ACLY activation, which thereby stimulating de novo lipogenesis and tumorigenesis. More importantly, USP7-induced G3BP2 deubiquitination is attenuated by PRMT5 depletion or inhibition. PRMT5-activity dependent methylation of G3BP2 is required for its deubiquitination and stabilization by USP7. Consistently, G3BP2, PRMT5 and G3BP2 R468me2 protein levels were found positively correlated in clinical patients and associated with poor prognosis. Altogether, these data suggest that PRMT5-USP7-G3BP2 regulatory axis serves as a lipid metabolism reprogramming mechanism in tumorigenesis, and unveil a promising therapeutic target in the metabolic treatment of head and neck squamous carcinoma.GTPase-activating protein-binding protein 2 (G3BP2) is a key stress granule-associated RNA-binding protein responsible for the formation of stress granules (SGs). Hyperactivation of G3BP2 is associated with various pathological conditions, especially cancers. Emerging evidence indicates that post-translational modifications (PTMs) play critical roles in gene transcription, integrate metabolism and immune surveillance. However, how PTMs directly regulate G3BP2 activity is lacking. Here, our analyses identify a novel mechanism that PRMT5-mediated G3BP2-R468me2 enhances the binding to deubiquitinase USP7, which ensures the deubiquitination and stabilization of G3BP2. Mechanistically, USP7- and PRMT5-dependent G3BP2 stabilization consequently guarantee robust  In addition to genetic and epigenetics alterations, aberrant lipid metabolism in cancer is becoming increasingly recognized as a hallmarker , 2. AccuGTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2) are originally recognized as core components that contributes to stress granules (SGs) assembly , 5. InhiProtein arginine methyltransferase 5 (PRMT5), a dominant type II protein arginine methyltransferase, plays an important role by interacting with cytoplasmic and nuclear molecules, such as transcription factors, elongation factors and splicing factors . ImportaIn the current work, we show that PRMT5 is responsible for G3BP2 accumulation by inducing its binding and deubiquitination by Ubiquitin-Specific Peptidase 7 (USP7). USP7-mediated G3BP2 deubiquitination and stabilization are attenuated by PRMT5 depletion or chemical inhibition of methyl-transferase activity of PRMT5. PRMT5 and USP7 serve as a G3BP2-sensitive ‘switch’ regulating G3BP2 stabilization and lipid metabolism reprogramming, which leads to lipogenesis and aggressive malignancy of head and neck squamous carcinoma (HNSC). PRMT5-USP7-G3BP2 regulatory complex is an essential driver for tumorigenesis.2. All of the cells were authenticated and no mycoplasma contamination was detected in the cell lines used in this study. Wild-type G3BP2, PRMT5, truncated and R468K-mutant G3BP2 plasmids were synthesized by GeneCopoeia . Constructs, siRNAs, and shRNAs were transfected into HEK293 and HNSC cell lines in accordance with the instructions of Lipofectamine 3000 reagents, respectively (Life Technologies/Invitrogen). Supplementary Table Human embryonic kidney cells (HEK293) were purchased from the type culture collection of the Chinese Academy of Sciences . HNSC cells  were purchased from Guangzhou Juyan Biological Technology . Cells were maintained in RPMI-1640 or in Dulbecco’s modified Eagle’s medium . All culture media were supplemented with 10% FBS and 1% Penicillin-Streptomycin in a 37 °C humidified incubator with 5% COThe indicated cells were lysed with RIPA buffer  supplemented with protease inhibitor cocktail. After incubation on ice for 30 min, 30 μl protein A/G magnetic beads was used to preclear cell extracts, followed by incubation with either IgG , USP7 primary antibody  or PRMT5  antibody overnight at 4 °C. The immune complexes were followed by incubation with 40 μl protein A/G beads for 2 h. Immunoprecipitates were washed and subjected to Western blot, silver staining and mass spectrometry analysis. Silver staining was performed using the Fast Silver Stain Kit (Beyotime) and mass spectrometry was carried out by Novogene, in Beijing, China. Immunoblotting assay was performed as previously described . AntibodACLY and FASN promoters were prepared by PCR and inserted into pGL3-Basic vectors. HEK293 and PRMT5 (KO) cells were grown in DMEM and transiently co-transfected with plasmids containing 3’-UTR of wild-type (WT) or R468K (MUT) from G3BP2 and PRMT5, along with ACLY or FASN luciferase reporter plasmids using Lipofectamine 3000. Luciferase activities were measured 60 h after transfection using the dual luciferase reporter assay system kit  and normalized to Renilla luciferase activity, each experiment was repeated in triplicate.The http://crispr.mit.edu).To generate PRMT5-Tu686 knockout cell lines, the sgRNA sequences were cloned into LentiCRISPRv2 plasmid, then the recombinant viral plasmids and viral packaging plasmids (psPAX2 and pMD2G) were co-transfected into HEK293 cells. After transfection for 48 h, the viral supernatants were harvested and filtered through a 0.45 μm filter. Targeted cells were then infected with the viral supernatant and selected with puromycin (1 mg/ml) for 2 weeks. sgRNA sequences targeting PRMT5 and USP7 were designed using the CRISPR designer  according to the manufacture’s instruction. The absorbance was measured using a Spark® multimode microplate reader . For colony formation assay, 500 cells were seeded into 6-well plates and cultured in medium for 10 days to 2 weeks. After macroclones (>50 cells) formed, colonies were fixed with methanol for 30 min and subsequently stained with 0.1% (m/v) crystal violet for 1 h at room temperature. The colonies were captured using microscope.For cell proliferation assay, indicated cells were plated in 96-well plates at 1 × 10Escherichia coli BL21-DE3 by induction with 1 mM IPTG . GST-tagged proteins were purified using Glutathione Sepharose 4B beads . G3BP2-His and G3BP2-His truncated proteins were purified with Ni-NTA agarose beads (Qiagen). Different purified GST- and His-tagged recombinant proteins were incubated with pull-down buffer  for 2 h at 4 °C before immunoblotting assay.GST, GST-PRMT5, GST-PRMT5 truncated recombinant proteins were expressed in Flag tagged PRMT5 full-length and its truncations were inserted into pcDNA3.1-Flag vector. The human G3BP2 cDNA were inserted into pcDNA3.1-HA vector. Indicated plasmids were transiently transfected into HEK293 cells, and after 48 h of incubation, cells were lysed in immunoprecipitation buffer as described. After washing three times, proteins in elutes were detected with anti-Flag or anti-HA antibody.P (FDR) values < 0.05 were considered statistically significant.Tu212 cells with ectopic G3BP2 were collected and flash-frozen in liquid nitrogen for extracting metabolites. Non-targeted liquid chromatography mass spectroscopy (LC-MS/MS) analysis and data preprocessing and annotation were performed at Shanghai Biotree Biotech Co. Ltd. Briefly, about 1000 μl extract sample (acetonitrile: methanol: water = 2: 2: 1) were homogenized and sonicated for 5 min in the ice-water bath. LC-MS/MS analyses were performed using UHPLC system  with a UPLC BEH Amide column  coupled to Q Exactive HFX mass spectrometer . The QE HFX mass spectrometer was used to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of acquisition software . The raw data were converted to mzXML format using ProteoWizard and processed by R package XCMS (version 3.2). R package CAMERA was used for peak annotation after XCMS data processing. The metabolites annotation were performed by accurate mass search and MS/MS spectral match using an in-house MS2 database, and adjusted 2, 5 mM β-mercaptoethanol, and 120 mM sucrose) at room temperature for 2 h and stopped by adding 5× SDS loading buffer and was resolved by SDS-PAGE.5 µg of recombinant Flag-PRMT5 proteins purified from HEK293 cells or GST-G3BP2-WT or R468K proteins purified from bacteria were incubated with immunoprecipitated Flag-PRMT5 in the presence of adenosyl-L-methionine, S-[methyl-3H]. The reactions were performed in the methylation buffer  for migration or invasion assay. The bottom chamber were filled with 500 μl medium containing 20% FBS. After culturing for 36 h, the migratory or invasive cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet solution. Five randomly selected fields from each well were imaged and counted under a microscope .For migration assay, 5 × 106 cell/well and incubated for 20 h. Then, a sterile 10-μl pipette tip was used to make the scratch line. The indicated cells were incubated with serum-free medium for 48 h. The images of each wound were observed by microscope and the wound healing rate was calculated using Image J software (http://rsb.info.nih.gov/ij/).Indicated cells were seeded in 6-well plates at a density of 2 × 107 Tu686 cells, with five mice per group. Tumor volumes were measured by the formula: length × width2 × 0.52. When the volume of xenograft tumors reached 800 mm3, mice were treated with indicated stable cell lines and shRNA for up to 8 weeks. Tumor volumes were measured every 3 days and at the end of the eighth weekend, and xenograft tumors were extracted and paraffin-embedded for further analysis. The animal study was conducted in accordance with protocols approved by the Animal Ethics Committee of Jiaying University.4–5-week-old nude mice were purchased from Vital River . The mice were randomly separated into four groups and subcutaneously injected with 1 × 10Immunohistochemistry assay was performed as previously described . ExperimRT-PCR analysis was carried out as previously described . The firt-test was performed to analyze the significance of the differences between two groups. All data were presented as mean ± SD. Survival curves of HNSC patients were plotted by the Kaplan–Meier method. Multivariate Cox regress analyses was performed to determine independent risk factors. All data were collected from at least three independent experiments. P values of less than 0.05 were considered to be statistically significant.All statistical analysis were performed using GraphPad Prism 7.0  and SPSS 19.0 . Student’s two-tailed Relatively few post-translational modifications of G3BP2 in human cancer cells have been reported. Our previous work identified PRMT5 is highly expressed in laryngeal cancer cells , and invNext, we performed a series of truncated fragments to examine which domain of PRMT5 is responsible for the binding with G3BP2. We generated three GST-tagged truncated PRMT5 for pull-down assays, which is composed from aa 1 to 292, 293 to 420, and 421 to 637 region. The results showed that both the FL-PRMT5 and the construct with enzymatic activity region (421-637aa) could specifically interact with G3BP2 Fig. . These dRattus norvegicus to Homo sapiens  was required for G3BP2 interacts with USP7 Fig. , suggestACLY, FASN, ACSL3, SCD1, and PPARγ in Tu212 cells. Immunoblotting analysis demonstrated that G3BP2 increased the protein levels of FASN, ACLY, PPARγ, SCD1, suggesting G3BP2-WT displayed a more stronger effect than G3BP2R468 in the de novo fatty-acid biosynthesis . USP7, a member of ubiquitin-specific processing proteases, was regarded as a biomarker for predicting metastasis and recurrence of several malignant tumors –44. USP7r system , 48. ConOur results extend the knowledge regarding of G3BP2 methylation and stabilization for tumor progression. High-level PRMT5 and USP7 are responsible for the accumulation of, and enhancing G3BP2 deubiquitination, which leads to lipogenesis and aggressive malignancy of HNSC. A PRMT5-USP7-G3BP2 regulatory complex is a potential therapeutic strategy for HNSC and other lipid metabolic diseases as well.Supplementary tablesSupplementary figure legendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6checklistOriginal Data File


Ror2-depleted HFSCs, we uncover that ROR2 is not only essential to regulate Wnt-activated signaling that is responsible for HFSC activation and self-renewal, but it is also required to maintain proper ATM/ATR-dependent DNA damage response, which is indispensable for the long-term maintenance of HFSCs. In analyzing HFSCs lacking β-catenin, we identify a compensatory role of ROR2-PKC signaling in protecting β-catenin-null HFSCs from the loss of stem cell pool. Collectively, our study unveils a previously unrecognized role of ROR2 in regulation of stem cell self-renewal and maintenance.Hair follicles undergo cycles of regeneration fueled by hair follicle stem cells (HFSCs). While β-catenin-dependent canonical Wnt signaling has been extensively studied and implicated in HFSC activation and fate determination, very little is known about the function of β-catenin-independent Wnt signaling in HFSCs. In this study, we investigate the functional role of ROR2, a Wnt receptor, in HFSCs. By analyzing  Wnt signaling functions in tissue homeostasis and tumorigenesis. Here the authors show that ROR2, a Wnt receptor, plays roles not only in transducing Wnt signaling, but also in regulation of DNA damage response critical for stem cell maintenance. The binding of secreted Wnt ligands to receptors and/or co-receptors initiates diverse signaling cascades that can be divided into β-catenin-dependent canonical and β-catenin-independent non-canonical Wnt signaling pathways1. These pathways might act independently or cooperatively to orchestrate various cellular functions.In mammals, Wnt signaling functions in tissue morphogenesis, stem cell activation, and tumor development4. Stabilized β-catenin protein then translocates to the nucleus where it binds to lymphoid-enhancing factor/T-cell factor (LEF/TCF) proteins to activate target gene expression5. Unlike Wnt/β-catenin signaling, β-catenin-independent Wnt pathways involve multiple intracellular signaling cascades that might be cross-connected. The induction of non-canonical Wnt signaling may trigger the release of intracellular calcium, which in turn activates downstream protein kinases, such as calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC)8. Non-canonical Wnt signaling can also be transduced via Rho family of the small GTPases, which activate c-Jun N-terminal kinase (JNK) and the downstream activating protein-1 (AP-1) complex for transcriptional regulation, or directly modulate cytoskeleton organization that orchestrates planar cell polarity (PCP) and cell migration13.Canonical Wnt signaling  is activated when a Wnt ligand binds to Frizzled (Fzd) and LRP-5/6, which triggers activation of Dishevelled (Dvl) proteins, leading to the inhibition of the destruction complex, composed by Axin, caseine kinase 1α (CK1α), adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β), thereby stabilizing β-catenin14, and then recognized as one of Wnt (co-)receptors due to its ability to interact with non-canonical Wnts, including Wnt4, Wnt5a, and Wnt1116. Genetic studies show that Ror2/−− mice displayed striking similarities to Wnt5a/−− mice, suggesting they may function in the same signaling pathway17. In vertebrates, ROR2 is required for Wnt5a-induced cell migration, a function that involves activation of JNK, PKC, actin-binding protein Filamin A and Rho-family of the GTPase21. The interaction of Wnt-ROR2 leads to phosphorylation of Dvl that induces the activation of AP-1 and Rac123. In addition, ROR2 was shown to interact with and be phosphorylated by CK1and GSK3, both of kinases that also play imperative roles in Wnt/β-catenin signaling26. In multiple systems, Wnt5a was shown to inhibit β-catenin-mediated canonical Wnt signaling29. The nature by which ROR2 mediates Wnt5a-dependent antagonism of Wnt/β-catenin signaling remains controversial. Under certain circumstances, Wnt5a inhibits Wnt3a-induced β-catenin signaling via ROR233; in others, ROR2 is not required for this inhibition34. In contrast, ROR2 has also been reported to potentiate Wnt/β-catenin signaling. In osteosarcoma cells, ROR2 enhances the transcriptional response to Wnt135; in lung carcinoma cells, ROR2 activates Wnt3a-induced canonical Wnt signaling as a co-receptor with Fzd231. Overexpressing ROR2 enhances β-catenin-mediated transcription; conversely, knocking down ROR2 decreases it in renal cancer cells36. It is noteworthy that studies that analyzed the effect of ROR2 in Wnt/β-catenin signaling have mostly depended on protein overexpression and the reporter assay for Wnt/β-catenin signaling activity. The physiological effect of ROR2 in Wnt signaling activities requires further investigation.Receptor tyrosine kinase-like orphan receptor 2 (ROR2) was initially identified along with ROR1 as a tyrosine kinase of the Trk family38. Ataxia Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) are DDR kinases that are primarily activated by genomic damage; however, the functions of ATM and ATR in DNA damage specificities are distinct40. In response to DNA damage, ATM and ATR get activated and phosphorylate downstream signaling proteins, such as checkpoint kinase 2 (CHK2) for ATM, and checkpoint kinase 1 (CHK1) for ATR, thereby regulating cell cycle checkpoints, DNA repair or apoptosis41. Independently from the DDR, both ATM and ATR can also be activated by the excess production of reactive oxygen species (ROS), which could be caused by imbalanced oxidative metabolism and/or accumulation of unrepaired DNA damage45. In response to ROS, ATM induces activation of 5′ AMP-activated protein kinase (AMPK)46 and nuclear factor erythroid 2-related factor 2 (NRF2)48 to modulate oxidative metabolism and induce antioxidant response to combat ROS, respectively. Deficiency in ATM-dependent DDR in SCs can lead to impaired self-renewal or apoptosis that could influence the numbers of stem cells and their functionality51.All cells in the body are exposed to DNA damage caused by exogenous factors, including mutagens, or endogenous processes, such as oxidative stress. Thus, in order to maintain genomic integrity that ensures tissue homeostasis and prevents the development of deleterious diseases such as cancer, DNA repair mediated by DNA damage response (DDR) is of vital importance, particularly for adult stem cells (SCs) as they persist for extended periods in adult tissues52. Mature HFs progress through cycles of growth (anagen), degeneration (catagen), and then rest (telogen)54. Hair follicle stem cells (HFSCs), located at bulge of the resting HF, remain quiescent during telogen phase of the hair cycle. At the onset of anagen, some of HFSCs become proliferative and migrate downward to replenish the lower HF55. The activation of HFSCs is tightly regulated by microenvironmental signals coming from their niche cells61. At anagen onset, upregulated Wnt/β-catenin signaling directs transcriptional regulation that governs HFSC activation64. HFSCs lacking β-catenin are unable to undergo hair regeneration65, but they can be maintained in their native niche without losing HFSC identity63. However, it remains unclear what is the mechanism that maintains β-catenin-null HFSC pool in a rich milieu where neighboring cells secrete various signal molecules along the hair cycle.Hair follicle (HF) is an excellent model system to study the underlying mechanisms that regulate stem cell activation and maintenanceRor2 in HFSCs caused a delay in HFSC activation, downregulation of HFSC stemness genes, and eventually led to loss of a HFSC population. By depleting Ror2 in primary cultured HFSCs, we uncovered that ROR2 is essential for the activation of Wnt-induced signaling and ATM/ATR-dependent DDR, thereby regulating HFSC migration, proliferation, and maintenance. Lastly, in analyzing HFSCs lacking β-catenin, we identified the necessity of ROR2 and the downstream PKC in protecting β-catenin-null HFSCs from the loss of stem cell pool and wrong fate differentiation. Together our results reveal that ROR2 not only regulates Wnt signaling that governs HFSC activation and migration, but also serves as a protective mechanism to ensure long-term maintenance of HFSCs.In this study, we identify a surprising role for ROR2 in regulation of stem cell self-renewal and maintenance. Using a genetic mouse model, we showed that depletion of 66, very little is known about the function of Wnt signaling in HFSCs apart from β-catenin-dependent regulation. To tackle this unsolved question, we focused on a Wnt receptor, ROR2, which is capable to transduce β-catenin-dependent and -independent Wnt signals35. We first examined the expression of ROR2 in HFs using immunostaining of whole-mount skin. As shown, ROR2 is fairly well expressed in HFSCs and neighboring cells in the bulge (Bu) but relatively low in the secondary hair germ (HG)  and anagen onset  using fluorescence-activated cell sorting (FACS), we confirmed that ROR2 protein level was elevated in aHFSCs as compared to qHFSCs  to the mice carrying conditional Ror2 alleles (Ror2fl/fl) and a Cre-activated YFP reporter (ROSA26LSL−YFP), and then depleted Ror2 gene in HFSCs and their progenies at postnatal (P) days 18–25  HFSCs  HFs  Fig. . NotablyHG) Fig. . By puriSCs Fig. . To dete–25 Fig. . We obseSCs Fig. , as wellHFs Fig. . The delHFs Fig. , suggestRor2 depletion in HFSCs that were synchronized and activated by hair depilation at the 2nd telogen phase  skin by performing quantitative reverse transcription PCR (RT-qPCR) analysis. As shown in Fig. Ror2 cKO HFSCs. This downregulation was also detected in Ror2 cKO HFSCs at anagen onset during hair cycling , corresponding to their lower proliferation, but also lost the expression of HFSC markers, Cd34 and Krt15  and YFP reporter (ROSA26LSL−YFP), and transducing them with lentiviral Cre recombinase in culture  HFSCs while the expression of ROR1 was getting elevated in Ror2/−− HFSCs during passaging  and Ror2/−− HFSCs for Wnt5a-induced activation of JNK and PKC, as well as for the activities of Rac1 and Cdc42. By performing western blotting analysis with Wnt-stimulated HFSCs, we showed that Dvl2 was phosphorylated 71, and 8-oxoguanine (8-oxoG), a biomarker for measuring the effect of oxidative DNA damage72. As shown in Fig. Ror2/−− HFSCs showing over 6 γH2AX foci than Ror2+/+ HFSCs indicated the accumulation of DNA DSBs in Ror2/−− HFSCs. We also found a significant percentage of Ror2/−− HFSCs displaying nuclear staining of 8-oxoG, something not detected in the nuclei of Ror2+/+ HFSCs , we depleted β-catenin in HFSCs and their progenies at P18–25, and found that β-catenin cKO (β-cat KO) HFs displayed a delay in anagen entry at the 1st hair cycle and then stayed at the 2nd telogen throughout the rest of the life as evidenced by the delay and inability of hair coat recovery, respectively , referred as Ror2/β-cat dKO. Depletion of Ror2 and/or β-catenin was initiated by administration of RU486 to mice at P18-25. Similar to Ror2 cKO and β-cat cKO mice, Ror2/β-cat dKO mice also showed the delay of anagen entry at the 1st hair cycle  was only observed in Ror2/β-cat dKO HFs and that more than a half of Ror2/β-cat dKO HFs showed enlarged SGs accompanied with aberrant HF structure or diminished bulge compartments  and GF109203X , to suppress the activities of JNK and PKC of cultured HFSCs, respectively, and then examined their effects in HFSC proliferation by performing EdU labeling experiments. As shown, inhibition of PKC, but not JNK, erased the differences in cell proliferation between Ror2+/+ and Ror2/−− HFSCs  littermates, or wildtype (Cre-) littermates only when the littermates of Ror2 cKO mice did not contain any Ror2 heterozygous mouse. Cre-recombinase activity was induced by intraperitoneal injection of RU486  at P18, P21, and P24, and daily topical administration of 4% RU486 in ethanol from P21–P25. To induce synchronized HFSC activation, HF depilation was performed on anesthetized mice at P55. For PKC inhibitor treatment, 100 μg of GF109203X (R&D Systems) in acetone (1 μg/μl) or acetone alone was applied to P55 mouse back skin every other day for 3 days. Experiments were designed based on sex-, age- and strain-matched pairs, mainly littermates, and were repeated on ≥3 pairs of sample sets. Phenotype and obtained results were reproducible in both male and female pairs.Mice were housed and treated according to the guidelines of the University Animal Ethics Committee, Université Catholique de Louvain. All experimental procedures were conducted in compliance with animal welfare regulations of Belgium. All laboratory mice are housed in 12:12 light:dark light cycles at ambient temperature 20–24 °C and humidity ranges of 45–65%. 78. Briefly, dorsal skin was harvested, scrapped for fat removal, and incubated in 0.25% trypsin for 30 min  or 40 min  at 37 °C. Epidermal and HF cells were scrapped off and cell suspension was filtered through 70 and 40 μm cell strainers (Fischer Scientific). Collected cells were suspended in PBS containing 4% FBS and incubated with antibodies before FACS. Stained cells were then sorted using a FACS Aria III (BD Biosciences) with FACS Diva v8.0.1 software. Fixable Viability Dye eFluor 780 was used to exclude death cells. Living HFSCs were then sorted based on surface expression of integrin α6, CD34 and cytosolic YFP. FACS gating scheme is provided in Supplementary Fig. FACS purification procedures were as described previouslySkin biopsies were collected and fixed in 4% paraformaldehyde at 4 °C for 4 h, and then immersed in 30% sucrose at 4 °C overnight before embedded in OCT compound. For skin whole-mounts, fat from the back skin was removed, and then incubated in 2 mg/ml dispase (Thermo Fisher Scientific) supplemented with 20 mM EDTA at 4 °C overnight. Skin epidermis with attached hair follicles was then peeled off from dermis and fixed in 4% PFA for 30 min at room temperature (RT). Washed whole mount skins were stored in PBS at 4 °C for further use.63. Images of immunofluorescence staining were acquired using Zen software v3.0 (Zeiss) on a Axio Vert.A1 fluorescence microscope (Zeiss) or Zen software v2.6 (Zeiss) on a Axio observer Z1 confocal microscope (Zeiss). Fluorescence intensity was measured using ImageJ.For immunofluorescence, 7 µm tissue sections were fixed, washed, and permeabilized in 0.5% Triton-X100 for 20 min. Sections were then incubated in immunofluorescence blocking buffer  at RT for 1 h, and then with primary antibodies at 4 °C overnight. When applicable, the MOM Fluorescein Kit (Vector Laboratories) was used to prevent nonspecific binding of primary mouse monoclonal antibodies. Tissue sections were washed and incubated with Alexa Fluor-conjugated secondary antibody (Jackson ImmunoResearch) at RT for 1 h before mounted with mounting medium containing DAPI (Roth). For whole-mount tissues, immunostaining was performed as described previously with overnight incubation of primary antibodies at 4 °CTo detect HFSC proliferation in vivo, 1 mg of EdU  per mouse was injected intraperitoneally twice at 12 h intervals for 24 h before skin samples were collected. For EdU-labeling assay in vitro, HFSCs were grown on Nunc Lab-Tek II Chamber Slides (Thermo Fisher Scientific) coated with 10 μg/ml fibronectin (Merck Millipore). Cells were incubated with DMSO (vehicle), 2 μM of SP600125 (JNKi), or 0.5 μM of GF109203X (PKCi) for 24 h and then labeled with 10 µM EdU for 4 h prior to detection. Incorporated EdU was detected by a Click-iT EdU Alexa Fluor 647 Imaging Kit (Thermo Fisher Scientific) based on manufacturer’s instructions.2 were counted for analysis. For long-term passaging experiments, three independent groups of 6 colonies per group were picked with clone cylinders (Bel-Art) and passaged onto mitomycin C-treated fibroblast feeders in 24-well plate. HFSCs were trypsinized and passaged at 1:10 dilution onto feeders every 10 days for 10 passages. The expression of Ror2 from every single colony was validated by RT-qPCR.For colony formation assays, FACS-purified HFSCs were seeded at 20,000 cells/well (for anagen onset HFs) or 33,000 cells/well (for telogen-phase HFs) of six-well plate containing mitomycin C-treated J2 3T3 mouse fibroblast feeders (kind gift from E. Fuchs lab upon agreement of H. Green lab). After two weeks of co-culture, colonies were then fixed with 4% of paraformaldehyde and stained with 1% Rhodamine B. Numbers of colonies which sizes ≥ 3 mmRor2fl/fl/ROSA26LSL-YFP mice were cultured and expanded on mitomycin C-treated fibroblast feeders in medium containing 0.3 mM calcium as described82. After 10 passages, HFSCs were taken off from feeders and ready for lentiviral infection. For the treatment of Wnts, HFSCs were starved with medium containing 0.5% of FBS for 16 h and then treated with vehicle (PBS), carrier-free 100 ng/ml Wnt3a (R&D Systems) or 300 ng/ml Wnt5a (R&D Systems) for 30 or 60 min prior to harvesting for protein purification, or for 6 h for mRNA purification. For the treatment of GSK3 or PKC inhibitor, HFSCs were treated with vehicle (DMSO) or 1 µM of CHIR99021 (R&D Systems) or 1 µM of GF109203X (R&D Systems) for 24 h prior to harvesting for protein extraction.FACS-purified HFSCs from the back skin of For directional cell migration assay, QCM 24-well colorimetric cell migration assay (Merck) was performed according to the manufacturer protocol. To analyze collective cell migration ability, scratch wound migration assay was performed. Briefly, feeder-free HFSCs were cultured to grow into a confluent monolayer, and then HFSC monolayer in the medium containing 1% of FBS with or without 8 µg/ml of mitomycin C (Merck) was scraped in a straight line using a pipet tip to create a gap. Images of the gap closing were acquired using a phase-contrast microscope at 0, 8, and 24 h post-scratching, and the gaps at each time points were measured using ImageJ. Presented data are the averages of three independent experiments.For cell cycle analysis, DNA of HFSCs was stained with 10 µg/ml DAPI at 37 °C for 1 h, and DNA content was measured by LSR Fortessa flow cytometer with FACS Diva v8.0.1 software (BD Biosciences) and analyzed by using FlowJo v10.7.2 software. To measure the level of intracellular ROS, HFSCs were stained with CellROX Deep Red reagent (Thermo Fisher Scientific) and analyzed by LSR Fortessa flow cytometer according to the manufacturer’s instruction.2-transfected with a plasmid mix containing pLKO.1 lentiviral expressing plasmid, PAX2 and MD2 viral packing plasmids. 48 h post-transfection, virus-containing supernatant from transfected HEK293FT cells was collected, filtered and mixed with polybrene (Sigma-Aldrich) and an additional 10% FBS before applying to Ror2fl/fl/ROSA26LSL-YFP HFSCs. Viral infection was performed by centrifugation of HFSCs with virus-containing mix at 1100 × g at 35 °C for 1 h. 5 days after infection, HFSCs were purified by FACS based on RFP and/or YFP expression. The following lentiviral expressing plasmids were used: pLKO.1-NLS-iCre-mRFP (kind gift from S. Beronja)83, pLKO.1-MCS-mRFP (derived from pLKO.1-NLS-iCre-mRFP where NLS-iCre was replaced by multiple cloning sites).HEK293FT cells (Thermo Fisher Scientific) were CaClPpib2 or Rps16. Primers used for mRNA expression are list in Supplementary Table n ≥ 3) indicated in each data set.Total RNAs from FACS-sorted cells or cultured cells were purified with the Direct-zol RNA MiniPrep kit (Zymo Research) and reverse transcribed with ProtoScript(r) II First Strand cDNA Synthesis Kit (New England Biolabs). cDNAs were amplified with indicated primers in SYBR Green (Bio-Rad) quantitative PCR assay on the Thermoblock 96 Silver Block Real-Time PCR system (Bio-Rad) and cycle numbers were recorded by CFX Manager v3.1 software. The relative expression levels were normalized to PCR amplification of housekeeping gene HFSCs were starved and then treated with 300 ng/ml of Wnt5a for 2 h prior to harvesting for small GTPase activation assay. For small GTPase activation assay, Rac1/Cdc42 activation assay combo kit (Cell Biolabs) was performed according to the manufacturer protocol. For immunoblotting analysis, proteins were extracted from cells with RIPA buffer . Protein lysates were then loaded into a 4–12% Bis-Tris gel (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific). Transferred membranes were blocked with 5% BSA in TBST buffer at RT for 1 h, and then incubated with primary antibodies at 4 °C overnight. After incubation, blots were washed and then incubated with the HRP-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h. The blots were developed using chemiluminescence detection reagent , and signals were then detected using the Fusion SL imaging system (Vilber) with Fusion-Capt Advance Solo 4S v16.16b software.T183/Y185 , JNK , p-PKCS660 (9371), PKCα (2056), Dvl2 , p-LRP6S1490 (2568), LRP6 , p-GSK3βS9 (9336), GSK3β , p-β-cateninS33/37/T41 (9561), Axin1 , CK1 (2655), p-ATMS1981 , ATM , p-ATRS428 (2853), ATR (2790), p-CHK2T68 (2661), CHK2 (2662), p-CHK1S345 , CHK1 , p-AMPKT172 , AMPKα (2532), α-tubulin (2144), Lamin B1 , Vinculin  and p-ATM/ATR substrates (2851) from Cell Signaling Technology; p-NRF2S40  and NRF2 (ab137550) from Abcam; β-catenin  and β-actin  from Sigma-Aldrich; CD34  from eBiosciences; Rac1 , Cdc42 (07-1466) and GAPDH  from Merck-Millipore; Krt5 (905901) and Krt15 (833901) from Biolegend; p-GSK3βY216  from BD Biosciences; HRP-conjugated secondary antibodies  from Jackson ImmunoResearch. All primary antibodies used for immunoblotting were at 1:1000 dilution, and secondary antibodies at 1:4000. The following Abs were used for immunostaining: ROR2  from Developmental Studies Hybridoma Bank; ROR1 , p-JNKT183/Y185 , JNK , p-PKCS660 , PKCα , γH2AX  from Cell Signaling Technology; GFP  from Abcam; β-catenin  from Sigma-Aldrich; CD34  from eBiosciences; 8-oxoG  from Merck-Millipore. Fluorescent dye-conjugated secondary antibodies  from Jackson ImmunoResearch.The following antibodies (Abs) were used for FACS: Viability dye F780 , integrin α6 , and CD34  from eBiosciences. The following Abs were used for immunoblotting: ROR2 (Ror2) from Developmental Studies Hybridoma Bank; ROR1 , p-JNKn ≥ 3). Experiments were not randomized and investigators were not blinded during data collection. All RT-qPCR results are generated from at least three biological independently animals or independent experiments. All results from histological analysis, immunoblotting and immunofluorescence staining are representative of at least two independent experiments. Graphs were presented using GraphPad Prism 8. Box-whisker plots are presented as the median (the line within the box), the 25th to 75th percentiles (bottom and top lines of the box) and the 10th to 90th percentiles (bottom and top whiskers). Dot plots were presented as the mean ± SEM. Other data were reported as average ± s.d.; * p < 0.05, ** p < 0.005, *** p < 0.0005, or specified p values in figure legends. Statistical analyses were determined by the unpaired two-tailed Student’s t-test for all experiments.Animal experiments were performed in equal numbers of male and female mice. Phenotypes are representative of a minimum of three sex-paired littermates (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Intestinal fibrosis, one of the complications of inflammatory bowel disease (IBD), is associated with fistula and intestinal stricture formation. There are currently no treatments for fibrosis. Mesenchymal stem cell-derived exosomes have been proven to exert inhibitory and reversal effects in IBD and other organ fibrosis. In this study, we explored the role of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) in IBD-related fibrosis and its associated mechanism to provide new ideas for the prevention and treatment of IBD-related intestinal fibrosis.  We established a DSS-induced mouse IBD-related intestinal fibrosis model and observed the effect of hucMSC-Ex on the mouse model. We also used the TGF-induced human intestinal fibroblast CCD-18Co to observe the role of hucMSC-Ex in the proliferation, migration, and activation of intestinal fibroblasts. Having observed that the extracellular-signal-regulated kinase (ERK) pathway in intestinal fibrosis can be inhibited by hucMSC-Ex, we treated intestinal fibroblasts with an ERK inhibitor to emphasize the potential target of ERK phosphorylation in the treatment of IBD-associated intestinal fibrosis. β-induced proliferation, migration, and activation of human intestinal fibroblasts, and ERK phosphorylation played a key role in IBD-associated fibrosis. The inhibition of ERK decreased the expression of fibrosis-related indicators such as α-SMA, fibronectin, and collagen I.  In the animal model of IBD-related fibrosis, hucMSC-Ex alleviated inflammation-related fibrosis as evident in the thinning of the mice's intestinal wall and decreased expression of related molecules. Moreover, hucMSC-Ex inhibited TGF- hucMSC-Ex alleviates DSS-induced IBD-related intestinal fibrosis by inhibiting profibrotic molecules and intestinal fibroblast proliferation and migration by decreasing ERK phosphorylation. Inflammation bowel disease (IBD) is a severe intestinal inflammation, including ulcerative colitis (UC) and Crohn's disease (CD). Intestinal fibrosis is a complication in IBD, especially in CD, which triggers the occurrence of stricture in the locations involved. Apart from stricture in the intestinal lumen, IBD-related fibrosis can induce anal fistula, an important complication in CD, when the inflammation appears in the rectum .β (TGF-β) is a key molecule in the progression of intestinal fibrosis. TGF-β functions in fibrosis by influencing downstream signaling pathways such as the TGF-β/smad pathway and the wnt/β-catenin pathway [The mechanism of intestinal fibrosis is not completely confirmed. In a recent study, it is widely accepted that transforming growth factor- pathway . Several pathway .In addition to not fully understanding the mechanism involved in intestinal fibrosis, there is also no ideal therapeutic method. Although some of the existing anti-inflammation drugs have been shown to prevent fibrosis to a certain extent, those therapies are not always useful as antifibrotic. Mesenchymal stem cells (MSCs) have the ability to self-renew and multidifferentiate . There aα and IL-1β and increasing the level of IL-10 [Exosomes are small vesicles secreted by cells and are considered to be a type of extracellular vesicle (EV). They have a diameter between 30 and 150 nm and a density between 1.13 and 1.19 g/ml . For exaof IL-10 . Moreoveof IL-10 . RegardlTherefore, in this study, we explored the role and mechanism of hucMSC-Ex in mitigating IBD-related intestinal fibrosis and provide new insights into intestinal fibrosis treatment approaches.The study was approved by the Ethical Committee of Jiangsu University (2012258).2 environment, a small amount of α-MEM (Invitrogen) containing 15% FBS  and 5% penicillin-streptomycin (Invitrogen) was used to cover segments in dishes, and the solution was replaced every 3 days. When observation under the microscope revealed long spindle cells near the small pieces of tissues and fused to 80%, it indicates that hucMSCs could be digested.Fresh umbilical cords were obtained from the Zhenjiang Fourth People's Hospital. The umbilical cords were washed in PBS for 30 minutes and were then cut into pieces and placed in clean 3.5 cm small dishes to adhere to the wall. After incubation at 37°C in a 5% COα-MEM with 10% fetal bovine serum  and 1% penicillin-streptomycin (Invitrogen). When the hucMSCs in the Petri dish grew to about 70% confluence, the nutrient solution was replaced by a new α-MEM (Invitrogen) nutrient solution, which is made up of 10% exosome-free FBS and 5% penicillin-streptomycin (Invitrogen). After culturing in the incubator for 48 hours, the supernatant was collected and the above process was repeated until the P7 passage. Human intestinal fibroblasts CCD-18Co were purchased from Beiner Biotechnology and cultured in RPMI 1640 medium (Invitrogen), which constitutes 10% FBS  and 5% penicillin-streptomycin (Invitrogen).hucMSCs were isolated and cultured in μm filter and distributed into EP tubes for storage in a -80°C refrigerator.hucMSC-Ex was extracted by ultracentrifugation. The supernatant of hucMSC culture was centrifuged at 4°C, 2000g for 30 minutes. The supernatant of the previous step was centrifuged at 4°C, 10000g for 30 minutes. Next, the obtained supernatant was concentrated to 100 ml using 100KD ultrafiltration tubes. The concentrated liquid was further centrifuged at 4°C, 100,000g for 70 minutes. The resultant precipitate was saved, and an appropriate amount of PBS was added to dissolve it. The dissolved solution was centrifuged at 4°C, 100,000g for 70 minutes, the precipitate was collected, and an appropriate amount of PBS was added to dissolve the precipitate. Finally, the resultant solution was filtered with a 0.22 The standard solution was used to generate a standard curve, and diluted hucMSC-Ex was added to the sample chamber and read the data. Use the software for image capture and analysis.μl hucMSC-Ex solution was dripped onto a 2 mm copper mesh. The copper mesh was allowed to stand, and excess liquid was wiped off the edge. hucMSC-Ex was counterstained with 3% phosphotungstic acid, and pictures were taken with the transmission electron microscope after drying.About 20 2O), while the DSS group was given 2% DSS solution throughout the first week and two weeks later and given food for three cycles and ddH2O when 2% DSS solution is not being administered. The hucMSC-Ex group received 100 μl (1 × 109 particles) hucMSC-Ex by tail vein injection on the 3rd, 10th, 17th, 24th, 31st, 38th, 45th, 52nd, and 59th days. All mice were weighed every 3 days, and fecal appearance and consistency were observed. On the 63rd day, the mice were sacrificed.The study used 6-8-week-old female C57BL/6 mice purchased from the Animal Experiment Center of Jiangsu University. The mice were weighed and randomly divided into three groups: NC group, DSS group, and hucMSC-Ex group Sup. . The NC The disease activity index (DAI) was calculated based on the criteria shown in Staining kits (Solarbio) were used for hematoxylin and eosin (H&E), Masson, and Sirius red staining of paraffin sections of mouse intestinal tissues.β-actin , anti-α-SMA , anti-collagen I , anti-collagen III , anti-fibronectin , and anti-t-ERK  overnight. After washing with 1x TBS/T buffer, the membranes were incubated with the secondary antibody  for 30 min at 37°C. Pictures were taken with a chemical gel imaging system (GE).A moderate volume of RIPA solution was added to cells and mixed thoroughly. The solution was shaken on a vortex shaker for 1 minute and allowed to stand on ice for 5 minutes. The process was repeated 5 times in total. The solution was centrifuged at 4°C, 12,000g for 15 minutes to obtain the supernatant, which is the protein solution. SDS-PAGE electrophoresis was used to separate target proteins and transfer them to a PVDF membrane. The PVDF membranes were soaked in 5% skim milk to block nonspecific proteins. The membranes were incubated with corresponding primary antibodies: anti-CD9 , anti-CD81 , anti-HSP70 , anti-calnexin , anti-RNA was extracted by TRIzol (Vazyme) and dissolved in DEPC water. cDNA was obtained by using the Vazyme kit on the PCR machine . Obtained cDNA was used to perform QRT-PCR using the Vazyme kit on Step One Plus Real-Time PCR System (ABI). The reaction process was 95°C, 5 minutes; 95°C, 30 seconds; 58~60°C (flexible setting according to different primers), 30 seconds; and 72°C, 30 seconds, for a total of 40 cycles. The specific system reagents and volumes are shown in Tables Mouse colon tissues were fixed in 4% paraformaldehyde. The tissues were dehydrated, cleared, and embedded in paraffin, followed by microtome sectioning at the First People's Hospital of Zhenjiang City. The slices were kept at 56°C for 4 hours to prevent the tissue from falling off in subsequent procedures. The tissue slices were deparaffinized, and the sections were placed in a hydrogen oxide-methanol solution (1 : 9 mixed with 30% hydrogen peroxide and methanol) and kept at room temperature for 30 minutes. Slices were washed 3 times with histochemical PBS, immersed in citrate solution, and steamed in an electric oven for 30 minutes. After cooling, the histochemical PBS was washed 3 times, followed by blocking nonspecific proteins in sections with BSA for at least 30 minutes. The protein antibody (Proteintech) was diluted to 1 : 200 with 5% BSA and used to cover the tissue at 4°C overnight. It was then washed 3 times with PBS, incubated with the secondary antibody  and SABC , and finally observed under the microscope after adding DAB (Bio-Rad), looking for golden yellow positive areas. The hematoxylin staining solution was used to counterstain the slide, and pictures were taken under the microscope.β-induced cell models  nutrient solution was added to the well. Three groups of cells in the six-well plate were digested and resuspended in FBS-free RPMI 1640 nutrient solution. A certain amount of resuspension was added to different Transwell wells, and the 24-well plate was cultured at 37°C and 5% CO2 for 18 hours. Cells were stained with 0.5% crystal violet, and images were obtained under the microscope.The TGF-els Sup.  were conβ  and anti-fibronectin  overnight. A fluorescent secondary antibody (SAB) was added to the slide and incubate for 1 hour at 37°C in the dark and then washed with PBS 3 times. Nuclei were stained with Hoechst 33342, the slides were fixed with an antifluorescence quencher, and images were taken under the laser scanning confocal microscope.Cell slides were placed in a 24-well plate, and cells were added to construct a cell model induced by TGF-β Sup. . After tP values of < 0.05 were considered statistically significant.Data for all results were shown as mean ± standard error. Analysis of variance was performed by using the GraphPad Prism software, and 10/ml.The extracted hucMSC-Ex were examined with nanoparticle tracking analysis (NTA), which revealed particle diameter between 50 and 150 nm . Transmiμl (1 × 109 particles) of hucMSC-Ex via the tail vein on the third day of every week, starting from the first week. The results showed variations in the weight of the different groups. With time, the weight of mice in the NC group gradually increased while that of the DSS group showed a downward trend with the administration cycle. However, body weight loss was effectively suppressed after the hucMSC-Ex injection  group, a DSS group, and a hucMSC-Ex group. The hucMSC-Ex group received 100 njection . At the njection . On day njection . To assenjection .β, and α-SMA was down-regulated in the hucMSC-Ex group . However, the expression of COL3A1 genes increased in the TGF-β group, and the changes were not obvious or even continued to increase after hucMSC-Ex treatment  and ERK phosphorylation. Moreover, treatment of TGF-β-induced fibroblasts with ERK phosphorylation inhibitors showed that the expression of α-SMA, collagen I, and fibronectin decreased. All the above results indicated that hucMSC-Ex could inhibit the ERK phosphorylation level in fibroblasts and thus inhibit the development of fibrosis.ERK is the final component of the Raf-MEK-ERK signaling module, which acts downstream of receptor tyrosine kinases and controls multiple cellular processes, including cell proliferation and differentiation. Activated bi-phosphorylated ERKs transmit pathway activation to cells through the phosphorylation of multiple substrates. The ERK pathway plays a regulatory role in a variety of cellular activities. Abnormalities in the ERK pathway could cause neurodevelopmental disorders. On one hand, ERK activation causes the proliferation of cancer cells. ERK has a proapoptotic effect, and enhanced ERK1/2 signaling can lead to tumor cell death . In addire aging . TherefoThis study demonstrates that exosomes can affect the occurrence of IBD-associated intestinal fibrosis by regulating ERK phosphorylation in human intestinal fibroblasts. However, our study has shortcomings, including the lack of a detailed mechanism of ERK phosphorylation in fibrosis inhibition and the use of only intestinal fibroblasts in the ERK inhibitor PD98059 experiment, which makes the evidence weak. In addition, there is a lack of data on clinical samples. There is still some gap between the simulated disease occurrence process and the real disease occurrence; thus, clinical studies are needed, which could lead to the safe use of exosomes in humans for IBD-related intestinal fibrosis treatment. Many cells play a role in the development of fibrosis. Studies have proved that hucMSC-Ex can regulate the proliferation and activation of intestinal fibroblasts. Whether other intestinal cells can also be regulated by hucMSC-Ex can also be further investigated. Nonetheless, our findings are significant and inspire further exploration in this area. We expect these findings to be linked to previous results in other organ fibrosis and IBD and provide new insights for inhibiting and reversing fibrosis.β-induced intestinal fibroblast proliferation, migration, and activation. The hucMSC-Ex relieve-effect is associated with its inhibition of ERK phosphorylation in intestinal fibroblasts, leading to reduced expression of fibrotic molecules to alleviate intestinal fibrosis.hucMSC-Ex alleviates DSS-induced IBD-related intestinal fibrosis in mice by inhibiting TGF-


Hyperactivation of Wnt signaling is crucial in tumor formation. Fully elucidating the molecular details of how the cancer-specific Wnt signaling pathway is activated or contributes to tumorigenesis will help in determining future treatment strategies. Here, we aimed to explore the contribution of CUEDC2, a novel CUE-domain-containing protein, to the activation of Wnt signaling and the tumorigenesis of triple-negative breast cancer (TNBC) and to determine the underlying mechanisms. TNBC patient samples and disease-free survival (DFS) data were used to determine the association between CUEDC2 and TNBC progression. The effects of CUEDC2 on TNBC were examined in TNBC cells in vitro and in subcutaneous xenograft tumors in vivo. Gene knockdown, immunoprecipitation plus liquid chromatography–tandem mass spectrometry, pull-down, co-immunoprecipitation, localized surface plasmon resonance, and nuclear translocation analysis were used to uncover the mechanisms of CUEDC2 in regulating Wnt signaling and TNBC development. CUEDC2 is sufficient to maintain the hyperactivation of Wnt signaling required for TNBC tumorigenesis. The contribution of CUEDC2 plays a major role in determining the outcome of oncogenic Wnt signaling both in vitro and in vivo. Mechanistically, the CUE domain in CUEDC2 directly bound to the ARM (7–9) domain in β-catenin, promoted β-catenin nuclear translocation and enhanced the expression of β-catenin targeted genes. More importantly, an 11-amino-acid competitive peptide targeting the CUE domain in CUEDC2 blocked the interactions of CUEDC2 and β-catenin and abrogated the malignant phenotype of TNBC cells in vitro and in vivo. We observed that TNBC patients who exhibited higher levels of CUEDC2 showed marked hyperactivation of the Wnt signaling pathway and poor clinical outcomes, highlighting the clinical relevance of our findings. CUEDC2 promotes TNBC tumor growth by enhancing Wnt signaling through directly binding to β-catenin and accelerating its nuclear translocation. Targeting the interactions of CUEDC2 and β-catenin may be a valuable strategy for combating TNBC. Wnt/β-catenin signaling becomes dysregulated in many cancer types. Hyperactivation of Wnt signaling has been demonstrated as a critical regulator controlling tumor-initiating cell self-renewal and differentiation, tumor metastasis, and drug resistance to cancer therapy. The β-catenin-dependent signaling pathway is initiated through Wnt ligands binding to FZD and LRP/5/6, which disrupts the core complex, including AXIN, APC, and GSK3, leading to the stabilization of β-catenin. Subsequently, β-catenin is accumulated in the cytosol, translocated to the nuclear, and initiates the oncogenic transcription of its target genes . β-catenCTNBB1 mutations. Undefined mechanisms may contribute to the hyperactivation of Wnt signaling in TNBC tumor cells [Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen receptors (ER) and progesterone receptors (PR) or overexpressed human epidermal growth factor receptor 2 (HER2) [or cells . CUE domor cells . CUEDC2 or cells . Consistor cells . Findingor cells ,8,9. CUEor cells ,11,12. Tor cells ,14. As wIn the present study, we determined the impact of CUEDC2 on Wnt/β-catenin signaling in TNBC. We further examined the mechanisms by which CUEDC2 activates Wnt/β-catenin signaling in TNBC. To translate our findings into a new therapeutic strategy, we developed a small competitive peptide to abrogate the malignant phenotype of TNBC cells by disrupting the interactions of CUEDC2 and β-catenin, providing a novel therapeutic strategy for TNBC treatment.n = 168 cases) containing tumors and matched adjacent normal tissues were obtained from the Peking University Cancer Hospital from patients who underwent surgery from 2003 to 2008 at the hospital. The median follow-up period was 3727.5 days. The following inclusion/exclusion criteria were applied: (a) pathological diagnosis of resectable stage I–III TNBC; (b) detailed follow-up information. Written and informed consent was obtained from all patients before the surgical operation and specimens’ collection. This project was approved by the ethical committee of the Peking University Cancer Hospital & Institute (2018KT98).Primary TNBC specimens (n = 5/group), and 2 × 106 MDA-MB-231 cells with stable CUEDC2 knockdown (shRNA) in 100 μL of Matrigel / PBS were injected into the mammary pad of mice. Tumor growth was monitored twice a week, and they were finally harvested at 3–4 weeks after tumor cell injection. Animal studies were approved by the Peking University Cancer Hospital Animal Care Committee (EAEC 2018-23) and were carried out by following the institutional guidelines.Here, 6–8-week-old NPG female mice  were randomized into groups . HEK 293FT was purchased from the Chinese Academy of Medical Sciences & Peking Union Medical College . Cells were cultured in DMEM high glucose medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% COFor details of the methods of plasmid construction and cell transfection, the cell viability and plate colony formation assays, the cell migration and invasion assays, co-immunoprecipitation (co-ip) and IP-LC/MS/MS spectrometry, the pull-down assay, the establishment of the CUEDC2 and β-catenin interaction model, immunoblotting and immunohistochemistry, the peptide competition assay (PCA) and localized surface plasmon resonance (LSPR) analysis, and statistical analysis, please see the online p = 0.0071, p = 0.0094, p = 0.1614, CUEDC2 is responsible for resistance to estrogen therapy in ER-positive breast cancer ,6. HowevAs the clinical data suggested that CUEDC2 contributes to TNBC progression , we deciTo determine the impact of CUEDC2 on the phenotype of TNBC cells in vitro, CUEDC2 was overexpressed in MDA-MB-231 and MDA-MB-468 cells . The resWe next identified the molecular mechanism by which CUEDC2 accelerated TNBC tumor cell growth. Previous studies showed that CUEDC2 preferred to bind to a target protein for the modulation of signaling pathways ,16,17,18Wnt-induced target genes represent major regulators of cancer stem cells responsible for the control of tumorigenesis. Since CUEDC2 directly interacts with β-catenin, we decided to determine if CUEDC2 functions through the activation of Wnt/β-catenin signaling. CUEDC2 knockdown significantly inhibited tumor cell proliferation in vitro and strongly reduced tumor formation in in vivo B,F. We tNuclear translocation is critical for β-catenin to promote tumorigenesis by initiating gene transcription . As CUEDhttps://github.com/sokrypton/ColabFold, accessed on 27 July 2022) and is shown in Next, we examined whether disrupting the interactions of CUEDC2 and β-catenin was a promising approach to restrain TNBC tumor formation. Based on our findings E, we synp = 0.0286, p = 0.0076, p = 0.0495, Finally, we aimed to confirm whether high levels of CUEDC2 and β-catenin correlate with poor prognosis for TNBC patients. We discovered that the CUEDC2 protein level is positively correlated with the β-catenin protein level in TNBC tumors . In FiguPrecise control of Wnt signaling is required to promote TNBC growth, progression, and the formation of metastasis. Aberrations in CUEDC2 gene expression have been reported in several human cancers; however, the role of CUEDC2 in the establishment of the hyperactivation of Wnt signaling in TNBC remains to be defined. We show here that CUEDC2 expression is negatively related to the DFS of TNBC patients. We further reveal that CUEDC2 promotes TNBC cell proliferation, migration, and invasion. Mechanistically, CUEDC2 accelerated Wnt/β-catenin signaling by directly binding to β-catenin and facilitating the nuclear translocation of β-catenin. A peptide targeting the binding sites between CUEDC2 and β-catenin disrupted the combination of CUEDC2 and β-catenin, and it suppressed the progression of TNBC in vitro and in vivo. Altogether, this study introduced a new role of CUEDC2 in TNBC, discovered its new interacting protein—β-catenin—and the novel action of CUEDC2 in regulating Wnt/β-catenin signaling, and presented a promising strategy for combating TNBC by targeting CUEDC2.We found that an 11-amino-acid competitive peptide targeting the CUE domain in CUEDC2 blocked the interactions of CUEDC2 and β-catenin and abrogated the malignant phenotype of TNBC cells in vitro and in vivo. We did not test if the peptide can block the interaction between beta-catenin and other proteins such as Tcf/Lef. We are aware that the interaction between β-catenin and Lef extends along the armadillo repeats ; therefoIt should be noted that CUEDC2 has multiple functions as an independent driver in different types of cancer . Our cliCdh1) or heat shock protein 70 (HSP70) by binding to APC/CCdh1 or HSP70, respectively [The CUE domain in CUEDC2 binds to a target protein and subsequently degrades the target protein in a ubiquitin- or phosphorylation-dependent manner ,6,7,18 Cectively ,24. In oThe present study has revealed a significant role of CUEDC2 in promoting TNBC. CUEDC2 fulfills its function by binding to β-catenin and facilitating β-catenin’s nuclear translocation. Targeting the CUEDC2-β-catenin interaction with a small peptide could effectively suppress TNBC development as well as its malignant phenotype . Our fin


Tg) is increased by 11 °C at most at filler concentrations from 1 to 7 wt%. Moreover, the room-temperature relative permittivity and dielectric loss factor of the composites at 50 Hz are decreased by 0.22 and 1.3‰, respectively. Conductivity at 10 mHz to 1 kHz and dc conductivity are also significantly decreased when the operating temperature is below Tg. The polarization relaxation process of the nanocomposite is dominated by regional carrier migration, interfacial and dipole polarization. The relaxation frequency of dipole polarization at high temperature (>Tg) is transformed to satisfy the Vogel–Tammann–Fulcher law. This research suggests that both the dielectric and the polarization relaxation properties of the epoxy resin composites can be modified by filling hyperbranched-polyester-treated nanosilica, because it enhances the bond strength of the inorganic–organic interface and enlarges the molecular scale of the composites via cross-linking reactions.Hyperbranched polyester is effective for enhancing molecular bond strength and improving the mechanical behavior of nanofilled polymers. This study examines the dielectric and polarization relaxation characteristics of epoxy resin composites filled with nanosilica 30 nm in diameter, which is treated by terminal carboxyl hyperbranched polyester. TEM and SEM analysis indicate that the nanosilica surface is grafted with a functional polymer layer ranging in thickness from several to tens of nanometers, and the nanosilica agglomeration in epoxy resin is remarkably inhibited. Measurements of thermally stimulated depolarization current and differential scanning calorimetry show that, deep traps with an energy of 1.09 eV are present in the nanocomposites, and the glass transition temperature ( Dielectric and relaxation properties of a nanofilled epoxy composite can be effectively modified by nanofiller treatment with a terminal carboxyl hyperbranched polyester. Among many surface-modification methods for the nanofiller, surface grafting is one of the most popular chemical methods. Silane or other similar coupling agents are widely used to treat inorganic nanofillers because they contain both hydrophilic and hydrophobic groups, located at opposite terminals. They can act as bridges and combine inorganic particles with organic polymer molecules. However, owing to the linear structures and low molecular weights of these conventional coupling agents, the interaction or modification area is not wide, and the bond strength is inadequate, which limits the ability to improve the behavior of the composites. Regarding this issue, considerable research has been conducted to find more effective surface modifiers and modification methods for inorganic nanofillers.9,10Nanosized inorganic fillers are often used to modify the insulating,3,11,12 In our previous study, the surface modification treated by carboxyl-terminated hyperbranched polyester (CHBP) significantly inhibited the agglomeration of nanosilica in epoxy resin and enhanced the tension and alternating-current (ac) breakdown strength of the composite.13 Owing to its dendritic molecular structure and active terminal groups, the hyperbranched polyester endows nanofillers with a higher probability of interacting with the epoxy matrix and a stronger bond force. However, little is known about the influence of hyperbranched polyester surface modification on the dielectric properties of nanofilled polymers. It is believed that the trap distribution and charge mobility of nanofilled composites are remarkably influenced by the interaction zone around fillers.14–17 In addition to the enhancement of the toughness of epoxy resin, the higher bond energy between nanofillers and epoxy molecules should exert various influences on the dielectric properties and molecular motions.18 The purpose of this study is to investigate the dielectric and relaxation properties of epoxy composites filled with nanosilica, focusing on the effects of surface treatment with the hyperbranched polyester.Hyperbranched polyester is a kind of highly branched three-dimensional polyester possessing numerous dendritic structures and active terminal radicals. It has been used to modify inorganic fillers of polymer composites in recent years. In particular, the improvement of the mechanical and thermal properties has been most actively investigated.2.We purchased nanosilica, of a spherical shape with a nominal diameter of 30 nm, from Aluminum Corporation of China. The nanosilica was treated in two steps. As shown in p-toluenesulfonic acid (PTSA). Here, the weight of the dried silica particles is regarded as 100%. After reacting with the solution for 40 min, silica was dried again under 100 °C.Nanosilica particles were mixed with ethanol and aminopropyltriethoxysilane (3-APTES). The weights of ethanol and 3-APTES are double and 10% of silica's. After mixing, the hybrid solution was stirred for 40 min at 20 °C, and then heated to 100 °C for 20 min to remove the solvent. Next, the silane-treated silica was gradually added into the mixture of 0.5 wt% of CHBP, 300 wt% of acetone and 0.1 wt% of via the reaction between amino and carboxyl groups. After two steps of surface modification, the surface activity of nanosilica particles is modified significantly by the presence of the dendritic structures and terminal carboxyl acids.During the first step of surface treatment, silane chains with a molecular weight of approximately 260 react with surface hydroxyl radicals on the surfaces of the nanosilica and are grafted on the surfaces. Owing to the existence of amino groups in the grafted silane molecule, CHBP can be successfully grafted onto the nanosilica surfaces As a next step, epoxy resin nanocomposites were fabricated using the silica nanoparticles modified as previously mentioned. Bisphenol-A epoxy resin  was used as base matrix resin. Methyl tetrahydrophthalic anhydride and 2,4,6-tris(dimethylaminomethyl)-phenol were used as a hardener and a curing accelerator, respectively. The nanosilica content was set to 0, 1, 3, 5, or 7 weight percent (wt%) of epoxy resin. After complete curing, the epoxy resin composites were shaped into circular sheets 0.5 mm thick and 30 mm in diameter.Before electric testing of epoxy composite sample, its surface composition was scanned by XPS . Distribution of nanofillers in epoxy composite was also observed by SEM .19,20 Therefore, the composite samples were polarized at a direct-current (dc) electric field of 20 kV mm−1 at 100 °C and then heated again from 10 to 200 °C to measure their TSDC. Using a digital electrometer , the dc conductivity of the composite was measured at an applied electric field of 10 kV mm−1 and 20 °C. The complex permittivity and glass transition temperature (Tg) of the composite samples were observed using a dielectric spectrometer  and a differential scanning calorimeter , respectively. For the DSC test, the temperature was increased at a rate of 10 °C min−1.For insulating polymers, measurements of thermally stimulated depolarization current (TSDC) often provides useful information about structural, thermal, and electrical properties.3.via grafted silane and CHPB molecules, and the thickness of CHBP functional layer ranges from several to tens of nanometers. After two steps of grafting reactions, nanosilica surface would be covered by silane and CHBP layers. Epoxy resin composites filled with 0, 1, 3, 5, and 7 wt% of nanosilica were prepared for the TSDC test. The experimental results are shown in QTSDC) is not simple, the values of QTSDC emitted from the composite filled with nanosilica are always smaller than the value observed for the pristine epoxy resin. These results suggest that the TSDC is significantly suppressed by the addition of CHBP-treated nanosilica.21 Using the initial rise method,22 the activation energy of the thermal process can be calculated. It is known that the TSDC in an insulating polymer arises mainly as a result of the thermally assisted detrapping of injected charges or the thermal relaxation of the dipolar orientation.The results shown in  and dielectric loss factor (tg δ) of nanocomposites at 50 Hz and 20 °C. It shows that with the increase in filler content, the permittivity constant and dielectric loss both decrease obviously. At 3 wt%, the nanocomposites own the lowest permittivity and dielectric loss. At 7 wt% of filler content, the value of  and tg δ approaches and exceeds that of pristine epoxy resin, respectively. between 100 and 1 kHz. While  decreases with the increase in frequency (f), the loading of CHBP-treated nanosilica seems to suppress the appearance of a very high  at a low frequency and below 120 °C. However, at a high temperature, the  of the composites filled with nanosilica is far larger than that of the pristine epoxy resin. measured for pristine and nanofilled samples. As discussed in the next section, the imaginary value of the relative permittivity mainly reflects the transport properties of charge carriers, especially in insulating polymers at a low frequency and a high temperature.23 of the nanofiller epoxy resin at a low frequency is smaller than that for pristine epoxy resin at a temperature below 120 °C. and  of the composite samples at 0.01 Hz. Clearly, the composites filled with CHBP-treated nanosilica exhibit a reduced relative permittivity and dielectric loss at a low temperature. This phenomenon becomes more obvious for the 3 and 5 wt% samples. The value of  of the nanofilled composites is approximately 0.2 smaller than that of the unfilled epoxy resin. However, at a high temperature, values of  and  of the pristine epoxy resin are remarkably smaller than those of all the nanofilled composites. and  satisfy the Cole–Cole relation, the electric modulus (M*) can be expressed by 24 is the complex relative permittivity, εr∞ and εrs are the values of the relative permittivity at infinitely high and near-zero frequencies, respectively, τ is the relaxation time, β is the shape parameter, and ω is the angular frequency.For obtaining the transition frequency of the dielectric permittivity at different temperatures, the use of the electric modulus, which is the inverse of the complex relative permittivity, is more effective than the use of the original permittivity. In the case where M′′), and the loss peak is wider than the one at a higher temperature. Moreover, with the addition of CHBP-treated nanosilica, the relaxation peak frequency decreases, so that the M′′ spectra appear to move towards the low-frequency direction. Moreover, if the filler content is more than 5 wt%, the decrease of relaxation peak frequency is not obvious any more.Considering that the real part does not exhibit clear features, which is similar to our previous studies, only the imaginary part of the electric modulus is shown in Tg of the epoxy resin composite is obviously improved by filling nanosilica by 3, 5 or 7 wt% of. For the 1 wt% sample, Tg is similar to that of pristine epoxy resin. The heat-flow curve of the pristine epoxy resin clearly indicates two separated glass transition processes and a wide transition-temperature range. However, for the nanofilled samples, only one drop of the heat flow is observed, and the transition-temperature width is reduced.σac) values for the composite filled with 3 wt% of nanosilica. The conductivity σac increases with an increase in frequency of the applied electric field. Additionally, if the temperature is <100 °C, the logarithmic value of σac is proportional to the field frequency. If the temperature of the composite exceeds 100 °C, within the low-frequency section, a platform is present in the log(σac)–frequency curve. σac to that of pristine epoxy resin (σac Pri) at 20 °C. It shows that nanocomposites own lower ac conductivities than pristine epoxy resin at low frequency. σdc values of all the composite samples under a dc electric field. Clearly, at a low temperature, σdc in the pristine epoxy resin is higher than in the nanofilled composites. Furthermore, σdc of the 3 wt% nanofilled composite is only 14% of that of the pristine sample at 80 °C. Nevertheless, if the sample temperature is >120 °C, σdc becomes the smallest in the pristine epoxy resin.4.25 the nanosilica can not only combine with sidechains of the epoxy resin but also can be embedded in the main chains of the epoxy resin. This can significantly change the linking status of the nanofiller and epoxy resin molecules, modifying the dielectric behavior and relaxation properties.The molecular weight of the CHBP used in this study is approximately 2600, which is greater than the molecular weight of most frequently-used silane coupling agents. Moreover, there are 12 terminal carboxyl groups. Because the carboxyl radicals can participate in the addition polymerization reaction,26 The thermally assisted detrapping of trapped charges is inevitably introduced, and these charges act as carriers. The dipolar relaxation and the charge-detrapping process maintain a close relationship with the composite temperature and molecular bond strength.As indicated by the TSDC experiments, the epoxy resin filled with CHBP-treated nanosilica releases a smaller TSDC and less charge than the unfilled epoxy resin after polarization under a dc electric field. Generally, two dominant reasons are identified for the appearance of the TSDC in insulating polymers such as epoxy resin. The first is the relaxation of the dipolar orientation. Because the epoxy resin matrix is composed of polar molecules, many dipoles are present, and relaxation of these dipoles can generate the TSDC. The second reason is the detrapping of space charges. During the manufacturing process of the composites, defects, interfaces, and impurity carriers should generate traps with different energies.Tg of the epoxy resin composite increases remarkably by the addition of 3 to 7 wt% of the CHBP-treated nanofiller. Because the CHBP molecules react with epoxy resin radicals and increase the cross-linking degree, the diversity of epoxy resin anisotropy is reduced, and only one DSC peak is present. Additionally, trap-energy calculation suggests that the trap energy of the epoxy resin becomes obviously deeper by the CHBP-treated nanofiller. All of the aforementioned improvements are ascribed to the introduction of the bridge linkage of CHBP molecules, which enhanced the bond strength of the nanosilica and epoxy resin molecules. Deep traps help to inhibit the charge injection from electrodes and increase the energy needed for charge detrapping. The increased Tg also makes the relaxation or jumping of charges in epoxy resin molecules difficult.The DSC test indicates that  curve of nanocomposites shows that obvious conduction process is present at high temperature.  curve also shows a giant permittivity at low frequency (<1 kHz) and an almost constant permittivity at high frequency (>1 kHz). Moreover, the real and imaginary permittivity curves approximately obey a log-linear frequency dependency at low frequency. Considering that the conduction process could not influence the real permittivity, the giant permittivity at very low frequency should be ascribed to low-frequency dispersion of nanocomposites. This means that at the low frequency range, the migration of charges limited in some local regions dominates the process of composite dielectric response.27 In  curves, it also can be found that the initial dispersion frequency of epoxy resin composites is significantly decreased by introduction of nanosilica.σac of nanocomposites is lower than that of pristine epoxy resin at low temperature (<120 °C). This may be deduced by deep traps and reinforced interface structure. As a result, the relative permittivity and dielectric loss of nanocomposite at low temperature are lower than those of pristine epoxy resin.The experiment of ac conductivity shows that with addition of CHBP-treated nanosilica,  and  curves are quite different, and clear relaxation peaks are present in the curve of  and M′′(f). This suggests that the local electric conduction existing in composites is not the only one process which is responsible for nanocomposite dielectric response at low frequency.28 Owing to the existence of CHBP-treated nanosilica, interfacial polarization and dipole relaxation should be other two important processes which influence the dielectric response of nanocomposites.However, note that the slopes of 29 However, if the filler content reaches a certain value, 7 wt% in this study, the adjacent overlapping of the nanofiller interface area occurs, reducing the bond strength of the nanofiller surface,30 and the regional interfacial polarization of high permittivity is enhanced.31,32 As a result, the composite relative permittivity is increased.Since the interfacial polarization of nanocomposites is sensitive to nanofiller content, when the filler content is kept below 5 wt%, the nanosilica is not close enough to the neighbouring filler for the nanofiller interface areas to overlap with each other, and the enhanced bond between the nanofiller and epoxy resin molecules can effectively inhibit the orientation of polar molecular chains or side groups in interfacial areas.fm)–1/T and log(σdc)–1/T do not satisfy the Arrhenius relation at high temperature.Moreover, the CHBP modifier changes the interfacial properties and influences the total relaxation mode of epoxy resin composites. 33 the relaxation of epoxy resin filled with CHBP-treated nanosilica may also satisfy the VTF law at high temperature. According to the VTF law, the temperature dependence of the peak frequency of relaxation and the dc conductivity can be expressed by 34 Here, fm0, Af, Tf0, σdc0, Adc, and Tdc0 are positive constants.As it reported that the relaxation process of epoxy resin at high temperature can be described by the Vogel–Tammann–Fulcher (VTF) law,fm) and dc conductivity (σdc) as a function of the constructed temperature factor described by σdc are proportional to the exponential term, and the VTF law is valid in this case. This suggests that the molecular structure of the epoxy resin composites is thermally stretched to allow molecular chains to move at a high temperature, and the energies of the carriers are sufficient for transport in the composites. Because the Tf0 or Tdc0 calculated via VTF linear fitting is approximately 50 K lower than Tg of the composites,35 the VTF-fitted parameters are consistent with the DSC results.Tg, the advantages of the low permittivity, low dielectric loss, and low dc conductivity due to the CHBP-treated nanosilica filler gradually disappear. This may be because of the composite structural change to the amorphous state, wherein it is easy for polymer molecular chains to move or turn. Thus, the structural equilibrium of the nanofiller interface is broken, and the CHBP polar molecules join in the interface polarization. Moreover, the bond strength of the nanofiller surface is reduced or undermined, thus, the detrapping and charge transport are enhanced. These thermal charges result in the increase in , , σ in the composites at a high temperature.Furthermore, the experiments in this study also indicate that if the operating temperature of the epoxy resin composite is higher than 5.Because of the terminal carboxyl radicals and the long molecular chain, CHBP is effective for the surface modification of nanosized inorganic fillers. In this study, the dielectric and relaxation properties of composites of epoxy resin and CHBP-treated nanosilica are investigated, and the following conclusions are drawn.Processed by silane and hyperbranched polyester, nanosilica surface can be grafted with a functional polymer layer ranging in thickness from several to tens of nanometers. Owing to its big matrix molecular weight, the surface polyester layer can effectively inhibit the agglomeration of nanosilica particles in epoxy resin.Tg.With addition of CHBP-treated nanosilica of 30 nm in diameter, deep traps with an energy of 1.09 eV are introduced in the epoxy resin composites. Real permittivity and dielectric loss of the nanocomposites of 10 mHz to 1 kHz is obviously decreased under low temperature, and the glass transition temperature of the composites is increased by 11 °C at most. Conductivity under ac and dc electric field are also lower than those of pristine epoxy resin, if the operating temperature is <Tg) is transformed to satisfy the VTF relation.The polarization process of epoxy resin nanocomposites filled with hyperbranched-polyester-treated nanosilica is dominated by regional carrier migration, interfacial and dipole polarization. Owing to the enhancing effect of hyperbranched polyester surface layer on bond strength of nanosized interfaces, the low-frequency dispersion deduced by regional charge movement and interfacial polarization in nanocomposites both are inhibited at low temperature. The relaxation frequency of dipole polarization in nanocomposites is also decreased, and the dipole relaxation procedure at high temperature (>There are no conflicts to declare.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2–5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS. •MiR-139 is downregulated in FUS ALS iPSC-derived motor neurons (iPSC-MNs)•MiR-139 inhibits β-catenin in iPSC-MNs•Elevated levels of β-catenin drive apoptosis in iPSC-MNs•Activation of WNT signaling and miR-139 loss is also seen in sporadic ALS MNs In this article, Hawkins and colleagues show that miR-139 is a motor neuron (MN)-enriched microRNA that is downregulated in FUS and sporadic ALS MNs. They discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the loss of upper and lower motor neurons (MNs) that affects 1–2/100,000 individuals worldwide . From clC9ORF72, TARDBP, and FUS, are involved in RNA processing  and are termed sporadic (sALS). Many genes linked to fALS, including ocessing , with thocessing . Moleculocessing . TDP-43 ocessing . Signifiocessing  and mRNAocessing , have bein vitro models of disorders like ALS that are otherwise difficult to study due to diseased cell type inaccessibility. These models provide a powerful platform to study pathogenesis in the context of the genetic background of a patient, without the need to overexpress the mutant proteins (Patient-derived induced pluripotent stem cells (iPSCs) offer an excellent opportunity to create human proteins .In this study, we deployed iPSC-based disease modeling with transcriptomic and phenotypic analysis to investigate molecular drivers of FUS ALS MN degeneration. Our results indicate that the downregulation of the MN-specific miRNA miR-139 contributes to the activation of canonical WNT signaling that drives MN degeneration. Importantly, these molecular defects are also observed in sALS MN, highlighting the shared dysregulation of RNA metabolism in fALS and sALS.FUS gene (H517Q/H517Q) and CRISPR-Cas9-edited heterozygous iPSC (H517Q/+) derived from this line  in FUS ALS MN. These results contrast with miRNA profiling data obtained in sALS MNs, where most miRNAs were observed as downregulated . One reaWe compared our expression data with three miRNA datasets generated from sALS tissue, and our previous study in which we profiled miRNAs dysregulated in FUS P525L MNs C. To ideThe overlap of differentially expressed miRNAs between ALS datasets was low, highlighting the well-known polygenic architecture of ALS C. MiR-13Downstream targets of a miRNA can reveal its functional role within a particular cell type. To determine the downstream targets of miR-139 in MNs, we overexpressed miR-139 in the H517Q/H517Q line and analyzed downstream changes in genome-wide gene expression using RNA-seq. PCA confirmed well-separated experimental conditions, indicating that miR-139 overexpression (OE) induced significant changes in gene expression A. We obsβ-Catenin is the major effector/transcriptional mediator of the canonical WNT signaling pathway A. PrevioSince miR-139 is downregulated in sALS, we hypothesized that WNT pathway genes targeted by miR-139 (miR-139-WNT gene set) would be upregulated in sALS datasets. We observed a significant enrichment of the miR-139-WNT gene set in genes upregulated in sALS MN K, suggesAs miR-139 is downregulated in ALS MNs and inhibits WNT signaling, we expected WNT signaling to be overactivated in our FUS H517Q MN lines. Accordingly, we found upregulation of several WNT ligands and WNT target genes in the FUS MN by qRT-PCR A. In addTo expand our observations beyond the H517Q FUS mutation, we evaluated WNT activity in our previously characterized FUS P525L iPSC MN A, alongsWe hypothesized that inhibiting WNT signaling should improve FUS ALS MN survival. We also targeted pathways known to synergistically interact with WNT signaling and activated in ALS, including mitogen-activated protein (MAP) kinase family members p38 (MAPK14), ERK1 and -2, and JNK1, -2, and -3, as well as TP53 . FurtherUntreated (vehicle-only control) FUS MN showed a significant loss of survival, with ∼50% of the MN lost at day 45 compared to day 30 E. InhibiTo further validate the role of canonical WNT signaling in MN degeneration, we overexpressed β-catenin in healthy 80A MN and observed a more than 2-fold increase in caspase activity, indicating increased apoptosis F and S4BTo investigate the relevance of the identified pathways to sALS, we performed GSEA on the sALS MN datasets using gene sets related to WNT/β-catenin or p38/MAPK perturbation. Enrichment analysis showed significant upregulation of several WNT and p38/MAPK gene sets in sALS MNs G. OveralFUS and TDP-43 are involved in different stages of the miRNA biogenesis pathway . To asseWe quantified primary miRNA (pri-miRNA) levels from RNA-seq data by measuring intronic reads mapping to host genes  Gaidatz. We testPDE2A gene , we did not observe any decrease in the intronic reads of PDE2A. Our analysis suggests that downregulation of pri-miR-139 transcription could be specific to FUS mutations only.These results demonstrate the potential of using deep RNA-seq data to predict transcriptional changes in pri-miRNAs and indicate that mutant FUS may impede transcription of the pri-miR-139 transcript. However, when we performed a similar analysis on RNA-seq data obtained from sALS MNs , in which inhibition of canonical WNT and activation of the non-canonical WNT/PCP leads to synaptic disassembly . Howeverin vitro and zebrafish MN in vivo (ALS-associated mutant Ubiquilin 4 (UBQLN4) has been linked to the upregulation of β-catenin when expressed in mouse spinal MN  in vivo . Pharmac in vivo . β -Cate in vivo . These o in vivo , as well in vivo . Our dat in vivo . MiR-218 in vivo . ConsequHow does WNT hyperactivation lead to MN death? WNT signaling is typically implicated in cell growth, proliferation, and specification . PreviouPDE2A levels, for both H517Q and P525L datasets. Therefore, in FUS-ALS, the downregulation of miR-139 could be due to a combination of reduced pri-miR-139 transcription and downstream miRNA processing defects. However, in sALS, we could not detect significant changes in the miR-139 pri-miRNA, suggesting that defects in miRNA processing are likely to be the primary cause of miR-139 downregulation.A related question is what causes miR-139 downregulation in ALS MNs. FUS-ALS accounts for only a small proportion of ALS cases, yet we also see miR-139 downregulated in sALS without FUS or TDP-43 mutation. However, defects in TDP43 and FUS localization are observed in sALS, while both proteins regulate aspects of miRNA biogenesis . Hence, Overall, our results highlight the importance of RNA processing in ALS and demonstrate the knock-on effect a single miRNA can have on transcriptomic equilibrium that can disrupt MN homeostasis.OCT4 were generated using the AAVpro Helper Free System (Takara). OCT4 is not expressed in MN; hence, this shRNA was used as a non-targeting control. The mature miR-139-5p sequence was expressed using a shRNA design to ensure the expression of miR-139-5p and not miR-139-3p (Adeno-associated viruses (AAVs) expressing miR-139 or small hairpin RNA (shRNA) against R-139-3p   24 h before transfection with a negative or miR-139-5p inhibitor (IDT) using lipofectamine STEM reagent (Thermo Fisher Scientific). Inhibitors were used at a final concentration of 20 nM. For luciferase assay, inhibitors were co-transfected with 50 ng STF19 plasmid  modified to contain 19 binding sites for β-catenin/TCF upstream of the firefly luciferase gene, and 5 ng Renilla luciferase plasmid  as an internal control. Media was replaced 24 h post-transfection and the luciferase assay completed 72 h post-transfection as per protocol . Readings for Firefly and Renilla luciferase were taken on the CLARIOstar Plus microplate reader (BMG Labtech).2 was used per well, and transfection mixes were 1/10 of the volume covering cells. Plasmid and CaCl2 master mixes were mixed gradually into Hank’s balanced salt solution (HBSS) to prevent the formation of large transfection complexes, and incubated for 20 min at room temperature before addition to wells. Cells were incubated with transfection mixtures at 37°C, 5% CO2 for 4.5 h, then washed once with 300 mM sodium acetate dissolved in DMEM:F12 medium for 5 min, 37°C to dissolve transfection complexes. Cultures were maintained in neuronal differentiation media supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) for a further 72 h, then lysed according to the Dual-Glo Luciferase Assay System (Promega).Day 30 MNs were plated in 96-well optically clear white tissue culture plates (Greiner). Cells were transfected with 700 ng STF19 plasmid  and 100 ng Renilla luciferase plasmid . Cells were transfected using a calcium-phosphate method modified from The miR-139 binding site in the 3′ UTR of the CTNNB1 transcript was identified using Targetscan  and exteAAVs expressing β-catenin-EGFP or EGFP only were generated using the AAVpro Helper Free System (Takara). β-Catenin-EGFP plasmid was a gift from Alpha Yap (Addgene plasmid #71367) . HealthyGSE203173.The accession number for the sequencing data reported in this paper is GEO: 2+ imaging assays. A. Rosa provided the FUS P525L iPSC lines. A.B. and L.W.S. funded the project. A.B. conceptualized the project. S.H. and A.B. wrote the manuscript. All of the authors contributed toward editing the manuscript.S.H., S.C.N., A.T., C.B., and A.B. designed and performed the experiments and analyzed the data. S.H. analyzed the RNA-seq and microarray data. P.H. analyzed the microarray data. C.F. and N.S.D. helped with imaging. A. Randall performed the CaThe authors declare no competing interests.