Abnormal regulation of β-catenin initiates an oncogenic program that serves as a main driver of many cancers. Albeit challenging, β-catenin is an attractive drug target due to its role in maintenance of cancer stem cells and potential to eliminate cancer relapse. We have identified C2, a novel β-catenin inhibitor, which is a small molecule that binds to a novel allosteric site on the surface of β-catenin. C2 selectively inhibits β-catenin, lowers its cellular load and significantly reduces viability of β-catenin-driven cancer cells. Through direct binding to β-catenin, C2 renders the target inactive that eventually activates proteasome system for its removal. Here we report a novel pharmacologic approach for selective inhibition of β-catenin via targeting a cryptic allosteric modulation site. Our findings may provide a new perspective for therapeutic targeting of β-catenin. The APC complex tightly regulates the cellular concentration of β-catenin. This is a dynamic assembly that is composed of GSK3β, CK1α, APC and AXIN. Phosphorylation activity of GSK3β and CK1α marks β-catenin at S33, S37 and T41, which in turn signals its recycling through ubiquitin-proteasome pathway5. Alternative phosphorylation by AKT and PKM1/2 at residues S552, Y654 and S675 may on the other hand enhance the activity of β-catenin7. Binding of Wnt ligand to Frizzled and LRP5/6 is known to halt the APC complex, leading to translocation of β-catenin into the nucleus. Increased nuclear presence of β-catenin activates the TCF/LEF1-mediated oncogenic events leading to increased stem cell-like behavior in cancers and poor clinical prognosis9.Wnt signaling pathway regulates expression of numerous genes that control development and tissue homeostasis10. Mutations in the pathway components, including APC, Axin, and β-catenin, have been closely linked with various cancers14. APC plays a pivotal role in β-catenin-dependent tumorigenesis, such that partial or complete loss of its function is recognized as a hallmark of many colon cancers. Impaired APC function leads to over-expression of β-catenin, which in turn renders cancer cells sensitive to its inhibition16. This vulnerability of many colon cancers is at the core of new therapeutic approaches for β-catenin, and may serve as a source of selectivity for novel β-catenin inhibitors. Indeed inhibition of β-catenin has been in the focus of a number of recent studies17. Some of the early Wnt pathway inhibitors are ICG-001, XAV939 and pyrvinium that target CREB protein (CBP), tankyrase and CK1α, respectively. Although these are valuable as research tools, they have limited therapeutic utility17. Recent efforts have been spent on targeting the β-catenin/TCF4 complex, i.e. the catenin responsive transcription (CRT), with an expectation to inhibit its downstream signaling. The CRT complex interacts with several gene regulators through a conserved mechanism, thus raising questions about specificity of this approach17. One recent study reports discovery of inhibitors iCRT-3, -5 and -14 that target the CRT complex18. While this provides hope for direct targeting of β-catenin, it is not clear whether they trigger degradation of β-catenin, thus raising concerns about the possibility of its re-localization back into the nucleus. Yet another promising study reports discovery of a small molecule MSAB, which allegedly promotes degradation of β-catenin and slows its oncogenic activity at high concentrations19. A more specific inhibitor, stapled peptide StAx35R, is reported to block AXIN from binding to β-catenin20. Whereas the peptide is large enough to cover a significant surface area on the target, it carries the drawback of low cell penetration thus necessitating its use at high doses. The clinical advantage of the stapled peptide class of molecules has yet to be established17.Aberrant Wnt signaling has been closely associated to carcinogenesis21. We report discovery of C2, an experimental small molecule that inhibits β-catenin via direct binding to an allosteric site. C2 reduces the oncogenic pressure of β-catenin and triggers its degradation in β-catenin-overexpressing cancer cells. We anticipate that our findings will contribute to the understanding of basic β-catenin biology, as well as encourage further drug discovery.While there has been an encouraging increase in the number of Wnt inhibitors, their mechanism of action of these compounds needs to be further elucidated to assess suitability for therapeutic use. As much as Wnt signaling remains attractive for therapeutic intervention, much attention needs to be allocated to therapeutic index and unwanted cell toxicity. It is therefore pharmacologically more meaningful to directly target β-catenin, rather than interfere with its upstream partners that are involved in other essential cellular functions. Direct targeting of β-catenin will eliminate toxicity and increase therapeutic benefit23.Focused computational analysis has revealed a cryptic allosteric site, site C, which is not involved in canonical interactions known to drive Wnt signaling pathway . This method relies on the hydration shell, charge or size of molecules by measuring changes of the mobility of molecules in microscopic temperature gradientsin silico approximations, we estimated the binding site of C2 to be in the junction of Armadillo domain and C-terminal Tans-Activation Domain (CTAD), spanning between residues 531–722. Therefore we questioned domain-specific interaction of C2 with β-catenin, in order to gain deeper insight into its molecular mechanism. We designed several TopFlash constructs carrying various domains of β-catenin, particularly Wild-type (WT), S33A, ΔNTAD, ΔCTAD, ΔNTAD/ΔCTAD and ΔARM  using SPR. We were able to measure binding affinity for each domain: 11 nM, 12 nM, 14 nM and 108 µM for domains ΔNTAD, ΔCTAD, ΔNTAD/ΔNTAD and ΔARM, respectively  of C2 for 24 hours. Efficiency of transformation and cell viability was checked across all reporters and was found to be consistent  for all molecular modeling, virtual ligand screening, cheminformatics and other computational biology tasks. All modeling and simulations were performed on a single desktop machine running Debian Squeeze Linux system , and in Q-MOL cloud services.We used Q-MOL molecular modeling software from Q-MOL LLC , were used as molecular probes to systematically scan the molecular surface of β-catenin by applying protein-ligand docking methodology as implemented in the Q-MOL molecular modeling platform . BCL9 and TCF4 peptides were removed from the complex before docking simulations. The protein molecule preparation included adding of hydrogen atoms and the assignment of the OPLS atom types. The ligand-binding center for site C  was performed as previously describedin vitro validated ligands were built using full-atom flexible protein-ligand docking in the internal coordinates as implemented by the Q-MOL program. Briefly, the initial ligand conformations were taken from the VLS experiments. The protein-ligand complex was then globally optimized in the OPLS39 force field using the Monte Carlo simulation in internal coordinate space. The protein Phi, Psi and Xi angles were allowed to change. In the case of a ligand molecule, its positional and rotable torsion variables were unfixed.The predicted binding modes of the Selected compound candidates were obtained from the NCI DTP; 20 mM stocks were prepared in DMSO. HEK293 cells were seeded in 6-well plates and transfected with a cocktail of the following plasmids: Cells were cultured in DMEM and 2% FBS at 37 °C for 1 day, then transferred into 96-well plate format (10.000 cells per well). After 12 hours, DMEM medium with diluted compounds was added to the cells. Initially, single dose (3 µM) per compound. Similarly, for TopFlash/FopFlash assay, HEK293 cells were transfected with a cocktail of these plasmids: 1 µg FopFlash, 1 µg pcDNA3-β-catenin and 50 ng pRL-TK using Lipofectamine 2000 (Invitrogen). For this assay, a dose range between 20 µM and 0.3 µM was tested for each compound. Luciferase reporter activity was measured using the Dual-Glo system (Promega) after 24 hours of incubation. Relative luciferase activity is normalized to DMSO control. All measurements were done in triplicates (n = 3).25, and the final constructs were confirmed with sequencing. HEK293 cells were cultured and transfected using Lipofectamine 2000 with a cocktail of these plasmids: 1 µg TopFlash, 1 µg of corresponding pcDNA3 construct and 50 ng pRL-TK. Cells were treated with a dose range (10–1.25 µM) of C2, incubated for 24 hours, then luciferase signal was measured. Signal was normalized to DMSO treatment of WT control. All measurements were done in triplicates.Domains of β-catenin were cloned into pcDNA3 plasmid using primers described beforeBL21 cells harboring the pPET28a-TEV-full-length human β-catenin (Addgene) plasmid were cultured at 37 °C until OD600 = 0.8 and were then induced with 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG). Cells were cultured for another 5 h at 20 °C. Cell pellets were collected and sonicated in buffer with 300 mM NaCl, 20 mM Tris (pH 8.8). Soluble His-tagged β-catenin was purified from cell lysate using a HiTrap column packed with Sepharose resin , with a gradient of 20–200 mM imidazole. Purified protein was dialyzed into the same buffer without imidazole. Domains of β-catenin  were cloned into the same plasmid. Expression and purification of these proteins were done using the same protocol.Thermal shift assays (TSA) were conducted with the 96-well-based CFX-96 real-time fluorescence plate reader . The fluorescent dye Sypro Orange  was used to monitor the protein folding-unfolding transition. Protein-ligand binding was gauged by shift in the unfolding transition temperature (ΔTm) acquired with protein alone or with protein in the presence of the inhibitor. 2.5 μM of purified wild-type human β-catenin protein was mixed with 20 μM of candidate compound in PBS buffer containing 2X SYPRO Orange dye. Negative control: protein plus corresponding amount of DMSO. Positive controls: StAx35R and iCRT3, 10 μM each. The sample plate was heated from 25 °C to 95 °C with a thermal ramping rate of 1 °C/min. The fluorescence signals were acquired with excitation and emission wavelengths centered at 490 and 560 nm, respectively. Differences in melting temperatures (ΔTm) were calculated via subtraction of Tm of the negative control.on for association rate, Koff for dissociation rate) were calculated via fitting of data to 1:1 Langmuir model.Interaction of with C2 with His-tagged β-catenin and its domains was measured using surface plasmon resonance (SPR) on a ProteOn instrument (Bio-Rad). First, protein was captured on the activated surface of HTE chip. Binding experiments were run in PBS buffer supplemented with 0.01% P-20 and 1% DMSO. C2 was dissolved in the same buffer and passed over His–β-catenin functionalized surface as well as activated blank reference. Measurements were performed in triplicates with three-fold dilutions (0.2 µM to 0.0025 µM). Binding constants  was incubated with 50 nM purified β-catenin protein for 5 min in assay buffer (PBST with 2.5% DMSO). The samples were loaded into NanoTemper glass capillaries and micro-thermophoresis was carried out using 60% excitation power and 40% MST, 25 °C. K2, cells were stained with Alamar blue and counted at wavelength of 560/590. Viability curves were plotted and IC50 values calculated using Prism8 GraphPad software.Fully tested and authenticated cancer cell lines  were purchased from ATCC (American Type Culture Collection). Cells were plated in 96-well plates with seeding density of 5000 cells per well in RPMI 1640 medium supplemented with 2% FBS. After 24 hours of incubation, a dose range of C2 under 20 µM was tested in triplicates. After 72 hours of incubator at 37 °C/and 5% CODLD1 and SW48 cells were plated in 6-well plates with seeding density of 400 cells per well in RPMI 1640 medium supplemented with 2% FBS. 24 hours later C2 (1 µM and 3 µM) or DMSO alone was added to the cells. Medium and treatment was renewed every other day. After 7 days of incubation, cells were stained with crystal violet and colonies were counted.Renilla luciferase. Negative control carried a non-inducible firefly luciferase costruct and a constitutively expressing Renilla luciferase construct, while positive control carried a constitutively expressing luciferase construct and a constitutively expressing Renilla luciferase construct. HEK293 cells transfected with the reporters were treated with a dose range (10–0.3 µM) of C2 for 24 hours.Signal Finder Cancer 10-Pathway Reporter Array included reporters for WNT (TCF/LEF), Notch (RBP-Jκ), p53/DNA damage, TGFβ (SMAD2/SMAD3/SMAD4), cell cycle (E3R/DP1), NFκB, Myc/Max, Hypoxia-inducible factor 1 (HIF-1), MAPK/ERK (Elk1/SRF), MAPK/JNK (AP1), together with negative control and positive control (Qiagen). All reporters carried luciferase construct with the corresponding transcription regulatory element together with constitutively expressing Cells were grown in RPMI 1640 medium supplemented with 2% FBS and 5 µM C2 or DMSO for 24 hours. For whole-cell western analysis, cell lysates were prepared in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl and 1 mM PMSF), and equal amount of cell lysate was loaded onto SDS-PAGE gel. Detection and quantitation of protein signal was done on LI-COR C-Digit Blot Scanner. For immuno-precipitation experiments, cells were treated with DMSO, C2 (5 µM), MG132 (10 µM) and combination of C2 (5 µM) and MG132 (10 µM) for 3 hours. Subsequently cells were lysed in TBS [150 mM NaCl and 20 mM Tris (pH 7.4)] with 1 mM PMSF and 1% Triton X-100 at 4 °C for 30 min, followed by removal of insoluble debris by centrifugation at 13,000 × g. Lysate with total protein amount of 500 µg was initially cleared with IgG, then incubated with 5 µg of anti-β-catenin antibody at 4 °C for 4 h. Then, protein A/G agarose beads (Santa Cruz Biotech) were added to the sample at 4 °C for 2 hours. Beads were washed 3× with TBS + 0.01% Tween 20, then boiled in SDS gel loading buffer at 95 °C for 5 min. Western blot analysis was performed as described above. 5% of total protein used for immune-precipitation was loaded as input. For time-course analysis, cells were treated with C2 at 1, 3, 6, 12 and 24 hour intervals, after which a standard western analysis protocol was applied. For nuclear-cytoplasmic fractionation, cultured DLD1 cells were resuspended in PBS buffer supplemented with 5% glycerol and protease inhibitors, then passed through 26 G needle multiple times on ice. Nuclear and cytoplasmic fractions were separated via centrifugation at 10.000 rpm. The fractions were then denatured in RIPA buffer and loaded onto SDS-PAGE gel for subsequent Western blot analysis. All primary and secondary antibodies were purchased from Cell Signaling Technologies.40. Co-localization of β-catenin and DAPI was compared for C2 and DMSO.To investigate cellular distribution of β-catenin, HCT116 cells were seeded in chamber slides (LAB-TEK) and incubated in 0.5 ml RPMI 1640 medium supplemented with 2% FBS and 1 µM C2. After 12 hours of incubation at 37 °C, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. Cells were then probed with anti-β-catenin primary antibody , washed and then probed with Alexa Fluor-tagged anti-rabbit IgG secondary antibody (Life Technologies). Subsequently, cells were incubated with phalloidin rhodamine (Life Technologies). Finally, mounting media with DAPI was used to attach No 1.5 cover slides. Confocal fluorescence microscopy was performed with a Zeiss LSM 710 inverted confocal microscope . The level of co-localization of β-catenin and DAPI was quantified using double-stained confocal sections (Alexa fluor/DAPI). Sections were processed using software packages ZEN lite and Fiji. To determine Pearson co-localization coefficient for twenty nuclei per sample, we used EzColocalization plugin for ImageJ image analysis software+ GFPhi intestinal organoid cultures from APCfl/flLGR5-EGFP-cre-ERT2 mice, as previously described31. To assess the ability of the inhibitor C2 to inhibit clonogenicity of APC-deficient intestinal organoids, we seeded 1000 live GFPhi tumor stem cells into a three-dimensional collagen matrix (Matrigel), and then incubated with organoid growth media  containing C2 or DMSO. Four days later, clonogenicity was calculated as number of organoids per cell. Percentage viability of formed organoids was compared for C2 and DMSO.We derived LGR56 DLD1 cells, resuspended in matrigel, into the left and right flanks. Xenografts were allowed to grow until the average tumor volume reached 100 mm3; then the mice were divided into two groups of six animals. One group received 25 mg/kg C2 and the other received vehicle (PEG400 + solutol + DMA) only. Doses were administered intra-peritoneally, and tumor volume and mouse weight were measured daily using external calipers. The mice were treated for a period of 35 days. At the end of the experiment (day 35), the mice were euthanized, tumors were excised, and tumors were weighed. All vertebrate experiments were conducted with the approval of Animal Care and Use Committee of University of California San Diego. Likewise, all vertebrate experiments were performed in compliance with guidelines set forth by the National Institute of Health and the Principles for the Use and Care of Vertebrate Animals.6-week-old female NSG (NOD-SCID) mice were injected with 3 × 10Supplementary Materials.


Metastasis is the main cause of laryngeal cancer-related death; its molecular mechanism remains unknown. Here we identify protein arginine methyltransferase 5 (PRMT5) as a new metastasis-promoting factor in laryngeal carcinoma, and explore its underlying mechanism of action in regulating laryngeal cancer progression. We illustrated that PRMT5 expression was positively correlated with tumor stages, lymphatic metastasis, and unfavorable outcome. Functional assays revealed that PRMT5 promoted laryngeal carcinoma cell proliferation, migration, and invasive capacity in vitro, as well as lymph-node metastasis in vivo. The ectopic expression of PRMT5 induced EMT with downregulation of E-cadherin and upregulation of N-cadherin, snail, and MMP9. Mechanistic results revealed that the metastatic effects could be attributed to PRMT5-mediated activation of Wnt signaling, and Wnt4 is an important driver of Wnt/β-catenin signaling pathway. Wnt4 silencing could reverse PRMT5-induced cell proliferation, migration, and invasion capacities. Furthermore, inhibition of the Wnt/β-catenin signaling pathway abolished the effect of PRMT5-induced proliferation, whereas activation of the pathway enhanced the effect of PRMT5 overexpression on cell proliferation. These results demonstrated that the oncogenic role of PRMT5 could be attributed to PRMT5/Wnt4 axis-mediated activation of the Wnt/β-catenin signaling pathway. PRMT5 may serve as a novel prognostic marker and a therapeutic target for lymphatic metastasis of laryngeal carcinoma. Despite major advances in diagnosis and treatment, the prognosis of patients with advanced laryngeal carcinoma remains dismal, and lymph-node metastasis is the leading cause of laryngeal carcinoma-related mortality3. Nevertheless, the molecular mechanism of laryngeal carcinoma lymph-node metastasis is not yet fully understood.As a malignancy in the head and neck region, laryngeal carcinoma represents the second most commonly occurring cancer of the respiratory tract, >95% of which is squamous cell carcinoma. In 2018, data statistics showed that there were 177,422 new cases and 94,771 deaths worldwide6 as well as nonhistone proteins 10. PRMT5 is overexpressed in a variety of cancers, and its activity is associated with cell transformation. Recent evidence suggests that PRMT5 contributes to tumorigenesis. For instance, PRMT5 overexpression or hyperactivation is observed in several tumor types, including acute myeloid leukemia, breast cancer, glioblastoma, lung, and prostate cancer, which facilitates tumor initiation and progression16. PRMT5 overexpression positively correlates with astrocytoma progression, and inversely correlates with survival13. Kryukov et al.17 reported that PRMT5 repression induced tumor shrinkage and cell apoptosis. Depletion of PRMT5 in glioma cell lines failed to form tumors in mice engrafted intracranially with GBM xenografts13, suggesting that PRMT5 serves as a potential therapeutic target for cancer therapy. Moreover, PRMT5 affects epithelial–mesenchymal transition (EMT) and is overexpressed during oncogenesis and progression in oral squamous cell carcinoma18. Tarighat et al.11 found that PRMT5 was recruited to the snail complex via interaction with the scaffold protein AJUBA to repress E-cadherin expression, resulting in EMT process. PRMT5 also regulates cell-cycle progression by increasing the expression of positive regulators of the G1 phase 19.Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, can epigenetically regulate gene transcription by symmetrically dimethylated histones 20. EMT is hijacked by cancer cells to leave the primary tumor site, invading the surrounding tissue, which is frequently associated with distant metastasis and tumor dissemination21. EMT is induced by genetic and epigenetic changes within the transforming cells, as well as signals from the tumor microenvironment22. A variety of signaling pathways are involved in EMT, including nuclear factor-ĸB (NF-ĸB), transforming growth factor-β (TGF-β), Notch, and Wnt/β-catenin pathways23. The canonical Wnt pathway plays critical roles in developmental processes, human tumorigenesis, and metastasis25. The Wnt family consists of 19 conserved genes that encode for secreted glycoprotein ligands for Frizzled receptors26. Wnt4 is a member of the Wnt family, and previous reports have shown that abnormal Wnt4 expression is associated with carcinogenesis, and it regulates the proliferation of cancer stem cells in response to progesterone29.EMT is a physiological process in which the epithelial cells lose their polarity and adhesion and can be converted into mesenchymal statesIn this study, we set out to reveal the contributions of PRMT5 in metastasis formation of laryngeal carcinoma via the Wnt/β-catenin pathway. Using both in vivo and in vitro models, we demonstrated that PRMT5 can facilitate EMT and lymph-node metastasis of laryngeal carcinoma cells via modulating the Wnt4/β-catenin pathway. PRMT5 may serve as a promising therapeutic target for highly invasive laryngeal carcinoma patients.2 at 37 °C. All cell lines used in this study tested negative for mycoplasma contamination.A total of 176 formalin-fixed, paraffin-embedded tissues, including primary laryngeal carcinoma, lymph-node metastatic carcinoma, and normal tissues, were collected from the Sun Yat-sen University Cancer Center of Sun Yat-sen University  between January 2010 and December 2018. A tissue microarray consisting of 66-case laryngeal carcinoma and lymph-node metastatic carcinoma tissues was purchased from Alenabio . This study was performed in accordance with the institutional ethical guidelines and was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center. Informed consent was obtained from the patients before the study began, none of whom had received chemotherapy or radiotherapy prior to surgery. The laryngeal cancer cell lines Tu212 and Tu686 were purchased from Guangzhou Juyan Biological Technology  and cultured in RPMI 1640 . H2e, M4e, and NOK cell lines were purchased from Shanghai Institute of Life Sciences Cell Resource Center, China Academy of Sciences  and cultured in Dulbecco’s modified Eagle’s medium . All media were supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin–streptomycin solution . Cells were grown in a humidified atmosphere of 5% COThe paraffin-embedded tissues were sectioned and then deparaffinized and rehydrated. The primary antibodies against PRMT5 (#79998) were obtained from Cell Signaling Technology . Anti-Ki67 (M00254-1) was obtained from BOSTER Biotech . Wnt4 (sc-376279) was obtained from Santa Cruz Biotechnology . Next, the sections were probed with the secondary antibody at 37 °C for 30 min and developed by diaminobenzidine staining solution . The expression of PRMT5 was blindly quantified by two pathologists according to a staining scoring system. Five high-power visual fields were randomly selected from each slice (×200) with 100 cells per field. The PRMT5-positive expression results were determined based on their respective percentage of positive cells: number of PRMT5-positive tumor cells/number of total tumor cells ≥10% was considered to be positive (+) and <10% was considered to be negative (−).siRNA oligonucleotides targeting PRMT5, Wnt4, and negative control siRNA were purchased from Ribobio . The siRNA sequences for PRMT5 were 5′-GCACCAGTCTGTTCTGCTA-3′ and 5′-GAGGTGCAGTTCATCATCA-3′. The sequences for Wnt4 siRNA were 5′-GCCAAGUCCAGACUUCUGUT-3′ and 5′-GCAGACGCTCTGGTTGTTA-3′. siRNA transfection used was Lipofectamine RNAimax (Life Technologies) according to the manufacturer’s instructions. After a 48-h period of transfection, the cells were collected for subsequent experimentation. The plasmids were transfected in strict accordance with the instructions of lipofectamine 3000 .ASprofile was used to analyze the differentially alternative splicing events. Differentially expressed genes were screened by the threshold of 2.0-fold change and p value lower than 0.05. All RNA-seq raw data have been deposited in the GEO database (accession no. GSE154449).Cells were transfected with siPRMT5 (siRNA-2) or control siRNA for 48 h. TRIzol  was used to extract the total RNA from laryngeal carcinoma cells and tissues. Library construction and sequencing were performed by Sinotech Genomics . The libraries were sequenced on an Illumina HiSeq 2500 platform, and 100-bp paired-end reads were generated. https://portal.gdc.cancer.gov/projects/). Microarray datasets of laryngeal carcinoma patients for gene expression analysis were acquired from online data repository  dataset (GSE51985).The Cancer Genome Atlas (TCGA) data regarding laryngeal carcinoma patients, including genomic alterations, gene expression, and clinical information, were obtained from the TCGA Data Portal website . Stable cell lines were obtained after selection with 1 μg/ml puromycin (Invitrogen) for 14 days. The efficiency of PRMT5 knockdown and overexpression were evaluated using quantitative reverse transcription polymerase chain reaction and western blot.−∆∆Ct method. The housekeeping gene GAPDH was used as an internal control. The aforementioned experiments were repeated three times independently. All specific primers used for RT-PCR are listed in Table Total RNA was extracted from the tissues and transfected cells in each group in strict accordance with the instructions of TRIzol reagent . RNA quality and quantity were assessed by Nano Drop 2000 analysis. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis kit  according to the manufacturer’s instructions. The SYBR green PCR kit (Roche) was used for PCR amplification. Relative gene expression quantification was determined using the 2Following 48 h of transfection, total protein lysates were prepared using RIPA buffer  followed by the addition of protease inhibitor cocktail (Roche) and PMSF (Solarbio), according to the manufacturers’ instructions. The protein concentration was determined using a bicinchoninic acid Protein Assay kit . Overall, 40 μg of protein was separated by 10% SDS-PAGE and then transferred to PVDF membranes . After being blocked with 5% bovine serum albumin (BSA)  for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C. Then the membranes were incubated with HRP-conjugated IgG for 1 h; immunoreactivity was detected with enhanced chemiluminescence  by ChemiDoc™ XRS+ . Primary antibodies used in the experiment were as follows: PRMT5 , E-cadherin , N-cadherin , β-catenin , snail , Lamin B1 , MMP9 , Wnt4 , c-myc , Cyclin D1 , Wnt7A , Wnt9A , and GAPDH .3 per well for 24 h. Cell proliferation was evaluated using the CellTiter96®AQueousOne Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions. MTS reagent (20 µl) was added to 100-µl culture medium after seeding for 24, 48, 72, and 96 h, and incubated in a CO2 incubator at 37 °C for 2.5 h each time. The absorbance at 490 nm was measured using a Spark® multimode microplate reader . Each experiment with five replicates was repeated three times. For colony-formation assays, 500 cells were seeded in six-well culture dishes and incubated until visible colonies (≥50 cells) formed in complete growth medium (10 days–2 weeks). Megascopic cell colonies were fixed with methanol, stained with 0.1% crystal violet , and counted manually.Cell proliferation was detected by MTS assay. Laryngeal cancer cells were seeded in 96-well plates at 2 × 104/well) were suspended in 300 μl of serum-free medium and plated into the upper chambers , and 500 μl of medium containing 20% FBS was placed into the lower chambers. After 24 or 36 h, the cells under the bottom membrane were stained with crystal violet. The migrating cells in five randomly selected fields at a magnification of ×200 were imaged using digital microscopy and counted. For invasion assays, the upper chamber was precoated with 50 μl of Matrigel ; the remaining steps were as described above for the migration assay. All assays were performed in triplicate.For migration assays, Tu686 and Tu212 cells  at ×100. The migration of cells was assessed by the changes in the distances. Three duplicate wells were set in each group, and the experiment was repeated in triplicate.3 Tu212 cells were seeded into 24-well dishes. After 24 h, the cells were transiently transfected with either 2 μg of pTOP flash or pFOP flash plasmids  and pSV40-Renilla plasmid  as an internal control by using Lipofectamine 3000 (Invitrogen). Following transfection for 48 h, both Renilla and firefly luciferase were analyzed using a Dual-Luciferase Reporter Assay kit (Promega) according to the instructions, with a Glomax luminometer (Promega). The luciferase activity of each well was normalized to Renilla luciferase activity. The experiment was repeated in triplicate.In order to estimate the Wnt/β-catenin activity, β-catenin/TCF was transfected according to the instructions . The 5 × 10® 488 Conjugate . The nuclei of the cells in the confocal dishes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and photographs were taken by a Zeiss LSM880 microscope .Frozen sections of tissue samples or cell slides were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with goat serum for 1 h, and incubated with anti-E-cadherin  and anti-β-catenin  at 4 °C overnight. After washing with PBS, the sections were then incubated with Alexa Fluor6 cells) stably expressing firefly luciferase were inoculated into the lower-right footpads of mice. Lymphatic metastasis was monitored and imaged by the Bruker In-Vivo FX PRO System  every 10 days after tumor cell injection. Six weeks later, the mice were euthanized, and the number of metastatic nodules was recorded under the guidance of a microscope followed by tumor volume calculation. For tumorigenesis assay, 1 × 107 cells were injected subcutaneously into the left flank of mice (n = 18). The tumor size was measured by using a caliper every 3 days, and the volume was calculated. After 33 days, the mice were euthanized, and tumors were surgically dissected. The tumor specimens were fixed in 4% paraformaldehyde.Animal experiments were approved by the Institute of Animal Care and Use Committee of Cancer center of Sun Yat-sen University. We confirm that all procedures involving animals were performed in accordance with relevant guidelines and regulations. Four- to five-week-old BALB/c nude mice were randomly assigned into two groups with five mice placed in each group. Lentivirus-transduced Tu212 cells  between the two independent groups. All statistical analyses were performed using GraphPad Prism 6 . All data are presented as the mean ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.The SPSS 19.0 software  was used for statistical analysis. Samples were analyzed with unpaired Student’s n = 88, Table To identify whether PRMT5 was expressed in laryngeal carcinoma and correlated with patient prognosis, a cohort of clinical samples  and overall survival time in our cohort Fig. . The assTo investigate the potential effect of PRMT5 in laryngeal carcinoma, first, we examined the expression status of PRMT5 in laryngeal carcinoma cell lines, including Tu212, Tu686, M2e, M4e, and normal oral mucosa epithelial cell (NOK). The results with anti-PRMT5 antibody demonstrated that the PRMT5 level was higher in Tu686 cells, while Tu212 cells with a lower basal level of PRMT5 Fig. . We esta30. Next, to further elucidate the biological effects of PRMT5 on laryngeal carcinoma progression, western blotting and immunofluorescent staining were used to examine the effect of PRMT5 on the expression of EMT-related markers. E-cadherin was downregulated, and N-cadherin, snail, and MMP9 were upregulated in PRMT5-overexpressed Tu212 cells compared with control  that stably overexpressed PRMT5, generated by lentiviral transfection, and also stably expressed firefly luciferase, were inoculated into the footpads of nude mice . To further explore the function of PRMT5 in gene regulation, we conducted Gene Ontology (GO) analysis to identify general functional features implemented by the PRMT5-inhibited group using downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that the overlapped 721 downregulated genes were significantly associated with Hippo, Wnt, MAPK signaling pathway, and pathways in cancer  in vitro and facilitated tumor metastasis in vivo. On the other hand, lentivirus-mediated knockdown of PRMT5 significantly inhibited cell proliferation and clonogenicity. Moreover, we observed that PRMT5 expression significantly correlated with EMT marker. Western blotting and IF analyses showed that the ectopic expression of PRMT5 induced EMT with downregulation of E-cadherin and upregulation of N-cadherin, snail, and MMP9 in laryngeal carcinoma cells. Knockdown of PRMT5 impairs its ability in downregulating E-cadherin and upregulating N-cadherin, snail, and MMP9. This is the first report on the role of PRMT5 in EMT induction.Here we performed retrospective and prospective studies in clinical samples, and found that PRMT5 was abnormally upregulated in high-grade laryngeal carcinoma tissues. The expression of PRMT5 was positively correlated with tumor stages, lymphatic metastasis, and poor outcome. The expression of PRMT5 was significantly higher in the laryngeal carcinoma tissues than that in the adjacent normal tissues. This finding is consistent with the TCGA database and other reports in breast cancer and glioblastoma34, and regulates important cellular functions, including cell growth, proliferation, and differentiation37. Interestingly, the subcellular localization and role of PRMT5 vary with the tumor type. In other types of tumors, the localization of PRMT5 and its target substrates in each cell chamber may be significantly different41. PRMT5 has been reported as an oncogene and is always complexed with MEP50/WDR77 that catalyzes arginine methylation on histones and other proteins. PRMT5 and MEP50 are required to maintain expression of metastasis and EMT markers42. In this study, we further elucidated the downstream signaling pathway using RNA-seq analysis; the pathway analysis of the gene expression profiling indicated that the PRMT5-mediated tumor growth and metastasis could be partially attributed to the activation of Wnt/β-catenin signaling pathway. The PRMT5-induced Wnt/β-catenin signaling was validated by TOP/FOP-flash reporter assay in Tu686 and Tu212 cells. Furthermore, the Wnt/β-catenin signaling pathway inhibitor XAV939 abolished the effect of PRMT5-induced proliferation, whereas the pathway activator LiCl enhanced the effect of PRMT5 overexpression on cell proliferation. We further found that knockdown of PRMT5 inhibited the expression of β-catenin accumulation in the nucleus, whereas the total β-catenin did not show any changes, indicating that PRMT5 might contribute to the activation of the Wnt/β-catenin pathway.PRMT5, a type II arginine methyltransferase, functions as a tumor initiator to regulate cancer progression by symmetric dimethylation of arginine residues on histone proteins29. Meanwhile, translocation of β-catenin into the nucleus may result in the loss of E-cadherin and subsequent induction of EMT. Inhibition of Wnt/β-catenin signaling can block EMT transcription factors and promote epithelial differentiation44. Nevertheless, the mechanism underlying PRMT5- overexpression-induced EMT in laryngeal carcinoma remains unknown. In this report, we elucidate that PRMT5 overexpression can facilitate lymph-node metastasis in vivo and activate the Wnt/β-catenin signaling pathway. This was confirmed by the upregulation of β-catenin, Wnt4, cyclin D1, and MYC of the Wnt/β-catenin signaling pathway. Wnt4, a member of the Wnt family, has been shown to participate in tumor carcinogenesis45. In this study, we demonstrated that PRMT5 is a coactivator to enhance the formation of β-catenin in the nucleus of laryngeal carcinoma cells, this signaling might be at least partly mediated by Wnt4, and the PRMT5/Wnt4 axis could be a new mechanism in laryngeal carcinoma development. However, in the canonical pathway, β-catenin combined with the LEF/TCF complex to activate Wnt signaling. Further investigation is needed to indicate whether PRMT5 and LEF/TCF interacted with each other, or whether PRMT5 could form a complex with TCF/LEF in the nucleus. We thus questioned whether PRMT5 could also form a complex with β-catenin in regulating the activity of the Wnt/β-catenin pathway.Loss of E-cadherin, a cell–cell adhesion protein responsible for intercellular attachment, is widely considered the hallmark of EMT, and is usually accompanied by deregulation of the Wnt signaling pathway. Currently, the role of EMT program in laryngeal carcinoma metastasis is poorly annotated. To gain further insight into PRMT5-mediated cancer metastasis and invasion, we investigate the downstream signaling pathway responsible for PRMT5-mediated tumor growth and metastasis, at least partly, attributed to Wnt4-increased β-catenin nuclear translocation; the induction of β-catenin signaling is critical for the proliferation and migrationIn conclusion, our present results identified the novel discovery that PRMT5 clinically and functionally participates in lymph-node metastasis and proliferation of laryngeal carcinoma by enhancing the Wnt/β-catenin pathway. Uncovering the precise role of PRMT5/Wnt4/β-catenin in the progression of laryngeal carcinoma will not only increase our knowledge of PRMT5-induced tumorigenesis, but also accelerate the development of novel biomarkers and therapeutic strategies for laryngeal carcinoma patients with lymphatic metastasis.Supplementary tablesSupplementary figure S1Supplementary figure S2Supplementary figure legends


Heat shock transcription factor1 (HSF1) was overexpressed to promote glutaminolysis and activate mTOR in colorectal cancer (CRC). Here, we investigated the mechanism for cancer-specific overexpression of HSF1.min/+ and APCmin/+ HSF1+/−mice.HSF1 expression was analyzed by chromatin immunoprecipitation, qRT-PCR, immunohistochemistry staining and immunoblotting. HSF1 translation was explored by polysome profiling and nascent protein analysis. Biotin pulldown and m6A RNA immunoprecipitation were applied to investigate RNA/RNA interaction and m6A modification. The relevance of HSF1 to CRC was analyzed in APCmin/+ mice when HSF1 was genetically knocked-out or chemically inhibited.HSF1 expression and activity were reduced after the inhibition of WNT/β-catenin signaling by pyrvinium or β-catenin knockdown, but elevated upon its activation by lithium chloride (LiCl) or β-catenin overexpression. There are much less upregulated genes in HSF1-KO MEF treated with LiCl when compared with LiCl-treated WT MEF. HSF1 protein expression was positively correlated with β-catenin expression in cell lines and primary tissues. After β-catenin depletion, HSF1 mRNA translation was impaired, accompanied by the reduction of its m6A modification and the upregulation of miR455-3p, which can interact with 3′-UTR of HSF1 mRNA to repress its translation. Interestingly, inhibition of miR455-3p rescued β-catenin depletion-induced reduction of HSF1 m6A modification and METTL3 interaction. Both the size and number of tumors were significantly reduced in APCβ-catenin suppresses miR455-3p generation to stimulate m6A modification and subsequent translation of HSF1 mRNA. HSF1 is important for β-catenin to promote CRC development. Targeting HSF1 could be a potential strategy for the intervention of β-catenin-driven cancers. Colorectal cancer (CRC) is the third most common cancer with high mortality rate globally . The accIn addition to proliferation activation and apoptosis resistance, metabolism reprogramming is one of important hallmarks of cancer cells . For exaGene expression can be controlled by multiple processes including transcription, mRNA degradation, translation and protein degradation. While gene translation and protein degradation have been extensively investigated, more and more studies focused on mRNA translation by exploring the effect of non-coding RNAs such as microRNAs (miRNAs) and new modifications of mRNA including N6-methyladenine (m6A) modification , 9. MiRNIn this study, we found that activated WNT/β-catenin signaling stimulated HSF1 translation to promote CRC development by repressing HSF1 mRNA-targeting miR455-3p to increase m6A modification of HSF1 mRNA. Therefore, targeting HSF1 translation could be a new strategy for the intervention of CRC and other cancers driven by activated WNT/β-catenin signaling.Human CRC cell lines SW480, SW620, DLD1, RKO were obtained from the American Type Culture Collection (ATCC). All cells were routinely cultured in RPMI 1640  or DMEM  supplemented with 10% fetal bovine serum. All cells were incubated at 37 °C with 5% CO2 and 95% humidity. The following antibodies were used for western blotting and IHC: HSF1 ; β-catenin ; β-actin ; FLAG ; cyclinD1 ; Cleaved PARP1 ; METTL3 ; GLS1 . Pyrvinium (P0027), LiCl (793620), cycloheximide (R750107), chloroquine (C6628), MG132 (474790) and PD150606 (D5946) were purchased from Sigma-Aldrich.Small interfering RNA (siRNA) targeting β-catenin, METTL3 and microRNAs were synthesized by Genepharma  and RiboBio . The sequence of these siRNAs and miRNAs were listed in Additional file The plasmid of β-catenin reporter was gifted from Prof. Ximei Wu (Zhejiang University). For HSF1 activity assay, a fragment containing 3 X HSE were synthesized and inserted into the PGL3-basic vectors . The plasmid was co-transfected with pRL renilla and β-catenin siRNA by using lipo2000  or treatment with pyrvinium by X-treme GENE HP DNA Transfection Reagent . 3′-UTR segment of the HSF1 was cloned by PCR and inserted into the vector pMIR-REPORTER (Promega). The mutation of miR455 binding sites in HSF1 3′-UTR was generated by Quick Site-Directed Mutagenesis . The resultant plasmids were co-transfected with pRL renilla and miR455 mimics by using lipo2000 . 48 h post-transfection, the luciferase activity was measured by the Dual-GLO Luciferase Assay System .ChIP analysis was conducted with the SimpleChIP™ Enzymatic Chromatin IP Kit . Antibodies used were anti-HSF1 , TCF7L2 and FLAG . The primers used for the PCR analysis of precipitated DNA were shown in Additional file Immunoblotting and immunohistochemistry (IHC) assays were performed as previously reported . For ImmFor IHC assay was performed in a tissue array containing 80 cases of colonic tissues. Primary antibodies used were listed above. The degree of immunostaining was assessed by 2 independent pathologists and evaluated by assigning a score of 0–3. Scores were defined as follows: 0, no staining; 1, faint staining; 2, moderate staining and 3, strong staining. Final scores of 0 and 1 were regarded as low expression, whereas scores of 2 and 3 considered as high expression.Cell apoptosis was measured by flow cytometry analysis and western blotting. For flow cytometry analysis of apoptosis, cells were harvested and re-suspended in 100 μl 1 x binding buffer. 5 μl fluorescein isothiocyanate (FITC) annexin V and propidium iodide (PI)  were added to the cell suspension and then incubated for 15 min at room temperature. After that, the samples were attenuated with 400ul 1 x binding buffer and analyzed by ACS Calibur flow cytometer.6 cells were plated in 10 cm dish. After the given treatment, cells were incubated with 1:1000 Biotin-dC-puromycin  for 24 h. Cells were lysed with 1% NP40 buffer  containing 1X protease inhibitor cocktail. After adequate centrifugation, the supernatant was incubated with 80ul streptavidin sepharose beads  by rotating at 4 °C for 6 h to overnight. The mixture was washed by 1% NP40 buffer for 5 times and subjected to western blotting using HSF1 antibody.To detect the change of nascent HSF1 synthesis, 2 X 10Polysome profiling separates translating or non-translating mRNAs on a sucrose gradient according to the number of bound ribosomes as previously described . In brieBiotin pull down assay was performed as described previously . Cells w7 cells were lysed in 100 μl RIP lysis buffer and immunoprecipitated with antibodies of interest and protein G magnetic beads at 4 °C overnight, followed by six times of washes in Washing Buffer and protein digestion at 55 °C. Total RNA was isolated and subjected to RT-PCR analysis. Following antibodies were used for RIP: N6-methyladenosine ; METTL3 ; IgG .RIP assay was performed by Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit . Briefly, 2 × 106 WT MEF and HSF1 KO MEF were plated and cultured overnight. Following day, cells were treated with 20 mM LiCl for 48 h. Cells were collected with Trizol reagent. The total RNA was processed by NEBNext® Poly(A) mRNA Magnetic Isolation Module to enrich mRNA, and the product RNA was used for construction Library, via KAPA Stranded RNA-Seq Library Prep Kit (Illumina). Sequencing libraries, denatured by 0.1 M NaOH to generate single-stranded DNA, as amplified in situ Illumina cBot ). The ends of the generated fragments were used to run 150 Cycles by the Illumina HiSeq 4000 Sequencer. All the experimental steps after the RNA extraction were conducted in Kangcheng Biotechnology Co., Ltd. (Aksomics), Shanghai, China. RNA-sequencing was performed three times.2X10min/+ mice were purchased from Model Animal Research Center of Nanjing University . HSF1 KO mice reported previously were used to generate Apcmin/+ mice HSF1+/− [min/+, Apcmin/+ HSF1+/− and Apcmin/+ treated with KNK437 as previously reported [Animal care and experiments were conducted in compliance with Institutional Animal Care and Use Committee and NIH guidelines. The C57BL/6 J mice and ApcP value < 0.05 was considered as statistically significant.All data were expressed as mean ± SD. Unless specified, the Student’s t-test was performed for statistical significance analysis. http://portals.broadinstitute.org/cmap/) [In an effort to explore potential regulations of HSF1, we screened chemicals generating a gene expression pattern similar to HSF1 depletion by connective map (g/cmap/) , 15, 16.g/cmap/)    was posig/cmap/) , 20  and HSF1 knock-out MEF (HSF1 KO MEF) before and after LiCl treatment (NCBI GEO: GSE151119). While only 750 genes were upregulated in LiCl treated-HSF1 KO MEF, there were 1435 genes significantly upregulated in WT MEF after LiCl treatment Fig. , Fig.S1Gp < 0.01, Chi-Square Test). All of these data indicated that β-catenin upregulates HSF1 protein expression.To delineate how β-catenin regulates HSF1, we quantitated protein levels of HSF1 before and after inhibiting β-catenin. Both pyrvinium and β-catenin depletion reduced the protein level of HSF1 Fig. a and b. However, there were no apparent alterations in HSF1 mRNA level after β-catenin knockdown or pyrvinium treatment , the expression of miR455-3p is lower in colon adenocarcinoma than in normal tissues  play an important role in regulating the efficiency of protein translation, we wondered whether HSF1 protein translation was regulated by microRNAs. Based on bioinformatics screening by TargetScan, miRDB and StarBase, some microRNAs including miR455-3p, miR214-5p, miR431-5p, miR184, miR490-3p, and miR375 were proposed to target 3′-UTR of HSF1 mRNA   play important roles in the regulation of HSF1 translation. Interestingly, we noticed that the matching sites of miR455-3p seed sequence in HSF1 mRNA 3′-UTR contains a typical motif of m6A modification  Fig. , Fig.S4Bmp) Fig. , Fig.S4CNext, we explored the interplay between miR455-3p and m6A modification of HSF1 mRNA. Both HSF1 mRNA m6A modification and its binding to METTL3 were decreased by the overexpression of wild type miR455-3p but not its mutant unable to bind to HSF1 mRNA 3′-UTR Fig. , Fig.S5AIndeed, miR455-3p inhibitor rescued β-catenin depletion-induced decrease of HSF1 mRNA m6A modification Fig. , Fig.S5Fhttp://linkedomics.org/)  . The expression of HSF1 and its downstream target GLS1 were increased, while miR455-3p expression was reduced, in intestine tissues from Apcmin/+ mice compared with normal C57BL/6 mice (Fig. min/+ mice developed multiple tumors in the intestine (Fig. min/+ mice treated with a chemical inhibitor of HSF1, KNK437 and Apcmin/+ HSF1+/− mice (Fig. In light of these in vitro findings, we further explored the relevance of HSF1 to colorectal carcinogenesis in ApcGenetic changes in components of WNT/β-catenin signaling such as deletion of the APC gene and CTNNB1 mutations have been frequently detected in many types of human cancers . All of Recently, RNA modifications such as m6A modification were important for fate decision of target mRNAs , 33 . HoAs a result, the abundance of miR455-3p is critical to regulate m6A modification of HSF1 mRNA. Interestingly, we found that β-catenin inhibited the biogenesis of miR455-3p by suppressing the transcription of its host gene COL27A. While β-catenin was well-known to be a transcriptional co-activator, it can also function as a transcriptional repressor to directly repress gene transcription , 38. ForTaken together, β-catenin suppressed the biogenesis of miR455-3p to allow METTL3 binding for m6A modification of HSF1 mRNA, thus stimulating HSF1 mRNA translation. In cancer cells, HSF1 can drive a transcriptional program distinct from heat shock response to support malignant phenotypes , 43. ForIn summary, β-catenin suppressed the biogenesis of HSF1-targeting miR455-3p to favor METTL3 binding and m6A modification of HSF1 mRNA, thus promoting HSF1 translation. HSF1 expression was upregulated in murine tumors driven by activated WNT/β-catenin signaling and human CRC tissues. Genetic and chemical inhibition of HSF1 significantly attenuated the pathogenesis of CRC in mice. Targeting HSF1 is a potential strategy for the management of human cancers associated with activated WNT/β-catenin signaling.Additional file 1: Table S1. RNA oligonucleotide sequences. Table S2. Primer sequences.Additional file 2: Supplemnetary Figures 1-6. Fig. S1. WNT/β-catenin signaling activates HSF1. Fig. S2. β-catenin has no effects on the RNA and protein half-life of HSF1. Fig. S3. The effect of microRNAs on HSF1 expression in CRC. Fig. S4. m6A modification of HSF1 mRNA. Fig. S5. The effects of miR455-3p and m6A modification on HSF1. Fig. S6. Volcano plot displays differentially regulated genes upon LiCl treatment.Additional file 3. The list of genes containing HSE in their promoters.


The osteoclast bone resorption is critical in aseptic loosening after joint replacement. The balance between activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) is considered to play a central role in osteoclast maturation. Fibroblasts from the periprosthetic membrane express RANKL and promote osteoclast formation. Studies have demonstrated that naringin inhibited osteoclastogenesis and wear particle-induced osteolysis. In this study, the naringin-induced OPG/RANKL effects and its underlying mechanism were studied in fibroblasts from periprosthetic membrane.Fibroblasts were isolated from the periprosthetic membrane during hip arthroplasty for revision due to aseptic loosening. Fibroblasts were cultured and treated with or without naringin and DKK-1 . OPG and RANKL mRNA and protein levels, gene expression of β-catenin, and cyclin D1, which participate in the Wnt signaling pathway, were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.The mRNA and protein levels of OPG were enhanced by naringin in a dose-dependent manner compared to that of the non-treated control. In contrast, naringin did not affect the expression of RANKL. Importantly, DKK-1 attenuated OPG expression in fibroblasts under naringin treatment. Moreover, naringin stimulated the gene expression of β-catenin and cyclin D1 in fibroblasts, and the effect could be inhibited by DKK-1.The results indicated that naringin enhanced OPG expression through Wnt/β-catenin signaling pathway in fibroblasts from periprosthetic membrane, which may be useful to inhibit periprosthetic osteolysis during aseptic loosening after total joint arthroplasty. Total hip replacement is one of the most effective treatments for advanced joint diseases. Periprosthetic osteolysis and subsequent aseptic prosthetic loosening are still the most common complications that limit the life of prostheses. The osteolysis is related to a chronic inflammation caused by wear debris gathering between the implant and bone. The inflammation is characterized by a granulomatous membrane at the prosthetic interface that is infiltrated with macrophages, fibroblasts, and giant cells .The RANKL, which is a member of the tumor necrosis factor family, has been shown to support osteoclast differentiation and maturation. It was found that the RANKL expression was enhanced in the tissues around the failed prostheses . The pseAlthough fibroblasts make up most of the cells at the membrane interface, there is less information about the role of fibroblasts in aseptic loosening than other cell types such as macrophages, osteoblasts, and osteoclasts. It has been shown that fibroblasts of periprosthetic membrane are an important source of RANKL , 4–8. ThThe Wnt/β-catenin signaling pathway may play an important role in regulating RANKL/OPG expression. Several studies have shown that OPG was increased through Wnt/β-catenin signaling pathway –15.Bisphosphonates are considered to be effective drugs to control osteolytic diseases. We previously reported that alendronate stimulated OPG expression in fibroblasts from periprosthetic membrane . HoweverNaringin is a natural product, chemically 4′,5,7-trihydroxyflavanone-7-rhamnoglucoside. It is a major flavanone glycoside extracted from tomatoes, grapefruits, and many other citrus fruits. Naringin has diverse biological and pharmacological properties, including anti-inflammatory, anti-oxidant and anti-apoptotic activities. Previous studies have shown that naringin could promote osteoblastogenesis and inhibited osteoclastogenesis –25. ReceMost of studies on naringin have concentrated on its effects on osteoblasts and osteoclasts. However, the effect of naringin on fibroblasts is rarely concerned. In previous studies, we found that fibroblasts in the periprosthetic membrane played a role in aseptic loosening and expressed RANKL and OPG –28. In tThe femoral and acetabular pseudomembrane samples were collected from eight patients , near osteolytic lesions between the implants or cement and bone, who underwent revision due to aseptic loosening of total hip arthroplasty. Every patient presented with a loose prothesis with radiological osteolysis. No patient had clinical or laboratory signs of infection. The indication of primary total hip replacement was osteoarthritis in all patients. Three of the implants were cemented, and five were uncemented. The mean years in situ were 11 ± 4 years. Informed consent was obtained from all donors, and the institutional ethical committee approved the procedures. The samples were washed with PBS, cut into small pieces, and digested in α-minimal essential medium (α-MEM) (Gibco) which contained 1 mg/mL collagenase (Sigma), added with 100 mg/mL of penicillin-G (Sigma), 50 mg/mL of gentamicin sulfate (Sigma), at 37 °C for 30 min. They were then incubated in Versene (Invitrogen) for 1 h . The celThe periprosthetic membrane-derived cells were immunochemically stained with the following antibodies to determine the phenotype evaluation and purity of the culture: fibroblast marker vimentin (Abcam), leukocyte/macrophage marker, CD45 and CD14 (Dako), endothelial cell marker CD31 (Dako). In order to determine the presence of osteoblasts in cultured cells, the histochemical evaluation of alkaline phosphatase expression in the cultures was also carried out.Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) method. Fibroblasts (1 × 104/well) were seeded into a 96-well plate and treated at various concentrations  of narinign for 24 or 48 h. The cells were washed 3 times in PBS and cultured for another 4 h with 10% CCK-8-containing medium. Then, the absorbance at 450 nm was measured using a microtablet reader.Confluent-stage fibroblasts were cultured in fresh medium containing 2% heat-inactivated fetal bovine serum, with or without naringin  and DKK-1 .The fibroblasts were cultured and treated with 0, 50, 100, or 150 μM naringin in the presence or absence of 100 ng/ml DKK-1 for 6 h. Total RNA was isolated with TRIzol reagent (Invitrogen) and converted to cDNA using the SuperScript First-Strand Synthesis Kit (Invitrogen). PCR was performed using the QuantiTect SYBR Green PCR Kit  and the 7500 Real-Time PCR Detection System . OPG was amplified using 5′-CGC CTC CAA GCC CCT GAG GT-3′, 5′-CAA GGG GCG CAC ACG GTC TT-3′; RANKL was amplified using 5′-GTC TGC AGC GTC GCC CTG TT-3′, 5′-ACC ATG AGC CAT CCA CCA TCG C-3′; β-catenin was amplified using 5′-TTG AAA ATC CAG CGT GGA CA-3′, 5′-TCG AGT CAT TGC ATA CTG TC-3′; Cyclin D1 was amplified using 5′-ACA AAC AGA TCA TCC GCA AAC AC-3′, 5′ TGT TGG GGC TCC TCA GGT TC-3′; and β-actin was amplified using 5′-AGG CCA ACC GCG AGA AGA TGA CC-3′, 5′-GAA GTC CAG GGC GAC GTA GCA C -3′ primer sets.For RANKL and OPG detection, fibroblasts were treated with naringin  in the presence or absence of 100 ng/ml DKK-1 for 24 h. The levels of RANKL and OPG in the culture supernatants were determined by commercially available enzyme-linked immunosorbent assay kits (R&D Systems) in accordance with the manufacturer’s instructions.t test was used to determine the statistical significance. The normal distribution was determined by the Kolmogorov-Smirnov test. The data were analyzed using one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant.Results were representative of at least three independent experiments. The data were expressed as means ± standard deviations (SD). Student’s After four passages, almost all of the cultures were spindle mononuclear cells. These cells were positive for vimentin and negative for CD45, CD14, CD31, and alkaline phosphatase.The result of cell proliferation is presented in Fig. The fibroblasts were cultured with naringin  or only in the medium (control group) for 6 h. As shown in Fig. The fibroblasts were cultured with naringin  or only in the medium (control group) for 24 h. As shown in Fig. The fibroblasts were treated with 100 μM naringin in the presence or absence of 100 ng/ml DKK-1. As shown in Fig. The fibroblasts were treated with 100 μM naringin in the presence or absence of 100 ng/ml DKK-1 for 6 h. As shown in Fig. Homeostasis of bone metabolism is maintained through a balance between bone resorption and bone formation. When osteoblast bone formation is less than osteoclast bone resorption, aseptic loosening occurs. The ratio of OPG to RANKL controls the process of osteogenesis and osteoclastogenesis . RANKL, Wnt/β-catenin signaling plays a crucial role in bone metabolism. The activation of Wnt/β-catenin signaling pathway has been extensively studied in bone biology and has been proved to promote bone formation. Research showed this pathway played a role in the development of osteolytic disease . The impThe agents that increase the activity of the Wnt/beta catenin pathway may serve as promising agents for the treatment of osteolysis. Naringin is a natural compound in citrus fruits, which has been proved to promote bone development and maintenance. The effect of naringin on Wnt/β-catenin signaling has been investigated because Wnt/β-catenin signaling is related to osteoblastogenesis. It was found that naringin stimulated Wnt/β-Catenin signaling in osteoblast-like UMR-106 cells , naringiThese results indicated that naringin activates Wnt/β-catenin signaling molecules, thereby enhancing the OPG expression in fibroblasts. However, the details of the mechanism of OPG expression induced by naringin are not fully understood. Recent studies have suggested that naringin activity may be mediated not only by the Wnt/β-catenin signaling pathway but also by the NF-κB, ERK, and other signaling pathways –40. BecaIn summary, this study showed that naringin might regulate OPG expression through the Wnt/β-catenin signaling pathway in fibroblasts from periprosthetic membrane. Our findings indicated that naringin could be used as a potential therapeutic drug for the treatment of periprosthetic osteolysis and aseptic loosening.


Osteosarcoma (OS) is the most common bone malignancy in adolescents and has poor clinical outcomes. Protein arginine methyltransferase 5 (PRMT5) has recently been shown to be aberrantly expressed in various cancers, yet its role in OS remains elusive. Here, we found that PRMT5 was overexpressed in OS and its overexpression predicted poor clinical outcomes. PRMT5 knockdown significantly triggered pronounced senescence in OS cells, as evidenced by the increase in senescence-associated β-galactosidase -stained cells, induction of p21 expression, and upregulation of senescence-associated secretory phenotype (SASP) gene expression. In addition, we found that PRMT5 plays a key role in regulating DNA damaging agents-induced OS cell senescence, possibly, via affecting the repair of DNA damage. Furthermore, we found that TXNIP acts as a key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. Mechanistically, TRIM21, which interacts with PRMT5, was essential for the regulation of TXNIP/p21 expression. In summary, we propose a model in which PRMT5, by interaction with TRIM21, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells. The present findings suggest that PRMT5 overexpression in OS cells might confer resistance to chemotherapy and that targeting the PRMT5/TRIM21/TXNIP signaling may enhance the therapeutic efficacy in OS. Osteosarcoma (OS) is the most common primary bone malignancy and occurs primarily in children and adolescents , 2. ChemProtein arginine methyltransferase 5 (PRMT5) is a druggable enzyme that has been shown to be aberrantly expressed in various cancers, and it acts as a putative oncogene in maintaining cancer cell survival –9, yet iCellular senescence is a state of permanent cell cycle arrest characterized by an accumulation of senescence-associated β-galactosidase  and the appearance of a senescence-associated secretory phenotype (SASP). Several stimuli, such as oxidative stress and DNA damage, have been demonstrated to cause cellular senescence , 20. TheIn the present study, we found that PRMT5 is highly expressed in OS tissues and that its overexpression predicts poor clinical outcomes. Knockdown of PRMT5 induces pronounced senescence in OS cells while overexpression of PRMT5 in OS cells inhibits DNA damaging agents-induced senescence, presumably through a mechanism of regulating the DDR. Finally, we elucidated that TRIM21, an E3 ubiquitin-protein ligase, by interacting with PRMT5, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells.PRMT5 has previously been shown to be overexpressed in multiple human cancers, including prostate, lung, and colon cancers , 8, yet Next, we sought to investigate the possible effects of PRMT5 on the growth of OS cells. As shown in In contrast, overexpression of PRMT5 by transiently transfection of the plasmid encoding HA-PRMT5 markedly reduced the percentage of senescent cells and the expression of p21 triggered by PRMT5 depletion, indicating the specific role of PRMT5 in regulating cellular senescence . To explIncreasing evidence suggests that DNA damage is a common mediator of cellular senescence , 32; theCDDP has been reported to induce cancer cells undergoing senescence , 36. NexNext, we sought to identify the key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. TXNIP has recently been reported as a key regulator of oxidative stress, DNA damage, and cellular senescence ; thus, wIt is well characterized that PRMT5 plays a key role in regulating transcription via methylating histone or non-histone substrates . Given tNext, we aimed to explore the possible role of TRIM21 in PRMT5 depletion-regulated TXNIP/p21 axis in the U2 OS cells. As shown in in vitro and in vivo [In recent years, PRMT5, as an oncoprotein, has gained increasing attention in terms of cancer prevention and therapy. PRMT5 is aberrantly expressed in various cancers and that inhibition or knockdown of PRMT5 suppresses cancer cell proliferation, induces cell cycle arrest, and abolishes cancer metastasis both  in vivo , 8. In tRecent studies have suggested that induction of cellular senescence is a promising strategy for enhancing therapeutic efficacy in various cancers, including OS , 25, 40.PRMT5 performs its function mainly through epigenetic silencing or direct methylation of histone and nonhistone molecules . A serieTRIM21, which belongs to the TRIM family, is an E3 ubiquitin ligase with a RING domain. TRIM21 is widely involved in cell proliferation, differentiation, autophagy, innate immunity and migration –53. Our In summary, we found that PRMT5 is overexpressed in OS and its overexpression is correlated with OS progression. In addition, we found that PRMT5 plays a key role in regulating senescence of OS cells either in the presence or absence of DNA damaging agents including CDDP. Mechanistically, TRIM21 was found to interact with PRMT5 to regulate senescence in OS cells in response to DNA damage by modulating the expression of TXNIP/p21 . These fHuman U2 OS and Saos-2 cells were cultured in McCoy's 5A medium  supplemented with 10% or 15% fetal bovine serum, respectively . HEK293TTissue microarray (OS208) containing samples of normal bone and various grades of OS were obtained from Alenabio  and used for IHC with an antibody against PRMT5 . A total of 34 OS sections were collected from patients at the First Affiliated Hospital of Jinan University and the Third Affiliated Hospital of Sun Yat-sen University. Briefly, paraffin sections were deparaffinized in xylene and rehydrated through graded ethanol washes , followed by incubation with 3% hydrogen peroxide for 10 min. Antigen retrieval was performed by heating slides in 10 mM Tris-HCl (pH = 10) for 15 min in a microwave. After three washes with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST), slides were blocked in 5% nonfat milk in PBST at room temperature (RT) for 1 h. Slides were incubated with primary antibody against PRMT5 at 4°C overnight, followed by three washes with PBST and incubation with HRP-conjugated anti-rabbit secondary antibodies at RT for 1 h. The signal was developed with diaminobenzidine for 10 min, and sections were counterstained with hematoxylin. Another set of 11 OS fresh sections was used for IHC to correlate the expression of PRMT5 and Ki67 . Semiquantitative analysis of PRMT5 expression was performed as previously described .http://r2.amc.nl) [The expression of PRMT5 in OS was extracted from the mixed osteosarcoma-Kuijjer dataset in the R2 Genomics Analysis and Visualization Platform (.amc.nl) . A KaplaLentiviruses expressing shRNA against PRMT5 (shP5) and TRIM21 (shT21) or SC shRNA were purchased from GenePharma . Lentiviruses expressing inducible TRIM21 were generated by co-transfected HEK293T cells with H125 pLenti-TRE-EGFP-EF1-rtTA3-IRES-Puro-TRIM21, pLP1, pLP2, and pLP-VSVG using at a ratio of 1:1:1:2. U2 OS cells were infected with the above lentiviruses by the addition of viral supernatant  for 24 h and were then selected with 2 μg/ml of puromycin for 3 days. Puromycin (1 μg/ml) was used to maintain stable cells. The targeting sequences of shPRMT5 and shTRIM21 are specified in U2 OS cell senescence was detected by Senescence β-Galactosidase Staining Kits  according to the manufacturer’s instructions. Briefly, U2 OS cells were seeded into six-well plates and cultured for 72 h. Cells were then fixed for 20 min at RT with 1 ml of fixative solution and were then washed twice with PBS. Next, cells were stained with a staining solution mixture containing X-gal overnight at 37°C.Briefly, U2 OS cells were collected and lysed using cell lysis buffer for WB and IP  supplemented with phenylmethylsulfonyl fluoride  and protease inhibitor cocktail . Proteins were then electrophoresed using SDS-PAGE gels  and transferred to polyvinylidene difluoride membranes . After blocking, membranes were incubated with primary antibodies and then with HRP-conjugated secondary antibodies at RT. The primary antibodies used were as follows: anti-β-actin , anti-PRMT5 , anti-p21 , anti-PCNA , anti-p16 , anti-γ-H2A.X , anti-p53 , anti-TXNIP , anti-TRIM21 , anti-Lamin B1 , and anti-RAD51 . Secondary HRP-conjugated antibodies (1:1000) were purchased from CST . Immunoreactive bands were visualized using Clarity Western ECL Substrate .5 cells/per dish in 6 cm dishes and incubated for 72 h. For the preparation of nuclear and cytoplasmic protein extracts, NE-PER Nuclear and Cytoplasmic Extraction Reagents  were used according to the manufacturer’s instructions. Western blot was used to analyze the subcellular localization of p21 and TXNIP.Cells were plated at a density of 4×10EcoRI and KpnI; the plasmids were verified by sequencing and expression analysis.The coding regions of HA-VC155-TRIM21, TRIM21, and mTRIM21 were amplified from cDNA of OS cells by PCR with Phusion High-Fidelity DNA Polymerase . The amplification primers used are specified in A DNA Damage Detection Kit was purchased from KeyGEN BioTECH (KGA240-50), and the experiments were performed according to the manufacturer’s instructions. Cells were harvested and suspended in PBS, mixed with agarose and placed on slides at 4°C for 30 min. Slides were immersed in cold lysis buffer and then incubated with alkaline  for 40 min. After electrophoresis, slides were washed using 0.4 mM Tris-HCl buffer (pH = 7.5) and stained with propidium iodide (PI). DNA damage was then detected using fluorescence microscopy at an excitation wavelength of 515~560 nm. The OTM was analyzed by Open Comet software .U2 OS cells were cultured on glass coverslips one day before treatment with DMF or CDDP (20 μM) for 3 h, and the medium was then replaced with fresh complete medium for 12 h or 24 h to provide the appropriate conditions for DNA repair. Sides with cells were fixed using 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking, cells were incubated with primary antibodies (1:200) against γ-H2A.X, 53BP1, RAD51, PRMT5, and TRIM21 for 1 h, followed by incubation with Alexa Fluor 555-labeled anti-rabbit IgG , 4’,6-diamidino-2-phenylindole (DAPI) and phalloidin-Alexa Fluor 573 (Life Technologies) overnight. Images were then acquired using a laser scanning confocal microscope .5 cells/well in a 6-well plate  overnight. siRNAs targeting PRMT5 and TXNIP were transfected into cells using Lipofectamine 2000  for 4 h. Cells were then cultured in fresh complete medium for 3 days. The targeting sequences of the above siRNAs are specified in the supplementary information . Cells were cultured at a density of 2 × 10ormation .BiFC was performed to analyze the interaction between PRMT5 and TRIM21 in U2 OS cells, as previously described , 39. Bri3VO4 and protease inhibitors. After incubation on ice for 30 min, 40 μl of 50% protein A agarose bead slurry  was used to preclear cell extracts for 30 min at 4°C, followed by incubation with 5 μg of either IgG  or antibody against PRMT5 overnight at 4°C. Immune complexes were then incubated with 40 μl of 25% protein A-Sepharose slurry for 2 h. Immunoprecipitates were subjected to WB after being washed five times with lysis buffer. Notably, IPKine HRP AffiniPure Mouse Anti-Rabbit IgG Light Chain  was used as the secondary HRP-conjugated antibody to eliminate interference from heavy chain fragments, which have a similar molecular weight as TRIM21.Co-IP assays were performed to explore the endogenous interaction between PRMT5 and TRIM21 in U2 OS cells. Briefly, cells were incubated in lysis buffer  supplemented with NaF, PMSF, Na−ΔΔCT) and was normalized to control gene expression. Primers used for real-time PCR are specified in the supplementary information ; p< 0.05 was considered statistically significant (*).All experiments were performed at least three times, and the data are expressed as the means ± SDs. Statistical analysis was performed using GraphPad Prism 6 software . Comparisons between two groups were performed by using Student's Supplementary MethodsSupplementary FiguresSupplementary Tables


Emerging evidence suggests the involvement of caudal-related homoeobox transcription factor 2 (CDX2) in tumorigenesis of various cancers. Although CDX2 functions in cancer invasion and metastasis, fewer studies focus on the role of CDX2 during the induction of epithelial–mesenchymal transition (EMT) in colorectal cancer (CRC).Immunohistochemical analysis of CDX2 was performed. A series of in vitro and in vivo experiments were conducted to reveal the role of CDX2 in the invasion and metastasis of CRC.CDX2 was downregulated in CRC tissues and reduced CDX2 correlated with poor prognosis. Knockdown of CDX2 promoted colon cancer cell invasion in vitro and facilitated liver metastasis in vivo with inducing EMT phenotypes. Further investigation indicated that CDX2 retarded Akt and GSK-3β phosphorylation, and thereby diminished Snail expression, β-catenin stabilisation and nuclear translocation. The depletion of β-catenin neutralised the regulation of Slug and ZEB1 by CDX2 knockdown. Mechanistically, CDX2 antagonised PI3K/Akt activity in CRC by modulating PTEN expression. CDX2 directly bound to the promoter of PTEN and transactivated its expression.Our study first uncovered that CDX2 inhibits EMT and metastasis of CRC by regulation of Snail expression and β-catenin stabilisation via transactivation of PTEN expression. Although great progress has been achieved in early detection and multimodality treatment of CRC,3 most advanced CRC patients have a poor prognosis. Distant metastasis and relapse are the main cause of death for CRC patients.5 Emerging evidence confirmed that multiple genes and cellular pathways participate in the tumorigenesis and metastasis of CRC.6 Elucidating the underlying molecular pathways might highlight better therapeutic strategies for improving the prognosis of patients with CRC.Colorectal cancer (CRC) is the second most common cause of cancer-related death.7 EMT plays pivotal and intricate roles in malignancy-related phenomena, including the cancer stem cell phenotype, drug resistance, circulating tumour cells and tumour-budding production.9 The loss of functional E-cadherin is considered as the hallmark of EMT.10 E-cadherin is negatively regulated by several transcriptional factors, including Snail, Slug, Twist and ZEB1/2.12Epithelial–mesenchymal transition (EMT) is a transdifferentiation process, with the cells losing their polarity and contacts with neighbouring cells and subsequently acquiring mesenchymal-like and motile phenotypes.15 Aberrant activation of Wnt signalling is associated with colorectal carcinogenesis.16 Mutations or dysregulation of the β-catenin destruction complex  results in activation of Wnt signalling.19 Phospho Akt (active Akt) inhibits GSK-3β activity by phosphorylation and subsequently decreases GSK-3β-mediated β-catenin degradation and stabilises β-catenin.20 An elevated nuclear β-catenin level leads to the activation of Wnt-related targets, including c-Myc, Slug, ZEB1 and MMP-7, thereby promoting cell proliferative, invasive and migratory potential.23In recent years, several signalling pathways have been implicated in EMT, including the Wnt, Hedgehog, transforming growth factor-β (TGF-β), phosphoinositide 3-kinase (PI3K) and Notch pathways.24 Emerging evidence supports a crucial role for CDX2 as a tumour suppressor during colorectal carcinogenesis. CDX2 expression is absent in ~30% of human CRC and is inversely associated with tumour grade.26 Mice with a CDX2+/− genotype are susceptible to developing colon cancer.27 Our previous study indicated that CDX2 inhibited proliferation, colony formation and cell motility in CRC.28 Reduction of CDX2 by EGF/bFGF induces sLex/a expression by transcriptionally downregulating FUT3 during EMT induction.29 However, CDX2 can cooperate with β-catenin to regulate tight junctions by increasing claudin-1 expression, which promotes invasion and EMT in CRC.30 Therefore, the exact role of CDX2 during the induction of EMT in CRC remains controversial. In the present study, we aim to investigate the function of CDX2 in EMT and metastasis of CRC.Caudal-related homoeobox transcription factor 2 (CDX2), an intestine-specific transcriptional factor, has been strongly implicated in the development and maintenance of intestinal mucosa.One-hundred-and-sixty-one CRC and paired normal colorectal (NC) tissue samples were randomly selected from CRC patients who had not received radiotherapy or chemotherapy before excision between February 2010 and September 2013. All the patients underwent surgery at the First Affiliated Hospital of Xi’an Jiaotong University. A tissue microarray of 90 pairs of primary CRC tissues was purchased from Shaanxi Kexin Biotechnology Co., Ltd. Informed consent forms were signed by all patients. Our study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University.2 atmosphere.All colon cancer cells  were maintained in RPMI-1640 medium  supplemented with 10% foetal bovine serum (FBS) (Gibco BRL) in a humidified 5% COLentiviral vectors with CDX2 shRNA or CDX2 overexpression were purchased from GeneChem Co., Ltd. . The target short-hairpin RNA (shRNA) sequences were 5′-ACAAATATCGAGTGGTGTA-3′ and 5′-GACAAATATCGAGTGGTGTAC-3′. Lentiviral infection referred to the manufacturer’s protocol.32 Briefly, after anaesthesia, abdominal surgeries were conducted to expose the spleen and slowly inject it with 1 × 107 cells. The spleen was then returned to the abdominal cavity, and the abdomen was closed. After 50 days of intrasplenic injection, the nude mice were anaesthetised with diethyl ether and sacrificed by cervical dislocation. All animal procedures were in accordance with the Helsinki Declaration, and approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University.The female BALB/c-nude mice (4-week-old) were purchased from a corporation of Shanghai . The mice were divided into four groups (HT-29-shCDX2/HT-29-shCtrl and SW480-shCDX2/SW480-shCtrl). The detailed protocol referred to the previous studies.33 Cells were cultured in six-well plates until 90% confluence. Pipette tips (10 µL) were then utilised to scratch artificial vertical lines. The cells were maintained in FBS-free medium for an additional 48 h. The images of wound closure were captured under a microscope at 0, 24 and 48 h.The would-healing assays were carried out as described previously.33 Briefly, cells, suspended in FBS-free medium, were seeded into Transwell  inserts coated with Matrigel  or not. The lower chamber contained 600 μl of RPMI-1640 medium with 20% FBS. Twenty-four hours later, the migratory or invading cells were imaged and counted under an inverted microscope after crystal violet staining.The transwell assays were carried out as described previously.34 The primers for qRT-PCR were listed in Supplementary Table Total RNA extraction, complementary DNA (ctDNA) synthesis and qRT-PCR were carried out as described previously.Microarray analysis was carried out to compare gene expression profile in SW480-shCDX2 and the control cells. Total RNA was extracted with TRIzol reagent and prepared for subsequent analysis by Affymetrix GeneChip system .34 Briefly, the extent of stained cells  and the staining intensity  were recorded. The immunoreactivity scores (IRSs) were defined as the product of extent and intensity scores. An IRS of >3 was considered as positive expression.The IHC staining was carried out as described previously.35 Nuclear extracts were prepared for subsequent analysis. The western blotting analysis was performed as described previously.34 Detailed information regarding these antibodies is shown in Supplementary Table Nuclear protein was extracted using a Nuclear Extraction Kit  as described previously.34For promoter analyses, a fragment of the PTEN 5′-flanking sequence (from –912 bp to +207 bp) and other truncated fragments were cloned into the pGL3.0 Basic Vector  to generate a PTEN full promoter reporter construct and the truncated ones  according to the method of the manufacturer’s instructions.33 The sample was observed using a fluorescence microscope to measure E-cadherin and vimentin expression and β-catenin subcellular localisation.The IF assay was carried out as described previously.For ICC assay, the cells were fixed with 4% paraformaldehyde for 20 min, punched with 0.2% Triton X-100 for 10 min and then incubated with the primary antibodies. The following protocol is the same as the IHC analysis.t test or one-way ANOVA. Correlations in the protein levels were conducted using Pearson linear-regression analysis. Survival rate was calculated using Kaplan–Meier method, and the difference in survival was analysed by log-rank test. Univariate and multivariate analyses were conducted using a Cox proportional hazard model. P < 0.05 was defined as statistically significant. All data were analysed using SPSS 18.0 software . All in vitro experiments were carried out in triplicate.The differences between the experimental and control groups were compared by the Student’s To determine the role of CDX2 in colorectal tumorigenesis, an IHC assay was first performed using 161 pairs of CRC versus adjacent NC tissues. CDX2 staining was observed in the nuclei of positive cells Fig. . The posNext, western blotting assay was performed to investigate differences in the expression levels of CDX2 in five colon cancer cell lines: RKO, Caco-2, HT-29, SW480 and Lovo , while ectopic expression of CDX2 enhanced phosphorylated β-catenin  reporter construct and the other three truncated ones were constructed and transfected into Lovo-CDX2 and Caco-2-CDX2 and the control cells, respectively. The luciferase activity of the PTEN promoter was detected by dual-luciferase reporter assay. Ectopic expression of CDX2 resulted in an elevated luciferase activity of the full-length fragments, but did not affect the luciferase activity of the other truncated fragments Fig. , suggestThe clinical relevance of CDX2, PTEN and EMT marker proteins in 46 randomly selected CRC tissues was explored (Supplementary Fig. 46 Moreover, lack of CDX2 expression was proposed as a poor prognostic and predictive biomarker for the response to chemotherapy in stages II and III CRC.47 Consistently, our data indicated that reduced CDX2 correlated with worse OS and RFS of patients with CRC. Moreover, CDX2 was validated as an independent predictor of OS but not RFS. In summary, CDX2 is likely to be an important biomarker for guiding evaluation of tumour progression and prognosis.Emerging evidence supports a crucial role for CDX2 as a tumour suppressor in colorectal tumorigenesis. However, the exact function of CDX2 during the induction of EMT in CRC remains to be elucidated. Our results gain new insight into the role of CDX2 in EMT. The present study firstly revealed that CDX2 expression was lower in CRC than NC tissue samples. Moreover, the expression of CDX2 is inversely correlated with lymphatic and distant metastasis and TNM staging. In stage IV unresectable CRC, lack of CDX2 expression predicted poor survival.48 In this study, the results obtained in liver metastasis models showed that knockdown of CDX2 promoted CRC liver metastasis in vivo. Subsequently, we demonstrated that CDX2 inhibited colon cancer cell invasion and migration in vitro. EMT has been linked to the mobility and dissemination of CRC by conferring increased invasiveness and metastatic potential to cells.50 By microarray analysis, real-time PCR and western blotting validation, we observed that colon cancer cells with CDX2 knockdown expressed high levels of fibronectin, vimentin and MMP-9, and low levels of ZO-1 and E-cadherin. Enhancing CDX2 expression had the reversed EMT programme. These data support that deficiency of CDX2 might be involved in metastasis of CRC through promoting EMT.Distant metastasis and relapse directly lead to a poor prognosis of CRC patients.15 Our study revealed that knockdown of CDX2 induced the phosphorylation of Akt and GSK-3β and promoted the expression of Snail, while ectopic expression of CDX2 had the opposite effect. It has been reported that active Akt impedes GSK-3β phosphorylation, which results in the stabilisation of Snail.43 In our study, blockade of PI3K/Akt pathway by MK-2206 in CDX2-depleted cells increased phosphorylation of GSK-3β, which was accompanied by Snail suppression. In summary, we concluded that CDX2 antagonises EMT in CRC by suppressing Snail expression through PI3K/Akt/GSK-3β pathway. Consistently, depletion of CDX2 in lung cancer promoted cell invasion and metastasis by increasing Snail expression.51 Intriguingly, Gross et al. reported that Snail could repress CDX2 transcription in colon cancer cells.52 Our previous study also indicated that Snail is involved in the HIF-α-induced downregulation of CDX2,53 implying that a positive feedback mechanism might exist between CDX2 and Snail; however, the exact mechanism remains to be further elucidated.Next, the molecular mechanism of EMT phenotypic changes was explored. There were a variety of signalling pathways responsible in regulating EMT, including Wnt/β-catenin, tumour growth factor, Notch and PI3K/Akt pathways.20 Here, we reported that CDX2 knockdown promoted the stabilisation and nuclear translocation of β-catenin in CRC, while ectopic expression of CDX2 had the reserved alterations. Nuclear accumulation of β-catenin interacts with LEF/Tcf transcriptional factors, which induces the transcription of EMT-related genes.54 Expectedly, the levels of Slug and ZEB1, two target genes of β-catenin, were increased in response to CDX2 knockdown, and this effect can be reversed by the depletion of β-catenin. Previous study indicated that direct phosphorylation of β-catenin by AKT also promotes its nuclear translocation and increases the transcriptional activity.55 Snail, Slug and ZEB1 acts as a strong inducer of EMT by repressing E-cadherin transcription.56 In our study, an elevation of Snail, Slug and ZEB1 followed by CDX2 knockdown might retard E-cadherin/β-catenin complex and induce β-catenin release from the membrane, which was confirmed by the results of IF assay showing a decreased β-catenin in the membrane of CDX2-depleted cells. Deficiency of membrane localisation of β-catenin causes the disassociation of cell–cell contracts and enhanced the metastatic potential of CDX2-depleted cells. Collectively, our study demonstrated that CDX2 inhibits EMT and metastasis of CRC by regulation of Snail expression and β-catenin stabilisation through PI3K/Akt/GSK-3β signalling. During gastrin-induced migration, Fas-induced EMT and endothelin-1-mediated EMT and tumour invasion, the inactivation of GSK-3β by PI3k/Akt signalling promotes Snail expression and β-catenin stabilisation.58The PI3K/Akt pathway impedes the activity of GSK-3β, leading to the stabilisation and nuclear translocation of β-catenin to promote cell proliferation, differentiation and EMT.59 Inactivation of PTEN has been reported to participate in EMT acquisition during the process of tumour metastasis.60 Emerging evidence indicated that PTEN can be regulated at the transcriptional level as well as by numerous post-transcriptional modifications.59 We observed that CDX2 regulated PTEN expression at both mRNA and protein levels. Moreover, ectopic expression of PTEN attenuated tumour cell invasion as well as Akt and GSK-3β phosphorylation and EMT marker proteins enhanced by CDX2 knockdown, while knockdown of PTEN antagonised the tumour-suppressive effect of CDX2 overexpression. The dual-luciferase reporter assay confirmed that CDX2 could promote PTEN transcription by binding to the −912-bp to −651-bp region of the PTEN promoter. Mechanistically, CDX2 specifically and directly binds to the P1 fragment of the PTEN promoter detected by qChIP. This is the first study to identify that CDX2 could directly regulate PTEN transcription.PTEN acts as negative regulator of PI3K-mediated AKT activation in cell homoeostasis.14 However, Elumalai et al. reported that the level of total β-catenin was reduced in PTEN-inactivated lung cancer cells, despite the accumulation of β-catenin in cell nucleus.60 These studies unanimously concluded that PTEN inactivation in tumour induces the nuclear accumulation of β-catenin, although the total β-catenin level might exhibit the inconsistent alteration. This might be due to loss of β-catenin substrate protein E-cadherin or proteasomal degradation of β-catenin substrate by AKT-mediated phosphorylation. Intriguingly, inhibition of PTEN by Hes-1 resulted in a decrease in the level of membrane and cytoplasmic β-catenin, but not nuclear accumulation.61This study indicated that depletion of PTEN elevated the expression of both total and nuclear β-catenin in CDX2-overexpressed cells. The direct target of PTEN by EBV-miR-BART7-3p exhibits a similar alteration in nasopharyngeal carcinoma.In this study, by conducting in vitro and in vivo assays and using a group of CRC patients, we first showed that CDX2 is a major inhibitor of the invasion-prone phenotype and EMT in colon cancer cells. Second, we demonstrated that CDX2 could directly transactivate PTEN expression and thereby suppress PI3K/Akt/GSK-3β signalling, which results in decreasing Snail expression and destabilising β-catenin. Decreased nuclear β-catenin suppresses Slug and ZEB1 transcription, and the reduction of Snail, Slug and ZEB1 induces E-cadherin transcription, which consequently enhances cell–cell interaction and retards cell migration and invasion. Finally, in clinical CRC samples, we observed that CDX2 was positively correlated with PTEN and E-cadherin expression, and was negatively correlated with Snail and vimentin expression. In summary, these findings reveal that CDX2 exerted an inhibitory impact on EMT and metastasis in CRC. The pivotal signalling pathway involved in this process was identified, as were suitable candidates for therapeutic targets in CRC patients.Supplementary file