Staphylococcus aureus (MRSA) cause pneumonia and empyema thoraces. TLR9 activation provides protection against bacterial infections and Heme oxygenase-1 (HO-1) is known to enhance host innate immunity against bacterial infections. However, it is still unclear whether HO-1 regulates TLR-9 expression in the pleura and modulates the host innate defenses during MRSA empyema. In order to determine if HO-1 regulates host innate immune functions via modulating TLR expression, in MRSA empyema, HO-1+/+ and HO-1-/- mouse pleural mesothelial cells (PMCs) were infected with MRSA  in the presence or absence of Cobalt Protoporphyrin (CoPP) and Zinc Protoporphyrin (ZnPP) or CORM-2 (a Carbon monoxide donor) and the expression of mTLR9 and mBD14 was assessed by RT-PCR. In vivo, HO-1+/+ and HO-1-/- mice were inoculated with MRSA (5x106 CFU) intra-pleurally and host bacterial load was measured by CFU, and TLR9 expression in the pleura was determined by histochemical-immunostaining. We noticed MRSA inducing differential expression of TLR9 in HO-1+/+ and HO-1 -/- PMCs. In MRSA infected HO-1+/+ PMCs, TLR1, TLR4, and TLR9 expression was several fold higher than MRSA infected HO-1-/- PMCs. Particularly TLR9 expression was very low in MRSA infected HO-1-/- PMCs both in vivo and in vitro. Bacterial clearance was significantly higher in HO-1+/+ PMCs than compared to HO-1-/- PMCs in vitro, and blocking TLR9 activation diminished MRSA clearance significantly. In addition, HO-1-/- mice were unable to clear the MRSA bacterial load in vivo. MRSA induced TLR9 and mBD14 expression was significantly high in HO-1+/+ PMCs and it was dependent on HO-1 activity. Our findings suggest that HO-1 by modulating TLR9 expression in PMCs promotes pleural innate immunity in MRSA empyema.Methicillin Resistant  Staphylococcus aureus (MRSA) causes necrotizing pneumonia and empyema thoraces[S. aureus pathogenesis [Pneumonia is a common reason for the infection-related deaths. Methicillin-resistant  thoraces. Pleural thoraces,4,5,6,7. thoraces,3. RecenPathogen associated molecular patterns are detected by the host innate immune cells via toll-like receptors (TLRs) among other pattern recognition receptors. TLRs upon ligand binding, transmit signals to intracellular milieu and stimulate important signaling pathways such as mitogen-activated protein kinase pathways and, nuclear factor-κB. TLR signaling contribute to the expression of several inflammatory and antimicrobial genes involved in various pathogenesis including Staphylococcal infections. The CpG-DNA induced TLR9 activation has been demonstrated to provide protection against various infections . The CpGS. aureus are susceptible to human β-defensin-3 (hBD3) [S. aureus [S. aureus the β-defensins are considered as promising candidates, particularly against the systemic infection underlying syndromes such as severe sepsis or empyema.The natural antimicrobial peptides released by host cells upon microbial infections are Defensins, Histatins and Cathelicidins. On the basis of the structure and the cells producing them the defensins are parted into α-defensins and β-defensins. The α-defensins are basically produced by neutrophils and Paneth cells in gastro intestinal tract. Histatins are mainly secreted in human saliva . However3 (hBD3) . RecentlS. aureus sepsis[Heme oxygenase-1 (HO-1), a 32-kDa inducible protein is cytoprotective against oxidative stress. Heme is degraded by the first-rate limiting catalytic activity of HO-1. HO-1 severs the alpha-meso carbon bridge of the b-type Heme molecules via oxidation to yield Biliverdin, carbon monoxide (CO) and free iron. Furtherus sepsis. Furtherus sepsis, and alsus sepsis. However+/+ and HO-1-/- mice and cultures were established as reported earlier [TLR9 murine agonist CpG ODN1826), TLR9 murine antagonist CpG (ODN2088), monoclonal anti-mTLR9 antibody, mouse monoclonal anti-hemagglutinin-tag (HA) Ab, pUNO-mTLR9-HA, pUNO-mcs purchased from Invivogen . Avian Myeloblastosis Virus-Reverse Transcriptase (AMV-RT) enzyme, SYBR Green Jump Start ready mix, Lysostaphin, actinomycin-D, and Tricarbonyl-dichloro-ruthenium (II) dimer (CORM2) were obtained from Sigma Aldrich . The Micro-Bicinchoninic Acid assay (BCA) protein assay kit was purchased from Pierce Biotechnology . The Cobalt Protoporphyrin-IX (CoPP), Zinc Protoporphyrin-IX (ZnPP), Biliverdin was from Frontier Scientific, Logan, UT. The RNA isolation kit was acquired from Qiagen . Murine PMCs were isolated from HO-1, TLR9 muMRSA (USA-3000) was inoculated into Mueller Hinton agar broth complemented with 6 μg/ml of oxacillin and 4% sodium chloride. The cultures were incubated at 35°C for 24 hours and the number of colonies were enumerated by plating on Mueller Hinton agar plates and expressed as CFU/ml. All experimental procedures with MRSA were performed inside the biosafety cabinet with stringent BSL-II precautions and containment.-/- mice (C57BL/6 back ground) were purchased from Jackson laboratories and required mice were breed in our laboratory. In this study 8 weeks age, (n = 6 mice per group) both male and female mice were used. Mice were maintained in micro isolator cages with a Hepa-filter barrier protection in Biosafety level-II housing suite. Animals received sterile food and water ad libitum. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Malcom Randal Veterans Affairs Medical Center Institutional Animal Care and Use Committee , North Florida/South Georgia Veterans Health System, Gainesville, FL; and all efforts were made to minimize their suffering. Pleural inoculations were performed under anesthesia with 2.5% isoflurane, and the mice were euthanized by exposing them to 2.5% to 5% isoflurane inhalation, followed by cervical dislocation upon confirming lack of toe pinch reflex. HO-1+/+ and HO-1-/- mice were intrapleurally infected with 5x106 MRSA CFU in PBS as reported earlier [+/+ and HO-1-/- mice were inoculated with equal volume of PBS to serve as controls. The mice were euthanized over time and lungs were harvested for further analysis.We established HO-1+/+, HO-1-/- PMCs were plated in 60mm culture dishes at a concentration of 5x105 cells per dish and maintained at 37°C with 5% CO2 in F-12K complete medium supplemented with streptomycin (100μg/ml), penicillin (100 U/ml), and 10% FBS. PMCs when reached to ∼70% confluence they were transferred to serum-free F-12K medium without antibiotics 16 h before treating them with agonists. The PMC cultures were pre-treated with ZnPP (10μM), CoPP (10μM), CORM2 (100μM) and Biliverdin (20μM) for 1h followed by exposure to MRSA at 10 MOI in serum-free medium (SFM) in vitro for indicated time points.HO-1Table 1) to amplify mouse TLRs and mouse Beta-Denfensin-14 (mBD14) were synthesized from Sigma. Mouse Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was amplified as an endogenous control.After treatments, PMC total RNA was isolated using RNeasy Mini Kit, . Using oligo-dT primers and enhanced AMV-RT enzyme, 1 μg of total RNA was used to synthesize cDNA by reverse transcription in 20-μl. Real time PCR was performed using 100 ng of cDNA template in triplicate using SYBR Green JumpStart ready mix in a 7500 real-time PCR system  as reported earlier. Primers+/+ and HO-1-/- PMCs were cultured in 60mm dishes, when there were near confluent they were infected with MRSA  for 6, 18 and 24 hours along with respective uninfected controls and TLR9 expression was evaluated by flow cytometry as reported earlier[HO-1d earlier. Brieflyin vivo the mice were infected with 5 x106 CFU of MRSA. The lungs, peripheral blood (PB) and pleural fluid (PF) were harvested aseptically at 24h, 48h and 72h post MRSA infection; Lung tissue was minced, viable bacteria (CFU) in the host was determined by plating serial dilutions of the lung tissue homogenate, PB, PF onto Mueller Hinton agar plates in triplicates as reported earlier [+/+ and HO-1-/- PMCs were exposed to MRSA (MOI 10) in the presence or absence of CoPP and CORM2 or Biliverdin respectively for 24h and CFU were determined as reported earlier[5 CFU of MRSA in 100μl volume for 3h at 37°C as reported earlier[To assess MRSA bacterial load in mice +/+ PMCs were infected with MRSA in the absence or presence of either CoPP or ZnPP. HO-1-/- PMCs were infected with MRSA in absence and presence of CORM2 or Biliverdin. After 6h of exposure cultures were harvested and TLR9 expression was detected using rabbit polyclonal anti-mouse TLR9 Ab  upon SDS-PAGE as reported earlier[In order to determine the TLR9 expression in PMCs, HO-1d earlier.2O2 followed by antigen retrieval for 10 min at 95°C . Sections were blocked with normal goat serum for 30 min at RT. Thereafter, sections were incubated with primary anti-mouse TLR9 antibody  for 45 minutes in humid chamber at 37°C. After washing, diluted biotinylated secondary antibody was applied for 30 minutes followed by conjugation with Streptavidin/Horseradish Peroxidase for another 30 minutes using Avidin-Biotin Complex (ABC) staining kit . Freshly prepared 3’3’-diaminobenzidine (DAB) substrate was applied and monitored for the brown color development. Slides were rinsed, counterstained with hematoxylin for 2 minutes and washed in running water, dehydrated and mounted in VectaMount to observe under microscope.TLR9 expression in the pleura and in PMCs cultures was determined by immunohistochemistry as reported earlier. Briefly+/+ PMCs were transfected with TLR9 vector as reported earlier[In order to delineate the TLR9 mediated signaling, HO-1d earlier. The genU’ tests. If the p-values were <0.05 the differences were considered statistically significant.Statistical analyses accomplished using SigmaStat 3.5 . Results were expressed as mean ±SD. Experiments with more than one treatment were assessed using the Kruskal-Wallis and the Mann-Whitney ‘+/+ and HO-1-/- PMCs, cells were infected with MRSA at MOI 10 for 3, 6, 18 and 24 hours. Total RNA was isolated and real- time PCR was performed to determine the TLR specific mRNA expression. The qPCR results showed a significant difference in TLR1, TLR2, TLR3, TLR4, and TLR6, and TLR9 mRNA the expression in MRSA infected both HO-1+/+ and HO-1-/- PMCs than compared to resting PMCs  data also revealed significantly higher expression of TLR9 in MRSA infected PMCs than compared to uninfected cells. MRSA induced TLR9 expression was significantly higher in HO-1+/+ PMC than compared to HO-1-/- PMCs and the maximum MFI was noticed at 6h post MRSA infection  in HO-1 control . Highestnfection .+/+ and HO-1-/- PMCs were infected with MRSA at MOI 1:10 in the absence or presence of either ZnPP-IX (HO-1 inhibitor) or CoPP (HO-1 inducer). In case of HO-1-/- PMCs, cells were treated with CORM-2  followed by MRSA infection. Under indicated conditions transcriptional and translational TLR9 expression was evaluated by quantitative RT-PCR and by Western-blot analysis respectively. Our qPCR results clearly demonstrated the drastic down regulation of TLR9 gene expression by ZnPP-IX whereas the CoPP significantly up-regulated the MRSA mediated TLR9 expression in HO-1+/+ PMCs  and HO-1+/+ mice (n = 6) were infected with 5x106 MRSA intra-pleurally, and the lungs, pleural fluid (PF), and peripheral blood (PB) were harvested upon euthanasia 24h, 48h and 72h post MRSA infection. Homogenized lung sample, PF, and PB were plated on Muller Hinton agar plates and the number of MRSA colonies enumerated after 24h of incubation. In HO-1-/- mice, we noticed significantly (p<0.001) high bacterial load, in the lungs, and PF, up to 72h post MRSA infection. Whereas in HO-1+/+ mice, significantly (p<0.001) low MRSA colonies were noticed in the lungs and pleural fluids up to 48h of infection  low MRSA colonies were recovered in CpG-ODN-1826 activated cultures than compared to the culture media obtained from TLR9 specific inhibitor the CpG-ODN2088 treated PMCs  and initiate innate immune defenses against invading pathogens. TLRs signaling triggers β-defensins release and participate in host defenses against invading pathogens. The β-defensins released through TLR signaling, in addition to their antimicrobial function, promote immune cell chemotaxis and thus participate in adaptive immunity. The β-defensins are mostly expressed by the epithelial cells in various tissues and could be induced by pro-inflammatory stimuli . In earls sepsis . Recentls sepsis . Impaire. aureus , and perthe skin . The mBDthe skin . Moreovethe skin . The mBDeus/MRSA . FurtherS. aureus infection, elucidating the significance of TLR9 in host-innate immunity to counter S. aureus [-/- mice were found to be susceptible to meningococcal sepsis due to high bacteremia [Empyema pleural fluids contain excess microbial pathogens and bacterial DNA ,13. TLR9. aureus . TLR9-/-cteremia . These oS. aureus activates innate immune function via multiple mechanisms including the TLRs, TNFR1; and NLRP3 and NOD2 [Chlamydia [+/+ PMCs and CORM2 treatment significantly increasing mBD14 expression in MRSA infected HO-1-/- PMCs suggesting HO-1 regulates mBD14 expression in PMCs and thus modulates host innate immune responses in the pleura.and NOD2 ,40,41. Thlamydia . Furtherhlamydia ,44. Nevehlamydia . We noti+/+ mice. Moreover, TLR9-mediated responses may serve as a means to augment antibacterial immunity in MRSA pneumonia or empyema, and these data may facilitate move forward in the development of new treatment strategies against MRSA infection.Here we have demonstrated the relevance of HO-1 in the PMCs innate immune defenses against MRSA infection. Our experiments elucidated the mechanisms of HO-1 axis in TLR9/mBD14 regulation in PMCs and its protective effect in MRSA empyema. The HO-1 deficient mice displayed reduced bacterial clearance compared to the HO-1


CRISP1, was investigated further by ChIP-PCR. The transcriptionally repressive function of H3K9me3 was confirmed in CRISP1. Our results provide mechanistic insight into the pathobiology of the “lipogenic phenotype of cancer.” Here, FASN reprograms the Ut-LMS epigenome through chromatin remodeling to promote the “malignant phenotype.”SK-UT-1 uterine leiomyosarcomas (Ut-LMS) cells were transduced with a fatty acid synthase (FASN)-containing retroviral vector to recapitulate the “lipogenic phenotype of cancer.” Consistent with this model, forced expression of FASN enhanced SK-UT-1 proliferation, migration, and cellular motion. Further investigation showed FASN promotes trimethylation of H3K9 (H3K9me3) and acetylation of H3K27 (H3K27ac) in SK-UT-1 cells. In contrast, siRNA targeting of FASN in high endogenous FASN expressing SK-LMS-1 Ut-LMS cells inhibits trimethylation of H3K9 and acetylation of H3K27. Palmitate, the predominant fatty acid product of FASN, increased H3K9me3, H3K27ac and H3K27me3 detection in SK-UT-1 cells. FASN promoted histone 3 methylation and acetylation through alteration of histone 3-modifying enzymatic activities . ChIP-seq in SK-UT-1-FASN cells with anti-H3K9me3 antibody identified regions of enriched binding compared to vector-only cells. One differentially-enriched gene,  Soft-tissue sarcomas (STS) are uncommon tumors of mesenchymal origin that consist of 70 subtypes . Uterinede novo long-chain fatty acid (FA) synthesis from acetyl–coenzyme A (acetyl-CoA), malonyl-CoA and NADPH. Another rate-limiting lipogenic enzyme is acetyl-CoA carboxylase (ACC), which catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA [de novo FA biosynthesis may regulate other cancer-related signaling networks.The “lipogenic phenotype” of cancer occurs as lipogenic enzymes, including fatty acid synthase (FASN), are upregulated during oncogenesis-mediated metabolic reprogramming , 4. FASNonyl-CoA . The FAsLipogenic enzymes including FASN are upregulated in epithelial cancers, and correlate with a higher risk of both disease recurrence and death , 4. SimiIn the current study, we developed a model of the “lipogenic phenotype” in Ut-LMS through FASN overexpression, which generated features characteristic of the malignant phenotype. We further explored the interrelationship between enhanced lipogenesis and epigenetic reprogramming in Ut-LMS. We found that FASN induced histone 3 (H3) remodeling by altering H3-modifying enzymatic activities. These results demonstrate that FASN reprograms the Ut-LMS epigenome through mediating histone modification signatures and chromatin remodeling to promote the “malignant phenotype” .Human Ut-LMS SK-UT-1 and SK-LMS-1 cell lines were purchased from the American Type Culture Collection , and were maintained in Eagle’s Minimum Essential Medium  with 10% fetal bovine serum. FASN-expressing (-FASN) or empty vector (-EV) SK-UT-1 cell lines were produced by transduction with retrovirus prepared from pBabe-FASN-HA-Flag and pBabe-EV-HA-Flag plasmids .Non-transformed “conditionally reprogrammed cells” (CRCs) have been established from patient-derived Ut-LMS in collaboration with Dr. Xuefeng Liu . CRC-2 cells were maintained in complete F medium: 3:1 of DMEM: F12 medium with hydrocortisone, EGF, insulin, cholera toxin and ROCK inhibitor Y-27632 as described [Palmitic acid was purchased from Sigma-Aldrich . FlexiTube GeneSolution GS2194 for FASN and All Stars control siRNA were purchased from Qiagen , and used for transfection with RNAiMAX transfection reagent . FASN antibodies were purchased from Santa Cruz Biotechnology, Inc.  and H3K4me3, H3K9me3, H3K27me3 and H3K27ac antibodies was were purchased from Abcam, Inc. . Human fibronectin was purchased from Corning, Inc .Stable FASN-expressing (-FASN) or empty vector (-EV) SK-UT-1 cell lines were selected and cultured for Western blot analysis. Parental SK-UT-1 cells were treated with 10, 100, 1000 μM palmitate for 24 hr, and cell lysates were collected to detect expression of histone 3 methylated and acetylated lysines by Western blot. FASN or scramble control siRNA were transfected into SK-LMS-1 cells and lysates were collected at the indicated time points to detect FASN and histone 3 expression.Histone-modifying enzymatic activity assay kits including histone deacetylase (HDAC), demethylase (HDM), acetyl transferase (HAT) and methyl transferase (H3K9 HMT) were purchased from Epigentek . Nuclear lysates from SK-UT-1-EV and -FASN cells were extracted and analyzed by ELISA according to the manufacturer’s protocols.Chromatin immunoprecipitation (ChIP) were performed on SK-UT-1-FASN and SK-UT-1-EV cells using the Magna ChIP A/G kit from  following the manufacturer’s protocol. Anti-H3K9me3 antibody was used to immunoprecipitate cross-linked and fragmented chromatin DNA. This was followed by elution of protein/DNA complexes, reversal of the cross-links, and DNA purification. Library preparation and massively parallel DNA sequencing (ChIP-seq) was performed with eluted DNA using an Illumina Hiseq 2500 sequencer by the City of Hope Integrative Genomics Core . Reads were aligned to hg19 genome assembly using Novoalign and only reads aligned to a unique genome location were reported. The aligned reads were then subject to peak calling using custom R scripts and Bioconductor package “chipseq”. Reads were extended to 200bp and regions that are longer than 100bp, with at least 10 reads in the enriched sample, and with 2.5-fold enrichment compared to the input sample, were considered as peaks. Peaks were annotated by overlapping to RefSeq mRNA database, and visualized by Integrated Genomics Viewer (IGV). The reads falling into these peaks in each sample were counted and differential peaks with 2-fold or more enrichment differences between the two cell lines were identified.Chromatin immunoprecipitation quantitative PCR (ChIP-PCR) was conducted using above the ChIP-eluted DNA as template. Quantitative reverse transcriptional PCR (q-RT-PCR) was carried out in total RNA extracted from SK-UT-1-FASN and SK-UT-1-EV cells. Correlative primers are shown in 2000 SK-UT-1-FASN and SK-UT-1-EV cells were seeded in 96 well plates for 72 hrs. Proliferation was determined by fluorescence-based digital image microscopy  as previously described . SurviviSK-UT-1-FASN and SK-UT-1-EV cells were grown in 6-well plates until 90% confluent. They were then scratched with a 1 ml pipette tip. Selected fields were marked, and microscopic images (magnification 4X) were taken at 0, 24, and 48 hrs post wound introduction. Similarly, SK-LMS-1 cells were transfected with FASN or scramble siRNA at 40% confluency. On the second day following transfection, a wound line was introduced, and microscopic images were taken at the same time points.t test. A p value of less than 0.05 was considered significant.Results were presented as the mean ± SD of three independent experiments. The significance of differences was evaluated by paired or unpaired Student's Endogenous FASN expression was detected in SK-UT-1and SK-LMS-1 Ut-LMS cell lines by immunoblotting. As shown in Histone methylation and acetylation patterns in two common repressive histone 3 (H3) marks, lysine 9 of histone 3 (H3K9me3) and lysine 27 (H3K27me3), and two activating H3 marks, lysine 27 (H3K27ac) and lysine 4 (H3K4me3), were examined in SK-UT-1-FASN cells by immunoblotting. The predominant FA product of FASN is palmitate. To evaluate FAs as signals for FASN-mediated chromatin remodeling, we examined whether palmitate could reproduce the same H3 modifications as FASN overexpression in low FASN-expressing parental SK-UT-1 cells. Due to the limited availability of Ut-LMS cell lines for study, non-transformed “conditionally reprogrammed cells” (CRCs) have recently been established from patient-derived Ut-LMS in collaboration with Dr. Xuefeng Liu  in a City of Hope Institutional Review Board (IRB)-approved protocol. Here, primary Ut-LMS cells are combined with a feeder layer of irradiated murine fibroblasts and a Rho kinase (ROCK) inhibitor . EventuaFASN expression was detected by immunohistochemistry (IHC) in an 80 core LMS tissue microarray (TMA) containing 30 cases of Ut-LMS. FASN expression was graded 0–3+ by a blinded pathologist. 37/80 (46%) of the LMS revealed 1–3+ expression, and 13/30 (43%) Ut-LMS showed 1–2+ expression. Histone-modifying enzymatic activities were measured in SK-UT-1-FASN and -EV cells, SK-UT-1, and SK-LMS-1 parental cells. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=yzcheumixbchpmn&acc=GSE98557). We focused on the greatest differentially-regulated exonic and intergenic regions enriched in proximal and distal regulatory regions (-8 to +2 kb) of adjoining genes  [CRISP1. CRISP1 is primarily expressed in male, and to lesser extent in female reproductive tracts, where it functions to modulate fertilization [CRISP1 promoter, and confirmed by quantitative polymerase chain reaction (qPCR)  in SK-UT-1-FASN and SK-UT-1-EV cell lines was performed with anti-H3K9me3 antibody to identify genomic regions differentially-enriched for H3K9me3 binding. 289 genomic regions were enriched ≥ 2-fold, and 329 regions were reduced ≥ 2-fold in SK-UT-1-FASN compared to SK-UT-1-EV cells in genome-wide analyses  . We choslization . Fig 4A n (qPCR) . Consistpression . FurtherCellular motion was quantified by time-lapse microscopy on plastic and fibronectin-coated plates, and migration of Ut-LMS cells was examined by scratch-wound assay. FASN is an attractive therapeutic target. FASN inhibitor-mediated cytotoxicity results from accumulation of toxic intermediary metabolite malonyl-CoA, induction of endoplasmic reticulum (ER) stress, accumulation of ceramide, and caspase activation –20. A phde novo lipogenesis and epigenetic reprogramming promote the sarcoma phenotype [Drosophila, Homolog 2 (EZH2) to promote expression in ES [HDAC1) mutations in 8.3% of cases [CEBPA) methylation in 24% of cases. Treatment with demethylating agents restores CEBPA expression in DLPS, is antiproliferative and proapoptotic in vitro and reduces tumor growth in vivo. Further, DLPS show elevated H3K9me3, and integrated chromatin immunoprecipitation-sequencing (ChIP-seq) with gene expression analyses show H3K9me3 mediates differential regulation of genes involved in cellular differentiation and migration through inhibition of Kruppel-like factor 6 (KLF6) [Both henotype , 23–26. on in ES . Dediffeof cases , and lipCRISP1. These results strikingly implicate the mechanistic pathway through which the “lipogenic phenotype of cancer” mediates its cellular effect is through altered chromatin remodeling.We report here that FASN enhances the malignant phenotype in Ut-LMS as demonstrated by increased proliferation, migration, and cellular motion. FASN promotes selected H3 trimethylation and acetylation through alteration of H3-modifying enzymatic activities. Palmitate, the principle end-product of FASN, recapitulates the FASN-mediated H3 modifications in a dose-dependent fashion, which links palmitate to FASN-mediated epigenetic signaling. Finally, ChIP-seq targeting H3K9me3 identified multiple regions of enriched binding, which we have shown to be functionally important as demonstrated by the transcriptional repression of S1 Fig(A) FASN overexpression does not alter H3K4me3 and H3K27me3 in SK-UT-1 cells. (B) Palmitate rescues FASN expression in SK-LMS-1 cells transfected with siRNA targeting FASN.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Gene expression analysis of The Cancer Genome Atlas (TCGA) breast cancer data set show that miR-20a is upregulated in human breast cancer, especially in triple-negative subtype. Gene Set Enrichment Analysis suggests that miR-20a expression negatively correlates with the autophagy/lysosome pathway. We report here that miR-20a inhibits the basal and nutrient starvation-induced autophagic flux and lysosomal proteolytic activity, increases intracellular reactive oxygen species levels and DNA damage response by targeting several key regulators of autophagy, including BECN1, ATG16L1 and SQSTM1. Re-introduction of exogenous BECN1, ATG16L1 or SQSTM1 reverses the inhibitory effect of miR-20a on autophagy and decreases DNA damage. A negative correlation between miR-20a and its target genes is observed in breast cancer tissues. Lower levels of BECN1, ATG16L1 and SQSTM1 are more common in triple-negative cancers than in other subtypes. High levels of miR-20a also associate with higher frequency of copy-number alterations and DNA mutations in breast cancer patients. Further studies in a xenograft mouse model show that miR-20a promotes tumor initiation and tumor growth. Collectively, these findings suggest that miR-20a-mediated autophagy defect might be a new mechanism underlying the oncogenic function of miRNA during breast tumorigenesis. Autophagy performs homeostatic ‘quality control’ functions to eliminate unfolded proteins or damaged organelles. Under conditions of high cellular stress, such as during nutrient starvation, bulk autophagy is induced to degrade intracellular components for recycling and building blocks necessary for cell survival.8 Loss of autophagy genes perturbs homeostasis and potentially primes cells for tumor development. Many core autophagy genes, including BECN1, UVRAG, SH3GLB1 (Bif-1), ATG2B, ATG5, ATG9B, ATG12 and RAB7A, are frequently mutated in human cancers.9 In various murine models, depletion of essential autophagy genes promotes tumorigenesis. Heterozygous disruption of BECN1 leads to spontaneous tumor formation in mice, indicating BECN1 is a halpoinsufficient tumor suppressor,12 mice lacking ATG5, ATG7 or AMBRA1 show similar effect.15 However, autophagy is also involved in oncogenic process in some circumstances. Autophagy promotes tumor cell survival and growth within tumor microenvironment, such as hypoxia or metabolic stress. Dysfunction in autophagy pathway suppresses tumor proliferation, dissemination and metastasis.15 Inhibition of autophagy increases sensitivity of tumor cells to chemotherapeutic agents as well as radiation therapy.18 Deletion of FIP200, a component of the ULK1–ATG13–FIP200 complex that is required for autophagosome formation, suppresses tumor initiation and progression. These findings strongly support the role of autophagy in oncogenesis in vivo.19In the context of cancer, the role of autophagy is controversial and resembles the mythical ‘janus face’. Autophagy plays tumor suppressive function by clearing unfolded proteins or damaged organelles, thereby preventing genome instability.23 miRNAs have crucial function in cancer; more than 50% of miRNA genes are located at chromosomal regions that include fragile sites or regions that exhibit genetic deletions or amplifications.26 miR-17/92 is the best-characterized oncogenic miRNA cluster.27 Human miR-17/92 gene is frequently amplified in solid tumors and several hematopoietic malignancies.29 We examined the expression patterns of miR-20a, a member of the miR-17/92 cluster, from TCGA breast cancer data set. A significant upregulation of miR-20a is observed in breast cancer, especially in triple-negative breast cancer. Gene Set Enrichment Analysis (GESA) suggests that miR-20a negatively correlates with the autophagy/lysosome pathway. Studies herein show that miR-20a inhibits basal and nutrient starvation-induced autophagic flux and lysosomal proteolytic activity, increases cellular reactive oxygen species (ROS) levels and DNA damage response by downregulating several key regulators of autophagy, including BECN1, ATG16L1 and SQSTM1. Low levels of BECN1, ATG16L1 and SQSTM1 are predominantly observed in triple-negative cancers than in other subtypes. A large fraction of copy-number-altered genome and DNA mutations are also detected in breast cancer patients with elevated levels of miR-20a. Collectively, our results suggest that miR-20a-mediated dysfunction of autophagy contributes to mammary tumorigenesis by promoting genomic damage and instability.MicroRNAs (miRNAs) contributes to almost all physiological and pathological processes. It is well established that miRNAs control gene expression through a post-transcriptional mechanism that involves mRNA degradation and translation repression.30 Here, we analyzed miR-20a expression from TCGA miRNA-seq data set; miR-20a expression was markedly higher in breast cancer tissues (n=109), ER positive (n=384), PR negative (n=164), PR positive (n=328), HER2 negative (n=411) or HER2 positive (n=71) based on immunohistochemical expression of hormone receptors. A significant upregulation of miR-20a expression was observed in ER-negative or PR-negative tumors (n=82) and other subtypes (n=391) revealed that miR-20a is upregulated in triple-negative subtype  medium to induce nutrient starvation. miR-20a significantly reduced GFP-LC3 puncta formation in nutrient-starved cells. Immunofluorescence assay with anti-LC3 confirmed these results . Next, wBECN1, ATG16L1 and SQSTM1 were identified as putative targets. To validate, we performed qPCR and immunoblotting analysis. Overexpression of miR-20a markedly reduced the transcript abundance of BECN1, ATG16L1 and SQSTM1 in both MDA-MB-231 and MCF7 cells  restored miR-20a-induced inhibition of luciferase activity  3′-UTRs of activity . Because32 Immunoblotting revealed that starvation-induced LC3-II turnover was inhibited by bafilomycin A1 (Baf A1) or hydroxychloroquine (HCQ), miR-20a inhibits basal level and starvation-induced LC3-II turnover  and breast cancer tissues (n=30) by in situ hybridization. Consistent with the TCGA miRNA-seq data set analysis, miR-20a expression was markedly higher in tumor tissues . miR-20a expression correlates negatively with that of BECN1, ATG16L1 or SQSTM1 in breast cancer  compared with other subtypes (n=391)  .Immunohistochemistry showed low levels of miR-20a targets  in tumor tissues. However, γH2AX foci formation was dramatically increased in tumor tissues, whereas the staining of γH2AX in normal tissues was nearly undetectable . In TCGAin vivo.To investigate the role of miR-20a in tumorigenesis, we generated MDA-MB-231 cell lines stably expressing NC or miR-20a through the lentivirus expression system. Stable transfection of miR-20a markedly suppressed mRNA and protein expression of ATG16L1, SQSTM1 and BECN1 in MDA-MB-231 cells. miR-20a stable transfection also led to reduced LC3-II level and increased OPTN expression, indicating that miR-20a inhibits autophagic flux . Nude mi29 Analysis of miRNA expression profiles revealed a three-fold increase in miR-17/92 expression in triple-negative breast carcinoma. However, comparison of absolute miRNA levels between normal breast tissue and tumor tissues did not show any significant differences.30 Here, we analyzed TCGA miRNA-seq data set, upregulation of miR-20a was observed in breast cancer, especially in triple-negative subtype.37Several studies report that the oncogenic miR-17/92 cluster is frequently deregulated in human cancers.c-MYC directly activates miR-17/92 gene expression. In addition, E2F1 and E2F3 also activate the transcription of miR-17/92 cluster, suggesting that miR-17/92 plays key roles in regulating cell proliferation.39 By contrast, the tumor suppressor TP53 transcriptionally represses miR-17/92 expression, through binding to the TATA box of the miR-17/92 promoter.40 A recent study report that both two members of the miR-17/92 cluster, miR-20a and miR-17, target the TP53-activating kinase DAPK3, which leads to inactivation of TP53 and upregulation of the miR-17/92 cluster.41 Several studies demonstrate that miR-20a promotes proliferation, sustains cell survival and increases angiogenesis by targeting a number of genes, including the E2F family members.43 However, in some circumstances, which depends on the physiological context and the cell type, miR-20a plays tumor suppressive function.46 Our results show that miR-20a inhibits nutrient starvation-induced autophagic flux. miR-20a suppresses autophagy and lysosomal activity through downregulating the expression of ATG16L1, BECN1 and SQSTM1. Autophagy-deficient cells have deregulated ROS and accumulate DNA damage, which possibly contribute to genome instability and tumor-initiating capacity . Thus, we propose that in cancers with higher miR-20a, there is parallel inactivation of autophagy and impairment of DNA repair; this may impair the ability of the cells to adapt to environmental stress, which may subsequently enable gene amplification, mutation and accumulation of DNA damage, thereby promoting genomic instability and cancer progression. Future work should address these questions.Autophagy-deficient cells are impaired in DNA repair by the error-free process of homologous recombination. Cells lacking autophagy machinery largely depend on the non-homologous end joining, the error-prone repair process of DNA double-strand breaks. Sustained reliance on non-homologous end joining results in loss of genetic integrity.http://tcga-data.nci.nih.gov/tcga/. miRNA-seq analysis was performed from data of 694 breast cancer tissues and 83 normal tissues. RNA-seq analysis was performed from data of 500 breast cancer cohorts. The fraction of copy-number altered genome and mutation counts data were from 347 breast cancer cohorts.We downloaded TCGA breast cancer data sets from 52 The expression levels of miR-20a were used as phenotype label, and ‘Metric for ranking genes’ was set to Pearson Correlation. All other basic and advanced fields were set to default.Gene Set Enrichment Analysis (version 2.0) was used to investigate the biological processes that correlate with miR-20a expression in breast cancer as described in the references.MDA-MB-231 and MCF7 cells were purchased from ATCC . MCF7 cells were grown in Dulbecco’s modified Eagle’s medium , MDA-MB-231 cells were cultured in ATCC-formulated Leibovitz’s L-15 medium (ATCC), supplemented with 10% fetal bovine serum  and 10 U/ml penicillin/streptomycin (Invitrogen). Cells were cultured in Earle’s Balanced Salt Solution  to induce nutrient starvation.Lipofectamine 2000 (Invitrogen) was used to transfect miRNAs or siRNAs. miRNAs were purchased from GenePharma Co. Ltd . siRNA pools of SQSTM1 (sc-29679) and ATG16L1 (sc-72580) were purchased from Santa Cruz Biotechnology . LNA-NC (199020-00) and LNA-20a (426943-00) were purchased from Exiqon . BECN1 siRNA is a pool of two target specific siRNA designed by GenePharm. BECN1 siRNAs: 5′-CAGUUUGGCACAAUCAAUATT-3′ and 5′-AAGAUCCUGGACCGUGUCACCTT3′ Scramble control: 5′-UUCUCCGAACGUGUCACGU-3′.SQSTM1 plasmid was purchased from Addgene ; BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd . Plasmids were transfected into MCF7 cells by Lipofectamine 3000 (Invitrogen).53 mRNA expression was normalized to GAPDH and miRNA was normalized to U6 snoRNA. The following primer are used for PCR analysis: GAPDH forward 5′-AAGGCTGTGGGCAAGG-3′, GAPDH reverse 5′-TGGAGGAGTGGGTGTCG-3′ BECN1 forward 5′-ACCTCAGCCGAAGACTGAAG-3′, BECN1 reverse 5′-AACAGCGTTTGTAGTTCTGACA-3′ ATG16L1 forward 5′-AACGCTGTGCAGTTCAGTCC-3′, Atg16L1 reverse 5′-AGCTGCTAAGAGGTAAGATCCA-3′ SQSTM1 forward 5′-GATGAGGAAGATCGCCTTGGA-3′, SQSTM1 reverse 5′-TTCGGATTCTGGCATCTGTAGG-3′.mRNA or miRNA expression was determined by quantitative reverse transcriptase–PCR analysis as previously described.miR-20a and LC3 plasmids (GFP-LC3 or mCherry-GFP-LC3) were co-transfected into MDA-MB-231 or MCF7 cells. Nutrient starvation was induced by treating cells with EBSS for 4 h. Fluorescent images of GFP-LC3 or mCherry-GFP-LC3 were acquired by confocal microscope . LC3 dots per cell was calculated from 150 cells in each sample.BECN1, ATG16L1 or SQSTM1 genes were cloned into pRL-TK luciferase vector , the resulting plasmids were called wild-type reporters (WT). To ablate the miR-20 bindings sites, we used site-directed mutagenesis kit  to generate mutated vectors (Mut). MCF7 cells were transfected with luciferase vectors and miR-20a or NC using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured and normalized by protein concentration.The 3′ UTRs fragments of human 53 Antibodies used are LC3 , ATG16L1 , BECN1 , SQSTM1/p62 , OPTN , γH2AX , GAPDH , ACTB , HRP-conjugated secondary antibodies . Protein bands were visualized by chemiluminescence .Cells were lysed in ice-cold whole-cell extract buffer as described previously.Cells were fixed in freshly prepared 4% paraformaldehyde solution for 15 min, washed three times with phosphate-buffered saline (PBS) and then treated with 0.25% Triton X-100/PBS for 5 min. The fixed preparations were blocked in 3% BSA/PBS for 1 h, incubated with primary antibodies for 1 h at room temperature, washed three times with PBS and incubated with Alexa Fluor 555 or Alexa Fluor 488 conjugated secondary antibody for another 1 h (Invitrogen). Fluorescent images were acquired by confocal microscopy.http://imagej.nih.gov/ij/).MCF7 cells transfected with miRNAs, siRNAs or LNAs were treated with 10 μg/ml DQ Red BSA  for 12 h at 37 °C. Samples were cultured in normal medium or EBSS solution to induce nutrient starvation. The fluorescent images of DQ Red BSA were acquired by confocal microscopy and the fluorescence intensity was measured by Image J software ; DNA damage was quantified by measuring the Tail Moment (TM) using CASP software (http://casplab.com).MCF cells were transfected with NC or miR-20a, cultured in normal medium or EBSS for 24 h. Cells suspension was mixed with low melting agarose  and plated onto comet slide to perform single-cell gel electrophoresis.MCF7 cells transfected with miRNAs or siRNAs were incubated in complete medium or EBSS solution for 4 h. Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit  was used to analyzing intracellular ROS levels.n=30) and breast cancer tissues (n=30) were obtained from Shanghai Outdo Biotech Co., Ltd . For in situ detection of miR-20a, the slides were hybridized with DIG-labeled oligonucleotide (Exiqon) complementary to miR-20a. Hybridization was visualized by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color substrate . The staining intensity of miR-20a was determined using a visual grading system.Paraffin-embedded consecutive slides of normal tissues , anti-ATG6L1 , anti-SQSTM1  or anti-γH2AX  overnight at 4°C. The slides were mounted and the images were captured and analyzed by a fluorescence microscope.n=12 for each group). miR-20a or NC was transfected into MDA-MB-231 cells by lentivirus infection. Stable cells were trypsinized and suspended in PBS. Each mouse was subcutaneously injected with approximately 5 × 106/0.2 ml cells at the abdominal region. The tumor size was measured using digital calipers daily for 3 weeks.Four-week-old female BALB/c nude mice were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine  and kept in pathogen-free conditions for 1 week. The mice were randomly divided into two groups (t-test.All data were expressed as mean±s.d. of three independent experiments. Measurement data between two groups were performed using nonparametric Mann–Whitney test. The correlation of miR-20a and target gene expression was examined by Spearman correlation test. Statistical analyses were performed using two-tailed Student’s


Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the shams phenotype is not known. Considering the role of Delta-Notch signaling in wing vein formation, we hypothesized that Shams might affect Delta-mediated Notch signaling in Drosophila. Using genetic interaction studies, we find that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation. Further, Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams. In agreement with the above in vivo observations, cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch. Loss of Shams does not impair the cis-inhibition of Notch by ectopic overexpression of ligands in vivo or the interaction of Notch and cis-ligands in S2 cells. Nevertheless, removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta. This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams. Taken together, our data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor’s response to cis-ligands vs. trans-Delta during Drosophila development.The  trans-activation. In contrast, interaction of Notch with ligands expressed in the same cell has an inhibitory effect on signaling and is known as cis-inhibition. The balance between trans- and cis- interactions ensures optimum Notch signaling in some contexts during animal development. We have used the model organism Drosophila (fruit fly) to decipher the mechanism through which a carbohydrate residue, xylose, regulates Notch signaling in specific contexts. We provide evidence that addition of xylose residues to the Notch receptor decreases its interaction with trans-Delta ligand without affecting its interaction with cis-ligands. Thereby, xylose tunes the Notch pathway by modulating the balance between Notch trans-activation by Delta and Notch cis-inhibition by same-cell ligands. Misregulation of Notch signaling causes a number of human diseases including cancer and developmental disorders. Therefore, understanding the role of xylosylation in Notch signaling can potentially establish a new framework for therapeutic targeting of this pathway.One of the key mechanisms used by neighboring cells to communicate with each other in animals is signaling through an evolutionarily conserved receptor family called Notch. Binding of Notch to ligands of the Delta/DLL and Serrate/JAG families from neighboring cells leads to activation of the signaling pathway and is called  O-linked sugar modifications [trans-activation and results in the induction of Notch downstream targets [cis-inhibition of the pathway [cis and trans interactions of Notch with ligands is thought to determine whether each cell assumes a signal-sending or a signal-receiving role with regard to a given ligand [Notch signaling is a juxtacrine signaling pathway broadly used during animal development and tissue homeostasis . Both Noications ,3. Inter targets ,5. In co pathway –10. The n ligand ,12.trans-activatory and cis-inhibitory interactions between Delta and Notch are important for demarcation of the boundary between vein and intervein tissues [cis-inhibit Notch and allow the cells to assume the wing vein fate. Delta from central provein cells also trans-activates Notch in neighboring cells to prevent the adoption of vein fate and to establish the wing-intervein boundary. The haploinsufficient wing vein phenotypes exhibited by Notch+/–and Delta+/–animals and their mutual suppression in Notch+/–; Delta+/–double-heterozygous flies [cis-inhibition and trans-activation of Notch by Delta are required for proper wing vein formation, and that altering the relative expression levels of these two proteins can tip the balance between cis- and trans-interactions between them. However, it remains to be determined whether other mechanisms exist to regulate this balance.The Notch ligands Delta and Serrate function redundantly in several contexts during fly development . However tissues ,18. Specus flies  further N-acetylglucosamine (GlcNAc) to O-fucose residues on Notch EGF repeats by Fringe proteins differentially regulates the response of Notch to its ligands by regulating Notch-ligand interactions [cis and in trans configurations [O-glucose onto Notch EGF repeats by the protein O-glucosyltransferase 1 (Poglut1). In Drosophila, Poglut1 is encoded by rumi [O-glucosylated EGF repeats also serve as a substrate for the addition of xylose by glucoside xylosyltransferase (GXYLT) enzymes [shams loss-of-function phenotype.Sugar modifications of the Notch receptors are known to affect Notch pathway activation at various steps, including folding, trafficking, ligand binding and potentially cleavage –22. For ractions –26. It iurations  and as s by rumi  and prom by rumi –30. O-gl enzymes ,32. Functrans-activation of Notch by reducing the binding of Notch to trans-Delta without affecting the binding of Notch to cis-ligands. The effect of loss of Shams on trans-activation of Notch by overexpressed Delta can be mimicked by decreasing the level of endogenous cis-ligands, suggesting that upon loss of Notch xylosylation, Delta trans-activation overcomes Notch cis-inhibition by ligands. Altogether, our observations indicate that Shams regulates the balance between trans-activation and cis-inhibition of Notch by Delta to ensure optimal Notch activation in several contexts during Drosophila development.Here, we provide evidence that Notch xylosylation by Shams decreases Delta-mediated shams mutants, we performed gene-dosage experiments using Delta genomic rescue transgenes [Delta in a wild-type background does not generate any adult wing phenotypes at 30°C  animals results is extra wing vein material  and ocellar (OC) bristles in the adult head [Delta in shams mutants rescues the loss of head bristles . Removing one copy of Serrate does not affect the loss of wing vein and head bristles in shams mutants , which induces FRT-mediated recombination in the wing imaginal discs with a high efficiency [shams on Notch activity by staining for Notch targets Cut and Wingless (Wg) in shamsΔ34 mutants clones. These mutant clones do not exhibit clear abnormalities in the expression of Cut and Wg when compared to adjacent wild-type or heterozygous cells  and double-mutant clones for ligands and shams (shamsΔ34Serrev6-1 and DlRevF10shamsΔ34). To analyze the effects of loss of shams on each ligand without interference from the other ligand, we focused on mutant clones which cross the dorsal-ventral (DV) boundary. Serrev6-1 clones show a highly penetrant loss of Cut, except for the cells that directly abut the wild-type tissue . In contrast, the average area of wings harboring DlRevF10 clones was not significantly different from the wings harboring DlRevF10shamsΔ34 double mutant clones . These results are in agreement with the data obtained from clonal analysis in larval wing discs and further support the notion that loss of shams partially suppresses the phenotypes caused by the loss of Serrate, likely by enhancing the Delta-mediated Notch activation in the developing wing.In addition to wing disc stainings, we examined the effects of ein loss . Adult walloping , in agre alleles –38. Howe1 clones . Indeed,trans-activation and cis-inhibition by its ligands [trans and cis functions of ligands and potential feedback mechanisms, we used the GAL4-UAS system [shams on the cis and trans effects of each ligand on Notch. When raised at 25°C, animals overexpressing Delta within the Dpp expression domain (dpp>Dl) exhibit two phenotypes in wing imaginal discs: loss of the Cut-positive cells and disruption of the endogenous wing margin within the Dpp expression domain due to cis-inhibition  [Tom results in cis-inhibition of Notch at the endogenous wing margin region and trans-activation of Notch outside of the Dpp expression domain in the ventral compartment  . As expepartment . In cont animals . These ddpp>Dl to trans-activate Notch upon loss of Shams, we decided to further explore the mechanism that prevents dpp>Dl from activating Notch in a shams+/+ background. In the sensory bristle lineage, decreasing the endogenous levels of Delta enhances the trans-activation of Notch by overexpressed Delta [Delta and/or Serrate on the expression of Cut in dpp>Dl larvae. In a DlRevF10 or Serrev2-11 heterozygous background, dpp>Dl induces moderate levels of Notch trans-activation in the dorsal-anterior quadrant  on the rate of aggregation formation and size of the aggregates formed between S2-Notch (S2-N) and S2-Delta (S2-Dl) or S2-SerrateTom (S2-SerTom) cells, which are indications for Notch-ligand binding [EGFP dsRNA. When co-cultured with plain S2 cells treated with shams dsRNA, S2-Dl cells form small aggregates which do not increase with time (shams dsRNA (P<0.01). qRT-PCR experiments indicate that shams dsRNA decreases shams mRNA levels greater than 80% in S2 cells  or equal amounts of Notch and cis-ligand expression vectors (S2-N&Dltransient or S2-N&Sertransient). The relative aggregation between S2-Dl and S2-Ntransient cells in the presence and absence of cis-ligands was used as an indication for the degree of cis-inhibitory effect of each ligand. For example, if the number of aggregates between S2-Dl and S2-N&Sertransient cells is 25% of the number of aggregates between S2-Dl and S2-Ntransient cells (i.e. relative aggregation 25%), we would conclude that cis-Serrate was able to block the interaction between Notch and trans-Delta by 75%. Co-culture of S2-Dl and control S2-Ntransient cells formed small aggregates, which grew in size and number with time, similar to the co-culture between S2-Dl and stable S2-N cells  also decreased their aggregation with S2-Dl cells . To ensure similar baseline levels of trans-Dl/Notch binding, we used the same amount of Notch expression plasmid in all experiments and only changed the amount of cis-ligand expression plasmids. Aggregation between S2-Dl and S2-Ntransient cells was once again inhibited by expression of cis-ligands (both cis-Delta and cis-Serrate) even at lowest levels. Quantitation of the number of cell aggregates greater than six cells at five minutes of co-culture showed that the magnitude of inhibition by cis-ligands was concentration-dependent: the lower the ratio of cis-ligand to Notch expression plasmid, the lower the ability of cis-ligands to decrease the number of aggregates between S2-Dl and S2-Ntransient cells BSC494/TM6C, Sb1, Dl9P/TM6, Tb1, DlRevF10SerRX82/TM6, Tb1, UAS-CD8::GFP (Bloomington Drosophila Stock Center), shamsΔ34/TM6, Tb1 [PBac{Ngt-wt}VK22, PBac{Ngt-16_20}VK22, [P{Dlgt-wt}attP2, PBac{Dlgt-wt}VK37, PBac{Sergt-wt}VK37 [y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; FRT82B y+tub-GAL80/TM6, Ubx [w; UAS-Dl (Gary Struhl), DlRevF10/TM6, Tb1 [Serrev6-1/TM6, Tb1 [Serrev2-11/TM6, Tb1 [SerRX106 [UAS-Serwt-Tomato (UAS-SerTom) [FRT82B shamsΔ34Serrev6-1/TM6, Tb1, Dl9PshamsΔ34/TM6, Tb1, FRT82B shamsΔ34DlRevF10/TM6, Tb1, FRT82B DlRevF10/TM6, Tb1, dpp-GAL4 shamsΔ34/TM6, Tb1, UAS-Dl; shamsΔ34/TM6b,Tb1, UAS-SerTom/CyO; shamsΔ34/TM6b, Tb1, y w Ubx-FLP/FM7; FRT82B Sb63y+/TM6, Ubx (this study).The following strains were used in this study: TM6, Tb1 , PBac{Ng20}VK22, , P{Dlgt--wt}VK37 , y w UbxTM6, Ubx , w; UAS-TM6, Tb1 , Serrev2TM6, Tb1 , SerRX10SerRX106 , UAS-Ser-SerTom) , FRT82B y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; FRT82B y+tub-GAL80/TM6, Ubx females were crossed to FRT82B mutant/TM6, Tb1 males, wherein mutant stands for shamsΔ34, Serrev6-1, DeltaRevF10, DeltaRevF10shamsΔ34, or shamsΔ34Serrev6-1. Crosses were kept at 25°C and mosaic Tb+ 3rd instar larvae were selected for dissection. To generate clones in adult wings, FRT82B shamsΔ34/TM6, Tb1, FRT82B Serrev6-1/TM6, Tb1, FRT82B shamsΔ34Serrev6-1/TM6, Tb1, FRT82B DeltaRevF10/TM6, Tb1, and FRT82B DeltaRevF10shamsΔ34/TM6, Tb1 males were crossed to y w Ubx-FLP/FM7; FRT82B Sb63y+/TM6, Ubx females and raised at 25°C. Sb63, Tb+ flies were selected for scoring the wings. All of the scored flies had regions of Sb+ microchaete on the thorax, confirming the generation of mutant clones in wing imaginal discs. dpp>Dl shamsΔ34/Δ34 animals were generated by crossing animals harboring a dpp-GAL4 shamsΔ34 recombinant chromosome to UAS-Dl; shamsΔ34/TM6b,Tb1 animals. Tb+ 3rd instar larvae were selected for dissection. To generate dpp>SerTomshamsΔ34/Δ34 animals, dpp-GAL4 shamsΔ34 animals were crossed to UAS-SerTom/CyO; shamsΔ34/TM6b, Tb1 animals. Tb+ 3rd instar larvae expressing Tomato were selected for dissection. To examine the expression pattern of cDNAs driven by dpp-GAL4 in larval wing imaginal disc, dpp-GAL4/TM6, Tb1 males were crossed to UAS-CD8::GFP females. Late 2nd and late 3rd instar Tb+ larvae were selected and dissected.All crosses were performed on standard media. All crosses were incubated at 25°C except for adult wings listed at 30°C, which were incubated at 25°C until late larval stage and shifted to 30°C during pupal stages. To generate MARCM clones  in 3rd iDissection and staining were performed by using standard methods. For surface staining, S2 cells were incubated with antibodies against the Notch extracellular domain (NECD) or the Delta extracellular domain (Dl-ECD) in the absence of detergent. A similar detergent-free protocol was used for Delta surface staining of wing imaginal discs. Antibodies used were mouse α-Cut (2B10) 1:500, mouse anti-Wg (4D4) 1:100, mouse anti-NECD (C458.2H) 1:100 , guinea pig anti-Dl-ECD 1:3000 (Gift from M. Muskavitch) , goat α-Drosophila Medium (Lonza) supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U/mL). For S2-N and S2-Dl stable cell lines , 200 nM methotrexate (Sigma-Aldrich) was added. S2-SerTom stable cells [EGFP dsRNA (control) or shams dsRNA was added to the culture medium of S2, S2-N or S2-Dl cells, and the cells were cultured for 24 hours at 25°C prior to induction with CuS04 (0.7 mM).S2 cells (Invitrogen) were cultured in Schneider's le cells  were cul6 cells/well) were transiently transfected with the constructs expressing the extracellular domain of Delta or Serrate fused to alkaline phosphatase (6.0 μg of either pMT-Delta-AP or pMT-Serrate-AP per well). After 24 hours, the transfected cells were induced by adding 0.7mM CuSO4 to the media. After three days of induction, conditioned media were collected and the amount of AP was determined by quantifying AP activity in each medium by following the manufacturer’s instructions  and using FLUOstar OPTIMA (BMG Labtech). In parallel, plain S2 or S2-N cells (2 x 106 cells/well) were induced by 0.7mM CuSO4 after 24 hours of treatment with EGFP dsRNA or shams dsRNA. After three days of induction, cells (S2 or S2-N cells) were collected and incubated with conditioned media (containing defined concentrations of Delta-AP or Serrate-AP) for 90 minutes on rotary shaker. After three times washing and cell lysis, the endogenous AP activity was heat inactivated (60°C for 10 min). The amount of AP-tagged ligand bound to S2-N cells was assayed as per the manufacturer’s instructions  and using FLUOstar OPTIMA (BMG Labtech). Binding of each ligand (Delta-AP or Serrate-AP) with plain S2 cells (which lack endogenous Notch protein [Ligand-receptor binding assays were performed as described previously  with min protein ) was use4 for 1–2 days and then used in aggregation assays. For each aggregation assay, 2.5 x 105 of the dsRNA-treated cells  were mixed with 5 x 105 S2-Dl, S2-SerTom or S2-N cells (induced with 0.7 mM CuSO4 for 3 hours prior to co-culture) in a total volume of 200 μl medium in a 24-well plate.Cells were incubated with CuSOcis-ligands, after 24 hours of treatment with either EGFP or shams dsRNA, S2 cells were transiently transfected with 2 μg total of either pBluescript (control), pBluescript and pMT-Notch (S2-Ntransient), pMT-Notch and pMT-Delta (S2-N&Dltransient), or pMT-Notch and pMT-Serrate (S2-N&Sertransient) using 3 μL of FuGENE HD (Promega). One μg of pMT-Notch was used in all assays. The remaining 1 μg was either pBluescript alone, pMT-Delta alone, pMT-Serrate alone, or a mixture of 0.5 μg, 0.2 μg or 0.1 μg of pMT-Delta or pMT-Serrate and pBluescript  in a total volume of 200μL in a 24-well plate. Co-cultured cells were gently shaken at 150 rpm to allow aggregation. Images of aggregate formation were taken at reported time points. The number of cell aggregates was quantified using a hemocytometer after 5 minutes of co-culture. Each assay was repeated at least three times. P-values were determined either by Student’s t-test or by One-Way ANOVA with Tukey’s multiple comparisons test.For co-culture assays expressing ipt Figs  and 8C. EGFP dsRNA primers:Forward primer-GAAATTAATACGACTCACTATAGGGGGTGAGCAAGGGCGAGGAGReverse primer-GAAATTAATACGACTCACTATAGGGGGTCTTTGCTCAGGGCGGshams dsRNA Primers:Forward primer-TTAATACGACTCACTATAGGGGAGATGCTGTATGTGGACACGGATReverse Primer-TTAATACGACTCACTATAGGGGAGATCCGTGGATAACCTTAACGApAct-EGFP plasmid DNA or y w genomic DNA. Purified PCR products were used as template for in vitro transcription reactions using the T7 MEGAscript kit (Ambion). Double-stranded RNA was purified with RNeasy Mini kit (Qiagen).Template DNAs were generated by PCR amplification from shams and Rp49 (RpL32) mRNA expression in S2 cells treated with either EGFP or shams dsRNA were assayed by qRT-PCR using TaqMan One-Step RT-PCR Master Mix and TaqMan primers/probe sets (Life Technologies Dm02144576_g1 and Dm02151827_g). Relative shams mRNA levels were compared using the 2–ΔΔCT method. P-values were determined by Student’s t-test.S1 FigSerrev6-1/+shamsΔ34/Δ34 animals (n = 73). (B) Adding one copy of a Serrate genomic transgene rescues the wing margin loss in these animals (n = 31).(A-B) All animals raised at 25°C. (A) Wing margin loss is observed in 21% of (TIF)Click here for additional data file.S2 FigEGFP or shams dsRNA). No difference in expression levels is apparent. Scale bar = 25 μm. (B) Graph shows the average fluorescence intensity in dsRNA (EGFP or shams) treated S2-N cells (n = 40 cells in each group). Error bars indicate standard error. NS: not significant.(A) Representative images showing cell surface Notch expression (red) in S2 cell (control) and S2-N cells (treated with (TIF)Click here for additional data file.S3 Figshams dsRNA (Shams KD), S2-Dl cells treated with a control dsRNA (EGFP) or S2-Dl cells treated with shams dsRNA. Representative images of each co-culture at 1, 5 and 15 minutes are shown. (B) Quantification of number of cell aggregates greater than 6 cells after 5 minutes of co-culture. Error bars indicate standard error. Note that Shams KD does not change the number of aggregates significantly (P>0.05). NS: not significant. (C) Representative images showing cell surface Delta expression (red) in S2 cells (control) and S2-Dl cells (treated with EGFP or shams dsRNA). No difference in expression levels is apparent. Scale bar = 25 μm. (D) Graph shows the average fluorescence intensity of surface Delta in dsRNA (EGFP or shams) treated S2-Dl cells (n = 40 cells for each group). Error bars indicate standard error. NS: not significant. (E) Detergent-free immunostaining for Delta extracellular domain (Dl-ECD) in third instar wing imaginal discs harboring shamsΔ34 MARCM clones . No difference in surface level of Delta between wild-type and mutant cells is apparent. Scale bar = 25 μm.(A) Cell aggregation assays were performed between S2-N cells with S2 cells treated with (TIF)Click here for additional data file.


A special focus will be placed on the role of HA/CD44-induced oncogenic signaling and histone methyltransferase, DOT1L activities in regulating histone modifications (via epigenetic changes) and miRNA activation. Many of these events are essential for the CSC properties such as Nanog/Oct4/Sox2 expression, spheroid/clone formation, self-renewal, tumor cell migration/invasion, survival and chemotherapeutic drug resistance in HA-activated head and neck cancer. These newly-discovered HA/CD44-mediated oncogenic signaling pathways delineate unique tumor dynamics with implications for defining the drivers of HNSCC progression processes. Most importantly, the important knowledge obtained from HA/CD44-regulated CSC signaling and functional activation could provide new information regarding the design of novel drug targets to overcome current therapeutic drug resistance which will have significant treatment implications for head and neck cancer patients.Head and neck squamous cell carcinoma (HNSCC) is a solid tumor composed by a genotypically and phenotypically heterogeneous population of neoplastic cells types. High recurrence rate and regional metastases lead to major morbidity and mortality. Recently, many studies have focused on cellular and molecular mechanisms of tumor progression that can help to predict prognosis and to choose the best therapeutic approach for HNSCC patients. Hyaluronan (HA), an important glycosaminoglycan component of the extracellular matrix (ECM), and its major cell surface receptor, CD44, have been suggested to be important cellular mediators influencing tumor progression and treatment resistance in head and neck cancer. HNSCC contains a small subpopulation of cells that exhibit a hallmark of CD44-expressing cancer stem cell (CSC) properties with self-renewal, multipotency, and a unique potential for tumor initiation. HA has been shown to stimulate a variety of CSC functions including self-renewal, clone formation and differentiation. This review article will present current evidence for the existence of a unique small population of CD44v3 Head and Neck Squamous cell carcinoma (HNSCC) is an aggressive malignant neoplasm associated with major morbidity and mortality. The three-year survival rate for patients with advanced-stage HNSCC who are treated with standard therapy is only 30% to 50% ,2,3,4. Rd-glucuronic acid and N-acetyl-d-glucosamine (6–1 × 107 Da) . I,6. Id-gl 107 Da) ,12 which 107 Da) . HAS3 sy) [5 Da) ,12 which) [5 Da) ,12. HAS2) [5 Da) . Downreg) [5 Da) . Overexp) [5 Da) . These oPHYAL-1, and PH20 (or Spam1)) have been reported [PHYAL-1, a pseudogene appears to be untranslated in humans [Large-size HA polymers can be digested or degraded into smaller-sized HA fragments by acid-active hyaluronidases  Figure . Overexpreported . For exareported . Hyal-2 reported ,20. Intereported . Hyal-3 reported ,22. Hyaln humans . PH20 ” or “Tumor-Initiating Cells (TICs)” share several common properties of normal stem cells ,44. Howehigh-expressing CSCs which can generate phenotypically distinct cells [high-expressing CSCs may exhibit stem cell-like properties  which are responsible for the generation of heterogeneous tumor cell populations. In fact, CD44s has been widely used as one of the most important stem cell surface markers in several studies [highALDH1high cells) was isolated using anti-CD44v3 antibody plus ALDH1-specific reagent and FACS-fluorescence-activated cell sorter [highALDH1high-expressing cells into NOD/SCID mice generated phenotypically distinct cells in heterogeneous tumors [highALDH1high cells (but not CD44v3lowALDH11ow cells or unsorted cells) into NOD/SCID mice [highALDH1high cells (but not CD44v3lowALDH11ow cells or unsorted cells) may be caused by unique tumor initiation properties of these subpopulations of HNSCC cells [Head and neck cancer (HNSCC) tumors contain a subpopulation of CD44sct cells . When thct cells . These o studies ,45. Alde studies ,47. Rece studies ,31,46,47l sorter ,49,50  which are known to function as transcriptional factors [One of the common properties of CD44v3 factors ,49,50. T factors . Overexp factors ,54,55,56 factors . It is e factors ,53. In H factors ,53,55,56 factors ,59,60. D factors . Increas factors . Oct4 al factors ,62. More factors . Most imhighALDH1high head and neck CSCs and breast tumor cells, respectively [highALDH1high head and neck CSCs [It has been reported that self-renewal, proliferation and differentiation regulated by Nanog/Sox2/Oct4 are the hallmark of stem cell properties that allow CSCs to produce both new cancer stem cells and phenotypically diverse cancer cells with different proliferative potential ,44. All ectively ,64,65  subpopulation isolated from HNSCC (HSC-3) [It has been well-documented that cytoskeleton functions are directly involved in tumor cell invasion of surrounding tissue, and metastasis . During  (HSC-3) ,49,50. HhighALDH1high (CSC-like) subpopulation from HNSCC cells [One general concept that has emerged from recent studies is that HA fragments  and their larger precursor molecules  may be involved in regulating distinct biological activities including miRNAs ,50,76,77CC cells . Most noCC cells . In a prCC cells . Most imCC cells ,50. TreaCC cells ,50. TheshighALDH1high head and neck cancer CSCs treated with 200 kDa-HA [Treatment of head and neck cancer CSCs with 200 kDa-HA also causes cisplatin resistance . Surviva0 kDa-HA . There i0 kDa-HA . These f0 kDa-HA . These o0 kDa-HA . Moreove0 kDa-HA . These fSaccharomyces cerevisiae, DOT1 was initially identified as a disruptor of telomeric silencing. The orthologs of DOT1 appear to be evolutionarily conserved from yeast to mammals [MLL-rearranged leukemia with EPZ00477 or EPZ-5676 (potent and selective amino nucleoside inhibitors of DOT1L histone methyltransferase activity) causes cell death in acute leukemia lines bearing MLL translocations [Epigenetic changes such as histone methylation have emerged as one of the important regulatory processes in the chromatin structural modifications and the gene expression alterations during cancer progression . Methyla mammals ,81. Both mammals . Specifi mammals . An earl mammals . It has  mammals . Treatmeocations ,87. Furtocations . These fhighALDH1high head and neck CSCs [highALDH1high CSCs. This process subsequently results in the overexpression of RhoGTPases and survival proteins leading to tumor cell invasion and cisplatin resistance. These findings suggest that HA/CD44-activated miR-10 production plays an important role in upregulating the cytoskeleton regulator, RhoC in CSC-like cells. Most importantly, we found that downregulation of HA-activated DOT1L signaling or miR-10b production by treating CSCs with DOT1L siRNA or anti-miR-10b inhibitor, respectively, not only reduces RhoGTPase (RhoC) expression and impairs tumor cell migration/invasion but also enhances chemosensitivity [highALDH1high head and neck cancer CSCs , which displays CSC characteristics with high tumorigenic potential and chemoresistance. These CD44v3highALDH1high-expressing CSCs appear to be closely involved in the pathobiology of head and neck squamous cell carcinomas. Most importantly, matrix hyaluronan (HA) interaction with CD44v3 plays a pivotal role in regulating stem cell marker expression, stemness properties  and chemoresistance in these head and neck cancer stem cells. Moreover, histone methyltransferase, DOT1L/H3K79 methylation-associated epigenetic changes induced by matrix HA has been shown to directly regulate miR-10 production leading to up-regulation of RhoGTPase and survival proteins as well as migration/invasion and chemoresistance in CD44v3highALDH1high-expressing CSCs. In the future, cancer stem cell-based signaling perturbation therapies may be developed to downregulate stem cell transcription factors and/or histone methyltransferase, DOT1L (using Nanog/Oct4/Sox2/DOT1L-specific siRNA and shRNA vector approaches or DOT1L inhibitors) and/or to silence miR-10b (using anti-miR-10b inhibitor) for blocking HA/CD44v3-mediated Nanog/Oct4/Sox2 signaling and DOT1L/miR-10b expression/function as well as chemoresistance and HNSCC progression. Taken together, these recently discovered matrix hyaluronan (HA)/CD44v3-mediated signaling pathways and DOT1L-specific epigenetic regulation in head and neck CSCs could provide unique molecular targets and specific therapeutic drugs for the treatment of head and neck cancer.This review has placed a special focus on the properties of a unique subpopulation of HNSCC cells (i.e., CD44v3


Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells  However, humans bearing mutations in the PCSK genes as well as PCSK-deficient experimental animal models have gene-specific phenotypes, which argues for substrate specificity in vivo .–52.Il1b,rophages . FinallyThe production of several cytokines secreted by macrophages, including TNF-α, IL-1β and TGF-β1, is dependent on proteolytic processing in the cell . SpecifiTgfb1 mRNA levels were not affected  mice in vivo  mice. Understanding the underlying mechanism(s) of aberrant cytokine expressions by FURIN deficient macrophages clearly requires further studies, but at least reduced TGFβ-1 maturation is likely to play a role [The analyses of proteolytically activated macrophage cytokines thus showed that a FURIN deficiency reduces the production of anti-inflammatory TGF-β1 but upregulates the activation of the TACE and Caspase-1 enzymes. These findings together with the inherent upregulation of pro-inflammatory mRNAs collectively contribute to the pro-inflammatory phenotype of LysMCre-y a role . In concfur alleles [fur(fl/fl) animals to generate myeloid-specific FURIN knockout mice LysMCre-fur(fl/fl). Mice were housed under pathogen-free standard conditions. All mouse experiments were performed in accordance with the National Animal Experiment Board, Finland, (Permit# ESAVI/2837/04.10.07/2015).Mice bearing floxed  alleles  were bacE. coli 0127:B8 serotype; Sigma-Aldrich, St Louis, MO, USA), TLR ligands  and/or cytokines . After stimulation, the cells were scraped to dislodge them and collected for RNA extraction . Neutrophils were isolated from the bone marrow of LysMCre-fur(fl/fl) and wild-type mice with the Anti-Ly-6G MicroBead Kit  and were left unstimulated or were stimulated with 100 μM of fMLP for 1 hour at 37°C.Peritoneal cells were extracted from the peritoneal cavity and cultured following a previously published protocol . After 1Peritoneal, splenic and bone marrow cells were analyzed using flow cytometry (BD FACSCanto™ II) and the FlowJo software . Anti-CD16/CD32 (Clone: 93)  was used to block Fc receptors and cell populations were surface stained following the eBioscience FACS protocol with PerCP-Cy5.5- labeled anti- F4/80 (BM8), PE-labeled anti-Gr-1 (RB6-8C5), PE-Cy7-labeled anti-CD11b (M1/70), FITC-labeled anti-B220 (RA3-6B2), APC-labeled anti-CD3, PE-Cy7 labeled anti-CD4 (RM4-5), APC-H7 labeled anti-CD8 (53-6.7), PE-labeled anti-CD62L (MEL-14), PE-labeled anti-Ly-6G (RB6-8C5), APC labeled anti-Ly-6C (HK1.4), PE-Cy7-labeled anti-CD11c (N418), FITC-labeled anti-CD11b (M1/70) .fur(fl/fl) and WT littermate mice (6-8 weeks of age) were injected intraperitoneally with NaCl (0.9%) or LPS  and serum was collected at the 0, 1, 3 h time points. The serum cytokines TNF-α, IL-6, MCP-1 and IL-10 were quantified using the BD™ Cytometry Bead Array Mouse Inflammation Kit  according to the manufacturer's instructions . IL-1β levels were measured with mouse IL-1 beta ELISA Ready-SET-Go!®  . Bioactive TGF-β1 was measured in the supernatants of cultured peritoneal macrophages isolated from LysMCre-fur(fl/fl) and WT littermate mice using Human/Mouse TGF beta 1 ELISA Ready-SET-Go! (2nd Generation)  .Serum cytokines were measured in both steady state and LPS injected mice. Briefly, LysMCre-fur(fl/fl) and WT littermate mice (6-8 weeks of age) were injected intraperitoneally with a lethal dose of LPS  or with NaCl (0.9%) as a control. Survival of the mice was monitored for 72 h at 3-hour intervals.LysMCre-The mRNA expression in stimulated and non-stimulated peritoneal macrophages was assessed using qRT-PCR. Briefly, total RNA was extracted using the RNeasy kit , quantified using a NanoDrop ND-2000  and transcribed into cDNA with the iScript Select cDNA Synthesis Kit . Gene expression levels were examined using the Bio-Rad CFX96 Real-Time System and the Sso Fast Eva Green Supermix . The primers used for quantitative real-time PCR are shown in the E. coli 0127:B8 serotype; Sigma Aldrich, St Louis, MO, USA). The samples were collected and the RNA was isolated using Qiagen RNeasy on-column DNase . 50 ng of purified RNA was amplified and dye labeled using Agilent's Low Input Quick Amp Labeling kit and RNA Spike In kit . Initial and purified RNA contents along with dyed and amplified cRNA contents were measured with a NanoDrop ND-2000 . RNA and cRNA quality was inspected using the Agilent 2100 Bioanalyzer RNA 6000 Nano kit . 300ng of Cy-3 and 300ng of Cy-5 labeled sample were hybridized together on an Agilent Mouse Chip 8×60K (Design ID 028005)  overnight at 65°C using the Gene Expression Hybridization kit. The chips were washed with the Gene Expression Wash Pack according to the instructions. The chips were scanned using an Agilent Technologies Scanner model G2565CA using the scan profile AgilentG3_GX_2Color. Scan results were converted into numerical data by the Agilent Feature Extraction software version 10.7.3. The data was analyzed using the R software [Two biological replicates of FURIN KO and WT peritoneal macrophages were left unstimulated or were stimulated for 1, 4 and 24 hours with LPS (1 μg/ml) pathway database was used to obtain a gene set involved in Toll-like receptor signaling. [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ililkmqqvvqlryn&acc=GSE84117.Differential gene expression was evaluated using the log2-transformed fold change difference and statistical testing using the two-sample Student's gnaling. . The rawE. coli 0111:B4, 500 ng/ml; InvivoGen, San Diego, CA, USA). Cells were washed twice with PBS and incubated with ATP . Next, the cells were collected and lysed in a buffer specific for Caspase-1  pH: 8.2 mM Ethyleneglycoltetraacetic acid (EGTA) pH: 7.5, 10% Glycerol, 1% Triton X-100, 50 mM Sodium fluoride, 200 μM Sodium vanadate) [E. coli 0127:B8 serotype; Sigma Aldrich, St Louis, MO, USA). Next, the cells were collected and lysed in a buffer specific for TACE , 10mM 1,10-phenanthroline) [For the Caspase-1 experiment bone marrow macrophages were cultured using a standard protocol  and wereanadate) . For thehroline) . Equal at-test. P < 0.05 indicates statistical significance and the variability is depicted using the standard error of the mean (SEM). The Kaplan Meier test was applied to estimate survival in the LPS induced endotoxemia experiment.Statistical analyses were performed using the two-tailed unpaired Student's


However, the effect of regorafenib on NF-κB-modulated tumor progression in HCC in vivo is ambiguous. The aim of the present study is to investigate the effect of regorafenib on NF-κB-modulated tumor progression in HCC bearing mouse model. pGL4.50 luciferase reporter vector transfected SK-Hep1 (SK-Hep1/luc2) and Hep3B 2.1-7 tumor bearing mice were established and used for the present study. Mice were treated with vehicle or regorafenib (20 mg/kg/day by gavage) for 14 days. Effects of regorafenib on tumor growth and protein expression together with toxicity of regorafenib were evaluated with digital caliper and bioluminescence imaging (BLI), ex vivo Western blotting immunohistochemistry (IHC) staining, and measurement of body weight and pathological examination of liver tissue, respectively, in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice. The results indicated regorafenib significantly reduced tumor growth and expression of phosphorylated ERK, NF-κB p65 (Ser536), phosphorylated AKT, and tumor progression-associated proteins. In addition, we found regorafenib induced both extrinsic and intrinsic apoptotic pathways. Body weight and liver morphology were not affected by regorafenib treatment. Our findings present the mechanism of tumor progression inhibition by regorafenib is linked to suppression of ERK/NF-κB signaling in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice.Regorafenib has been demonstrated in our previous study to trigger apoptosis through suppression of extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) activation in hepatocellular carcinoma (HCC) SK-Hep1 cells  Human hepatocellular carcinoma (HCC), or primary malignancy of the liver, is notorious for the high mortality rate . Progresin vitro and in vivo ) vector was purchased from Promega . SK-Hep1 cell was transfected with pGL4.50 [luc2/CMV] vector using JetPEI™ transfection reagent as described previously . SK-Hep1luc2 (1 × 107) or Hep3B 2.1-7 cells (2 × 107) in 150 μl of mixture containing serum-free DMEM and matrigel (2:1) were inoculated subcutaneously into the right legs of nude mice  and regorafenib group (treated with 20 mg/kg/day by gavage for 14 days)  in Taipei Medical University, Taipei, Taiwan (IAUCU number: LAC-2016-0029). Four-week-old nude mice were obtained from the National Laboratory Animal Center, Taipei, Taiwan. SK-Hep1/ude mice . When tu14 days) . TreatmeMice were anesthetized using 1–3% isoflurane and injected intraperitoneally with 150 mg/kg D-luciferin 15 min before acquiring image. The photons emitted from tumor were detected with Xtreme . The image acquisition period was 10 s. Regions of interest (ROIs) of tumor were drawn and quantified by using Molecular Imaging Software (MI) version 7.2  .Mice were killed on day 14 after treatments. Tumors were removed from mice and fixed in 4% paraformaldehyde (PFA) at 4°C. Paraffin-embedded tumor tissues were sectioned in 5 μm-thick slices by Bio-Check Laboratories Ltd . Protein levels in tumor tissues were evaluated with IHC staining. The instructions provided with the kit were followed to perform IHC staining. The sectioned slices were immunohistochemically stained with MMP-9, VEGF, MCL-1, XIAP, C-FLIP, Cyclin-D1, anti-P-ERK, NF-κB p65 (Ser536), P-AKT (Ser473), Caspase-9, -8, and -3 antibodies respectively. The stained slides were scanned using the microscopy-based TissueFAXS platform  and images were captured at 100× magnification . ImageJ Proteins were extracted from mice tumors using nuclear and cytoplasmic extraction kits . Equal amount proteins were loaded on 10% sodium dodecyl sulfate/PAGE for electrophoresis assay under 100 voltage. After running step, proteins were transferred onto 0.2 μm polyvinylidene difluoride membrane and then blocking with 3% bovine serum albumin for 1 h. Primary antibodies of P-ERK (Thr202/Tyr204), T-ERK, NF-κB p65 (Ser536), P-AKT (Ser473), T-AKT, caspase-3, caspase-8 and caspase-9 were added on membrane and both were gently shaked on shaker in 4°C refrigerator overnight. The levels of protein were finally colored by enhanced chemiluminescence (ECL) and detected by ChemiDoc MP Imaging System . Quantification of protein level was performed by Bio-Rad Image Lab .Mice were sacrificed on day 14 after treatments. Lobes of each mouse liver were removed and fixed in 4% PFA at 4°C. Paraffin-embedded liver tissues were sectioned in 5 μm-thick slices and then stained with hematoxylin and eosin (HE) by Bio-Check Laboratories Ltd . The stained slides were scanned using the microscopy-based Tissue FAXS platform  and images were captured at 100× magnification.t-test was used to verify significance of difference between treatment and control group. Data presented were means ± standard error. Statistical significance was reached when P-value was less than 0.05. All experiments were repeated independently for three times.Student’s luc2 and Hep3B 2.1-7 were prepared for animal studies respectively. The study procedure of animal was displayed as luc2 and Hep3B 2.1-7 tumor bearing mice in the experiment. We found regorafenib significantly inhibited both SK-Hep1/luc2 and Hep3B 2.1-7 tumor growth as compared with vehicle group  via inhibition of STAT-3 activation. Tai et al. also presented tumor size was significant diminished by regorafenib treatment (20 mg/kg/day) as compared with vehicle group in HCC PLC tumor bearing mice [luc2 and Hep3B 2.1-7 tumor bearing mice , MMP-9, VEGF, MCL-1, XIAP, C-FLIP, and Cyclin-D1 /mitogen-activated protein kinase kinase (MEK)/ERK and phosphoinositide 3-kinase (PI3K)/AKT signaling cascades both play critical roles in the progression of HCC. NF-κB activation can be regulated by P-ERK and AKT in cancer cells ,22. In pin vitro . In contin vitro  presentein vivo. We performed experiment on two different HCC bearing mice and suggested anticancer effect of regorafenib is mediated by the inhibition of ERK/NF-κB activation in HCC in vivo.In conclusion, the present study demonstrated regorafenib inhibited tumor progression through suppression of ERK/NF-κB activation in HCC


We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer.E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their ‘core’ cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners  in vivo, in the living mouse. To address this question we developed a system, which combines gene targeting in embryonic stem cells with high-throughput mass spectrometry. This approach allowed us to determine the identity of cyclin E protein partners, as well as phosphorylation substrates of cyclin E and its associated kinase, Cdk2, in different mouse organs. These analyses identified several novel, previously unanticipated interactors and phosphorylation substrates of cyclin E-Cdk2, and revealed that cyclin E plays distinct molecular functions in different compartments in vivo. This approach can be used to study the in vivo function of essentially any protein, in any model organism, and in any physiological and pathological state.The proliferation of mammalian cells is driven by proteins called cyclins, which bind and activate their catalytic partners, the cyclin-dependent kinases (Cdks). Cyclin-Cdk complexes drive cell proliferation by phosphorylating several cellular proteins. This study focuses on the E-type cyclins (cyclins E1 and E2). These proteins were shown to play important roles in cell cycle progression, and are often overexpressed in human cancers. It remained unclear, however, whether in addition to their well-established cell cycle roles E-type cyclins perform distinct tissue-specific functions  Kip1), centrosome duplication , histone biosynthesis (p220NPAT) and DNA replication , thereby driving cell proliferation -ATP. For inhibition of Cdk2 kinase activity, 20 μM CVT-313 was added to both reaction and washing buffer.GST-Lin9, GST-Lin37, GST-Lin52, GST-Lin54, GST-Mybl1 and GST-Dmrtc2, were constructed as described above. For kinase assays, GST proteins (1 μg) or histone H1  were incubated with recombinant cyclin E-CDK2  at 30°C for 30 min in 20 mM Tris-HCl pH 8.0, 1 mM EGTA, 10 mM MgCl2, 1 mM EGTA, 10 mM MgCl2, 0.5 mM DTT, 30 μg/ml digitonin, 5 mM GTP, 0.1 mM ATP, 0.1 mM N6-(phenethyl) ATPγS (Biolog), 1X phosphatase inhibitor cocktail I and II (Sigma), and 1X complete protease inhibitors, EDTA-Free (Roche)], as described previously [To generate an analog-sensitive (AS) version of Cdk2, we introduced a mutation in mouse Cdk2 cDNA that changes phenylalanine 80 to a glycine as described previously . Human eeviously . After tCdk2-/- mice were homogenized in Trizol (Invitrogen), and total RNA was extracted. Reverse transcription and qPCR were performed using SuperScript III SYBR Green One-Step kit . Data were normalized to Gapdh levels, and calculations were made based on the ΔΔCT method.Testes dissected from wild-type or Rb-/-p107-/-p130-/-) MEFs [All cells were cultured in DMEM with 10% fetal bovine serum (Sigma). Early passage wild type MEFs and TKO (/-) MEFs  . Approximately 250 μL of samples were loaded onto the Superdex size exclusion column in buffer .All statistical analyses were performed by using R or MATLAB.S1 Fig(A) Sections of adult brains, testes, spleens and thymuses, as well as of embryonic brains, stained for Ki67. (B) Quantification of the fraction of proliferating (Ki67-positive) cells from (A). (C) The levels of Ser807/811-phosphorylated pRB (Phospho-Rb), total pRB, and cyclin A2 in the five mouse organs. Tubulin served as a loading control.(TIF)Click here for additional data file.S2 Fig(A) Selected cyclin E1-interactors identified in our mass spectrometric analyses were verified by immunoprecipitating cyclin E1 from spleens of tagged cyclin E1 knock-in mice, followed by immunoblotting with the indicated antibodies. (B) Verification of the interaction between endogenous Cdk2 or endogenous wild-type cyclin E1 and selected novel interactors. (C) Verification of the interaction between cyclin E1 and selected novel interactors in human HeLa cells.(TIF)Click here for additional data file.S3 FigCdk2+/+/cyclin E1Ntag/Ntag (KI), and Cdk2-/-/cyclin E1Ntag/Ntag (Cdk2-/-) mice was gauged by immunoprecipitation with an anti-Flag antibody and immunoblotting with the indicated antibodies. (B) Ablation of Cdk2 does not trigger re-distribution of cyclin-bound Cdk4 and Cdk1. Cyclins D1, A2 or B1 were immunoprecipitated from lysates prepared from wild-type (Ctrl) or Cdk2-/- spleens and immunoblots were probed with the indicated antibodies. Whole, whole cell lysates. Tubulin served as a loading control.(A) The amount of cyclin E1-associated Cdk1, Cdk2, Cdk4 and Cdk5 in the thymuses of wild-type (Ctrl), (TIF)Click here for additional data file.S4 Fig-/-p107-/-p130-/- MEFs lacking pRB, p107 and p130 were immunoprecipitated with an anti-Lin37 antibody. The immunoprecipitates and 10% input (whole cell lysates) were resolved on SDS-PAGE gel and probed with indicated antibodies. (D) T98G cells were transfected with HA-tagged wild-type p130 (WT) or pan-phosphorylation mutant (PM), with or without HA-tagged Cdk2 (HA-Cdk2) and Myc-tagged cyclin E1 (Myc-Cyclin E1), as indicated. Cells were lysed 24 hrs post transfection and immunoprecipitated with an anti-Lin37 antibody or, for control, with IgG. Whole cell lysates (10% of input) and immunoprecipitates were resolved on a SDS-PAGE gel and analyzed by immunoblotting with the indicated antibodies.(A) Immunoprecipitation (IP) followed by re-IP-immunoblotting to demonstrate that cyclin E1, Cdk2 and the DREAM complex components are present within the same multi-protein complex. Protein lysates from wild-type (Ctrl) or KI mouse embryonic fibroblasts (MEFs) were immunoprecipitated with anti-Flag antibody, eluted with Flag peptide, re-immunoprecipitated with anti-p130 or -Lin9 antibodies, and then immunoblotted with the indicated antibodies. (B) The DREAM complex subunits were immunoprecipitated from human glioblastoma T98G cell extracts with the indicated antibodies and immunoblots were probed with the antibodies against p130, cyclin E1 and Lin9. (C) Extracts prepared from wild-type (WT) and triple-knockout (TKO) Rb(TIF)Click here for additional data file.S5 FigAmino acid sequences of human Lin proteins are shown. Peptide fragments that were detected by mass spectrometry are highlighted in yellow, and identified cyclin E-Cdk2 phosphorylation sites are labeled in red.(TIF)Click here for additional data file.S6 FigAmino acid sequences of human Mybl1 and Dmrtc2 are shown. Peptide fragments that were detected by mass spectrometry are highlighted in yellow, and identified cyclin E-Cdk2 phosphorylation sites are labeled in red. Note that the recombinant protein used to examine phosphorylation sites in Dmrtc2 was an N-terminal fragment protein (aa 1–201).(TIF)Click here for additional data file.S1 TableThis table contains six separate worksheets, first five of which show lists of proteins identified in LC-MS/MS analyses from knock-in (KI) or wild-type control (WT) embryonic brains (EB), spleens, testes, thymuses, and adult brains (AB), respectively. Proteins in the ‘core’ (Category 1) group are highlighted in green, and those in Categories 2 and 3 in yellow and blue, respectively. Each row lists protein name, gene name, STRING name (mouse and human), gene ID, the number of peptides found in KI (Peptides KI) and WT (Peptides WT), the number of detections across experiments for KI (KI detected) and for WT (WT detected), p-value tissue, ratio tissue, p-value all, and ratio all see . The las(XLSX)Click here for additional data file.S2 TableThis table contains three separate worksheets. The first shows the list of proteins present in the cyclin E1 interactome in all organs. For each cyclin E1- interactor, gene name is listed in the first column, and the major Gene Ontology term assigned to generate (XLSX)Click here for additional data file.S3 TableThis table contains six separate worksheets and lists Gene Ontology terms enriched among cyclin E1-interactors detected in the indicated organs. EB, embryonic brain; AB, adult brain.(XLSX)Click here for additional data file.S4 Tablecyclin E1Ntag/Ntag purification products (KI) versus mock purification (WT); (D) Ratio of the relative abundance of a given protein between Cdk2-/-/cyclin E1Ntag/Ntag purification products (Cdk2KO) versus cyclin E1Ntag/Ntag purification products (KI); (E) For each protein, Cdk2KO:KI ratio was normalized against the abundance of cyclin E1 in Cdk2KO and KI purification products.Each row corresponds to a different protein. Shown are: (A) Accession number; (B) Protein name; (C) Ratio of the relative abundance of a given protein between (XLSX)Click here for additional data file.S5 TableThe table lists the forward and reverse primers used for RT-qPCR .(XLSX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.