Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants  was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.Consolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen  Lignocellulosic biomass is an abundant feedstock and attractive source of sugars for biofuel production. Large-scale utilisation is however challenged by the general lack of low-cost technologies that can overcome cellulose recalcitrance . One potSaccharomyces cerevisiae, Pichia stipitis, Candida shehatae, Pachysolen tannophilus) and bacteria  have been engineered for CBP ethanol production however their capacity to secrete saccharification and fermentation enzymes at sufficient yield remains an obstacle  or butanol ] with a single treatment tested per 96-well plate (see 6 ml-1) (eight wells per treatment per wild type/transformant strain). Plates were sealed with a lid and parafilm and incubated at 25°C for 7 days. Fungal growth was measured using a spectrophotometer OD600nm , and percentage increase or decrease in growth relative to wild-type control was determined. For data analysis see In the tertiary screen, tolerance to both ethanol and butanol was assessed, with the goal of examining the potential for cross-correlation between ethanol and butanol tolerance. Conidial inoculum for the tertiary assessment was generated by individually inoculating each transformant into Mung bean broth   values obtained from RT-PCR were used to calculate the accumulation of the target gene (relative mRNA accumulation) relative to β-tubulin transcript by the 2-ΔΔCT method, where ΔΔCT =  [An experiment was designed to investigate the temporal accumulation of a putative hexose transporter under alcohol stress  see Met. Each extubulin) . ResultsF. oxysporum strain 4287 sugar transporters was constructed using amino acid sequences from transcripts showing ≥30% homology to FOXG_09625  and a Box and Whisker plot was used to depict the distribution of transformant response across various treatments. The primary/secondary screen dataset was non-normally distributed as determined within Minitab . The significance between treatments at each time point (48 and 96 hours respectively) or ethanol treatments was analysed within the Statistical Package for the Social Sciences  using Kruskal-Wallis H-test for non-parametrical data. The significance between treatments and transformants amongst the 29 individuals selected from secondary screening was determined using one-way ANOVA as the data could be transformed to fit a normal distribution using Johnson transformation within Minitab. For tertiary screen analysis see F. oxysporum strain 11C to generate a library of gene disruption transformants  (-1)) response levels measured across 48 and 96 hours  . The lib96 hours . Transfohph transgene (P<0.001) (Over 90% (402/443) of the monoconidial lines generated from primary transformants were PCR-positive for the ransgene . On re-sP<0.001) . A totalP<0.001) (-1) ethanol the highest level of hyper-or hypo-tolerance relative to parent strain 11C was observed for Tr. 230 and Tr. 259 (-1), Tr. 259 continued to demonstrate a substantial hypo-tolerance to ethanol relative to the parental strain (-43.01%±6.40) . Similar to results recorded for ethanol, at the highest butanol concentration (0.75% vv-1) Tr. 259 (-30.61%±13.49) showed the highest hypo-butanol phenotype and Tr. 185 (12.57%±13.16) the most hyper-butanol phenotype relative to strain 11C (Tertiary screening was used to assess tolerance of the 29 selected transformants to ethanol and cross-tolerance to butanol . A signiP<0.001) . At 2 an Tr. 259 . At the 1%±6.40) . A stronrain 11C . hph probe indicated an average transgene copy number of 1.55 (P<0.05) altered tolerance relative to the positive control (wild type strain 11C) to ethanol , butanol  or significantly(P<0.05) for both alcohols, Tr. 259. Southern blot analysis of a random sub-set of nine transformants using an  of 1.55 . PCA ana of 1.55  highlighr≥-0.967, n=5, P≤0.02) and butanol  concentration whereas 11C showed significantly better growth than Tr. 259 up to 6% (vv-1) ethanol and 1% (vv-1) butanol (P<0.05) (-1) ethanol and 0.75% (vv-1) butanol a substantial difference in growth (≥42%) was recorded between Tr. 259 and 11C prompting the selection of these concentrations for further studies  . At 4% ( studies . P≤0.01)   & S6E-F.In silico analysis of the recovered coding sequence showed the highest homology in the ORF of a putative sugar transporter gene FOXG_09625 in F. oxysporum f.sp.lycospersici (strain 4287) at both the protein (68% identity) and nucleotide (83% identity) level respectively  (FGCD). Five of these proteins were annotated as hexose transporters  including FOXG_09625, and the remaining as hypothetical proteins. Comparison of these proteins showed considerable sequence diversity indicating potentially diverse roles of these proteins in F. oxysporum (F. oxysporum f.sp. lycospersici (strain 4287) encodes 13 proteins annotated as hexose transporters ( FOXG_09625 showed homology (percent identity ≥ 30%) with 30 different proteins all containing MFS and sugar transporter domains respectively, encoded within the genome of xysporum . In totasporters .P>0.05). (P<0.01) (P<0.001) with transcript levels highest at 0.5 hours  cultivation of germinated spores (pre-cultured for 12 hours) in the absence or presence of alcohol . In the P>0.05). . Reduced(P<0.01) . In the .5 hours .F. oxysporum strain 11C. Although the generation of fungal insertion libraries is well documented, the integration of a three-tier alcohol screen into an ATMT platform is novel, and led to the generation and isolation of phenotypically diverse transformants from the library, indicating genotype-dependent variation and a functional ATMT system.This study has shown that ATMT is an effective tool to generate phenotypic diversity in response to alcohol stress within -1) ethanol) was high compared to Candida albicans, which like F. oxysporum can ferment glucose to ethanol but can only tolerate 1% (vv-1) ethanol stress [-1)), however at lower concentrations (4-6% (vv-1)) a 50% reduction in yeast’s growth has been reported [-1) exists for most microbes with the exception of some Lactobacillus strains (3% (vv-1)) [Bacillus subtilis (5% (vv-1)) [ Ethanol tolerance of strain 11C (>6% (vvl stress . In contreported . Unsurpr (vv-1))  and Baci (vv-1)) .F. oxysporum ATMT-derived transformants for alcohol tolerance has not yet been described. In other ascomycetous fungi notably the rice blast fungus Magnaporthe grisea, large-scale ATMT mutagenesis coupled with phenotypic plate-based screens has been used in pathogenicity related studies [ Large-scale screening of filamentous fungi can be challenging as manipulating fungal material (e.g. mycelium) can interfere with pipette-aided transfer between plates . Fungal  studies ,45.hph-PCR positive transformants. It cannot be ruled out that false negatives were lost to the analysis during these two stages. These primary rounds of screening were based on single reads to accelerate the phenotyping process and isolate transformants of interest for further analysis using an intensive confirmatory tertiary screen. This latter screen highlighted transformant Tr. 259 which underwent phenotypic and genomic analysis to confirm its hypo-alcohol tolerant phenotype and the ATMT-based disruption of a coding region showing homology to a putative sugar transporter (FOXG_09625) in F. oxysporum f.sp lycospersici (strain 4287). Southern hybridisation suggested a single insertion site within Tr. 259 with genome walking indicating partial T-DNA integration but it cannot be ruled out that additional T-DNA insertion events have also occurred through the genome of Tr. 259.  The three-tier screening system was designed to specifically discriminate either alcohol sensitive or tolerant phenotypes. Integration of the primary screen into the ATMT protocol provided a course but effective method to instigate a logistically feasible sorting of the library, thereby avoiding the laborious and time-consuming task of purifying thousands of putative transformants prior to screening. Non-purified, putative transformants underwent primary screening in contrast to the secondary screen, which used single-spored S. cerevisiae, Pichia stipitis, Schizosaccharomyces pombe, Kluveromyces lactis), few sugar transporters have been identified and characterised in filamentous fungi [F. oxysporum f.sp lycospersici (strain 4287) has at least 13 putative hexose transporters including FOXG_09625, similar to Aspergillus nidulans reported to have 17 [-1) ethanol stress led to the proposal that the cell enters a pseudo-starvation state during ethanol stress, whereby sugar-based nutrients are no longer accessible to the cell despite being present in the culture medium [ Eukaryotic transporters are best characterised in the yeast 20-member family of major hexose transporter genes  which includes two sensor-coding genes  and the GAL2 gene . In contus fungi ,48. The  have 17 . RT-PCR  have 17 . It was e medium ,51. In te medium . A previous study  identifiE. coli), a knowledge deficit exists for filamentous fungi [ Filamentous fungal transporters appear to hold diverse functional roles notably in plant-fungi symbiosis -57 pathous fungi -68. A beus fungi  coupled F. oxysporum via ATMT, which has led to the generation of a collection of transformants, which are now available to the research community for future studies. This is all the more relevant in light of the recent advances and application of RNA sequencing to elucidate complex biological processes at the transcript level in an unlimited array of organisms [F. oxysporum. In conclusion, the work completed in this study represents a first step in the investigation of alcohol tolerance in rganisms . On the Figure S1Microplate  layout for three-tier screening.Fusarium oxysporum wild-type strain 11C (WT) and putative transformants were inoculated into columns (1-12) and screened across five treatments: T1; no ethanol, T2; hygryomcinB , T3; 0.5% (vv-1) ethanol , T4; 6% (vv-1) ethanol T5; 10% (vv-1) ethanol with treatments positioned in rows (A-F) (Primary-tier) (A) or into rows (A-H) and then transferred and inoculated into corresponding rows in a second microplate and screened across six treatments: T1; no ethanol, T2; 6% (vv-1) ethanol , T3; 7% (vv-1) ethanol T4; 8% (vv-1) ethanol, T5; 9% (vv-1) ethanol, T6; 10% (vv-1) ethanol (Second-tier) (B) or into columns (1-12) with eight replicates per strain tested (column A-H) and screened across a single treatment including media control (MC) per plate: either no alcohol, 2% (vv-1) ethanol , 2% (vv-1) ethanol, 4% (vv-1) ethanol, 6% (vv-1) ethanol or 0.25% (vv-1) butanol, 0.5% (vv-1) butanol and 0.75% (vv-1) butanol (C). Grey shading indicates microplate well not used during experiment.(TIF)Click here for additional data file.Figure S2Fusarium oxysporum strain 11C and transformant Tr. 259.Experimental design flow chart to investigate alcohol-induced stress in (TIF)Click here for additional data file.Figure S3Fusarium oxysporum strain 11C transformants.Generating and screening  Inoculation of Fusarium oxysporum strain 11C into Mung bean broth [4 conidia per ml (A) Inoculation of Agrobacteriumtumefaciens strain AGL-1 to minimal medium (MM) [-1) for 2 days at 28°C (OD600nm 0.4-0.6) followed by dilution to OD600nm of 0.15 in induction medium (IM) [B) Co-cultivation of an equal volume of bacterial and fungal cells for 30 minutes at 28°C in liquid co-cultivation medium [F. oxysporum strain 11C and randomly integration into the fungal genome results (C) Transfer of cellulose filter membrane to modified selection medium (SM) on large Petri plates (150x20mm) supplemented with 60 hygromycinB for 7-9 days at 25°C followed by isolation of hygromycinB (60 μg ml-1) resistant putatively transformed colonies into microtiter (96-well) plates with minimal medium [-1) for 3 days at 25°C (D) Transfer of putative transformants to microtiter (96-well) plate with minimal medium supplemented with ethanol and butanol selection for primary alcohol tolerance screening followed by purification and PCR prior to two additional rounds of screening (2° and 3°) and selection of candidates for future analysis (E).an broth  for 5 daium (MM)  supplemeium (IM)  supplemen medium  followedl medium  suppleme(TIF)Click here for additional data file.Figure S4Fusarium oxysporum strain 11C transformants.Molecular analysis of  PCR was used to determine if fungal transformant genomic DNA (gDNA) extracts contained hph gene present in pSK1019. Lanes 2-6 represent gDNA from transformants, 7; PC – positive control (plasmid DNA pSKI019), 8; NTC – no template control (water), 9; WT - gDNA from wild-type strain 11C. Arrow indicates PCR product size (576 bp) based on 100bp ladder  (Lane 1) (A). Southern blot analysis was used to confirm transgene integration in EcoR1 digested fungal gDNA and to determine copy number, using a 741nt fragment of the hygromycin phosphotransferase gene (hph) as a probe. Lanes 1-11 represent DNA digested with EcoR1; 1, gDNA from wild type fungal strain 11C, 2-10, gDNA from strain 11C transformants Tr. 144, Tr. 175, Tr. 230, Tr. 51, Tr. 43, Tr. 133, Tr. 145, Tr. 55 and Tr. 168, 11, plasmid DNA from pSK1019. Red dots indicate bands and arrows indicate molecular size (Kb) based on 1 Kb DNA ladder  (B).(TIF)Click here for additional data file.Figure S5Fusarium oxysporum growth.Effect of increasing alcohol concentration on  Fungal conidial inoculum was produced in Mung bean broth [6 ml-1. A volume 100 µl conidia (106 ml-1) was added to microtiter  plates supplemented with either ethanol  (A) or butanol  (B). Fungi were maintained at 25°C for 7 days. Fungal growth (OD600nm) was measured using a spectrophotometer . Bars indicate SEM. Growth of Tr. 259 significantly different from 11C is highlighted with an asterisk . an broth  and resuan broth  at a con(TIF)Click here for additional data file.Figure S6F. oxysporum.Effect of alcohol stress on spore germination and biomass production in  Spore germination of F. oxysporum strain 11C and transformant Tr. 259 in the presence of no alcohol (A) 4% (vv-1) ethanol (B) and 0.75% (vv-1) butanol (C); fungal conidial inoculum was produced in Mung bean broth [6 ml-1. Fungi were maintained at 25°C for 24 hours shaking at 150 rpm and percentage germination was measured every 2 hours. Bars indicate SEM. Biomass production of F. oxysporum strain 11C and transformant Tr. 259 post – treatment with either; no alcohol (D) 4% (vv-1) ethanol (E) or 0.75% (vv-1) butanol (F). Minimal medium (48 ml) was inoculated with fungal conidia (2ml of 106 ml-1) and flasks were incubated at 25°C, 150 rpm. After 12 hours, flasks were amended with 2ml of water, ethanol or butanol to a final concentration of 0, 4 and 0.75% (vv-1), respectively. Fungal material was collected at 72, 120 and 168 hours post treatment. Bars indicate SEM. Spore germination or biomass production of Tr. 259 significantly different from 11C is highlighted with an asterisk .an broth  and resuan broth  at a con(TIF)Click here for additional data file.Figure S7F. oxysporum 11C transformant Tr. 259.Genome walking in  Genome walking analysis using APATM Gold Genome Walking Kit  to walk the right border (RB) of pSK1019. Lanes represent: 1 - 4; degenerate random tagging (DRT) primers A, B, C and D respectively with target specific primer (GSP) for RB of pSK1019 amplified with 500 ng of fungal genomic DNA from Fusarium oxysporum strain 11C transformant Tr. 259. Arrow indicates PCR fragment sizes (bp) on Hyperladder™ III  (Lane 5). Red dots indicate gel excised bands for subsequent cloning (A). Digestion of plasmid DNA from pGEMT ® easy  cloned PCR products to verify correct fragment insert size. Plasmid DNA (500 ng) was digested in 20 µl reaction and incubated at 37°C for 2 hours. Lanes represent: 1, Hyperladder™ III , 2 - 5, bands excised from B). PCR analysis of cloned PCR products to verify presence of target specific primer (GSP) used in genome walking prior to sequencing. Lanes represent: Lanes represent: 1, Hyperladder™ III , 2 - 5, bands excised from C).(TIF)Click here for additional data file.Figure S8Insilico analysis of identified coding sequence obtained via genome walking in Tr. 259. Sequence analysis of cloned PCR product obtained via genome walking within Fusarium oxysporum strain 11C transformant Tr. 259. Walking was from the right border (RB) of the transformation vector pSK1019 using the APATM Gold Genome Walking Kit , a universal kit primer (UAP-2) (red) and the gene-specific (GSP) primer (blue) (3’-5’) (A). Identified coding sequence was analysed against the F. oxysporum f.sp.lycospersici (strain 4287) genome ) using Blastn (B) and Blastx (C).(TIF)Click here for additional data file.Figure S9Fusarium oxysporum.Phylogenetic analysis of putative sugar transporters in  Homology (≥30%) of FOXG_09625 to putative sugar transporters in F. oxysporum 4287 (A). Comparison of putative hexose transporters in F. oxysporum 4287 compared to Saccharomyces cerevisiae and putative sugar transporters showing (≥30%) homology to FOXG_09625 in Aspergillus nidulans, Tricoderma reesei, Mucor circinelloides, Neurospora crassa (B). Amino acid sequences were obtained from FGCD (http://www.broadinstitute.org/annotation/genome/fusarium_group) SGD (http://www.yeastgenome.org) and NCBI (http://www.ncbi.nlm.nih.gov). All protein sequences showed sugar transporter domains and were aligned using ClustalW. A phylogram was constructed using the Neighbor-Joining method within Phylogeny.fr software. Numbers on internal nodes represent bootstrap values (100 re-sampling). Arrow indicates putative hexose transporter (FOXG_09625) used in RT-PCR analysis. Asterisk indicates transcripts annotated as hexose transporters in F. oxysporum 4287 (FGCD).(TIF)Click here for additional data file.Table S1Primers used in this study for genome walking and real-time PCR.(DOC)Click here for additional data file.Methods S1Expanded description of methods.(DOCX)Click here for additional data file.References S1(DOCX)Click here for additional data file.


HERV-K(HML-2) loci is a hallmark of ECCs, we asked if this HERV group was also reactivated in ESCs and induced pluripotent stem cells (iPSCs).Malignant human embryonal carcinoma cells (ECCs) rely on similar transcriptional networks as non-malignant embryonic stem cells (ESCs) to control selfrenewal, maintain pluripotency, and inhibit differentiation. Because re-activation of silenced HERV-K(HML-2) RNA and protein expression in undifferentiated human ESCs and iPSCs. Induction of differentiation by embryoid body formation resulted in rapid silencing of HERV-K(HML-2) provirus expression. Sequencing analysis of a conserved region of the gag gene showed that proviral expression in ESCs and iPSCs represents at least 11 of the 66 nearly full length HERV-K(HML-2) loci, with slightly varying patterns in individual cell lines. These proviruses are human specific integrations and harbor promoter competent long terminal repeats (LTR5hs subgroup). We observed high mRNA levels of the NP9 and Gag encoding proviruses K101(22q11.21) in all and K10(5q33.3) in most of the ECC, ESC, and iPSC lines tested, while K37(11q23.3) mRNA was detected only in ESCs and iPSCs. In addition, we detected expression of proviral mRNA encoding the RNA export adaptor Rec in all cell lines studied. Proviral mRNA originating from the K108(7p22.1) locus, which inter alia codes for functional Rec and Env proteins, was only reactivated in malignant ECC lines, not in benign ESCs or iPSCs.Using RT-PCR and Western Blot, we demonstrate HERV-K(HML-2) RNA and protein expression is a marker for pluripotent human stem cells. Initiation of differentiation results in rapid down-regulation. Further studies are needed to explore a putative functional role of HERV-K(HML-2) RNA and proteins in pluripotent stem cells. HERV-K(HML-2) harbor open reading frames for viral proteins, as well as promoter competent long terminal repeats (LTRs). In addHERV-K(HML-2) in three ESC lines , three fibroblast lines  and iPSC lines derived from these fibroblasts. All materials and methods are described in detail in Additional fileHERV-K(HML-2) transcripts derived from proviral promoters, we analyzed completely spliced viral RNA by RT-PCR. Full length and spliced transcripts and the location of all primer pairs used are depicted in Figure OCT4 and NANOG and absence of the differentiation marker gene LMNA in pluripotent stem cells, ACTB specific amplicons served as internal controls[HERV-K(HML-2) specific amplicons in any of the fibroblast cell lines, which readily expressed the differentiation marker LMNA. In contrast, all ESC and iPSC lines tested showed expression of all three types of completely spliced HERV-K(HML-2) transcripts, in addition to the pluripotency markers OCT4 and NANOG  type 2 transcripts in all pluripotent stem cell lines tested prompted us to investigate whether they encode functional Rec protein and thus enable translation of HERV-K(HML-2) Gag and Env proteins. Using a HERV-K(HML-2) Gag specific monoclonal antibody in Western blot analysis, we indeed detected low to moderate levels of this protein in the H9 ESCs and the hFib2-iPS5 iPSC line, but not in the parental fibroblasts hFib2. By contrast, the ECC line GH showed high levels of Gag protein expression . The differences in HERV-K(HML-2) Gag protein levels observed in H9 and hFib2-iPS5 correlated with the differences in signal intensities of their rec specific amplicons in RT-PCRs  expression in pluripotent stem cells changes during differentiation, we induced embryoid body formation of hFib2-iPS5 cells. We applied semi-qRT-PCR analysis using the primer pair K7/K21  RNA started to decrease from day 6 onwards in hFib2-iPS5 cells, concomitant with a decrease of NANOG and an increase of LMNA expression  expression levels dropped from 100% on day 0 to roughly 50% on day 6, and to 20% on day 10. Similarly, NANOG expression levels decreased from 100% on day 0 to roughly 75% on day 6, and 55% on day 10.To determine whether ERV-KHML- expressiERV-KHML- expressiHERV-K(HML-2) RNA expression during differentiation with significantly reduced transcription already detectable on day 2 of embryoid body differentiation  downregulation is even faster than repression of OCT4 and NANOG.To verify this with a second method and cell line, we performed real-time RT-PCR analysis using differentiating H9 ESCs,20. ThisThese slightly divergent results may simply reflect the greater accuracy of real time PCR compared to semi-quantitive PCR or may be rooted in the well-known subtle differences in the expression profiles of pluripotent stem cells especially those of ESC compared to iPSC lines.HERV-H[HERV-K(HML-2) proviruses is specific for certain proviral elements or occurs in a stochastic manner.The recent findings that high RNA levels of the endogenous retrovirus group HERV-H and exprHERV-H are alsoHERV-K(HML-2) proviruses in malignant and non-malignant pluripotent stem cells using an established protocol[gag gene  proviruses present in the human genome[HERV-K(HML-2) proviruses. Interestingly, phylogenetic classifications have shown that all human-specific retroviral integrations fall into this proviral subgroup[K37(11q23.3) may also be present in the genomes of great apes. Five of the re-activated proviruses belonged to type 1 and six to type 2 genotypes, respectively, but their number and expression levels varied between individual ESC/iPSC and ECC cell lines. Such variations in HERV-K(HML-2) proviral transcription profiles are commonly observed in somatic malignant and non-malignant tissues and cell lines[K101(22q11.21) in all pluripotent stem cell lines tested, suggesting a more directed re-activation. K101(22q11.21), K37(11q23.3) and K10(5q33.3) encode gag and protease genes and may account for the protein expression detected, e.g. in the H9 and hFib2-iPS5 lines  expression exclusively in the malignant ECC lines. Notably, a recent survey of HERV-K(HML-2) proviruses transcribed in melanomas in comparison to melanocytes also demonstrated K108(7p22.1) derived mRNA only in melanomas but not in the non-malignant precursor cells[We analyzed activation of protocol to ampliERV-KHML- provirusn genome, a subsen genome, deep sen genome as well subgroup. Indeed,ll lines,28 indicll lines. By controup[K371q23.3 maHERV-K(HML-2) is unique in its potential to code for viral and accessory proteins. We observed, in slightly varying patterns, re-activation at the RNA and protein levels of certain human specific, protein encoding HERV-K(HML-2) proviruses with promoter competent LTR sequences in pluripotent ESCs, iPSCs, and ECCs. The surprisingly constant activation of K101(22q11.21) across all cell lines tested may indicate a particular function in or association with pluripotency. We detected K108(7p22.1) only in the malignant ECC lines. This corresponds to up-regulated K108(7p22.1) transcription described in other germ cell tumours, in brain tumours but not in normal brain[HERV-K(HML-2) proviruses were rapidly silenced upon embryoid body differentiation. Re-activation of this HERV group thus represents another marker for the undifferentiated state of pluripotent stem cells.The endogenous betaretrovirus group al brain but not in vitro and germ line carcinoma in situ in transgenic mice[HERV-K(HML-2) proviruses were preferentially activated in pluripotent stem cells and that their expression was simultaneously silenced upon differentiation independent of their chromosomal localization is in favour of such an idea.Active type 2 proviruses produce Rec protein, which supports cell transformation nic mice,31. Typenic mice. Both Renic mice and Np9 nic mice which minic mice. Since tnic mice, it is iThe authors declare that no competing interests exist.HERV-K(HML-2) cloning, performed the sequence analysis and did the immunoblot assay. SL performed cell culture, embryoid body differentiation and RT-PCR analysis. GQD supplied the cells. ZI and GQD financially supported the study. JL helped interpret the results. NVF, SL, JL and RL wrote the manuscript. All authors have read and approved the submission of the manuscript.NVF, SL and RL conceived the study and performed the analyses. NVF did “Materials and Methods”.Click here for file“HKc10-B(10q24.2)”.Click here for file“K41(12q14.1)”.Click here for file“K109(6q14.1)”.Click here for file“KI(3q21.2)”.Click here for file“Alignments”.Click here for file“K101(22q11.21)”.Click here for file“K10(5q33.3)”.Click here for file“K106(3q13.2)”.Click here for file“K115(8p23.1)”.Click here for file“K37(11q23.3)”.Click here for file“K102(1q22)”.Click here for file“K108(7p22.1)”.Click here for file


BP-pm1 on chromosome 13, LOD 3.78; BP-pm2 on chromosome 11, LOD 2.76; BP-pm3 on chromosome 2, LOD 2.61; BP-pm4 on chromosome 4, LOD 2.50) and two suggestive BP-QTLs , four of which  were unique to this post-menopausal cohort. These data demonstrate distinct polygenic susceptibility underlying post-menopausal salt-sensitive hypertension providing a pathway towards the identification of mechanism-based therapy for post-menopausal hypertension and ensuing target-organ complications.Essential hypertension affects 75% of post-menopausal women in the United States causing greater cardiovascular complications compared with age-matched men and pre-menopausal women. Hormone replacement and current anti-hypertensive therapies do not correct this post-menopausal increased risk suggesting a distinct pathogenic framework. We investigated the hypothesis that distinct genetic determinants might underlie susceptibility to salt sensitive hypertension in pre-menopausal and post-menopausal states. To determine whether distinct genetic loci contribute to post-menopausal salt-sensitive hypertension, we performed a genome-wide scan for quantitative trait loci (QTLs) affecting blood pressure (BP) in 16-month old post-menopausal F2 (Dahl S×R)-intercross female rats characterized for blood pressure by radiotelemetry. Given identical environments and high salt challenge, post-menopausal BP levels were significantly higher than observed in pre-menopausal  and ovariectomized  F2-intercross female rats. We detected four significant to highly significant BP-QTLs ( Cumulative observations indicate that sex-specific susceptibility exists in various traits The increased prevalence of salt-sensitive hypertension in post-menopausal women To investigate the genetic determinants that contribute to post-menopausal salt-sensitive hypertension we performed a genome scan for QTLs affecting blood pressure in an F2 (Dahl S×R)-intercross post-menopausal female rat population characterized for blood pressure by radiotelemetry. We implemented the following experimental design: Female F2 hybrids were maintained from weaning on a low salt (0.008% NaCl) diet until challenge with high salt (8% NaCl) at post-menopause confirmation at 14 months of age. BP-implant surgery was done at 13.5 months of age; after 12 days, baseline BP levels were obtained. We characterized 130 subjects for blood pressure. The cohort was genotyped with 100 informative markers for our (Dahl S×R) intercross with an average density of 23.9 Mbp.16m) and previously reported pre-menopausal (6m) 6m) P<10−4), diastolic (DBP P<10−4) and mean arterial pressures (MAP P<10−4) significantly compared with BP in pre-menopausal rats  detected with suggestive linkage  using 16 m-old post-menopausal F2 (Dahl S×R)-intercross female rats.  linkage . AdditioBP-pm1 on rat chromosome 13, BP-pm2 on rat chromosome 11, BP-pm3 on rat chromosome 2 and BP-pm4 on rat chromosome 4 . All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.Inbred Dahl S/jrHsd and Dahl R/jrHsd rats were obtained from Harlan . Parental strains  were crossed to produce F1 progeny. The F2 subjects were derived from brother-to-sister mating of F1 hybrids to produce the F2 female (n = 130) segregating population.In order to ascertain post-menopausal status of the female F2 hybrids to be characterized for blood pressure daily vaginal smears were performed on females to determine the stage of their estrous cycle  essentially as described Blood pressure (BP) was measured essentially as described P value<0.01 for the interaction effect.Phenotype distributions were analyzed for normality; data transformations were done when necessary and datasets that passed Kolmogorov-Smirnov normality testing (SigmaStat) were used for linkage analysis. QTL analysis was performed using SBP and DBP as quantitative traits. Linkage maps, marker regression and composite interval mapping were done with the Map Manager QTXb19 (MMQTXb19) program for windows SNP genotyping was carried out on an Applied Biosystems 7900 Real-Time PCR System. SNPs (n = 97) and SSLP markers (n = 3) were selected from the RGD SNP database. SNP assays (TaqMan assays) were procured from Applied Biosystems and were validated in our laboratory.We performed one-way ANOVA followed by all pairwise multiple comparisons using Tukey test for blood pressures as indicated per experimental comparison.


DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4–6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in  Extension of axons towards their final target is a major requirement for the proper development of the nervous system. Axons encounter guidance cues along their growth routes, including four major canonical signaling pathways: DCC-Unc5-Netrin-1, Slit-Roundabout (Robo), Semaphorin-Plexin-Neuropilin, and Ephrin-Eph Here we focus on identifying interacting proteins for DCC. DCC is a 175–190 kDa member of the Ig superfamily consisting of a single transmembrane domain and a large extracellular domain (ECD) containing four C2-like Ig domains and six FNIII domains Drosophila, mutants of the DCC homologue frazzled lead to more severe axon midline guidance defects than netrinAB mutants and this is partially rescued by commissureless independent of netrin and suggestive of an additional frazzled ligand DCC or Netrin-1 in mice results in a failure of commissural axons to cross the spinal cord midline but the severity of the commissural axon defects in the developing spinal cord are greater in DCC knock-out mice compared to Netrin-1 mutant mice commissureless, these findings support the suggestion that there are additional ligands regulating DCC activity in vertebrates.In CBLN4 knock-out mouse CBLN4 knock-out.Here we report, using an unbiased large-scale protein-microarray screen, that in addition to Netrin-1, CBLN4 is a highly specific ligand for DCC. The four members of the CBLN family, CBLN1-4, are secreted, glycosylated proteins that contain a C-terminal globular C1q domain characteristic of the C1q-TNF superfamily The Genentech Institutional Animal Care and Use Committee approved all animal studies.Expression constructs were generated by standard PCR methods using an in-house clone collection or cDNA purchased from Origene as template and subcloned into pRK5 vector containing the appropriate N-terminal or C-terminal epitope tag. Mutants for DCC domain mapping studies were made using the Quick-Change II XL site-directed mutagenesis kit (Stratagene). All constructs were sequence verified before use. N-terminal flag-tagged (MGGTAARLGAVILFVVIVGLHGVRGKDYKDDDDKLE), full-length and deletion constructs for human DCC are as follows: Flag-DCC full-length (H26-I1442); Flag-DCC Ig1-4 (H26-S425-BamH1-L1098-I1442); Flag-DCC FN1-6 (S426-I1442), Flag-DCC FN4-6 (E722-I1442). Protein expression constructs were generated for soluble  Proteins were expressed in Chinese hamster ovary (CHO) cells and purified as described previously Protein microarrays were constructed on epoxysilane coated glass slides (Schott) as previously described 2). The AP signal was developed with nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) stock solution (Roche) diluted 50 fold in AP buffer in the dark for at least 1 h until the AP dependent formation of blue NBT/BCIP precipitate was visualized.COS-7 cells (ATCC) were transfected with cDNAs encoding DCC or, as a control, Nogo receptor (NgR) using PolyFect (Qiagen) according to manufacturer’s recommendations. After incubation at 37°C for 48 h, cells were washed twice in binding buffer , 0.05% sodium azide, 2 µg/ml heparin in PBS), and incubated with 20 nM Alkaline Phasphatase (AP), CBLN4-AP, or Nogo 66-AP from conditioned media for 90 min in binding buffer at room temperature. The cells were then washed three times in binding buffer, fixed for 7 min in 4% paraformaldehyde (PFA) in PBS, washed three times in HEPES buffered solution , heat inactivated for 30 min at 65°C, and washed three times in alkaline phosphatase buffer  experiments were performed on a Biacore 3000  at 25°C using a CM5 sensor chip in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (HBS-P) running buffer at a flow rate of 30 µl/min. Full-length N-terminally HA-tagged CBLN1 and CBLN2 were purchased from R&D systems. The extracellular region of Robo3 fused to Fc (Robo3-Fc) and full length CBLN3-Fc and CBLN4 C-terminal His were produced recombinantly  and puriHEK293T cells (ATCC) were transiently transfected with Flag-tagged human DCC or DCC domain mutants using Fugene-6 (Promega) according to manufacturer’s recommendations. Flow cytometric binding experiments were performed in PBS containing 2% FBS at 4°C. Expression of Flag-tagged DCC and domain mutants on the surface of HEK293T cells was confirmed 48 h post-transfection with either an anti-Flag M2 antibody (Sigma) or anti-DCC antibody  followed by incubation with anti-rabbit IgG or anti-mouse IgG phycoerythrin (PE)- or anti-mouse IgG allophycocyanin (APC)-labeled secondary antibody. Protein binding to Flag-tagged DCC, domain mutants, or non-transfected cells was evaluated by incubating cells for 30 min with recombinant CBLN4-Fc, UNC5C-Fc, gD-Netrin-1 or gD-BTLA at 10 µg/ml. Cells were then washed and binding was detected with either anti-human IgG-PE- or anti-human IgG-APC-labeled secondary antibody or anti-gD and anti-mouse IgG-PE- or anti-mouse IgG-APC-labeled secondary antibody.DCC domain mapping studies were performed in 384-well imaging and assay plates (Aurora Biotechnologies). For COS-7 transfections, DNA for DCC domain mutant constructs and negative control were dispensed into wells at 60 ng per well. Fugene 6 (Promega) transfection reagent was prepared at a ratio of 3 µl of Fugene to 1 µg of DNA in Opti-MEM (Invitrogen), incubated for 5 min, and mixed with DNA in the 384-well plate and incubated for 15–45 min to allow for complex formation. COS-7 cells were dispensed at a density of 3,000 cells per well into 384-well plates and incubated for 48 h. Transfected cells were washed three times and blocked with PBS containing 1% BSA for 1 h prior to incubation with CBLN4-Fc protein or anti-Flag M2 antibody (Sigma) for 1 h on ice. After washing, cells were fixed in 4% PFA for 30 min at room temperature and protein binding was detected with Alexa Fluor-488 labeled anti-human IgG (Invitrogen) or expression was detected with Alex Fluor-647 labeled anti-mouse IgG (Invitrogen) for 1 h. Fluorescent signal for the entire plate was acquired and analyzed on an Image Express Velos (Molecular Devices). Image analysis was performed using the analysis software for the Image Express Velos. Positively stained cells were counted as those above a background fluorescence intensity threshold and within the particle filter parameters for area and mean signal intensity. Images for publication purposes were collected from the same plate using an INCell Analyzer 2000 . The candidate interaction screen was also performed using the above staining protocol.125I-Na using the Iodogen method. Competition reactions were set-up in triplicate with a fixed concentration of iodinated ligand premixed with 3-fold serial dilutions of unlabeled ligand starting at 500 nM followed by addition of DCC cell suspension at a density of 250,000 cells per 200 µl to give a final reaction volume of 250 µl. Reactions were incubated for 2 h at room temperature prior to separation of free from bound iodinated ligand using Millipore multi-screen filter plates . Air-dried filters were counted using a Wallac Wizard 1470 gamma counter (PerkinElmer Life Sciences). The binding data were evaluated using NewLigand software (Genentech), which uses the fitting algorithm of Munson and Rodbard to determine the binding affinity 125I CBLN4-Fc (300 pM) or 125I gD-Netrin-1 (52 pM), respectively, was added and allowed to bind for 1 h at 4°C before washing and counting.Radio-ligand binding assays were performed with recombinant CBLN4-Fc and gD-Netrin-1 protein with DCC expressing HEK293T cells in 96-well U-bottom plates in DMEM media containing 2% FBS, 50 mM HEPES, pH 7.2 and 0.1% sodium azide. CBLN4-Fc and gD-Netrin-1 were iodinated with 2, 5 mM EGTA, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate in PBS) and the β-galactosidase activity staining was developed in washing buffer with 5 mM potassium ferrocyanide, and 1 mg/ml X-gal in Dimethylformamide (DMF) at 37°C in the dark.Whole embryo (E11.5) or 200 micron vibratome sections (E13.5) that were fixed in 4% PFA in PBS were washed three times (20 min each) in washing buffer  binding assay was performed. The results show that CBLN4-AP binds to COS-7 cells transiently transfected with DCC but not to cells transiently transfected with NgR that bind to Nogo66-AP . In addi125I-labeled CLBN4-Fc and gD-Netrin-1. The equilibrium dissociation constant, DK, for CBLN4 was calculated to be 117±34 nM compared to a dissociation constant of 22±6 nM for Netrin-1 .To determine if there were other CBLN4 receptors besides DCC, a protein-microarray screen was performed in reverse where CBLN4-Fc was used as bait, however, no clear hits were found (data not shown). Curiously, DCC itself was not identified as a hit. Prior studies indicate that proteins are largely active on the microarray substrate DCC is a single transmembrane protein containing four Ig repeats and six FNIII repeats in the ECD. To assess which domains in DCC are necessary for CBLN4 binding, deletion constructs of DCC were generated and transfected into COS-7 cells for binding analysis. Four DCC constructs were tested, constituting either the entire ECD (DCC.FL), the Ig1-4 repeats (DCC.Ig), the FN1-6 repeats (DCC.FN1-6), or only the C-terminal FN4-6 repeats (DCC.FN4-6). The Ig1-4 repeats (DCC.Ig) showed no binding to CBLN4-Fc, suggesting that primary binding is dependent on the FN domains . Consist125I-labeled CBLN4-Fc (300 pM) bound specifically to DCC expressing HEK293T cells and that binding was blocked by pre-incubation of the cells with Netrin-1 (1 µM) compared to no treatment control (125I-labeled Netrin-1 (52 pM) with CBLN4-Fc (2 µM) was observed in the reverse experiment and most likely can be attributed to the ∼5-fold difference in affinity of CBLN4 and Netrin-1 for DCC. A titration with increasing concentrations of Netrin-1 showed clear displacement of a fixed concentration of 125I CBLN4-Fc to DCC expressing HEK293T cells Click here for additional data file.Table S1Domain boundaries and amino acid sequences for protein reagents used in this study.(XLS)Click here for additional data file.Table S2Composition of the libraries used for the protein microarray screens.(XLS)Click here for additional data file.


The Runt-related transcription factor Runx2 is critical for skeletal development but is also aberrantly expressed in breast cancers, and promotes cell growth and invasion. A de-regulated serine/threonine kinase Akt signaling pathway is implicated in mammary carcinogenesis and cell survival; however, the mechanisms underlying Runx2 role in survival of invasive breast cancer cells are still unclear.The phenotypic analysis of Runx2 function in cell survival was performed by gene silencing and flow cytometric analysis in highly invasive MDA-MB-231 and SUM-159-PT mammary epithelial cell lines. The expression analysis of Runx2 and pAkt (serine 473) proteins in metastatic breast cancer specimens was performed by immunohistochemistry. The mRNA and protein levels of kinases and phosphatases functional in Akt signaling were determined by real-time PCR and Western blotting, while DNA-protein interaction was studied by chromatin immunoprecipitation assays.The high Runx2 levels in invasive mammary epithelial cell lines promoted cell survival in Akt phosphorylation (pAkt-serine 473) dependent manner. The analysis of kinases and phosphatases associated with pAkt regulation revealed that Runx2 promotes pAkt levels via mammalian target of rapamycin complex-2 (mTORC2). The recruitment of Runx2 on mTOR promoter coupled with Runx2-dependent expression of mTORC2 component Rictor defined Runx2 function in pAkt-mediated survival of invasive breast cancer cells.Our results identified a novel mechanism of Runx2 regulatory crosstalk in Akt signaling that could have important consequences in targeting invasive breast cancer-associated cell survival. Breast cancer is the most commonly diagnosed form of cancer and a serious health concern for women worldwide . One sigThe Runt-related transcription factor, Runx2, is a key regulator of normal bone development, homeostasis and remodeling ; howeverMMP9 and MMP13), osteocalcin, bone sialoprotein and genes related to cancer cell migration and invasion [The invasive breast cancer-derived MDA-MB-231 cells express increased levels of Runx2 compared to non-tumorigenic MCF-10A cells . The Runinvasion -20. ThesIn response to epidermal growth factor (EGF) stimulation, the PI3K signaling pathway is activated, resulting in phosphorylation of serine/threonine kinase Akt (pAkt). The phosphorylation of Akt at Serine 473 residue is regulated by mammalian target of rapamycin complex-2 (mTORC2) and at Threonine 308 residue by phosphoinositide dependent kinase-1 (PDK1) ,22. The In this study, we find that Runx2 suppression robustly enhances apoptotic cell death in invasive cancer cell lines in response to glucose- and growth factor-deprivation. We show that Runx2 is required for maintaining pAkt (Serine 473) levels in invasive mammary epithelial cell lines via mTORC2 complex proteins. Altogether, our results identify a novel mechanism implicating Runx2 regulatory network in the Akt cell survival pathway.ATCC, Manassas, VA, USA) were cultured at 37°C in humidified incubator with 5% CO2. For routine cell culture, the human MCF-10A cells  [Cellgro, Mediatech, Manassas, VA, USA) supplemented with 5% horse serum , insulin (10 μg/ml) , epidermal growth factor (EGF) (20 ng/ml) , cholera toxin (100 ng/ml)  and hydrocortisone (0.5 ug/ml) , and 50 U/ml penicillin and 50 μg/ml streptomycin (pen-strep) . The human MCF-7  [Cellgro, Mediatech) supplemented with 10% fetal bovine serum (FBS)  and pen-strep. The SUM-159 and SUM-159-PT  [Life Sciences Technology, Grand Island, NY, USA) supplemented with 5% FBS, HEPES (10 mM), Insulin (5 μg/ml), hydrocortisone (1 μg/ml) and pen-strep. Since the cell lines used in this study were commercially obtained, the experiments performed in this study did not require approval from our institution’s ethics committee.All cell lines ( WT-p53)  were cul WT-p53)  and MDA- WT-p53)  cells weant p53)  were culCayman Chemical, Ann Arbor, MI, USA) treatment, the serum-deprived cells were pre-treated with LY294002 for 10 minutes before treatment with EGF or LY294002.For EGF treatment, the cells were deprived of serum  and growth factors, where appropriate, for 16 hours. The cells were then treated with EGF (100 ng/ml) in serum-deprived media for multiple time points (10 minutes to 6 hours). In experiments requiring PI3K inhibitor LY294002 , Akt , Akt1, Akt2, pPdk1 (Serine 241), pmTOR (Serine 2448 and 2481), mTOR , Rictor, Raptor, GβL, pGSK-3β (Serine 9) and FOXO1 were purchased from Invitrogen, Grand Island, NY, USA). The following siRNA sequences were commercially  synthesized: PHLPP1: (Sense) CCG UUG GAG UGA UGC ACA ATT. (Antisense) UUG UGC AUC ACU CCA ACG GCT; Runx2: (Sense) CUU GAU GAC UCU AAA CCU ATT. (Antisense) UAG GUU UAG AGU CAU CAA GCT.The siRNA transfections for transient gene knockdown were carried out by using oligofectamine as per the manufacturer’s guidelines (Rictor shRNA was obtained from Addgene (plasmid #1853)  [tetO operator in the absence of doxycycline to suppress shRNA, while in the presence of doxycycline it cannot bind to tetO, thus permitting the shRNA-mediated gene knockdown. The cells expressing pLVTHM (control or Runx2-shRNA) vectors harboring tetO were transduced with lentivirus expressing pLV-tTR-KRAB to generate doxycycline-induced Runx2 knockdown.The stable cell lines were generated utilizing lentivirus vectors. The lentivirus vectors expressing WT-Runx2, pLVTHM (RNAi-control) and Runx2-shRNA (Runx2-RNAi) were described and generated previously . The lenMA, USA) . The doxMA, USA) . The tTRVector Laboratories Inc., Burlingame, CA, USA) but with some modifications. A human invasive carcinoma tissue microarray was obtained from US Biomax, Inc., (#BR1007) Rockville, MD, USA. This part of the study did not require approval from the Institutional Review Board as the tissue microarray was commercially sourced. Briefly, the standard histology procedures were used to deparaffinize the microarray slide in xylene and rehydrate it in graded ethanol series. The target retrieval was carried out by boiling the sections in citrate buffer (pH 6)  at 95°C for 15 minutes. The endogenous peroxidase activity was quenched by incubating slides in 3% hydrogen peroxide for 10 minutes. The blocking was performed with normal blocking serum (Vector Laboratories) for 30 minutes. The sections were subsequently incubated with control or anti-Runx2 or anti pAkt (Serine 473) antibody overnight at 4°C. The next day the sections were incubated in diluted biotinylated secondary antibody for 30 minutes at room temperature followed by 30-minute incubation with Vectastain Elite ABC reagent. The sections were thoroughly rinsed in PBS-T (PBS supplemented with 0.1% Tween-20) in between the above mentioned steps. The sections were finally incubated in peroxidase substrate solution to develop color, followed by washing in water, counter staining with hematoxylin (Vector Laboratories), clearing in xylene and cover-slipping with Permount . The staining intensity for Runx2 and pAkt (Serine 473) were graded semi-quantitatively from 0 to 3 grades  blindly by two investigators (MT and JP). The experiments were repeated three times.The immunohistochemistry procedure was performed according to instructions in the Vectastain Elite ABC kit , SDS (2% w/v), DTT (50 mM), glycerol (10%) and bromophenol blue (0.01% w/v). The nuclear lysates were prepared in direct lysis buffer as previously described [Runx2: (F) TGC CTG CCT GGG GTC TGT A (R) CGG GCC CTC CCT GAA CTC T; mTOR: (F) TCC GGC TGC TGT AGC TTA TTA (R) CGA GCA TAT GCC AAA GCA CT; Rictor: (F) CTT CGA GGA GGA CTA AAC AC (R) CTA CAT CAG CTC GCA CAT AC; PHLPP1 (1): (F) CCT CAT CCG CTT CTA TGC AGG (R) GCATCTTGCCTTTACGGACAT; PHLPP1 (2): (F) GCC AGT GAA CCG ATG GAC AA (R) GTC CCA CAT AGG ATG ACT TGG; GAPDH: (F) ATG TTC GTC ATG GGT GTG AA (R) TGT GGT CAT GAG TCC TTC CA; 28S: (F) GAA CTT TGA AGG CCG AAG TG (R) ATC TGA ACC CGA CTC CCT TT. All the experiments were repeated at least three times.The real time PCR with SYBR chemistry was performed as previously described . The folmTOR promoter  [mTOR promoter were used: Runx2: (F) GAA AGA GCA AGG GGG AAA AG (R) TGG AGA GGC AGA ATC ATG TG; mTOR promoter: (F) CAG TGG TGC AGT GGT GAG AT (R) AGG CAG GTG GAT TGT TTG AG. The experiment was repeated at least three times.The ChIP was performed as previously described . The TF 0 bases) . The folBD Biosciences). The cell cycle analysis with propidium iodide staining was performed as previously described [Sigma-Aldrich, St. Louis, MO, USA) and stained with propidium iodide (18 μg/ml) (Invitrogen) for one hour. The cell cycle analysis was performed in FACS Canto (BD Biosciences). The gating and data analysis were performed in FlowJo software . The Dean Jett Fox model was used to set gates for G1, S and G2 stage cells, while Sub-G1 was manually gated before G1 population. The experiments were repeated three times.The Annexin V and AAD staining was performed as per the manufacturer’s guidelines  was used to estimate cell number in triplicates at various time points in 96-well culture plates as previously described [An indirect cell proliferation assay  compared to control cells as examined by Western blot analysis  levels , a readout for the Akt signaling pathway activity  levels with Runx2 suppression was specific for invasive cells, we examined additional invasive cell lines  with Runx2 knockdown and response to EGF treatment. Of these cell lines tested, SUM-159 and SUM-159-PT showed similar regulation as observed in MDA-MB-231 cells. As these cell lines have higher levels of endogenous pAkt (Serine 473) compared to MDA-MB-231 cells  and normal (MCF-10A) cells, Runx2 knockdown  levels  by chromatin immunoprecipitation assays. Our result showed a direct binding of Runx2 on the mTOR promoter region (−2420 to −2441) containing two highly conserved Runx binding elements at −2420 and −2430 bp spanning 22 base pairs  [Previous studies have shown that functional mTORC2 complex is required for Akt phosphorylation at Serine 473 residue . Consistr HDACs)  or posttr HDACs) ,41 furthThe differential Akt regulation by Runx2 in non-invasive and invasive cancer cells could be due to altered Runx2 phosphorylation in invasive cells. Several reports indicate that in response to growth factor stimulation, phosphorylation and DNA binding activity of Runx2 is enhanced in osteogenic cell lines, endothelial cells and osteosarcoma cell lines -44. In nAll three Runx transcription factors  are shown to be expressed at varying levels in mouse and human normal or cancerous mammary epithelial cells ,54 and aOur studies in MDA-MB-231 and SUM-159-PT cells show that Runx2 knockdown increases cell death under glucose and growth factor-deprivation-induced stress. It has been shown in endothelial cells that glucose activates Runx2 phosphorylation, nuclear localization, DNA binding and cell migration . These fWe have demonstrated that Runx2 is required for phosphorylation of Akt via directly regulating sub-units of the mTORC2 complex. The Runx2-mediated pAkt regulation promotes survival of invasive mammary epithelial cells.Ad: Adenovirus; Akt: Serine/threonine-specific protein kinase (PKB); CA: Constitutively active; CBFβ: Core binding protein β subunit; CBP: CREB-binding protein; ChIP: Chromatin immuno precipitation; EGF: Epidermal growth factor; ER: Estrogen receptor; ERK: Extracellular signal regulated kinase; FBS: Fetal bovine serum; FOXO1: Forkhead box protein o1; GFP: Green fluorescent protein; GβL: G-protein β subunit-like protein (mTOR associated protein); HDAC: Histone deacetylase; Her2: Human epidermal growth factor receptor-2; MMP: Matrix metalloproteinase; mTOR: Mammalian target of rapamycin; mTORC2: Mammalian target of rapamycin complex-2; MTT: Tetrazolium dye for colorimetric assay; NLS: Nuclear localization signal; NMTS: Nuclear matrix targeting signal; p300: E1A binding 300 kDA protein; p53: Protein 53; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; PDK1: Phosphoinositide-dependent protein kinase-1; PHLPP1: PH domain leucine-rich repeat protein phosphatase-1; PI3K: Phosphatidyl inositol 3′ kinase; PR: Progesterone receptor; Runx2: Runt-related transcription factor-2; Ser: Serine; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; Thr: Threonine; WT: Wild type.The authors declare that they have no competing interests.All authors meet the authorship requirements. MT generated stable cell lines and analyzed cellular and molecular effects of Runx2 overexpression or knockdown . ZC performed real time RT-PCR analysis and Western blots of samples with Runx2 overexpression or knockdown. MT and JP participated in the data analysis. JP conceived and coordinated the study and MT and JP wrote the major part of the paper. All authors read and approved the final manuscript.Runx2 expression levels in non-invasive or invasive cell lines and the effect of high Runx2 expression in MDA-MB-231 cells on proliferation and survival.Click here for fileRelationship between Runx2 and pAkt (Serine 473) expression in invasive breast cancer specimens.Click here for fileRunx2 knockdown reduces pAkt (Serine 473) when stimulated with various doses of EGF in MDA-MB-231 cells.Click here for fileRunx2 knockdown increases pAkt (Serine 473) in non-invasive MCF-10A or MCF-7 cells in response to EGF stimulation in the presence of LY294002.Click here for fileRunx2 knockdown in MDA-MB-231 or SUM-159PT cells alters expression levels of mTORC2 proteins.Click here for file


Deleted in Colorectal Cancer (DCC) gene, has been linked to many human cancers. However, it is unclear if loss of DCC is the specific underlying cause of these cancers. The Drosophila imaginal discs are excellent systems in which to study DCC function, as it is possible to model human tumors through the generation of somatic clones of cells bearing multiple genetic lesions. Here, these attributes of the fly system were utilized to investigate the potential tumor suppressing functions of the Drosophila DCC homologue frazzled (fra) during eye-antennal disc development.Loss of heterozygosity at 18q, which includes the fra loss of function clones are eliminated during development. However, when mutant clone cells generated in the developing eye were rescued from death, partially differentiated eye cells were found outside of the normal eye field, and in extreme cases distant sites of the body. Characterization of these cells during development indicates that fra mutant cells display characteristics of invasive tumor cells, including increased levels of phospho-ERK, phospho-JNK, and Mmp-1, changes in cadherin expression, remodeling of the actin cytoskeleton, and loss of polarity. Mutation of fra promotes basement membrane degradation and invasion which are repressed by inhibition of Rho1 signaling. Although inhibition of JNK signaling blocks invasive phenotypes in some metastatic cancer models in flies, blocking JNK signaling inhibits fra mutant cell death, thereby enhancing the fra mutant phenotype.Most fra/DCC and metastatic phenotypes in an animal model and suggest that Fra functions as an invasive tumor suppressor during Drosophila development.The results of this investigation provide the first direct link between point mutations in  DCC gene, was identified in a large percentage of colorectal cancers )}G13 P{Ubi-GFP.nls}2R1 P{UbiGFP.nls}2R2/P{w[+mW.hs]=FRT(w[hs])}G13 fra[3]. P35-rescued fra[4]mutant clones were generated via the MARCM system )}G13 P{piM}45F P{tubP-GAL80}LL2/P{w[+mW.hs]=FRT(w[hs])}G13 fra[4]; P{tubP-GAL4}LL7/UAS-p35.H.BH3 (clones were detected with an anti-P35 antibody) or eyFLP; P{FRT(w[24])}G13 P{piM}45F P{tubP-GAL80}LL2/P{w[+mW.hs]=FRT(w[hs])}G13 fra[4]; P{tubP-GAL4}LL7/UAS-p35.H.BH3 UAS-EGFP-34 (clones were positively marked by GFP expression). Rescue by DN-JNK or DN-Rho1 was assessed in the following genotypes: eyFLP/UAS-DN-JNK; P{FRT(w[24])}G13 P{piM}45F P{tubPGAL80}LL2/P{w[+mW.hs]=FRT(w[hs])}G13 fra[4]; P{tubP-GAL4}LL7/UAS-p35.H.BH3 UAS-EGFP-34 and eyFLP/UAS-DN-Rho1; P{FRT(w[24])}G13 P{piM}45F P{tubPGAL80}LL2/P{w[+mW.hs]=FRT(w[hs])}G13 fra[4]; P{tubP-GAL4}LL7/UAS-p35.H.BH3 UAS-EGFP-34. GFP-positive control clones in which P35 expression was driven ectopically were produced in flies of the following genotype: hsp70-FLP; GAL4-Act5C(FRT.CD2).P}S UAS-GFP/UAS-P35. Additional information about all of these fly strains is available at Flybase (http://flybase.bio.indiana.edu).M system  in fliesImmunohistochemistry was performed generally as described by Patel . The folLive imaging was performed with a Zeiss LSM 710 laser scanning microscope. Third instar larvae were dissected and placed in a drop of saline solution on a microscope slide under a number 1 glass cover slip. Motile GFP-expressing clones were checked for auto-fluorescence, which would rule out that they were hemocytes. In Additional File AVD performed genetic crosses, immunohistochemistry and microscopy, analyzed data, and helped draft the manuscript. JS performed live imaging assays and assisted with genetic crosses, data analysis, and drafting the manuscript. EF assisted with experimental design, immunohistochemistry, microscopy, data analysis, and drafting of the manuscript. MDS conceived of the study, participated in its design and coordination, performed experiments, evaluated data, and prepared the manuscript. The authors have read and approved the final manuscript.fra mutant cellsStill image accompanying live imaging movies of invasive . A stationary GFP-positive P35-rescued fra4 mutant clone in the ventral portion of the eye disc is marked by an * in the still image. Migratory mutant cells observed in the accompanying live imaging movies are marked by arrows.Click here for filefra mutant cells, 55×Live imaging of invasive . GFP-positive P35-rescued fra4 mutant cells exiting the eye-antennal disc were imaged in live tissue preparations. In this video, two migratory cells  have exited the ventral portion of the eye disc.Click here for filefra mutant cell, 200×Live imaging of invasive . The migratory P35-rescued fra4 mutant cell marked by the large arrow in the lower portion of Figure S1 is captured at higher magnification. Here, projections extending and retracting as the cell moves can be observed.Click here for file


The B cell antigen receptor (BCR) and pathogen recognition receptors, such as Toll-like receptor 4 (TLR4), act in concert to control adaptive B cell responses. However, little is known about the signaling pathways that integrate BCR activation with intrinsic TLR4 stimulation. Antigen receptors initialize activation of the inducible transcription factor nuclear factor-κB (NF-κB) via recruitment of the membrane-associated guanylate kinase caspase recruitment domain protein 11 (CARD11), the adapter molecule B cell CLL/lymphoma 10 (BCL10), and the "paracaspase" mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) into lipid rafts. Upon BCR triggering, this activation strictly depends on BCL10, but not on MALT1, leading to the hypothesis that a MALT1-independent NF-κB activation pathway contributes to BCR-induced NF-κB activation downstream of BCL10. The identity of this pathway has remained elusive.Using genetic and biochemical approaches, we demonstrate that the IRAK4- and IRAK1-dependent TLR signaling branch is activated upon BCR triggering to induce partial NF-κB activation. BCR-induced MALT1-independent IκB degradation and B cell proliferation were inhibited in MALT1/IRAK4 double knockout B cells. Moreover, IRAK1 was recruited into lipid rafts upon BCR stimulation and activated following transient recruitment of IRAK4.We propose that the observed crosstalk between BCR and TLR signaling components may contribute to the discrimination of signals that emanate from single and dual receptor engagement to control adaptive B cell responses. Activation and survival of B cells in response to antigen receptor (AgR) engagement depends on the activation of the inducible transcription factor NF-κB. BCR-induced NF-κB activation is mediated by components of the so-called CBM signaling complex. The CBM complex consists of the CARD-containing membrane-associated guanylate kinase CARD11, the CARD-containing adaptor protein BCL10, and the death domain (DD)-containing "paracaspase" MALT1 . ComplexParadoxical to the established requirement of MALT1 for T cell AgR (TCR)-mediated proliferation and NF-κB activation, BCR-driven proliferation and IκB degradation are reduced, but not abrogated in MALT1-deficient B cells, even though the impact on B cell proliferation was contradictory among previous reports -5,14. InTLRs are responsible for the recognition of pathogen-associated molecular patterns expressed by extracellular pathogens. Toll-like receptor 4 (TLR4) is the prototypic TLR that recognizes lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria . It relaex vivo proliferation of IRAK4-/- T cells  with or without 5 μg ml-1 anti-CD40 , or 20 μg ml-1 ultra pure E. coli LPS (InvivoGen). [3H]thymidine incorporation was measured as described before ATP and 10 μM cold ATP. Reactions were incubated at 37°C for 30 min and stopped by addition of SDS-PAGE loading buffer. Western blot analysis was performed using antibodies recognizing phospho-p44/42 MAP kinase (Thr202/Tyr204) , IκBα , or phospho-IκBα (Ser32) , actin , IRAK1 , IRAK4 (Acris), MALT1  and secondary horseradish peroxidase-coupled antibodies. Monosialotetrahexosylganglioside (GM1) was visualized with the FITC-conjugated Cholera Toxin B Subunit . The EMSA was performed using the Odyssey Infrared EMSA Kit and AP-1 probes from LI-COR.Cells were lysed as described previously . ProteinB-cells (6 spleens per stimulation) were purified with IMag Streptavidin Particles Plus by anti-CD43  depletion and were either left untreated or stimulated with anti-IgM for the indicated times. The cells were lysed in 750 μl cold 1% Brij lysis buffer and mixed with 750 μl 85% sucrose made with MNE buffer . After transfer of the lysate to the centrifuge tube, 1.5 ml 35% sucrose in MNE buffer was overlaid, then 1 ml 5% sucrose in MNE buffer. After centrifugation for 16 h at 39,000 rpm in a Sorvall Ultracentrifuge OTD-C , 0.4 ml fractions were collected from the top of the gradient.LPS: lipopolysaccharide; BM: bone marrow; TLR, Toll-like receptor; AgR: antigen receptor; BAFF: B cell activation factor of the TNF family; BCR: B cell AgR; TCR: T cell AgR; PRR: pathogen recognition receptor; NF-κB: nuclear factor-κB; CARD: caspase recruitment domain; DD: death domain; BCL10: B cell CLL/lymphoma 10; MALT1: mucosa-associated lymphoid tissue lymphoma translocation gene 1; NOD: nucleotide-binding oligomerization domain containing; TACI: transmembrane activator and calcium modulator and cyclophilin ligand interactor; TNFR: tumor necrosis factor receptor; TRAF6: tumor necrosis factor receptor-associated factor 6; TAK1: transforming growth factor β-activated kinase 1; TLR: Toll-like receptor; IL: interleukin; TIR: Toll-IL-1 receptor; TIRAP: TIR domain-containing adaptor protein; TRIF: TIR domain-containing adaptor inducing interferon-β; IRAK: IL receptor-associated kinase; RIP: receptor-interacting protein; WT: wild type; (D)KO: (double) knockout; PKC: protein kinase C; RAG: recombination activating gene; TPA: 12-O-Tetradecanoyl-phorbol 13-acetate; GM1: monosialotetrahexosylganglioside.The authors declare that they have no competing interests.AD designed the study, performed experiments, analyzed the data and wrote the manuscript. WWS revised the manuscript and provided final approval for publication. All authors have read and approved the final manuscript.


In patients with hypertrophic cardiomyopathy (HCM), positron emission tomography (PET) and cardiac magnetic resonance (CMR) are potentially useful tools for risk stratification. Parameters which could be linked to clinical ourcomes are accurate measures of myocardial ischemia and fibrosis.We sought to measure simultaneously abnormalities in myocardial blood flow (MBF) and cardiac sympathetic activity using PET and investigate their correlation with the patterns of left ventricular (LV) hypertrophy and myocardial fibrosis assessed with CMR.11C-hydroxyephedrine (11C-HED) and 15O-labeled water (15O-H2O), and delayed enhancement (DE) CMR on a 1.5 T scanner (Siemens Avanto). LGE images were acquired after intravenous gadolinium-diethylenetriamine penta-acetic acid (0.1 mmol/kg) in short-axis planes identical to cine SSFP images with a breath-hold inversion-recovery gradient echo sequence. The amount of DE was quantitatively assessed on short-axis CMR images with MRI-MASS (Medis) the fibrotic boundaries were identified by “full width half maximum” (FWHM) technique, and fibrosis extent was quantified as a percentage of total left ventricular mass. Myocardial presynaptic catecholamine reuptake/turnover was assessed measuring volumes of distribution (Vd) of 11C-HED, and MBF was determined from dynamic 15O-H2O PET scans at rest and during adenosine stress (140 mcg/Kg/min). PET and CMR data were analysed on a co-registered segmental basis.Thirteen patients (pts) with HCM (age 53±8 years) and 12 healthy age-matched controls (52±10 years) underwent PET with 2, and ejection fraction 68±9%. None of the patients had known coronary artery disease. Resting MBF was similar in pts and controls (0.9±0.3 vs. 1.1±0.2 mL/min/g). HCM pts had significantly lower stress MBF  and 11C-HED_Vd  than normal subjects. In HCM pts 104 out of 205 evaluable LV segments showed DE-CMR > 10% ; in these segments stress MBF was significantly lower . Binary logistic regression showed a significant association of LGE (p=0.019) and 11C-HED_Vd (p=0.0003) with a coronary flow reserve < 2.The maximal septal thickness in HCM pts was 21±4 mm, LV mass index (LVMI) 89±19 g/m11C-HED_Vd was correlated with stress MBF (p<0.001) and inversely with the amount of myocardial fibrosis on DE-CMR (p<0.001).Our preliminary findings suggest that in HCM pts the presence of fibrosis and the reduced presynaptic nor-adrenaline reuptake, which results in increased sympathetic activity could contribute to aggravate the inducible ischemia due to the anatomical abnormality of the intramural coronary arterioles.


Left atrial (LA) radiofrequency (RF) ablation has become routine treatment for atrial fibrillation (AF) but still suffers from AF recurrence requiring a repeat procedure. LA-RF ablation success rates vary between 53% and 85%. Delayed-enhancement Cardiac Magnetic Resonance (DE-CMR) can be used to noninvasively visualize LA hyperenhancement (scar). We have utilized DE-CMR to quantify LA scar extent post LA-RF-ablation and related this measure to AF recurrence.Twenty-seven patients  with paroxysmal and chronic AF underwent LA-RF-ablation and subsequent DE-CMR, an average of 260.7±314.7 days post procedure. The DE-CMR procedure was performed utilizing a navigated 3D inversion recovery gradient echo sequence (Siemens 1.5T Avanto or 3.0T Verio) approximately 15 minutes after administration of 0.2 mmol/kg Diethylenetriaminepentaacetic Acid−Gadolinium . All scans were electrocardiographically (ECG)-gated and acquired during a 150 ms window in mid-diastole with navigator-gating and fat suppression. We have developed an image analysis method and graphical user interface to semi-automatically quantify hyperenhanced regions in the LA wall (scar). LA scar was quantified by a single experienced observer blinded to patient data. LA-scar measurements were normalized by LA size. The intra-class correlation coefficient (ICC) was used to assess intra-observer variability of 4 randomly selected scans which were re-read one week later. Variables were tested for normality with the Shapiro-Wilk test and a p-value<0.05 was considered statistically significant . All patients provided informed consent.The DE-CMR scans were performed 260.7±314.7 days after the initial LA-RF- ablation procedure . AF recurrence was noted to occur in 13 (48%) patients whereas 14 (52%) patients demonstrated no AF recurrence. There was a trend toward a larger LA-volume in the AF-recurrence group (128.49±44.0 ml vs. 96.0±38.5 ml; p=0.06, see Table LA scar extent can be reproducibly quantified with DE-CMR; and a lower scar burden post LA-RF-ablation is associated with AF recurrence.This work was supported in part by NIH grant T32HL07812.


Estrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation.in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547.Inhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies Treatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/β and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation.These data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer. PIP3 then recruits pleckstrin homology (PH) domain-containing proteins such as PDK1, SGK and AKT to the plasma membrane, where AKT is phosphorylated at T308 by PDK-1 and, subsequently, at S473 by TORC2, becoming fully activated [AKT is a serine/threonine kinase downstream of phosphatidylinositol-3 kinase (PI3K) that plays a critical role in cellular survival, proliferation, metabolism and resistance to apoptosis . Upon acPIK3CA) and regulatory subunit p85α (PIK3R1), amplification of wild type PIK3CA, p110β (PIK3CB) and PDK1, loss/inactivation of the PIP3 phosphatases PTEN and INPP4B, mutation and/or amplification of AKT1-3 and amplification of RTKs, such as HER2, IGF-IR, MET, FGFR1 and EGFR [The PI3K/AKT signaling pathway is the most frequently mutated pathway in breast cancer -4. PI3K de novo or acquired resistance to endocrine therapies. Overexpression of the ErbB2/HER2 protooncogene has been shown to promote clinical resistance to antiestrogen therapy [About 80% of breast cancers express estrogen receptor α (ER) and/or progesterone receptor (PR), biomarkers indicative of hormone dependence . Therapi therapy ,8. HowevThe PI3K pathway has been causally associated with resistance to endocrine therapy -14. UponCell lines  were maintained in improved minimum essential medium (IMEM)/10% fetal bovine serum (FBS)  and authenticated by short tandem repeat profiling using Sanger sequencing (sequenced in March 2011). Long-term estrogen deprived (LTED) cells were generated and maintained in phenol red-free IMEM with 10% dextran/charcoal-treated FBS (DCC-FBS) .S473, P-AKTT308, AKT, P-PRAS40, P-GSK-3α/β, P-S6S240/244, S6, P-IGF-IRβY1131/P-InsRβY1146, P-HER3Y1197, P-HER2Y1248, P-SrcY416, P-FRS2-αY436, EGFR , InsRβ, IGF-IRβ, ERα (F-10), HER3, HER4, FGFR2 , HER2 , PR , IRS-1 , and actin . Densitometric analysis was performed using ImageJ. Phospho-RTK arrays were performed using the Human Phospho-RTK Array Kit according to the manufacturer's protocol (R&D Systems).Lysates from cells treated with AZD5363 , IGF-I, 4 cells/well for MCF-7/LTED and 4 × 104 cells/well for other lines) were treated in 10% DCC-FBS ± AZD5363, selumetinib  [50 of 14 nM. In cellular assays, the compound prevents autophosphorylation of IGF-IR in fibroblasts from IGF-IR knockout mice stably transfected with human IGF-IR with an IC50 of 48 nM; it inhibits autophosphorylation of human InsR in CHO-T cells with an IC50 of 186 nM. AZD9362, dosed at 25 mg/kg qd, also inhibits phosphorylation of IGF-IR by >50% for at least six hours and induces >70% inhibition of tumor volume in NIH3T3 fibroblasts stably transfected with IGF-IR. Media and inhibitors for proliferation assays were replenished every three days; after five to ten days, adherent cells were trypsinized and counted using a Coulter Counter or fixed/stained with crystal violet [IGF-IR, InsR, HER3, or non-silencing control were obtained from Qiagen.Cells seeded in triplicate in 12-well plates (2.5 × 10-142886)  (SelleckFoxO3) (Life Technologies), ER or non-silencing control (Qiagen) using Dharmafect 1 according to the manufacturer's protocol . Two days later cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours followed by RNA isolation and RT qPCR.Cells grown in 10% DCC-FBS ± AZD5363 were harvested and their RNA extracted using the RNeasy Mini Kit (Qiagen). Using the iScript cDNA Synthesis Kit , 1 µg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SABiosciences (Qiagen). For siRNA experiments, cells were transfected with siRNA targeting forkhead box class O  were transfected with 2.5 µg of an AKT-PH-GFP plasmid  using Lipofectamine 2000 according to the manufacturer's protocol . On day four, cells were treated with 10% DCC-FBS ± AZD5363, AEW541 or BKM120 for four hours. Cells were viewed on an LSM 510Meta confocal microscope at 40x magnification at the Vanderbilt University Cell Imaging Shared Resource.3 were randomized to treatment with vehicle (25% (2-hydroxypropyl)-β-cyclodextrin), AZD5363 (150 or 100 mg/kg bid p.o.), fulvestrant (5 mg/week i.p.), AZD9362 (25 mg/kg/day p.o.) or AZD4547 [3 calculated as volume = width2 x length/2. Tumors were harvested one or four hours after the last dose of AZD5363 or 24 hours after the last dose of fulvestrant and flash-frozen in liquid nitrogen or fixed in 10% formalin prior to paraffin-embedding. Frozen tumors were homogenized using the TissueLyser II (Qiagen). Tumor lysates were prepared, subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblot analysis [Animal experiments were approved by the Vanderbilt Institutional Animal Care and Use Committee. Female ovariectomized athymic mice were implanted s.c. with a 14-day-release E2 pellet . The next day, 107 MCF-7 cells suspended in IMEM and mixed with matrigel  at 1:1 ratio were injected s.c. into the right flank of each mouse. After >2 weeks, mice bearing tumors ≥150 mm AZD4547   histoscores were determined by unpaired t-tests. P <0.05 was considered significant.In cell proliferation assays, significant differences were determined by one-way analysis of variance (ANOVA) (one cell line) or two-way ANOVA with Bonferroni post-hoc tests corrected for multiple comparisons. Unpaired [See Additional file We previously established a panel of ER+ breast cancer cell lines with acquired resistance to LTED . TreatmeUpon escape from hormone deprivation, some ER+ tumor cells retain estrogen (ligand)-independent ER function. PI3K/AKT can phosphorylate and activate ER transcription in the absence of estradiol . Estrogein vivo. ER+/PIK3CA mutant MCF-7 xenografts were established in ovariectomized athymic female mice supplemented with a 14-day-release E2 pellet. The E2 pellets expired after 14 days. Four weeks after tumor cell inoculation, mice with tumors measuring ≥150 mm3 were randomized to treatment with vehicle, AZD5363, fulvestrant or the combination. AZD5363 and fulvestrant significantly inhibited tumor growth compared to vehicle-treated controls  pathway when only one pathway is inhibited.We next assessed the effects of the AKT inhibitor ± fulvestrant on tumor growth s Figure . Treatmes Figure . AZD5363s Figure . Treatmes Figure . Finallyin vitro  upregulated mRNA levels of IGF-I and IGF-II in three of the four LTED cell lines Figure , as well4 hours uEstrogen is known to modulate IGF-I signaling in breast cancer, and ER induces IGF-IR and IGF-II expression . The IGFWe then postulated that the phosphorylation of IGF-IR/InsR upon inhibition of AKT would be inhibited by blocking ligand binding to receptors with IGFBP-3. Treatment of MCF-7/LTED cells with IGFBP-3 inhibited IGF-I and IGF-II-induced phosphorylation of IGF-IR/InsR, as well as AKT Figure . IGFBP-350 of 48 nM), as well as autophosphorylation of InsR in CHO cells transfected with human InsR . Treatment with AZD9362 also significantly sensitized cells to the AKT inhibitor , but displays weaker activity against FGFR4 (IC50 of 165 nM) [P <0.05; Figure in vitro [P = 0.004), inducing a complete tumor regression in one mouse  . Treatmein vitro . Additioe Figure . Overallin vitro and in vivo, including IGF-IR, InsR, HER3 and FGFRs. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and AKT phosphorylation following treatment with AZD5363. Inhibition of AKT resulted in upregulation of ER- and FoxO-dependent IGF-IR, IGF-I, and IGF-II. Treatment with IGFBP-3 blocked the AZD5363-induced phosphorylation of IGF-IR/InsR and AKT, suggesting that the induced ligands activated IGF-IR/InsR. Finally, inhibition of IGF-IR/InsR enhanced the antitumor effect of the AKT inhibitor both in vitro and in vivo.PI3K/AKT/mTOR pathway activation has been implicated in endocrine resistance in breast cancer ,10,12-14in vitro and in vivo  sections in Figure S4 in Additional file Supplementary materials and methods for Supplementary figures. A pdf file presenting the Supplementary materials and methods.Click here for fileSupplementary figures S1-S9. Figure S1 showing inhibition of AKT with AZD5363 reduces phosphorylation of AKT/TORC1 substrates in ER+ breast cancer cells. Figure S2 showing inhibition of AKT with AZD5363 prevents the emergence of hormone-independent ER+ breast cancer cells. Figure S3 showing inhibition of PI3K with BKM120 upregulates ER expression and activity. Figure S4 showing treatment with AZD5363 and fulvestrant synergistically inhibits proliferation in vivo. Figure S5 showing AKT inhibition suppresses the growth of HBCx-3 ER+ luminal B breast cancer xenografts. Figure S6 showing the Src inhibitor dasatinib suppresses AZD5363-induced upregulation of HER3 phosphorylation and enhances its growth inhibitory effects. Figure S7 showing inhibition of AKT is followed by phosphorylation of multiple RTKs. Figure S8 showing inhibition of AKT with AZD5363 upregulates IGF-I and IGF-II protein levels. Figure S9 showing mice exhibit minimal weight loss when treated with pharmacological inhibitors.Click here for file


Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell–related genes , and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1− cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide–like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin It has been reported that Ulex europaeus agglutinin-1 (UEA-1) binds some enteroendocrine cells, but it also reacts with other epithelial cell components, such as M cells in the follicle-associated epithelium (FAE) and exocrine goblet cells Gcg and Gip promoters have enabled the identification and isolation of L cells and K cells, respectively The intestinal epithelial cell layer consists of functionally heterogeneous cell populations, including absorptive epithelial cells, goblet cells, paneth cells, M cells, cup cells, tuft cells, and enteroendocrine cells, all of which are derived from Lgr5Claudins (Clds), integral membrane proteins with four transmembrane domains, are crucial components of tight junctions (TJs) that function as a primary barrier to solutes and water as well as charge-selective channels between the apical and basal sides of epithelial cellular sheets + TJs formed with neighboring epithelial cells . Fluorescence-activated cell sorting (FACS) analysis revealed that approximately 8% of the total viable epithelial cell fraction (PI−CD45−Ter119−EpCAM+) of the intestinal single-cell preparation from wild-type (WT) mice showed positive staining with anti-Cld4 mAb (HKH-189), whereas virtually no positive signal was detected in that from Cldn4−/− mice  and Cld3 (Cldn3) transcripts (Chga) were confined exclusively to the Cld4+ cell fraction  were also expressed preferentially in the Cld4+ cell fraction ; Cld4−UEA-1+ cells (13%); Cld4+UEA-1− cells (6%); and Cld4+UEA-1+ cells (1%)  of Cld4+UEA-1+ cells expressed GLP-1. On the other hand, the expression of GIP and GLP-1 was also detected in a minor fraction of Cld4+UEA-1+  and Cld4+UEA-1−  cells, respectively, suggesting an overlap of endocrine hormone expression as reported previously -producing K cells are physically separable from other enteroendocrine cells including GLP-1–producing L cells by means of a flow cytometric cell sorter.By using multicolor FACS analysis with anti-Cld4 antibody (HKH-189) and UEA-1, we were able to identify four distinct populations in the intestinal EpCAMlls (1%) . We thenpression . It was ne cells . Among tfraction . In partne genes . We conf4+ cells , whereas1+ cells . By immu−CgA− epithelial cells, most likely representing goblet cells  after dextran administration. FACS analysis confirmed that about 23% of Cld4– and 32% of Cld4+ epithelial cells were labeled with fluorescein after fluorescein–dextran administration, whereas no staining was detected in the epithelial cells from untreated mice  of UEA-1+ cells revealed a strong fluorescein staining, although only a minor proportion (17.4%) of UEA-1– cells representing K cells showed a marginal staining, if any  that recognizes the extracellular portion indicated that approximately 8% of total small intestinal epithelial cells expressed Cld4, and that the isolated Cld4+ cells exclusively exhibited the expression of the genes associated with enteroendocrine cells, such as Chga, Ffar1, Gpr119, and Ffar4. The proportion could be an overestimate for enteroendocrine cells, because the Cld4+ cell population may also include other minor epithelial cells such as FAE in Peyer’s patches and villous tip enterocytes Cldn4 transcripts could be detected in the Cld4− cell population. Thus, in practice, it is strongly suggested that Cld4 serves as a specific cell surface marker covering entire enteroendocrine cells, providing a useful means for the isolation of this minor epithelial cell population as viable cells.Enteroendocrine cells comprise a very small population of gut epithelial cells that are scattered solitarily throughout the gastrointestinal tract, and the physical isolation of these cells has been hampered by the lack of specific cell surface markers. In the current study, we found that CgA+ epithelial cells consisted of UEA-1+ and UEA-1− populations, both of which comparably expressed Chga. The Cld4+UEA-1− cell population almost exclusively expressed Gip, with undetectable level of Cck or Sct, thus representing K cells. The population expressed lower levels of Gcg and Pyy, and it remains to be determined whether this was attributable to a minor contamination of UEA-1low cells, or to overlapping hormone expression in a single cell type as suggested previously +UEA-1+ cell population contained abundant transcripts of a series of hormones, including Gcg, Pyy, Cck, Sct, and Tph1, but no detectable Gip. Thus, although the cell population was much smaller than the Cld4+UEA-1− cell population, it was suggested that the Cld4+UEA-1+ cell population comprises functionally heterogeneous enteroendocrine cells, including L cells. The results of gene expression analysis were confirmed at the protein level with immunostaining analysis; GIP expression was associated with Cld4+UEA-1− cells, whereas GLP-1 expression was detected with Cld4+UEA-1+ cells with a minor overlap expression. The combination of rat anti-Cld4 monoclonal antibody (HKH-189) and UEA-1 thus provides an effective means to isolate K cells and other enteroendocrine cell populations, including L cells, separately from the intestinal epithelium. It has been reported that UEA-1 binds to a portion of enteroendocrine cells +CgA+ enteroendocrine cells have an activity of taking up luminal materials as well. The antigen-capturing activity was found mainly in the minor Cld4+UEA-1+ cells population, with little activity in the Cld4+UEA-1− K cells. Although enteroendocrine cells apparently form ZO-1+Cld4+ TJs with adjacent absorptive epithelial cells, it is unclear whether the luminal antigen is delivered through a transcellular route like M cells Although M cells present in the FAE of Peyer’s patches are specialized for capturing luminal antigens and delivering them to lamina propria to initiate mucosal immunity This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by Animal Research Committee, Graduate School of Medicine, Kyoto University (MedKyo13045).Cldn4−/− mice were generated as described -free conditions at the Kyoto University Laboratory Animal Center in accordance with university guidelines. Mice were euthanized by cervical dislocation and used for experiments.8-week-old to 10-week-old C57BL/6 WT female mice were purchased from Japan SLC and kept for at least 1 week before experiments in our animal room. -Tek OCT compound (Sakura). Unless otherwise noted, cryostat sections (6 µm) were blocked with 2% (vol/vol) fetal calf serum in phosphate-buffered saline (PBS) for 30 minutes at room temperature, followed by being incubated with primary antibodies or reagents for 16 hours at 4°C. Samples were then washed with 0.1% PBS-Tween and PBS, each for 5 minutes, followed by staining with secondary reagents for 30 minutes at room temperature. Samples were washed with PBS twice and then counterstained with DAPI (Sigma-Aldrich) for 10 minutes at room temperature to visualize the nucleus. Finally, samples were washed with PBS twice, mounted in Mowiol , and examined under a fluorescence photo microscope .Histological analysis was performed as described, with some modifications −CD45−TER119−EpCAM+ gating and biotinylated HKH-189 was used for Cld4 staining. Samples were analyzed by FACSCanto (Becton Dickinson) or sorted by FACSAria (Becton Dickinson).FACS analysis and cell sorting were performed as described previously -Cld4 mAb (HKH-189) -conjugated or biotinylated UEA-1 (Vector Laboratories); phycoerythrin (PE)-conjugated rat anti-CD45 mAb (30-F11) (Becton Dickinson); PE-conjugated rat anti-TER119 mAb (TER-119) (eBioscience); PE-Cy7–conjugated rat anti-EpCAM mAb (G8.8) (Biolegend); purified rabbit anti-chromogranin A pAb (ImmunoStar); purified rabbit anti-GIP pAb (Abbiotec); purified rabbit anti-GLP-1 mAb (EPR4043) (Epitomics); purified rabbit anti-ZO-1 pAb (Invitrogen); purified rat anti-ZO-1 mAb (R26.4C) ; purified rabbit anti-Cld3 pAb (Abcam); purified rabbit anti-Cld4 pAb (invitrogen); purified rabbit anti-Cld10 pAb (invitrogen);Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch); Alexa Fluor 488–conjugated or Alexa Fluor 647–conjugated anti-rabbit IgG (Invitrogen); and allophycocyanin-conjugated streptavidin (Becton Dickinson).The antibodies and reagents used for immunohistological and flow cytometry analyses were as follows: purified or biotinylated rat anti-time polymerase chain reaction (PCR) using QuantiTect STBR Green PCR mix (Qiagen) on a LyghtCycler 480 Real-Time PCR System (Roche). The transcripts of each gene were normalized to those of cyclophilin. The following primer sequences were used: CgA, (sense) -CAGGCTACAAAGCGATCCAG-3′5′ and (antisense) -GCCTCTGTCTTTCCATCTCC-3′5′; Gip, (sense) -CAGGTAGGAGGAGAAGACCTCAT-3′5′ and (antisense) -CCTAGATTGTGTCCCCTAGCC-3′5′; Gcg, (sense) -CACGCCCTTCAAGACACAG-3′5′ and (antisense) -GTCCTCATGCGCTTCTGTC-3′5′; Ffar1, (sense) -TAGGACTGGCTTCTGGTGCT-3′5′ and (antisense) -CTGCAGAGAAAAGAATGTCACAA-3′5′; Gpr119, (sense) -CCAGGAATCGTGGTCCAG-3′5′ and (antisense) -TGCATGTTCTTGAGAGAAGTCC-3′5′; Ffar4, (sense) -TTCTGGGGCTCATCTTTGTC-3′5′ and (antisense) -GCCACCAGCACTAGAGCAC-3′5′; Cck, (sense) -TGATTTCCCCATCCAAAGC-3′5′ and (antisense) -GCTTCTGCAGGGACTACCG-3′5′; Sct, (sense) -GCTGTGGTCGAACACTCAGA-3′5′ and (antisense) -GAGACAGGGACCCATCCAG-3′5′; Pyy, (sense) -CCTACCCTGCCAAACCAG-3′5′ and (antisense) -GGACATCTCTTTTTCCATACCG-3′5′; Tph1, (sense) -CACAGTTCAGATCCCCTCTACA-3′5′ and (antisense) -GAACGTGGCCTAGGAGTTCA-3′5′; Cldn3, (sense) -TGGGAGCTGGGTTGTACG-3′5′ and (antisense) -CAGGAGCAACACAGCAAGG-3′5′; Cldn4, (sense) -GGCGTCTATGGGACTACAGG-3′5′ and (antisense) -GAGCGCACAACTCAGGATG-3′5′; Tjp1, (sense) -CGCGGAGAGAGAGACAAGATGT-3′5′ and (antisense) -TCCTCCATTGCTGTGCTCTT-3′5′; and cyclophilin, (sense) -GACGAAGGTAGCCAGTCACAAG-3′5′ and (antisense) -AATCAGGCCTGTGGAATGTGAG-3′5′.RNA was extracted from isolated cells with Trizol (Invitrogen), followed by synthesis of cDNA with SuperScript III (Invitrogen). Gene expression level was assayed by real-conjugated lysine-fixable dextran (Life Technologies). Thirty minutes after oral challenge, the small intestine was examined by immunohistochemistry or FACS as described above..C57BL/6 WT mice were administered oral 5 mg/500 µl of 10-kDa tetramethylrhodamine or fluoresceint test or ANOVA.Data are presented as mean values ± standard deviation. Statistical significance was determined with the Student Figure S1Cld4 signals detected by independent anti-Cld4 antibodies were absent in −/−Cldn4 mice. Small intestine of WT and −/−Cldn4 mice were immunostained with anti-Cld4 polyclonal antibody or monoclonal antibody (HKH-189) (green) and DAPI (white). Bars, 50 µm.(TIF)Click here for additional data file.