Autophagy is an essential process involved in the pathogenesis of inflammatory bowel disease (IBD). Although there are many data showing the roles of autophagy in intestinal epithelial cells (IECs), the mechanisms involved remain to be fully elucidated. We investigated the influence of autophagy in IECs on gastrointestinal tract inflammation.Atg5 in IECs (Atg5flox/flox/villin-Cre mice) were subjected to dextran sulfate sodium (DSS)-induced colitis and analyzed for colitis susceptibility. Additionally, we used Atg5-silenced rat IECs (IEC6shAtg5 cells) for in vitro assays.Mice with conditional knockout of Atg5flox/flox/villin-Cre mice compared to that in wild-type mice. In IEC6shAtg5 cells, apoptosis was enhanced, and cell viability significantly decreased compared to IEC-6 cells. The expression of proinflammatory cytokines increased upon suppression of autophagy. Furthermore, silencing of Atg5 was associated with inflammation of IECs, activation of the mitogen-activated protein kinase (MAPK) signaling pathway by the intracellular reactive oxygen species accumulation, and NF-κB p65 phosphorylation.Sensitivity to DSS markedly increased in Autophagy in IECs plays an essential role in the maintenance of intestinal homeostasis, and autophagy deficiency triggers inflammation. Development of methods targeting autophagy might be beneficial in the treatment of IBD. ATG16L1) and immunity-related GTPase family M (IRGM) genes, as genes associated with susceptibility to Crohn’s disease [Inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis (UC), is an intractable condition that causes chronic inflammation in the mucosa of the small and large intestine. Numerous studies have attempted to characterize the pathogenic mechanism of IBD. Interestingly, genome-wide association studies (GWAS) have identified the autophagy-related 16-like 1  production in g5 cells . This stBafilomycin (Cat. No. 88899-55-2) and MEK inhibitor (PD98059) (Cat. No. 1213) were purchased from Wako Pure Chemical Inc. . Lipopolysaccharides (Cat. No. L4130) and N-Acetyl-L-Cysteine (NAC) (Cat. No. 9165) were purchased from Sigma-Aldrich . 3-methyladenine (3-MA) (Cat. No. SC-205596) was purchased from Santa Cruz . NF-κB transcriptional activity inhibitor (JSH23) (Cat. No. ab144824) was purchased from Abcam .Atg5 allele were purchased from the Jackson Laboratory  and RIKEN BioResource Center , respectively. Male Atg5flox/flox/villin-Cre mice, which were IEC-specific Atg5 conditional knockout mice, were generated by crossing Atg5flox/+ mice and villin-Cre-expressing mice as described previously[Villin-Cre mice and mice with a floxed reviously.Atg5flox/+/villin-Cre, and Atg5flox/flox/villin-Cre mice, all aged 8–10 weeks, were given 2.5% DSS  in drinking water for six days, and plain drinking water afterward. The body weights of the mice were measured every day, and, on day eight after DSS administration, the mice were sacrificed. The histological scores of the Hematoxylin and eosin (HE)-stained rectal specimens were determined as described previously[To create colitis model mice (C57BL/6), wild-type (WT), IEC-6 cells, which are rat IECs, were obtained from RIKEN BioResource Center  and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Atg5 knockdown IEC-6 cells (IEC6shAtg5 cells) were generated as described previously. IEC6shATNF-α, IL-1β, IL-6, and GAPDH are depicted in To measure the expression levels of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α, qPCR was performed as described previously. BrieflyWestern blot analysis was performed as described previously. Briefly2O2). The cells were next incubated with Cell Counting Kit-8  for 1 h, and the absorbance in each well was measured at a wavelength of 450 nm using a microplate reader . Inhibition of viability was determined relative to IEC-6 cells without H2O2.treatment.Cells were plated in 96-well plates and incubated for 24 h with or without hydrogen peroxide  was used to evaluate the levels of oxidative stress in IEC-6 and IEC6shAtg5 cells before at-test for parametric data, which are presented as mean ± the standard deviation (SD). Non-parametric data  were analyzed using the Mann-Whitney test. Statistical significance was defined as p < 0.05.Statistical significance was evaluated using the Student’s Atg5 deficiency in IECs on colitis, we prepared DSS colitis models using WT, Atg5flox/+/villin-Cre, and Atg5flox/flox/villin-Cre mice. Atg5flox/+/villin-Cre mice exhibited the same weight loss as WT mice, but the weight loss in Atg5flox/flox/villin-Cre mice was significantly lower than that in WT mice (Atg5flox/flox/villin-Cre mice compared with controls (flox/flox/villin-Cre mice (control), no epithelial lesions were observed. Although loss of crypts and inflammatory cells infiltration to the lamina propria were observed in all mice after DSS administration, the severity was higher in Atg5flox/flox/villin-Cre mice than in WT mice. We next evaluated the histological scores indicating the extent of inflammation in the intestinal tissues. The histological score was significantly higher in Atg5flox/flox/villin-Cre mice (5.76±1.15) than in the WT (3.53±0.64) and Atg5flox/+/villin-Cre mice (3.81±0.72)  . Next, wCre mice . ConversTo further investigate the role of autophagy in IECs, we exploited IEC6shAtg5 cells. As we reported previously, autophagy in the cells was almost completely suppressed judging by the amount of the lipidated form of LC3, LC3-II. To conf2O2. As we previously reported that IEC6shAtg5 cells produced higher ROS than IEC-6 cells did [2O2 as an inducer of oxidative stress. Although the viability of IEC6shAtg5 cells was lower than that of IEC6 cells before H2O2 exposure, the difference was not significant. In contrast, the difference became significant upon H2O2 exposure (2O2 concentration (10 μM) (2O2 (100 μM). Furthermore, we also performed TUNEL assay to confirm that the susceptibility to oxidative stress might be due to apoptosis. The result showed that the number of TUNEL positive cells was significantly higher in IEC6shAtg5 cells compared with in IEC-6 cell  . PARP1 cC-6 cell . These fIL-1β and IL-6 mRNA expression levels were markedly elevated in IEC6shAtg5 cells compared to those in IEC-6 cells Click here for additional data file.


Scientific Reports 10.1038/s41598-019-42601-3, published online 03 May 2019Correction to: This Article contains an error in the order of the Figure legends. The legends for Figures 1 and 2 were incorrectly given as those for 2 and 1 respectively.The legend for Figure 1 should read:(A) MicroRNA database top predicted targets for microRNA hsa-miR-125b-5p. The number in the center of each circle represents target score of prediction. Full names of all genes are available at https://www.ncbi.nlm.nih.gov/gene.  Bioinformatics analysis of hsa-miR-125b-5p binding to 3′UTR of TRAF6 mRNA. (B) Seed-Matched sequences of hsa-miR-125b-5p in the 3′UTR of TRAF6 mRNA. (C) Target Scan prediction. (D) Cross-species conservation by Target Scan algorithm.”“The legend for Figure 2 should read:(A) Smooth and damaged cartilage tissues from OA patients (B) Expression of hsa-mir-125b-5p in smooth and damaged cartilage tissues. #p < 0.01 versus smooth cartilage tissue. (C) Gene Expression of TRAF6 in smooth and damaged cartilage tissues. @p < 0.01 versus smooth cartilage tissue. (D) Protein expression of TRAF6 in smooth and damaged cartilage tissues (full length blot is provided in Supplementary Information File). *p < 0.01 versus SC. SC, smooth cartilage; DC, damaged cartilage. Band images were digitally captured by the Un-Scan-It software and the band intensities of TRAF6 proteins were divided by band intensities of β-actin and are expressed in average pixels.”“Expression of hsa-miR-125b-5p and TRAF6 in smooth and damaged OA cartilage tissues.


Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.  Ebola virus (EBOV) is in the family Filoviridae within the order Mononegavirales . The vir1,2, VP30, and VP24 [The negative-sense, single-stranded RNA (ssRNA) genome of EBOV is approximately 19 kb and encodes seven genes: Nucleoprotein (NP), virion protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and the RNA-dependent RNA polymerase (L) [and VP24 ,14,15,16and VP24 . Besidesand VP24 ,19,20,21and VP24 . In addiand VP24 . Beyond and VP24 .NP is the critical protein that drives the assembly of the nucleocapsid (NC) ,26,27. IIn the current study, we aimed to further elucidate NP biology by identifying NP-host PPIs. We used affinity purification-mass spectrometry (AP-MS) to identify candidate NP cellular interactors, including RUVBL1 and RUVBL2. RUVBL1 and RUVBL2 interactions were verified by co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Furthermore, the utilization of an EBOV minigenome system showed that the siRNA-mediated knockdown of RUVBL1, but not RUVBL2, modestly decreased minigenome activity. In addition, the overexpression of RUVBL1 and RUVBL2 in the EBOV minigenome showed no effect on minigenome activity. Super resolution structured illumination microscopy (SIM) was then used to confirm the colocalization of NP with RPAP3 and PIH1D1, two other components of the RUVBL1/2-containing R2TP complex. The siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 expression also had no effect on minigenome activity, instead suggesting a role in capsid assembly. Taken together, we identify RUVBL1/2 as novel interactors of EBOV NP and show recruitment of the R2TP complex, which may offer novel therapeutic targets for EBOV.2 incubator. A conventional calcium phosphate transfection was used for initial AP-MS transfections. Subsequent experiments used a jetPRIME transfection reagent from Polyplus-transfection according to manufacturer’s recommendations .HeLa cells and HEK293T cells from the American Type Culture Collection  were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% COpCAGGS-HA-EBOV-NP was purchased from BEI (NR-49343). pCAGGS-NP-V5, pCAGGS-V5-VP30, and pcDNA3 vectors were generated at the USAMRIID. pCAGGS-FLAG-VP35 was a kind gift from Dr. Christopher F. Basler (Georgia State University). pCDNA-3xFLAG-Pontin and pCDNA-3xHA-Reptin were gifts from Dr. Steven E. Artandi  . pCAGGS_Mouse anti-HA (H3663), rabbit anti-HA (H6908), mouse anti-FLAG (F3165), rabbit anti-FLAG (F7425), mouse anti-V5 (V8012), rabbit anti-V5 (V8137), mouse anti-RPAP3 (WH0079657M1), rabbit anti-GAPDH (SAB2108668), and mouse anti-Calnexin (C7617) antisera were purchased from Sigma-Aldrich . Rabbit anti-PIH1D1 (ab238862) antisera was purchased from Abcam . Rabbit anti-RUVBL1 (NBP2-20245) and rabbit anti-RUVBL2 (NBP1-40354) antisera were purchased from Novus Biologicals . Goat anti-HA (A00168) antisera was purchased from GenScript . Goat anti-rabbit HRP (65-6120), goat anti-mouse HRP (32430), goat anti-mouse Alexa Fluor 488 (A32723), goat anti-rabbit Alexa Fluor 568 (A11011), donkey anti-goat Alexa Fluor 647 (A21447), and Hoechst 33342 (H3570) were purchased from ThermoFisher Scientific . Silencer Select Negative Control No. 1 siRNA (4390843), Silencer Validated siRNA RUVBL1 (AM1331), Silencer Pre-designed siRNA RUVBL2 (AM16704), and Silencer Pre-designed siRNA RPAP3 (AM16708) were purchased from ThermoFisher Scientific. The Dual-Glo Luciferase Assay System (E2920) was purchased from Promega  and used according to manufacturer’s recommendations.p-values less than 0.01) were used to identify unknown host proteins.Affinity purified material and the empty vector control were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10–20% gradient polyacrylamide gels (ThermoFisher Scientific). Protein was visualized by Imperial Coomassie blue staining (ThermoFisher Scientific), and equal sized sections from the affinity purified sample were further analyzed by liquid chromatography/mass spectrometry (LC/MS/MS). Equal sized gel pieces were excised from this immune-precipitated sample and destained, dehydrated, and reductively alkylated by incubating samples in 2.5 mM DTT for 1 h at 60 °C and adding iodoacetamide to 10 mM for 1 h incubation in the dark. Samples were in-gel digested with trypsin during overnight incubations at 37 °C and individual digestates analyzed directly by LC/MS/MS. All LC/MS/MS experiments were conducted on an AmaZon ion trap mass spectrometer from Bruker Daltonics  and interfaced with a nanoflow HPLC system . Tryptic peptides were separated under nanoflow conditions (400 nL/min) using a trap loading column  and resolved with a linear acetonitrile gradient  on a monolithic c18 reverse phase column . Tandem MS/MS data was acquired using data dependent acquisition by fragmenting the 4 most abundant ions in each spectral scan by collision induced dissociation . Raw data was deconvoluted by an automated charge state and mass assignments using Bruker Daltonics software , and the MASCOT algorithm was used to search the SwissProt database (species: Homo Sapiens). Statistical cutoffs (6 cells) were transfected with the indicated plasmids using 3 µL of the transfection reagent jetPRIME (Polyplus) per 1 µg DNA per manufacturer’s instructions. The total amount of DNA for each transfection was kept constant in each experiment by complementing with empty vector. Twenty-four hours post-transfection, cells were lysed in a modified RIPA buffer  containing protease and a phosphatase inhibitor cocktail . A portion of ~10% whole cell lysate was reserved, and immunoprecipitation was performed using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma), EZview Red Anti-HA Affinity Gel (Sigma), or Anti-V5 Agarose Affinity Gel (Sigma) according to manufacturer’s recommendations. After 1 h of bead incubation at 4 °C, the beads were left untreated or treated with 20 mg/mL of RNase A (Qiagen) and incubated at 37 °C for 30 min. Beads were washed 4 to 5 times with TBS, re-suspended in 2X Lane Marker Reducing Sample Buffer (Thermo Scientific), and then subjected to SDS-PAGE and immunoblotting.HeLa cells  was used to visualize protein on an Azure c600 imaging system.5 cells) were grown on Fisherbrand Microscope Coverglass slides (Fisher Scientific) and transfected with the indicated plasmids using jetPRIME, according to manufacturer’s instructions. Vector controls were included for endogenous samples in the absence of HA-NP. Twenty-four hours post-transfection, cells were fixed in 4% PFA, permeabilized in 0.01% Triton X-100 (Fisher Bioreagents), and blocked in 3% bovine serum albumin (Fisher Bioreagents). The primary antibodies mouse anti-HA or goat anti-HA, rabbit anti-FLAG, rabbit anti-V5, rabbit anti-RUVBL1, rabbit anti-RUVBL2, mouse anti-RPAP3, and rabbit anti-PIH1D1 were used along with the secondary antibodies goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 568, and donkey anti-goat Alexa Fluor 647. Slides were probed with primary antibodies either for 1–2 h at room temperature or overnight at 4 °C. Secondary incubations were performed for 1–2 h at room temperature. Hoechst 33342 was used for a nuclear stain. Slides were mounted using Fluoromount Aqueous Mounting Medium (Sigma). Colocalization images were collected by a Zeiss LSM800 confocal microscope (Carl Zeiss MicroImaging). SIM images were collected with a Zeiss ELYRA PS.1 illumination system (Carl Zeiss MicroImaging), and SIM processing was performed with the SIM module of Zen BLACK software (Carl Zeiss MicroImaging).HeLa cells (1 × 105) were seeded in 12-well plates and transfected in suspension with 30 nM of siRNA against RUVBL1, 50 nM of siRNA against RUVBL2, a combination of siRNA against RUVBL1 and RUVBL2, or 80 nM of Silencer Select Negative Control No. 1 siRNA. For RPAP3 knockdown experiments, 30 nM of siRNA against RPAP3 was used, along with a siRNA control of 30 nM of Silencer Select Negative Control No. 1. The siRNA concentrations used were determined by previous optimization experiments. The minigenome assay was performed 24 h post-siRNA transfection using a RNA polymerase II-driven EBOV minigenome as previously described [Renilla luciferase values and plotted as fold activity calculated relative to the no L control. Standard error of the mean (SEM) values and paired, two-tailed t tests were calculated using GraphPad Prism 7.05 software . HeLa cells ; empty vector, transfected control cells were also processed by identical AP procedures. The control and EBOV NP samples were then visualized by SDS-PAGE A. To ideTo validate candidate interactors of NP, we performed co-IPs. The NP candidate interactors selected for further investigation were RUVBL1 and RUVBL2. RUVBL2 was recently reported as a potential but unconfirmed NP interactor in a MS study . RUVBL1/To validate further the mass spectrometry and IP results, we used confocal microscopy to examine the localization of endogenous RUVBL1 and RUVBL2 in the absence or presence of HA-NP. HeLa cells were transfected with vector control or HA-NP. In the absence of HA-NP, both RUVBL1 and RUVBL2 showed strong nuclear localization as has been previously shown ,48  E1A was recently identified to be important for suppression of interferon response . RUVBL2 The identification of novel interactions between NP and RUVBL1/2 and the association observed between NP and the R2TP complex suggest potential novel therapeutic targets of EBOV. RUVBL1 and RUVBL2 are related AAA+ proteins that often exist in complex with each other. In fact, their ATPase activity has been shown to require the catalytic activity of both in complex, with ATP hydrolysis synergistically increasing upon hetero-oligomerization compared to that of the constituent proteins ,78. BecaComparing interactor candidates with two previous studies that used similar AP-MS approaches revealed an overlap of seven interactions including TROVE2, TUBA1B, TUBB2C, IGF2BP1, PDHB, RUVBL2, and RUVBL1 ,22. HoweIn recent years, more cellular interactors of EBOV NP have been identified ,22,23. T


Several studies have established a correlation between the VEGF–VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers.We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry.+ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA−FOXP3highCD4+ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8+ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2+ eTreg cell proliferation, and this effect could be inhibited by RAM.Within the tumor microenvironment, both PD-L1 expression and CD8This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.The online version of this article (10.1186/s40425-018-0403-1) contains supplementary material, which is available to authorized users. Recent advances in cancer immunotherapy, including the development of immune checkpoint blockades (ICBs), such as those targeting PD-1, PD-L1, and CTLA-4, have resulted in a paradigm shift in cancer treatment. In fact, the U.S. Food and Drug Administration has granted approvals for ICB use across several tumor types . The capRamucirumab (RAM), a fully humanized IgG1 monoclonal anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody, has been shown to exhibit significant efficacy as an anti-angiogenic agent in several cancers, including non-small cell lung cancer (NSCLC), gastric cancer (GC), colorectal cancer, and bladder cancer –15. FurtIn this study, we prospectively enrolled patients with advanced GC who were scheduled to receive RAM-containing chemotherapy between January 2016 and August 2016. In addition, we obtained pre- and post-treatment paired blood and primary tumor samples by endoscopic biopsy to assess immune profiles  which allowed to detect gene mutations, copy number variants and fusions across multiple genes  mAb, anti-mutS homolog 2 (MSH2) mAb, anti-postmeiotic segregation increased 2 (PMS2) mAb, and anti-mutShomolog 6 (MSH6) mAb using archived FFPE tumor samples. Tumors were considered negative for MLH1, MSH2, PMS2, or MSH6 expression only if there was a complete absence of nuclear staining in the tumor cells, and normal epithelial cells and lymphocytes were employed as an internal control. Tumors lacking MLH1, MSH2, PMS2, or MSH6 expression were defined as MMR deficient, whereas tumors that maintained expression of all markers were considered MMR proficient.Chromogenic in situ hybridization (ISH) for EBV-encoded RNA was performed with fluorescein-labeled oligonucleotide probes (INFORM EBER probe) with enzymatic digestion  and an iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system. Archived FFPE tumor samples were used for the analysis.Biopsy specimens were formalin-fixed, paraffin-embedded, and sectioned onto slides for IHC, which was conducted using monoclonal antibodies against PD-L1 and CD8 using an automatic staining instrument . PD-L1 positivity was defined as staining in ≥1% in tumor cells or immune cells. CD8 staining intensity was assessed as follows: no staining (0), weak staining (1+), moderate staining (2+), and strong staining (3+). Two researchers (D. Kotani and T. Kuwata) independently evaluated the stained slides. Multiplexed fluorescent immunohistochemistry was performed by the TSA method using an Opal IHC kit  according to the manufacturer’s instructions. Multiplexed fluorescent labeled images of three randomly selected fields (669 × 500 micrometer) were captured with an automated imaging system .We washed cells in PBS with 2% fetal calf serum and then stained with mAbs specific for CD3, CD4, CD8, CD45RA, PD-1, LAG-3, ICOS, VEGFR2, or CTLA-4 as well as a fixable viability dye. In addition, intracellular Ki67 and FOXP3 staining was performed with an anti-FOXP3 mAb and FOXP3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. After washing, we analyzed all cells with the LSRFortessa instrument (BD Biosciences) and FlowJo software . Antibody dilutions were performed per the manufacturer’s instructions.Alexa Fluor 700–conjugated anti-CD3 (UCTH1) mAb, violet 500-conjugated anti-CD4 (RPA-T4) mAb, and Brilliant Violet 421 (BV421)–conjugated anti-PD-1 (EH12) mAb were obtained from BD Biosciences . BV785-conjugated anti-CD8 (RPA-T8) mAb, BV711-conjugated anti-CD45RA (HI100) mAb, and APC-conjugated anti-CTLA-4 (L3D10) mAb were purchased from BioLegend . Phycoerythrin (PE)-conjugated anti-FOXP3  mAb, PE- and Cy7-conjugated anti-ICOS (3D12) mAbs, peridinin–chlorophyll–protein complex (PerCP)- and Cy5.5-conjugated anti-Ki67 (20Raj1) mAb, and eFluor 780-conjugated fixable viability dye were obtained from eBioscience . Fluorescein isothiocyanate (FITC)–conjugated anti-LAG-3 (17B4) mAb was obtained from ENZO Life Science . Anti-CD4 (4B12) mAb, anti-Ki67 (MIB-1) mAb, anti-MLH1 (ES05) mAb, anti-MSH2 (FE11) mAb, anti-PMS2 (EP51) mAb, and anti-MSH6 (EP49) mAb for IHC were obtained from Dako . Anti-PD-L1 (SP263) mAb and anti-CD8 (SP57) mAb for IHC were obtained from Ventana. Anti-CD3 (SP7) mAb and anti-FOXP3 (236A/E7) mAb for IHC was purchased from Abcam; RAM was obtained from Eli Lilly and Company ; and VEGFA was obtained from Wako . In addition, APC was conjugated to RAM to detect VEGFR2.t-tests to analyze continuous variables. All survival curves were estimated using the Kaplan–Meier method and compared using the log-rank test. All statistical analyses were two-tailed and were performed using Prism version 7 software . P < 0.05 was considered statistically significant.We used Welch’s or paired A total of 20 patients  with advanced GC and with available pre- and post-treatment samples that were scheduled to receive RAM-containing chemotherapy were enrolled in this study  and ERBB2, MET, FGFR2, or KRAS, amplification, or a RHOA mutation were also identified, which was in line with a previous study . Fu. Fu+ T c+ T cells was significantly reduced in TILs after RAM-containing therapies , we observed no trends in the eTreg kinetic changes . Although not significant, patients with PR tended to have a lower PD-1 expression by CD8+ T cells in pre-treatment TILs than those with PD  proliferated . This indicates a probable correlation between CD8+ T cells and PD-L1 expression. VEGF is related to the immunosuppressive microenvironment in that it inhibits T-cell proliferation and infiltration; this inhibition can be reversed by anti-angiogenic reagents, including RAM [+ T-cell infiltration and PD-L1 expression were induced by RAM-containing therapies. In addition, TIL eTreg cells and PD-1 expression by CD8+ T cells are decreased by RAM-containing therapies. To the best of our knowledge, this is the first study to demonstrate that an anti-angiogenic reagent can cause an increase in CD8+ T-cell infiltration and PD-L1 expression and a decrease in eTreg cells and PD-1 expression by CD8+ T cells in the tumor microenvironment in humans. PD-1 expression by TIL CD8+ T cells was decreased by RAM-containing therapies, which is in line with findings in an animal model demonstrating the effects of VEGFA/VEGFR2 on the expression of IC molecules [Recent studies have demonstrated that cancer immunotherapies, particularly those that modulate immune co-inhibitory signals, can be beneficial for patients with various types of cancers –7. Howevesponses , 34. Intding RAM , 36. Botolecules . Furtherolecules . Thus, tolecules .While systemic chemotherapy is the primary treatment option for patients with unresectable advanced GC, which is the fifth leading cancer worldwide , their p+ T cells suggests the importance of analyzing TILs, in which T cells directly attack tumor cells. The decrease of eTreg cells and PD-1 expression by CD8+ T cells in TILs in some PR patients may be a reflection of reduced tumor burdens, whereas this tendency was also observed at PD state, suggesting that RAM-containing therapies can reduce eTreg cells and PD-1 expression by CD8+ T cells in TILs independently of clinical responses. eTreg cells are a highly immunosuppressive subset of CD4+ T cells characterized by high expression of the master regulatory transcription factor FOXP3 [We employed flow cytometry to investigate the detailed immune profiles of hosts, where significant differences exist between PBMCs and TILs. The RAM-containing therapy-induced reduction in TIL eTreg cells and PD-1 expression by CD8or FOXP3 –46. Our Myeloid cells including myeloid-derived suppressor cells in tumors has been implicated in suppression of antitumor immunity , and VEG+ Treg cells, thus corroborating the results of our study [+FOXP3+CD4+ T cells were defined as “Treg cells” in the previous study, we defined these more precisely as “eTreg cells” (CD45RA−FOXP3highCD4+ T cell). These eTreg cells have a suppressive function, while non-Treg CD45RA−FOXP3lowCD4+ cells, which produce inflammatory cytokines such as IFN-γ are included in the CD25+FOXP3+CD4+ T cell population. Thus, our reported “eTreg cell” frequency in PBMCs was lower than the previously reported “Treg cell” frequency in PBMCs.A previous study reported that Treg cells express high levels of VEGFR2 and that RAM therapy could reduce the frequency of VEGFR2ur study . Anotherur study , althougur study  could po+ T-cell infiltration were increased following RAM-containing therapy in patients with advanced GC. Based on more detailed immune profiles, both TIL eTreg cells and PD-1 expression by CD8+ T cells were decreased by RAM-containing therapies, an effect not observed in PBMCs. Further, high eTreg frequencies in TILs correlate with favorable responses to RAM-containing therapies. Thus, our study is the first to utilize human clinical samples to highlight the significance of TIL analyses and to propose RAM as an immuno-modulator in combination with ICB.We observed a significant difference in immune profiles between PBMCs and TILs and determined that PD-L1 expression and CD8Additional file 1:Table S1. Gene list of the Oncomine™ Cancer Research Panel (OCP143). (DOCX 18 kb)Additional file 2:Table S2. Patient characteristics. (DOCX 19 kb)Additional file 3:Table S3. Detailed clinical characteristics and immune cell data. (DOCX 21 kb)Additional file 4:Figure S1. Genomic features of patients with GC who received RAM-containing therapies. (DOCX 137 kb)Additional file 5:Figure S2. Difference in patient characteristics according to eTreg-cell frequency in TIL. (DOCX 129 kb)Additional file 6:Figure S3. Kinetic changes of eTreg cells and PD-1 expression by CD8+ T cells according to therapies in TILs. (DOCX 242 kb)Additional file 7:Figure S4. Kinetic changes of CD45RA−FOXP3−CD4+ T cells and eTreg cells in CD3+ T cells. (DOCX 137 kb)Additional file 8:Figure S5. Kinetic changes of IC molecule expression by CD8+ T cells in both PBMCs and TILs. (DOCX 203 kb)Additional file 9:Figure S6. Kinetic changes of IC molecule expression by CD45RA−FOXP3−CD4+ T cells in both PBMCs and TILs. (DOCX 208 kb)Additional file 10:Figure S7. Kinetic changes of IC molecule expression by eTreg cells in both PBMCs and TILs. (DOCX 108 kb)Additional file 11:Figure S8. Comparison of IC expression by eTreg cells between pre-and post-treatment in both PBMCs and TILs. (DOCX 240 kb)Additional file 12:Figure S9. % of eTreg-cell reduction and % of PD-1 reduction on CD8+ T cells and clinical responses. (DOCX 77 kb)Additional file 13:Figure S10. Impact of anti-VEGFR2 blockade on PBMCs in vitro. (DOCX 266 kb)


Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1β, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection. Innate and adaptive immune activation is one of the strongest predictors of HIV disease progression to AIDS and non-AIDS morbidity and mortality. Type I interferon (IFN-I) signaling is a major driver of such activation even in ART-treated people. Therefore, manipulation of IFN-I signaling has been proposed as a therapeutic approach to improve HIV disease outcome. However, we previously reported that blockade of IFN-I signaling in rhesus macaques during acute SIV infection results in increased SIV reservoir size, accelerated CD4 T cell depletion and faster progression to AIDS. Our present study addresses the more clinically relevant effects of IFN-I signaling blockade in chronic SIV infection during treatment with antiretroviral therapy. We found that administration of a novel, long half-life, PASylated IFN-I receptor antagonist suppressed IFN-I-related inflammatory pathways but, in contrast to the case of acute SIV infection, did not result in loss of control of SIV replication. Thus, our findings provide a rationale for the safe and effective use of such interventions to reduce inflammation in HIV-infected people during ART. Persistent inflammation during chronic HIV infection is a central contributing factor to immune exhaustion, CD4 T cell depletion and progression to AIDS –3. PreviMAC251 challenge, 25 rhesus macaques initiated daily ART while 10 remained untreated  and 6 IFN-1ant-treated animals (group 2) received 3x weekly injections from week 16 to week 24 and had no significant difference in plasma virus load (VL) before administration of the antagonist  whereas a 3x per week regimen did not result in significant changes , pre-IFN-1ant (week 14), during IFN-1ant  and post-IFN-1ant (week 26). For this purpose, we chose a set of 19 ISGs previously demonstrated to be reduced by IFN-1ant treatment during acute SIV infection . As meas changes . Overall changes , suggestTo assess the risk that blocking type I IFN signaling could pose for the control of SIV replication, we measured plasma VL during and after antagonist treatment. In the absence of ART, there was no significant difference between the plasma VL of animals receiving antagonist or placebo from antagonist administration at week 16 p.i. up to week 50 post-infection . In the + animals or the plasma virus loads of animals that were CMV infected prior to antagonist administration  and effector memory (EM) cells. While some EM cells express CCR5 [+ cells were gated prior to EM in order to measure virus DNA in the total fraction of cells expressing CCR5 given its role SIV infection. As expected, ART initiation at week 8 significantly reduced the amount of SIV gag DNA copies measured in all cell subsets in all animals in each experimental group (gag DNA copies in CCR5+ T cells only (P = 0.02). However, administration of 2x or 3x weekly IFN-1ant did not show significant effects on CM, EM or total SIV gag copies. In the absence of ART, SIV gag DNA copies remained high from week 8 through 24 in PB cell subsets, with no difference between antagonist and placebo groups. In the LN, CD4 T cells were sorted into CM, EM, germinal center follicular helper (GC Tfh) and non-germinal center Tfh (non-GC Tfh) subsets. After ART initiation, the amount of SIV gag DNA copies continuously decreased up to week 50 p.i. in all LN subsets in all animals, with no effect of the antagonist treatment given from week 16 to week 24 . At week 8 post-infection, 30 animals were started on antiretrovirals while 10 animals were left untreated. The ART regimen was subcutaneous injections of 20mg/kg/day Tenofovir and 30mg/kg/day Emtricitabine (Gilead), as well as orally administered drugs including 100mg BID Raltegravir (Merck), 800mg BID Darunavir  and 100mg BID Ritonavir (Abbvie), all given mixed with food. To investigate the effect of blocking IFN-I signaling, infected animals received i.m. injection of 3.5mg per injection given 2 or 3 times per week with a type I interferon receptor antagonist (IFN-1ant) used previously [E. coli according to a published procedure [in vivo administration of the antagonist to healthy macaques, the chosen regimen of 2 and 3 times per week should result in minimum plasma concentrations of 5 nM and 10–20 nM respectively  were socially housed at the National Institutes of Health with oversight from facility behavioral management staff. Primary enclosures consisted of stainless steel primate caging provided by a commercial vendor. Animal body weights and cage dimensions were regularly monitored. Overall dimensions of primary enclosures (floor area and height) met the specifications of The Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Regulations (AWR's). All primary enclosures were sanitized every 14 days at a minimum, in compliance with AWRs. Secondary enclosures (room level) met specifications. All animals were housed under controlled conditions of humidity, temperature and light (12-hour light/12-hour dark cycles). Animals were fed commercial monkey chow, twice daily, with supplemental fruit or other produce at least three times per week. Filtered water was available ad libitum. Animals were observed at least twice daily by trained personnel, including behavioral assessments. Environmental enrichment included provision of species appropriate manipulatives, and foraging opportunities, as well music and video watching opportunities multiple times per week. No adverse events have been associated with study interventions. For procedures requiring chemical immobilization and sedation, different anesthetics were used at the discretion of the attending veterinarian according to the IACUC approved protocol. Prior to immunization, drug treatments or blood draws, anesthetics included Ketamine Hydrochloride 5.0–25.0 mg/kg IM with xylazine 0.5–1.0 mg/kg. For technical procedures, Buprenorphine Hydrochloride 0.015 mg/kg was administered. For euthanasia according to endpoints specified in the IACUC approved protocol, animals were initially sedated with ketamine  followed by lethal overdose of sodium pentobarbital to effect.Animal use and all study procedures  were approved by the Vaccine Research Center (VRC) Animal Care and Use Committee (ACUC), meeting National Institutes of Health standards; and in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines, all federal, state, and local regulations, and in compliance with Plasma was separated from EDTA blood by centrifugation and PBMCs were isolated by density centrifugation using Ficoll-Paque Plus  and Leucosep Centrifuge Tubes (Grenier Bio-One). Lymph nodes (LN) were collected into RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin-Glutamine (Gibco) and cell suspensions were passed through a 70μm filter to remove debris.-1, Sigma Aldrich) for SIV gag qPCR. For assessment of T cell activation and exhaustion, cryopreserved PBMC were thawed and stained with fluorochrome-labelled mAbs anti-CD38-FITC (Stem Cell), anti-Ki67-CY7PE, anti-CD28-CY5PE, anti-CD3-CY7APC (BD Biosciences), anti-HLA-DR-TRPE (Life Technologies), anti-PD-1-BV711, anti-CD95-BV785 (Biolegend), anti-TIGIT-APC (ThermoFisher) and anti-LAG3-PE (R&D). All samples were stained with Aqua LIVE/DEAD Fixable Dead Cell Stain.For CD4 T cell subsets sort, PBMC were stained with fluorochrome-labelled mAbs anti-CD28-CY5PE, anti-CCR5-PE, anti-CD3-CY7APC, anti-CD4-BV605, anti-CD8-Pacific blue (BD Biosciences) and anti-CD95-BV785 . LN cells were stained with anti-CD28-CY5PE, anti-CD3-CY7APC, anti-CD4-BV605 (BD Biosciences), anti-CD8-BV570, anti-CXCR5-PE (eBioscience) and anti-CD95-BV785 . PBMC and LN CD4 subsets of interest were sorted and lyzed in proteinase K  according to manufacturer’s instructions. Isolated DNA was then analyzed using an Applied Biosystems QuantStudio Real-Time PCR system (12K Flex) with Cytomegalovirus (CMV) specific primers  and a TaqMan Probe (6FAM CCT TCC CGG CTA TGG MGBNFQ). Each sample was run in triplicate for 40 cycles along with positive controls. Copy number was calculated by comparison to a standard curve and the viral load was reported as CMV copies/mL of plasma.RNA was extracted from cryopreserved PBMCs using RNAzolRT (Molecular Research Center) according to the manufacturers’ instructions. Purified RNA was used for transcriptome analysis. Briefly, polyadenylated transcripts were purified on oligo-dT magnetic beads, fragmented, reverse transcribed using random hexamers and incorporated into barcoded cDNA libraries based on the Illumina TruSeq platform. Libraries validated by microelectrophoresis were sequenced on an Illumina HiSeq 4000 in 100-base single-read reactions. RNA-seq analysis was conducted at the Yerkes Nonhuman Primate Genomics Core Laboratory. Estimates of gene-wise and isoform-wise expression levels for individual genes were performed using DESeq2 . The RNAwww.broadinstitute.org/gsea/). GSEA was performed on the ranked transcript lists using 1,000 phenotype permutations, and random seeding. Gene sets used included the MSigDB (http://software.broadinstitute.org/gsea/msigdb/collections.jsp) H , C5 (GO), C2 (curated), C7 (immunologic) gene sets  Plasma concentrations of IFN-I as measured by ELISA in healthy macaques following a single i.m. injection with either 1 mg (49.8 nmol) unmodified IFN-1ant (N = 4) or 3.5 mg (51.5 nmol) of PASylated IFN-1ant . Based on PK parameters measured for unmodified IFN-1ant and its PASylated version, plasma concentration profiles were simulated for both proteins according to a 2 or 3 times per week dosing schemes; showing a minimum plasma concentration of 5nM in a 2x per week regimen (b), 10-20nM in a 3x per week regimen (c) and both superior to the unmodified antagonist.((TIF)Click here for additional data file.S2 Fig10 Plasma SIV RNA levels at weeks 4–5, 8 and 14 p.i. in animals treated from week 16–24 p.i. with placebo saline or PASylated IFN-1ant 2 times per week (IFN-1ant 2x) or 3 times per week (IFN-1ant 3x). ART-treated animals started antiretrovirals on week 8 p.i. Horizontal lines indicate medians.Log(TIF)Click here for additional data file.S3 FigP values were calculated by unpaired t test.Fold change of the median read counts of the 19 ISGs in ART-treated and ART-untreated macaques from week 6 to week 14 p.i. Error bars indicate lower and upper quartiles. (TIF)Click here for additional data file.S4 Fig10 Plasma SIV RNA levels measured from SIV challenge at baseline up to 25 weeks after administration of PASylated IFN-1ant. ART-untreated animals received placebo saline (n = 4) or 3 times weekly IFN-1ant injections (n = 6). In ART-treated animals, antiretroviral treatment was initiated at week 8 p.i. and animals received placebo saline (n = 9), IFN-1ant injections 2 times weekly  or 3 times weekly . Shading indicates IFN-1ant treatment period from week 16 to week 24 p.i.Log(TIF)Click here for additional data file.S5 Figa) PBMC CD4 T cells were sorted into CCR5+ defined as Aqua-CD3+CD8-CD4+CCR5+, central memory (CM) defined as Aqua-CD3+CD8-CD4+CCR5-CD28+CD95+ and CCR5- effector memory (EM) defined as Aqua-CD3+CD8-CD4+CCR5-CD28+CD95-. (b) LN CD4 T cells were sorted into effector memory (EM) defined as Aqua-CD3+CD8-CD4+CD28+CD95-, central memory (CM) defined as Aqua-CD3+CD8-CD4+CD28+CD95+CXCR5loPD-1lo, germinal center T follicular helper (GC Tfh) defined as Aqua-CD3+CD8-CD4+CD28+CD95+CXCR5hiPD-1hi and non-GC Tfh defined as Aqua-CD3+CD8-CD4+CD28+CD95+CXCR5intPD-1int.((TIF)Click here for additional data file.S6 Fig2 transformed  and normalized to the Week 14 median expression level of each transcript. Statistical significance of differences between Week 14 and Week 19 expression levels for each transcript was determined using negative binomial generalized linear models (DESeq2).Heatmaps indicate expression of select genes associated with T cell activation, or exhaustion/inhibition at time points prior to (Week 14 post-infection) and following (Week 19 post-infection) IFN-1ant treatment. Heatmap colors represent gene expression levels that were log(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Endoplasmic reticulum (ER) stress causes an adaptive response initiated by protein kinase RNA-like ERkinase (PERK), Ire1 and ATF6. It has been reported that these upstream regulators induce microRNAs. The currentstudy was designed to find a novel microRNA that mediates ER stress components and finally contributes to cell fatedecision. In this experimental study, miR-706 levels were checked under different conditions ofER stress induced by Thapsigargin, Tunicamycin or low glucose media. PERK and ATF4 were knocked-downby administration of lentivirus-mediated short hairpin RNA to explore the effect of ER stress related proteins onmiR-706 expression. The effect of miR-706 on caspase activity and apoptosis inhibitor 1 (CAAP1) levels wereexamined by using mimic-miR-706. The role of CAAP1 in inhibiting cell death (measured by Annexin V staining)and contributing to patient overall survival (measured by Kaplan-Meier estimate) were further confirmed by anti-miR-706 and CAAP1 knock-down. We showed that Thapsigargin or Tunicamycin triggered ER stress leading to the induction of miR-706.miR-706 induction is dependent on PERK and its downstream regulator ATF4, as knocking-down of PERK and ATF4suppressed miR-706 induction in response to ER stress. Knocking-down of miR-706 reduces cell death triggered byER stress, indicating that miR-706 is pro-cell death microRNA. We further identified CAAP1 as a miR-706 target inregulating ER stress initiated cell death.Collectively, our results pointed to an ER signaling network consisting of proteins, microRNA and noveltarget. Unfolded protein response (UPR) or endoplasmicreticulum (ER) stress is a signaling pathway elicitedin response to various stimuli such as hypoxia duringlow oxygen pressure in tumors, virus infection andother stresses which disturb cell homeostasis -3.DurinPERK activation leads to translational suppressionvia eIF2 alpha serine 51 phosphorylation and this causesATF4 production . ATF4 ismicroRNAs (miR) are almost 22 nucleotides RNAs coding no protein. However, they perform diversefunctions in a variety of biological processes by posttranscriptional regulation of gene expression -24.ReguIn this experimental study, NIH3T3 mouseembryonic fibroblasts  andAML12 mouse hepatic cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10%fetal bovine serum  at 37˚C ina humidified atmosphere of 5% CO2. The cells werepassaged every two days at 1:6 ratios. The cells weretreated with 500 nM Thapsigargin  or 100 ng/ml Tunicamycin  unless otherwise noted. Human lung cancer cellline H1299 , human ovarian cancerline SKOV3 , human ovarian surfaceepithelial cell line HOSE  andhuman lung fibroblast HFL1  cellswere cultured in RPMI 1640  with10% fetal bovine serum.Short hairpin RNA targeting PERK, ATF4 or CAAP1from Dharmacon (USA) was transfected together withpackaging plasmids  into 293T cells by Lipofectamine 2000. For lentiviral transduction,NIH3T3 cells were incubated with 2 ml virus and10 μg/ml polybrene  for threehours .Mimic miR-706 (MmiR3117-MR03) was purchasedfrom Genecopoeia (USA). Oligos targeting miR-706 were transfectedusing Lipofectamine 2000 as previously reported .Ambion microRNA purification kit  andmicroRNA reverse transcription kit  were used for total RNA preparation and RNAreverse transcription. Quantitative reverse transcriptionPCR (qRT-PCR) was performed on an Applied Biosystems7900 apparatus . Primers formiR-706, ATF4, and CHOP included:miR-706-F: 5´-ACACTCCAGCTGGGACAGAAACCCTGTCTC-3´R: 5´-TGGTGTCGTGGAGTCG-3´ ATF4-F: 5´-TCCTGAACAGCGAAGTGTTG-3´R: 5´-ACCCATGAGGTTTCAAGTGC-3´CHOP- F: 5´-CTGCCTTTCACCTTGGAGAC-3´R: 5´-CGTTTCCTGGGGATGAGATA-3´.Western blot was also conducted as previously reported. The antCells were trypsinized, spun and stained with APCannexin V  BD Bios.To determine if ER stress triggers miR-706, NIH3T3cells were incubated with Thapsigargin for 5 and 10hours. We used these time points as they are early enoughto induce in full gear pro-apoptotic machinery , 24.TotWe next treated hepatic cell line AML12 withThapsigargin for ER stress induction. In these cells, therewas also remarkable elevation of miR-706 expression. To checCHOPtranscription . TherefoSince miR-706 is induced by PERK and the lattermolecule has dual pro-survival as well as pro-apoptoticfunctions , we studUPR or UPR has been shown to regulate severalmicroRNAs. PERK up-regulates miR-30-c-2* which in turnrepresses Xbp-1. CHOP , as a doER stress signaling has dual purposes. In the initial stage,UPR signaling tries to preserve homeostasis by suppressingprotein synthesis, launching antioxidant response andinducing pro-survival signals such as miR-211 . In themiR-706 protects oxidative stress induced hepaticfibrogenesis through blocking PKCα/TAOK1 signaling .Lian etBlast search with mmu-miR-706 showed that it isclosely matched with Homo sapiens uncharacterizedLOC105372576. In future, role of this microRNA in humanphysiology in the context of ER stress could be investigated.Our results identified the fundamental role for miR-706in regulating cell death induced by ER stress and suggestedthat miR-706 might be a novel therapeutic target for humancancers. We also provided evidence that CAAP1 is the directtarget of miR-706 in response to PERK/AFT4 pathwaymediated ER stress.


Doxorubicin-loaded nanoparticles displayed increased anticancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were re-sensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles successfully circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.Resistance to systemic drug therapy is a major reason for the failure of anticancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3 According to Globocan there "were 14.1 million new cancer cases, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis) in 2012 worldwide" [Drug efflux mediated by transporters including adenosine triphosphate (ATP)-binding cassette (ABC) transporters has been shown to play a crucial role in cancer cell drug resistance ,5. ABCB1Here, we here investigated the effects of doxorubicin-loaded human serum albumin (HSA) nanoparticles in ABCB1-expressing neuroblastoma cells. HSA nanoparticles are easy to produce –17, and HSA nanoparticles were prepared by desolvation as previously described –17. The n = 6) was detected, indicating only a moderate stabilisation by electrostatic repulsion.The spherical shape and narrow size distribution of HSA nanoparticles was confirmed by scanning electron microscopy (SEM) as depicted for nanoparticles stabilised by a 100% cross-linking degree . For theWhile HSA (40%), HSA (100%), and HSA (200%) nanoparticles displayed similar drug loads between 152 and 191 µg doxorubicin/mg nanoparticle, HSA (0%) nanoparticles had bound 371 µg doxorubicin/mg HSA . This prrDOX20) and vincristine-adapted (UKF-NB-3rVCR1) sub-lines substantially differed in their doxorubicin sensitivity (50 (3.8 ng/mL). UKF-NB-3rVCR1 was 4-fold more resistant to doxorubicin than UKF-NB-3 (doxorubicin IC50: 15.5 ng/mL). UKF-NB-3rDOX20 showed the highest doxorubicin IC50 (89.0 ng/mL), resulting in a 23-fold increase in doxorubicin resistance compared to UKF-NB-3 , HSA (40%), HSA (100%), or HSA (200%) nanoparticles on neuroblastoma cell viability are shown in rDOX20, although the difference between doxorubicin-loaded HSA (200%) nanoparticles and doxorubicin solution reached statistical significance , HSA (100%), and HSA (200%) nanoparticles potentially showing a trend towards a slightly increased activity . Howeverificance . NotablyrVCR1 (50 doxorubicin solution/doxorubicin IC50 nanoparticle-bound doxorubicin for HSA (40%), HSA (100%), and HSA (200%) nanoparticles (3.6–4.5-fold) was higher than for UKF-NB-3 (1.9–2.5-fold), and UKF-NB-3rDOX20 (2.1–2.9-fold). The differences between doxorubicin-loaded HSA (40%) nanoparticles, HSA (100%) nanoparticles, and HSA (200%) nanoparticles and doxorubicin solution reached statistical significance (P < 0.05)  higher in UKF-NB-3rDOX20 cells than the doxorubicin IC50 of doxorubicin solution in parental UKF-NB-3 cells.The vincristine-adapted UKF-NB-3 sub-line UKF-NB-3rVCR1 displayed decreased doxorubicin sensitivity. However, doxorubicin-loaded HSA (40%), HSA (100%), and HSA (200%) nanoparticles displayed a higher relative potency compared to doxorubicin solution in UKF-NB-3rVCR1 . The fol < 0.05) . DoxorubrDOX20 cells are characterised by high ABCB1 expression [rVCR1 cells are sensitised by the ABCB1 inhibitor zosuquidar [rVCR1 cells.Doxorubicin is an ABCB1 substrate, and UKF-NB-3pression . Vincrispression –23. Accosuquidar  to doxorrVCR1 cells, combining both doxorubicin with zosuquidar and doxorubicin encapsulation into HSA nanoparticles reduced the doxorubicin IC50 to the level of parental UKF-NB-3 cells  nanoparticles  did not affect the efficacy of doxorubicin solution or nanoparticle-bound doxorubicin in parental UKF-NB-3 cells  (rDOX20.In UKF-NB-3orubicin . The doxrDOX20 cells (rDOX20 cells than doxorubicin solution. Zosuquidar only moderately increased the efficacy of doxorubicin nanoparticles further (1.1–1.8-fold) in UKF-NB-3rDOX20 cells   and vincristine (UKF-NB-3rVCR1), which both display ABCB1 activity and resistance to doxorubicin. The HSA nanoparticles were prepared by desolvation and stabilised by glutaraldehyde, which crosslinks amino groups present in albumin molecules [The occurrence of drug resistance is the major reason for the failure of systemic anticancer therapies . Here, wolecules –17. Glutolecules . The resrVCR1 cells were similarly sensitive to doxorubicin-loaded nanoparticles as parental UKF-NB-3 cells to doxorubicin solution (and doxorubicin-loaded nanoparticles). This suggests that the doxorubicin resistance of UKF-NB-3rVCR1 cells exclusively depends on ABCB1 expression. In concordance, the ABCB1 inhibitor zosuquidar re-sensitised UKF-NB-3rVCR1 cells to the level of parental UKF-NB-3 cells.Doxorubicin-loaded nanoparticles displayed similar activity as doxorubicin solution in the parental UKF-NB-3 cell line, but exerted stronger effects than doxorubicin solution in the ABCB1-expressing UKF-NB-3 sub-lines. The UKF-NB-3rDOX20 cells displayed a more pronounced doxorubicin resistance phenotype than UKF-NB-3rVCR1 cells and were neither re-sensitised by nanoparticle-encapsulated doxorubicin nor by zosuquidar to the level of UKF-NB-3 cells. This suggests that UKF-NB-3rDOX20 cells have developed multiple doxorubicin resistance mechanisms. In contrast, adaptation of UKF-NB-3rVCR1 cells to vincristine, a tubulin-binding agent with an anticancer mechanism of action that is not related to that of the topoisomerase II inhibitor doxorubicin [The UKF-NB-3orubicin –26, did Furthermore, zosuquidar did not increase the efficacy of doxorubicin-loaded HSA (100%) and HSA (200%) nanoparticles and only modestly enhanced the efficacy of doxorubicin-loaded HSA (40%) nanoparticles. Together, these data confirm that administration of doxorubicin as HSA nanoparticles resulted in the circumvention of ABCB1-mediated drug efflux. The difference between HSA (40%) nanoparticles and the other two preparations may be explained by elevated drug release due to the lower degree of cross-linking.Interestingly, high concentrations of the cross-linker glutaraldehyde did not affect the efficacy of the resulting doxorubicin-loaded nanoparticles although high glutaraldehyde concentrations might have been expected to affect drug release and/or to covalently bind to doxorubicin via its amino group.Notably, the results differ from a recent similar study in which nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) or polylactic acid (PLA), two other biodegradable materials approved by the FDA and EMA for human use –28, did rDOX20 cells cannot be fully re-sensitised to doxorubicin by zosuquidar) [Despite the prominent role of ABCB1 as a drug resistance mechanism, attempts to exploit it as drug target have failed so far, despite the development of highly specific allosteric ABCB1 inhibitors (of which zosuquidar is one) ,21. One uquidar) ,21. HencIn conclusion, doxorubicin-loaded HSA nanoparticles produced by desolvation and cross-linking using glutaraldehyde overcome (in contrast to other nanoparticle systems) transporter-mediated drug resistance in drug-adapted neuroblastoma cells. However, our data also show that bypassing of transporter-mediated drug efflux may not be sufficient to sensitise cancer cells, which have developed multiple resistance mechanisms, to the level of sensitive parental cells.HSA and glutaraldehyde were obtained from Sigma-Aldrich Chemie GmbH . Dulbecco's phosphate buffered saline (PBS) was purchased from Biochrom GmbH . Doxorubicin was obtained from LGC Standards GmbH . All chemicals were of analytical grade and used as received.g for 12 min) and resuspension steps performed three times in purified water. During the particle purification the supernatants were collected and the drug content was measured by high-performance liquid chromatography (HPLC) as described below. The loading efficiency of doxorubicin in the nanoparticles was calculated based on the difference between the doxorubicin amount used for nanoparticle preparation and the unbound amount detected in the collected supernatants.HSA nanoparticles were prepared by desolvation as previously described –17. 100 The average particle size and the polydispersity were measured by photon correlation spectroscopy (PCS) using a Malvern zetasizer nano instrument . The resulting particle suspensions were diluted 1:100 with purified water and measured at a temperature of 22 °C using a backscattering angle of 173°.The zeta potential was measured in the same instrument by laser Doppler microelectrophoresis to provide information about the surface charge of the nanoparticles. Thus, the nanoparticle dilutions described above were transferred into a folded capillary cell and the experiment was conducted at 22 °C.TM membrane filter, Merck Millipore, Darmstadt, Germany) and dried overnight in a desiccator. Afterwards, the membrane filter was sputtered with gold  under argon atmosphere. SEM was performed on a CamScan CS4 microscope  and the sample was visualised with an accelerating voltage of 10 kV, a working distance of 10 mm, and 10,000-fold magnification.3 µL of diluted HAS nanoparticle suspension (0.25 mg/mL) was applied on a 0.1 µm membrane filter  using a LiChroCART 250 × 4 mm LiChrospher 100 RP 18 column . The mobile phase was a mixture of water and acetonitrile (70:30) containing 0.1% trifluoroacetic acid . In ordef 485 nm .rDOX20) [rVCR1) were established by continuous exposure to step-wise increasing drug concentrations as previously described [The neuroblastoma cell line UKF-NB-3, which harbours a MYCN amplification (a major indicator of high-risk disease and poor prognosis ), was esescribed  and deriescribed .All cells were propagated in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% foetal calf serum, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 °C. The drug-adapted sub-lines were continuously cultured in the presence of the indicated drug concentrations. The cells were routinely tested for mycoplasma contamination and authenticated by short tandem repeat profiling.4 cells suspended in 100 µL of cell culture medium were plated per well in 96-well plates and incubated in the presence of various doxorubicin concentrations (free or nanoparticle-encapsulated) for 120 h. Where indicated, free or nanoparticle-encapsulated doxorubicin was combined with a fixed concentration of 1 µM of the ABCB1 inhibitor zosuquidar. Then, 25 µL of MTT solution (2 mg/mL (w/v) in PBS) was added per well, and the plates were incubated at 37 °C for an additional 4 h. After this, the cells were lysed using 200 µL of a buffer containing 20% (w/v) sodium dodecylsulfate and 50% (v/v) N,N-dimethylformamide with the pH adjusted to 4.7 at 37 °C for 4 h. The absorbance was determined at 570 nm for each well using a 96-well multiscanner. After subtracting of the background absorption, the results are expressed as percentage viability relative to control cultures which received no drug. The drug concentrations that inhibited cell viability by 50% (IC50) were determined using CalcuSyn .Cell viability was determined by 3--2,5-diphenyltetrazolium bromide (MTT) assay modified after Mosman , as prevt-test was used for comparing two groups. Three and more groups were compared by ANOVA followed by the Student–Newman–Keuls test. P-values lower than 0.05 were considered to be significant.The results are expressed as the mean ± standard deviation of at least three experiments. The Student’s 50s in neuroblastoma cells in the absence or presence of the ABCB1 inhibitor zosuquidar. Effects of doxorubicin applied as solution or incorporated into HSA nanoparticles on neuroblastoma cell viability. Effects of doxorubicin solution or doxorubicin HSA nanoparticles on neuroblastoma cells with or without zosuquidar.Doxorubicin ICFile 1Additional experimental details.


NKL homeobox genes encode basic transcriptional regulators of cell and tissue differentiation. Recently, we described a hematopoietic NKL-code comprising nine specific NKL homeobox genes expressed in normal hematopoietic stem cells, lymphoid progenitors and during lymphopoiesis, highlighting their physiological role in the development of T-, B- and NK-cells. Here, we identified aberrant expression of the non-hematopoietic neural NKL homeobox gene NKX2-2 in about 12% of both, classical Hodgkin lymphoma (HL) and nodular lymphocyte predominant (NLP) HL patients. The NKX2-2 expressing NLPHL-derived cell line DEV served as a model by analysing chromosomal configurations and expression profiling data to reveal activating mechanisms and downstream targets of this developmental regulator. While excluding chromosomal rearrangements at the locus of NKX2-2 we identified t resulting in overexpression of the IL17 receptor gene IL17RB via juxtaposition with the IGH-locus. SiRNA-mediated knockdown experiments demonstrated that IL17RB activated NKX2-2 transcription. Overexpression of IL17RB-cofactor DAZAP2 via chromosomal gain of 12q13 and deletion of its proteasomal inhibitor SMURF2 at 17q24 supported expression of NKX2-2. IL17RB activated transcription factors FLI1 and FOXG1 which in turn mediated NKX2-2 expression. In addition, overexpressed chromatin-modulator AUTS2 contributed to NKX2-2 activation as well. Downstream analyses indicated that NKX2-2 inhibits transcription of lymphoid NKL homeobox gene MSX1 and activates expression of basic helix-loop-helix factor NEUROD1 which may disturb B-cell differentiation processes via reported interaction with TCF3/E2A. Taken together, our data reveal ectopic activation of a neural gene network in HL placing NKX2-2 at its hub, highlighting a novel oncogenic impact of NKL homeobox genes in B-cell malignancies. Hodgkin lymphoma (HL) is a B-cell malignancy comprising some 10% of all lymphomas. This cancer is subclassified into two distinct entities according to clinical and histopathological features, namely classical HL (cHL) and nodular lymphocyte predominant (NLP) HL . This piCompromised B-cell development has been highlighted as a major aspect of the pathogenesis in HL by analyses of cell lines and micro-dissected HRS cells whose main TFs important for B-cell development are absent or inactivated, resulting in B-cells with incomplete phenotypes , 9. AberNKL homeobox genes encode TFs which regulate fundamental processes in both embryonal development and differentiation in the adult. Some represent master genes for specific cell types/organs like NKX2-3 (spleen), NKX2-5 (heart), or NKX3-1 (prostate) –18. OtheMalignant cells of T-cell acute lymphoblastic leukemia (T-ALL) are developmentally arrested thymocytes showing disturbed T-cell differentiation due to aberrant expression of particular oncogenes including NKL homeobox genes . This hoHere, we analyzed NKL homeobox gene NKX2-2 which is aberrantly activated in subsets of cHL and NLPHL patients. We used a NKX2-2 expressing HL cell line as a model and identified upstream regulators and downstream targets. Our data assemble a pathological gene regulatory network surrounding NKX2-2 and extend the oncogenic role of NKL homeobox genes to this type of B-cell lymphoma.Recently, we systematically analyzed NKL homeobox gene expression in normal B-cell development and in B-cell lymphoma patients, thereby identifying a physiological NKL-code in B-cell differentiation and aberrant activities in HL patient samples . Datasethttps://www.proteinatlas.org/) showed normal NKX2-2 expression in the brain which coincides with the first report of this gene in mice , showing NKX2-2 overexpression in NLPHL-derived DEV Figure . Subsequ in mice  29, 30], 30https in mice . Taken tIn T-ALL and splenic marginal zone lymphoma (SMZL) aberrant activation of particular NKL homeobox genes is conducted via chromosomal rearrangements , 31. TheThe SKY data indicated additional chromosomal rearrangements including t which might contribute, albeit indirectly, to NKX2-2 activation. Genomic profiling data showed absence of copy number alterations on the short arm of chromosome 3 and indicated a B-cell specific deletion at the rearranged IGH locus on the long arm of chromosome 14 Figure . FISH anTo analyze the role of IL17RB in NKX2-2 expression we performed siRNA-mediated knockdown experiments. Subsequent quantification of IL17RB and NKX2-2 transcripts by RQ-PCR demonstrated concomitant downregulation, indicating that IL17RB-signalling activates NKX2-2 expression Figure . HoweverThe activity of IL17RB is supported by the cofactor DAZAP2 which in turn is regulated via proteasomal degradation mediated by SMURF2 . RQ-PCR The expression level of SMURF2 was strongly reduced in DEV as compared to HL control cell lines and primary hematopoietic cell/tissue samples Figure . InteresNKX2-2 is aberrantly expressed in Ewing sarcoma. In this malignancy the TF encoding gene FLI1 is fused to EWS and the resultant hybrid protein directly activates NKX2-2 transcription . To examPhysiologically, NKX2-2 operates in the development of the brain and additionally in the pancreas , 37. In To identify additional activators of NKX2-2 we performed comparative expression profiling analysis of dataset GSE40160 using R-based online tools. This examination revealed 250 significant differentially expressed genes in DEV as compared to the four associated HL control cell lines, including IL17RB, SYK, INPP5D, FOXG1 and AUTS2 . Of notePublic RNA-sequencing data showed overlapping expression patterns of NKX2-2, IL17RB, FLI1, NEUROD1 and FOXG1 in the brain , suggestAdditionally, our comparative expression profiling approach revealed significantly enhanced activity of AUTS2 in DEV . AUTS2 eNKL homeobox gene MSX1 is downregulated in several HL cell lines . ReducedNKL homeodomain proteins interact with corepressors of the TLE/Groucho family via the conserved EH1-motif in their N-terminal region . ExpressFinally, to uncover potential oncogenic functions of aberrant NKL homeobox gene NKX2-2 activities in HL we analyzed selected public expression profiling datasets using R-based online tools. We compared NKX2-2 expressing DEV with four control cell lines -signalling activates NKX2-2 expression in the ventral neural tube development , 51. HowNKL homeobox gene NKX2-2 is aberrantly expressed in T-ALL as well . In thisHere, we identified and characterized a novel non-recurrent aberration in DEV, t, which activates IL17RB. Our data also demonstrated normal IL17RB expression in the brain. However, several reports show that this receptor gene plays a role in T-cell development. Accordingly, IL17RB is expressed in invariant NK/T-cells and in double negative developing T-cells where it is regulated by GATA3 –62. Of nNormal expression of NKX2-2 is additionally regulated via chromatin-modifications. Bivalent chromatin contains both activating and repressive histone-marks and is established in embryonal stem cells (ESCs) at key developmental genes including NKX2-2 . This maDeregulations of bHLH factors play a fundamental role in the development of HL . InteracIn conclusion, we identified aberrant expression of NKL homeobox gene NKX2-2 in HL which supports the oncogenic role of this gene subclass in lymphoid malignancies. Their widespread activity in leukemia and lymphoma indicates a common function of this group of homeobox genes which might hence represent a promising target for therapies. Their particular expression patterns may serve as diagnostic markers as well. In addition, our data document the fitness of DEV to model NLPHL at the molecular level, the only cell line hitherto available for investigating this entity in the laboratory.https://www.ncbi.nlm.nih.gov/gds): GSE12453  [https://www.bioconductor.org/). Statistical significance was calculated using online tools provided by GEO, or using R-based Students T-Test. The obtained p-values are indicated.Public expression profiling datasets of lymphoma/leukemia patients were obtained from Gene Expression Omnibus  , GSE3913B-cells) , GSE7788B-cells) , GSE4016B-cells) . These ghttp://derisilab.ucsf.edu/microarray/software/ClusterTreeView.pdf). To reveal potential biological function of shortlisted genes, gene-annotation enrichment analysis was performed using DAVID bioinformatics resources [Expression profiling datasets of seven HL cell lines using HG U133 Plus 2.0 gene chip (Affymetrix) were generated by Prof. Andreas Rosenwald  and by Dr. Robert Geffers . The datasets are available at GEO (GSE115191). Expression profiling data for HL cell line DEV were obtained from dataset GSE40160. After RMA-background correction and quantile normalization of the spot intensities, the profiling data were expressed as ratios of the sample mean and subsequently log2 transformed. Data processing was performed via R/Bioconductor using limma and affy packages. For creation of heat maps we used CLUSTER version 2.11 and TREEVIEW version 1.60  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. For functional examinations treated DEV cells were stained by trypan blue (Sigma) and counted microscopically (Neubauer chamber) or analysed by the IncuCyte S3 Live-Cell Analysis System . For detection of apoptotic cells we used the IncuCyte Caspase-3/7 Green Apoptosis Assay (Essen Bioscience).HL-cell lines HDLM-2, KM-H2, L-428, L-540, L-1236, SUP-HD1, U-HO1 and PMBL cell line U-2940 are held by the DSMZ  and were cultivated as described . HL cellDEV cells were transfected as indicated and and treated for 20 h with 100 μM Etoposide (Sigma) which has been dissolved in dimethylsulfoxide, and subsequently prepared for standardized MTT -2,5-diphenyltetrazolium bromide; obtained from Sigma) assays. The measurement was performed twice in triplicates. The absorbance was determined at 540 nm and at 620 nm as background control using ELISA reader Multiskan EX .For genomic profiling genomic HL-cell line DNA was prepared using the Qiagen Gentra Puregene Kit (Qiagen). Labelling, hybridization and scanning of HD Cytoscan arrays were performed at the Genome Analytics Facility, Helmholtz Centre for Infection Research , according to the manufacturer’s protocols (Affymetrix). Data were interpreted using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix).Chromosomal analysis by Spectral Karyotyping (SKY) and fluorescence in situ hybridization (FISH) was performed as described previously . SKY proTotal RNA was extracted from cell line samples using TRIzol reagent . Primary human total RNA used in this study was commercially obtained - isolated from peripheral blood mononuclear cells (PBC), thymus, lymph node (LN), spleen, bone marrow (BM), NK-cells, and brain from Biochain/BioCat , and RNA from peripheral CD19-positive B-cells, CD3-positive T-cells, and CD34-positive stem cells from Miltenyi Biotec . cDNA was synthesized from 5 μg RNA by random priming using Superscript II (Invitrogen). Real-time quantitative (RQ)-PCR analysis was performed with the 7500 Real-time System, using commercial buffer and primer sets . Quantification of MSX1 was performed as described previously . For norFor genomic copy number quantification of exon 1 from SMURF2 we extracted genomic DNA using the High Pure PCR Template Preparation Kit . The following oligonucleotides were used for Q-PCR analysis: SMURF2-1 5’-’CTCATGGCGACGACTCTCGGCAC-3’, SMURF2-2 5’-GGCGAGGCGCGGCGGAGTCACC-3. For normalization we used MEF2C as described previously .T-Test and the calculated p-values indicated by asterisks .Quantitative analyses were performed in triplicate. Standard deviations are presented in the figures as error bars. Statistical significance was assessed by Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-tubulin (Sigma), NKX2-2 , ERK1 , and phospho-ERK (Santa Cruz Biotechnology). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was performed using the digital system ChemoStar Imager .


Elevated aerobic glycolysis rate is a biochemical alteration associated with malignant transformation and cancer progression. This metabolic shift unavoidably generates methylglyoxal (MG), a potent inducer of dicarbonyl stress through the formation of advanced glycation end products (AGEs). We have previously shown that the silencing of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, generates endogenous dicarbonyl stress resulting in enhanced growth and metastasis in vivo. However, the molecular mechanisms through which MG stress promotes metastasis development remain to be unveiled.In this study, we used RNA sequencing analysis to investigate gene-expression profiling of GLO1-depleted breast cancer cells and we validated the regulated expression of selected genes of interest by RT-qPCR. Using in vitro and in vivo assays, we demonstrated the acquisition of a pro-metastatic phenotype related to dicarbonyl stress in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cellular models. Hyperactivation of MEK/ERK/SMAD1 pathway was evidenced using western blotting upon endogenous MG stress and exogenous MG treatment conditions. MEK and SMAD1 regulation of MG pro-metastatic signature genes in breast cancer cells was demonstrated by RT-qPCR.High-throughput transcriptome profiling of GLO1-depleted breast cancer cells highlighted a pro-metastatic signature that establishes novel connections between MG dicarbonyl stress, extracellular matrix (ECM) remodeling by neoplastic cells and enhanced cell migration. Mechanistically, we showed that these metastasis-related processes are functionally linked to MEK/ERK/SMAD1 cascade activation in breast cancer cells. We showed that sustained MEK/ERK activation in GLO1-depleted cells notably occurred through the down-regulation of the expression of dual specificity phosphatases in MG-stressed breast cancer cells. The use of carnosine and aminoguanidine, two potent MG scavengers, reversed MG stress effects in in vitro and in vivo experimental settings.These results uncover for the first time the key role of MG dicarbonyl stress in the induction of ECM remodeling and the activation of migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells. Importantly, the efficient inhibition of mitogen-activated protein kinase (MAPK) signaling using MG scavengers further emphasizes the need to investigate their therapeutic potential across different malignancies.The online version of this article (10.1186/s13058-018-1095-7) contains supplementary material, which is available to authorized users. A key hallmark of cancer cells is their metabolic reprogramming consisting of enhanced aerobic glycolysis over oxidative respiration. This so called “Warburg effect” leads to the accumulation of methylglyoxal (MG), a highly toxic and reactive dicarbonyl, that spontaneously glycates proteins, nucleic acids and lipids . The reaFurthermore, it remains controversial whether GLO1 in cancer acts as a tumor suppressor or oncogene. Several studies depicted GLO1 as an amplified and/or overexpressed oncogene associated with a poor prognosis in various types of malignant tumors, thus considering the inhibition of GLO1 activity as a potential anti-cancer therapy (for review ). ZenderComprehensive RNA-sequencing (RNASeq) analysis of GLO1-depleted cancer cells identified a metastatic transcriptomic signature involving genes tightly associated with cell migration and extracellular matrix (ECM) remodeling, such as collagens and tenascin C. We have demonstrated that MG-induced pro-metastatic signature is linked with the activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-related protein kinase (ERK) pathway, which signals through activated SMAD1. We have further demonstrated that MEK/ERK-sustained activation in GLO1-depleted cells notably occurs through the down-regulation of dual specificity phosphate 5 (DUSP5) phosphatase expression upon MG stress in breast cancer cells. Overall, our study demonstrated for the first time that MG stress is able to change the ECM and to regulate migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells.MDA-MB-231 and MCF7 cancer cell lines were obtained from the American Type Culture Collection . MDA-MB-468 cells were kindly provided by Dr C. Gilles . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) containing 10% fetal bovine serum  and 2 mM L-glutamine (Lonza). For cell signaling analysis, cells were cultured in DMEM with L-glutamine and without FBS. L-carnosine (C9625), aminoguanidine (396494) and methylglyoxal  were from Sigma. We excluded the presence of significant formaldehyde contamination (< 3%) in MG (lot #BCBQ9416V) by nuclear magnetic resonance (NMR) analysis. Transforming growth factor (TGF)β (#10021) was from Peprotech. U0126 (S1102) was from Selleckchem. Anti-argpyrimidine antibody (mAb6B) specificity has been previously confirmed by competitive enzyme-linked immunosorbent assay (ELISA) and shown to not react with other MG-arginine adducts .MG measurements were performed as previously described . Brieflyn = 12–14 per condition). Lungs were collected and embedded in paraffin for immunohistochemical analysis (IHC).All animal experimental procedures were performed according to the Federation of European Laboratory Animal Sciences Associations (FELASA) and were reviewed and approved by the Institutional Animal Care and Ethics Committee of the University of Liège. Animals were housed in the GIGA-accredited animal facility (University of Liège). Eight-week-old female NOD-SCID mice were inoculated via the tail vein with MDA-MB-231 shNT, shGLO1#1 and #2 cells ) and were monitored for 6 weeks for development of metastases. Mice received intra-peritoneal (IP) injection of carnosine (100 mg/kg) three times per week from the day of the engraftment until the end of the experiment . Vimentin-positive metastatic cells were quantified by computerized counts (QuPath 0.1.2 open source software) and verified by manual counting. The number of positive cells to lung area was reported, yielding a count expressed as the number of cells per square millimeter. For tenascin C IHC and Masson’s trichrome staining, a score of 0 was associated with negative staining and a score of 1 with positive staining in tumor cells.Standard protocols are detailed in Additional file Collagen gels were made as previously described . Briefly5 cells) were suspended in serum-free DMEM medium , 1% penicillin/streptomycin (pen/strep)) and seeded into the upper part of Transwell filters  for migration or Transwell filters precoated with Matrigel for invasion (BD Biosciences). The lower compartment was filled with DMEM containing 1% BSA, 1% pen/strep and 10% FBS. After 16 h or 24 h incubation at 37 °C for migration and invasion, respectively, migrating cells were stained with Diff-Quick (Medion Diagnostics). Inserts were scanned at 10× magnification . The area covered by migrating/invading cells was quantified by densitometry (ImageJ). Data are expressed as relative migration/invasion ability compared to control cells.MDA-MB-231 and MDA-MB-468 cells  and MCF7-M  cells were seeded in Seahorse XFp mini-plates (Agilent) and analyzed using the Seahorse glycolysis stress test according to manufacturer’s recommendations. Cells were first challenged with glucose (10 mM) then successively stressed with oligomycin (1 μM) and 2-deoxyglucose (50 mM). All results were normalized to protein quantification.Cells were incubated in glucose-free medium in the presence of 2-NBDG (ThermoFisher Scientific) for 30 min and further analyzed by flow cytometry (BD Biosciences). Median fluorescence intensity (MFI) was calculated.GLO1 activity was assessed as previously described . Brieflyt test with or without Welsch’s correction according to homoscedasticity. Experimental data from more than two groups were compared using one-way or two-way analysis of variance (ANOVA) depending on the number of grouping factors. Dunnett’s test was applied for simple comparisons while Student-Newman-Keul’s (one-way ANOVA) or Bonferroni’s (two-way ANOVA) tests were used for multiple comparisons. In the case of discrete variables (IHC scores) or non-normally distributed variables, groups were compared using Mann-Whitney’s U test. Outliers were detected using whisker box plots. A bilateral p value <0.05 was considered statistically significant with a 95% confidence interval.Data from two groups were compared using the unpaired Student’s In an attempt to unveil the mechanisms by which MG stress enhances the metastatic potential of breast cancer cells, we performed genome-wide messenger RNA (mRNA) profiling of MDA-MB-231 GLO1-depleted cells. We first validated GLO1 depletion by western blot and the subsequent increased MG production in stably GLO1-silenced MDA-MB-231 cells  independently from the growth of the primary tumor. We observed that GLO1-depleted cells injected into the tail vein of NOD-SCID mice induced a significant increase in pulmonary tumor burden when compared with control , PI3K/AKT, ERK, c-Jun N terminal kinase (JNK), Src-focal adhesion kinase (FAK) and Rho-GTPases . FollowiWe therefore explored alternative signaling pathways downstream of tyrosine kinase receptors and found that the level of phosphorylated MEK and ERK was increased in GLO1-depleted MDA-MB-231 cells Fig. b. KnowinTo consolidate the existence of a link between MG stress and MEK/ERK activation, we cultured MDA-MB-231 and MDA-MB-468 cells in the presence of increasing concentrations of exogenous MG. MG treatment induced P-MEK and P-ERK in both cell lines Fig. c. Along To understand how (hyper)activation of the MEK/ERK signaling cascade in GLO1-depleted breast cancer cells relates to their metastatic potential, we first considered that MEK/ERK signaling could be mediated by the physical translocation of ERK in the nucleus. Using immunofluorescence, we excluded this possibility as GLO1-silenced cells showed P-ERK levels that were comparable to control cells using both MDA-MB-231 and MDA-MB-468 cells  that fulfill pro-tumoral and pro-metastatic functions at both primary and metastatic sites. On the other hand, an interesting parallel can be drawn with diabetes mellitus, where hyperglycemia results in the accumulation of MG and its protein adducts. In fact, GLO1-depleted myoblasts grown under a hyperglycemic condition display MG-induced elevated expression of collagens and increased fibrosis . ElevateSeveral pro-tumorigenic signaling pathways that are activated by ECM stiffness promote aerobic glycolysis . EmerginSMAD4 is a common transcriptional partner for activated SMADs, the loss of which predicts liver metastasis in colon cancer  and bad The role of GLO1 in cancer progression remains controversial. In this study, GLO1 silencing favored a pro-migratory and metastatic phenotype, suggesting a tumor-suppressing role of GLO1. A recent study published by Guo and colleagues reported that inhibition of GLO1 promoted apoptosis and decreased invasion of MDA-MB-231 cells . These sThe significant anti-metastatic effect of carnosine designates MG stress as a promising target for therapeutic intervention in aggressive tumor cells that have undergone energy metabolic switch. To date, inhibitors of glycolysis have had only modest therapeutic efficacy in cancer. These unsatisfactory results may be related to the large heterogeneity reported for several glycolytic enzymes, as a consequence of different genes, splice forms and post- translational modifications. These latter make cancer cells react differently when challenged with anti-glycolytic agents . Based oThe main results of this study further emphasize the relevance of MG stress in the acquisition of a metastatic phenotype by breast cancer cells. Our data point to direct ECM remodeling by breast cancer cells and sustained activation of MAPK signaling as key molecular events underlying MG stress. This study improves our understanding of the connection between glycolytic switch and breast cancer aggressiveness and proposes MG scavenger molecules (such as carnosine and aminoguanidine) as potential novel treatment options in the management of metastatic breast cancer.Additional file 1:Supplementary methods: RNA sequencing, shRNA transfection, siRNA transfection, Plasmids transfection, Immunofluorescence, RNA isolation and quantitative reverse transcription-PCR (qRT-PCR), Western blot, Flow cytometry analysis and Soft agar colony formation assays. (PDF 560 kb)Additional file 2:Figure S1. RNA sequencing analysis of GLO1-depleted MDA-MB-231 cells. (A) MG extracellular concentrations were assessed over 48 h in conditioned medium of GLO1-depleted MDA-MB-231 cells using UPLC-MS/MS. (B) Volcano plots highlighting differentially expressed genes in shGLO1#1 and shGLO1#2 MDA-MB-231 cells. Orange and red dots represent genes differentially expressed significantly (q < 0.05 and log2 fold change (FC) > 1) for shGLO1 clones. Red dots represent genes of the pro-metastatic signature. (C) Tenascin C, Lumican and CD24 mRNA levels were assessed by qRT-PCR in MDA-MB-231 cells treated with MG 300 and 500 μM for 1 h. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S2. Dicarbonyl stress promotes anchorage-independent growth and invasion of breast cancer cells. Representative pictures of the colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-231 cells treated with carnosine. Figure S3. Dicarbonyl stress promotes invasion of breast cancer cells. (A) Migration ability toward serum of GLO1-overexpressing shNT and shGLO1#1 MDA-MB-231 cells was assessed using Transwell filters. Representative filters are shown for each condition. (B) Quantification of migration assays of MDA-MB-231 shGLO1 cells upon GLO1 overexpression. (C) GLO1 protein level was assessed using immunoblot in GLO1-silenced MDA-MB-231 upon GLO1 overexpression. β-actin protein is used as loading control. Western blot is representative of three independent experiments. (D) Invasiveness ability of GLO1-depleted MDA-MB-231 toward serum was assessed using Transwell filters. Where indicated, cells were pre-treated with MG scavengers, carnosine and aminoguanidine, 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. (E) Quantification of invasiveness ability of GLO1-silenced MDA-MB-231 cells treated with carnosine and aminoguanidine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S4. Highly migratory MCF7 cells (MCF7-M) display enhanced aerobic glycolysis. (A) Representative pictures of cultured MCF7 and MCF7-M cells. Scale bar represents 400 μm. (B) Extracellular acidification rates (ECAR) in MCF7 and MCF7-M cells using Seahorse flux analyzer. Glycolytic-related ECAR was calculated. Data were analyzed using Student’s t test and shown as mean values ± SEM of two independent experiments. **p < 0.01. Figure S5. Dicarbonyl stress promotes migration and anchorage-independent growth of MDA-MB-468 breast cancer cells. (A) GLO1 protein level in MDA-MB-468 shNT control and shGLO1#2 and #3 cells. β-actin protein is used as loading control. Western blot is representative of three independent experiments. (B) Lumican and CD24 mRNA levels were assessed by qRT-PCR in GLO1-depleted MDA-MB-468 cells. (C) Lumican and CD24 mRNA levels were assessed by qRT-PCR in MDA-MB-468 cells treated with MG 300 and 500 μM for 1 h. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. (D) Migration ability of GLO1-depleted MDA-MB-468 toward serum was assessed using Transwell filters. Where indicated, cells were pre-treated with carnosine 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. (E) Quantification of migratory ability of GLO1-silenced MDA-MB-468 cells treated with carnosine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. (F) Representative pictures of the colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-468 cells treated with carnosine. (G) Quantification of colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-468 cells treated with carnosine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S6. P-ERK localization in GLO1-depleted breast cancer cells. P-ERK immunofluorescence staining in GLO1-silenced MDA-MB-231 (A) and MDA-MB-468 (B) cells cultured in serum-free conditions. Data are representative of three independent experiments. Magnification × 630. Zoomed pictures (white square) are shown where indicated. Figure S7. GLO1 rescue impairs increased SMAD1 phosphorylation in GLO1-depleted MDA-MB-231 cells and SMAD1 activation favors the enhanced migration ability of MCF7-M cells. (A) (P-)SMAD1 (ser206) protein levels were assessed using immunoblot in shNT and shGLO1#1 MDA-MB-231 cells upon GLO1 overexpression. (B) P-SMAD1 (ser206) and SMAD1 protein level in MCF7 and MCF7-M cells cultured in serum-free conditions. (C) Migration ability toward serum of SMAD1-silenced MCF7 and MCF7-M cells was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. (D) Quantification of migration assays of SMAD1-silenced MCF7 and MCF7-M cells. (E) SMAD1 protein level assessed by immunoblot to validate SMAD1-silencing using siRNAs in MCF7 and MCF7-M cells (related to panels B and C). β-actin or HSC70 were used as loading control. Immunoblots were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. All western blots are representative of three independent experiments. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S8. SMAD1 activation promotes the pro-metastatic phenotype in MDA-MB-468 cells. A. SMAD1 target genes mRNA levels were assessed using qRT-PCR in SMAD1-silenced MDA-MB-468 shGLO1 cells. SMAD1 mRNA level was assessed to validate efficient SMAD1 siRNA silencing. (B) Migration ability toward serum of SMAD1-silenced MDA-MB-468 shGLO1 cells was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. (C) Quantification of migration assays of SMAD1-silenced MDA-MB-468 shGLO1 cells. (D) SMAD1 protein level assessed by immunoblot to validate SMAD1-silencing using siRNAs in GLO1-depleted MDA-MB-468 cells (related to panels B and C). Western blots are representative of three independent experiments. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S9. GLO1 depletion is associated with a global decrease of phosphatases expression in breast cancer cells. (A) mRNA levels of PPP2 phosphatases  in GLO1-depleted MDA-MB-231 and MDA-MB-468 cells were assessed by qRT-PCR. (B) DUSP1, 5, 8, 10, 12, 14 and 16 phosphatases mRNA levels in GLO1-depleted MDA-MB-468 cells were quantified by qRT-PCR. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S10. DUSP5 overexpression decreases the migratory capacity of GLO1-depleted breast cancer cells. Migration ability toward serum of DUSP5-overexpressing MDA-MB-231 (A) and MDA-MB-468 (B) cells was assessed using Transwell filters. Representative filters are shown for each condition. Table S1. Remarkable genes coding for ECM components and ECM regulators, the expression of which is significantly modulated in GLO1-depleted MDA-MB-231 breast cancer cells. Genes shown in bold have been validated at the protein and/or mRNA expression levels. ns, not significant. Table S2. DUSPs and PPPs subunits expression data from RNASeq in GLO1-depleted MDA-MB-231 cells. Fold change and false discovery rate (FDR) are shown for both shGLO1 clones. ns, not significant. Table S3. Primer sequences and probes used for quantitative reverse transcription-PCR (qRT-PCR). Table S4. Antibodies and dilution used for western blot experiments. (PDF 2523 kb)