Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the  Drosophila as a model, we now identify a conserved regulatory logic that underlies cell-type specific removal of uncommitted cells by apoptosis. We find that the transcription factor Cut activates differentiation, while it simultaneously represses cell death via the direct regulation of a pro-apoptotic gene. We show that this regulatory interaction occurs in many diverse cell types and is essential for normal development. Using in vivo Drosophila cancer models, we demonstrate that apoptosis activation in differentiation-compromised cells is an immediate-early cancer prevention mechanism. Importantly, we show that this type of regulatory wiring is also found in vertebrates and that other cell-type specification factors might employ a similar mechanism for tumor suppression. Thus, our findings suggest that the coupling of differentiation and apoptosis by individual transcription factors is a widely used and evolutionarily conserved cancer prevention module, which is hard-wired into the developmental program.Apoptosis is a highly conserved cellular function to remove excessive or unstable cells in diverse developmental processes and disease-responses. An important example is the elimination of cells unable to differentiate, which have the potential to generate tumors. Despite the significance of this process, the mechanisms coupling loss of differentiation and apoptosis have remained elusive. Using cell-type specification in  It has been a long-standing paradigm that impaired cell fate commitment is a key initiator of cancer development Drosophila posterior spiracle (PS) as a model to analyze the interplay of differentiation and apoptosis at the mechanistic level. By studying the morphogenesis of this organ, we have identified a hard-wired program through which the cell-type specifying transcription factor Cut (Ct) controls in a subset of PS cells, the filzkörper cells, initiation of differentiation and simultaneous repression of apoptosis via the direct transcriptional regulation of the pro-apoptotic gene rpr. Using two well-established Drosophila in vivo eye cancer models, we demonstrate that this regulatory circuit instructed by the transcription factor Ct is a very potent mechanism to prevent and/or reduce tumor growth, as it allows the lineage-specific removal of abnormal cells at the time of their genesis. Moreover, our data show that a related regulatory wiring is used in vertebrates and that other cell-type specification factors might employ a similar mechanism for tumor suppression, thus suggesting that the coupling of differentiation and apoptosis by individual transcription factors is a widely used and evolutionary conserved cancer prevention module, which is hard-wired into the developmental program.Since loss of terminal differentiation and the inability to activate apoptosis are crucial steps in cancer development, the existence of regulatory mechanisms preventing the accumulation of cells harboring mutations in both pathways seems essential for the survival of multi-cellular organisms. Consistently, mutations in differentiation genes very often result in the activation of the programmed cell death machinery Drosophila respiratory system to the environment and consists of an internal tube, the spiracular chamber with a refractile filter, the filzkörper, which is specified by the transcription factor Ct, and an external protrusion in which the spiracular chamber is located, the stigmatophore, which is under the control of the transcription factor Spalt   (head involution defective (hid), grim and sickle (skl) , thereby releasing inhibition of caspases and promoting apoptosis ct deficient filzkörper precursor cells of stage 11 embryos using the genetically-encoded caspase reporter Apoliner rpr de-repression is followed by apoptosis induction in ct mutant embryos. To study the interplay between cell-type specification and cell death at the mechanistic level, we identified conserved Ct-dependent regulatory regions in the rpr intergenic regions using computational methods. Due to the principal requirement of the Hox transcription factor Abdominal-B (Abd-B) for PS development rpr coding region, which we termed rpr-HRE-571  –S1D [9].tectable , which aer (rpr)  but not le (skl) –S1N tranor cells , evidenc-HRE-571 . Sequenc-HRE-571 . Immunosatophore  but not atophore , that doore   and the cell adhesion molecule DE-Cadherin  . HoweverCadherin . ConsistCadherin . These d embryos  was indi embryos . Local amination . Taken tDrosophila eye using RNAi   . Co-exprqRT-PCR) , or of aCadherin . HoweverCadherin , these cCadherin . SimilarCadherin , suggestrpr interaction in two well-established in vivo Drosophila cancer models, we asked whether the combined transcriptional regulation of differentiation and apoptosis repression by Ct could represent a cancer prevention mechanism. In the oncogenic “eyeful” model Delta (Dl) and the two epigenetic regulators longitudinals lacking (lola) and pipsqueak (psq) ey::Dl flies over-expressing Dl exclusively in eye tissue ey::Dl;RNAi2xct)  , demonstey::Dl;RNAi2xct eye imaginal discs  upon Ct depletion  in comparison to control animals (ey::Dl) (mys) ey::Dl pre-oncogenic background. We observed an increase in primary and secondary tumor formation in both situations, while reducing the activity of a related but Ct-independent integrin, the α-PS2 integrin (if), did not have any effect , also induced an increase in secondary tumors  in comparison to control animals (ey::GFP)  . To test(ey::Dl) , by reduy effect . Since dy tumors , we aske  , suggestin vivopuma were consistently elevated, whereas the anti-apoptotic gene Bcl-2 was down-regulated upon Cux1 depletion  drove expression in CNS midline cells of stage 14 embryos    resulted707+156) . Using t embryos , and con mutants . Due to  mutants , we assu compare . Thus, cse of Ct , primaryse of Ct . Furtherse of Ct . Togetheg motifs , these fg motifs , which cC. elegans, Drosophila and mouse have shown that apoptosis is used to sculpt tissues and to remove excessive and unwanted cells, thus defining the morphology required for diverse physiological functions rpr in these cells. Due to its immediate effect, the coupling of differentiation and apoptosis on the transcriptional level represents one of the fastest and most direct mechanisms to eliminate abnormal cells in status nascendi and thereby immediately interferes with their potential to develop into harmful cells.Programmed cell death is an integral aspect of animal development rpr but also by hidDrosophila early developmental mutants only the expression of the pro-apoptotic gene hid is up-regulated rpr is induced when a factor specifying a distinct cell type is lost. Although it is currently unknown how hid expression is regulated at the transcriptional level, this raises the possibility that the apoptosis gene hid acts a safeguard when broad positional information at the onset of embryogenesis is absent, whereas rpr might take over this function later in development when individual and specific cell types are defined by transcription factors restricting cell fate choices.Interestingly, apoptosis induction as a consequence of aberrant cell-type specification is not only mediated by the cell death promoting gene Drosophila PS or the developing eye, only results in the survival of the Cut deprived cells, but not in tumor development. These results demonstrate that cells, which are unable to undergo the cell lineage-specific differentiation program, have to be eliminated, since they have the potential to develop into cancerous cells when other genetic or micro-environmental changes accumulate Given the well-known role of the vertebrate homologue of Cut, Cux1, in tumor initiation and progression in specific cancer types Drosophila Cut Drosophila Cut, they act as downstream effectors of the Notch signaling pathway in vivopuma and the down-regulation of the anti-apoptotic gene Bcl-2. Thus, two crucial regulators for Smac/DIABLO release are controlled by Cux1 on the transcriptional level, showing that the basic design principle of the Cut-Rpr regulatory wiring is conserved but has been adapted to the system requirements in evolution. In future, it will be intriguing to study this mechanism in diverse cellular backgrounds, including stem cells, which neither die nor differentiate.Several lines of evidence suggest that the dual role of Cut in differentiation and apoptosis for cancer prevention is conserved in evolution. First of all, the two vertebrate homologues of Cut, Cux1 and Cux2, code for homeobox-containing transcription factors, which are crucially involved in cell-type specific terminal differentiation http://jaspar.genereg.net/) and 90% cut-off. Abd-B binding site clusters were identified if at least three Abd-B sites were present in a 400 bp window. Conserved enhancers were identified using PhastCon score http://jaspar.genereg.net/).To identify Abd-B binding sites we used the method of Wasserman and Sandelin (2004) Drosophila melanogaster strain Oregon R was used as wild type. Amorphic allele db7/FM7ctems-Gal4 and ems-Gal4, UAS-GFP/TM6BUAS-Dcr2; ey-Gal4 from B. Dickson, eq-Gal4/TM6BUAS-Apoliner5EHK2/CyOUAS-ctRNAi; UAS-ctRNAiUAS-ct  from Y.N. Jan and ey-Gal4, UAS-Dl/CyO and eyeful flies  from M. Domiguez UAS-Dcr2; C96-Gal4 (BL-25757), UAS-CD8::GFP (BL-5130) from Bloomington stock center. GMR-Gal4, UAS-p35, UAS-Abd-B, arm-Gal4, UAS-rpr, UAS-lacZ were described elsewhere DE-Cad (v8024), rpr (v12045), vvl (JF02126), gro (v6316), H (v24466), αPS2 (if) (BL27544), αPS4 (v109783), βPS (mys) (HMS00043), PI3K (v107390) and Timp (v109427). Five independent transgenic lines were analyzed for each reporter construct.Panc-1 human pancreatic cancer cells  and 293 T cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, non-essential amino acids, 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml amphotericin B. shRNA directed against human Cux1 was generated using the following complementary oligonucleotides (forward and reverse):Cux1_KDa:5′CCGGAAGAAGAACACTCCAGAGGATCTCGAGATCCTCTGGAGTGTTCTTCTTTTTTTG3′ and5′AATTCAAAAAAAGAAGAACACTCCAGAGGATCTCGAGATCCTCTGGAGTGTTCTTCTT3′;Cux1_KDb:5′CCGGAAGAATCTTCTCGTTTGAAACCTCGAGGTTTCAAACGAGAAGATTCTTTTTTTG3′ and5′AATTCAAAAAAAGAATCTTCTCGTTTGAAACCTCGAGGTTTCAAACGAGAAGATTCTT3′;shRNA control (C),5′CCGGAATTGCCAGCTGGTTCCATCACTCGAGTGATGGAACCAGCTGGCAATTTTTTTG3′ and5′CCGGAATTGCCAGCTGGTTCCATCACTCGAGTGATGGAACCAGCTGGCAATTTTTTTG3′.pLKO lentiviral vectors containing shRNA were transfected into 293 T cells together with psPAX2 (packaging vector) and pMD2.G (VSV-G envelope protein expression vector) using the calcium-phosphate transfection kit (Sigma). Panc-1 cells were infected using lentivirus-containing 293 T cell supernatant and Cux1 protein levels were assessed by Western blotting using anti-Cux1 (Sigma-Aldrich) and anti-β-Actin (GeneTex) antibodies.pHPelican-GFPpHPelican-GFP_DESTrpr-HRE-571-S2 enhancer fragment, mutation introduced into the Cut consensus sequences were identical to the ones introduced into the EMSA probes (see below). Primer sequences are available upon request.All enhancer fragments were PCR amplified from genomic DNA and cloned in actin5C mRNA for imaginal disc analysis. Relative expression levels are based on three biological replicates.Real Time PCR was performed following standard protocols using SYBR green. Expression was normalized to GAPDH for mammalian cells and to endogenous Drosophila embryos were fixed as described In situ hybridization and immunochemistry were performed as described in situ hybridizations were done as described previously pMAL2-c2x vector (NEB) and expressed as Maltose Binding Protein (MBP) fusion proteins. EMSAs were carried out as described in Stöbe et al. (2009) Cut CR3HD (4849–5412 of ctRA from Y.N. Jan) was cloned into Wild-type.TCAAGTCTCGTCAGTTCGGGTTTGTGTCATAAAAAATC3′5′GCACTTTTGCCTGCAMutated.GGAAGTCTCGGGAGTGGGGGTTTGTGTCATAAAAAATC3′5′GCACTTTTGCCTGCACut consensus sequences are underlined, exchanged nucleotides in the mutated versus the wild-type sequence are shown in bold.ey::CD8-GFP or eyeful+CD8-GFP;RNAict;p35 3rd instar larvae were dissected by rupturing the larval cuticle at the posterior end with a pair of fine forceps, the hemolymph was collected in ice-cold Schneider's medium (Invitrogen GIBCO) containing 1× Complete protease inhibitor mixture (Roche). Hemolymph cells were analyzed via FACSAria to quantify GFP-labeled cells circulating within the hemolymph. In addition, GFP mRNA levels within the hemolymph were measured by qRT-PCR.ey::Dl or RNAiey::Dl::2xct 3rd instar larvae were dissected in cold PBS, total RNA was extracted using standard procedures. Microarray analysis was conducted at the Genomics Core Facility, EMBL, Heidelberg, Germany. Microarray data analysis was performed using the R package as described previously Eye-antennal discs of Figure S1rpr transcription in a cell-autonomous manner. (A–D) Expression of the posterior spiracle markers Spalt  (green), which labels the stigmatophore precursor cells, and Cut (Ct) (red), which marks the spiracular chamber and filzkörper precursor cells, in stage 11 (A), stage 13 (B), stage 14 (C) and stage 16 (D) embryos. In (A), (B) and (D) lateral views of the posterior spiracle primordia are shown, whereas in (C) a dorsal view is presented.  rpr mRNA expression (green) in posterior spiracle primordia of stage 14 wild-type (E) and ct mutant (F) embryos is shown . Spalt  protein (blue) labels stigmatophore precursor cells, Cut (Ct) protein  marks spiracular chamber and filzkörper precursor cells and the apical membrane marker Crb (red) outlines the cells. Small, white arrow in (F) marks additional tracheal cells found at the posterior end in ct mutant embryos; yellow circle in (F) highlights rpr expression in ct mutant embryos. (G) EMSA using S2 sub-fragment with Ct binding sites either in wild-type (wt probe) or mutated (mut. probe) version and no protein (−), purified MBP protein (M), and purified Cut-MBP fusion protein consisting of the Cut repeat 3 and the Cut homeodomain (C). The black arrowheads indicate the specific DNA-protein complexes, the black arrow highlights unspecific DNA-protein complex. Loading of equal amounts of labeled wild-type and mutated oligonucleotides is illustrated by formation of comparable amounts of unspecific DNA-protein complex (indicated by black arrow).Cut directly represses (JPG)Click here for additional data file.Figure S2rpr transcription and apoptosis activation. (A–F) rpr RNA expression in stage 11 wild-type , db7ct  and arm::ct (F) embryos. rpr transcription is ectopically activated in the posterior spiracle primordium (B) and the gut primordium (D) in ct mutant embryos (marked by red arrowheads), and is globally repressed when Ct is ubiquitously mis-expressed (F).  Acridine Orange (AO) staining of stage 13 wild-type (G) and ct mutant (H) embryos highlights up-regulation of programmed cell death in the PS primordium of ct mutant embryos. (I–N) hid , grim , skl  RNA expression in stage 11 wild-type  and db7ct  mutant embryos. Red boxes indicate posterior spiracle primordium in respective embryos.Ct represses (JPG)Click here for additional data file.Figure S3rpr and apoptosis in the PS primordium. (A–D) GFP expression in the posterior spiracle primordium of different reporter lines at developmental stage 15. Spalt  and Cut (Ct) proteins label stigmatophore (blue) or spiracular chamber and filzkörper precursor cells (red). Closed, yellow arrowheads in (C) and (D) mark reporter gene expression in filzkörper cells, whereas open, yellow arrowheads in (A) and (B) mark missing GFP expression. (E–H) Single color images of the different reporter lines showing only Sal expression. (I–L) Single color images of the different reporter lines showing only Ct expression. (M–P) Single color images of the different reporter lines showing only GFP expression.Cut directly represses (JPG)Click here for additional data file.Figure S4rpr-HRE-571 element. (A) Conservation graph of the rpr intergenic region obtained from the UCSC genome browser (http://genome.ucsc.edu/). The rpr-HRE-571 element, which is marked by a light-red box (3 L: 18384438..18385008), is located 6 kb downstream of the rpr coding sequence (marked by a dark-red box). The coding region of the pro-apoptotic gene grim is marked by a dark-blue box. (B) Alignment of the rpr-HRE-571 region from five different Drosophila species . Abd-B binding sites are marked by purple, Ct binding sites by orange boxes.Location and conservation of the (JPG)Click here for additional data file.Figure S5db7; ems::GFPct  and db7; ems::p35;GFPct . (A–C) Cuticle preparations of the different genotypes with focus on the posterior spiracle of 1st instar Drosophila larvae. Closed, orange arrowhead in (A) marks the filzkörper, whereas open, orange arrowheads in (B) and (C) indicate the absence of this structure in the respective genotypes. (D–F) Ct and Crb stainings in the respective embryos are shown to highlight the morphology of the filzkörper in the different genotypes. (G–I) DE-Cad staining in the respective genotypes. Closed, red arrowheads in (G) indicate the presence of the filzkörper, whereas open, red arrowheads in (H) and (I) highlight the absence of this structure in db7; ems::GFPct (H) and db7; ems::p35;GFPct (I) embryos.Ct function is required for filzkörper differentiation. The following genotypes are shown: wild type , (JPG)Click here for additional data file.Figure S6Drosophila notum in wild-type (A), Eq::RNAict (B), Eq::RNAict; p35 (C) and in Eq::RNAict; RNAirpr animals (D). Open, yellow circles in  mark the absence of external sensory organs, whereas closed, yellow circle in (A) highlights the presence of these structures in the different genotypes. Note that in (B) most bristles are missing, whereas in (C) and (D) some bristles form, which have cell polarity defects. Expression of Cut in mechanosensory organs of the notum has been shown before Drosophila adult wing with focus on wing margin between the wing veins L2 and L3 in wild-type (E), C96::RNAict (F), C96::RNAict; p35 (G) and in C96::RNAict; RNAirpr (H) animals. Closed, blue arrowheads in (E), (G) and (H) highlight the presence of mechanosensory bristles at the wing margin, whereas the open, blue arrowhead in (F) marks their absence in the respective genotype. Importance of Cut function for wing margin development has been shown before Ct represses apoptosis in wing margin bristles and in external sensory organs of the notum. (A–D) Close-up of (JPG)Click here for additional data file.Figure S7eyeful::RNAict 3rd instar eye-antennal discs, yellow circle in (B) marks ELAV-positive cells at ectopic location in eyeful::RNAi;p35ct 3rd instar eye-antennal discs.Invasiveness of Ct depleted cells. (A–D) Co-localization of the eye differentiation marker Embryonic Lethal Abnormal Vision (ELAV) and the proliferation marker Phosphorylated histone H3 (PH3) in 3rd instar eye-antennal discs. Blue circle in (D) marks loss of ELAV expression in (JPG)Click here for additional data file.Figure S8Drosophila eye. Genes: vvl: ventral veins lacking; gro: groucho; ct: cut; H: Hairless.Simultaneous regulation of differentiation and apoptosis represents a general cancer prevention mechanism. Top: Representative pictures of tumorous eye growth in flies of indicated genotypes. Bottom: Quantification of primary tumor growth in the respective genotypes. Genes tested were selected based on their function as cell-type specifying transcriptional regulators active in the (JPG)Click here for additional data file.Table S1puma gene.Putative binding sites for vertebrate Cux1 within the non-coding regions of the (DOC)Click here for additional data file.


ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.Six different somatic missense mutations in the human  ERBB2 gene amplification), high expression of ADAM12-L, but not ADAM12-S, mRNA is associated with poor prognosis ADAM12-L and ADAM12-S are two different splice variants that encode the long, transmembrane protein isoform ADAM12-L and the short, secreted ADAM12-S, respectively Disintegrin and metalloproteinase domain-containing protein ADAM12 is a member of the ADAM family of proteins that mediate cleavage of substrates at the cell surface and/or modulate intracellular signaling pathways ADAM genes that encode catalytically active proteases ADAM12 is the most frequently somatically mutated gene in human breast cancers. As of September 2013, the COSMIC database  listed 6 confirmed somatic missense mutations in the ADAM12 gene per a total of 1104 unique breast carcinoma samples analyzed. The frequencies of breast cancer-associated missense mutations in other genes encoding catalytically active ADAMs were: 1/973 in ADAM9, 1/973 in ADAM10, 1/1147 in ADAM17, 1/1010 in ADAM19, 1/973 (plus one nonsense mutation) in ADAM20, and 1/973 in ADAM30. No other missense/nonsense somatic mutations were reported for the remaining ADAM genes encoding catalytically active proteases . A relatively high frequency of mutations in the ADAM12 gene can be attributed to the fact that ADAM12 is located on human chromosome 10q26.2, in a region capable of forming highly stable secondary structures Among thirteen different The six breast cancer-associated mutations in the ADAM12-L protein include the D301H mutation in the metalloproteinase domain, G479E in the disintegrin domain, T596A and R612Q in the cysteine-rich domain, G668A in the epidermal growth factor (EGF)-like domain, and L792F in the cytoplasmic tail Here, we show that there is functional diversity between the three recently identified mutations. While ADAM12-L containing the G668A mutation is largely retained in the endoplasmic reticulum (ER) and is not proteolytically activated in the Golgi, the T596A mutant is properly trafficked and proteolytically processed but is still catalytically inactive. The R612Q mutant is trafficked, processed, active, and is indistinguishable from the wild-type (WT) ADAM12-L. Taking into consideration all six known breast cancer-associated somatic mutations in ADAM12-L, we note an apparent association between the catalytic activity of ADAM12-L mutants and the molecular characteristic of the tumor. The two mutations that do not have any effect on ADAM12-L activity, R612Q and L792F, occurred in TNBCs. The four mutations that render ADAM12-L inactive, i.e., D301H, G479E, G668A, and T596A, were described in non-TNBCs. This observation further suggests an important role of the catalytically active ADAM12-L in the triple negative breast cancer disease.ADAM12-L-pBABEpuro was used for the expression of the wild-type (WT) ADAM12-L protein. The D301H, G479E, T596A, R612Q and G668A point mutations were introduced by site-directed mutagenesis using the QuickChange kit (Stratagene). The entire lengths of all DNA inserts were sequenced to confirm that no other mutations were introduced during mutagenesis. The expression construct of Delta-like 1 (DLL1) in pIRESpuro vector was described earlier Retroviral expression vector Human MCF10A mammary epithelial cells (ATCC) were cultured in DMEM/F12 (1∶1) supplemented with 5% horse serum, 0.5 μg/ml hydrocortisone, 20 ng/ml human EGF, 10 μg/ml insulin, 100 ng/ml cholera toxin, and 1% penicillin/streptomycin. Retrovirus production and stable transduction of MCF10A cells with viruses encoding WT or mutant ADAM12-L, or with control viruses bearing empty pBABEpuro vector, were performed as described previously Anti-ADAM12 rabbit polyclonal antibody (Ab 3394) specific for the cytoplasmic domain of human ADAM12-L was developed in our laboratory, as described Stably transduced MCF10A cells were plated on glass coverslips placed in 6-well plates. Two days later, cells were fixed with 3.7% paraformaldehyde/DPBS for 20 min, followed by permeabilization with 0.1% Triton X-100/DPBS for 5 min. Coverslips were incubated with anti-ADAM12-L polyclonal antibody (1∶500 dilution) and anti-KDEL antibody (1∶200 dilution), followed by incubation with rhodamine Red-X-conjugated anti-rabbit IgG antibody, Alexa 488-conjugated anti-mouse IgG antibody, and DAPI. Immunofluorescence was examined using an Axiovert 200 inverted fluorescent microscope.Cells were trypsinized into a single cell suspension, washed with DPBS containing 3% BSA, and incubated with anti-ADAM12 monoclonal antibody or isotype control antibody  for 30 minutes on ice. Cells were then washed 3 times, incubated with allophycocyanin (APC)-conjugated anti-mouse antibodies  for 30 min on ice, washed again, and then incubated with 1 μg/ml propidium iodide  for viability. Analysis was performed using a BD FACSCalibur flow cytometer. Only the cells negative for PI staining (viable cells) were selected for the ADAM12 analysis.12-biotin (Thermo Scientific), and then washed with ice-cold 100 mM glycine/DPBS. Cellular proteins were extracted with extraction buffer -benzene-sulfonylfluoride hydrochloride (AEBSF), 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 10 mM 1,10-phenanthroline; 0.5 ml buffer/well in a 6-well plate). Cell extracts were centrifuged at 21,000×g for 15 min at 4°C, and supernatants were incubated for 1 h at 4°C with NeutrAvidin sepharose . The resin was washed three times with extraction buffer, eluted with SDS sample buffer; samples were then resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Western blotting was performed using anti-ADAM12 polyclonal , anti-DLL1 , anti-EGFR , anti-pY1173 EGFR  primary antibodies and HRP-conjugated secondary antibodies, as described Cells were washed with DPBS, incubated for 60 min at 4°C with 2.5 mM EZ-link NHS-PEGf denaturing buffer (New England BioLabs), boiled, and then treated with Endo Hf , according to the manufacturer’s instructions.Stably transduced cells were treated with extraction buffer and centrifuged at 21,000×g for 15 min at 4°C. Supernatants were treated with EndoHStably transduced cells were seeded in 6-well plates at the density of 60,000 cells/well. After 24, 48, and 72 h, cells were detached and counted with Cellometer AutoT4 (Nexcelom Bioscience), in duplicates. Exponential growth curves were fitted to each dataset and the doubling times were calculated using a nonlinear regression function in the GraphPad Prism 5.0 software.3VO4, and 10 mM Na4P2O7, and analyzed by SDS-PAGE and Western blotting using anti-phospho-EGFR (pY1173) and anti-EGFR antibodies.MCF10A cells stably overexpressing WT or mutant ADAM12-L proteins, or cells stably transduced with empty pBABEpuro vector, were incubated for 16 h in serum-free media. Conditioned media were then collected, pre-cleared by centrifugation, and added to the duplicate wells containing “reporter” empty vector-transduced MCF10A cells that were pre-incubated for 16 h in serum-free medium. After 30 min, cells were washed with DPBS, treated with extraction buffer containing phosphatase inhibitors 50 mM NaF, 2 mM Na4 cells/chamber. The lower chambers contained the full culture medium supplemented with 5% horse serum and 20 ng/ml EGF. After incubation at 37°C for 18 h, cells were fixed with 3% glutaraldehyde in DPBS for 20 min, washed twice with DPBS, and stained with 0.5% crystal violet in 20% methanol for 10 min. Cells at the upper face of the membrane were removed with cotton swabs, and cells at the lower face were examined with an inverted microscope using a 10× magnification, and photographed. Numbers of cells in five random fields were counted, and the mean number of migrated cells for each insert was determined.MCF10A cells with stable overexpression of WT or mutant ADAM12-L proteins, or control empty vector-transduced cells, were suspended in MCF10A medium containing 0.1% BSA instead of horse serum, and seeded in the upper chambers of Transwell inserts with a 8-μm pore size polyethylene terephthalate membrane (BD Biosciences), at 2.5×10http://zhanglab.ccmb.med.umich.edu/I-TASSER/, refs. The I-TASSER software  and compared to the WT ADAM12-L. When the lysate of WT-expressing cells was treated with Endo H, the mobility of the nascent ADAM12-L increased, while the mature form was more resistant to the Endo H treatment . This reThe next question might be: Why is the G668A mutant inefficiently trafficked to the cell surface and predominantly retained in the ER? We reasoned that this mutant might be misfolded and retained by the protein quality control system operating in the ER. However, using co-immunoprecipitation, we did not detect interaction between the G668A mutant (or WT ADAM12-L) with ER chaperones BiP, Grp94, and calnexin, or with ER stress proteins that assist in proper disulfide formation ERp44, ERp57, ERp72, Ero1, PDI (results not shown). Also, cellular levels of these chaperones/stress proteins were not elevated in G668A-expressing cells. Cycloheximide chase experiments further demonstrated that the G668A mutant was in fact more stable than the WT ADAM12-L protein. Estimated half-life of the G668A mutant was 5.9 h, which was significantly larger than the half-life of WT ADAM12-L 3.3 h, . CollectNext, we asked about the catalytic activity of ADAM12-L mutants. While a mutation causing impaired intracellular trafficking of ADAM12-L is naturally expected to cause a decrease in the enzyme activity at the cell surface , a mutation that does not affect intracellular trafficking and processing can still have an impact on the catalysis or substrate recognition. For comparison, we also included the D301H and G479E mutants in the current analysis. The corresponding D299H and G477E mutations in mouse ADAM12 were shown previously to block the intracellular trafficking and processing of the protein, as well as ADAM12-mediated cleavage of the substrate protein DLL1 We used three different approaches to evaluate the catalytic activity of the D301H, G479E, T596A, R612Q, and G668A mutants at the surface of MCF10A cells . In the In the second approach, we focused on ADAM12-L-mediated shedding of EGFR ligands, as this function of ADAM12-L has been recently shown to be important in the biology of TNBC In the third approach, we investigated the effect of WT and mutant ADAM12-L expression on cell migration. We showed previously that overexpression of mouse ADAM12 in NIH3T3 cells increased cell migration using scratch wound assay, and that the D299H and G477E mouse ADAM12 mutants were inactive www.ensembl.org). SIFT is a sequence homology-based tool that sorts intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will have a phenotypic effect .A summary of the functional characterization of breast-cancer associated ADAM12-L mutations, as well as the properties of breast tumors in which each mutation was identified, is provided in The current study, together with two other previous reports The G668A mutation adds to the other two mutations that were previously found to cause ER retention and a lack of proteolytic processing of mouse ADAM12, and are now confirmed to inhibit the processing of human ADAM12-L, i.e., D301H and G479E. While we do not find any evidence that these mutants are unfolded and rapidly degraded, they must assume a significantly different conformation from the WT ADAM12-L to be retained in the ER. Clearly, since these three mutants exist mostly as the ∼120-kDa precursors, with the inhibitory pro-domain intact, they are expected to be catalytically inactive. The lack of catalytic activity of the D301H, G479E, and G668A mutants has been confirmed here by the DLL1 cleavage assay, by the EGFR activation assay, and by the Transwell migration assay.The effects of D301H, G479E, and G668A mutations on the structure/function of ADAM12-L agree well with the effects predicted by the SIFT and PolyPhen tools see . InteresADAM12 gene is located in the genomic region that has been recently found to be significantly deleted in Luminal B tumors  ADAM12 gene was hypermethylated and showed lower expression in Luminal B tumors than in other types of breast cancer The most striking observation emerging from this study is the apparent association between the catalytic activity of ADAM12-L mutants and the type of breast cancer. Two mutations that did not have any impact on the catalytic activity of ADAM12-L, R612Q and L792F, were found in triple-negative tumors. In contrast, all four loss-of-function mutations, D301H, G479E, T596A, and G668A, occurred in non-triple-negative tumors. Thus, it appears that loss-of-function mutations in ADAM12-L tend to be excluded from TNBCs. However, a larger patient population and possibly a broader mutation spectrum are needed to test whether there is indeed a significant association between the type of ADAM12-L mutations and the triple-negative status of breast tumors. Interestingly, the ADAM12-L, increased EGFR phosphorylation in a mouse xenograft model of breast cancer, and a strong correlation between the level of anti-ADAM12-L and anti-phospho-EGFR immunostaining in human breast tumor samples. We have also noticed a positive correlation between ADAM12-L and HB-EGF and EGFR in TNBCs, but not in receptor-negative non-TNBCs We have recently postulated that ADAM12-L may be the primary protease responsible for the activation of EGFR in early stage, lymph node-negative TNBCs


A recently developed T2-preparation module for cardiac imaging at high field is insensitive to B0 and B1 inhomogeneity, cardiac motion and flow. Obtained T2 weighted images exhibit bright fat signal that can obscure the signal of myocardium and other muscles. Therefore, a chemical selective saturation (CHESS) is usually applied for fat suppression after the T2-preparation, e.g. in coronary artery MRI. Its quality is often poor and it is unsuited for longer readout durations. In this study, we propose a new fat suppressed T2-preparation module for 3T using binomial composite RF pulses as 90° pulses to more efficiently suppress fat, maintaining robustness towards inhomogeneity, cardiac motion, and flow.An oil and water phantom was scanned on a Siemens MAGNETOM Trio 3T MR scanner (16-channel coil) to compare the proposed fat-suppression method with the existing T2-preparation using CHESS fat-suppression.The proposed T2-preparation method as shown figure Figure The proposed T2-preparation method with integrated fat-suppression has superior fat suppression performance than existing techniques.This research was supported by Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (K21002001824-11E0100-02910).


Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines , tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines  may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models. Salmonella enterica serovar Typhi (S. Typhi), a human restricted pathogen, is the causative agent of typhoid fever. S. Typhi is a Gram-negative, facultative intracellular pathogen that poses a significant threat to public health, particularly in the developing world S. Typhi isolates has further exacerbated the problem S. Typhi by producing interferon-γ (IFN-γ) and other T helper (Th) 1/T cytotoxic (Tc) 1 cytokines S. Typhi infected cells Interleukin-17A is a pro-inflammatory cytokine that is involved in protection against extracellular and intracellular bacterial pathogens, as well as fungi and viruses S. Typhi infection, we investigated the presence of IL-17A producing CD8+ T cells in peripheral blood mononuclear cells (PBMC) obtained from Ty21a recipients following ex vivo stimulation with S. Typhi-infected autologous B-lymphoblastoid cell lines (B-LCL) or an S. Typhi-infected HLA-E restricted cell line. Furthermore, given the prominent role that multifunctional T cells have been shown to have in the host defense from other human infections S. Typhi infection.Therefore, to evaluate the hypothesis that IL-17A may play a role in protection from PBMC collected from 6 healthy adult volunteers, recruited from the Baltimore-Washington area and University of Maryland, Baltimore campus, were used in this study. Written informed consent was obtained and all procedures were approved by the University of Maryland, Baltimore IRB. PBMC were isolated immediately after blood draws by density gradient centrifugation and cryopreserved in liquid nitrogen following standard techniques 2 for 2–3 weeks. B-LCL were maintained in culture or cryopreserved until use.Epstein Barr virus (EBV)-transformed B-LCL were generated from PBMC obtained from Ty21a vaccinees and control volunteers as previously described An HLA class I-defective B cell line transfected with HLA-E fused to the HLA-A2 leader peptide , 721.221.AEH (AEH cells), were provided by Dr. D. Geraghty S. Typhi strain ISP1820 in RPMI without antibiotics for 3 hours at 37°C with 5% CO2 as previously described S. Typhi, cells were stained with anti-Salmonella common structural Ag (CSA-1)-FITC  and analyzed by flow cytometry using an LSR-II instrument (BD). The percentage of cells infected with S. Typhi was recorded for each experiment. Infected targets were only used if infection was >30% of viable cells.Target cells were infected by incubation with wild-type oC, 5% CO2 in complete media (RPMI 1640 media (Gibco) supplemented with 100 U/mL penicillin (Sigma), 100 µg/mL streptomycin (Sigma), 50 µg/mL gentamicin (Gibco), 2 mM L-glutamine (Gibco), 2.5 mM sodium pyruvate (Gibco), 10 mM HEPES buffer (Gibco), and 10% fetal bovine serum ) at a concentration of 1×106 cells/mL in 6 or 12 well plates. Cells were then resuspended in complete media and stimulated with S. Typhi-infected autologous B-LCL or S. Typhi-infected HLA-E restricted AEH cells. Media alone and uninfected autologous B-LCL or HLA-E restricted AEH cells were used as negative controls. Staphylococcal enterotoxin B (SEB)  was used as a positive control. Cells were incubated at 37°C in 5% CO2. After 2 hours, Golgi Stop (containing monensin) and Golgi Plug (containing brefeldin A) from BD were added at concentrations of 0.5 µl/mL and cultures continued overnight at 37°C in 5% CO2.PBMC were thawed and rested overnight at 37www.immport.org) software packages. Graphs were generated using GraphPad Prism version 5.03 .Following stimulation as described above, cells were plated in 96-well V-bottom plates for staining. Cells were washed once with staining buffer  and stained for live/dead discrimination using Invitrogen LIVE/DEAD fixable violet dead cell stain kit . Fc receptor blocking was performed with human immunoglobulin  followed by surface staining, performed as previously described S. Typhi-infected) and negative control (non-infected) cultures was significantly increased by Chi-square tests. P values <0.05 were considered significant.All tests were performed using GraphPad Prism version 5.03 (Graphpad Software). Comparisons between groups were performed using Mann-Whitney and Kruskal-Wallis tests. In flow cytometry experiments a response was considered significant if the differential in the number of positive events between experimental . PBMC were isolated before immunization and at 11 time-points up to 1 year after immunization . Following isolation, PBMC were stimulated with S. Typhi-infected autologous B-LCL. Uninfected B-LCL and SEB were used as negative and positive controls respectively. PBMC were stained with monoclonal antibodies against CD3, CD4, CD8, CD14, CD19, CD45RA, and CD62L followed by intracellular staining for CD69, IL-10, IL-17A, IL-2, IFN-γ, TNF-α, and MIP-1β. Due to unavailability of the antibody, MIP-1β staining was not performed in 2 of the volunteers (48 s and 50 s). The samples were then analyzed by multichromatic flow cytometry. The gating strategy is shown in N; CD62L+ CD45RA+), T central memory cells , T effector memory , and T effector memory CD45RA+  as previously described  was used to normalize the data. We found that 3 of the 5 immunized volunteers (60%) showed detectable intracellular cytokine responses above baseline responsiveness at day 0  produced high levels of TNF-α and IFN-γ (5–15% of circulating TEM at the peak times), followed by IL-2 (4–8% of circulating TEM at the peak times), and then IL-17A (2–5% of circulating TEM at the peak times)  . Intracek times) . The kink times) . In voluk times) . The kink times) . Voluntek times) . In addiEMRA responses were generally lower in magnitude than TEM responses in volunteer 53 s, essentially absent in volunteer 54 s, and substantially lower than TEM responses in volunteer 50 s , stained, and analyzed by multichromatic flow cytometry. The same gating strategy was applied . With the exception of MIP-1β production by volunteer 53 s, the levels of detectable intracellular cytokines were lower in response to HLA-E restricted stimulation compared to autologous B-LCL stimulation  and HLA-E restricted (53 s and 54 s) cells. TEM were the predominant source of IL-17A, with intracellular IL-17A also detected in TEMRA of volunteer 53 s  are shown in response to stimulation with S. Typhi-infected autologous cells , and IL-17A+ IL-2- IFN-γ+ TNF-α+ MIP-1β+ (“quadruple positive”). IL-17A production by volunteer 53 s was predominantly by quintuple-positive cells co-producing IL-17A, IL-2, IFN-γ, TNF-α, and MIP-1β at most time points. The peak of the quintuple-positive cells was on day 42 post immunization following stimulation with S. Typhi-infected autologous B-LCL (3.91% of CD8+ CD69+ TEM) and day 56 post immunization following stimulation with S. Typhi-infected HLA-E restricted cells (2.01% of CD8+ CD69+ TEM)  . Quadrup69+ TEM) . Volunte69+ TEM) . IL-17A+nization . MIP-1β nization .S. Typhi-infected B-LCL were analyzed. The same data that were analyzed for intracellular detection of IL-10, IL-17A, IL-2, IFN-γ, TNF-α, and MIP-1β by conventional, user-guided methods (EM events. Data for the 4 selected time-points for each volunteer were uploaded to the ImmPort website (http://immport.niaid.nih.gov) and FLOCK analyses performed. The number of unique populations varied at different time-points and between volunteers . In order to compare data across time-points and between volunteers, a cross-sample analysis was performed. In this cross sample analysis, the populations identified in a single sample (volunteer 53 s day 10 post infection following stimulation with S. Typhi-infected B-LCL) were applied to all samples. Six unique CD69+  populations were identified  and dim (+) populations of IFN-γ and TNF-α based on mean fluorescence intensity (MFI) . BrighteThe net percentage of positive cells for each population at the 3 time-points, as determined by cross-sample analysis, is shown in S. Typhi-specific CD8+ T cells in response to both S. Typhi-infected autologous B-LCL and HLA-E restricted stimulation. Multifunctional IL-17A+ cells demonstrated multiphasic kinetics and were still detectable one year after immunization. We identified quadruple and quintuple positive CD8+ TEM and TEMRA IL-17A producing cells that co-produce pro-inflammatory cytokines/chemokines IL-2, IFN-γ, TNF-α, and/or MIP-1β but not IL-10. These multifunctional populations were confirmed using unsupervised flow cytometric analysis with FLOCK.Here we demonstrate the presence and persistence of multifunctional IL-17A producing Salmonella Typhimurium from the gut Salmonella Enteriditis infection and IL-17A knockout mice had an elevated bacterial burden in the liver and spleen as compared to wild-type mice S. Typhi. Because the gastrointestinal mucosa is the first point of contact for S. Typhi, mucosal immune responses are likely to play an important role in protection. This is to our knowledge, the first report of IL-17A production in response to S. Typhi, both to S. Typhi-infected autologous targets, as well as HLA-E restricted stimulation. The presence of S. Typhi-specific CD8+ T cells producing IL-17A suggests that these cells may play a role in the mucosal response. CD8+ T cells with characteristics of the Th17 lineage, Tc17, have been described in the setting of chronic hepatitis C virus infection + Tc17 cells responsive to antigens from infectious agents.IL-17 has been increasingly implicated in host responses against intracellular pathogens i.e., those producing 2 or more cytokines simultaneously, have been shown to produce higher levels of individual cytokines, have enhanced function, and are more likely to correlate with protection from disease, when compared to single cytokine producing cells in vitro and animal studies have previously suggested.Multifunctional T cells, S. Typhi-infected autologous B-LCL, they represent only a portion of these responses. It will be important to confirm these findings using different targets such as autologous blasts. However, these data are supported by the fact that in previous studies, monoclonal antibodies against HLA-E were only able to partially block CTL activity against S. Typhi-infected targets, while monoclonal antibody against a pan-MHC class I marker (W6/32) showed almost complete abrogation of CTL against S. Typhi-infected targets S. Typhi-infected cells raises the likely possibility that various effector cells might contribute differently to the host defense at various times after exposure. These observations also point to the importance of the timing of specimen collection following immunization and its impact on the magnitude of effector immune responses which can be measured. Finally, it is important to mention that these data confirm and extend previous observations on the longevity of CMI responses (at least 1 year) elicited following immunization with Ty21a and other attenuated typhoid vaccine candidates Multiphasic kinetics have previously been demonstrated in response to Ty21a immunization following stimulation in an HLA-E restricted manner S. Typhi-specific CD8+ T cells at various times could represent these cells homing cells to the gut. Previous studies have, in fact, shown expression of gut-homing molecules on S. Typhi-specific CD8+ T cells S. Typhi will provide key information to assess the role of multifunctional cells producing high levels of IL-17A and other cytokines in protection from infection. These studies are likely to contribute to accelerate the development of much needed novel typhoid vaccines.The significance of the multiphasic responses observed is not entirely clear; however, it is possible that the decline in the levels of cytokine producing Figure S1Gating strategy used for flow cytometry analyses. Lymphocytes were gated based on forward and side scatter, followed by doublet exclusion. Live (ViViD negative) CD14- CD19- CD3+ T cells were then selected. CD8+ CD4- cells were further divided into memory subsets based on CD62L and CD45RA expression. Naïve T cells , T central memory cells , T effector memory , and T effector memory CD45RA+ (TIF)Click here for additional data file.Figure S2Kinetics of cytokine/chemokine production following stimulation of PBMC with S. Typhi-infected autologous B-LCL (non-responders and a non-immunized control). Responses are expressed as net values with day 0 subtracted to normalize for differences in base-line responses to S. Typhi in different volunteers. A) CD8+ TEM cells for volunteer 48 s B) CD8+ TEMRA cells for volunteer 48 s C) CD8+ TEM cells for volunteer 52 s D) CD8+ TEMRA cells for volunteer 52 s E) CD8+ TEM cells for volunteer 196 s F) CD8+ TEMRA cells for volunteer 196 s * MIP-1b not measured(TIF)Click here for additional data file.Figure S3EM responses to Multifunctional CD8+ TS. Typhi-infected autologous B-LCL. A) Scatter plot showing all combinations of the 6 cytokines/chemokines measured that were positive at one or more time points for volunteer 54 s. Increases of >0.5% cytokine positive cells over uninfected targets were found to be statistically significant (P<0.01). Each point represents a single time-point and the median value is denoted by a horizontal bar. The cyokine/chemokine combinations with the top 2 median values are indicated by red squares. Median values of the subsets of multifunctional cells that show a significant difference , when compared to the subsets without asterisks, are indicated. B) Kinetics of the top 2 populations  over time.(TIF)Click here for additional data file.Figure S4EM responses to Multifunctional CD8+ TS. Typhi-infected HLA-E restricted cells. A) Scatter plot showing all combinations of the 6 cytokines/chemokines measured that were positive at one or more time points for volunteer 54 s. Net increases of >0.5% cytokine positive cells over uninfected targets were found to be statistically significant (P<0.01). Each point represents a single time-point and the median value is denoted by a horizontal bar. The cyokine/chemokine combinations with the top 2 median values are indicated by red rectangles. Median values of the subsets of multifunctional cells that show a significant difference , when compared to the subsets without asterisks, are indicated. B) Kinetics of the top 2 populations  over time.(TIF)Click here for additional data file.Figure S5EM responses to Multifunctional CD8+ TS. Typhi-infected autologous B-LCL. A) Scatter plot showing all combinations of the 5 cytokines/chemokines measured (MIP-1β was not measured) that were positive at one or more time points for volunteer 50 s. Net increases of >0.5% cytokine positive cells over uninfected targets were found to be statistically significant (P<0.01). Each point represents a single time-point and the median value is denoted by a horizontal bar. The cyokine/chemokine combinations with the top 3 median values are indicated by red rectangles. Median values of the subsets of multifunctional cells that show a significant difference , when compared to the subsets without asterisks, are indicated. B) Kinetics of the top 3 populations  over time.(TIF)Click here for additional data file.


Alpha B-crystallin (CRYAB) is a protein that functions as “molecular chaperone” in preserving intracellular architecture and cell membrane. Also, CRYAB is highly antiapoptotic. Abnormal CRYAB expression is a prognostic biomarker for oral cancer, while its genomic variations and the association with carcinogenesis have never been studied.CRYAB single nucleotide polymorphisms may be associated with oral cancer risk. In this hospital-based study, the association of CRYAB A-1215G (rs2228387), C-802G (rs14133) and intron2 (rs2070894) polymorphisms with oral cancer in a Taiwan population was investigated. In total, 496 oral cancer patients and 992 age- and gender-matched healthy controls were genotyped and analyzed. A significantly different frequency distribution was found in CRYAB C-802G genotypes, but not in A-1215G and intron2 genotypes, between the oral cancer and control groups. The CRYAB C-802G G allele conferred an increased risk of oral cancer (P = 1.49×10−5). Patients carrying CG/GG at CRYAB C-802G were of lower 5-year survival and higher recurrence rate than those of CC (P<0.05).Therefore, we hypothesized that CRYAB C-802G is correlated with oral cancer risk and this polymorphism may be a useful marker for oral cancer recurrence and survival prediction for clinical reference.Our results provide the first evidence that the G allele of  In addition, the two clinical outcomes contribute to the highest death rate of oral cancer, metastasis and recurrence, were analyzed of their associations with CRYAB genotypes.In order to understand the genomic role of CRYAB A-1215G, C-802G, and intron 2 for the participants are shown in CRYAB C-802G is significantly different between oral cancer and control groups (P<0.05), while those for A-1215G or intron 2 were not significant (P>0.05) , not those of A-1215G  or intron2 , is found to be associated with the susceptibility for oral cancer  . Also, tl cancer . To sum CRYAB genotypes, the relationships among disease-free survival, recurrence, metastasis and CRYAB C-802G genotypes were analyzed. First, the oral cancer patients carrying the CRYAB C-802G CG had a significant trend toward decreased disease-free survival, and the patients carrying CRYAB C-802G GG had the shortest disease-free survival period  at the first diagnosis. Interestingly, the patients would have frequent recurrence and high second primary tumor rates within the following five years. Second, compared with those with CC genotype, the patients carrying CRYAB C-802G CG or GG genotype had a higher recurrence rate within the following five years , but not a higher metastasis rate  and one at the intron 2 (intron2), to investigate their associations with oral cancer risk and prognosis. We found that CRYAB C-802G polymorphism, not A-1215G or intron2, was associated with increased risk of oral cancer : 5′-ACCTGTTGGAGTCTGATCTT-3′ and 5′-ATGCACCTCAATCACATCTC-3′; for CRYAB C-802G (rs14133): 5′-TTGACCATCACTGCTCTCTT-3′ and 5′-TTGGCAATGTGACACATACC-3′; for CRYAB intron 2 (rs2070894): 5′-GTCTAGAAGACTAAGTTAGG-3′ and 5′-AGAGAAGTCACAACTCAAGT-3′; The following cycling conditions were performed: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The PCR products were studied after digestion with Fau I, Fat I, and DPN I, restriction enzymes for A-1215G (cut from 212 bp A type into 67+145 bp G type), C-802G (cut from 363 bp G type into 85+278 bp C type), and intron 2 (cut from 363 bp T type into 74+339 bp C type), respectively.Genomic DNA from oral cancer and health control subjects were prepared from peripheral blood leucocytes using a QIAamp Blood Mini Kit  and further processed according to previous studies only those matches with all SNPs data (case/control  = 496/992) were selected into final analyzing. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of CRYAB SNPs in the control subjects from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's two-sided Chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the CRYAB genotypes between cases and controls.In our study, P<0.05.The primary outcome was disease-free survival. The endpoints included local cancer recurrence and metastasis. Follow-up information was available for all patients at the 5-year time point. Disease-free survival time was calculated from the date of treatment until the time of recurrence, defined as disease recurrence at the same site or the detection of metastases, including recurrence in the neck lymph nodes. The genotypes were coded assuming an allele dose-effect . Disease-free survival curves were generated by the Kaplan-Meier method and verified by the log-rank test. The significance level was set at


The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR−/− mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.We show reduced NDRG2 mRNA in livers of FXR The Farnesoid X Receptor  is a member of the nuclear hormone receptor superfamily, predominantly expressed in tissues exposed to high levels of bile acids, such as the entire gastrointestinal tract, the liver, the bile duct and gallbladder. FXR mRNA can also be detected in tissues such as the adrenals, kidneys and adipose tissues −/− mice spontaneously develop hepatocellular carcinoma beyond 12 months of age, suggesting that FXR has a prominent function as a tumor suppressor against liver tumor formation FXRin vivo FXR binding sites in liver and intestine −/− mice do also spontaneously develop liver tumors beyond 12 months of age, similar to what is found in FXR−/− mice Using genome-wide Chromatin Immunoprecipitation followed by sequencing (ChIP-Seq), two groups have identified numerous genes containing N-myc downstream regulated gene 2 (NDRG2) was recently reported as a candidate tumor suppressor in human liver cancer metastasis and it is transcriptionally reduced in HCC −/− mice compared to wild type mice and Ndrg2 mRNA can be induced by non-steroidal FXR agonists in livers of wild type mice. In humans, both, NDRG2 and FXR mRNA's are reduced in primary hepatocellular carcinoma samples of different tumor stages compared to normal human liver or non-HCC liver diseased human liver. Finally, we provide evidence that stable over-expression of FXR in SK-Hep-1 human hepatoma cells does reduce proliferation and migration of these cells in an FXR agonist dependent fashion in vitro and tumor growth and metastasis in an orthotopic xenograft mouse model in vivo.Here we show that NDRG2 is a direct transcriptional target of FXR in mouse liver and human hepatoma cell lines. We further demonstrate that Ndrg2 mRNA is reduced in livers of FXRGA]GGT[TC]A-N-T[AG]ACC[TC] that is known to be recognized by FXR in identified target genes GGGTTAGTGACCC) within the first intron in all three species (http://biogps.org). Interestingly, there is a second IR1-like sequence (AGGTTGATGACCC) just downstream of the first sequence in the human NDRG2 gene, which is absent in the mouse and rat genes , we could amplify a 196 bp DNA fragment by PCR, encompassing the predicted IR1 (http://genome.ucsc.edu/cgi-bin/hgGateway), we identified a strong FXRα binding signal that was located right on the predicted IR1 sequence in the first intron of mouse Ndrg2  and/or to differentially phosphorylated forms. The structurally related FXR agonist PX20606 , an inhibitor of protein synthesis. Similar to the established direct FXR target gene SHP, NDRG2 mRNA induction was not reduced by the protein synthesis inhibitor Cycloheximide, suggesting that NDRG2 is a direct target gene of FXR . InteresIn order to address if Ndrg2 is controlled by FXR in mouse liver, we analysed the expression levels of Ndrg2 mRNA in livers of wild type mice 4 hours after oral gavage of 30 mg/kg of either PX20350  or the F−/− mice might result in reduced expression of Ndrg2 mRNA in the liver. Quantitative Real Time PCR (RT-qPCR) indicated a significant reduction of Ndrg2 mRNA in livers of FXR−/− mice compared to livers of wild type mice  showed robust reduction of NDRG2 in presence and absence of PX20606 compared to the negative control small hairpin  3 days after implantation and fluorescence was determined using the In-Vivo Imaging System FX from Kodak on the following day. X-ray photographs were taken in parallel to allow for both quantification (in vivo fluorescence imaging) and localization (X-ray) of the tumor cell load, at the primary sites of injection in the liver as well as at sites of metastasis . Animals were randomized on day 4 and subsequently treatment was started by daily oral gavage with vehicle or compounds suspended in vehicle. In a first pilot experiment with four animals per group, we observed a strongly reduced growth of tumors in the livers of mice that received SK-GI-18 cells, overexpressing FXR, compared to mice that received SK-Hep-1, during an observation period of 32 days (data not shown).Female nude mice were injected with either 5×106 SK-Hep-1 or 5×106 SK-GI-18 cells by injection into one lobe of the liver, but group sizes were increased . After 3 days of recovery, and starting with day 4, mice were daily gavaged with vehicle or 10 mg/kg/d of the FXR agonists PX20606 in vehicle or 100 mg/kg/d of Sorafenib (Nexavar™) in vehicle until the animals were finally sacrificed on day 56. For this experiment, PX20606 was chosen because of its improved pharmacokinetic properties compared to PX20350  did, as expected, significantly reduce the growth of tumors in the liver and metastases in lymph nodes derived from both SK-Hep-1 and SK-GI-18 cells when compared to vehicle treatment . The synThese data suggest that Sorafenib might be particularly effective in preventing metastasis to lymph nodes with hepatoma cells ectopically expressing FXR.Accordingly, in a separate experiment, SK-GI-18 cells were orthotopically implanted into nude mice and daily gavaged with vehicle, PX20606 (10 mg/kg/d), Sorafenib (50 mg/kg/d) or a combination of Px20606 and Sorafenib (10 and 50 mg/kg/d respectively) starting on day 4 until the end of this experiment on day 51. Tumor load (fluorescence intensity) in the liver was again significantly reduced by treatment with PX20606 (P), Sorafenib (S) and the combination of Px20606 and Sorafenib (P+S) as shown in −/− mice compared to livers from wild type mice. It is therefore tempting to speculate, that the downregulation of Ndrg2 mRNA in FXR−/− mice might contribute to the time dependent development of hepatocellular carcinoma in these mice. This hypothesis could be directly tested in the future by liver specific deletion of NDRG2 in adult mice or shRNA-mediated knock-down of NDRG2. However, loss of FXR also results in downregulation of SHP, and SHP−/− mice also do develop HCC in a time-dependent fashion Herein we show that the bile acid receptor FXR, most likely as a heterodimer with RXR, controls transcription of NDRG2 though IR1 response elements identified in the first introns of mouse, human and rat NDRG2 genes, respectively. We show that FXR, when activated by a non-steroidal agonist, induced the transcription of the liver tumor suppressor candidate NDRG2 in vitro. We also showed that interference with NDRG2 expression by small hairpin RNA expression blunted the FXR agonist dependent reduction in proliferation in vitro, suggesting that NDRG2 might mediate at least to some extent the growth reducing effects of FXR agonists in the respective cell lines. We employed an orthotopic xenograft tumor model in nude mice combined with in vivo imaging to follow growth of hepatoma cell-derived tumors at the primary site of injection in the liver and metastasis into lymph nodes, gut and bone marrow. The growth of tumors derived from FXR over-expressing SK-GI-18 cells was significantly reduced in the liver and in the gut compared to tumors derived from the parental SK-Hep-1 cells. In contrast, there was no significant difference in metastasis of SK-GI-18 cells into lymph nodes compared to the parental SK-Hep-1 cells. After oral application of the strong FXR agonist PX20606, however, both, growth of tumors in the liver and metastases in the lymph nodes were significantly reduced both compared to vehicle as well as compared to SK-Hep-1. This might be explained by a higher availability/activity of the non-steroidal FXR agonist Px20606 at these peripheral sites compared to the endogenous bile acids which are more concentrated in enterohepatic tissues. The growth and metastasis suppressing activity of FXR agonist Px20606 (10 mg/kg/d) in this orthotopic xenograft model with either SK-Hep-1 or SK-GI-18 is only slightly less efficacious compared to Sorafenib (Nexavar™), presently the only medicament approved for treatment of advanced HCC We could demonstrate that ectopic expression of FXR in the SK-Hep-1 derivative SK-GI-18 resulted in an increase in basal and FXR agonist-induced expression of NDRG2 and in an FXR agonist-dependent reduction in growth rate and migration potential Interestingly, Sorafenib showed a higher efficacy to interfere with metastasis of SK-GI-18 cells to lymph nodes compared to the parental SK-Hep-1 cells. Moreover, the combined treatment with Sorafenib (50 mg/kg/d) and Px20606 (10 mg/kg/d) did further reduce the metastasis of SK-GI-18 cells to lymph nodes in the orthotopic xenograft model. These findings suggest that pathways activated by FXR agonists and inhibited by the pan-tyrosine kinase inhibitor Sorafenib may synergize to interfere with effective metastasis of hepatoma cells. Future studies will have to address in detail the mechanism for this synergistic activity on tumor metastasis.−/− mice −/− mice on a nude background in order to directly test the involvement of mouse FXR on the observed behaviour. More advanced stages of human HCC seem to go along with progressive de-differentiation of malignant liver cells and progressively reduced FXR expression and/or activity. However, residual FXR expression in malignant liver cells and FXR expression/activity in non-malignant surrounding tissue could both represent targets for antitumor activities of FXR agonists. This may be the basis for novel medicines that could not only prevent or slow down the development of a primary tumor or progression of tumor growth at early stages but potentially also tumor growth or metastasis at later clinical stages of HCC. The absence of strong cytotoxicity of FXR agonists like PX20350 or PX20606 and their hepatoprotective activities may also make them possible candidates for a combination with classical chemotherapy or multi-kinase-targeted anti-tumor therapies such as Sorafenib.In view of our results and previously published studies we propose that FXR functionally acts as a tumor suppressor in liver and presumably other enterohepatic tissues. We propose that reduction of FXR expression or FXR function over an extended time period could lead to a reduced expression of direct and indirect target genes, including NDRG2 and SHP, mediating the anti-tumorigenic functions of FXR. NDRG2 has been implicated as a tumor suppressor in a number of malignancies nuFoxn1 mice (nude mice) were purchased from Charles River, Erkrath, Germany and obtained in microisolators in barrier conditions. At 6–8 weeks of age, mice were injected with SK-HEP-1 or SK-GI-18 cells.All animal experiments were conducted according to the guidelines of the German Animal Welfare Act (Tierschutzgesetz) and under the permission numbers of the Regierungspräsidium Freiburg, Germany G-10/05 and 35-9185.64/1. Animal health was examined prior to tumor implantation and randomization to ensure that only animals without any symptoms of disease were selected to enter testing procedures. During the experiments, animals were monitored twice daily regarding tumor burden, general condition, food and water supply. Crl:NU-1H-NMR and LC-MS before being tested in FXR activity assays. All other chemicals used were of analytical grade or higher. Sorafenib  was derived from prescription strength Nexavar 200 mg tablets . Compounds were dissolved in 100% DMSO  and diluted in growth medium to a final concentration containing 0.2% DMSO for in vitro studies. DMSO was added to cultures at a concentration of 0.2% (v/v) as a vehicle control. For in vivo experiments in nude mice, compounds were suspended in 0.5% Polyvinylpyrrolidone K30 , 0.1% Polysorbat 80  in 150 mM NaCl  as a vehicle and administered by oral gavage at 2.5 ml/kg dosing volume. Sorafenib  was gavaged twice a day with a 6 hour time lag.GW4064, PX20350 and PX20606 were synthesized according to published synthesis protocols Oris™ Cell Migration Assay with Fibronectin coated 96 well plate was obtained from Platyplus Technologies, Madison, U.S.A.. Mouse monoclonal anti-FXR antibody  was purchased from R&D Systems, Wiesbaden-Nordenstadt, Germany. Goat anti-NDRG2 antibodies (E-20) were purchased from Santa Cruz, Heidelberg, Germany. Mouse anti FLAG M2 antibodies (F1804), Mouse-anti GAPDH antibodies (G8795), goat anti-mouse IgG-Alkaline Phosphatase Conjugate (A3688) and rabbit anti-goat IgG-Alkaline Phosphatase Conjugate (A4062) were all obtained from Sigma-Aldrich, Munich, Germany and Lumi-Phos WB Chemiluminescent Substrate (34150) was obtained via Thermo Scientific, Bonn, Germany.For short term induction experiments (4 hours) in C57Bl/6 mice see , a diffeHep-G2 (ACC-180), SK-Hep-1 (ACC-141) and Hek-293 (ACC 305) cells were obtained from DSZM, Braunschweig, Germany. HuH-7 (JCRB0403) cells were obtained from the Health Science Research Resources Bank, Osaka, Japan. Cells were cultivated in RPMI-1640 (Hep-G2 and SK-Hep-1 cells), MEM (Hek-293) or DMEM (HuH-7) containing 8.6% fetal calf serum supplemented with 2 mM L-Alanyl-L-Glutamine .in vitro proliferation cells were plated at 1000 cells per well of a 96 well plate and after 16 h, growth medium with different concentrations of the respective FXR agonist was added. After 72 hours, the DNA content was determined using a spectro-fluorometer  using the CyQuant Direct Proliferation Assay .For determination of GGGGACAAGTTTGTACAAAAAAGCAGGC-ATGGGATCAAAAATGAATCTCTCG and GGGGACCACTTTGTACAAGAAAGCTGGG-TGTAATCCCCATCACTGCACTC (letters in bold refer to the FXR cDNA) and after generation of an Entry Clone  recombined into pTRex-Dest30  to yield pTRex-Dest30-hFXRα2, in which the coding region of human FXRα2 is under control of the CMVtetO2 promoter. For expression of FXR via an episomal vector, a 3751 bp SnaBI-SfiI fragment from pTReX-Dest30-hFXRα2 carrying the CMVteto2-hFXR-SV40polyA unit was inserted into the corresponding sites in pCEP4  to yield pCEP4-TReX-hFXRα2. To generate stable SK-Hep-1 and Huh7 clones (SK-GI-18 and Huh7-37) overexpressing human FXR, pTReX-Dest30-hFXRα2 was microporated into cells using microporation  and stable clones selected in RPMI growth medium supplemented with 8.6% FCS and 1 mg/ml of G-418  and extracts of cells tested for overexpression of FXR in Western Blot using the mouse-anti-FXR antibody A9033A . HepG2-FXR5 cells overexpressing human FXRα2 were generated by transfecting HepG2 cells with pCEP4-TReX-hFXRα2 and selecting a stable cell line in RPMI medium supplemented with 8.6% FCS and 200 µg/ml of Hygromycin B .For overexpression of human FXR , the coding region was amplified using Gateway compatible primers The plasmid pMC3TRex is a derivative of pMEP4  in which the Metallothionine promoter was replaced by an expression cassette consisting of the E.coli tetracycline repressor controlled by the CMV promoter and the 3054 bp Sbf1-AscI fragment from pTReX-Dest30 which carries a gateway recombination cassette under control of the CMV-tetO2 promoter . An N-terminal FLAG-tagged human FXR was cloned into pDONR201 and then recombined into pMC3TRex using a LR reaction  to yield pMC3TRex-FLAG-FXR. Stable HepG2 cells carrying episomal pMC3TRex or pMC3TRex-FLAG-FXR were generated using microporation  and selection of stable clones was done in RPMI growth medium supplemented with 8.6% FCS and 200 µg/ml of Hygromycin B .CAACCTGGATAACATTGAATT; 2: GGACCAGCTTGCAGACATGAT, 3: ACCAGTGCAGCATCCGTTCAT, 4: ATGTGGGACTCAACTATAAAT and as negative control NC: GGAATCTCATTCGATGCATAC under control of the U1 promoter and a hygromycin resistance marker gene. Stable SK-GI-18 cells were generated using microporation  and stable clones selected in RPMI growth medium supplemented with 8.6% FCS and 200 µg/ml of Hygromycin B  followed by growth in medium containing 1 mg/ml of G418 .For knock-down of NDRG2 expression, SureSilencing shRNA plasmids were purchased from Qiagen, Hilden Germany with the insert sequences 1: Total protein was prepared from cells growing in 6 well tissue plates (Corning) by scraping using RLN buffer  Nonidet P-40). Protease and Phosphatase inhibitors  at a concentration of 1∶100 were freshly added to the RLN buffer before use.Total cell lysates made in RLN buffer (10 µg) were separated by electrophoresis on 12% SDS-PAA gels, then electroblotted to Protran membranes (Whatman) in Tris transfer buffer containing 20% methanol. Membranes were stained with Ponceau S to verify loading and transfer efficiency. Membranes were probed with primary and secondary antibodies in Tris-buffered saline with Tween-20  containing 5% Soybean Hydrolysate . Signals were visualized by incubating the blots in Lumi-Phos WB Chemiluminescent Substrate  and exposing to X-ray film . Membranes were reprobed with anti-GAPDH antibodies after stripping with 0.2M Glycine for 2 hours.th of the isolated total RNA from one 96 well. Quantitative PCR was performed and analyzed using Absolute QPCR Rox Mix  and a 384-format ABI 7900HT Sequence Detection System . Primer and probe sequences  are listed in supporting Frozen tissues were ground to fine powder under liquid nitrogen with a mortar. Total RNA from 25 mg powdered tissue was isolated using RNAzol RT Reagent  and an RNA Isolation Kit  following instructions provided by the manufacturers. The concentration and integrity of the total RNA was assessed by gel electrophoresis and cDNA's were synthesized from 0.2 µg of total RNA using SuperscriptII™ reverse transcriptase  primed with 50 pmol of random nonamers. Total RNA from cells grown in 96 well tissue plates were isolated following the instructions of the RNeasy 96 RNA isolation kit  and cDNA's were synthesized from 1/25http://www.origene.com/assets/documents/TissueScan/LVRT101.xls;). To study FXR, NDRG2, SHP and TBP expression, PrimeTime qPCR assays  were used using the 384-format ABI 7900HT Sequence Detection System . FXR, BSEP, and SHP gene expression was normalized to TBP gene expression. The PrimeTime assays used in this study can be found in supporting FXR (NR1H4), NDRG2 and small heterodimer partner  mRNA levels were quantified in human normal liver, HCC and non-malignant liver disease cDNA's purchased from Origene . These cDNA samples have been generated from a total of 34 HCC, 8 normal livers and 12 non-HCC liver disease livers verified by pathologists before isolation of RNA and conversion to cDNA. The HCC samples included seven samples of stage I HCC, eight samples of stage II and IIIA HCC each, and three samples of stage IV HCC and 13 samples of non HCC liver diseases (The IR1-type FXR response element from the human IBABP gene was cloned as a 68 bp KpnI-BglII fragment containing two IR1 elements (shown below) into the KpnI-BglII site of pGL2-promoter  resulting in pGL2-IBABPx2.GGGTGAaTAACCTcggggctctgtccctccaatcccaGGGTGAaTAACCTcgggA5′CcccaCCCACTtATTGGAgccccgagacagggaggttagggtCCCACTtATTGGAgcccTCTAG 5CATGGgggt′The NDRG2 IR1-type response element shown below was cloned as a 66 bp Asp718-XhoI fragment into the Asp718 and XhoI sites of pGL4.30  replacing the NFAT response element resulting in pGL4-NDRG2-RE.GGGTTaGTGACCCGAGGAGGTTGaTGACCCTGGGAGTCTGGGTCTTGGCTCGTACCACACTGGGGACCCAATCACTGGGCTCCTCCAACtACTGGGACCCTCAGACCCAGAACCGAGAGCTGTGTGACCCCTThe NDRG2 IR1mut sequence shown below was cloned as a 66 bp Asp718-XhoI fragment into the Asp718 and XhoI sites of pGL4.30  replacing the NFAT response element resulting in pGL4-NDRG2mut.TTAGTGAGGGGAGGAGGTTGaTGAGGGTGGGAGTCTGGGTCTTGGCTCGTACCACACTGGGGACCCAATCACTCCCCTCCTCCAACtACTCCCACCCTCAGACCCAGAACCGAGAGCTGTGTGACCCCTGGGRenilla luciferase under control of a CMV promotor as an internal control) into HEK293 cells and cultivated in presence of the FXR agonist Px20350 (0.5 µM) of DMSO for 16 h followed by cell lysis with Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured sequentially in the same cell extract using a Dual-Light-Luciferase-Assay system and microplate luminometer . Relative luciferase activity (RLA) was calculated as ratio between firefly luciferase and Renilla luciferase activity and relative fold induction calculated a RLA in presence of Px20350 divided by RLA in DMSO from at least three individual experiments.The reporter plasmids  were cotransfected with pTRex-Dest30-hFXR (50 ng/well) and pCMV-RL  in 25 µL assay buffer . After 60 min of incubation the FRET efficiency was determined using a spectro-fluorometer . Measurements were done in duplicates. FRET values are given in nM and Eff (%) is the maximum efficacy of the compound relative to the control FXR agonist GW4064.To assess the FXR agonist activity by TR-FRET a biotinylated SRC-1 peptide b-CPSSHSSLTERHKILHRLLQEGSPS-COOH (0.4 µM), 200 ng streptavidine-allophyco-cyanine and 6 ng europium labeled-anti-GST were mixed with either purified human FXRaa187–472, mouse FXRaa202–484) expression plasmid fused C-terminally to a Gal4 DNA-binding domain under transcriptional control of the CMV promoter in pCMV-BD . Additionally a second reporter plasmid pRL-TK constitutively expressing the Renilla luciferase under control of a TK promotor was included as an internal control. 4 h after transfection cells were treated with serial dilutions of the test FXR agonists. 16 h after treatment cells were lysed with Passive Lysis Buffer  and firefly and Renilla luciferase activities were measured sequentially in the same cell extract using a Dual-Light-Luciferase-Assay system and microplate luminometer . Relative luciferase activity was calculated and reported as ratio between firefly luciferase and Renilla luciferase activity. EC50 values were calculated from at least three independent experiments.As cellular assay for FXR agonist activity, a Gal4-FXR fusion construct reporter system was used. HEK293 cells were transiently transfected with a reporter plasmid pFRluc  encoding a Gal4 promotor driven firefly luciferase and a given FXR-LBD  in 300 µl ChIP buffer with mild sonication . PCR was done with forward GAACTGATGCCCTTGTAGCC and reverse CAACGAGGTGAATGACATGG primers, for 41 cycles (15 sec 95°C and 60 sec 60°C) to amplify a 196 bp DNA fragment harbouring the IR1 sequence.HepG2 cells stably transfected with pMC3TRex or pMC3TRex-FLAG-FXR were grown in 10 cm cell culture dishes to 70% confluency and cells fixed and chromatin sheared using the Shearing ChIP Kit and the Bioruptor . For chromatin immunoprecipitation, the One-day ChIP kit was used according to the recommended protocol . One 206 SK-Hep-1 or 5×106 SK-GI-18 cells were resuspended in a volume of 100 µl PBS and injected into one liver lobe, respectively, according to the SOP established at Oncotest Freiburg, Germany.Mice were anesthetized with isoflurane and 5×10Mice were injected intravenously with 0.02 mg anti-human-CD10-Antibody , that is labelled with fluorescence dye Xenolight CF750 . Mice were anesthetised by isoflurane inhalation 20 hours after the injection, and pictures (fluorescence and x-ray) were taken with a Kodak in vivo imaging system (Kodak Image Station in vivo FX).Data were expressed as the means ± SEM from at least three experimental measurements.Differences between experimental groups were evaluated for statistical significance using Student's t-test: *, p<0.05; **, p<0.01; ***, p<0.001. using the software Prism5 . where * p<0.05 is considered to be statistically significant.Figure S1Small hairpin mediated knock-down of NDRG2 mRNA in SK-GI-18-sh3-14 cells compared to SK-GI-18-NC16 cells. Cells were grown in triplicates in 6 well plates in medium containing DMSO (D) or 1 µM PX20606 (PX) in DMSO for 18 h before the relative NDRG2 mRNA levels were determined by RT-qPCR from isolated total RNA. The NDRG2 mRNA levels were normalized to TBP and the relative NDRG2 mRNA levels in SK-GI-18-NC16 cells grown with DMSO set to 1 and Students ttest performed.(DOCX)Click here for additional data file.Table S1Sequences of primers for PrimeTime-Assays (qPCR) supplied by IDT DNA Technologies.(DOCX)Click here for additional data file.