Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics.Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian  and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5–2% O2) and compared to normoxia (21% O2). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively.Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro. As the tumors  have deme tumors . Shahrzae tumors  publishee tumors . Methione tumors . SAM is e tumors . Liu et e tumors  have shoxia (1%) . GATA3, xia (1%) . Interesh2 cells .N-linked glycosylation sites on integrin-α3 are important for cell to cell adhesion, and that modification to these glycans results in a potential EMT phenotype , is associated with fucosylation , 14. Shihenotype . DNA methenotype –24. It ihenotype .in-vitro, and determine the epigenetic alterations that may underlie these altered glycosylation profiles.In this study, our objective was to demonstrate the impact of hypoxia on glycosylation profiles from the secreted glycoproteins of ovarian and breast cancer cells 2. A2780cis cells were treated with 1 μM cisplatin every 2/3 passages, to ensure chemo-resistance was maintained. All cell lines were also routinely tested for Mycoplasma contamination in the UCD Conway Institute of Biomolecular and Biomedical Research UCD, Dublin.A2780, PEO1, PEO4, MDA-MB-231, MDA-MB-436 were authenticated in 2015/16 by DDC medical, via short tandem repeat (STR) profiling. A2780cis cells were obtained from The European Collection of Authenticated Cell Cultures (ECACC). All cell lines were maintained in RPMI 1640 medium supplemented with 10% v/v fetal calf serum (FCS) and 2 mM L-glutamine. PEO1 and PEO4 were further supplemented with 1 mM sodium pyruvate. All cells were cultured at 37°C with 5% CO2 for a 24 and 48 h period, respectively, with media preconditioned to the relative oxygen environment for at least 12 h prior to culturing. The 48 h hypoxia exposed cell line media was replenished with preconditioned media after 24 h. Following incubation, the cells were immediately harvested and depending on the analysis being performed, cell pellets were fixed in 70% methanol, or frozen at −20°C, for up to 6 weeks or −80°C for longer periods for western blot and subsequent TaqMan® RT-qPCR analyses.To ensure a sustainable deficiency of oxygen, cells were incubated in a hypoxic chamber under 0.5, 1, and 2% OCells were harvested by trypsinisation, fixed in 70% methanol and subsequently stained with Propidium Iodide (PI) (Sigma) and an anti-5′methylCytidine (5′MeC) (Eurogentec) primary antibody. Prior to staining with 5′MeC, the cells were pre-incubated with 1 M HCl at 37°C for 1 h. IgG negative controls were used at the same concentration as the primary antibody. Secondary antibody staining was conducted using an FITC conjugated rabbit anti-mouse secondary antibody (Dako). Analyses were performed on an Accuri C6 flow cytometer and results assessed using FCS Express software (De Novo).N-glycan analysis.Supernatants were spun down and concentrated using Amicon Ultra-15 10K ultrafiltration (Millipore), to a final volume of below 200 μL. Proteins were precipitated with a half volume of 50:50 TCA: acetone (w/v) on ice. The mixture was then incubated for 45 min on ice and centrifuged at 13,000 rpm for 5 min. The resultant pellet was washed with cold acetone and centrifuged again at 13,000 rpm for 5 min. This final pellet was dried and resuspended in sample buffer  for subsequent N-glycans were released from glycoproteins in samples, by in situ digestion with Peptide N-glycosidase F  in-gel blocks  by reductive amination (l blocks . Brieflymination . The samHILIC-UPLC was carried out on a BEH Glycan 1.7 μM 2.1 × 150 mm column (Waters), on an Acquity UPLC H-Class (Waters), coupled with an Acquity fluorescence detector. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 40°C. The following conditions were used; 30 min method using a linear gradient of 30–47% A at 0.56 mL/min in 23 min . Samplesnegative-sensitivity mode with a capillary voltage of 1.80 kV. The ion source block and nitrogen desolvation gas temperatures were set at 120 and 400°C, respectively. The desolvation gas was set to a flow rate of 600 L/h. The cone voltage was maintained at 50V. Fullscan data for glycans were acquired over m/z range of 450–2,500. Data collection and processing were controlled by MassLynx 4.1 software . The fluorescence detector settings were as follows ex = 330 nm, em = 420 nm. Sample injection volume was 8 μL. The flow rate was 0.150 mL/min and column temperature was maintained at 60°C; solvent A was 50 mM ammonium formate in water (pH 4.4) and solvent B was acetonitrile. A 40 min linear gradient was used, and was as follows: 28% (v/v) A for 1 min, 28–43% (v/v) A for 30 min, 43–70% (v/v) A for 1 min, 70% (v/v) A for 3 min, 70–28% (v/v) solvent A for 1 min and finally 28% (v/v) A for 4 min.Lyophilised cell line secretome samples, were reconstituted in 3 μl of water and 9 μl acetonitrile. Online coupled fluorescence (FLR)-mass spectrometry detection was performed using a Waters Xevo G2 QTof with Acquity® UPLC  and BEH Glycan column . For MS acquisition data, the instrument was operated in 2+, pH 5.0), using arrays of the following enzymes: Arthrobacter ureafaciens sialidase (ABS), 0.5 U/mL; Streptococcus pneumoniae sialidase (NAN1), 1 U/mL; bovine testes β-galactosidase (BTG), 1 U/mL; Streptococcus pneumoniae β-galactosidase (SPG), 0.4 U/mL; bovine kidney α-fucosidase (BKF), 1 U/mL; β-N-acetylglucosaminidase cloned from Streptococcus pneumonia, expressed in E. coli (GUH), 8 U/mL (Prozyme) or 400 U/mL (NEB); jack bean α-mannosidase (JBM), 60 U/mL; almond meal α-fucosidase (AMF), 0.4 mU/mL. All enzymes were purchased from Prozyme and GUH, was also purchased from NEB. Following digestion, the enzymes were removed by filtration through a Pall 10 kDa MWCO microcentrifuge filtration device , and the oligosaccharides were analyzed by HILIC-UPLC.The 2AB-labeled oligosaccharides were digested in a volume of 10 μL for 18 h at 37°C in 50 mM sodium acetate buffer, pH 5.5 (with the exception of jack bean α-mannosidase (JBM), where the buffer was 100 mM sodium acetate, 2 mM ZnGlycan peaks were pooled based on similar structural or compositional features of the peak glycan members. Features pertaining to a peak were determined based on the major glycan members of that peak. See .PARP , p21 , p16 , N-Cadherin , GATA2 and GATA3 ; rabbit polyclonal antibody targeting LC3 ; and mouse monoclonal antibodies targeting E-Cadherin , Rb , ST3GAL4  and MGAT5 . The membranes were then incubated with a secondary goat anti-mouse or anti-rabbit antibody . The blots were developed using TMB for enzymatic colourimetric detection. To analyse protein loading, the mouse monoclonal, α-Tubulin antibody  was used. Western blots were quantified using ImageJTM software (FIJI).Whole cell extracts were separated by SDS-PAGE using 4–15% precast TGX gels (BioRad), and transferred onto PVDF membranes using the Trans-Blot® Turbo™ system (Biorad). Blots were blocked and incubated with rabbit monoclonal antibodies targeting (i) glycosyltransferases mannosyl--glycoprotein β1,6-N-acetyl-glucosaminyltransferase MGAT5), (ii)( and tissue specific transplantation antigen P35B TSTA3)( and (iii) TFs, GATA1, GATA2 and GATA3 identified in a preliminary in-silico study . Firstly, total RNA was isolated from the cells using an RNeasy Mini kit . The RNA (500 ng) was subsequently reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR was then performed using the following TaqMan assay probes ST3GAL4: Hs00920871 m1, MGAT5: Hs00159136 m1, TSTA3: Hs00163023 m1, GMDS: Hs00155276 m1, MPI: Hs00159228 m1, GATA1: Hs01085823 m1, GATA2: Hs00231119 m1, GATA3: Hs00231122 m1 (Thermo Fisher), on a 7500 Fast Real-Time PCR System (Applied Biosystems) to analyse the expression of each RNA by the comparative delta Ct method, normalized using the TATA box binding protein (TBP): Hs99999910 m1. TATA is a constitutive gene and does not vary in the treatments  at 595 nm. As a negative control, a set of stoppers were left in place until the experiment was completed, so the cells were contained within the stoppers and no migration occurred in this sample.The Oris™ Cell Migration Assay (Platypus Technologies) was used to assess cell migration in all cell lines under normoxia and hypoxia conditions. Conditions were optimized based on the manufacturer's instructions. Briefly, 2.5 × 105 cells/well the day before transfection and for PEO1 and PEO4 cells this was 6 × 105 cells/well. On the day of transfection, separate tubes were prepared with the appropriate volume of DharmaFECT1 (2 μL/well), scrambled siRNA  and targeted siRNA , diluted in serum free RPMI media. The DharmaFECT1 was then mixed with the siRNA and allowed to stand at room temperature for 20 min. At the same time, the media was removed from the 12 well-plates, and replaced with 800 μL/well of fresh complete growth media. 200 μL of DharmaFECT1/siRNA mix was then added to the appropriate wells. The cells were incubated at 37°C for 24 and 48 h.Cells were transiently transfected with 100 nM of either siRNA targeting GATA2  or GATA3  (Dharmacon) transcripts, and 25nM of scrambled control siRNA using DharmaFECT1 (Dharmacon) transfection reagent. DharmaFECT1 volumes and siRNA concentrations were optimized for a 12 well-plate according to the manufacturer's protocol . Brieflypost-hoc analysis using Tukey's Honest Significant difference. For other data, such as the RT-qPCR, Western blot densitometry and migration assays, t-tests, one- or two-way ANOVAs were used as was deemed appropriate. A p < 0.05 was considered statistically significant.All data are expressed as the means ± the standard deviation (SD). Statistical analyses were performed using SPSS statistical software for Windows . For significances in glycan data, individual glycan peaks and features, the HILIC-UPLC data were logit transformed, and examined using a MANOVA with Histograms indicating fold changes in hypoxia conditions in comparison to normoxia in Figures were created using Hierarchial Clustering Explorer HCE 3.5 software.in-vivo. All cells were cultured in 0.5, 1, and 2% O2 percentages and compared to normoxic conditions (21% O2) for 24 and 48 h, to determine the impact if any that hypoxia had on global cellular DNA methylation. Hypoxic exposure (0.5 and 1% O2 levels), significantly altered the global DNA methylation profile compared with the cells cultured under normoxic conditions (21% O2) (p ≤ 0.05). There was a significant increase in DNA methylation (p ≤ 0.05) (up to 60%) after 24 h and significant decrease in DNA methylation (p ≤ 0.05) (up to 60%) after 48 h incubation under these hypoxic conditions for all cell lines  and two TNBC (MDA-MB-231 and MDA-MB-436) cell lines were selected for this study. A2780/A2780cis and PEO1/PEO4 are pairs of chemosensitive/chemoresistant ovarian cancer cell lines and were selected to determine if our observed results differ between chemosensitive/chemoresistant cancer cell lines. The inclusion of two TNBC cell lines, allowed for comparison with another tumor type, which like ovarian cancer, is very prone to a hypoxic tumor microenvironment ll lines .2) and normoxia controls (21% O2), at the 24and 48 h timepoint, were analyzed for detailed N-glycan composition from secreted glycoproteins. Profiles from each cell type are represented in https://glycostore.org/) (p ≤ 0.05).All differential hypoxia cultures (0.5–2% Ore.org/) , exoglycp ≤ 0.05), 0.08–62.02-fold, in all cell lines. To see the specific type of glycans affected, the glycans were pooled into common features (p ≤ 0.05) (p ≤ 0.05). The results for PEO1 were different from all other cell lines, but were not statistically significant  , Table 1 ≤ 0.05) . The mosnificant .p ≤ 0.05) , on triantennary glycans, containing core and outer arm fucose, was found  . This pe ≤ 0.05) .After the effect of hypoxia on DNA methylation and glycosylation of secreted glycoproteins was evaluated, additional cancer cell hallmarks were examined in relation to potential metastatic capability under hypoxic conditions, which could be related to the observed glycomics changes. These features were the epithelial to mesenchymal transition (EMT) and cellular migration. Cellular apoptosis, senescence and autophagy were also examined.2) compared to normoxia cultured cells, migration and EMT were assessed to determine if there was a link between altered glycan structures and changes in migratory capability. Firstly, markers related to EMT were analyzed in all cell lines. Specifically, E-cadherin, which is downregulated in EMT and N-cadherin and vimentin, both known to be upregulated in EMT (p ≤ 0.05) (up to 30.27 times), with an increased trend observed in all other cell lines with the exception of PEO4. Specifically for PEO4, E-cadherin expression was seen to increase (*), indicating a decrease in EMT. The two TNBC cell lines MDA-MB-231 and MDA-MB-436, demonstrated variability in E-cadherin protein expression following hypoxia exposure. E-cadherin protein expression was not detectable in A2780, A2780cis, MDA-MB-231 and MDA-MB-436. N-cadherin expression was not detected in PEO1, PEO4 and MDA-MB-231. Vimentin was not detected in PEO4.Following determination of the glycosylation changes with exposure to hypoxia  except for MDA-MB-436. A 1% hypoxia exposure for 48 h, also increased migration in A2780 and MDA-MB-231 (p ≤ 0.005) and 2% for 48 h in MDA-MB-231 cells (p ≤ 0.05).The Oris™ Cell Migration Assay was used to assess changes in migration potential  followin2), the ovarian PEO1 and TNBC MDA-MB-436 cell lines were the least affected by hypoxia (Looking at both the EMT and migration in hypoxia conditions (0.5–2% O hypoxia .2 percentages applied (0.5%) resulted in more significant increases observed in their EMT and migration potential (p ≤ 0.05)  and lower O ≤ 0.05) .p ≤ 0.05, up to 11.94 times)  . There w4 times) .2, p ≤ 0.05, up to 2.66 times) compared to A2780 cis. Although not significant, PARP cleavage increased in the chemosensitive PEO1 and decreased in the chemoresistant (PEO4) ovarian cells. For the 2 TNBC cell lines, although not reaching significance, there was increased PARP cleavage in MDA-MB-231 with increasing hypoxia, with no PARP cleavage evident in the MDA-MB-436 cells, these changes were from 0.43 up to 2.66 times (PARP cleavage was significantly increased only in A2780 in hypoxia conditions (0.5–2% O66 times .2) , and it was not expressed in MDA-MB-436. The autophagy marker LC3 increased in all cell lines . However, the TNBC cell line MDA- MDA-MB-231, did not express LC3 under these conditions.The senescence marker p21 was significantly decreased in all hypoxic exposure in PEO4 cells with a concomitant increase in the autophagy marker LC3. The senescence markers p16, Rb, and p21 were variably expressed in the cell lines in hypoxia (0.5–2% O2) . For thehttp://genome-euro.ucsc.edu/), we were able to identify transcription factors (GATA2 and GATA3) that potentially regulate MGAT5 and ST3GAL4 in our ovarian cancer cell lines. These tracks included transcription factor CHIP-seq clusters from the ENCODE project [Encyclopedia of DNA Elements,  might have in altering DNA methylation was assessed. Results demonstrated that following differential exposure to hypoxia for 24 and 48 h, significant changes in global DNA methylation were observed in all six cell lines, in comparison to cells cultured in normoxic (21% O2) conditions. Specifically, DNA methylation increased significantly after 24 h, but decreased significantly after 48 h (p ≤ 0.05). This was observed predominantly for the 0.5 and 1% O2 exposure. Observed changes in DNA methylation following 2% O2 exposure were small and not significant  (2 percentages (0.5% O2) was associated with more significant increases in EMT  and cell migration in all cell lines. It is well-documented that hypoxia induces migration and increases tumor cell aggressiveness reflecting the many pathways that are HIF-regulated. The main contributor in the literature appears to be HIF1-α .The metastatic capability of all 6 cell lines was assessed using a combination of the migration assay (Oris cell assay) and determining the relative expression of proteins known to be involved in cell adhesion and cell motility. All cell lines grown in 0.5% hypoxia demonstrated a significant increase in migration compared to cells grown in normoxia (21% O2) . This in2) , 3. In ge HIF1-α , which he HIF1-α . Anothere HIF1-α . The UPRlability . The ER lability –51. The Alterations in markers which are well-established with other cellular events such as apoptosis and senescence were examined. Interestingly, PARP cleavage, indicative of cellular apoptosis, showed an increase in chemosensitive cells (A2780 and PEO1), but a decrease in chemoresistant cell lines  . LC3 autN-linked glycans, specifically MGAT5 and ST3GAL4. Potential TFs, GATA1-3 for these glycosyltransferase enzymes, had previously been identified in silico. An siRNA targeted inhibitory approach was undertaken to see how the expression of these glycosyltransferases was affected. Results demonstrated that the protein expression levels of the GATAs and the glycosyltransferase levels were decreased at 48 h 0.5% hypoxia exposure, but not at 24 h , the expression of the glycosylation enzymes, previously found dysregulated in the ovarian cancer cell line OVCAR3 under demethylation conditions, was investigated  Figure . This li at 24 h .p ≤ 0.05)  . Interes ≤ 0.05) . This wa ≤ 0.05) , where D ≤ 0.05) . GATA3 i ≤ 0.05) . GATA2 h ≤ 0.05) . TherefoN-glycan branching and sialylation, were also decreased at 48 h hypoxia (0.5%) exposure. Potentially, hypoxia may act as a driver of GATA regulation through its altered DNA methylation. While it has be shown that GATA3 is required for the stabilization of a hypoxic microenvironment through binding with HIF1-α, this current study shows the possible role of GATA 2 and GATA3 in altering the glycosylation of tumor glycoproteins, thereby potentially contributing to cell invasiveness. In a recent study, a GATA3-specific DNAzyme (hgd40) was used to inhibit GATA3, which resulted in successfully protecting mice from chronic inflammation caused by ulcerative colitis (in vitro study to investigate DNA methylation, hypoxia and N-glycan profiling together in the in vitro setting, and highlights the complexity of these key integrated tumor microenvironmental features.Global hypermethylation was observed at 24 h and global hypomethylation at 48 h in all cell lines exposed to 0.5–2% hypoxia. Concomitantly, glycosylation on secreted cell glycoproteins changed to highly branched and sialylated glycan structures. These same structures are known to be involved in increased aggressiveness and increased metastatic capabilities . In hypo colitis . A similThe original contributions presented in the study are included in the article/PR, AM, and RS conceived and initiated this study. GG, EL, CH, JC-F, SP, RO'F, and RS carried out the experiments. GG, EL, CH, RO'F, and RS generated and interpreted the results. EL, RP, SM, and RS conducted the statistical analyses. GG, EL, RP, PR, AM, and RS drew the conclusions. GG and RS with significant input from AM wrote the manuscript which was reviewed by all co-authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DKK3 is a secreted protein, which belongs to a family of Wnt antagonists and acts as a potential tumor suppressor in gallbladder cancer. To further understand its tumor suppressor functions, we overexpressed DKK3 in 3 GBC cell lines. We have employed high-resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides to check the downstream regulators. In this study, we reported for the first time the alteration in the phosphorylation of 14 kinases upon DKK3 overexpression. In addition, we observed DKK3 induced hyper phosphorylation of 2 phosphatases: PPP1R12A and PTPRA, which have not been reported previously. Canonical pathway analysis of altered molecules indicated differential enrichment of signaling cascades upon DKK3 overexpression in all the 3 cell lines. Protein kinase A signaling, Sirtuin signaling pathway, and Cell Cycle Control of Chromosomal Replication were observed to be differentially activated in the GBC cell lines. Our study revealed, DKK3 overexpression has differential effect based on the aggressive behavior of the cell lines. This study expands the understanding of DKK3-mediated signaling events and can be used as a primary factor for understanding the complex nature of this molecule. The family of dickoppfs are majorly involved in embryonic development through its interaction and regulation of the Wnt signaling pathway . Multiplmyopathy . In HEK2myopathy . DKK3 ovmyopathy ,8,9. AltProteins account for the larger part of the cell molecular machinery and as a part of organized functional modules and networks. It performs various cellular activities which determine the cell fate . In receGallbladder cancer (GBC) is highly invasive and molecularly heterogenous cancer. The molecular mechanisms underlying the progression of this disease are not well studied. Isocitrate dehydrogenase (IDH), fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (ERBB), KRAS, ROS1 remain to be the most promising therapeutic targets to date ,19. Iden2.3 gallbladder cancer cell lines TGBC24TKB, OCUG-1, G-415 were grown and maintained in DMEM (dulbecco’s modified eagle’s medium) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% COpCS2-hDKK3  and X-tremeGENE HP DNA transfection reagent . Briefly, DNA was diluted in opti-MEM to a final concentration of 1 µg plasmid DNA per 100 µL medium (0.01 µg/µL) and mixed gently. X-tremeGENE HP DNA transfection reagent was added in 1:2 (DNA:reagent) ratio directly into the medium containing the diluted DNA without coming into contact with the walls of the tube. The complex was mixed gently and incubated for 15 min and added to the cell lines with swirling the wells to ensure even distribution over the entire plate surface [TGBC24TKB, OCUG-1, G-415 were used to determine the optimum concentration of  surface . Cell liPart of transiently overexpressed cell lines: TGBC24TKB, OCUG-1, G-415 were harvested using RIPA lysis buffer , 1% Triton-X-100, 0.1% SDS containing protease and phosphatase inhibitor cocktails) after serum starvation and sonicated to extract proteins. Western blot analysis was performed, as previously described, using 30 µg protein lysates . NitrocepCS2-hDKK3  was overexpressed transiently in TGBC24TKB, OCUG-1, G-415 cells for 72 h. The cells were serum starved in serum-free medium for 12 h, and then lysed in cell lysis buffer (2% SDS in 50 mM TEABC (triethyl ammonium bicarbonate). Protein concentration was estimated using the BCA method . Equal amount of protein from control and overexpressed TGBC24TKB, OCUG-1, G415 cells was precipitated using ice-cold acetone.Cell lysis and protein extraction was performed as previously described . pCS2-hDProteins were reduced and alkylated using 5mM DTT (dithiothreitol) and 20 mM IAA (iodoacetamide), respectively. Samples were then digested overnight at 37 °C using trypsin (1:20) . DigesteThe TMT labeled peptides were subjected to stage tip based SDB-RP (styrenedivinylbenzene-reverse phase sulphonate) fractionation to generate 3 fractions, as described previously . For pac3 . Dried peptide fractions were reconstituted in 80% acetonitrile/0.1% TFA. Peptide mixtures were enriched for phosphorylated peptides with 10 µL IMAC beads for 40 min. Enriched IMAC beads were loaded on Empore C18 silica packed stage tips. Stage tips were equilibrated with methanol followed by 50% acetonitrile in 0.1% formic acid and 1% formic acid. The beads with enriched peptide were loaded onto C18 stage tips and washed with 80% acetonitrile in 0.1% trifluoroacetic acid. Phosphorylated peptides were eluted from IMAC beads with 500 mM dibasic sodium phosphate, pH 7.0 .IMAC beads were prepared from Ni-NTA superflow agarose beads  that were stripped of Nickel with 100 mM EDTA and incubated in an aqueous solution of 10 mM FeCl5 and ion filling time set 50 milliseconds. The most intense ions with charge state ≥2 was isolated and fragmented using HCD (higher collision dissociation) fragmentation with 34% normalized collision energy and detected at a mass resolution of 50,000 at 200 m/z. The AGC target for MS/MS was set as 1 × 105 and ion filling time set 100 milliseconds dynamic exclusion was set for 30 s.Enriched phosphopeptides and peptides were analyzed on an Orbitrap Fusion Tribrid mass spectrometer  interfaced with Easy-nLC II nanoflow liquid chromatography system . Peptides were separated on an analytical column  C18) at a flow rate of 280 NL/min using a step gradient of 8–22% solvent B (0.1% formic acid in 90% acetonitrile) for first 70 min, followed by 22–35% up to 103 min. The total run time was set to 120 min. The mass spectrometer was operated in a data-dependent acquisition mode. A survey full scan MS (from m/z 350–1600) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z. The AGC (automatic gain control) target for MS1 was set as 4 × 10The mass spectrometry raw data were searched using Sequest HT search engines with Proteome Discoverer 2.1 (Thermo Fisher Scientific). Phosphopeptide-enriched fractions from each replicate were searched against the RefSeq protein database (version 89). The search parameter used as carbamidomethylation of cysteine residues as a fixed modification. Oxidation of methionine, the phosphorylations of serine, threonine, and tyrosine, were selected as dynamic modifications. Trypsin was set as the protease and a maximum of two missed cleavage were allowed. Precursor mass tolerance was set to 10 ppm, and a fragment mass tolerance of 0.05 Da was allowed. All peptide-spectrum matches (PSM) were identified at a 1% false-discovery rate (FDR). The probability of phosphorylation for each site was calculated by the phosphoRS node in Proteome Discoverer. Only phosphopeptides with >75% site localization probability were considered for further analysis. A 1.5-fold cut-off was used for dysregulated phosphopeptides compared to control cell line. High-confidence PTMs were profiled using PTM-Pro tool .https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis (accessed on 15 August 2020)). Significantly altered phosphopeptides in all the 3 cell lines were used to do the analysis. To generate the core analysis, Ingenuity Knowledge Base (genes only) repository was used. Core analysis was created using direct and indirect relationships and species human criteria. The cutoffs were set to 0.58 and −0.58 for hyperphosphorylated and hypophosphorylated molecules respectively. The rest of the filters were set to default to avoid loss of information and relationships. Core analyses identified the top canonical pathways, upstream regulators, etc., significantly enriched in our datasets. After performing core analysis for each cell line, comparison analysis was performed on the output of core analysis for all the 3 cell lines. The analysis results were schematically replicated using Adobe Illustrator .The core and comparison analysis were generated using IPA . Available online: http://www.kinhub.org/kinmap/index.html (accessed on 9 June 2020)) was used to build the kinome map. The list of identified kinases were plotted and highlighted on the kinome map as described previously [The KinMap tool (KinMap beta. Available online: eviously . Dysreguhttps://casecpb.shinyapps.io/ksea/ (accessed on 1 August 2020)) [z-scores are reported. NetworKIN predictions with scores 2 or greater. A negative score corresponds to a collective dephosphorylation of the kinase’s substrates; the inverse is true for a positive score. Bars that are colored blue or red are kinases whose score met the p < 0.05 score cutoff.The kinase-substrate enrichment analysis was done using the online KSEA tool ) . ProteinOur previous study , identifp-value significant in total dataset. Out of these proteins, 12, 41, 47 of molecules were found to be significantly up-regulated upon DKK3 overexpression in TGBC24TKB, OCUG-1, G-415 cells, respectively, compared to control cells. In addition, 6, 7, 27 of molecules were found to be significantly down-regulated upon DKK3 overexpression in TGBC24TKB, OCUG-1, G-415 cells, respectively, compared to control cells. The list of dysregulated proteins for each cell line are shown in Quantitative proteomic analysis of TGBC24TKB, OCUG-1, G-415 control, and DKK3+ cells resulted in identification of 4646, 4717, and 4419 proteins, respectively. Overall, 1904, 1800, and 2361 molecules were found to be The TMT labeled peptides from control and DKK3 overexpressed cells were enriched using IMAC beads as per manufacturers’ protocol and LC MS/MS analysis was carried out as described in methods section. To increase the reliability of our phosphoproteomic analyses, we included technical replicates in our study. The MS data were processed and searched against databases by SEQUEST-HT algorithms using the Proteome Discoverer 2.1 platform. The probability of Ser/Thr/Tyr phosphorylation sites on each peptide was calculated by the PTM-pro algorithm using a cut-off of >75%. We identified 2382 unique peptides with PTM modifications corresponding to 1110 proteins in TGBC24TKB, with a total of 2130 phosphosites identified: 1907 Ser residues, 211 Thr residues, and 12 Tyr residues . Overallp-value 2.5 × 10−4), Sertoli Cell-Sertoli Cell Junction Signaling , Role of p14/p19ARF in Tumor Suppression  , Sumoylation Pathway , BER pathway , DNA Double-Strand Break Repair by Non-Homologous End Joining , HOTAIR Regulatory Pathway  , Sirtuin Signaling Pathway , ATM Signaling , Cell Cycle: G2/M DNA Damage Checkpoint Regulation , Granzyme B Signaling   A. The to × 10−2) B. The to × 10−3) C.z score = 2) and inactivated in G-415 (z score = −1.667). Simultaneously, pathway leading to Cell Cycle Control of Chromosomal Replication was activated only in OCUG1 cell line with z score of 2.646. The Protein Kinase A Signaling pathway was activated in OCUG-1 (z score = 0.816) and inactivated in G-415 (z score = −1.633)  A. To fur −1.633) B. The meIn our phosphoproteomic dataset, we identified 79 non redundant kinases. In TGBC24TKB, 13 kinases were identified. In OCUG-1, 45 kinases were identified of which, 10 were hyperphosphorylated. In G-415 cell line, 49 kinases were identified, out of which 1 was hyperphosphorylated and 5 were hypophosphorylated. The list of dysregulated kinases along with their phosphorylation sites are provided in DKK3 also known as REIC plays an important role in several biological processes. Dysregulated DKK3 expression is linked to the development of a number of diseases like cardiac hypertrophy , systemiMultiple groups have applied conventional approaches to study the effects of DKK3 in different model organisms, and there are no studies till date which have analyzed downstream effects of DKK3 using high throughput platform. Our proteomics and phosphoproteomics data analysis revealed that alteration was majorly seen at phosphorylation level upon DKK3 overexpression in gallbladder cancer cells. To understand the downstream dysregulated pathways, we analyzed phosphoproteomic data for all the 3 cell lines distinctively. Ingenuity pathway analysis identified molecules based on their functional annotation were significantly associated with 9 molecular and cellular functional categories; RNA Post-Transcriptional Modification, Cellular Development, Cell Death and Survival, Protein Synthesis, Cell Morphology, Cellular Assembly and Organization, Cell Cycle, Gene expression, and Cellular Growth and Proliferation . DKK3 isThe IPA analysis compared the effect of DKK3 overexpression with respect to the invasiveness of the cell lines. Our comparison analysis identified the set of pathways predicted to be differentially activated in GBC cell lines. We identified several pathways which are not linked with the expression of DKK3 before. The Sirtuin signaling pathway and Protein kinase A signaling pathway were predicted to be activated in OCUG-1 cell line compared to G-415 cell line. However, the exact role of DKK3 in these pathways is not fully understood. Among the list of upstream regulators, we identified novel molecules that showed differential activation and are never linked to DKK3 expression before. From our analysis, we identified molecules such as P-TEFb, PPME, SET, CIP2A which showed differential activation pattern upon DKK3 overexpression with respect to the invasiveness of the cell lines. P-TEFb is a kinase that phosphorylates the C-terminal domain of the large subunit (RPB1) of RNA polymerase II and subsequently stimulates productive transcription elongation of multiple cellular genes. It is a master regulator involved in transcription elongation and its dysregulation is involved in multiple diseases including cancer. P-TEFb release and activation by CDK9 inhibitors or by other releasers and activators is known to promote cell growth arrest . ActivatWe further identified potential altered kinases upon DKK3 overexpression that could be validated to deduce the mechanism by which DKK3 acts. Among all the dysregulated kinases, CDKs (cyclin-dependent kinases) were majority. It is well established, a fundamental aspect of cancer is dysregulated cell cycle control and CDK being a major part regulating cell cycle control, our data identified 6 CDKs to be differentially phosphorylated. Among the differentially phosphorylated CDK’s CDK1 is only the kinase, which was hypophosphorylated in our data while CDK16, CDK11A/B, CDK2, and CDK18 were hyperphosphorylated. The exact mechanism for the differential phosphorylation for these kinases needs to be validated. Nevertheless, the result of these alterations is important in studying the tumor suppression function of DKK3. We also observed hyperphosphorylation of serine/threonine-protein kinase PRP4 homolog (PRPF4B) at 3 different positions S257, S292; S294, which is involved in pre-mRNA splicing. We also observed the hyperphosphorylation of kinase involved in myocyte cytoskeletal development: (SPEG), Oxidative Stress Responsive Kinase 1 (OXSR1), etc. A consolidated list for the available small molecule inhibitors that target against dysregulated kinases was adapted from Moret et al., and is depicted in Overall, the data generated from this study greatly expands the understanding of the downstream modulators upon DKK3 overexpression. Our study highlights the list of dysregulated molecules to be important in regulation of various process initiated by overexpressing DKK3. For the very first time the complexities and responses involved upon DKK3 overexpression and its effect on biological functions and cellular phenotype are identified using high-throughput method. We have characterized differential protein expression and phosphorylation events that occur upon DKK3 overexpression. We identified differential activation of regulators and pathways that hold potential significance with respect to DKK3 signaling and needs to be unfolded in time ahead. Our findings help in unraveling the complex biological nature of DKK3 molecule. However, as the next step of this study, a detailed analysis of the molecules identified in the study is needed, which is indeed a limitation for this study. Our study highlights downstream effect of DKK3 overexpression at global proteomic and phosphoproteomic levels in gallbladder cancer cell lines, however the deeper validation of these integrated signaling molecules would provide a more explicit visualization. Furthermore, assessing the involvement of regulatory networks described in our study needs to be widened across multiple cellular and biological models.


Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants  was selected as target and both nucleases were expressed by an egg‐cell specific promoter to assure efficient germline transmission  conferring Imazapyr resistance and one silent point mutation to avoid DSB induction within the donor. For donor excision, the donor molecule is flanked by the respective spacer sequence (Figure LbCas12a or ttLbCas12a, were transformed as T‐DNAs into Arabidopsis thaliana Col‐0 plants via the floral dip method. After transformation, the T0 plants were grown either at 22°C or at 28°C until maturity and the harvested seeds subsequently sown out on Kanamycin selection medium. The resistant T1 primary transformants were grown until maturity at either 22°C or 28°C, respectively. The harvested T2 seeds were sown out on Imazapyr medium to verify ipGT events. At least 90 lines were tested per approach (Figure LbCas12a outperforms the standard enzyme at 22°C by almost twofold. At 28°C, still one third more lines with GT events were detected for ttLbCas12a. The effect is even more pronounced if we take the number of GT events per line into account. For all lines, the individual GT frequencies were calculated by setting the total number of seeds into relation with the number of Imazapyr resistant seedlings. Mean GT frequencies were determined over all lines per approach. At 22°C, ttLbCas12a outperforms the native enzyme by 2.4‐fold and at 28°C still by 1.7‐fold. This is also obvious when we compare the targeting frequencies for the single lines individually for 28°C and for 22°C (Figure ALS gene as target (Wolter et al., LbCas12a are in about half of the cases true GT events indicating that the modified enzyme, ttLbCas12a, is at least as efficient as the native one, which showed perfect GT events in about one third of the cases. Furthermore, for ttLbCas12a at 28°C molecular analysis indicated that 3% of the analysed T2 lines already comprised a homozygous GT event, which did not occur in any other approach in this study.Therefore, we performed ipGT experiments in Arabidopsis testing e Figure . The GT h Figure . As expeC Figure . In prinLbCas12a‐mediated GT in plants (van Vu et al., LbCas12a (Schindele and Puchta, LbCas12a for GT might be even more promising as our data indicate. We were recently able to show that ttLbCas12a provides access to target sites that could hardly be edited at all before by LbCas12a even with a high‐temperature treatment (Schindele and Puchta, LbCas12a (Wolter and Puchta, LbCas12a might increase GT efficiencies at those loci that are more difficult to access even much stronger.Recent results already demonstrated the beneficial effect of increased temperature on The authors declare no conflict of interest.H.P. conceived the research. P.S., F.W., T‐K.H. and L.M. designed and L.M. executed the experiments. H.P., P.S. and L.M. wrote the article.


Dendrobium chryseum Rolfe, which is used in traditional and folk Chinese medicine, the protocorms were raised in Murashige and Skoog (MS) media in three strengths, full strength (FMS), half strength (1/2 MS), and quarter strength (1/4 MS), with or without the phytohormones 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) and coconut water (CW). The comparative cytotoxic activities of the wild and in vitro-raised protocorms were evaluated in human cervical carcinoma (HeLa) and human glioblastoma (U251) cell lines by MTT assay. In in vivo and in vitro, the methanol extracts of D. chryseum showed significant cytotoxic activities. Significant growth inhibition (%) and potent IC50 values were demonstrated in HeLa cell lines (49.79% (210.5 μg/mL) for in vitro-raised Dendrobium chryseum (DCT) versus 46.97% (226.5 μg/mL) for wild Dendrobium chryseum (DCW)). Similarly, activities against U251 cell lines exhibited also significant inhibition (28.76% (612.54 μg/mL) for DCW and 17.15% (1059.92 μg/mL) for DCT). The cytotoxic activities of both, wild and tissue-cultured samples, were superior in HeLa cells. In U251 cells, the wild sample was more active than the tissue-cultured one with a moderate cytotoxic effect. Hence, protocorm culture may therefore be a promising future tool for producing pharmacologically bioactive compounds in medicinal orchids. Such sustainable technology approach will minimize the pressure on the natural population of threatened but commercially important medicinal orchids.From the medicinal orchid  Orchidaceae is a highly developed and extensively distributed monocotyledonous family consisting of terrestrial, saprophytic, and epiphytic species , 2. OrchDendrobium is the second largest genus of the family Orchidaceae in Nepal. Thirty Dendrobium species are found in Nepal, distributed across the tropical, subtropical, and temperate climatic regions [Dendrobium chryseum is an epiphytic herb found throughout the western and eastern Himalayas at elevations from 1000 to 2150 m. It is popularly used in traditional and folk Chinese medicine for its antipyretic, eye benefiting, and immunomodulatory effects. Yang et al. reported the presence of particular compounds such as bibenzyl-phenanthrene and coumarin derivatives in D. chryseum. While the chemical compound isolated from the stem of D. chryseum exhibited antioxidant activity [D. aurantiacum var denneanum (D. chryseum non Rolfe.) possess tumor-inhibitory and blood glucose-reducing effects in vivo [D. chryseum Rolfe demonstrated antioxidant properties and an antidiabetic indication in various studies [in vitro techniques in plant conservation efforts designed to counter the rapid decline in the world's biodiversity [ex situ cultivation is one of the key elements in modern conservation strategies adopted to address the threats of increasing urbanization, population growth, and industrialization [in vitro techniques are highly useful for providing sustainable sources of optimal plant-derived natural products [ regions –17. Dendactivity , polysac in vivo . D. chry studies . Its wil studies –23. Receiversity , 24, 25.lization . Furtherproducts .in vitro [D. chryseum was cultured in vitro to produce protocorms for the assessment of potential cytotoxic activity and comparison of such activities with its wild counterparts. Protocorm formation is the peculiar stage in orchids in which an embryo transforms and develops into a specific spherical structure with unique characteristics. Protocorms undergo growth and differentiation stages during their development into mature plants [D. chryseum, especially for its potential as a source for antineoplastic agents. The objectives of the present study were to establish protocorm culture for the production of bioactive secondary metabolites. Subsequently, isolated extract material thereof was examined for its effect between substances originated from protocorms compared to wild plant material in terms of cell proliferation against two representative cancer cell lines: human cervical carcinoma (HeLa) and human glioblastoma (U251), respectively.In particular, orchid species have been cultured with various techniques to synthesize bioactive molecules in vitro , 35. In e plants , 36. Proe plants . This isD. chryseum, collected from Godavari, Lalitpur, Central Nepal, at 1579 m above sea level, were used as explants to produce protocorms in vitro. The plant was identified by the Nepalese taxonomist Dr. Keshav Rajbhandari as well as from the literature. A voucher specimen of this plant was deposited in the Tribhuvan University Central Herbarium (TUCH) (voucher number P07) in Kathmandu, Nepal. Material from the stems and leaves of the wild plants and the in vitro protocorms were subsequently analyzed for their cytotoxic activities.Immature capsules of D. chryseum for protocorm formation is already described and reported by us and is followed in the present experiment as well [D. chryseum were first washed under running water added by 2-3 drops of Tween-20 for half an hour to remove dust particles and then rinsed off with distilled water for 10 min. Before inoculation, the explants were surface-sterilized by immersing them first in a solution of 1% sodium hypochlorite and then 70% ethyl alcohol for 2 min each. Finally, they were rinsed off with sterile distilled water at least three times. The surface-sterilized capsules were transferred into Petri dishes and dried on a filter paper in a laminar airflow cabinet. Next, the sterile capsules were cut longitudinally into two equal halves using a sterile surgical blade exposing the seeds within  and placed in a sonicator for extraction. Then, the concentrated plant extract was placed in a rotary evaporator until the solvent was dry and the resultant crude extracts of plant materials were stored in a refrigerator at 4°C for later use.The stems of wild plants and protocorms of 2 incubator [4 U251 cells/well and 2 × 104 HeLa cells/well) in 100 μL of the medium and incubated in a 5% CO2 incubator at 37°C for 24 h. Following attachment and cell confluence, the cells were treated with different concentrations of the plant extracts  for 48 h. Crude extracts were diluted by dissolving them in 0.1% DMSO  and the EMEM culture medium. The assay was performed in triplicate. Following 48 h of incubation, the supernatant was removed and 150 μL of the medium was combined with 50 μL of MTT for each well. Following 4 h of incubation, a purple formazan crystal was produced. Then, 100 μL of 0.1% DMSO was added and the plates were incubated for a further 15 min at room temperature to dissolve the formazan crystals. The absorbance of samples was measured with a microplate reader  at a wavelength of 595 nm. A commercially available cis-platin drug was used as positive control. The percentage of cytotoxic activity was calculated by the following formula:The cytotoxic activity of extracts was evaluated in 96-well flat-bottomed microtiter plates (Corning) by using the standard MTT -2,5-diphenyl-tetrazolium bromide) colorimetric assay with a slight modification. For this purpose, HeLa human cervical carcinoma cells and U251 human glioblastoma cells were cultured in T25 flasks in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 1% of penicillin/streptomycin, and 1% L-glutamine and kept at 37°C in a 5% COncubator . The cel50 μg/mL of the extract). The cytotoxic capacities of extracts (IC50) were calculated using a regression equation.A dose-response curve was plotted for each extract to calculate the 50% inhibition of cell growth , FMS fortified with CW (5% and 10%) and MS medium supplemented with 0.5 mg/L BAP alone and in combination with 0.5 mg/L NAA, 1 mg/L BAP + 0.5 mg/L NAA, 1.5 mg/L BAP + 0.5 mg/L NAA, and 2 mg/L BAP + 0.5 mg/L NAA  for DCT and 46.97% (226.5 μg/mL) for DCW while the cytotoxic activities of wild and in vitro samples were nearly the same, being slightly better in the in vitro-grown samples  for DCW and 17.15% (1059.92 μg/mL) for DCT. The cytotoxic activities of wild and in vitro samples differed in such a way that the inhibitory action of samples derived from wild-grown plant material was better than that from in vitro-grown samples. However, in vitro-grown samples still provided substantial inhibition of the investigated human glioblastoma cell proliferation  and NAA (0.5 mg/L) took the most time to germinate. The size of protocorms varied depending upon the nature of the nutrient. Small protocorms were observed on 1/4 MS, 1/2 MS, and FMS media with both 5% and 10% CW. These small protocorms developed directly into shoots. In contrast, the protocorms which developed on FMS supplemented with NAA (0.5 mg/L) and BAP (0.5 mg/L) grew quite large by the 24th week but did not develop shoots. Maximum seed germination occurred on 1/2 MS medium which later developed into young protocorms. The result of mass propagation of D. chryseum by using protocorm culture is already reported by us [Dendrobium species with a high percentage of germination and protocorm formation included 67% MS [D. transparens seeds germinated in vitro on a 1/2 MS recipe with 2.0 mg/L of 6-benzyladenine (BA) [Vanda dearei (45%) and Taprobanea spathulata (92.73%). Based on the germination result, the present study revealed that all different strengths of MS medium favored seed germination up to the protocorm development. A similar result was also found for the in vitro germination of orchid seeds of Phaius tankervilleae and Cymbidium iridioides [In the present investigation, seeds of ed by us . A simild 67% MS . D. tranine (BA) . Half stidioides , 49.D. nobile showed cytotoxicity against human lung carcinoma, human ovary adenocarcinoma, and human promyelocytic leukemia cell lines [D. signatum exhibited cytotoxic activities against human MDA-23 1, HepG2, and HT-29 cancer cell lines [D. chrysanthum inhibited the proliferation of leukemia HL-60 cells [D. longicornu and D. moniliforme showed cytotoxic activities against HeLa and U251 cancer cell lines [Bulbophyllum kwangtungense showed antitumor activities against HeLa and K562 human tumor cell lines [Bulbophyllum odoratissimum was found to have cytotoxic activities against human cancer cell lines, such as human leukemia cell lines K562 and HL-60, human hepatoma BEL-7402, human lung adenocarcinoma A549, and human stomach cancer cell line SGC-7901 [in vitro versus wild plants has been reported.Previous studies revealed that orchids can be a potent source of anticancer agents. For instance, ll lines , 51. D. ll lines . D. chry60 cells . D. longll lines , 5, 35. ll lines  while Buin vitro grown plant samples of D. chryseum on HeLa cells revealed that tissue-cultured protocorms (in vitro samples) were slightly more active than wild plants. Both, however, showed a significant percentage of growth inhibition and highly potent IC50 values in a dose-dependent manner. Upon microscopic investigation, initially cultured HeLa cancer cells before the treatment regimen were elongated and spindle-shaped (D. chryseum extracts play a pivotal role in inhibiting the growth of HeLa cancer cells, whereas, against the U251 cell line, the activity of the wild sample was comparatively better than the in vitro-raised protocorms. Nevertheless, also for this cellular glioblastoma model system, significant percentage of cancer cell proliferation inhibition and corresponding IC50 values were demonstrated.A comparison of the cytotoxic activity of both wild and e-shaped . Soon afe-shaped . This cycis-platin drug was found active against HeLa cells and U251 cells with the lowest IC50 value and applied as the assay control.Similarly, a commercially available in vitro-grown protocorms may provide a good source for obtaining bioactive compounds efficiently.Protocorm suspension cultures have been established for the mass propagation of valuable orchid species , 29, 36 D. chryseum [D. chryseum. One of them, isolated from the stem, exhibited antioxidant activity [in vitro propagation of medicinal orchids and the application of other plant tissue culture techniques provide an avenue for the efficient production of selective metabolites, with medicinal use.A number of studies about the pharmacological properties of plant metabolites support the findings of this study. The majority of plant-based secondary metabolites are phenols, alkaloids, flavonoids, and tannins , 58. Allchryseum . Furtheractivity . The in D. chryseum, as a novel source of active agents with the potential for treating human malignancies. Both wild samples and in vitro-grown protocorm mass of D. chryseum exhibited cytotoxic activities in a dose-dependent manner against selected cancer cell lines of human cervical carcinoma and human glioblastoma, a type of brain tumor. In vitro-cultured protocorms were comparable to wild stems in inhibiting cancer cell proliferation in HeLa and U251 cell lines, respectively. Therefore, protocorm in vitro culture offers a highly suitable process for the production of the investigated pharmacologically useful compounds contained in protocorm samples. Further work on the isolation and pharmacological validation against a range of targets and medical indications of bioactive compounds derived from protocorms is needed. However, this may underscore the general attractive utility that protocorms are a potential source for the commercial production of desirable metabolites from in vitro culture. Such technology implementation would certainly augment to vast extent very much needed efforts to minimize the pressure on the natural population and their entire habitats of threatened but commercially important medicinal orchids in Nepal.The present findings, based on these first cellular studies, establish the orchid species,


To enable computational analysis of regulatory networks within the cancer cell in its natural tumor microenvironment, we develop a two-stage histoepigenetic analysis method. The first stage involves iterative computational deconvolution to estimate sample-specific cancer-cell intrinsic expression of a gene of interest. The second stage places the gene within a network module. We validate the method in simulation experiments, show improved performance relative to differential expression analysis from bulk samples, and apply it to illuminate the role of the mesothelin (MSLN) network in pancreatic ductal adenocarcinoma (PDAC). The network analysis and subsequent experimental validation in a panel of PDAC cell lines suggests AKT activation by MSLN through two known activators, retinoic acid receptor gamma (RARG) and tyrosine kinase non receptor 2 (TNK2). Taken together, these results demonstrate the potential of histoepigenetic analysis to reveal cancer-cell specific molecular interactions directly from patient tumor profiles. The lack of correspondence of experimental models with tumor biology in vivo may lead to only partial understanding of oncogenic mechanisms, ultimately resulting in failures of experimental therapies targeting the oncogene. One illustrative example is the glycosylphosphatidylinositol-anchored cell-surface protein mesothelin (MSLN), whose expression is elevated in many pancreatic ductal adenocarcinoma (PDAC) cases1 and is associated with shorter overall survival2. Most of the current knowledge about MSLN’s function in the context of PDAC comes from mouse or cell-line models, which have shown its upregulation increases cancer cell proliferation, cell cycle progression, and cell survival5; while its interaction with MUC16 contributes to cell motility and invasion7. These models have shown limited utility for developing therapies targeting MSLN: while preclinical studies targeting MSLN in pancreatic cancer cell lines and patient-derived xenograft mouse models have resulted in inhibition of cancer growth9, early-stage clinical trials have shown stabilized disease or increase in survival for only a few patients11, with partial and overall responses being mostly low or absent13.The study of oncogene expression in unperturbed cancer cells within their tumor microenvironment provides insights that may not be gained from other more accessible models that may not reflect as closely the tumor biology in vivo15. This is a particularly important concern for PDAC tumors as they can show an abundant and highly variable stromal component, leading to ambiguous interpretation of bulk expression profiles16. For example, a sample with higher bulk expression of MSLN may either contain cancer cells with higher intrinsic expression of MSLN or the expression of MSLN may be constant and the sample may just have higher purity (proportion of cancer cells), resulting in higher bulk expression of the gene. This ambiguity hampers not only cancer-cell intrinsic differential gene expression analysis, but also the analysis of genes of interest in the context of their cancer-cell-specific regulatory networks.One obvious step toward addressing this problem is to study the oncogene and its network within its natural tumor microenvironment. However, transcriptional profiling of tumors is confounded by sample-to-sample differences in tumor purity and cell-type composition14, but cannot explicitly trace differential expression to specific cell types nor place the gene within its network module. Our previously developed Epigenomic Deconvolution (EDec) method17 partially addresses this problem by comparing cell-type-specific profiles starting from two groups of predefined groups of samples. One obvious limitation of EDec is the requirement that the two groups be predefined. This limitation precludes grouping by cell-intrinsic expression of genes of interest such as MSLN. To address this “chicken and egg” problem, we develop a histoepigenetic analysis method that expands on the EDec method in two ways: (1) It integrates within an iterative procedure a two-way clustering step with deconvolution to separate PDAC tumors based on cancer-cell-specific expression of a gene of interest such as MSLN; (2) It estimates a network of genes potentially interacting with the gene of interest. We here validate the method in simulation experiments, compare it to alternative approaches, and apply it to elucidate MSLN’s network within PDAC cancer cells.One approach toward addressing this problem would be to computationally tease apart the associated cancer cell-type-specific differences between groups of tumors. Numerous computational methods have been developed to account for tumor purity differences15. Per sample estimates of cancer cell proportions for these 150 tumors showed high Spearman’s correlation with previous tumor purity estimates calculated using InfiniumPurify18  and ABSOLUTE15    collection. EDec estimated four distinct cell type profiles, provided their per sample proportions, and provided estimates of cell-type-specific DNA methylation and expression profiles. Based on expression of known cell-type-specific marker genes, the profiles could be unambiguously recognized as belonging to stromal, immune, normal epithelial/acinar, and cancer cell types Fig. . Compari16) Fig. . Likewis16) Fig. . These rMSLN expression—addressing complications introduced by tumor purity differences  differences in several cell-type proportions—notably cancer—between groups .We next compared the result of iterative grouping to grouping based on bulk profiles. For this purpose, we split samples into three quartiles for each method and compared the samples in the top (first) and bottom (third) quartile for each method. While the overlap was high Fig. , the excMSLN high expression by both groupings and the sample set showing concordant MSLN low expression also by both groupings , TNK2 (ref.22), CAPG23, and MUC1 (ref.24). Gene set enrichment analyses (GSEA) of these 33 genes identified a highly enriched subset of 11 genes bound by retinoic acid receptor gamma (RARγ/ RARG) in mice25, including MSLN and RARG itself  reduced RARG transcription  stood out for their previously demonstrated ability to phosphorylate and activate AKT. Specifically, ACK1 activates AKT in pancreatic cancer where it also promotes cell survival and correlates with disease progression22. On the other hand, RARγ is known to activate AKT in hepatocellular carcinoma28. These findings were intriguing in light of the previous results implicating MSLN in the activation of AKT in PDAC cell lines via phosphorylation5 by an unknown mechanism.Among the 33 genes in the MSLN in the cell lines with high MSLN expression. As predicted, the knockdown reduced RARγ and ACK1 levels  at both residues (S473 and T308) necessary for AKT activation29. Upon MSLN overexpression in cell lines with low MSLN expression, protein levels of RARγ and ACK1 were increased  overlap was found with genes previously shown to be activated by AKT in vitro, suggesting that the in vitro cell line modeling indeed reflects the behavior of cancer cells in vivo.We next asked if the downstream effects of MSLN-mediated activation of AKT observed in cell line studiesMSLN network module, validate direct interactions of MSLN with TNK2 and RARG within the module, and identify additional genes relevant for understanding MSLN function in cancer cells. Toward this goal, we inferred a Gaussian graphical model (GGM) from the 150 bulk PDAC tumor expression profiles in the TCGA collection . To eliminate confounding due to variations in tumor purity, cell-type proportions (inferred by deconvolution) were added as covariates . The final GGM included the 367 nodes corresponding to the genes differentially expressed between MSLN-high and low tumors within any of the four estimated cell types  stage of histoepigenetic analysis  enrichment of connections between RARG and genes with known motifs for RARγ30 termed retinoic acid response elements (RAREs)  .We then focused on the 88 genes showing differential cancer cell expression between 47) Fig. . Moreove30 and those associated with parvin-beta (PARVB) by GSEA. Significant connectedness was indeed found for both sets , suggesting that the method is sensitive. In contrast, as expected, three random gene sets did not show significant connectedness . Specifically, RARγ regulates the balance of self-renewal and differentiation32. In the developing pancreas, retinoids affect ductal versus acinar differentiation27. Treatment of some pancreatic cancer cell lines with retinoids induces apoptosis, epithelial differentiation, cell cycle arrest, and reduces cell viability35. Some of these effects are mediated by RARγ as observed in the BxPC3 and Capan2 cell lines33, the same cell lines in which we showed reduction of RARγ upon MSLN knockdown. Conversely, cell lines with low RARγ appear resistant to this effect33. Moreover, patient PDAC tumors show reduced levels of retinoids compared to normal counterparts36. Thus, MSLN’s observed effect on pancreatic cancer cell proliferation in vitro4 may partly be mediated by RARγ and its ability to regulate the balance of self-renewal and differentiation depending on the concentrations of retinoids present.Our findings complement current understanding of RARγ and retinoids as important players in PDAC biology and normal development. Because RARγ expression is reduced upon 7, which is associated with worse prognosis in PDAC patients37. Similar to the effects of MSLN in PDAC cell lines5, activated AKT inhibits apoptosis, is involved in cell cycle progression, and mediates cell proliferation38. Our results expand upon the notion that MSLN may activate AKT, suggesting that this activation may be mediated through RARγ or ACK1. Moreover, because MSLN inhibition decreases phospho-AKT levels in other cancers with elevated MSLN levels39, our results may also be relevant for cancers other than PDAC.Another important member of the MSLN network is AKT. There are conflicting reports in the literature regarding the ability of MSLN to affect the phosphorylation and activation of AKT in PDAC cell linesMSLN network have significant implications for current PDAC drug discovery efforts targeting MSLN, RARγ, and their network neighborhood. Specifically, the limited benefit of targeting MSLN may be examined in the context of the tumor-specific state of the downstream mediators suggested by our study. Moreover, the pattern of interactions within the MSLN module has implications for utilizing retinoids and ACK1 inhibition as therapeutic strategies. Pilot clinical trials utilizing retinoids—most not selective for RARγ—have shown limited or no effects, with only some pancreatic cancer patients showing prolonged stable disease41. Preclinical trials showing that inhibition of ACK1 in PDAC cell lines has resulted in AKT suppression and subsequent decrease in cell proliferation, cell cycle arrest, and induction of apoptosis22. Our results suggest that these therapeutic targets should not be viewed in isolation but rather in relation to one another. In addition to the already tested targets, the MSLN network module includes new candidates that may play important roles. Finally, the suggested interactions within the MSLN network can be utilized to identify candidate biomarkers for stratification of patients or as targets for combination therapies.The interactions within the 15, were downloaded from UCSC Xena42. For the methylation dataset, probes with a missing beta value for any individual sample or falling on sex chromosomes were removed. Problematic 450 K probes43 were filtered using the mapping, typeINextBaseSwitch, snp5.common, extBase, and sub30.copy masks from http://zwdzwd.io/InfiniumAnnotation/current/hm450/hm450.hg38.manifest.tsv.gz and multi-mapping, cross-reactive, and probes containing common SNPs from https://github.com/sirselim/illumina450k_filtering. Probes associated (FDR < 0.01) with gender, radiation therapy history, history of diabetes, history of pancreatitis, smoking status, and history of targeted molecular therapy were determined by fitting an analysis of variance model using R’s aov function and removed. As were those associated with age and weight, determined by fitting linear models using R’s lm function.Level 3 DNA methylation, RNA-seq (HiSeqV2), and clinical metadata datasets from the TCGA Pancreatic Cancer cohort (PAAD), not flagged during previous reviewEDec R package was used to infer cell-type-specific methylation and gene expression profiles as previously described17. 500 informative probes were chosen by running the run_edec_stage_0 command with the “one.vs.rest” option and p value < 0.00001 for six selected reference groups using available methylation profiles from NCBI’s Gene Expression Omnibus44 (GEO)  were downloaded from GEO, QC’d using FastQC, processed using Trim Galore, and aligned using RSEM. The RNA-seq profiles were multiplied by the same proportions as their matched methylation mixtures for all 100 mixtures and reads for each gene per mixture summed to create the mixture’s bulk profile. The iterative group fit method was used to group the RNA-seq mixtures around MSLN expression. Splitting was performed 20 times to check stability and all possible pairwise correlations of the final group members across these runs were compared. Groups were then deconvoluted separately.Methylation datasets for four cell types  were downloaded from GEO . Noisy profiles were generated for each dataset and then the immune, fibroblast, and one of two cancer profiles were mixed in random proportions as previously describedMSLN high (n = 30) and low (n = 35) groups were determined by overlapping samples classified by both the bulk MSLN and the iterative group fit models. The deconvoluted cancer cell profiles for the groups were compared for differential expression using t-tests with the estimated means and standard errors of genes as previously described and with the same caveats17. Bulk differential expression between groups was done with raw  RSEM gene counts downloaded from NCI’s GDC Legacy Archive  using the DESeq2 R package45 with groups (MSLN high or low) with or without EDec-predicted cancer cell proportions as factors. All comparisons used a fold change ≥ 2 and false discovery rate (FDR) q value < 5%.For the TCGA samples, MSLN high group were used for gene set enrichment analysis using the Molecular Signatures Database (MsigDB) web site version 6.3 (ref. 46). The top ten overlaps of these genes were computed against each of the C1–C6 gene set collections using an FDR q value < 0.05. The 689 genes that showed higher expression in the MSLN high group at a p value < 0.05 were compared to the gene list AKT_expression_UP from MsigDB. Enrichment for the 13 gene overlap with the 172 AKT genes was computed using a hypergeometric test.The 33 genes with higher expression in the cancer cell profile of the huge47  and GeneNet48  were used to build networks over log2 transformed RNA-seq values for all 150 TCGA tumor samples on the 367 differentially expressed genes and the proportions of each cell type per sample. Positive controls were a set of 7 RARG related genes and a set of 21 genes related to PARVB expression and negative controls were randomly selected gene sets of size 7, 21, and 88  method (λ = 0.125). GeneNet created two networks, one with all edges and one with the same number of edges present in the huge network. To eliminate confounding due to variation in cell type composition, a node representing cell proportion was added  of a subset of nodes in a network, giving highly connected subsets in the network stronger significance compared to sparsely connected node subsets.The R package MSLN high cancer cells and MSLN and (2) 5 genes with RAREs in their regulatory regions30 and RARG  by at least one standard deviation (σ = 0.0297) were considered as being strong.To assess the directionality of the edge weights, two sets of edge weights were evaluated between: (1) Genes predicted to be differentially expressed in CTD was used to query the cancer, stromal, and immune specific dysregulated genes, as well as the 3-gene subset of MSLN, RARG, and TNK2 for modularity. Enrichment of edges was computed using a hypergeometric test.MSLN overexpression stable cell lines of Panc1, Panc28, and Panc48, as well as MSLN shRNA knockdown cell lines of Capan2, CFPAC1, and PL45 were established and maintained as previously described50. ASPC1, BxPC3, and SU86.86 cells were cultured in RPMI 1640 ; CFPAC1, HPAF-2, MIA Paca2, Panc1, Panc28, Panc48, and PL45 cells were cultured in DMEM ; Capan2 cells were cultured in McCoy’s 5 A medium . All cells were supplemented with 10% fetal bovine serum  and cultured at 37 °C with a humidity of 90–95% and 5% CO2.PDAC cell lines were purchased from ATCC, authenticated by short tandem repeat profiling, and propagated for less than 6 months after authentication. 51. RARG and MSLN measurements were normalized to GAPDH in the PDAC cell panel and relative to controls in the overexpression and knockdown comparisons. Primer sequences (5′–3′):Total RNA for each cell line was extracted using TRIzol reagent  according to the product’s manual. cDNA synthesis was performed with a reverse transcription-PCR kit (Bio-Rad) and real-time PCR was performed by standard procedures as previously describedMSLN forward CTATTCCTCAACCCAGATGCGT and reverse GCACATCAGCCTCGCTCA,Human RARG forward CAAGTGCATCATCAAGATCGTG and reverse GATACGCAGCATCAGGATATCT,Human GAPDH forward TCGACAGTCAGCCGCATCT and reverse CCGTTGACTCCGACCTTCA.Human Cell lysates were prepared using RIPA lysis buffer with protease and phosphatase inhibitor cocktail and were quantitated for protein abundance using the Pierce BCA Protein Assay Kit from Thermo Fisher Scientific. PVDF membranes were blocked in 5% nonfat dry milk/TBST solution and incubated at 1:1000 dilution with: MSLN mAb (D9R5G), RARγ1 mAb (D3A4), AKT Ab #9272, Phospho-AKT (Ser473) mAb (D9E), Phospho-AKT (Thr308) mAb #2965, and Phospho-ACK1 (Tyr284) Ab#3138 from Cell Signaling Technology and ACK1 antibody (A-11) from Santa Cruz Biotechnology. Anti-β-actin Ab (A2228) was at 1:2,000 dilution (Sigma-Aldrich). Immunodetection was performed using SuperSignal West Pico (34577) and Femto (34095) Chemiluminescent Substrate according to the manufacturer’s instructions .https://www.R-project.org/). P values were adjusted for multiple comparisons using the Benjamini & Hochberg method. Survival analyses were done with the R package survival, including multivariate Cox regression using variables (Supplementary Table p values < 0.05 in univariate Cox regression. Real-time PCR results were representative of three independent experiments and compared with two-tailed unpaired Student’s t-test with Welch correction when sample variances were not equal as defined by the Brown–Forsythe test using GraphPad Prism Software Version 6 and 7.Unless otherwise noted, all statistical analyses and bioinformatics were performed using the R programming language version 3.4.0 (Supplementary InformationSupplementary dataSupplementary dataSupplementary dataSupplementary data


Expression of telomerase reverse transcriptase (TERT) and telomerase activity (TA) is a main feature of cancer, contributing to cell immortalization by causing telomeres dysfunction. BIBR1532 is a potent telomerase inhibitor that showed potential anti-tumor activities in several types of cancer, by triggering replicative senescence and apoptosis. In a previous work, we detected, for the first time, TERT expression and TA in preclinical models of feline oral squamous cell carcinoma (FOSCC); therefore, we aimed at extending our investigation by testing the effects of treatment with BIBR1532, in order to explore the role of telomerase in this tumor and foreshadow the possibility of it being considered as a future therapeutic target. In the present study, treatment of FOSCC cell lines SCCF1, SCCF2, and SCCF3 with BIBR1532 resulted in successful inhibition of TA, with subsequent cell growth stoppage and decrease in cell viability. Molecular data showed that up-regulation of cell cycle inhibitor p21, unbalancing of Bax/Bcl-2 ratio, and down-regulation of survival gene Survivin were mostly involved in the observed cellular events. Moreover, BIBR1532 diminished the expression of TERT and its transcriptional activator cMyc, resulting in the down-regulation of epidermal growth factor receptor (EGFR), phospho-ERK/ERK ratio, and matrix metalloproteinases (MMPs)-1/-2 and−9, likely as a consequence of an impairment of TERT extra-telomeric functions. Taken together, our data suggest that BIBR1532 exerts multiple anti-cancer activities in FOSCC by inhibiting telomerase pathway and interfering with signaling routes involved in cell proliferation, cell survival, and invasion, paving the way for future translational studies aimed at evaluating its possible employment in the treatment of this severe tumor of cats. Telomerase is a holoenzyme that extends telomeres, the terminal DNA sequences of chromosomes . This fuIn somatic cells, TERT gene is not transcribed, and telomerase is not active; therefore, telomeric ends shorten at each cell duplication until, after a scheduled number of replications, they reach a critical point of erosion . The celInstead, cancer cells are prone to maintain a highly proliferative rate; therefore, it is not surprising that TERT expression and telomerase activity (TA) represent a common feature of most of human malignancies . A main Due to this multilevel role in carcinogenesis, different strategies are under investigations for telomerase inhibition, such as gene silencing through siRNA or treatment with small molecules . BIBR153Telomerase inhibition represents a promising therapeutic approach for human oral squamous cell carcinoma (hOSCC), where the role of this enzyme is prominent –18. Then5 density in 6-well plates, and after 24 h, BIBR1532 diluted at 25, 50, and 100 μM in culture medium was added to wells. In control plates, drug was replaced with the same amount of DMSO for each dose. After 48 h, wells were read under ZOE Fluorescent Cell Imager (Bio-Rad Laboratories) for scanning and phase contrast photography, and then cells were harvested by trypsinization and analyzed as described below.FOSCC cell lines SCCF1, SCCF2, and SCCF3 developed in the Rosol laboratory are a kind gift from Professor T.J. Rosol (The Ohio State University) and were cultured as previously described , 25, 27.Cell pellets from treatments at 25, 50, and 100 μM for 48 h were resuspended in phosphate buffered saline , and then equal amounts of cell suspension and 0.4% trypan blue (Lonza #17-942E) were mixed, incubated for 1–2 min at room temperature (rt), and counted by the use of Cell Counter (Bio-Rad). Changes in cell viability and growth at each dose of treatment with BIBR1532 were calculated and expressed as percentage compared with the respective control plate treated with DMSO set as 100%.TA in FOSCC cell lines treated at 25, 50, and 100 μM for 48 h was assessed by a telomeric repeat amplification protocol (TRAP) assay by using TRAPeze® Telomerase Detection Kit (Merck #S7700) as previously described . ReactioTotal protein extraction, protein quantification, sodium dodecyl sulfate (SDS)-PAGE, and Western blotting (WB) on cells treated at 50 or 100 μM for 48 h were performed as reported elsewhere . Detecti−ΔΔCt method by using Bio-Rad CFX Manager software  to cDNA, and real-time quantitative PCR (qPCR) for feline EGFR, TERT, and cMyc were performed on cells treated at 50 μM for 48 h, as previously described , 31. Forsoftware . Changest-test was performed using SPSS 17.0 software (SPSS Inc.), and differences were considered to be statistically significant for *P < 0.05 or **P < 0.01.For statistical analysis, Student's BIBR1532 blocks TA by specifically limiting the amount of nucleotide repeats available for substrate elongation; this activity has been detected in numerous preclinical models of cancer; however, it has never been tested in FOSCC cell lines . In ordeThen, to investigate the impact of telomerase inhibition by BIBR1532 on these cell lines, cell growth and viability were evaluated under the same experimental conditions. The treatment induced growth inhibition in SCCF1, SCCF2, and SCCF3 in a dose-dependent manner ; notablyTo further evaluate the possible effects of BIBR1532 on cell morphology, cells were scanned by phase contrast microscope. In agreement with growth arrest data, the number of cells observable in treated wells decreased in a dose-dependent manner, and interestingly, morphologic changes were also visible upon BIBR1532 incubation in all cell lines: unlike untreated cells, growth-arrested SCCFs became flattened, enlarged, with intercellular bridges and increased granularity, often multi-nucleated and had vacuolated cytoplasm .Treatment with BIBR1532 of different cancer cell lines causes cell cycle arrest and apoptosis through activation of transcriptional pathways triggered by telomeres dysfunction, such as the increase of cell cycle inhibitor p21 and the unbalancing of Bax/Bcl-2 ratio –38. To fIn SCCF1, the highest increase of p21 gene expression was yielded at 25 μM, and this was followed by a gradual decrease at 50 and 100 μM, whereas a dose-dependent effect was observed in treated SCCF2 and SCCF3 cells ; howeverWhen analyzing Bax and Bcl-2 expression and their molecular ratio, the following results were observed: SCCF1 showed a dose-related reduction of Bcl-2 at 25 and 50 μM but not at 100 μM ; howeverBIBR1532 affects cell survival in leukemia cell lines by reducing the expression of the Survivin gene; therefore, its mRNA levels were also investigated in treated SCCF cells , 38. In In addition to the canonical inhibitory effects on TA, treatment with BIBR1532 has been shown to down-regulate the expression of cMyc in a series of cancer cell lines, resulting in decreased TERT transcription –39. ConsBeyond its role as catalytic subunit of telomerase, TERT exerts several extra-telomeric functions; among these, transcriptional regulation of EGFR is of particular relevance, especially in cancer , 5, 6. HTreatment with BIBR1532 decreases the protein expression of several MMPs in different types of cancer cells, through down-regulation of TERT , 9. To aWith the present study, we aimed at expanding our ongoing investigation on the role of TERT and TA in FOSCC by treating tumor cell lines with the specific inhibitor BIBR1532 and evaluating its potential anti-cancer activities . In SCCFFelis catus Papillomavirus type-2 E6, thus providing a likely explanation to the different degrees of activation of downstream genes between the two cell lines (Replicative senescence is triggered by telomeres attrition through up-regulation of the cell cycle inhibitor p21, and optionally, it may shift to apoptosis through unbalancing of Bax/Bcl-2 ratio . Insteadll lines . Howeverll lines . Neverthll lines , 38; ourll lines .Taken together, these data suggest that inhibition of TA by BIBR1532 in SCCF cells switches off cell proliferation and, despite with variable degrees and molecular responses, promotes cell death mostly by engaging p53-dependent pathways triggered by telomeres shortening, in agreement with literature data , 49–51. TERT gene is under transcriptional regulation by cMyc, a well-known oncogene over-expressed in FOSCC and previously detected in SCCF cells , 26, 52.in vitro. Considering the main role of EGFR in the proliferation of FOSCC-derived cells (Regulation of EGFR is among the non-canonical functions of TERT , and inted cells , it is lFOSCC is characterized by highly invasive phenotype and metastatic potential, possibly due to the expression of MMP-1/-2 and−9 as in the human counterpart . It has in vitro models of cancer (In conclusion, this work demonstrates that BIBR1532 exerts a series of anti-cancer activities linked to the inhibition of canonical telomerase pathway and TERT extra-telomeric functions in FOSCC cell lines see , paving f cancer , 49, 53.The original contributions presented in the study are included in the article/GA, LL, KP, and GDL performed the experiments. GA, GB, and PM drafted the manuscript. GA, GDL, PM, GG, BDU, and GB conceived the study. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Klotho has been recognized as a gene involved in the aging process in mammals for over 30 years, where it regulates phosphate homeostasis and the activity of members of the fibroblast growth factor (FGF) family. The α-Klotho protein is the receptor for Fibroblast Growth Factor-23 (FGF23), regulating phosphate homeostasis and vitamin D metabolism. Phosphate toxicity is a hallmark of mammalian aging and correlates with diminution of Klotho levels with increasing age. As such, modulation of Klotho activity is an attractive target for therapeutic intervention in the diseasome of aging; in particular for chronic kidney disease (CKD), where Klotho has been implicated directly in the pathophysiology. A range of senotherapeutic strategies have been developed to directly or indirectly influence Klotho expression, with varying degrees of success. These include administration of exogenous Klotho, synthetic and natural Klotho agonists and indirect approaches, via modulation of the foodome and the gut microbiota. All these approaches have significant potential to mitigate loss of physiological function and resilience accompanying old age and to improve outcomes within the diseasome of aging. Klotho  (FGF23) ; it is t (FGF23) .The Klotho protein that was identified as the FGF23 co-receptor was the α-Klotho isoform, which hereafter is termed Klotho. Klotho is expressed mainly on the cell surface membrane of proximal and distal renal tubules –20, thouKlotho regulates many pathways involved in aging processes, such as the regulation of phosphate homeostasis, insulin signaling and Wnt signaling , 22. AddKlotho expression levels and its circulating level decline during aging. In humans, Klotho deficiency features medial calcification, intima hyperplasia, endothelial dysfunction, arterial stiffening, hypertension, impaired angiogenesis, and vasculogenesis  .As Klotho-deficient phenotypes have been attenuated and rescued by Klotho gene expression, or supplementation, it is suggestive that Klotho has a protective effect with regard to the vasculature .Mice deficient in Klotho exhibit a phosphate imbalance and hyperphosphatemia due to impaired urinary phosphate excretion, but significantly develop a complex progeric phenotype including poor growth, atrophy of multiple organs, vascular calcification (VC), sarcopenia, cardiac hypertrophy and fibrosis, cognitive impairment, and shortened lifespan , 2, 5. CAs Klotho has been regarded as an anti-aging gene, in order to more fully understand how Klotho might impact age-related disease, this review focuses on what we actually mean by the aging process, its relevance to chronic kidney disease (CKD) as a disease of accelerated aging, and how this is impacted by Klotho. Furthermore, we discuss how modulation of Klotho might be exploited to mitigate the effects of the diseasome of aging.Rather than simply being a collection of morbidities arising during the final decades of life, aging is an active process across the entire life-course. It has been described as an accumulation of physiological and molecular deficits accruing at different rates, both within different tissues and organs in the same individual and between different individuals . Aging lIn mammals, aging has a number of distinct additional features to its common hallmarks, comprising phosphate toxicity, diminished global Nrf2 expression, and microbial dysbiosis . These fSerum phosphate (Pi) levels shows an exceptionally strong negative correlation with lifespan in mammals , 36, PhoPertinent to these observations are observations of Klotho signaling independent of FGFR4 in the heart, liver, and lung , 42. In Cyp24a1, which encodes 24-hydroxylase, responsible for the conversion to inactive vitamin D by a Klotho dependent mechanism , which are colloids comprised of calcium-Pi bound to fetuin A, a circulating inhibitor of vascular calcification , 37, 44.echanism . Cy27b1 n of PTH . Serum ln of PTH , 51. Notn of PTH . This haCKD is part of the diseasome of aging and displays an overlapping phenotype with Klotho-deficient mice. It has been suggested that Klotho function is tightly correlated with the pathogenic mechanisms underpinning CKD , 53–55.CKD is a progressive systemic disease that affects 10–12% of the population . The preChronic low-grade inflammation also contributes to the premature aging phenotype in CKD  and othein vivo  treatment. Accordingly, the stable delivery of soluble Klotho has been reported to reduce chronic hyperphosphatemia and VC both in vitro and in vivo  and renal fibrosis have both been associated with diminished Klotho expression. Studies in a mouse model of CKD-MBD have indicated that loss of Klotho expression is a key event in renal and bone injuries and that restoration of Klotho expression attenuated CKD related bone defects . These oin vivo  in keepiThe weight of basic science, pre-clinical, and clinical evidence suggests that modulation of Klotho function is an exciting and viable target for therapeutic interventions. As such, it is a prime therapeutic target for intervention in a range of diseases within the diseasome of aging, including CKD , 123. GiAs such, Klotho-mediated therapies may be classed as geroprotective , and senomorphic (i.e. suppression of senescence effects without cytotoxicity). It is important to note, however, that the context of their application and time in the life course applied are key elements for any success. While most basic and pre-clinical studies have focussed on mid- to end of life application, data from early life interventions are sparse and require a more robust evaluation. This is pertinent to the concept of antagonistic pleiotropy, as aging is a continuous process occurring across the life course, thus suggesting that the effects of any interventions will be dependent on the context of the stage of the life course at which they are applied. Cellular senescence is likely to be subject to antagonistic pleiotropy, as senescent cells are not desirable in young individuals, but are essentially anti-oncogenic in the old. The potential for any intervention to generate adverse effects, especially subtle epigenetic or cryptic effects, thus needs evaluated from this perspective. Initial evidence, however, suggests that most interventions appear to be well-tolerated and have some beneficial effective. Indeed, over-expression of α-Klotho has been reported to reverse the phenotype and extended life-span in Klotho-deficient mice , 124.Targeting Klotho in CKD is particularly pertinent on a number of fronts, Firstly, accurate detection of CKD at an early stage has proven problematic and currently no biomarkers are available which are suitably accurate, easily measured and sensitive enough . CurrentSeveral studies have suggested that pharmaceutical replacement or supplementation of Klotho can improve complications associated with CKD . Pre-cliActivation of Klotho has been reported to reduce levels of oxidative stress , improvein vitro by modulation of Klotho promoter expression , a DNA methyltransferase inhibitor, to increase Klotho expression ~1.5- to 3-fold pression . While tDeacylation: Inhibition of histone deacetylases using HDAC inhibitors, such as trichostatin A or valproic acid have been shown to reduce inhibition of Klotho expression by TNF/TWEAK signaling in the kidney .in vitro. This induction was blocked by PPAR-c antagonists or by small-interfering RNA-mediated gene silencing of PPAR-c, suggesting this is a PPAR-c-dependent mechanism  receptor is also important in the aging process. Activation of the AT1 receptor by AngII is implicated in the age-related developments of hypertension, diabetes, and kidney disease. Disruption of the AT1 receptor gene in mice extends life-span and lowers levels of oxidative stress . AngioteVitamin D derivatives e.g. paricalcitol: which has been shown to induce Klotho expression in rodent CKD models , 146. AnExogenous recombinant peptide has also been used to raise soluble α-Klotho concentrations , 148 andBlockade of FGF23 using Burosumab, has been demonstrated to be effective in control and improvement of rickets in patients with X-linked hypophosphataemia , 151. UsNumerous studies have demonstrated the effectiveness of exercise to prevent, reverse, or attenuate premature aging , 154. ExNumerous compounds have been approved for use in CKD but with only some positive outcomes seen suggesting the extent of reactivating and or replacing Klotho and its effects are yet to be clarified .A number of studies have intimated that Klotho expression is amenable to nutritional intervention. Age-related changes in Klotho gene expression, particularly renal Klotho expression, and vitamin D metabolism can be affected by dietary Pi intake. As such, nutritional acquisition of Pi can radically affect age-related health, which opens up an easy avenue for therapy. There is good rationale for such an approach, as Pi has been linked to accelerated biological aging  and poorer renal function  Higher aOther pre-clinical studies have indicated that a high fat diet (HFD) as a surrogate for an energy rich and calorie dense western diet, results in age-related alterations in renal α-Klotho expression that could affect the responsiveness of dietary phosphate to vitamin D metabolism .Alternative nutritional interventions have been designed to modulate Klotho-FGF23 inter-activity in CKD and thus address significant features associated with its pathophysiology including hyperphosphatemia, iron deficiency, and anemia [reviewed by ]. Interv3 and calcium levels were unaffected. However, the high magnesium reduced bone mineral density and presented treated animals with possible osteomalacia security and FG23 levels. In young adults studied in the context of coronary artery risk development, this has been tested under the hypothesis that food insecurity would be associated with intake of food and drinks with higher levels of phosphate additives, with potential impact on increased levels of FGF23 as a compensatory response to higher dietary phosphate intake . Pool etp = 0.001; I2 of 36%) in vitamin D deficient individuals.Secondary analysis of the placebo-controlled Styrian Vitamin D Hypertension trial  trial inn = 74) enrolled in the FIT-AGEING study, a negative association has been demonstrated between total alcoholic intake and S-Klotho plasma levels , even when adjusted for body composition parameters (in vitro (NRK-52E cells) and in vivo (mouse kidney) with involvement of the ATF3/c-Jun pathway  has not featured as a mediator of such effects, though this is intuitive with respect to β-Klotho, as its FGF19 substrate is secreted by bile acids produced by gut microbiota (The gut microbiota has emerged as a significant contributory factor to age related health [reviewed in ], where crobiota .There is also an emerging relationship between the gut microbiota and the vitamin D/FGF23 axis. In murine models, Bora et al.  have demThe emerging field of Geroscience seeks to address the cellular and molecular mechanisms underpinning the aging process and to understand how, when and why these become dysregulated, as the diseasome of aging manifests. Modulation of Klotho has emerged as a prime target for interventions within this space, having the potential to complement and synergise with other emerging therapies, including senolytics and live biotherapeutics, as well as more conventional pharmaceuticals. The ability to couple any emerging therapies with nutritional and exercise based approaches, offers a more holistic means of translating these to the general population and thus improving healthspan. Applications to age related diseases, such as CKD are exciting, and have already demonstrated clinical benefit with more promised to come.All authors contributed to formulation, writing, discussion, and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest