of pCMV-HA-KCTD1 and 3 µg of pCMV-Myc-β-catenin or only transfected with either 3 µg of pCMV-HA-KCTD1 or 3 µg of pCMV-Myc-β-catenin. After 24 h, cells were treated as described previously HeLa cells were cotransfected with different amounts of expression plasmids pCMV-Myc-KCTD1 and the same amount of pCMV-Myc-β-catenin plasmids or cotransfected with either plasmids pCMV-Myc-KCTD1 and pCMV-Myc-β-catenin (WT) or with plasmids pCMV-Myc-KCTD1 and pCMV-Myc-β-catenin (Mut). To identify protein degradation pathway, transfected cells were treated with 20 µM of MG132, a specific proteasome inhibitor for 10 h prior to harvesting. Cells were collected and lysed in the RIPA buffer followed by Western blotting detected with mouse monoclonal antibodies against Myc-tag, AP-2α(sc-12726), β-actin (sc-47778) and CCND1 (sc-56302) (Santa Cruz Biotech). To detect the effects of APC and p53 on KCTD1-mediated β-catenin downregulation, HeLa cells were transfected with pCMV-Myc-β-catenin either alone or with pCMV-Myc-KCTD1, pCMV-Myc-APC (a.a. 1-453 and 454-1020) or pCMV-HA-p53 or in combination as indicated. The cell lysates were analyzed with mouse monoclonal antibodies against Myc-tag, HA-tag (sc-7392) and GAPDH (sc-32233) (Santa Cruz Biotech).HeLa cells were transfected with either expression plasmids pCMV-Myc-β-catenin alone or with pCMV-Myc-KCTD1, pCMV-Myc-ubiquitin or pCMV-Myc-β-TrCP as indicated. 24 h after transfection, cells were harvested and lysed. Myc-tagged β-catenin was immunoprecipitated with rabbit polyclonal antibodies against β-catenin and these immunoprecipitates were subjected to Western blotting with mouse monoclonal antibodies against Myc-tag and ubiquitin (sc-8017) (Santa Cruz Biotech) to detect β-catenin-ubiquitin conjugation.t test. Values of P<0.05 were considered statistically significant.Data are presented as mean ±SD from at least three independent experiments. The significance of difference was assessed using Student's Because Zebrafish Kctd15, the homolog of human KCTD1 gene, inhibits neural crest formation by antagonizing the canonical Wnt/β-catenin signaling in vivo. HeLa cells were transiently transfected with pCMV-Myc-KCTD1, the lysates were immunoprecipitated with the indicated antibodies, and the co-immunoprecipitated proteins were analyzed with Western blots. Endogenous β-catenin was detected in immune complexes of Myc-KCTD1, but control IgG did not precipitate any band , but KCTD1 suppressed TCF/LEF reporter activity of mutant β-catenin with S37F, suggesting KCTD1 mediates β-catenin degradation dependent on CK1/GSK-3β-mediated phosphorylation. However, mutation of the β-catenin gene most commonly occurs and ultimately results in stabilization and nuclear accumulation of β-catenin in cancer cells Our findings concur with the notion that APC fragments containing at least one SAMP repeat are efficient at promoting β-catenin degradation Taken together, our findings support a novel function that KCTD1 represses canonical Wnt/β-catenin pathway through its interaction with β-catenin, resulting in subsequent ubiquitin-dependent degradation of β-catenin dependent on CK1/GSK-3β-mediated phosphorylation and β-TrCP-mediated proteasome pathway, thus, facilitates the downregulation of Wnt downstream target genes. Although the physiological functions between KCTD1 and Wnt signaling pathway in human cancers are needed to clearly elucidate in future studies, KCTD1 might play a crucial role in particular tumors at least in part by promoting the degradation of wild-type β-catenin, a core player in canonical Wnt/β-catenin signaling pathway.


Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect β-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In β-catenin(−/−) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, β-catenin/TCF target genes are induced in β-catenin(−/−) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of β-catenin. However, β-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed.β-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The β-catenin homologue γ-catenin  is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use β-catenin However, ectopic expression of γ-catenin in WT mESCs robustly activates Wnt target genes, blocks neuronal differentiation and reinforces the retention of pluripotent stem cell markers Oct-4, Sox2 and Nanog, whereas knocking down its expression via shRNAs appears to promote exit from the pluripotent state. The overexpression of γ-catenin does not appear to be as potent as the overexpression of β-catenin in preventing differentiation to an EpiSC-like state, as assessed by marker analyses in short term differentiation assays. Intriguingly, ectopic expression of γ-catenin in β-catenin(−/−) cells is sufficient to enable TCF target gene activation upon the inhibition of GSK-3, suggesting that a threshold level of γ-catenin is required before it is accessible for regulation by the machinery of the β-catenin destruction complex. Collectively, our findings suggest partial functional overlap between γ-catenin and β-catenin in the regulation of mESC properties, and have implications for cancers in which γ-catenin is mutated and/or misexpressed.In this study, we utilized β-catenin+/+) were maintained in DMEM (Thermo) supplemented with 15% FBS (Fisher), 1× nonessential amino acids, 1× L-glutamine, 1× sodium pyruvate, 55 µM β-mercaptoethanol  and 1000 U/ml ESGRO® LIF (Millipore). All cells were kept in a humidified incubator at 37°C, 5% CO2. The medium used for EB experiments was identical to that above, except that it contained 5% FBS and lacked LIF. The N2B27 culture medium used to initiate neural differentiation consisted of a 1∶1 ratio of Neurobasal media and DMEM/F12 with N2 and B27 supplements Cell culture supplements were obtained from Gibco (Life Technologies) unless otherwise specified. Mouse ESCs ; mouse anti-β-catenin ; rabbit anti-β-catenin ; mouse anti-FLAG-tag ; mouse anti-GAPDH ; mouse anti-β-Tubulin 1 ; mouse anti-Myc-tag ; rabbit anti-Nanog ; mouse anti-Oct3/4 ; rabbit anti-Sox2 ; mouse anti-β-III-Tubulin .To generate myc-tagged γ-catenin expression constructs in pCAG-IP All chemicals were purchased from Sigma, unless otherwise specified. To prepare whole cell lysates, 60 mm dishes of cells were rinsed twice with PBS at room temperature and were then lysed with ice-cold RIPA buffer  on ice for 10 minutes. Lysed cells were transferred into 1.5 mL tubes on ice and centrifuged at 16, 100 × g for 10 minutes (4°C). The supernatants, containing soluble proteins, were isolated and the protein content was quantified using the Lowry method . The samples were then prepared for electrophoresis by diluting them to contain 1× LDS buffer (Invitrogen) with 5% TCEP Bond-Breaker solution (Thermo) and heating them for 5 minutes at 95°C prior to western blot analysis. Hypotonic (cytosolic) lysates were prepared by lysing PBS-washed cells in ice-cold 1 × hypotonic lysis buffer , incubating on ice for 30 minutes, and then centrifuging at 235, 000 × g in a Sorvall M150 ultracentrifuge for 30 minutes at 4°C. The supernatant (cytosol) was collected and the protein content was quantitated as described above.Protein extracts from nuclear and cytoplasmic fractions were performed as described previously Jup were purchased from Origene (Cat. No. TR501141). After a preliminary screen to identify the most effective shRNA sequence in mESCs, an shRNA construct harbouring the following sequence was used in these studies: γ-catSH: AGAGTGGCAACAGACATACACCTACGACT. The negative control constructs used were: 1) Vector (empty pRS vector); and 2) NegCtrlSH .HuSH 29mer shRNA constructs against (+/+)) and β-catenin(−/−) mESC cell lines were derived in Dr. Austin Cooney's laboratory, and have been described previously Transgenic and knockdown cell line generation using mESCs was performed as described previously mESCs were cotransfected with the constructs 8X TOPFlash (1.8 µg), driving firefly luciferase Total RNA was isolated using the PureLink RNA Mini Kit (Life Technology), and 1 µg was used to generate cDNA with SuperScript II reverse transcriptase (Invitrogen). The cDNA was diluted to 100 µl and 3 µl of this was used for each 20 µl PCR reaction with PerfeCTa® SYBR Green® FastMix®, ROX™ (Quanta Biosciences). β-actin was used as the housekeeping gene for all qRT-PCR assays. Stratagene's Mx3000P instrument and accompanying MxPro software or Bio-Rad's CFX96 instrument and CFX Manager™ software were used to determine relative gene expression levels using the delta-delta Ct method with data obtained after 40 cycles of PCR. Primer sequences were designed using IDT's online primer design software (idtdna.com) or were obtained from prior publications and are listed in EB formation and staining were performed as described previously 2O in a 2∶1∶1 ratio; Millipore, SCR004) for 15 minutes at room temperature in the dark. An additional wash with 0.2% TBS-T was performed before the cells were visualized using an Olympus IX-81 microscope and standard brightfield illumination.Cell lines were grown on iMEFs for 24 hours, prior to pre-plating to remove the iMEFs, as described above. Cells were subsequently counted and plated in the various conditions indicated. After 72 hours, cells were fixed in 4% paraformaldehyde for 2 minutes, washed in 0.2% TBS-T and incubated in AP staining reagent  and unpaired t-test analyses were performed using the statistical software package Prism (GraphPad).Although previous studies have linked γ-catenin to the regulation of Wnt signaling by virtue of its homology to β-catenin, few have directly assayed whether γ-catenin stability is regulated by GSK-3-mediated phosphorylation. To determine whether γ-catenin was stabilized in mESCs after inhibition of GSK-3, we treated cells with the highly specific GSK-3 inhibitor CHIR99021 (15 µM for 24 hours), and assayed for cytoplasmic stabilization of γ-catenin using western blotting . As expe(−/−) cell lines with efficent γ-catenin knockdown. In wild-type mESCs, compensatory upregulation of β-catenin was not observed in γ-catenin knockdown clones (Axin2 and Brachyury), in response to GSK-3 inhibition (15 µM for ∼24 hours). No readily detectable stabilized γ-catenin was observed in cytoplasmic extracts derived from γ-catenin knockdown lines (clones 2B and 2D) after treatment with CHIR99021, relative to controls (Axin2 and Brachyury were robustly upregulated in WT and control lines (Vector and NegSH) after treatment with CHIR99021 (Axin2 or Brachyury expression ((−/−) mESCs did not significantly  reduce basal TCF reporter activity or the expression of the general TCF target gene Axin2 or the pluripotency-related TCF3 target genes Nanog and Esrrb  mESCs ((−/−) mESCs (compare lanes 1 and 2 of (−/−) mESCs  mESCs ((−/−) mESCs.Although γ-catenin knockdown had no detectable effect on Wnt signaling in WT mESCs , we hypo−) mESCs . Indeed,−) mESCs . Accordi−) mESCs . We conc(−/−) mESCs ((−/−) mESCs; as expected, these cells exhibited elevated Axin2, Brachyury and Cdx1 expression following GSK-3 inhibition  mESCs expressing a mutant form of γ-catenin, lacking its C-terminal TCF transactivation domain (γ-catΔC), remained largely incapable of Wnt target gene activation, even after GSK-3 inhibition. γ-catS28A, harboring a serine to alanine point mutation at a putative GSK-3 phosphorylation site Axin2, Brachyury and Cdx1 expression in the β-catenin(−/−) mESCs in the presence or absence of GSK-3 inhibitor. The ability of γ-catS28A to induce target gene expression required an intact C-terminus, as γ-catS28AΔC-expressing cell lines displayed negligible target gene activation  mESCs , and ask−) mESCs . As a cohibition . Intrigu-catenin . This activation .Brachyury expression was similar to that obtained with stable β-catenin overexpression), suggesting that the ability of γ-catenin to activate gene expression is dependent on the context of target sequences.It has become increasingly clear that the inhibition of GSK-3 enhances the self-renewal of pluripotent mESCs in culture, and facilitates the derivation of ESC lines from resistant strains from different species It has been reported that overexpression of γ-catenin can be associated with concomitant increases in stabilized β-catenin in some tumor cell types We next examined the differentiation capabilities of γ-catS28A mESCs. To make an initial assessment of the ability of γ-catS28A mESCs to exit the pluripotent state, we used short-term loss of pluripotency assays, essentially as were performed previously for mESCs expressing β-catS33A Although we demonstrated that ectopic expression of β-catS33A and γ-catS28A delays the loss of pluripotency in N2B27 medium , we founEsrrb, Klf4, Nanog, Pecam-1, Rex1 and StellaFGF5 and Otx2Esrrb, Klf4, Nanog, Pecam-1 and Rex1, compared to the levels detected in WT control mESCs. By contrast, Stella expression was approximately 4-fold lower in β-catS33A and γ-catS28A ovexpressing mESCs than in WT mESCs, which was unexpected. Taken together, with the exception of Stella, the marker analyses indicate that mESCs overexpressing β-catS33A and γ-catS28A, when maintained in LIF-containing medium, reside in a state of naïve pluripotency. The unexpected Stella results suggest that this gene does not share the same regulatory controls that guide the expression of the other core pluripotency factors in response to Wnt/β-catenin signaling.As it has been reported that Wnt/β-catenin signaling serves to prevent the differentiation of naïve pluripotent mESCs to primed pluripotent mEpiSCs FGF5 (more than 200-fold higher expression in EB conditions relative to ES conditions). In the same differentiation conditions, mESCs overexpressing γ-catS28A displayed attenuated expression of FGF5 compared to the expression observed in WT mESCs (approx. 50% of WT levels) but overexpression of β-catS33A was much more efficient in suppressing FGF5 expression in cells maintained in EB medium (approx. 10% of WT levels). With regard to the EpiSC marker Otx2, γ-catS28A overexpression had no effect on the induction of Otx2 expression in EB culture conditions. That is, cell lines overexpressing γ-catS28A displayed an approximately 10-fold higher level of Otx2 expression in EB relative to ES conditions, which was also observed in the WT cells. By contrast, mESCs overexpressing β-catS33A showed levels of Otx2 expression in EB conditions that were approximately 50% of those detected in WT mESCs.Wild-type mESCs maintained in EB differentiation conditions for 72 hours  showed dramatically upregulated expression of the EpiSC marker Esrrb, Nanog, Pecam-1 and Rex1 to approximately 25% of the expression observed in ES medium, whereas the cell lines overexpressing stabilized β-catenin or stabilized γ-catenin displayed expression of these markers at levels similar to those obtained from analyses of WT mESCs in ES medium. The expression of Klf4, a marker of naïve pluripotent cells, was differentially affected by the expression of stabilized β-catenin versus stabilized γ-catenin. Cells expressing γ-catS28A displayed the same reduction in Klf4 expression as observed in WT mESCs in EB medium, whereas β-catS33A-expressing cells retained approximately 5-fold higher levels of Klf4 expression in the same conditions. In the N2B27 differentiation conditions, which promote neurectodermal differentiation Esrrb, Klf4, Nanog, Pecam-1 and Rex1, which were the same or higher than those detected in WT mESCs maintained in ES medium. By contrast, the expression of these markers was reduced to similar extents in WT mESCs and γ-catS28A mESCs maintained in N2B27 medium. Taken together, our data suggest that β-catenin overexpression is more effective than γ-catenin overexpression in sustaining the retention of markers of naοve pluripotency, and preventing EpiSC differentiation, in cells that have been subjected to differentiation-inducing conditions.In EB differentiation conditions, WT mESCs displayed a reduction in the expression of mESC markers, To assess the tri-lineage differentiation capacities of β-catS33A and γ-catS28A mESCs, we generated embryoid bodies using these lines, and examined the levels of neuronal markers by immunofluorescence  and qRT-Xenopus laevis embryos Xenopus laevis or immortalized mammalian cell lines as model systems. In our study, we used mESCs as a physiologically relevant cell culture model that more accurately reflects the signaling events in early mammalian development. In mESCs, we found that, similar to β-catenin, γ-catenin is robustly stabilized upon Wnt pathway stimulation, achieved either through small molecule-mediated inhibition of GSK-3, or through treatment with Wnt3a-conditioned medium  cells, γ-catenin would become stabilized in response to GSK-3 inhibition and thereby substitute for β-catenin's role in the activation of TCF target genes. However, consistent with a recent study (−/−) mESCs, we found that GSK-3 inhibition in these cells does not elicit the cytoplasmic stabilization of γ-catenin, suggesting that β-catenin is needed to somehow maintain and/or enhance γ-catenin stability in response to pathway activation. Various possibilities exist as to why this might be the case. Perhaps β-catenin is an imperative and irreplaceable component of the pathway, and its presence is needed to recruit γ-catenin to the β-catenin destruction complex, where its stability can be modulated by pathway activation. Alternatively, any cellular γ-catenin in β-cat(−/−) mESCs may be fully sequestered by desmosomes and adherens junctions, such that the critical signaling pool cannot be regulated via the destruction complex per se. Our findings with ectopically expressed γ-catenin in β-cat(−/−) mESCs support this second scenario, as γ-catenin overexpression partially rescues the ability of β-cat(−/−) cells to respond to GSK-3 inhibition by activating TCF target genes ((+/+) and β-cat(−/−) mESCs), might γ-catenin then contribute to the transactivation of Wnt target genes?We used stably expressed γ-catenin-specific shRNAs to demonstrate that, in mESCs, suppression of γ-catenin expression does not detectably affect the transactivation of β-catenin/TCF target genes in response to Wnt pathway stimulation via GSK-3 inhibition . This exet genes . Thus, iet al. ultimately reach similar conclusions regarding the role of endogenous γ-catenin in Wnt signaling, our studies differ in certain key ways. First, the F9 line employed by Shimizu et al. harbors chromosomal abnormalities et al. observed only very modest γ-catenin stabilization, suggesting that the Wnt pathway does not appreciably affect γ-catenin stability in this cell line. Furthermore, our studies are the first to describe the functional consequences of γ-catenin overexpression and knockdown in mESCs with respect to their differentiation capacities in short-term assays.A previous study by Shimizu and colleagues assessed the roles of β-catenin and γ-catenin in Wnt signaling using the murine embryonal carcinoma (EC) F9 line et al. recently showed that γ-catenin is frequently overexpressed and mislocalized to the nucleus in acute myeloid leukemia Survivin(−/−) mESCs rescues their ability to regulate TCF target genes. Kolligs et al. c-myc expression, whereas β-catenin does not. We directly interrogated whether a form of γ-catenin, analogous to that harboring the S28A missense mutation identified by Caca et al. Axin2, Brachyury, Cdx1) when stably expressed in mESCs. Indeed, this form of γ-catenin robustly activated the TCF reporter and Wnt target genes  mESCs (http://www.sanger.ac.uk/re-sources/databases/cosmic.html), mutations in the gene encoding γ-catenin (Jup) occur in a small proportion of lung, ovarian and breast cancers. Whether these mutations give rise to forms of γ-catenin with altered signaling activity and/or expression patterns, remains to be determined.Interestingly, previous studies have suggested that γ-catenin variants harboring the S28A mutation do not exhibit significantly enhanced stability relative to wild-type γ-catenin, which is itself inherently more stable than β-catenin −) mESCs . As propNanog, a key target of which, Esrrb, has been linked to the effects of GSK-3 inhibition in mESCs There is accumulating evidence that small molecule-mediated inhibition of GSK-3, or activation of Wnt/β-catenin signaling by other means, reinforces mESC self-renewal and enhances iPSC derivation The precise mechanisms through which overexpressed γ-catenin exerts different effects from overexpressed β-catenin on mESC pluripotency and differentiation remain to be elucidated. It has been previously reported that γ-catenin is less efficient than β-catenin in forming a ternary complex with LEF1 and the LEF1-binding sequence (−/−) mESCs. However, in WT mESCs, overexpression of γ-catenin is sufficient to activate Wnt signaling without a requirement for β-catenin stabilization and knocking down endogenous γ-catenin levels appears to accelerate the exit of WT mESCs from the pluripotent state, suggesting that γ-catenin may aid in the maintenance of pluripotency. Our data also show that γ-catenin is responsive to GSK-3 inhibition, and can activate TCF target genes when the levels of its expression reach a critical threshold. Although β-catenin and γ-catenin both appear to function similarly when overexpressed in mESCs, based on their ability to block neurectodermal differentiation in embryoid body assays, there are clear differences in the ability of these proteins to sustain the expression of pluripotency markers and to prevent ESC-to-EpiSC differentiation. Taken together, our data suggest a mechanism through which γ-catenin may play a role in the genesis of cancers in which it becomes mutated and/or misexpressed, whereby it may promote the sustenance of a more primitive and/or de-differentiated cellular state.Overall, our study has demonstrated that the β-catenin homologue, γ-catenin, is not an integral component of the Wnt signaling cascade in mESCs, nor can endogenous γ-catenin substitute for β-catenin in this pathway of β-catenin


Additionally, paracrine actions of 1,25(OH)2D3 on stromal cells may also repress this pathway in neighbouring tumor cells. Here we review the molecular basis for the various mechanisms by which 1,25(OH)2D3 antagonizes Wnt/β-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate. The effect of the vitamin D system on Wnt/β-catenin signaling and tumor growth in animal models will also be commented in detail. Finally, we revise existing data on the relation between vitamin D receptor expression and vitamin D status and the expression of Wnt/β-catenin pathway genes and targets in cancer patients.The Wnt/β-catenin signaling pathway is abnormally activated in most colorectal cancers and in a proportion of other neoplasias. This activation initiates or contributes to carcinogenesis by regulating the expression of a large number of genes in tumor cells. The active vitamin D metabolite 1α,25-dihydroxyvitamin D Subsequent hydroxylation in the liver, kidney and other tissues renders 1α,25-dihydroxyvitamin D3 2D3, calcitriol), the most active vitamin D metabolite. 1,25(OH)2D3 is a pleiotropic hormone with many regulatory effects. In addition to its classical action on intestinal calcium absorption and bone biology, 1,25(OH)2D3 inhibits proliferation, migration and anchorage-independent growth, and promotes differentiation of a variety of cultured cancer cells [Vitamin Der cells ,5.2D3 regulates gene expression via binding to the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors that is expressed in many normal and cancer cell types. VDR heterodimerizes with Retinoid X Receptor (RXR) and acts as a ligand-regulated transcription factor modulating the transcription rate of hundreds of target genes [2D3 on membrane and cytosolic signaling molecules  [2D3/VDR complexes [1,25(OH)et genes ,7. VDR ahatases) . These eomplexes .Human Wnts are a group of 19 secreted proteins that control proliferation, survival, migration, differentiation, and/or lineage decisions in many cell types during development and adult life . To thisThe Wnt/β-catenin pathway controls the intracellular levels of β-catenin. In the absence of Wnt signals, free β-catenin is targeted by a cytoplasmic protein complex known as the β-catenin destruction complex, which promotes its phosphorylation, ubiquitination and degradation by the proteasome. Components of this complex include the tumor suppressors AXIN and APC and the protein kinases CK1 and GSK3β. Wnt binding to membrane heterodimeric receptors (Frizzled and LRP5/6) results in inhibition of destruction complexes and subsequent accumulation of unphosphorylated β-catenin molecules in the cytoplasm, a part of which enters the nucleus and behaves as a co-activator of LEF/TCF transcription factors. β-catenin/LEF/TCF target genes encode proteins that are involved in most if not all cellular processes including proliferation, cell cycle regulation, metabolism, migration, lineage commitment, and differentiation ,13.The Wnt/β-catenin pathway is mainly active during embryonic development, although it also contributes to homeostasis in different tissues in adult life. Importantly, its abnormal activation in a series of epithelia is linked to generation or progression of carcinomas of the colon, breast, liver, pancreas and others. In particular, activation of the Wnt/β-catenin pathway is the initial event in a high proportion of colorectal carcinomas ,14, and APC gene are by far the most frequent insult leading to constitutive activation of the Wnt/β-catenin pathway in colorectal cancer. However, in a small percentage of cases, mutations in AXIN or in the CTNNB1/β-catenin proto-oncogene itself have also been found. In addition, 20%–40% of hepatocellular carcinomas present mutations in CTNNB1/β-catenin or AXIN [Mutations in the tumor suppressor  or AXIN ,22. By cThe Wnt/β-catenin pathway cannot be considered intrinsically tumorigenic, as it promotes differentiation of some cell types such as osteoblasts, myoblasts and neural precursors ,24,25,26Non-canonical Wnt pathways include the so-called planar cell polarity pathway which involves activation of Rho small GTPases and JNK and ROCK kinases, and the calcium pathway with activation of protein kinase C, calmodulin kinase, calcineurin and NFAT transcription factor ,30. The The first evidences of crosstalk between nuclear hormone receptors (NHR) and Wnt/β-catenin signaling appeared the late 1990s  and earl2D3 antagonizes the Wnt/β-catenin pathway in human colon cancer cells by three main mechanisms -1, an extracellular inhibitor of Wnt signaling [2D3 inhibits the expression of several β-catenin/TCF target genes such as c-MYC, TCF1, LEF1, AXIN2, PPARδ, CD44, ENC1 and EPHB2 in human colon cancer cells [Results from our group showed that 1,25(OH)chanisms . First, membrane . And thiignaling . As a reer cells ,39,40. Ter cells ,42.et al. in other cell types [C-terminal region of β-catenin and the activator function-2 domain of VDR. Interestingly, while 1,25(OH)2D3 antagonizes β-catenin/TCF transcriptional activity, β-catenin potentiates that of 1,25(OH)2D3/VDR [et al. have reported that wild-type APC enhances VDR/β-catenin interaction and that the VDR ligand lithocholic acid also promotes the interaction, albeit to a lesser extent than 1,25(OH)2D3 [VDR/β-catenin interaction was later confirmed by Shah ll types . In addi)2D3/VDR ,43,44. R5(OH)2D3 .2D3 seems to be cell-type specific, as the antagonism has also been observed in E-cadherin-negative cell lines such us LS174T [2D3 [et al. observed that 1,25(OH)2D3 induces E-cadherin expression and inhibits β-catenin/TCF transcriptional activity in rat Rama-37 mammary epithelial cells [The contribution of E-cadherin induction to Wnt/β-catenin inhibition by 1,25(OH)s LS174T . Support74T [2D3 ,46. In aal cells .2D3 is unclear. However, DKK-1 seems to have antitumor effects independent of β-catenin/TCF transcriptional inhibition, as DKK-1 overexpression in APC-mutant colon cancer cells decreases colony formation capacity in vitro and tumor growth in immunodeficient mice [DKK-1 belongs to the Dickkopf gene family that encodes secreted proteins that bind to LRP5/6 and function as extracellular inhibitors of Wnt/β-catenin signaling. DKK-1 binding to LRP5/6 blocks Wnt-Frizzled-LRP5/6 interaction and also induces the formation of a complex with another DKK receptor named Kremen that leads to LRP5/6 endocytosis ,49. As ment mice . 2D3 inhibits DKK-4 expression in human colon and breast cancer cells and that DKK-4 overexpression in human colon cancer cells increases their migratory, invasive and angiogenic capacities [2D3 may contribute to the antitumor effects of 1,25(OH)2D3 in colon cancer.DKK-4 is a weaker inhibitor of Wnt/β-catenin signaling than DKK-1. Surprisingly, we found that 1,25(OH)pacities . These r2D3 in cancer prevention and therapy is restricted by its hypercalcemic effects at therapeutic doses. This has led to the development of several 1,25(OH)2D3 analogues that retain the antitumor actions but lack the hypercalcemic effects of 1,25(OH)2D3. We have found that 1,25(OH)2D3 analogues EB1089, KH1060, MC903, WU515, CD578 and WY1113 inhibit β-catenin/TCF transcriptional activity in a similar or even greater extent than 1,25(OH)2D3 in human colon cancer cells [et al. obtained similar results with the QW and BTW analogues in colon and breast cancer cells [The use of 1,25(OH)er cells ,52. In aer cells .2D3 and Wnt/β-catenin pathway 2D3 induces the expression of TCF4 in several human colon cancer cell lines by a VDR-dependent indirect mechanism [2D3 may have a protective role in colon cancer.Notably, other authors have reported different mechanisms of crosstalk between 1,25(OH) pathway . Beildecechanism . Howeverechanism . Thus, tet al. have reported a role of tumor stroma in the interplay between Wnt/β-catenin signaling and 1,25(OH)2D3 [2D3 through the inhibition of IL-1β production in THP-1 cells and, if functional in tumor-associated macrophages, it may be another way to interfere with Wnt/β-catenin signaling in vivo. Kaler 5(OH)2D3 . They foet al. have recently identified VDR/RXR and TCF4/β-catenin cistromes using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-Seq) in a human colon cancer cell line [2D3 treatment, most of them distal to transcription start sites. ChIP-Seq analysis also revealed 828 β-catenin and 3,161 TCF4 binding sites: after treatment with 1,25(OH)2D3 these figures decreased slightly for the former, but increased significantly for the latter. Examination of the overlap between TCF4/β-catenin and VDR/RXR cistromes indicates that the two heterodimers colocalize at 74 sites located near a limited set of genes that included c-FOS and c-MYC. These data support a direct action of both complexes at certain gene loci [Interestingly, Meyer ell line . They foene loci .MYC is a key regulator of cell cycle progression and its expression is frequently elevated or deregulated in human cancer [2D3 has been reported to downregulate c-MYC expression by two mechanisms. On the one hand, ligand-activated VDR directly represses c-MYC expression by binding two vitamin D response elements (VDRE) in the c-MYC promoter [2D3 interferes with β-catenin/TCF-induced c-MYC transcription mediated by Wnt responsive elements (WRE) in the proto-oncogene promoter [et al. have recently demonstrated that 1,25(OH)2D3-dependent suppression of β-catenin function in a head and neck squamous cell carcinoma cell line is largely responsible for the inhibition of c-MYC RNA expression [2D3 enhanced the expression of the c-MYC antagonist partner Mad/Mxd1 [c-MYC target genes.c-n cancer ,58. In an cancer . 1,252D3 represses the expression of P-cadherin, smooth muscle α-actin and α6- and β4-integrins that are myoepithelial/basal markers [Vdr-deficient mice express abnormally high levels of P-cadherin and smooth muscle α-actin in the mammary gland [2D3 may protect against the triple-negative and basal-like breast cancers that are associated with poor prognosis, perhaps via the inhibition of the Wnt/β-catenin pathway.Although mutations in rognosis ,63. Morerognosis . Interes markers . In linery gland . These d2D3 analogue paricalcitol has also been observed in other cellular systems unrelated to cancer such as vascular smooth muscle cells and renal podocytes [Remarkably, the antagonism of Wnt/β-catenin signaling by the 1,25(OH)odocytes ,67.2D3 against several neoplasias [Data from several types of studies using experimental animals support a protective and therapeutic effect of 1,25(OH)oplasias ,69. in vivo approach in cancer research. Numerous studies have shown that 1,25(OH)2D3 and many analogues significantly reduce the growth of xenografts generated by tumor cells derived from several types of cancers [2D3 analogue EB1089 inhibits the growth of xenografts generated by SW480-ADH human colon cancer cells. Remarkably, this effect is associated with induction of E-cadherin and DKK-1 expression, β-catenin nuclear export and inhibition of the expression of the β-catenin/TCF target gene ENC1 in the xenografts [Human cancer cells injected subcutaneously into immunosuppressed mice (xenografts) are commonly used as an  cancers ,69. We fnografts ,39,41.2D3 or several analogues [2D3 is accompanied by the induction of E-cadherin expression and the repression of the β-catenin/TCF target genes c-Myc and Ccnd1/Cyclin D1 in the colonic crypts and tumors of these animals [Similar results were obtained when chemical carcinogens (azoxymethane or azoxymethane plus dextran sodium sulphate) were used to induce colon tumors in mice or rats. Several studies showed that tumor incidence in these models decreases following treatment with 1,25(OH)nalogues . In addi animals ,71.Apcmin/+ mice are commonly used animal model for intestinal cancer. They harbour a germ line inactivating mutation in one Apc allele and spontaneously develop multiple intestinal neoplasias at approximately three months of age [Apc allele (loss of heterozygosity) and the consequent activation of the Wnt/β-catenin pathway. Huerta et al. found that both 1,25(OH)2D3 and the 1,25(OH)2-16-ene-19-nor-24-oxo-D3 analogue reduce tumor load  along the entire gastrointestinal tract of Apcmin/+ mice [et al. confirmed these results and showed that treatment with 1,25(OH)2D3 or two of its analogues increases E-cadherin expression, reduces nuclear β-catenin levels and inhibits the expression of the β-catenin/TCF target genes c-Myc and Tcf1 in the small intestine and colon of these mice [et al. did not find any effect of two 1,25(OH)2D3 analogues on the growth rate of colonic tumors developed in Apcmin/+ mice treated with the tumor inducer dextran sodium sulphate. As mentioned by the authors, the length of treatment with the 1,25(OH)2D3 analogues or the putative loss of Vdr expression in the tumors might be the cause of the discrepancy with previous studies [s of age . This phn/+ mice . Xu et aese mice . However studies .Apcmin/+ mice and in other animal models for intestinal cancer [et al. used transcriptomic analyses to characterize the changes induced by the Western-style diet in colon epithelial cells of normal mice. They found that Wnt signaling is one of the functional categories that are significantly enriched among the genes whose expression is altered by Western-style diet and reversed to normal by calcium and vitamin D supplementation. These genes include those coding for β-catenin and for the Wnt receptors Frizzled-2 and -10 [et al. observed that calcium and vitamin D supplementation abrogates the increase of β-catenin/TCF transcriptional activity and of Frizzled-5 and Ephb2 expression promoted by Western-style diet in intestinal villi and colon crypt cells of normal mice [Several studies indicate that a Western-style diet that is high in fat and low in calcium and vitamin D increases the incidence of spontaneous intestinal tumors in normal mice and dramatically accelerates tumor formation in l cancer . In addil cancer . Yang et and -10 ,77. Simimal mice .Vdr-deficient mice do not show an increase in spontaneous cancer incidence but are more predisposed to oncogene- or carcinogen-induced breast and skin cancer and leukemia [Vdr gene deletion on intestinal tumorigenesis by generating Apcmin/+Vdr−/− mice [Apcmin/+Vdr−/− mice as compared to Apcmin/+Vdr+/+. Remarkably, the lesions of Apcmin/+Vdr−/− mice showed higher β-catenin nuclear levels and expression of its targets genes Ccnd1/Cyclin D1 and Lef1 than those of Apcmin/+Vdr+/+ mice, suggesting that Vdr deletion promotes intestinal tumor growth through the activation of the Wnt/β-catenin pathway [Genetically-modified mice have also been used to analyze the effects of VDR on carcinogenesis. leukemia . In the leukemia ,81. Thus−/− mice ,82. No d pathway ,82.Garland and Garland were the first to suggest that vitamin D deficiency may explain the higher colon cancer mortality rates found in latitudes with low solar radiation in USA . Since tet al. conducted a randomized, double-blinded, placebo-controlled, 2 × 2 factorial clinical trial to analyze the effect that daily supplementation of sporadic colorectal adenoma patients with vitamin D (800 IU) and/or elemental calcium (2 g) for 6 months has on the expression of APC, E-cadherin and β-catenin in crypts of the normal appearing rectal mucosa [At the molecular level, Ahearn l mucosa . Vitaminl mucosa .2D3 or its analogues. Our group found that the transcription factors Snail1 and Snail2 repress VDR expression and block the antitumoral actions of 1,25(OH)2D3 in cultured colon cancer cells and in xenografted mice, including the inhibition of Wnt/β-catenin signaling. In addition, VDR RNA expression in colon tumors inversely correlates with that of SNAIL1 and SNAIL2, suggesting that these transcription factors are responsible for the downregulation of VDR found in colon cancer [2D3 on this pathway. In addition to colon cancer, VDR downregulation by Snail factors has been also observed in osteoblasts, osteosarcoma, breast cancer and renal cells [VDR is expressed in the normal colon and upregulated at early stages of colon tumorigenesis , whereas it decreases at advanced stages (carcinomas) ,88,89,90n cancer ,84,93,94n cancer ,96,97,98al cells ,101,102.VDR and CDH1/E-cadherin RNA levels in human colon tumors, suggesting that VDR/1,25(OH)2D3 may contribute to the restoration of normal E-cadherin levels in human colon cancer [E-cadherin expression is lost during the progression of several types of carcinomas and its downregulation is frequently associated with the acquisition of invasive and metastatic properties by tumor cells. Thus, it is considered an invasion-suppressor gene ,104. Accn cancer ,105.DKK-1 expression is frequently downregulated in human colon cancer, which suggests that DKK-1 may act as a tumor suppressor gene in this neoplasia [DKK-1 downregulation is due to promoter methylation in 24% of advanced colon tumors (Dukes’ stages C and D) [DKK-1 expression by 1,25(OH)2D3 found in cultured cells and animal models may contribute to restore DKK-1 expression and antitumor effects in human colon cancer. Accordingly, we found a significant direct correlation between VDR and DKK-1 RNA expression in tumor biopsies from colon cancer patients [et al. have recently reported that dietary vitamin D intake was negatively associated with DKK-1 promoter methylation in a large cohort of human colorectal cancer patients [2D3 against colon carcinogenesis.Our group reported that eoplasia . DKK-1 dC and D) . Thus, tpatients , and Rawpatients . These rDKK-4 is overexpressed in human colon tumors and in colon samples from patients with inflammatory bowel disease [VDR and DKK-4 RNA levels in human colorectal tumors, suggesting that the regulation of DKK-4 observed in cell lines also occurs in patients [We and others found that  disease ,108,109.patients .2D3 inhibits β-catenin/TCF transcriptional activity in colon and other cancer cells, the upregulation of the Wnt/β-catenin pathway by either ligand-activated or unliganded VDR has been described in osteoblasts and keratinocytes, where it promotes bone formation and hair follicle differentiation, respectively. Thus, the interplay between Wnt/β-catenin pathway and 1,25(OH)2D3/VDR seems to depend on the cell or tissue type.Although 1,25(OH)2D3 effects in bone are similar to those of Wnt, suggesting a crosstalk between both pathways. Indeed, 1,25(OH)2D3 induces the expression of the Wnt co-receptor Lrp5 in mouse osteoblasts [2D3 stimulating Wnt signaling in normal bone. However, the interplay between these two pathways seems to change in tumor tissue, as it has been reported that 1,25(OH)2D3 inhibits Wnt/β-catenin signaling in SaOS2 osteosarcoma cells [Wnt signaling promotes the differentiation of bone marrow-derived mesenchymal stem cells to bone while it represses their differentiation to other cell types, such as adipocytes ,111. Someoblasts ,113, whieoblasts . These ema cells .Vdr, but not of 1,25(OH)2D3, results in alopecia in mice [et al. have shown that VDR is a Wnt effector and that β-catenin behaves as a VDR co-activator in the skin to induce transcription of genes associated with differentiation of hair follicle lineages [2D3-independent, it is enhanced by the hormone. More recently, Luderer et al. have reported a direct interaction between VDR and LEF1 that is independent of both ligand and β-catenin, and that is required for normal canonical Wnt signaling in keratinocytes [Lef1 knock-out mice develop alopecia at an early age [Vdr−/− mice [in vivo significance of the VDR-LEF1 interaction is not yet clear, it may contribute to at least some of the ligand-independent effects of VDR in the skin. Similarly to what happens in bone, this interplay seems to change in the tumoral context. The development of trichofolliculomas (benign hair follicle tumors) induced by prolonged activation of β-catenin in the skin is inhibited by the 1,25(OH)2D3 analogue EB1089 [Vdr−/− mice [2D3 treatment has the opposite effect [2D3/VDR suppresses epidermal tumor formation by limiting the hyperproliferative actions of β-catenin in the skin. Surprisingly, epidermal β-catenin ablation cannot reduce the increased number of UVB-induced tumors developed by epidermis-specific Vdr knock-out mice, suggesting that VDR has β-catenin-independent anticancer functions in this model [Ligand-independent actions of VDR on Wnt canonical signaling have also been reported. In the skin, absence of  in mice , and two in mice ,117. In inocytes . Interesarly age  and tran−/− mice . Therefoe effect ,122. TheEvidence from a wide series of biological systems shows that inhibition of Wnt/β-catenin pathway is one of the mechanisms of vitamin D action. Wnt/β-catenin signaling has many important regulatory effects in the organism and thus, it is conceivable that the role of vitamin D is the control of an adequate level of activation of the route in each tissue and developmental stage. This most probably includes the antagonism of Wnt/β-catenin signaling in colorectal and possibly other carcinomas in which this pathway has a crucial oncogenic action. The finding that the repressive action of vitamin D takes places at different levels of the Wnt/β-catenin pathway, by distinct mechanisms, and in several cell types reinforces the importance of this regulatory action.


A delicate balance between estrogen and progestagen signaling underlies proper functioning of the female reproductive tract and, in particular, the monthly re- and degenerative phases characteristic of the menstrual cycle. Here, we propose that the canonical Wnt/β-catenin signaling pathway may underlie this finely tuned hormonal equilibrium in endometrial homeostasis and, upon its constitutive activation, lead to neoplastic transformation of the endometrium. During the menstrual cycle, estradiol will enhance Wnt/β-catenin signaling in the proliferative phase, while progesterone inhibits Wnt/β-catenin signaling, thus restraining estrogens' proliferative actions, during the secretory phase. In case of enhanced or unopposed estrogen signaling, constitutive activation of Wnt/β-catenin signaling will trigger endometrial hyperplasia, which may develop further into endometrial cancer. Whereas the functionalis layer comprises the upper two-thirds of the endometrium and is shedded during menstruation, the basalis includes the lower one-third which remains intact and is responsible for producing a new functionalis during each subsequent menstrual cycle ..functionIGF-1), that stimulate endometrial proliferation . If . If IGF-e uterus .The inhibition of estrogens' mitotic activity exerted by progesterone is of clinical relevance as unopposed or increased estrogen action in women is a well-established risk factor for endometrial cancer  9]..9].Endometrial cancer is one of the most common cancers of the female genital tract . It accoPTEN and in DNA mismatch repair genes . Also, oncogenic mutations in KRAS and/or CTNNB1 (β-catenin) are recognized major alterations  [16 [16PTEN r-2/neu)  [15] . Th. Th22]. 2), the less active estradiol precursor, estrone (E1), as well as its inactivated form, sulfated estrone (E1S), were measured   whi whiFOXO1stradiol . Based oCCND1) and c-MYC, in addition to a broad spectrum of other cellular functions i.e. cellular migration (e.g. CD44), cell adhesion (CDH1), extracellular matrix (MMP7) and many others ..CCND1) abasalis stimulates endometrial cell proliferation during the first two weeks of the menstrual cycle, thus giving rise to the functionalis. In week three and four of the menstrual cycle, the corpus luteum will produce progesterone, which reduces estrogens-driven proliferation and induces differentiation of the functionalis thus preparing the endometrium for implantation of the fertilized oocyte around day 20 - 22 of the menstrual cycle.The central role of Wnt/β-catenin signaling in the regulation of tissue homeostasis has been extensively investigated for the gut  46]. Al. Al46]. SFRP2), estradiol-induced proliferation was abolished [Wnt/β-catenin signaling activity along the crypt-villus axis of the intestine follows a decreasing gradient from the stem cell (SC) and proliferative (TA) compartment to the more differentiated compartment . In the bolished . In two bolished  52], Li, LiSFRP2In summary, it seems likely that, during the menstrual cycle, sex hormones can modulate Wnt/β-catenin signaling to maintain the balance between proliferation and differentiation.WISP2  was shown to be upregulated through direct interaction of activated ERα with its promoter region in human breast cancer cells  [50 [5054]  56]; th; th56]; er cells .ERα, however, can also function as a transcriptional modulator without directly binding to DNA sequences in the promoter region of the affected genes. For example, Shi et al  could shFurthermore, ERα has also been observed to associate with important growth factor pathways such as the PI3K pathway thus indirectly cross-talking with canonical Wnt signaling  62]. Bi. Bi62]. DKK1 in endometrial stroma cells during the secretory phase of the menstrual cycle. Using the antiprogestagen mefipristone (RU486), Catalano et al. , th, thDKK1 al cells .IHH (Indian hedgehog), its receptor PTCH, and the transcription factor and target gene GLI1 are all up-regulated upon estrogen signaling and downregulated by progesterone during the menstrual cycle. Furthermore, it has been shown that when IHH expression is impaired, the downstream effects of progesterone are lost in the uterus . A . A 36]. staining . APC mut cancers . Moreove-catenin , often i-catenin . Notably-catenin . Hence, Enhanced or unopposed estrogen signaling is the most important risk factor for endometrial hyperplasia and endometrial cancer. In view of the observations according to which i. Wnt/β-catenin signaling plays a central role in endometrial homeostasis, ii. it possibly represents one of the downstream effectors of estrogen signaling, and iii. its constitutive activation is likely to underlie malignant transformation in the uterus, it is important to assess whether canonical Wnt can trigger endometrial hyperplasia and cancer in the absence of enhanced estrogen signaling. In other words, among the allegedly broad spectrum of downstream effectors of estrogen signaling, does constitutive Wnt/β-catenin activation play a rate-limiting role for endometrial malignancies?Amhr2-Cre-driven oncogenic activation of β-catenin (tm1Mmt/+Ctnnb1). The Amhr2 gene is expressed as of embryonic day 12.5 onwards in mesenchymal cells surrounding the Mullerian duct and in adult mice in the myometrium. The corresponding Cre/+;Ctnnb1tm1Mmt/+Amhr2 mice develop myometrial hyperplasia and mesenchymal tumors (similar to leiomyomas) and endometrial sarcomas. Furthermore, hyperplasia of endometrial glands was occasionally observed in the uterus, suggesting that mesenchymal activation of Wnt/β-catenin signaling plays a role in the early events of epithelial tumorigenesis in the endometrium. Jeong et al. , di, diAmhr2ometrium , we receIn this review we discussed the role of estrogens and progestagens in the regulation of Wnt/β-catenin signaling as well as the involvement of activated Wnt/β-catenin signaling in the development of endometrial cancer. Evidence indicates that estrogens can induce Wnt/β-catenin signaling and that enhanced or unopposed estrogen signaling, as well as activated Wnt/β-catenin signaling may underlie endometrial hyperplasia and cancer. Furthermore, progesterone was shown to be a strong inhibitor of Wnt/β-catenin signaling. The latter is of clinical relevance as it is well known that progesterone can inhibit endometrial hyperplasia and well differentiated endometrial cancer growth.These observations place Wnt/β-catenin signaling at the center of physiological regulation of the menstrual cycle and point at a central functional role for its uncontrolled activation in endometrial carcinogenesis. Although Wnt/β-catenin signaling inhibitors (e.g. CGP049090, PKF115-584  and XAV9