In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/β-catenin activity through blocking β-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this  Together these properties allow selective passage of molecules into and out of the brain and thus provides the proper microenvironment to support brain function6.Brain microvessel endothelial cells are the cellular interface separating the blood and its constituents from brain extracellular fluid. Together with astrocytic foot processes, pericytes, and basal lamina, the brain endothelial cells form the blood-brain barrier (BBB). Brain microvessel endothelial cells are characterized by the presence of tight junction proteins, an assortment of uptake and efflux transporters and reduced vesicular transport processes9. Dysregulation of Wnt/β-catenin pathways has also been implicated in various CNS disorders that involve BBB breakdown including multiple sclerosis10, stroke11, Alzheimer’s disease12, Huntington’s disease13 and brain tumors14. Modulating Wnt signaling pathways is of interest therapeutically, however as a target, Wnt remains challenging due to the complexity of the pathways and its multiple players. There are two different signaling pathways that Wnt can activate. The first is the canonical Wnt pathway, also known as the Wnt/β-catenin signaling pathway. The second is the non-canonical Wnt pathway that is divided into a Wnt/planar cell polarity and Wnt/Ca2+ utilization pathway15. The complexity scales up as the proteins involved in Wnt/β-catenin signaling have multiple isoforms. For example, there are ten Wnt receptors (Frizzled 1–10), four Wnt co-receptors , nineteen Wnt ligands (Wnt 1–16) and ten Wnt modulator peptides 16. Little information is available regarding the distribution, function, and action of each isoform in the brain microvasculature.Activation of the Wnt/β-catenin pathway promotes the formation of tight junction proteins and transporters in the developing brain capillaries20.Wnt canonical signaling involves binding of Wnt ligand to the Frizzled (Fzd) receptor and co-receptor LRP5/6. Upon binding, the cytoplasmic tail of LRP5/6 is phosphorylated. Binding of Wnt ligand initiates docking of Dishelleved (Dvl) to Fzd and further recruitment of the β-catenin destruction complex  to the plasma membrane. In the inactive state, the β-catenin destruction complex is located in the cytosol where it can efficiently process β-catenin for proteosomal degradation. Recruitment of β-catenin destruction complex to the plasma membrane upon Wnt activation leads to a stabilization of β-catenin. The increased levels of β-catenin in the cytosol results in its translocation to the nucleus where it acts as to modulate Wnt/β-catenin target gene transcription22, less is known regarding the role of Wnt in the maintenance of BBB integrity in mature animals as well as Wnt/β-catenin activity in human BBB. The immortalized human brain microvessel cell line, hCMEC/D3, is a commonly used human in vitro BBB model28. Using hCMEC/D3, several laboratories have determined that Wnt/β-catenin signaling regulates P-glycoprotein (Pgp) expression30. However, comprehensive characterization of the extent that Wnt/β-catenin influences the barrier properties of the hCMEC/D3 model, beyond changing of Pgp drug efflux, has not been reported. In the present studies, the expression profile of Wnt components including Wnt ligands, receptors, co-receptors and modulators were characterized. The studies dissected the contribution of endogenous Wnt ligands released from hCMEC/D3 in establishment of BBB phenotype and compared the alteration in the BBB phenotype of hCMEC/D3 following activation through natural Wnt ligands and downstream kinase inhibition. While hCMEC/D3 produced Wnt ligand, the autocrine Wnt/β-catenin signaling contribution toward brain endothelial barrier function in the present study was minimal. In contrast, hCMEC/D3 were more responsive both in term of expression of genes known to contribute to BBB phenotypes, as well as functional barrier properties, following to exogenous activation of Wnt/β-catenin signaling through natural Wnt ligand or the inhibition of GSK activity. The studies suggest that autocrine activation of Wnt/β-catenin activation in the cerebral vasculature alone is insufficient to induce BBB phenotype. However, activation of Wnt/β-catenin through pharmacological means such as ligand stimulation or modulation of downstream elements in the signaling pathway can impact on the barrier properties of these cells.While previous studies have established the role of Wnt/β-catenin signaling in BBB developmentUsing PCR and qPCR, the various Wnt receptors, activators and modulators were profiled in hCMEC/D3 monolayers. As depicted in Fig. Using the same method, the 19 Wnt ligands were also profiled. As depicted in Fig. 33.Proteomics profiling of hCMEC/D3 lysates using SOMAscan assay confirmed the expression of Wnt7a, Dkk-1, Dkk-3, SFRP-1 and SFRP-3 proteins . Inhibition of Pgp function by GF increased accumulation of Rhodamine 800 by ~30% (p < 0.01). A similar increase in BCRP drug efflux transporter activity was observed following Wnt3a treatment. Wnt3a reduced mitoxantrone accumulation inside the cell by ~30%  . This protein is associated with the ability of the brain endothelial cells to form stomatal caveolae or endothelial fenestrationsth) Fig. . Functioth) Fig. . A simil43. Although the Wnt/β-catenin signaling activity in the brain microvasculature is significantly reduced following maturation, its role in BBB maintenance appears vital9. Evidence in support of this are the studies linking loss of endothelial β-catenin activity in an adult animal model with seizures and the resulting depletion of claudin-1 in the brain microvasculature11. Another study observed widespread sulfo-NHS-biotin leakage into the brain upon LRP-5 and LRP-6 conditional knockout in 24-day old mice22.The importance of canonical Wnt signaling for BBB development has been widely studiedwww.genecards.org) confirms that many intracellular transporters and intercellular junction proteins important in maintaining a BBB phenotype have a binding site for TCF4, TCF7 and LEF-1, that are known transcripton factors in the Wnt/β-catenin pathway. Genes for Pgp, BCRP, MRP4, Glut-1, claudin-1, claudin-3 and PLVAP have a regulatory element that interacts with TCF-4. In addition, studies using a TCF4 dominant negative mutant mouse demonstrated reduction in claudin-5 expression22. Together these studies suggest that Wnt/β-catenn signaling may play an important role in regulating gene expression important for maintaining the BBB phenotype.It should be noted that public database analysis  the contribution of Wnt ligand, produced by brain endothelial cells themselves, towards maintaining the BBB phenotype is likely minimal, (2) modulation of Wnt/β-catenin signaling in brain endothelial cells from exogenous sources can significantly alter BBB function, and (3) the extent of BBB phenotype changes observed with Wnt activators correlated with the magnitude of Wnt/β-catenin produced.Current understanding suggests that astrocytes are a source for Wnt ligand in the BBBin-vitro BBB model due to its ability to retain the brain endothelial phenotype from passage to passage compared human primary brain endothelial cells (HBMEC)28. While canonical Wnt signaling in the hCMEC/D3 cell model has been reported29, the present study is the first to provide a comprehensive profiling of the effects of Wnt/β-catenin signaling, through both endogenous and exogenous activation routes, on the barrier properties of this cell. The current studies in the hCMEC/D3 culture model show the expression of multiple Wnt receptors, with eight frizzled receptors along with LRP-5 and LRP-6. The Wnt receptor profile observed in hCMEC/D3 was similar to previous reports in mouse brain microvessels7. Expression of frizzled-4 was particularly high in the hCMEC/D3 BBB culture model. Besides being a receptor for Wnt ligand, frizzled-4 is also important for Norrin, a non traditional ligand for Wnt canonical signaling49. Endothelial frizzled-4 deletion in the embryonic, postnatal and adulthood is associated with reduction of claudin-5 and increase expression of PLVAP in the cerebellum48. This suggested the important of Frizzled-4 not only for BBB development but also BBB maintenance.The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 is widely used as an 45. In agreement with hCMEC/D3, our RT-PCR examination of primary human brain endothelial cells (HBMECs) also indicated expression of Wnt2b and Wnt3 as the major canonical Wnt ligand  is a non traditional Wnt agonist capable of regulating Wnt/β-signaling strength through altering frizzled receptor turnover52. Previous studies suggested that brain endothelial cells were the major producer of R-spondin compared to brain pericytes51 and addition of RSPO together with Wnt3a increased Wnt/β-catenin activation by 10-fold compared to Wnt3a alone33. Thus, any small leak of Wnt release following WntC59 treatment could be potentiated by RSPO and result in some basal activity. In contrast, ICRT-3 blocks the TCF-4 transcription factor. Blocking at the level of the transcription factor would be expected to be more effective way of reducing the intrinsic Wnt activation as the pharmacological intervention occurs downstream of ligand interactions with the membrane receptors. This may explain why the effects observed with ICRT-3 appear to be more robust than the effect observed with WntC59.While these studies showed minimal effect of inhibition of Wnt ligand release on barrier properties of hCMEC/D3, the brain microvessel endothelium has the capability to adjust the level of Wnt activation through autocrine pathways involving Wnt modulators or R-spondin (RSPO). Our studies and others found relatively high expression of RSPO in the human brain endothelial cell at the mRNA and protein levelWhile blocking endogenous Wnt ligand release and downstream transcription factor interactions had minimal impact on the BBB properties of the cell culture model, exogenous activation of Wnt/β-catenin using either Wnt3a or LiCl, significantly improved the BBB phenotype in hCMEC/D3 cells. Using Axin-2 expression as a reporter of Wnt/β-catenin activity, the highest activation of Wnt signaling in hCMEC/D3 cells was achieved through its natural ligand (Wnt3a). Activation of canonical Wnt pathways with Wnt3a also produced a robust effects on gene and protein expression involved in the BBB phenotype. This included selected transporters, such as Pgp, Glut-1, BCRP, MRP-4, as well as tight junction proteins including claudin-1, claudin-3, claudin-5 and occludin.45. The source of Wnt3a in the brain is more likely from neuron, astrocytes and pericytes55. Thus the study with Wnt3a provided us with a way to examine the ability of the brain endothelial cells to respond to non-endothelial based Wnt ligands.Selection of Wnt3a, from the many Wnt ligands is based on the following. First, Wnt3a is not expressed in our hCMEC/D3 culture model of the BBB, nor in primary human brain microvessel endothelial cells. Studies in other peripheral endothelial cells also suggest that Wnt3a is not a ligand that would be produced by the endothelial cells themselves21. This could be explained by high potency of Wnt3a in activating Wnt/β-catenin signaling compared to other canonical Wnt ligands38. Unlike Wnt3a which activates the Wnt receptor, and triggers the binding of Axin and prevents the formation of the β-catenin destruction complex, LiCl activates Wnt indirectly through inhibition of GSK356. As only a small pool of GSK3 is associated with Axin, it takes substantial inhibition of GSK3 to impact Wnt/β-catenin signaling. Indeed, it has been reported that GSK3 has to be inhibited by at least 80% before activation of Wnt/β-catenin is observed57. Thus there is a limit to activation of Wnt through GSK3 blockers that is not encountered with the Wnt receptor ligands.The effects observed with exogenous Wnt3a were more robust compared to the activation of Wnt/β-catenin through GSK inhibition. Similar results have also been observed in the bEnd-3, mouse endothelial cell line where Wnt3a conditioned media improved the BBB phenotype compared to LiCl treatment41. Treatment with Wnt3a resulted in reductions in both PLVAP expression and fluorescently labeled albumin uptake in hCMEC/D3. As BSA is internalized through a caveoli-mediated process58, the reductions in PLVAP expression observed with Wnt3a resulted in reduced endocytosis in the cells. Inhibition of autocrine activation of Wnt signaling with ICRT-3 resulted in an increase in PLVAP gene expression, although no significant alteration in caveoli-mediated endocytosis was observed. These finding are support the previous studies reporting that Wnt/β-catenin signaling influences PLVAP expression in brain endothelial cell50.In the present studies we also examined the effects of Wnt/β-catenin signaling on plasmalemma vesicle-associated protein PLVAP/Mecca 32 gene expression. This protein is responsible for formation of stomatal fenestrae and caveolae involved in pore formation and vesicular transport, respectively, and is normally expressed at very low levels in brain endothelial cells53. The present studies characterized the impact of Wnt/β-catenin signaling in the hCMEC/D3 cell culture model of the blood brain barrier, demonstrating changes in transporter, paracellular barrier and vesicular endocytosis activity through altered expression of genes important for the BBB phenotype . Human Recombinant Wn3a, and GF120918 (GF) were purchased from R&D Systems . Rhodamine 800 and Rhodamine 123 were purchased from Sigma . IRDye 800 CW PEG were purchased from LICOR . EBM-2 media was purchased from Lonza . The E-plates for RTCA were obtained from ACEA Biosciences . Transwell and tissue culture plates were purchased from Corning . Trizol and tetramethylrhodamine BSA was purchased from Life Technologies . P-glycoprotein and Claudin-1 antibodies were purchased from Abcam . β-catenin, claudin-5 and β-actin antibodies were purchased from Sigma . Human fetal brain total RNA was purchased from Takara Bio USA, Inc. . Primers were obtained from Invitrogen  and primer sequence information is provided in Table hCMEC/D3 were obtained from Dr. Peter Couraud2 onto T75 flasks (Corning Inc) that had been coated with rat tail collagen 0.1 mg/ml for an hour. Cells were cultured at 37 °C and 5% CO2 in EBM-2 media supplemented with 5% FBS, 1% penicillin streptomycin, 1 ng/ml bFGF, 10 mM HEPES, 5 µg/ml ascorbic acid, 1/100 CD lipid concentrate, and 1.4 µM hydrocortisone. Cells were passaged when reaching approximately 80% confluency using 1 ml Trypsin/EDTA. For expression and functional studies, cell were seeded onto culture plates coated with rat tail collagen at a density of 25,000 cells/cm2 and used upon reaching confluency . To insure maintenance of BBB properties the hCMEC/D3 were used at passage 29 to 3423.For cell expansion, hCMEC/D3 were seeded at 10.000 cells/cmCell were solubilized using RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitors (Thermo Scientific). The lysates were subsequently centrifuged at 12.000 rpm for 8 minutes for isolating the whole cell lysate. The protein concentration was determined by Pierce BCA protein analysis (Thermo Scientific). Depending on the protein examined, 20–40 µg of protein was loaded into the SDS-polyacrylamide gel and run for separation at 110 Volt. The proteins were then transferred to PVDF membrane (BioRad) for 2 hour at 200 mAmp. The membrane was subsequently blocked in 5% non skim milk at TBST buffer for an hour at room temperature. Then, the membrane was incubated with the primary antibodies dissolved at 5% non skim milk TBST buffer at 4 °C on the rocking rack. Dilution 1: 1000 were applied for Claudin-1, Claudin-5, P-glycoprotein, β-catenin, GSK3β, phospho-GSK 3β, and phospho-β-catenin. In the next day, membranes were washed thrice with TBST buffer for ten minutes each before being incubated with secondary antibodies for 1 hour at room temperature. Bands were visualized using chemiluminescence at ChemiDoc Imager (Biorad) and analyzed using Image Lab software (Biorad).Expression of Wnt receptor, co-receptor, ligand, and modulator were examined in the human cerebral microvessels endothelial cell line (hCMEC/D3) using RT-PCR. Human fetal brain total RNA was used as a positive control for the primer as it expressed most of the Wnt component. Total RNA were isolated using Trizol®. RNA concentration was measured using Nanodrop UV Vis Spectrometer (Fisher Scientific). One microgram of total RNA was converted to cDNA using MLV Reverse Transcriptase enzyme (Invitrogen) with the final volume of 60uL according to the manufacturer’s protocol. 1uL of the cDNA were subjected to PCR using Platinum Taq polymerase (Invitrogen). The cycle was initial denaturation at 94 °C for 2 minutes followed by 30 PCR cycle that consist of 94 °C for 30 seconds for denaturation, 60 °C for 45 second for annealing and 72 °C for 45 second for extension using programmable thermal controller (MJ Research Inc.). Ten microliter of PCR product were eluted in the 1% agarose gel at 104 Volt in 1x TAE buffer. Before the gel solidified, 10 uL of GelGreen (Biotium) were added to the agarose gel for PCR product visualization. The image was visualized using ChemiDoc (BioRad).RNA isolation was done similar to the previous section. Total RNA was subjected to qPCR using ITaq Universal Syber Green (BioRad) according to manufacturer protocol using QuantStudio 5 (Thermo Fisher).61. The cells were treated with Wnt3a, LiCl, WntC59 and ICRT-3 for 15–20 hour before the experiment under serum free media. On the day of experiment, the cells were washed with sterile PBS. Subsequently the cells were equilibrated in 0.5 ml assay buffer for 30 minutes. In this 30 minutes pre-incubation, only the GF group received 3.2 μM GF. The uptake were done for two hour at 37 °C and 5% CO2 with 0.5 ml sterile assay buffer  containing Rhodamine 123 2.12 µM. At the end of the experiment, the plates were washed two times with 1 ml of ice cold PBS. To solubilize intercellular Rhodamine 123, an 0.4 ml of 1% Triton X in PBS were added to each well and placed to the −20 °C freezer overnight. Following day, 100 µL of the lysate were transferred to black 96 well plate and were analyzed using Biotek Synergy HT Microplate Reader at excitation 485 nm and emission 528 nm. The amount of intercellular Rhodamine 123 was normalized to protein content using Pierce BCA protein assay and expressed either as nanogram Rhodamine 123 per mg protein or as a percent of the control group.Standard P-glycoprotein functional assay were done at 24 well plate using Rhodamine 123 (2.12 µM) as a Pgp substrate and GF120918 or GF (3.2 µM), as a Pgp inhibitorAlternative P-glycoprotein functional assay were done using Rhodamine 800 0.1 µM as Pgp substrate with GF120918 (GF) 3.2 µM as an inhibitor. The protocol were similar to Rhodamine 123 studies. At the end of the experiment, the intercellular accumulation of Rhodamine 800 was quantified using Odyssey Near Infrared Imager  using 700 nm channel.62 using similar protocol as Rhodamine 123. Intracellular accumulation of mitoxantrone were quantified using Odyssey Near Infrared Imager (Licor) using 700 nm channel.BCRP functional studies used mitoxantrone 15 µM as a BCRP substrate and GF120918 (GF) 3.2 µM as a BCRP inhibitorRhodamine 123, Rhodamine 800, and mitoxantrone studies were done in separated sets of experiments. The assay was conducted at 15–20 hour post-treatment with Wnt ligands and modulators.58. Methyl β-cyclodextrin  and genestein 200 µM were used as inhibitors of endocytic pathways63. Similar protocol as Pgp and BCRP functional studies were used. After uptake for two hours, intercellular accumulation of tetramethyl conjugated BSA was quantified using Biotek Synergy HT Microplate Reader at excitation 530 nm and emission 590 nmTetramethylrhodamine conjugated BSA 5 μg/ml was used to study vesicular transport mediated endocytosis2. Apical and basolateral chamber were filled with media 1.5 ml and 2.5 ml complete EBM-2 subsequently. The monolayer was used upon reaching confluency, typically 4–5 days after seeding. For the permeability studies, the monolayers were treated with different Wnt activators or inhibitors for 15–20 hours in the absence of serum. On the day of experiment, the media was removed and replaced with assay buffer in both basolateral and apical compartments. The assay buffer in the apical compartment also contained 0.1 µM Rhodamine 800, 0.1 µM IR Dye CW PEG and 1 µM sodium fluorescein to assess monolayer permeability. A hundred microliters samples were removed from the basolateral compartment at various times  and replaced with equal volume of fresh assay buffer. Samples (10 μl) were also taken from apical compartment at the start and conclusion of permeability study. The samples from the apical and basolateral compartments were placed in black 96-well plates and diluted to 100 μl of assay buffer. Quantitative analysis of the various solutes were performed using an Odyssey Near Infrared Imager  and Biotek Synergy HT Microplate Reader at excitation 485 nm and emission 528 nm for sodium fluorescein. The amount of fluorescence activity was quantitated using standard curves for each fluorescent compound. Permeability data were presented as the percent flux.Permeability studies were done in the 6 well Transwell 0.4 µm pore polycarbonate membrane after pre-coated with rat tail collagen. hCMEC/D3 were seeded in the density of 25.000 cell/cmMonolayer electrical impedance were measured using xCELLigence RTCA system. Briefly, the cell was seeded in 16 well E-plate (ACEA Biosciences) that have been coated with rat tail collagen with the density 14,000 cell/well. The E-plate surface was covered with microelectrode that measure the electrical impedance of the monolayer resulting a dimensionless value called Cell Index (CI). The cell was seeded in 200 µL EBM-2 media and been replaced with new media every other day. The impedance figauto-mesurement interval was every 5 minutes. After the impedance reached the plateau for at least 24 hour, the media was removed and replaced with treatment drug with serum free EBM-2. Impedance monitoring was continued 24–48 hour post treatment.SupModulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells


Several studies have confirmed that gold nanoparticles (AuNPs) of specific concentration and size exert a boosting effect on cell proliferation; however, the mechanism through which this effect occurs remains unknown. This study explores the canonical Wnt signaling pathway in AuNP promotion of human periodontal ligament stem cell (hPDLSC) proliferation.LRP5 and β-catenin was steered by shRNA plasmids and siRNA, respectively, at which point the expression of MYC, CCND1, AXIN2, and POU5F1 had been estimated via real-time PCR. The expressions of LRP5 and β-catenin were detected via western blot assay.MTS was employed to evaluate hPDLSC proliferation. The interference of The proliferation of hPDLSCs treated with 60 nm AuNPs at 56 μM was clearly elevated. In contrast, β-catenin siRNA significantly decreased cell viability. The LRP5 shRNA plasmid did not consistently impact cells. The expressions of these four genes downstream of the Wnt/β-catenin signaling pathway were not significantly overexpressed in response to the interference of shRNA plasmid/siRNA with the treatment of AuNPs.These results suggest that the Wnt/β-catenin signaling pathway plays a significant role in the process of AuNP promotion of hPDLSC proliferation. As a result of the rapid development of tissue engineering, proliferation of human periodontal ligament stem cells (hPDLSCs)  as seed Our previous experiments confirmed that 60 nm AuNPs at a concentration of 56 μM significantly promoted the proliferation of hPDLSCs, while exerting no apparent side effects on the differentiation potential of cells. However, another study reported that AuNPs mainly promote the differentiation of human adipose-derived mesenchymal stem cells (MSCs) in osteogenesis via various signaling pathways . MoreoveMYC and CCND1 [Wnt/β-catenin signaling is crucial for regulating embryonic development and cell proliferation as well as for the determination of the cell fate . Prolifend CCND1 . Recentlnd CCND1 , 13.MYC, CCND1, AXIN2, and POU5F1) were evaluated via real-time PCR. The results indicate that AuNPs promote the proliferation of hPDLSCs, which is likely a result of the Wnt/β-catenin signaling pathway.Here, an in-vitro study was conducted to investigate the role of the Wnt/β-catenin signaling pathway for the proliferation of hPDLSCs treated with AuNPs (60 nm at 56 μM). LRP5 and β-catenin were interfered with by shRNA and siRNA, respectively. Changes in the expression of Wnt-related genes  were supplemented with an immunofluorescence identification using vimentin and cytokeratin. STRO-1+ hPDLCs were sorted via flow cytometry (FCM) and preliminary identification of the stem cell characteristics was achieved via a plate clone formation assay, multidirectional induction as osteoblasts, and adipocytic differentiation.Premolars from teenagers 12 and 13 years old, which had been extracted for orthodontic reasons, were freshly obtained from the Oral Surgery Department, Hospital of Stomatology, Tongji University with informed consent. The teeth were rinsed three times with PBS complemented with Antibiotic–Antimycotic solution. The periodontal ligament tissue in the middle third of the root was carefully removed with a surgical scalpel. The tissue was minced and enzymatically digested using type I collagenase  and Dispase II  for 1 h at 37 °C. A suspended cell solution was passed through a 70-mm cell strainer to prepare a single cell suspension. The cells were centrifuged and rinsed with PBS and the obtained cell pellet was resuspended and cultured in alpha-modified minimum essential medium  with 10% FBS (Gibco) at 37 °C in 5% COThe 60 nm AuNPs were acquired from British Biocell International Company . The final concentration of AuNPs was adjusted with α-MEM medium. The AuNPs were characterized using both a UV–Vis spectrophotometer  and a Zetasizer .6 cells per well in six-well culture plates and were cultured until 50–70% confluence. Cells were transfected according to an established shRNA transfection protocol [2 in humidified air, cells were cultured overnight with 1 ml α-MEM (Gibco) containing 20% FBS and 1% Antibiotic–Antimycotic. The medium was carefully removed and cells were washed with PBS. After culture for 3 h with AuNPs (60 nm at 56 μM), hPDLCs were washed with PBS. The culture medium was replaced by α-MEM, and the cells were cultured for 48 h.For both shRNA plasmid and siRNA interference, cells were seeded at a density of 2 × 10protocol . After iTo measure cell viability, hPDLCs were interfered with by either shRNA plasmid or siRNA, and were then plated in triplicate in 96-well tissue culture-treated plates at a density of 1000 cells per well and incubated overnight. The medium was carefully removed and cells were washed with PBS. The 60 nm AuNPs were diluted with fresh serum-free α-MEM medium at a concentration of 56 μM and were added to the hPDLCs. After 2 h, the medium was aspirated and replaced with full medium. Cells without AuNPs were used as control; wells without AuNPs and cells were used as blanks for spectrophotometric readings. MTS  was added to each well at 0, 24, 48, 72, and 96 h. After incubation at 37 °C for 2 h, the optical density (OD) value for each well was measured spectrophotometrically at 490 nm.The viability assay (Promega) was conducted according to the manufacturer’s instructions. Results were reported as percentage relative fold changes for each group and were internally normalized to the OD reading at 0 h.MYC, CCND1, AXIN2, POU5F1, LRP5) and β-catenin. All target gene expression results have been normalized to ACTB.After inducing interference with siRNA or shRNA (with or without AuNPs), hPDLSCs were washed with PBS. Total RNA was isolated using Trizol reagent following the manufacturer’s instructions and the RNA concentration was measured spectrophotometrically . First-strand cDNA was then synthesized using a PrimeScript First Strand cDNA Synthesis Kit . PCR was performed using 1 μl of the RT products as a template. For the amplification step, 40 cycles were conducted under the following conditions: denaturation at 94 °C for 2 min, annealing at 58 °C for 30 s, and extension at 72 °C for 45 s. The specific gene primers used for real-time PCR are presented in Table Cells were washed with ice-cold PBS twice and lysed in high-salt lysis buffer (45 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 6.25 mM NaF, 20 mM β-glycerophosphate, 1 mM DTT, 20 mM sodium butyrate, and 1× protease inhibitor cocktail; Roche). Equal amounts of protein extracts were resolved via 12% SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (BIO-RAD). The membranes were blocked with 5% (wt/vol) dry milk in PBS-Tween-20 (0.5% vol/vol) and probed with primary antibodies overnight. Anti-β-catenin (#9562) and anti-LRP5 (#5731) antibodies were purchased from Cell Signaling Technology . Secondary antibodies were conjugated to horseradish peroxidase  for 1 h at room temperature. The immunoreactivity was detected with an ECL kit . Anti-ACTB antibodies  were used as loading control. The quantification of western blot analysis was ushered by ImageJ software.p < 0.05 was considered statistically significant.Data were collected from three separate experiments and are shown as the mean ± standard deviation (SD). Statistical differences were analyzed with one-way analysis of variance (ANOVA) using SPSS 17.0. The 60 nm AuNPs peaked at 538 ± 14 nm Fig. . The ZetTo evaluate the proliferation of hPDLSCs in the cell viability assay, hPDLSCs were cultured with AuNPs, β-catenin siRNA, or AuNPs and β-catenin siRNA Fig. , or withLRP5 gene expression was detected via real-time PCR after interference with shRNA plasmids. Both 1.5 and 2.0 μg/μl concentration of the plasmid shRNA significantly downregulated the expression of the LRP5 gene (p < 0.05). No significant difference was found between 1.5 and 2.0 μg/μl  compared to the control group. Protein β-catenin of hPDLSCs did not increase after shRNA plasmid interference with LRP5 and even incubation with AuNPs, compared to the control group Fig. .p < 0.05) compared to 0.25 μg siRNA, while 0.5, 0.75, and 1 μg siRNA did not significantly downregulate β-catenin expression. Therefore, we chose 0.5 μg siRNA for further experiments. According to the results of western blot analysis . Interfering in the LRP5 gene expression with shRNA plasmids downregulated expression of these four genes (p < 0.05). Under combined AuNP and shRNA plasmid treatment, only AXIN2 gene expression was downregulated compared to control (p < 0.05).AuNPs (60 nm at 56 μM) and/or 1.5 μg shRNA plasmid were added to the hPDLSC culture system and the expressions of Wnt-related gens MYC Fig. , CCND1 . β-catenin was interfered with by siRNA and, except for MYC, the other three genes were downregulated (p < 0.05). Although β-catenin was part of the interference of siRNA, addition of AuNPs decreased the interference compared to the control.AuNPs (60 nm at 56 μM) and/or 0.5 μg siRNA to β-catenin were employed to investigate the resulting effects on hPDLSCs in 100 μl medium per well and the Wnt/β-catenin signaling pathway-related genes MYC Fig. , CCND1  in murine macrophages indicated no cytotoxicity under the exposure conditions tested, and neither do these elicit proinflammatory responses; however, the localization of AuNPs in intracellular vacuoles suggests endosomal containment and an uptake mechanism involving endocytosis . ConsideAuNPs with different surface charge were employed to treat human bone marrow-derived mesenchymal stem cells (hMSCs). Surface-functionalized AuNPs were without acute toxicity. Positively charged AuNPs showed higher cellular uptake. AuNPs did not inhibit osteogenesis but ALP activity and calcium deposition were remarkably reduced in response to negatively charged AuNP treatment. The proteins in the biological medium were likely coating AuNPs, leading to a change of the initial surface charge of nanoparticles . Therefo. Hence, the variation of the canonical Wnt signal pathway for the proliferation of hPDLSCs treated with AuNPs was investigated in this experimental study. Furthermore, AuNPs of size 30–74 nm had an uptake half-time of about 2 h, while the removal half-time was about 0.5 min, depending on cell types. Global gene expression analysis [Multiple signaling pathways, including SMAD, Notch, FGF, Hedgehog, and Wnt, have been closely associated with cell proliferation. Many related signaling pathways of stem cell proliferation have an intrusive cross-talk with Wnt signalinganalysis  and protanalysis  of AuNPsanalysis , 13. ThiMYC, CCND1, AXIN2, and POU5F1. Interfering with the gene expressions of LRP5 and β-catenin with shRNA plasmid and siRNA respectively, the expressions of the aforestated genes were downregulated. After interfering with the gene expressions of LRP5 and β-catenin with shRNA plasmid and siRNA, AuNPs were added and β-catenin protein did not increase. The expressions of the four tested genes were downregulated. The possible mechanisms with which AuNPs promote proliferation of human periodontal ligament stem cells were as follows: AuNPs outside the cells could promote LRP5, Wnt, and Frizzled protein aggregation and act on β-catenin in the cytoplasm. Phosphorylated β-catenin was dephosphorylated, transferred into the nuclei, and the expression levels of intracellular hPDLSC proliferation-related genes such as MYC, CCND1, AXIN2, and POU5F1 increased, thus promoting hPDLSC proliferation.Our experimental results showed that the AuNPs could activate LRP5 and/or β-catenin, and then activate downstream stem cell proliferation-related genes such as CCND1. A protein of the cell cycle, CCND1 regulates cells via G1 to S phase transformation, cell proliferation, and differentiation. Studies have shown that CCND1 expression and its upregulation have a close relationship with activated Wnt signaling pathways and increase the β-catenin expression level [MYC after combination [When Wnt/β-catenin signaling pathways were activated, β-catenin accumulated in the cytoplasm, entered the nucleus, and activated the downstream gene on level ; howeveron level . β-catenbination , thus rePOU5F1  belongs to the family of POU and plays an important role in the maintenance of self-renewal of stem cells. In addition to the differentiation of mature stem cells, the expression of POU5F1 gradually declines [POU5F1. AXIN2 participates in a β-catenin degradation compound. The AXIN family has two members: AXIN1 and AXIN2. AXIN2 has a very sensitive negative feedback effect on the Wnt signaling pathway. Whether AXIN2 expression increases is an important indicator of whether the Wnt signaling pathway is involved in the regulation of cellular physiological and pathological physiology change. AXIN2 as a Wnt signaling pathway inhibitory factor in the cell may regulate Wnt signal activation, where it leads to cell proliferation, prevents excessive cellular proliferation, and prevents malignant transformation [declines . Therefoormation , 25.Cell proliferation is a complex process in which many factors interact. Our results suggest that AuNPs promote hPDLSC proliferation effectively, which is closely related to the Wnt/β-catenin signaling pathway; however, their relationship and the underlying mechanism have yet to be further revealed. Further studies of AuNPs on the hPDLSC proliferation effect and the underlying mechanism will not only allow us to better understand AuNPs and their interaction with hPDLSCs, but can also provide excellent advancements of periodontal tissue engineering and thus lead to good seed cells.In a previous experiment, we suggested that AuNPs (60 nm at 56 μM) efficiently promote the proliferation of hPDLCs/hPDLSCs in vitro. However, the AuNP-induced proliferation-boosting effect on hPDLSCs was diminished as β-catenin siRNA or LRP5 shRNA plasmid was added. The AuNP treatment equalized the accumulation of β-catenin in hPDLSCs. The proliferation of hPDLSCs was initiated by the Wnt/β-catenin signaling pathway. Nevertheless, more details of the interaction between AuNP proliferation and the Wnt/β-catenin signaling pathway are required to suggest more potential applications.


Protein arginine methyltransferases (PRMTs) are known for their ability to catalyze methylation of specific arginine residues in a wide variety of cellular proteins, which are involved in a plethora of processes including signal transduction, transcription, and more recently DNA recombination. All members of the PRMT family can be grouped into three main classes depending on the type of methylation they catalyze. Type I PRMTs induce monomethylation and asymmetric dimethylation, while type II PRMTs catalyze monomethylation and symmetric dimethylation of specific arginine residues. In contrast, type III PRMTs carry out only monomethylation of arginine residues. In this review, we will focus on PRMT5, a type II PRMT essential for viability and normal development, which has been shown to be overexpressed in a wide variety of cancer cell types, owing it to the crucial role it plays in controlling key growth regulatory pathways. Furthermore, the role of PRMT5 in regulating expression and stability of key transcription factors that control normal stem cell function as well as cancer stem cell renewal will be discussed. We will review recent work that shows that through its ability to methylate various cellular proteins, PRMT5 functions as a master epigenetic regulator essential for growth and development, and we will highlight studies that have examined its dysregulation and the effects of its inhibition on cancer cell growth. G-monomethylation or ω-NG, NG-asymmetric dimethylation and ω-NG, N'G-symmetric dimethylation. Based on the type of modification introduced, PRMTs can be classified into three groups: Type I  catalyze monomethylation and asymmetric dimethylation, type II (PRMT5 and PRMT9) catalyze monomethylation and symmetric dimethylation, while type III (PRMT7) carry out monomethylation only [et al. (2005) [et al. (2012) showed that PRMT7 exclusively catalyzes monomethylation of arginine residues in substrate proteins [Normal cell growth and development rely on the proper regulation and interplay of many pathways that control cell growth and division, as well as cell differentiation and death. Over the last two decades, it has become abundantly clear that modification of chromatin plays a central role in triggering the appropriate gene expression programs required during different stages of cell growth and development. Among the various modifications that affect chromatin is arginine methylation, which has been implicated in a growing list of cellular processes including signaling, transcription, RNA processing, DNA recombination and repair –6. Prote III PRMT carry ouion only . It is w. (2005) ; howeverproteins .Suppressor of Tumorigenicity 7 (ST7), retinoblastoma (RB) family of tumor suppressors, and Protein Tyrosine Phosphatase Receptor-type O (PTPROt) [Iroquois homeobox 1 (IRX1), in gastric cancer cells. Furthermore, PRMT5 knock down in gastric cancer cell lines results in attenuated cell growth and reduced metastasis [This review will focus on work that has been done recently to enhance our understanding of the biological functions of PRMT5. PRMT5 is ubiquitously expressed in all eukaryotes and its catalytic domain is highly conserved from yeast through humans Figure . Based o(PTPROt) , 21. In tastasis . RegardlDelta Like Non-Canonical Notch Ligand 1 (Dlk-1) [Although extensive evidence indicates that PRMT5-induced H3R8 and H4R3 methylation leads to transcriptional repression of its target genes, cellular differentiation studies have shown that PRMT5-induced histone methylation is also involved in transcriptional activation. For instance, in concert with cooperator of PRMT5 (COPR5), PRMT5 has been shown to be involved in the control of adipocyte progenitor cell fate through transcriptional repression of the Wingless/Integration 1 (Wnt) target gene,  (Dlk-1) . However (Dlk-1) , suggestFibroblast-derived Growth Factor Receptor-3 (FGFR-3) and eukaryotic elongation Initiation Factor-4E (eIF4E) genes in colorectal cancer [FGFR-3 and eIF4E expression. The results from these experiments revealed that PRMT5-induced H3R8 and H4R3 symmetric methylation increases transcription of FGFR-3 and eIF4E, and that PRMT5 knockdown reduced the levels of methylated H3R8 and H4R3 as well as expression of both genes. More recently, Deng et al. (2017) showed that PRMT5 stimulates expression of the androgen receptor (AR) in prostate cancer cells. In this study, co-immunoprecipitation results showed that PRMT5 interacts with both BRG1 and Sp1 to induce H4R3 symmetric methylation in the promoter region of the androgen receptor gene, resulting in its activation and enhanced cell growth. The authors also showed that inducible knockdown of PRMT5 inhibits growth of AR-positive but not AR-negative prostate cancer cells as well as xenograft tumors in an AR-dependent manner. Similarly, pharmacological inhibition of PRMT5 decreased symmetric methylation of H4R3, expression of AR in AR-positive prostate cancer cells, and reduced cancer cell growth [Further evidence in support of the activator role played by PRMT5 comes from studies of the l cancer . In thisl growth .Besides its ability to methylate histones, PRMT5 is also capable of methylating several important transcription factors Table , highliget al. (2007) showed that down regulation of microRNAs (miR) 92b and 96 was responsible for enhanced translation of PRMT5 mRNA in various B cell lymphoma types [et al. (2015), the authors showed that, through a negative feedback loop and in combination with NF-κB/p65 and HDAC3, PRMT5 binds to the miR96 promoter and suppresses its transcription  2, which include Enhancer of Zeste Homolog (EZH) 2, Suppressor of Zeste (SUZ) 12 homolog, and Embryonic Ectoderm Development (EED). Previous work had shown that subunits of the PRC2 complex are transcriptionally regulated by the RB/E2F complex . More re and HRK . In ligh and HRK .A fascinating aspect of PRMT-induced arginine methylation is that different members of the PRMT family endow their target proteins with distinct properties. A good example of this regulation is illustrated by the mutually exclusive methylation of E2F1 by PRMT1 and PRMT5. In an elegant investigation led by La Thangue and co-workers, it was shown that PRMT1-mediated asymmetric dimethylation of E2F1 at arginine 109 (R109) enhances its transcriptional activity, which results in increased expression of E2F1 pro-apoptotic target genes during DNA damage . In starBAX, thereby promoting cancer cell growth and survival. In accord with these results, breast cancer gene array profiling indicated that elevated expression of KLF4 in both MCF10A and MCF7 breast cancer cell lines upregulates transcription of several oncogenes and cell cycle activators such as MAPK, EGF/EGFR, IGF1, CYCLIN D2, CDK1, and CYCLIN E1. Other factors involved in regulating stem cell renewal and metastasis were also upregulated in KLF4 overexpressing breast cancer cells including c-MYC, SOX9, BMI1, Z1B1, SLUG, TWIST and N-CADHERIN. More importantly, analysis of clinical samples showed that the cellular levels of both PRMT5 and KLF4 were elevated in highly aggressive breast tumor tissues compared to adjacent normal tissues. These results clearly demonstrate that PRMT5 regulates key stem cell transcription factors through cross-talk between arginine methylation and ubiquitylation, and that it is a master epigenetic regulator capable of targeting multiple pathways to promote cancer cell growth and survival [Another example of the relevant role played by PRMT5-mediatd arginine methylation in the control of genome stability and tumorigenesis is illustrated by the Kruppel-like factor 4 (KLF4), which is involved in regulating cell-fate decision and cell cycle progression . In thissurvival .in vitro and in vivo. Furthermore, histopathological analysis of excised tumors from NSG (NOD/Scid/IL-2Rγnull) mice injected with either control or PRMT5 knock down BCSCs showed that reduced PRMT5 expression results in a more differentiated and less aggressive breast cancer phenotype compared to control tumors, indicating that PRMT5 promotes breast cancer cell stemness by suppressing differentiation. While these initial experiments strongly suggested that PRMT5 is vital for growth and proliferation of BCSC, global RNA-Seq profiling in control and PRMT5 knock down BCSCs revealed differential expression of the forkhead box protein 1 (FOXP1) transcription factor, which is known to be associated with normal and cancer stem cell function. Further characterization of the relationship between PRMT5 and FOXP1 demonstrated that PRMT5 upregulates FOXP1 expression through symmetric methylation of H3R2, which serves as a binding site for the WDR5 subunit of the SET1/MLL methyltransferase complex. Consistent with these recruitment studies, either inactivation of PRMT5 with the GSK591 inhibitor or disruption of the interaction between WDR5 and the SET/MLL1 complex triggered a dramatic decrease in H3K4 methylation and FOXP1 expression. These results, in combination with the inhibitory effects that FOXP1 knock down has on breast cancer cell growth both in vitro and in xenografts, imply that some of the tumor promoting properties of PRMT5 are mediated through FOXP1, and further suggest that PRMT5 inhibition might be a viable therapeutic option that should be explored in breast cancer treatment.In line with its influence on proliferation and self-renewal of embryonic and adult stem cells, PRMT5 has recently been implicated in the regulation of breast cancer stem cell (BCSC) function . The finc-MYC and CYCLIN D1 [The role-played by PRMT5 in the onset and progression of cancer is becoming more and more clear as previous work by Wei and colleagues showed that ectopic expression of PRMT5 is associated with upregulation of the G1 phase cyclins D1, D2, and E1, as well as cyclin-dependent kinases (CDK) 4 and 6 . ExperimYCLIN D1 . The mecYCLIN D1 . Taken tAlthough the list of PRMT5 associated proteins has grown significantly over the last few years, insight into the biological relevance of these interactions has only begun. Programmed Cell Death 4 (PDCD4) is a tumor suppressor protein whose expression has been shown to correlate with better survival in various cancers; however, upon interaction with PRMT5, PDCD4 becomes methylated at R110 and loses its tumor suppressor activity in MCF-7 cells . ExperimCancer cells alter their metabolic pathways in order to support rapid growth and proliferation, and most of the acquired changes are optimized to promote transformed cell growth. Besides altering pro-survival and cell death pathways, PRMT5 has been implicated in reprogramming of lipogenesis in cancer cells, and in modulation of the G1 to S phase checkpoint under high glucose concentration. The gene expression program responsible for biosynthesis of fatty acids, triglycerides, and phospholipids is tightly controlled by the transcription factors, sterol regulatory element-binding protein (SREBP) 1a and c . Both trSimilarly, xenograft studies revealed that HepG2 cells expressing wild type SREBP1a induced tumors that were larger and heavier compared to cells expressing mutant R321K, which were less proliferative as measured by Ki67 expression. In addition, analysis of patient-derived hepatocellular carcinoma (HCC) samples revealed a strong correlation between the amount of SREBP1a symmetric methylation and aggressiveness of tumors as measured by size, stage and histologic grade. In another study, it was shown that SREBP1c overexpression correlates with high expression of lipogenic genes in clear cell Renal Cell Carcinoma (ccRCC), and that it is downregulated by the ring finger protein 20 (RNF20), which contains a ring finger-containing E3 ubiquitin ligase activity that promotes SREBP1c polyubiquitination and subsequent degradation . Since SINK4a), thereby facilitating G1-S transition in HCC cells [Among the many changes cancer cells acquire as they become transformed is to use aerobic glycolysis in order to generate ATP instead of oxidative phosphorylation . Cancer CC cells . PRMT5 hCC cells . Under fCC cells . When blCC cells . TherefoCDKN2A (p16INK4a and p14ARF) tumor suppressor gene deletion. The consequence of this frequent deletion is accumulation of methylthioadenosine (MTA), which selectively inhibits PRMT5 methyltransferase activity and sensitizes it to further inhibition [Deletion of metabolic enzyme 5-methylthioadenosine phosphorylase (MTAP) is a frequently observed phenomenon in many cancers harboring hibition . What ishibition , 58.FGF5, GATA6, LHX1, FOX2, HOXA3, HOXA7 and HOXD9 [OCT4, NANOG and REX1, whereas PRMT5 knockout in mouse models leads to abnormal embryonic growth followed by lethality [CYCLIN B and BUB1b [Nestin-Cre transgenic mouse results in CNS developmental defects and lethality within 14 days after birth [Id2 and Id4 leading to their silencing during glial cell differentiation.Studies investigating the role played by PRMT5 during mouse embryonic development have shown that abundant levels of PRMT5 protein are found in the cytoplasm of mouse embryonic stem (ES) cells, where it interacts with STAT3 to maintain embryonic stem cell pluripotency by repressing transcription of differentiation genes such as nd HOXD9 . Furtherethality . These fnd BUB1b . PRMT5 ier birth . PRMT5 ler birth . The autet al. (2018) showed the functional relevance of PRMT5-mediated histone H4R3 symmetric methylation in oligodendrocyte differentiation and developmental myelination. Evaluation of PRMT5 mRNA demonstrated that its levels are high in proliferating, but not in differentiating, oligodendrocyte progenitor cells (OPCs). In addition, symmetric methylation of histone H4R3 was found to be enriched in the cytoplasm of proliferating OPCs and in the nucleus of differentiating OPCs. Interestingly, conditional deletion of PRMT5 in OPCs results in a severe hypomyelination at postnatal day 14 due to a decrease in the number of oligodendrocytes, but without any effect on proliferation of OPCs. These findings were further confirmed using either the PRMT5 inhibitor GSK591 or a PRMT5-specific CRISPR/Cas9 lentivirus, suggesting that PRMT5 is more important for survival and differentiation of OPCs than for their proliferation. Mechanistically, the authors determined, using both brain sections of PRMT5 mutant mice and ex-vivo primary cultures of OPCs, that PRMT5 regulates differentiation of OPCs through induced symmetric methylation of H4R3, which precludes acetylation of nearby lysine residues [Recent work by Scaglione residues .et al. (2018) showed in a different study that loss of PRMT5 (Oligo2-Cre) triggers early post-natal mortality and altered brain development, which is marked by hypomyelination and lack of mature oligodendrocytes (OL). The root cause of this deficiency stems from the fact that conditional PRMT5 inactivation in FL/FL;Olig2CrePRMT5 mice leads to decreased expression and plasma membrane localization of PDGFRα protein in the OL lineage. Similar results were observed in rat primary OPCs when PRMT5 was either knocked down, using a PRMT5-specific siRNA, or inhibited, using the PRMT5 specific inhibitor EPZ015666. Moreover, functional characterization revealed that PRMT5 interacts with and methylates PDGFRα on R554 within a Cbl E3 ligase binding site (aa 553-568). Thus, PRMT5-mediated PDGFRα methylation prohibits its degradation, and promotes self-renewal and proliferation of OPCs. However, as PRMT5 expression diminishes during OL differentiation, symmetric methylation of PDGFRα/R554 decreases, unmasking the Cbl E3 ligase binding site and triggering PDGFRα proteasome degradation [Consistent with these findings, Calabretta radation .in vivo studies show that PRMT5 levels are elevated in mouse germ cells soon after sex determination during embryonic development, and its loss leads to cell death resulting in inefficient germ line development [STRA8, SCP3, and γH2AX in mouse ovaries soon after sex determination, and downregulation of these genes after PRMT5 inactivation leads to inactivation of meiosis, thereby, resulting in germ cell loss during oogenesis. Similarly, conditional PRMT5 knock down in testes of PRMT5Δ/f;STRA8-Cre transgenic mice results in aberrant spermatogenesis, which is accompanied by reduced expression of meiotic genes such as STRA8, SPO11, RAD51, SEP1, SEP3, DME1, and REC8. This ultimately leads to meiotic arrest and death of germ cells in adult testes [OCT4, SOX2 and NANOG in germ cells. Instead, expression of these genes becomes elevated, indicating that PRMT5 plays cell specific roles during embryonic development [et al. (2014) showed that loss of PRMT5 in human embryonic stem cells does not affect transcriptional activity of pluripotency genes OCT4, SOX2, and NANOG, suggesting that PRMT5 is not involved in maintaining pluripotency of human stem cells. The same study also showed that PRMT5 plays a crucial role in promoting human embryonic stem cell proliferation via transcriptional repression of the p57KIP2 cell cycle inhibitor during self-renewing conditions [More elopment . The rolt testes . Unlike elopment . In starnditions . Taken tMx1-Cre) in adult hematopoietic stem cells (HSC) [IL-2 and TNF-α, which become transcriptionally repressed under normal conditions [IL-2 and TNF-α.The role of PRMT5 extends beyond the germ cells, embryonic stem and neural stem cells compartments. PRMT5 plays an essential role in blood progenitor cell specification as demonstrated by the anemia and pancytopenia developed by conditional ablation of PRMT5 (ls (HSC) . Lack ofls (HSC) . Consequls (HSC) , suggestnditions . Thus, Pet al. (2014) showed that PRMT5 symmetrically methylates R30 and R35 of NF-κB/p65 in TNF-α activated endothelial cells, which in turn activates transcription of chemokine CXCL10/IP-10. This chemokine induces recruitment of various inflammatory cells such as Th1 (type-1 T helper) CD4+ T cells, CD8+ cytotoxic effector cells, natural killer, natural killer T cells, plasmocytoid dendritic cells, and some B-cell subsets to the site of inflammation [PRMT5 has also been shown to promote transcription of pro-inflammatory genes in activated endothelial cells during inflammation by methylating several transcription factors. PRMT5-mediated methylation of Homeobox transcription factor 9 (HOXA9) in TNF-α activated endothelial cells promotes transcription of pro-inflammatory genes such as the cytokine-induced expression of E-selectin and Vascular Cell Adhesion Molecule 1 (VCAM-1), which help leukocyte adhesion and infiltration at the site of inflammatory lesions . In a diammation . Furtherammation . Thus, uin vitro and in preclinical murine models of various aggressive and incurable malignancies. In light of these positive results, it is going to be important to develop an experimental therapeutic program that can facilitate translational research, which explores PRMT5 inhibition both alone and in combination with other drugs known to inhibit cancer cell growth.PRMT5 has emerged as an important biomarker for several types of cancer, and increasing evidence argues that enhanced expression of PRMT5 induces proliferative signaling, which drives cancer cell growth and survival. The oncogenic properties of PRMT5 are mediated by its ability to methylate both histone and non-histone proteins, which trigger changes necessary to reprogram cell growth from normal to malignant and ultimately metastatic. However, it is important to consider the fact that PRMT5 is also essential for normal development and differentiation. So, while it is justified to develop very potent PRMT5-specific inhibitors to treat various types of cancer, rigorous testing in cell culture and animal models should be done to rule out the adverse effects PRMT5 inhibition might have on other vital processes such as embryogenesis, spermatogenesis, neurogenesis, muscle regeneration, adult hematopoiesis and immune response. The ability of PRMT5 to methylate symmetrically specific and ubiquitous receptors, signaling molecules, transcription factors and histones, endows it with the capacity to transform the growth characteristics of almost any cell. Hence, as new information about the role played by PRMT5 in regulating cellular growth and proliferation becomes available, studies aimed at evaluating the efficacy of existing inhibitors, which can modulate other growth regulatory pathways, in combination with PRMT5 inhibitors should be conducted in cancer cells with elevated PRMT5 activity. While drug development efforts are ongoing to tailor more potent and specific inhibitors against different members of the PRMT family, which have been implicated in disease states, it is worth noting that encouraging results have been obtained with PRMT5 inhibition


Chronic inflammation and oxidative stress are common and co-substantial pathological processes accompanying and contributing to cancers. Numerous epidemiological studies have indicated that non-steroidal anti-inflammatory drugs (NSAIDs) could have a positive effect on both the prevention of cancer and tumor therapy. Numerous hypotheses have postulated that NSAIDs could slow tumor growth by acting on both chronic inflammation and oxidative stress. This review takes a closer look at these hypotheses. In the cancer process, one of the major signaling pathways involved is the WNT/β-catenin pathway, which appears to be upregulated. This pathway is closely associated with both chronic inflammation and oxidative stress in cancers. The administration of NSAIDs has been observed to help in the downregulation of the WNT/β-catenin pathway and thus in the control of tumor growth. NSAIDs act as PPARγ agonists. The WNT/β-catenin pathway and PPARγ act in opposing manners. PPARγ agonists can promote cell cycle arrest, cell differentiation, and apoptosis, and can reduce inflammation, oxidative stress, proliferation, invasion, and cell migration. In parallel, the dysregulation of circadian rhythms (CRs) contributes to cancer development through the upregulation of the canonical WNT/β-catenin pathway. By stimulating PPARγ expression, NSAIDs can control CRs through the regulation of many key circadian genes. The administration of NSAIDs in cancer treatment would thus appear to be an interesting therapeutic strategy, which acts through their role in regulating WNT/β-catenin pathway and PPARγ activity levels. The complex process of cancer can be defined in terms of three stages: initiation, promotion, and progression ,2,3. ManIn parallel, oxidative stress causes DNA damage in cancers . In the Several epidemiological studies have shown that non-steroidal anti-inflammatory drugs (NSAIDs) could have a positive effect on both the prevention of cancer and tumor therapy. Moreover, the regular administration of aspirin, a NSAID, has been found to be correlated with a reduction in cancer incidence . A regulNSAIDS act as peroxisome proliferator-activated receptor gamma (PPARγ) agonists and could thus downregulate the aberrant WNT/β-catenin pathway in cancers . PPARγ aThis review focuses on the interest of using NSAIDs in cancer therapy through their capacity to regulate the aberrant canonical WNT/β-catenin pathway and PPARγ, two systems that respond in an opposite manner.A strategy for reducing cancer risks could involve the use of NSAIDs  . Some stThe use of NSAID (aspirin) in ovarian cancer cells decreases Bcl-2 expression and increases Bax gene expression . The rolNSAIDs are known to protect cells from one step on the path to cancer through the inhibition of COX . COX preColorectal cancer presents epidermal growth factor receptor (EGFR) overexpression and the use of aspirin can downregulate EGFR . EGFR isCordia myxa fruit, would appear to be promising “NSAID-like” agents in that they inhibit cancer and inflammation [The long-term use of NSAIDs could result in the appearance of side effects such as renal failure and gastro-intestinal symptoms  . The admammation . NSAIDS act as PPARγ agonists by inhibiting COX-2 in gliomas  and coloThe ligand-activated transcriptional factor peroxisome proliferator receptor γ (PPARγ) is a component of the nuclear hormone receptor super family. It makes a heterodimer with retinoid X receptor (RXR), forming a PPARγ-RXR complex that binds to specific peroxisome proliferator response element (PPRE) regions in the DNA. It also activates numerous target genes involved in fatty acid transport (FABP3), cholesterol metabolism , glucose homeostasis , and lipid catabolism (SCD-1). This dimer acts on other coactivator proteins like PGC-1α, and leads to specific gene overexpressions . GlucoseSome positive effects have been observed with the administration of PPAR gamma agonists in the cancer process. PPAR gamma stimulation could reduce cancer development by the arrest of cell proliferation and the inhibition of the tumor growth factor . The decHowever, the use of PPAR gamma agonists does have some side effects, even if new molecules now have fewer disadvantages . RosigliSome studies have presented that chronic inflammation leads to DNA damage and tissue injury . ChronicThe immune system is also regulated by several inflammatory factors, such as the tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumor growth factor-β (TGF-β) ,91. TNF-Chronic inflammation is also responsible for an augmentation in cyclooxygenase 2  . SeveralOxidative stress is considered as an imbalance between the production and elimination of ROS and RNS ,102. CelNumerous studies have shown that the phenomenon by which oxidative stress can enhance chronic inflammation, which in a negative feedback could lead to cancers  Figure . The imbMany studies have observed that the canonical WNT/β-catenin pathway increases the inflammatory process . FurtherThe name WNT is derived from wingless drosophila melanogaster and its mouse homolog Int. The WNT pathway is implicated in several pathways and regulating signaling pathways, like embryogenesis, cell proliferation, migration and polarity, apoptosis, and organogenesis . During WNT ligands are lipoproteins that stimulate specific co-receptors . These WDuring the “off-state” of the WNT/β-catenin pathway, WNT ligands do not interact with FZD and LRP 5/6. The β-catenin destruction complex, composed by AXIN, APC (adenomatous polyposis coli) and GSK-3β (glycogen synthase kinase 3β), phosphorylates β-catenin. Then, phosphorylated β-catenin is destroyed into the proteasome.Many WNT inhibitors downregulate the canonical WNT/β-catenin pathway. GSK-3β is the main inhibitor of the WNT pathway. GSK-3β is a neuron-specific intracellular serine-threonine kinase that controls numerous signaling pathways like inflammation, neuronal polarity, and cell membrane signaling ,133,134.Positive interplay between WNT/β-catenin and NF-κB has been observed . The actNF-κB overexpression inactivates GSK-3β whereas it stimulates β-catenin signaling ,150. GSKThe over-activated PI3K/Akt pathway observed in the cancer process is stimulated by ROS production ,156. PTEThe role of PPARγ agonists remains unclear in cancer cells, even if their role is well understood in the regulation of differentiation and stemness programs . In physNSAIDs downregulate the activity of COX-2 and thereby inhibit the synthesis of prostaglandins (PGE2) and then the WNT pathway . Recent Several studies have also shown that NSAIDs can inhibit the canonical WNT/β-catenin pathway . Both asNumerous biological phenomena in the body are regulated by the circadian “clock” (circadian locomotors output cycles kaput). The circadian clock is in the hypothalamic suprachiasmatic nucleus (SCN). CRs are endogenous and entrainable free-running periods that last approximately 24 h. Numerous transcription factors can regulate CRs. These are called circadian locomotor output cycles kaput (Clock), brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 , Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), and Cryptochrome (Cry 1 and Cry 2) ,186 Fig. These fEpidemiological and fundamental evidence supports the idea of linking circadian disruption with cancer . DNA repMelatonin has been used in the treatment of chronic bowel inflammation resulting in decreasing inflammation through inhibition of COX-2 and iNOS . MoreoveRecent studies have indicated that the hypoxic response in cancer could be directly controlled by the circadian rhythm Clock/Bmal1 . In a siThe WNT/β-catenin pathway is the downstream target of the RORs control factors and has several putative Bmal1 clock-binding sites within its promoter  Figure . ThroughIn physiological circumstances, the core circadian genes work in accurate feedback circles and keep the molecular clockworks in the SCN. They permit the regulation of peripheral clocks ,188. PerFew studies have investigated the role of NSAIDs regarding the circadian clock. Nevertheless, Kowanko et al. observed that pain reported in rheumatoid arthritis after a twice a day therapy by flurbiprofen may be more efficient than four times daily flurbiprogen, and that regimen without an evening dose was the least efficient of three twice-daily therapies tested. Moreover, their results showed that morning stiffness in rheumatoid arthritis was not only the result of nocturnal inactivity but also a response to an appropriately timed medication . MoreovePPARγ acts directly with the core clock genes and presents diurnal variations in liver and blood vessels ,224 Fig. In miceThe numerous interactions between clock dysregulation and cancer underline the interest of circadian therapeutic actions . The temIn mouse ovaries, melatonin administration protects against ROS production and mitochondrial damage . In coloChronic inflammation, oxidative stress and the disruption of circadian rhythms are important factors in the cancer process and are enhanced by overstimulation of the WNT/β-catenin pathway. In cancers, the WNT/β-catenin pathway is generally activated whereas PPARγ is decreased. These two signaling pathways act in opposing manners and this could explain their unidirectional profile observed in cancers. The use of NSAIDs, which act as PPARγ agonists, could be interesting in the reduction of both chronic inflammation and oxidative stress and in the control of circadian rhythms by inhibiting the WNT/β-catenin pathway . Due to


Treponema denticola (Td), has been linked to orodigestive carcinogenesis, but its role in oropharyngeal squamous cell carcinoma (OPSCC) has remained open. We evaluated the presence of Td chymotrypsin-like protease (Td-CTLP) in a series of 201 unselected consecutive OPSCC patients, and the relation of the Td-CTLP to human papillomavirus (HPV) status, to expression of toll-like receptors (TLR) 5, 7, and 9, and to clinical parameters and patient outcome.An opportunistic oral pathogen, Td-CTLP was evaluated by immunohistochemistry. Immunoexpression of TLRs 5, 7, and 9, and HPV status we studied earlier in this patient series.Clinicopathological data came from hospital registries. The expression of cell surface-bound Td-CTLP in 81% of the OPSCC, and especially in HPV-negative tumours . Among the HPV-positive tumours , low Td-CTLP expression associated with low TLR 5 and high TLR 7 expression. Among those HPV-negative, higher TLR 5 and lower TLR 7 expression associated with high Td-CTLP expression. Strong Td-CTLP expression associated with poor disease-specific survival, but no similar association among HPV-positive and HPV-negative subgroups emerged.We detected Td-CTLP was highly expressed in OPSCC and was associated with the HPV status of tumour tissue. Recently, Td has been evident also in oral and gastrointestinal tumour samples.11 Furthermore, Fusobacterium nucleatum has been associated with colorectal cancer and shown to promote colorectal carcinogenesis.12 Oral carcinogenesis, on the other hand, was promoted by F. nucleatum and P. gingivalis based on in vitro and in vivo studies via interaction with oral epithelial cells through toll-like receptors (TLR).13 The prevalence and role of oral pathogens in various OPSCC HPV subgroups calls for investigation.Besides the known association of HPV with OPSCC, the role of several oral pathogens in oral and gastrointestinal tract cancers is evident. Epidemiological studies have shown an association between elevated periodontal pathogen 17 and with the development of diabetes mellitus type I and II complications.18 The mechanisms explaining the associations involve chronic low-grade systemic inflammation and oral microorganisms entering the bloodstream.21Td is an invasive opportunistic pathogen belonging to a red complex group of oral pathogens identified in severe forms of periodontitis.22 Although Td, as well as other oral anaerobic spirochetes, may be present in the normal oral flora, its proportion increases significantly in periodontal infection.23Td occurs in oesophageal cancer tissue10 and in squamous cell carcinomas (SCC) of the tongue, tonsils, and oesophagus, as well as in adenocarcinomas of the stomach, pancreas, and colon.11Td possesses several virulence factors including an ability to adhere to epithelial cells and extracellular matrix components,26 to produce degrading enzymes,30 to secrete cytotoxic substances,31 to activate host-derived tissue-destructive matrix metalloproteinases,11 and to suppress local immune responses.32 One of its key virulence factors, a chymotrypsin-like protease (Td-CTLP), degrades a number of basement membrane components, thus enabling Td to invade epithelial tissue.28 The role of Td in the carcinogenesis of OPSCC is thus far unknown. However, the host’s systemic inflammatory response to chronic exposure to Td and its toxins,33 in combination with the proteolytic and immunomodulatory activities triggered by the bacteria, may be of significance.11During the last decades periodontitis has been associated with several systemic diseases, e.g., cardiovascular diseases,Td-CTLP), a key virulence factor of the oral pathogen Td, in 201 unselected consecutive OPSCC patients, and this factor’s relationship to HPV status, TLR 5, 7, and 9 expression, clinical parameters, and patient outcome.Our aim was to determine the prevalence of a cell surface-bound chymotrypsin-like proteinase , concurrent head and neck squamous cell carcinoma (HNSCC) (n = 5), earlier treated HNSCC (n = 11), histology other than SCC (n = 18), or tumour tissue unavailability (n = 52).This patient cohort was previously described.35; primary treatment, i.e., surgery (Sx), radiotherapy (RT), and chemotherapy (CRT); tumour recurrence, treatment of recurrent disease, and status at the last follow-up.34 Follow-up of all patients was at minimum 3 years or until death. Data on dates and causes of death came from Statistics Finland. The study received institutional permission and the approval of The Research Ethics Board at the Hospital District of Helsinki and Uusimaa.Clinicopathological data from hospital registries, based on the earlier investigation on this same patient group, comprised patient age, sex, tumour histology, grade, UICC 7th edition TNM stageIn the group of 201 patients, 130 underwent primary surgery. Among these 130 patients 116 received also either RT or CRT as adjuvant oncological treatment. Five patients received no postoperative oncological therapy because of having Stage I–II disease and nine had none for case-related reasons. Definitive CRT or RT was delivered to 71 patients, and among them 11 underwent salvage surgery in the primary treatment phase . Tissue samples were collected before RT/CRT with the exception of two patients with only post-treatment samples available for immunohistochemistry.34 Briefly, HPV status was defined by Ventana Inform HPV in situ hybridisation (ISH) assay using a high-risk HPV probe and iVIEW Blue detection kit in Benchmark XT series stainer . This assay has affinity to these high-risk HPV subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 56, 58, and 66. As a pretreatment, 5-μm thick sample sections were incubated with an extended Ventana cell-conditioning solution including ISH protease 3 (Ventana) for 32 min. HPV status was regarded as positive if any spot in ISH assay was positive. TLR status was studied by immunohistochemical staining by monoclonal mouse anti-human TLR 5 , monoclonal rabbit anti-human TLR 7 , and polyclonal rabbit anti-human TLR 9  as described.14HPV status and the expression of TLRs 5, 7, and 9  had undergone analysis earlier by our research group.Td-CTLP antibody was prepared according to the method of Grenier et al.28 The IgG fraction of the CTLP antibody was purified and washed on a Protein A-Sepharose® CL-4B column . The resulting anti-CTLP IgG fraction was tested for its specificity to Treponema denticola in immunofluorescence and ELISA assays with a closely related oral spirochete Treponema vincentii as a reference by a method described by Nieminen et al.1111 Polyclonal anti-CTLP IgG (1:1500 dilution) served as the antibody against Td-CTLP. Negative-control staining involved a non-immune species-specific Rabbit IgG  and by omitting the primary antibody.Preparation of the tissue microarray (TMA) blocks and immunohistochemical sample staining were performed as described by Nieminen et al.Td-CTLP antibody were scored by two researchers (J.H. and A.K.K.) separately. Any discordance in scoring was solved by reassessment to achieve consensus. Td-CTLP scoring was based on intensity of the positivity in tumour tissue: none (0), mild (1), moderate (2), or strong (3). When several tumour spots were available for analysis, the choice was the highest positivity value for Td-CTLP. The positivity in Td-CTLP staining was localised in the cytoplasm of the carcinoma cells. In addition, immunopositivity was detectable in non-cancerous epithelial cells, secretory cells, and mononuclear inflammatory cells of some tissue samples, but these were not included in scoring evaluation.The decoded TMA blocks immunostained with P-value, whichever appropriate. The 5-year disease-specific survival (DSS) rates were calculated by Kaplan–Meier (KM) estimation. A log-rank test served in evaluation of statistical significance in the KM analysis. The follow-up time in the DSS evaluation was defined as the period between the last treatment day and the last day of follow-up or date of death from the disease. To minimise the bias in follow-up, the maximum follow-up period for the analysis was 5 years.Statistical analysis utilised SPSS version 20.0 . Statistical differences between categorical variables were evaluated based on Chi-square testing using an asymptotic or an exact P-value less than 0.1. The proportional hazard assumptions were tested with KM curves. A two-sided P-value less than 0.05 was considered statistically significant.Multivariate survival analysis used the Cox proportional hazards model. The analysis included clinically relevant variables with a Td-CTLP immunoexpression in the 201 samples occurred as follows: 19% had none (n = 39), 31% mild (n = 63), 31% moderate (n = 63), and 18% strong (n = 36) expression. Td-CTLP was expressed in the cytoplasm of carcinoma cells in granular and diffuse forms . Td-CTLP staining was more prominent in Grade-1 tumours than in Grade-3 tumours. High Td-CTLP immunopositivity was associated with class-N0 disease. Stage I–II disease had higher Td-CTLP immunoexpression than did Stage III–IV disease. Expression of Td-CTLP was higher among smokers and ex-smokers than among non-smokers  had a tumour arising from the lateral wall of the oropharynx, followed in prevalence by the anterior (30%), superior 10%), and posterior wall (1%). In anterior and lateral-wall carcinomas, none to moderate immunoexpression  of 0%, and pTd-CTLP immunoexpression existed between patients receiving primary surgery and RT/CRT as adjuvant treatment (n = 130) and those treated with definitive RT/CRT (n = 71). Furthermore, no difference in the 5-year DSS was observed between the treatment groups.No difference in the Td-CTLP immunoexpression showed a significant association with HPV status. Strong expression was associated with HPV negativity and mild expression with HPV positivity  than did patients with strong Td-CTLP immunoexpression (61%) (P = 0.013) . Among patients with HPV-negative OPSCC, the 5-year DSS was 73% among patients with Td-CTLP score 0–2 and was 54% among those with strong Td-CTLP expression, but this difference was not significant (P = 0.089). In multivariate analysis, Td-CTLP was never an independent prognostic factor (P = 0.115).In survival analysis, no difference in the 5-year DSS was observed when the patient groups stained with none to mild (0–1) and moderate to strong 2–3)  Td-CTLP Td-CTLP in the majority (87%) of all HPV-negative OPSCCs. According to the literature, the same common risk factors: smoking and poor oral hygiene, are associated with development of HPV-negative OPSCC36 and periodontitis.39 Our results are in line with these findings, as we found an association between regular smoking and high Td-CTLP immunoexpression. In addition, we detected Td-CTLP immunopositivity also among 74% of HPV-positive OPSCC samples. Its expression levels were, however, significantly lower than for the HPV-negative subgroup. Interestingly, some studies suggest that chronic periodontitis is a risk factor for HPV-positive base-of-tongue and head-and-neck cancers,41 and findings support the hypothesis of the periodontal pocket as a reservoir for latent HPV.42 Our investigation provides no information as to whether HPV infection is followed by Td infection or vice versa in the subgroup of HPV-positive and Td-CTLP-positive cancers. Thus, the role of Td in HPV-positive OPSCC remains open and requires further examination.We found Td-CTLP immunoexpression was associated with tumours arising from the anterior and the lateral wall of the oropharynx. In addition, low Td-CTLP immunopositivity was more prominent in poorly differentiated tumours. These findings are consistent with Goldberg et al.,43 who showed HPV-positive tumours as being more likely to have higher differentiation grade and to arise predominantly from the palatine or lingual tonsils. The present finding of an association between lymph node metastasis and low Td-CTLP immunoexpression is in line with the results linking HPV-positive OPSCC and higher N class.44 Further, we detected an association between lower Td-CTLP immunoexpression and stage III–IV tumours, which probably is explained by the fact that HPV-positive OPSCC is typically diagnosed at an advanced stage.5Low Td-CTLP immunoexpression in the HPV-positive subgroup was associated with low TLR 5 expression and high TLR 7 expression is in line with our earlier results. Those showed HPV-related OPSCC having typically low TLR 5 and high TLR 7 immunoexpression. In addition, earlier we proposed that a high TLR 5 and low TLR 7 expression in HPV-positive OPSCC is associated with poor DSS.34 Unfortunately, in the present study, the number of HPV-positive, strong Td-CTLP immunoscore samples with high TLR 5 and/or low TLR 7 expression was too small (n = 2) for survival evaluation. Nevertheless, our current finding of an association of strong Td-CTLP immunoexpression with high TLR 5 expression and low TLR 7 expression in the HPV-negative subgroup suggests that differences exist in the immunological responses between HPV-positive and HPV-negative OPSCC subgroups with Td infection, and that these may be linked to the presence of Td.TLRs and their role in carcinogenesis have been widely studied. TLRs participate in innate immune response cascades through recognition of pathogen-associated molecular patterns of bacteria, viruses, fungi, and parasites, and non-infectious structures, for example from dead or dying cells associated with tissue damage. That low Td-CTLP immunoexpression had significantly better 5-year DSS (79%) than did patients with strong Td-CTLP expression (61%). A similar trend, although not statistically significant, was apparent for DSS in HPV-negative OPSCC. The number of patients with strong Td-CTLP expression in HPV-positive OPSCC was too small for us to estimate the effect of strong Td-CTLP on survival in this subgroup. These findings, however, evoke further questions on the role of Td infection on survival of patients with HPV-positive as well as HPV-negative OPSCC, and need to be studied in larger patient series.We showed that patients with weaker Td-CTLP was highly expressed in OPSCC, and was associated with the HPV status of the tumour tissue. High immunoexpression of Td-CTLP in HPV-negative OPSCC may be a result of common risk factors for HPV-negative OPSCC and Td infection and periodontitis. The role of Td-CTLP in immunological responses, carcinogenesis, and clinical outcome needs further investigation both in HPV-negative and HPV-positive OPSCC.


FBXO16 complex that targets nuclear β‐catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant β‐catenin activation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Aberrant activation of β‐catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/β‐catenin‐signaling pathways is still poorly understood. In this study, we show that F‐box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1‐Cullin1‐F‐box protein) complex, which targets the nuclear β‐catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C‐terminal domain of β‐catenin and promotes its lysine 48‐linked polyubiquitination. In addition, it inhibits epithelial‐to‐mesenchymal transition (EMT) by attenuating the level of β‐catenin. Therefore, depletion of FBXO16 leads to increased levels of β‐catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and β‐catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCF Cancer is one of the leading causes of death, and breast cancer contributes to a majority of the mortality rate among women Previous reports suggested the activation of Wnt signaling in 50% of breast cancer cases, with a relatively lower frequency of somatic mutations in Wnt signaling components It is well known that the proteasomal degradation of cellular proteins is mostly controlled by SCF (SKP1‐Cullin1‐F‐box protein) E3 ubiquitin ligases. F‐box proteins are an integral component of the SCF complex and function as substrate receptors. Human genome encodes 69F‐box proteins; however, the function of a majority of the F‐box proteins remains elusive Here, we decipher the function of FBXO16, an F‐box protein, in cancer. In an attempt to identify interacting proteins of FBXO16, we adopted a mass spectrometry approach. We identified β‐catenin as a vital cellular substrate of FBXO16. Our results show that FBXO16 is a putative tumor suppressor that suppresses the growth, migration, invasion of cancer cells, and EMT by directing the proteasomal degradation of nuclear β‐catenin. Interestingly, the expression of FBXO16 and β‐catenin is conversely correlated in breast cancer patient samples, with the attenuation of FBXO16 expression with cancer progression. Altogether, these findings might contribute to developing new therapeutic targets for limiting nuclear β‐catenin‐mediated malignancy.m l‐glutamine and were cultured in 5% CO2 at 37 °C in a humidified atmosphere.The human cell lines MCF10A, MCF7, MDA‐MB‐231; T47D and HCT‐116; and HEK293 and HEK293T were kind gifts from Prof. Michael Green . MCF7, HEK‐293, and HEK‐293 T cells were cultured at 37 °C in DMEM . MDA‐MB‐231, T47D, and HCT‐116 were maintained in RPMI‐1640. MCF10A cells were grown in DMEM‐F12 (Gibco) media. All tissue culture media were supplemented with 10% FBS/horse serum and 2 mN‐β‐catenin, GFP‐ΔC‐β‐catenin, GFP‐ΔNC‐β‐catenin, and GFP‐T41A‐β‐catenin were kind gifts from Dr. Jomon Joseph, Pune, Maharashtra, India. psPAX2 and pMD2.G were obtained from Didier Trono . Human cDNA encoding FBXO16 was procured from Origene . The F‐box deletion mutant was generated using the primers shown in supplementary material, Table GFP‐WT‐β‐catenin, GFP‐Δg, and supernatants were collected. Cellular fractionation was performed as described previously e‐cadherin (610181) were from BD Biosciences . Antibodies to c‐Myc (9402), phospho GSK3β (8566), phospho PKCδ (9376), and K48‐linked ubiquitin antibodies (8081) were from Cell Signaling Technology ; to DDK (clone OTI4C5) from Origene; and FBXO16 (PA5‐31419) was from Thermo Fisher Scientific .Cells were harvested, washed with PBS, and lysed with buffer on ice for 30 min Immunoprecipitation was performed using 600 μg proteins and 2 μg of antibody in IP lysis buffer as described previously m MG132 for 6 h before harvesting. Cells were lysed, and protein extracts were immunoprecipitated with anti‐DDK antibody MCF7 cells expressing either vector or DDK‐FBXO16 were grown in 5 μLentiviral shRNAs  were a kind gift from Prof. Michael Green . Stable knockdown cells were generated by lentivirus transduction as described previously A total of 5000 cells (transduced/treated) were seeded in 35 mm‐diameter dishes and allowed to grow for 12–15 days to form colonies. Cells were fixed with 3.7% formaldehyde and stained with 0.05% crystal violet. Plates were photographed, and representative images are shown in Figure Soft agar assays were performed as described previously Scratch wound‐healing assay was performed as described previously Invasion assays were performed as described previously GAPDH was used to normalize the data. The primers used are listed in supplementary material, Table RT‐qPCR was performed using SYBRmix as described previously In vitro and in vivo ubiquitination assays were performed as described previously in vivo) and ubiquitylation reaction mixture (in vitro) were resolved by SDS‐PAGE and blots probed with an ubiquitin‐specific antibody .Tissue samples were obtained from SDM College of Medical Sciences following established protocols and with Institutional Ethical Board approval. Tissue samples were stained with H&E, and the histological type and grade of tumors were determined. β‐Catenin and FBXO16 levels in breast cancer patients, including malignant tissue and adjacent nonmalignant epithelium, were detected using immunohistochemical staining and were quantified as described previously Cells were seeded in a 24‐well plate. Cells were transduced/transfected with the indicated plasmids for 60 or 36 h, respectively. Cells were then treated with either EGF or Wnt3a‐conditioned medium for 12 h prior to harvesting. Luciferase activity was measured according to the manufacturer's protocol , and Renilla luciferase was used for normalization.m glycine. Cells were then washed with PBS and lysed. The cell lysates were sonicated and then incubated overnight with anti‐β‐catenin antibody at 4 °C. Preblocked protein A agarose beads were incubated with lysate–antibody complex for 2 h, followed by reversal of cross‐linking using proteinase K. DNA was extracted using a phenol–chloroform method. qPCR was performed using indicated primers listed in supplementary material, Table Chromatin immunoprecipitation (ChIP) assay was performed as described previously 6 cells/mice) stably expressing NS and FBXO16 shRNA were injected subcutaneously in the right flank of 5‐week‐old NOD‐SCID mice (n = 5). The length (L) and width (W) of tumors were measured with a digital Vernier caliper, and tumor volume was calculated using the formula: V = (L × W2)/2, with W being smaller than L. All animal experiments were approved by the Institutional Animal Ethics Committee of the National Centre for Cell Science.MCF7 cells  were overexpressed in MDA‐MB‐231 cells. Results demonstrated that ΔF‐FBXO16 is incompetent to reduce the expression of β‐catenin Figure A. IntereWnt ligands are the canonical activators of β‐catenin A previous study showed that EGF also activates β‐catenin signaling MYC and CCND1 .Supplementary materials and methodsSupplementary figure legendsFigure S1. Analysis of immunoprecipitatesFigure S2. FBXO16 regulates β‐cateninFigure S3. FBXO16 maintains basal levels of β‐cateninFigure S4. FBXO16 regulates β‐catenin in phosphorylation independent mannerFigure S5. Depletion of FBXO16 promotes EMTFigure S6. Depletion of FBXO16 promotes tumorigenesis.Table S1. Primers used for generating F‐box deleted FBXO16Table S2. Primers used for RT‐qPCRTable S3. Primers used for ChIP assaysTable S4. Interactomes of FBXO16 (minimum three unique peptides were identified for each protein)Supplementary materials and methodsClick here for additional data file.Supplementary figure legendsClick here for additional data file.Figure S1. Analysis of immunoprecipitatesClick here for additional data file.Figure S2. FBXO16 regulates β‐cateninClick here for additional data file.Figure S3. FBXO16 maintains basal levels of β‐cateninClick here for additional data file.Figure S4. FBXO16 regulates β‐catenin in phosphorylation independent mannerClick here for additional data file.Figure S5. Depletion of FBXO16 promotes EMTClick here for additional data file.Figure S6. Depletion of FBXO16 promotes tumorigenesisClick here for additional data file.Table S1. Primers used for generating F‐box‐deleted FBXO16Table S2. Primers used for RT‐qPCRTable S3. Primers used for ChIP assaysTable S4. Interactomes of FBXO16 (a minimum of three unique peptides were identified for each protein)Click here for additional data file.