Intriguingly, c-MYC could bind directly to the EphA2 and Wnt1 promoter to enhance their transcription. The entire process formed an EphA2-mediated feed-forward loop. A small molecular inhibitor of EphA2 potently inhibited the proliferation of gastric cancer in vitro and in vivo, including gastric cancer patient–derived xenografts. Thus, our data identify EphA2 as an excellent candidate for gastric cancer therapy.The erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) belongs to the Eph family of receptor tyrosine kinases. EphA2 is highly correlated with the formation of many solid tumors and has been linked to the dysregulation of signaling pathways that promote tumor cell proliferation, migration, and invasion as well as angiogenesis. Deregulation of Wnt signaling is implicated in many forms of human disease including gastric cancer. We previously reported that EphA2 promotes the epithelial–mesenchymal transition through Wnt/β-catenin signaling in gastric cancer. Herein, we present a novel mechanism by which EphA2 regulates Wnt/β-catenin signaling. EphA2 acts as a receptor for Wnt ligands and recruits Axin1 to the plasma membrane by directly binding Dvl2. The EphA2-Dvl2/Axin1 interaction was enhanced by Wnt3a treatment, suggesting that EphA2 acts as a functional receptor for the Wnt/β-catenin pathway and plays a vital role in downstream signaling. We showed that Dvl2 mediates the EphA2-Axin1 interaction by binding to the tyrosine kinase domain of EphA2. We propose that EphA2/Dvl2/Axin1 forms a complex that destabilizes the β-catenin destruction complex and allows β-catenin to translocate to the nucleus and initiate the transcription of  Members of this family enhance or suppress tumor development depending on their mode of activation. For instance, ligand-dependent signaling induced by EphrinA1, a ligand of the EPH receptor A2 (EphA2), is tumor suppressive. In contrast, EphA2 can be activated by interaction with other cell-surface molecules in cancer cells, thus amplifying MAPK, RAS, and AKT signalings, which enhances tumor development3. EphA2 overexpression has been observed in a wide variety of neoplasms such as gastric cancer, colorectal cancer, etc. EphA2 acts as a key driver of metastasis and is a predictor of poor prognosis in various cancers7. We previously reported that EphA2 overexpression is associated with poor prognosis for gastric cancer patients and promotes the epithelial-mesenchymal transition (EMT) through the Wnt/β-catenin pathway in gastric cancer cells11. However, the exact mechanisms of the EphA2 regulation of Wnt/β-catenin signaling in gastric cancer is unclear.The erythropoietin-producing hepatoma (EPH) family is the largest family of receptor tyrosine kinases, the dysfunction of which is recognized as a key initiator of carcinogenesis12. Moreover, the Wnt pathway plays a key role in the proliferation, differentiation, development and maintenance of cancer stem cells13. Dysregulation of Wnt signaling is implicated in many forms of human disease including gastric cancer14. When the Wnt pathway is “on”, Wnt ligands bind to the Frizzled receptor and LRP5/6 receptors, which convey the signal to intracellular components that then promote the recruitment of Disheveled (Dvl) to the plasma membrane. In turn, Dvl further recruits Axin1 to the membrane and forms a complex with Frizzled and LRP5/6. Finally, the β-catenin destruction complexes are disassembled, which allows unphosphorylated β-catenin to accumulate and subsequently translocate to the nucleus16.Wnt signaling is an evolutionarily conserved pathway that controls cell-to-cell interactions during embryogenesis and contributes to tissue homeostasis in most of the organ systems in adultIn this study, we discovered a novel mechanism by which EphA2 drives a feed-forward loop that regulates Wnt signaling; and targeting EphA2 significantly inhibits the proliferation of gastric cancer in vitro and in vivo.Antibodies to proteins were obtained from the following sources: Flag (#F1804): Sigma-Aldrich; Wnt1 (#sc-5630) and ubiquitin (#sc-9133): Santa Cruz Biotechnology; EphA2(#6997), GST , Dvl2 (#3224), c-Myc (#13887), Axin1 (#2087), β-catenin (#8480), histone H3 (#4499), phos-β-catenin (#9565), GSK3β (#9315), and β-TRCP (#4394): Cell Signaling Technology; HA : Origene; GAPDH (#D190090): Sangon ; α-tubulin (#66031-1-Ig): Proteintech. Reagent sources were as follows: recombinant human proteins Wnt3a (#5036-WN) and EphrinA1 (#6417-A1): R&D Systems; EphA2 inhibitor ALW-II-41-27(ALW): MedChem Express; MG132 proteasome inhibitor: Selleck; Dual-Luciferase Reporter Assay System: Promega.The human gastric cancer cell lines AGS and BGC823 were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FBS). HEK293 cells were cultured in Dulbecco’s modified Eagle medium (Gibco) with 1 g/l glucose and 10% FBS. All cell lines were obtained from ATCC. The cell lines tested negative for mycoplasma contamination. All cell lines were used for experiment within 3 months of thawing. All cell lines were authenticated by short tandem repeat profiling prior to use. DNA fragments encoding Flag-EphA2, GFP-EphA2, VN-EphA2, VC-Axin1, VC-Dvl2, RFP-Dvl2, RFP-Wnt1, RFP-Axin1, HA-c-MYC, and Flag-Wnt1 were generated by PCR and cloned into a Flag-tagged (p3xFLAG-CMV-10) or GFP-tagged (pEGFP-N1) empty vector. Different constructs for N-terminal–truncated EphA2 sequences were generated by PCR and cloned into Flag-tagged empty vectors and verified by sequencing. siRNAs were purchased from Ribobio . Plasmids and siRNAs were transfected into cells using Lipofectamine 3000 (Invitrogen). Stable EphA2 short hairpin RNA (shRNA) knockdown lines were generated by infecting AGS and BGC823 cells with three types of reagents: lentiviruses harboring pGLV3/H1/GFP + Puro vector , vectors harboring human EphA2 shRNA targeting sequences , or control GFP-targeting sequences (5′-TTCTCCGAACGTGTCACGT-3′). Selection was with puromycin (2–3 μg/ml). EphA2 knockdown was confirmed by western blotting.GAPDH was amplified in parallel as an internal control. Expression of each gene was quantified by measuring cycle threshold values, and the 2–ΔΔCt method was used to calculate relative changes in gene expression. Primers are listed in Supplementary Table cDNA was synthesized from 2 μg total RNA with a reverse transcription kit (Promega). mRNA level was evaluated using SYBR Green real-time qPCR (Takara). Human Extracts of cells lysed with RIPA buffer were cleared by centrifugation. Lysates (50 μg of protein) were subjected to SDS-PAGE, and the separated bands were transferred to a polyvinylidene difluoride membrane (Millipore) that was then probed with various antibodies.Cytoplasmic and nuclear fractions were isolated with nuclear and cytoplasmic protein extraction kits .17.Paraffin-embedded sections were cut 4 µm thick, then deparaffinized and rehydrated. EphA2, c-Myc, β-catenin, Ki67, and CD31 were detected by immunohistochemical staining as described18. Briefly, cell lysates were incubated with 2 μg antibody at 4 °C overnight. Protein A/G-Sepharose beads (Millipore) were added, and the mix was incubated for 2 h at 4 °C. The immunocomplexes were subsequently washed with lysis buffer three times and subjected to SDS-PAGE.Co-IP assays were performed as described19. Briefly, recombinant proteins were expressed as GST fusions in Escherichia coli BL21. Cells were lysed in phosphate-buffered saline (PBS) containing 1% Triton X-100 and inhibitors of proteases and phosphatases. Lysates containing GST fusion proteins were mixed with glutathione-coupled beads for 2 h at 4 °C, and the beads were washed with PBS three times. Proteins were added to the glutathione-beads and incubated overnight at 4 °C. Beads were washed with PBS three times, resolved by SDS-PAGE, and subjected to western blotting and Coomassie staining.GST pull-down assays were performed as described20. Briefly, AGS cells were treated with 1% formaldehyde and incubated for 10 min to generate DNA-protein crosslinks. Cell lysates were then sonicated to produce chromatin fragments of 200–300 bp and immunoprecipitated with anti-c-Myc or with IgG (control). Antibody-bound complexes were precipitated with Protein A/G-Sepharose beads. The DNA fragments in the immunoprecipitated complexes were released by reversing the crosslinks at 65 °C for 5 h, and purified DNA was analyzed by PCR and agarose gel electrophoresis. PCR was performed using promoter-specific primers for EphA2 or Wnt1 with amplification of the c-MYC-binding regions. Primers are listed in Supplementary Table ChIP assays were performed as described21 using the Cell Counting Kit-8 . The migration and invasion assay was as described21. Briefly, cells (1 × 105) were seeded onto the upper chamber in 200 μl serum-free RPMI-1640; the lower compartment was filled with 500 μl RPMI-1640 supplemented with 10% FBS. After 24 h of incubation, migrated and invasive cells on the lower surface of the filter were fixed and stained using crystal violet. Cells on the upper side were removed using a rubber scraper. Data represent counts of migrated and invasive cells. Experiments were performed in triplicate. For the colony formation assay, cells (1 × 103/ml per well) were seeded in 6-well plates and cultured for 14 days in RPMI-1640 supplemented with 10% FBS. Colonies were fixed with methanol and stained with crystal violet, then scored using Image J software.Cell proliferation assays were carried out as describedA cell-cycle and apoptosis analysis kit with propidium iodide staining reagent (Beyotime) was used for flow cytometric analysis. Cells were harvested by trypsinization, washed once with cold PBS, and suspended in 70% ethanol; cells were fixed by paraformaldehyde overnight with rotation. Before staining, the cells were washed with PBS. Then, cells were incubated with staining buffer (Beyotime) containing propidium iodide and RNase A in a 37 °C water bath for 0.5 h and then analyzed with flow cytometry.Cells were fixed in medium containing 3.7% paraformaldehyde for 1 h and then permeabilized using 0.5% Triton X-100 and blocked using normal goat serum. The primary antibodies were added and incubated at room temperature for 2 h. Alexa Fluor 488-conjugated or 568-conjugated secondary antibodies (Beyotime) were added and incubated for 1 h. Stained cells were examined using a fluorescence microscope.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed using DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s recommended protocol.22.Cells were serum-starved overnight and co-transfected with 200 ng TOP-flash or FOP-flash expression plasmid and 50 ng pRL-TK using Lipofectamine 3000. The activities of both firefly and Renilla luciferase reporters were determined at 48 h post-transfection using the Dual Luciferase Assay kit (Promega). The TOP-flash reporter activity is presented as the relative ratio of firefly-to-Renilla luciferase activities, and the TOP/FOP ratio was used as a measure of β-catenin–driven transcription23 into two groups based on EphA2 expression; specimens in the top 30% were designated as EphA2high and in the bottom 30% as EphA2low. GSEA24 was used to compare the gene set differences between the two groups.We divided primary gastric adenocarcinoma specimens25. Briefly, HEK293 cells were transiently co-transfected with VN- and VC-tagged plasmids at a ratio of 1:1 for 48 h. Images were taken with a fluorescence microscope using excitation (480 ± 30 nm) and emission (535 ± 25 nm) filters under the same conditions.The BiFC assay is based on the reconstitution of a fluorescent protein upon the reassociation of two split nonfluorescent fragments via their linkage to independent interacting proteins. When Venus green fluorescent protein  was cut into two fragments containing either the N-terminal (VN) or C-terminal (VC), neither of the fragments displayed fluorescent property when expressed alone. Coexpression of the two fragments linked to interacting proteins allowed the partial reformation of Venus with the concomitant appearance of the fluorescent signal. Details of the procedure were as described26.Flag-Wnt1 was transfected into HEK293 cells for 48 h, and then the cell culture medium (Wnt1 CM) was collected and concentrated with Amicon-Ultra-15 filters (Millipore). EphA2, Frizzled2, or an empty vector were transfected into HEK293 cells for 36 h and the cell surface-binding assay was performed as describedPLA was carried out using Duolink in situ fluorescence kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, HEK293 cells were transfected with pcDNA3.1-EphA2. Forty-eight hours later, cells were incubated with Flag-Wnt1 conditional medium (CM) for an additional 3 h, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking for blockage. Anti-Flag (mouse) and anti-EphA2 (Rabbit) antibodies were added and incubated at 4℃ overnight. Secondary antibodies conjugated with oligonucleotides (Rabbit antibody with PLA probe plus and Mouse antibody with PLA probe minus) were incubated for 1 h at 37 °C after primary antibody. After wash, ligation was taken place for 30 min at 37 °C, followed by amplification with polymerase for 100 min at 37 °C.6 EphA2 shRNA knockdown (or negative control shRNA) AGS cells in 100 μl RPMI-1640 that was mixed with Matrigel (1:1). Once tumors reached 100 mm3, mice received 20 mg/kg ALW in 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300 or the vehicle alone. ALW is a small-molecule tyrosine kinase inhibitor of EphA2 that effectively inhibits EphA2 function in lung cancer and breast cancer models29. Mice were treated once daily via intraperitoneal injection, and tumors were measured daily with digital calipers. Tumor volume was calculated using the following formula: volume = length × width2/2. Each experimental group had five mice.All animal care and euthanasia protocols were approved by the Institutional Animal Care and Use Committee of Central South University . For the cancer cell xenograft study, 4-week-old nude male mice were injected subcutaneously in the hind flanks with 5 × 103 portions of freshly resected gastric tumor tissues were implanted subcutaneously in 4-week-old athymic recipient male NOD/SCID mice. GC001, GC002, GC003, and GC004 gastric cancer-derived lines were established, and the information for the four gastric cancer patients is listed in Supplementary Table 3 in the mice, tumors were collected, cut into 2-mm3 portions, and serially transplanted subcutaneously in 4-week-old recipient male NOD/SCID mice to establish cohorts29. Tumors were allowed to grow to ~50–100 mm3. The mice were randomly divided into two groups: the control group was injected with PBS, and the ALW group was injected intraperitoneally once daily for 3 weeks with 20 mg/kg ALW in 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300. Each group had five mice. Tumors were measured four times weekly, and tumor volume was calculated as described above. At the end of the treatment period, tumors were collected and analyzed for proliferation, apoptosis, and microvascular density as described above. All animal procedures and were performed in accordance with institutional guidelines.For PDX (patient derived xenograft) mouse model, ~2 mmt-test, and P-values < 0.05 were considered to reflect statistical significance.Statistical significance was calculated using Prism (GraphPad Software) and SPSS17. All experiments were performed in triplicate. Data represent the mean ± s.d. Statistical differences were assessed with the unpaired Student EphA2 and CTNNB1 (encoding β-catenin) with the EphA2-expressing vector in gastric cancer cells BGC823 and AGS  data32, we noted significantly elevated EphA2 expression in gastric cancer tissues compared with corresponding normal tissues, which mirrored the expression profiles of c-MYC and β-catenin , and CTNNB1 in 295 gastric cancer specimens23 . After incubation with Flag-Wnt1 conditional medium (CM), Wnt1 bound to cells transfected with EphA2 or Frizzled2, whereas no or little Wnt1 binding was detected for empty vector-transfected cells, or soluble Ephrin A1 blocked cells  with Frizzled, a receptor for Wnt ligands. We thus performed a cell surface-binding assaylls Fig. . Moreovells Fig. . To furtlls Fig. . The co-lls Fig. . To testlls Fig. , which ills Fig. , and immlls Fig. . Taken t33. Axin1 acts as a scaffold protein for the β-catenin destruction complex and is an important negative regulator of Wnt/β-catenin signaling34. As EphA2 promotes Wnt/β-catenin signaling, we hypothesized that EphA2 may relay Wnt signaling through Dvl2 and Axin1. As shown in Fig. The best-characterized branch of the Wnt signaling pathway is mediated by the canonical Wnt/Frizzled-LRP6/Dvl2/Axin1-GSK3β-β-catenin. Dvl2 is the scaffold protein that relays Wnt signaling by bridging receptor and adapter proteins in both the canonical and non-canonical Wnt pathwaysTo further verify the interaction between EphA2 and Dvl2 in Wnt signaling, we established a bimolecular fluorescence complementation (BiFC) system for visualizing and analyzing the interaction between EphA2 and Dvl2. Co-expression of VN-tagged EphA2 and VC-tagged Dvl2  in cells resulted in a significantly more intense BiFC signal compared with empty-vector control Fig. . Co-exprEphA2 mutant fusion proteins tagged at their N-terminus with Flag or GST  showed that the transcription factor c-MYC  binds to the promoter of hA2 Fig. ; and EphhA2 Fig. .Fig. 5EpEphA2 through UCSC (http://genome.ucsc.edu/). We generated a series of primers corresponding to the putative EphA2 promoter  of the EphA2 promoter in the pGL3-enhancer vector  and sh-EphA2 cells with ALW or DMSO (as negative control) and evaluated whether the inhibitory effect of ALW on gastric cancer cells was dependent on the EphA2 level. Compared with DMSO treatment, ALW significantly reduced the proliferation of both AGS and BGC823 cells, but the decrease was less prominent in the sh-EphA2 group than the sh-NC group of cells  via intraperitoneal injection. Compared with vehicle treatment, administration of ALW to tumor-bearing animals for 17 days significantly inhibited tumor growth in vivo, but the decrease was less prominent in the sh-EphA2 group than in the sh-NC group  via intraperitoneal injection. Administration of ALW to tumor-bearing mice for 3 weeks significantly inhibited tumor growth  and Frizzled are canonical co-receptors in canonical Wnt signaling36. Frizzled binds Wnt proteins through its CRD37, and EphA2 also has this domain. We first guessed whether EphA2 binds to Wnt1 by forming a co-receptor with Frizzled/LRP6. However, our data show that there is no interaction between EphA2 and Frizzled2/LRP6  inhibits Dvl2/Axin1 recruitment to the membrane, which stabilizes the β-catenin destruction complex by binding Dvl2/Axin139. Dvl2 is the scaffold protein that relays Wnt signaling by bridging receptor and adapter proteins in both the canonical and non-canonical Wnt pathways40. In previous studies, certain Dvl proteins interact with EphB241. Our present results reveal that EphA2 conveys Wnt ligand signaling by interacting with Dvl2/Axin1, and this process is similar to the Frizzled-mediated membrane recruitment of Dvl2, which interacts with Axin through its DIX domain to interfere with the function of downstream β-catenin42. Our results indicate that EphA2 plays a similar role to the Frizzled receptor in regulating Wnt/β-catenin signaling. However, when, where, and how Wnt ligands use different receptors to relay signaling to the downstream effectors—and what changes take place when EphA2 binds to Wnt ligands—are issues that will necessitate further investigation.Although EphA2 has been extensively studied as a regulator of many signaling pathways43. Loss or inhibition of EphA2 results in reduced expression of c-MYC; conversely, EphA2 gain-of-function had the reverse effect. c-MYC overexpression rescued proliferation defects induced by the loss of EphA229. In this study, we demonstrated that EphA2 greatly increased the expression of c-MYC and that c-MYC significantly enhanced the transcription of EphA2 by directly binding to the EphA2 promoter. We identified that c-MYC directly binds to the Wnt1 promoter. These new data suggest that EphA2 may function to coordinate a feed-forward loop that ensures EphA2 effectiveness in regulating Wnt/β-catenin signaling  tumors, implying that tumor inhibition function of ALW is EphA2-dependent, at least partly.Previous studies showed that ALW inhibited EphA2 function in lung and breast cancersSupplementary Tables and Figures


Aberrant activation of the Wnt/β-catenin signaling pathway promotes the progression of osteoarthritis (OA). We previously reported that R-spondin 2 (Rspo2), an activator of the Wnt/β-catenin signaling, facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification. However, the role of Rspo2 in OA remains elusive. Here, we showed that the amounts of Rspo2 protein in synovial fluid were increased in OA patients. We searched for a preapproved drug that suppresses Rspo2-induced Wnt/β-catenin signaling in chondrogenic cells and reduces joint pathology in a rat model of OA. In Rspo2-treated ATDC5 cells, mianserin, a tetracyclic antidepressant, inhibited Wnt/β-catenin signaling, increased proteoglycan production, and upregulated chondrogenic marker genes. Mianserin suppressed Rspo2-induced accumulation of β-catenin and phosphorylation of Lrp6. We identified that mianserin blocked binding of Rspo2 to its receptor Lgr5. We also observed that intraarticular administration of mianserin suppressed β-catenin accumulation and prevented OA progression in a rat model of OA. We conclude that mianserin suppresses abnormally activated Wnt/β-catenin signaling in OA by inhibiting binding of Rspo2 to Lgr5. ECM is comprised of a highly hydrated fibrillar network of collagens embedded in a gel of negatively charged proteoglycans like aggrecan (Acan)3.Osteoarthritis (OA) is characterized by progressive loss of articular cartilage and concomitant loss of extracellular matrix (ECM), and causes pain and functional disorders in elderly people4. Binding of secreted Wnts to their cell-surface receptor comprised of Frizzled and low-density lipoprotein receptor-related proteins 5/6 (Lrp5/6) phosphorylates Lrp5/6 and accumulates β-catenin. Accumulated β-catenin is translocated into the nucleus, and interacts with transcriptional factors, T-cell factor/lymphoid enhancer factor (TCF/LEF), to regulate gene expressions. Excessive activity of Wnt/β-catenin signaling has been implicated in many human diseases including OA6. Activation of Wnt/β-catenin signaling promotes hypertrophic differentiation of articular chondrocytes which, in turn, induces cartilage degradation and subsequent OA aggravation7. Indeed, OA progression is mitigated by inhibiting the Wnt/β-catenin signaling10.Wnt/β-catenin signaling pathway plays crucial roles in determination of cell fate, and controls tissue homeostasisXenopus11 and often co-expressed with Wnts12. Rspos binds to two cell-surface receptors: Lgr4-6 (leucine-rich repeat containing G protein-coupled receptors) and RNF43 (ring finger protein 43)/ZNRF3 (zinc and ring finger 3)16. RNF43/ZNRF3 are E3 ubiquitin ligases that degrade the Wnt receptors, Frizzled and Lrp5/6. Rspos bridge Lgr4-6 and ZNRF3/RNF43, and induce endocytosis of Lgr4-6 and ZNRF3/RNF43, which suppresses the E3 ubiquitin ligase activity of ZNRF3/RNF4318. The effects of monoclonal antibodies against Rspo1, Rspo2, and Rspo3 to suppress Rspo-induced signaling are reported in human tumor xenografts19.R-spondins (Rspos) were originally identified as secreted positive-feedback activators of Wnt/β-catenin signaling in RSPO2 are associated with proliferative bone and soft tissue diseases in human21. We recently reported that Rspo2 activates Wnt/β-catenin signaling and reduces expressions of chondrogenic marker genes of Sox9 , Col2a1 (collagen type II α1), and Acan, which subsequently facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes in the growth cartilage22. Hypertrophic differentiation of articular chondrocytes is similarly observed in human OA and animal OA models24. Excessive Wnt/β-catenin signaling activity facilitates hypertrophic differentiation of articular chondrocytes, which, in turn, induces cartilage-degrading metalloproteinase expression and subsequent OA aggravation25. However, involvement of Rspo2 in OA remains to be elucidated. Here, we observed that Rspo2 is increased in synovial fluid in OA patients. We next searched for a drug to antagonize the effect of Rspo2 using the drug repositioning strategy, in which a drug currently used to treat a specific disease is applied to another disease27. We found that mianserin, a tetracyclic antidepressant, suppressed Rspo2-induced activation of Wnt/β-catenin signaling in chondrocytes, which subsequently ameliorated loss of proteoglycans and increased expressions of Sox9, Col2a1, and Acan. Mianserin exerted its anti-Rspo2 effect by directly blocking binding of Rspo2 to Lgr5. In a rat model of OA, intraarticular administration of mianserin reduced accumulation of β-catenin in articular chondrocytes and prevented OA progression.Rspo2 is an important factor for regulating cell proliferation and differentiation, as well as tissue development. Single nucleotide variations in 28, were increased in synovial fluid with increasing OA severities that was evaluated by the Kellgren-Lawrence (KL) radiographic grading system29 , which are degradation products of GAG chains in cartilage proteoglycansm29 Fig. . We nextm29 Fig. . The amom29 Fig. . In cont17. We thus performed drug screening with 120 ng/ml rhRspo2 and 20 ng/ml rhWnt3a, and found that a tetracyclic antidepressants (TeCA), mianserin, that is an antagonist or inverse agonist of the histaminergic H1 receptor, serotoninergic 5-HT1–7 receptors, and α2-adrenergic receptor, suppressed the TOPFlash reporter activity in a dose-dependent manner . Taken together, mianserin inhibited the TOPFlash reporter activity via Rspo2, and the effect is unlikely to be associated with its tetracyclic structure.We next attempted to identify a clinically applicable drug that inhibits Rspo2-induced activation of Wnt/β-catenin signaling and OA progression. We quantified Wnt/β-catenin signaling activity using the TOPFlash luciferase reporter assay in the presence of 1,271 FDA-approved drugs in HCS-2/8 cells, and searched for a drug that suppresses Rspo2-activated Wnt/β-catenin signaling. Recombinant human Rspo2 (rhRspo2) alone does not activate Wnt/β-catenin signaling in HCS-2/8 cells, but enhances the signaling in the presence of a low dose of recombinant human Wnt3a (rhWnt3a) , and β-catenin proteins in 48 h in differentiated ATDC5 cells  cells stated below and ATDC5 cells , we isolated OA cells from OA patients undergoing total knee replacement surgery. We first confirmed that We also examined the effects of mianserin on articular chondrocytes in a rat OA model. Rats were intraarticularly injected with 50 µl phosphate-buffered saline  or 50 µM mianserin once weekly for 8 weeks after OA induction by destabilization of the medial meniscus (DMM) surgery. At 8 weeks after surgery, mianserin improved Safranin-O staining on the articular surfaces and preserved articular cartilage structures in DMM-operated knees Fig. . We also40, a certain level of Wnt/β-catenin signaling is required for articular cartilage41. Optimization of administration protocols, scrutinized analysis of adverse effects, and examination of an additive effect of concomitant treatment for a rat OA model need to be performed before mianserin is applied to human OA patients. Evaluation of the suppressive effect of long-term mianserin in depressed patients on OA development in epidemiological studies will also help elucidate the effect of mianserin on OA patients. In addition to OA, dysregulation of Rspo2 is also reported in several cancers42 and in proliferative diseases of bone and soft tissue43. We expect that mianserin is a potential repositioned drug that ameliorates human diseases associated with Rspo2-mediated abnormal activation of Wnt/β-catenin signaling.Thus, intraarticular injection of mianserin suppresses Wnt/β-catenin signaling in articular chondrocytes and ameliorates OA progression in a rat OA model Fig. . As the 44. Two authors independently evaluated plain knee radiographs and severity of knee OA was decided using the Kellgren-Lawrence (KL) radiographic grading system 29. Each KL grade is characterized as follows: grade 1, doubtful OA with minimal osteophytes of dubious importance; grade 2, mild OA with definite osteophytes but unimpaired joint space; grade 3, moderate OA with moderate osteophytes and definite joint space narrowing; and grade 4, severe OA with considerably impaired joint space and sclerosis of subchondral bone45.All studies involving human synovial fluid were performed in accordance with the protocol in Institutional Review Board of Nagoya University Hospital (The protocol No. 2015-0239-2) and approved by the Ethical Review Committee of the Nagoya University Graduate School of Medicine. After an appropriate written informed consent was obtained at Nagoya University Hospital, synovial fluid samples were acquired from 52 patients with knee OA  with needle puncture at the outpatient clinic when the patients had hydrarthrosis. The diagnosis of knee OA was based on the criteria of the American Rheumatism Associationg for 20 min at 4 °C to remove cells and other debris, concentrations of Rspo2 and total protein in the collected synovial fluid samples were quantified. Concentrations of Rspo2 were determined by the Human R-Spondin 2 (RSPO2) ELISA Kit (MyBioSource) according to the manufacturer’s instructions and using PowerScan4  to measure absorbance at 450 nm. Concentrations of total protein were quantified by the Pierce 660 nm Protein Assay Kit (Pierce Biotechnology). We also evaluated concentrations of glycosaminoglycans (GAG) in synovial fluid by a spectrophotometric dye binding assay using 1,2, dimethylmethylene blue 46. A positive correlation between GAG concentration in synovial fluid and KL grade was demonstrated in a previous report28.All patients had knee effusion caused by synovitis and underwent an arthrocentesis of the knee under aseptic condition. After centrifugation at 18,000 × 47. HCS-2/8 cells were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum . After 60–70% cellular confluency in a 100-mm dish, HCS-2/8 cells were transfected with 10 µg TOPFlash firefly luciferase reporter vector  and 0.25 µg Renilla luciferase plasmid  using FuGENE 6 (Promega). At 24 h after transfection, the cells were seeded in a 96-well culture plate at 4 × 104 cells/well and incubated for 24 h in the presence of 10 μM of each drug. Recombinant human Rspo2 protein  and/or recombinant human Wnt3a protein  were added to the medium. Luciferase activity was measured with the Dual Luciferase Reporter Assay System (Promega) in PowerScan MX . Firefly luciferase activity was normalized by Renilla luciferase activity.We screened 1,271 FDA-approved chemical compounds  to identify a drug that specifically suppresses Rspo2-mediated activation of Wnt/β-catenin signaling. We quantified Wnt/β-catenin signaling activity with TOPFlash luciferase reporter assay. Human chondrosarcoma (HCS-2/8) cells were kindly provided by Dr. Masaharu Takigawa at Okayama UniversityDifferentiated ATDC5 cells and OAC cells were treated with 0–20 μM mianserin in the presence of 200 ng/ml rhRspo2 for the indicated times. Cells were harvested with ice-cold RIPA Lysis Buffer (Santa Cruz Biotechnology) with 0.1 mM dithiothreitol, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin and phosphatase inhibitors . Whole-cell lysates were separated on SDS-PAGE and transferred to a nitrocellulose membrane. Expression levels of β-catenin, β-actin, Lrp6, phosphorylated Lrp6, Lrp5, Frizzled 6 and Gapdh were determined using antibodies against β-catenin , β-actin , Lrp6 , phospho-Lrp6 at Ser1490 , Lrp5 , Frizzled 6 , and Gapdh , respectively. Signal intensities were normalized either to the β-actin or the total Lrp6 level. Three independent experiments were performed, and quantified using the NIH ImageJ software.RNF43 cDNA in pcDNA3-HA was kindly provided by Drs. Shigetsugu Hatakeyama and Tadasuke Tsukiyama at Hokkaido University, Japan48. We previously reported the full-length human LGR5 (BC099650) cDNA in pcDNA3.149. The C-terminal-deleted human RSPO2 cDNA (amino acids 1–218) and the C-terminal of rat Agrn cDNA (amino acids 1141–1937 of M64780.1) were cloned into APtag-5 (GenHunter Corp.) at the HindIII and SnaBI sites to make the myc-and-alkaline phosphatase-tagged Rspo2 (Rspo2-mycAP) and the myc-and-alkaline phosphatase-tagged Agrn (Agrn-mycAP) as described previously49.The full-length human 3, and 20 mM HEPES (pH 7.0) in Hank’s balanced salt solution), and then fixed in 60% acetone for 10 min on ice followed by 4% paraformaldehyde in 20 mM HEPES (pH 7.0) in Hank’s balanced salt solution for 10 min on ice. Fixed cells were washed once with 20 mM HEPES (pH 7.0) and 150 mM NaCl, incubated at 65 °C for 30 min, washed once with 0.1 M Tris-HCl (pH 8.0), and lysed with 0.1% Triton-X in Tris-HCl (pH 8.0). AP activity in the lysed solutions was measured using LabAssay ALP  to quantify bound Rspo2-mycAP or Agrn-mycAP. Agrn-mycAP was used to confirm that it did not bind to the cell surface of RNF43-transfected or Lgr5-transfected HEK293 cells (data not shown).HEK293 cells were transfected with Rspo2-mycAP to make Rspo2-CM. Rspo2-mycAP and Agrn-mycAP in the CM were concentrated ~100-fold using Amicon Ultra-4 filters (Merck Millipore) for the cell surface binding assay. HEK293 cells were transfected with 3 μg RNF43/pcDNA3-HA, 5 μg Lgr5/pcDNA3.1, or 5 µg empty pcDNA3.1 using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were incubated for 24 h with concentrated CM containing either Rspo2-mycAP or Agrn-mycAP then placed on ice for 1.5 h. Cells were washed with HABH buffer , and 50 µl PBS (control group) and 50 μM mianserin in 50 µl PBS (mianserin group) was intraarticularly injected into both knees once weekly, respectively. At 8 weeks after surgery, the rats were sacrificed and their knee joints were fixed overnight in 4% paraformaldehyde at 4 °C, decalcified, and embedded in paraffin. We made multiple sections around the middle portion of the medial femoral condyle (i.e. the weight-baring area) of each sample. A single section crossing the narrowest interarticular portion was stained with Safranin O and Fast-green. OA progressions were graded according to the modified Mankin histologic score51 on both tibial and femoral sides of articular cartilage.All animal experiments were performed in accordance with the recommendations in the Regulations on Animal Experiments in Nagoya University and approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine (The protocol No. 2017–29469). Ten-week-old male Wistar/ST rats were anesthetized with isoflurane and their right knee joints were induced to OA by surgical destabilization of the medial meniscus (DMM)The modified Mankin histological score is the sum of seven parameters. These include the articular cartilage structure (grades 0–11); tidemark duplication (grades 0–3); Safranin O staining (grades 0–8); fibrocartilage (grades 0–2); chondrocyte clones in uncalcified cartilage (grades 0–2); hypertrophic chondrocytes in calcified cartilage (grades 0–2); and subchondral bone (grades 0–2). Using a single well-cut and well-stained sagittal section crossing the narrowest interarticular portion in the medial compartment of the knee joint, the grades of OA were scored by a single blinded observer and averaged in each group of mice.2O2 for 15 min to inactivate endogenous peroxidases. They were then treated with a blocking buffer containing 5% goat serum in TBS-T for 1 h at room temperature and incubated with anti-β-catenin antibody  at 4 °C overnight. The sections were then washed with TBS-T and incubated with Alexa 488-conjugated goat anti-rabbit IgG  at room temperature for 1 h. The specimens were then mounted in VectaShield containing 2 µg/ml diamidino-2-phenylindole (DAPI)  and visualized with an A1Rsi microscope (Nikon Corp.). The average signal intensities of β-catenin in cytoplasm and nucleus of articular chondrocytes were blindly quantified with MetaMorph software . When the intensity of β-catenin in the nucleus was similar to or more than that in the cytoplasm, the cell was recognized as nuclear β-catenin-positive.For immunofluorescence staining, the paraffin-embedded sections were deparaffinized and rehydrated. They were unmasked with 10 mM sodium citrate buffer (pH 6.0) at 90 °C for 15 min and incubated in 3% Ht-test or one-way ANOVA followed by Tukey’s post-hoc test. P-values less than 0.05 was considered statistically significant. All statistical analyses were performed with SPSS ver. 23 (IBM Corp.).All continuous variables are expressed as mean ± standard deviation (SD). Statistical significance was estimated either by Student’s Details of other materials and methods are available in the Supplementary Information. This section includes detailed descriptions of total RNA extraction, real-time quantitative polymerase chain reaction (qPCR), Alcian blue staining, MTS assay, and isolation of human osteoarthritic chondrocyte (OAC) cells.Supplementary information


We present two cases of low-grade non-ITAC with squamoid morules coexpressing CDX2 and nuclear β-catenin by immunohistochemistry, both of which were found to harbor a mutation in CTNNB1, the gene encoding β-catenin. This finding provides support that an alteration in the β-catenin pathway, including mutations in the β-catenin gene itself, is responsible for this recently described morphologic phenomenon in non-ITAC.Sinonasal non-intestinal-type adenocarcinoma (non-ITAC) is a rare, morphologically diverse neoplasm of the head and neck. Squamoid morular metaplasia has recently been reported as an occasional finding in non-ITAC. Interestingly, these squamoid morules often show aberrant expression of CDX2 as well as nuclear expression of  We present two cases of non-ITAC with squamoid morules coexpressing CDX2 and nuclear β-catenin, which were found to have CTNNB1 mutations detected by next-generation sequencing (NGS).The sinonasal tract is affected by a diverse spectrum of neoplasms, which may be epithelial, mesenchymal, or neuroectodermal in origin . Adenocataplasia . AlthougAn 80-year-old man with a past medical history of diabetes mellitus presented to his primary care physician after several weeks of left-sided congestion and epistaxis that he had attributed to seasonal allergies. He denied significant weight loss, headaches, visual changes or weakness. A computed tomography (CT) scan of the sinuses showed abnormal soft tissue causing contiguous opacification of the left nasal cavity, frontal, maxillary and ethmoid sinuses .After a biopsy confirmed carcinoma, the patient was referred for further surgical management. A repeat CT confirmed a 1.5 cm polypoid opacity in the superior left nasal cavity with likely involvement of the anterior cranial base. The patient then underwent an endoscopic craniofacial resection of the sinonasal mass.β-catenin likewise showed nuclear staining restricted to squamoid morules with membranous staining throughout the rest of the tumor . The analysis identified a missense mutation (S37C) inThe patient received postoperative radiotherapy and has no evidence of disease at 10 months after surgery.A 25-year-old Asian female with a past medical history of myotonic muscular dystrophy presented to an otolaryngologist with complaints of otalgia. She reported intermittent right ear pain of mild severity, as well as sneezing, itchy nose, and watery eyes for one month. Nasal endoscopy revealed a lobulated, fleshy mass just medial to the right middle turbinate. A CT scan of the sinuses revealed a 3.0 cm mass in the right nasal cavity extending to the roof of the nasal cavity, without an obvious cranial base defect .β-catenin and CDX2 were strongly expressed in the areas of squamoid morular metaplasia  inThe patient subsequently underwent endonasal craniofacial resection of the tumor to negative margins. Given the low-grade histology and the pathologic stage of T1N0M0, the patient did not require adjuvant chemotherapy or radiation and will undergo close clinical surveillance.Sinonasal non-ITAC is a rare yet phenotypically diverse neoplasm of the head and neck which occurs over a wide age range . Low-graDiffuse and weak positivity for CDX2 has previously been noted in several other types of sinonasal neoplasms. Tilson et al.  found thβ-catenin and CDX2. β-catenin is part of the cadherin protein complex, which plays a role in cell-cell adhesion, and it is normally degraded through the ubiquitin-proteosome mechanism [β-catenin gene CTNNB1 is mutated or another process results in upregulation of the Wnt pathway, the degradation of β-catenin is reduced and it accumulates in the cytoplasm and nucleus and acts a transcription factor [β-catenin in morular metaplasia has also been noted in other neoplasms of various organs, including endometrioid ovarian carcinoma [β-catenin may play a role in squamoid morule formation at the molecular level.Purgina et al.  also notechanism . When thn factor . Interesarcinoma , endometarcinoma , and pularcinoma . This su CTNNB1 mutations in tumors demonstrating squamoid morules strongly positive for nuclear and cytoplasmic β-catenin immunohistochemical expression [β-catenin gene. As described by Sekine et al., these mutations are “considered to result in β-catenin stabilization and constitutive activation of Tcf/Lef-dependent transcription.” Additionally, Saegusa and Okayasu [β-catenin gene, involving codons 32, 33, 34, 37, 41, and 45, in 22 of 26 (84.6%) cases of endometrioid endometrial cancer with squamous morules. However, a genetic alteration in the β-catenin pathway to explain the morular formation in sinonasal non-ITAC, to our knowledge, has yet to be reported.Previous studies have detectedpression . Sekine pression  reported Okayasu  reported CTNNB1 are characteristic of sinonasal glomangiopericytoma, a distinctive mesenchymal neoplasm with a myoid phenotype [ KRAS, APC, CTNNB1, DNA mismatch repair genes, or TP53. Another study by Franchi et al. [ CTNNB1 and a unique morphology and immunophenotype.Molecular analysis has revealed defining mutations in other tumors occurring in the sinonasal tract. Similar gain-of-function mutations inhenotype . Additiohenotype , 13. Whihenotype . With cohenotype  noted a i et al.  reported CTNNB1 mutations may be responsive to mTOR inhibitors [Clearly, non-ITAC is diverse not only morphologically but at the molecular level. With an advanced understanding in the molecular basis of non-ITAC comes the potential improvement in treatment options for patients. Several clinical trials are currently underway and preclinical evidence suggests that tumors with activatinghibitors  and inhihibitors . With coβ-catenin that suggests a possible synergistic effect in squamoid morular formation. We report two cases with a confirmed mutation in β-catenin which can provide a molecular explanation for this recently described morphological phenomenon in non-ITAC.Squamoid morules are an interesting morphologic feature and can be present in non-ITAC, and they show a peculiar coexpression of CDX2 and


To investigate the correlation between the level of serum β-catenin with tumor marker carcinoembryonic antigen (CEA) and CRC clinicopathological parameters and the receiver operating characteristic (ROC) curve were analyzed.  A total of 327 clinical serum samples and their electronic medical records were collected. Detecting by enzyme-linked immunosorbent assay (ELISA), the correlations of serum β-catenin levels in the CRP and CRC patients were significantly higher than those in the healthy control (HC) group (P < 0.05 and P < 0.001). Compared with CRP, serum β-catenin level in CRC was also increased (P < 0.05). However, there was no significant difference in gender, age, location, tumor size, Dukes staging, or metastasis (P > 0.05) between serum β-catenin and clinical parameters of CRC. There was no correlation between serum β-catenin levels and CEA in CRC patients (P = 0.14). ROC curve analysis showed that serum β-catenin possessed the maximum diagnostic efficiency in CRP  with 86.41% sensitivity and 51.56% specificity. β-Catenin combined with CEA had the highest diagnostic efficiency  with 81.88% sensitivity and 73.44% specificity. With CRC patients from CRP patients, ROC analysis of the combining detection  had the 70% sensitivity and 84.5% specificity.  Serum β-catenin levels are gradually increased in CRP and CRC, while there is no correlation between its levels and CRC disease process. Single serum β-catenin or combined CEA would be one of the potential candidate biomarkers for colorectal disease diagnosis. The serum  Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system . GloballWnt)/β-catenin signaling pathway is a conserved pathway mediating the tumorigenesis and embryogenesis [Wnt signaling regulates signal transduction by controlling the levels of β-catenin through phosphorylation and proteasomal degradation [β-catenin destruction complexes  and then the accumulation of β-catenin in the cytoplasm and nucleus [β-catenin in CRC cells, which initiates the transcription of many target genes that related to proliferation and invasion and then leads to tumorigenesis [Wingless-related integration site  were recruited from the Second affiliated hospital of Chongqing Medical University from January 2015 to March 2018. The samples were divided into three groups, CRC group (n = 160), CRP group (n = 103), and normal healthy subjects with no history of CRC or CRP disease group (n = 64). CRC group included 86 males and 74 females: 95 patients below the age of 60 years and 65 patients beyond 60 years old. According to the tumor size, 78 cases of the tumors were less than 3 cm and 82 cases were over 3 cm. According to tumor location, 83 cases were with colon tumors and 77 cases were with rectal tumors. According to the Dukes staging system, colorectal tumors were divided into stages A, B, C, and D. 81 cases were in A+B stage, and 79 cases were in C+D stage. There were 95 cases of nonmetastasis and 65 cases of lymphatic metastasis in CRC patients   according to the manufacturer's instructions. The CEA values were measured by electrochemiluminescence immunoassay “ECLIA”.The serum 2O2) in methanol for 15 min to block endogenous peroxidase. The sections were then incubated with the anti-β-catenin polyclonal antibody  overnight at 4°C, following incubation with secondary antibody tagged with the peroxidase enzyme  for 30 min at room temperature, and were visualized with 0.05% 3,3-diamino-benzidine tetrachloride (DAB) till the desired brown reaction product was obtained. The sections were finally counterstained with hematoxylin. Control sections were performed using phosphate-buffered solution (PBS) without a primary antibody. All the slides were observed under a Nikon E400 Light microscope, and representative images were taken.This experiment was detected according to Wu et al.'s method , brieflyβ-catenin and CEA level in CRP and CRC patients and HC was analyzed by the Kruskal-Wallis test. The relationship between serum β-catenin and clinical pathological parameters of CRC was analyzed by the Mann–Whitney test. The ROC curve analysis was conducted to evaluate the diagnostic efficacy of serum β-catenin, CEA, and “β-catenin+CEA” in samples. The correlation between serum β-catenin and CEA level was estimated by Spearman's rank correlation coefficient. The statistical differences are presented at probability levels of P < 0.05 (∗), P < 0.01 (∗∗), and P < 0.001 (∗∗∗), indicating that the difference was statistically significant.All the data were processed with SPASS 20.0 software. The difference between serum β-catenin in sections of eight pairs of samples  and five CRP samples. Expression levels of β-catenin were significantly higher from either CRC or CRP tissues than from HCs. Of note, its expression was higher in CRC tissues than that in CRP tissues  and CRC group  was higher than that in the HC group . The difference was statistically significant  was higher than that of the CRP group  . The serum β-catenin level in the group younger than 60 was 30.22 ± 18.51 pg/mL and in the group older than 60 was 34.35 ± 22.57 pg/mL. This difference was not statistically significant (P = 0.34). The serum β-catenin level was 31.45 ± 19.9 pg/mL in the colon tumor group and 32.86 ± 21.25 pg/mL in the rectal tumor group. The difference was little statistically significant (P = 0.76). Also, we analyzed the relationship with essential parameters for the process of CRC disease, like tumor size, Dukes stages, and lymph node metastasis. The serum β-catenin level is 31.92 ± 21.7 pg/mL in the tumor group (size < 3 cm), and 32.33 ± 19.47 pg/mL in the tumor group (size ≥ 3 cm), with no significant difference (P = 0.57). We also found that the serum β-catenin level in Dukes stages (A+B) is 32.67 ± 20.36 pg/mL, and 31.58 ± 20.82 pg/mL in Dukes stages (C+D), with no significant difference (P = 0.7). The serum β-catenin level in the nontransfer (Absent) group is 31.79 ± 20.23 pg/mL, and 32.23 ± 21 pg/mL in the transfer (Present) group, with no significant difference (P = 0.86). These results were shown in Given the significant increase in the level of serum β-catenin? We used the electrochemiluminescence immunoassay “ECLIA” technique to detect the CEA level in the collected samples. We found that the CEA level in the CRC group (14.27 ± 26μg/L) is significantly higher than that in the CRP group (7.13 ± 7.52μg/L) and the HC group (4.34 ± 4.98μg/L)  by Spearman's rank correlation analysis ; cell-free DNA (cfDNA), which includes circulating tumor DNA (ctDNA); circulating cell-free RNA (cfRNA), which includes not only some small RNAs but also mRNAs; circulating extracellular vesicles (EVs); proteins; and metabolites . ctDNA cβ-Catenin is a crucial component of the Wingless-related integration site (Wnt) signaling pathway. High expression of β-catenin is considered a sign of aberrant β-catenin signaling pathway activation and is thought to promote tumor progression [Wnt signaling pathway were Wnt proteins, which bind to the frizzled (Fz) receptors and coreceptor LRP on the cell membrane [Wnt ligands caused the phosphorylation of β-catenin. The phosphorylated β-catenin fails to bind and disperse the components of the degraded complex. Thus, the β-catenin cannot be degraded, resulting in the accumulation of β-catenin in cells [Wnt signaling pathway to inhibit cell growth and tumor growth. Studies on β-catenin mainly involved in the accumulation in the cell and the transcription of the target gene into the nucleus, leading to tumorigenesis [β-catenin can be detected in serum and might have a certain relationship with the course of hepatitis C, hepatitis B, type 2 diabetes, PTEN hamartoma tumor syndrome, and early-onset ulcerative colitis [β-catenin might be used as a marker for serum disease-associated markers. Many studies focused on the initiation of target genes in the cell by β-catenin into the nucleus, but neglected that it may itself be a cytokine that can be secreted outside the extracellular. As a multifunctional protein molecule, β-catenin can also be combined with E-cadherin to mediate cell adhesion. Under normal circumstances, β-catenin is abundantly located on the cell membrane, while in the stages of tumor disease, EMT phenomenon is often accompanied by a decrease in E-cadherin [β-catenin to bind and release into the extracellular space. However, this hypothesis needs to be further confirmed.gression , especiagression . The stamembrane , 34. Thein cells , 35. Thiigenesis , 36–38.  colitis –23. Thescadherin . Thus, iβ-catenin level in CRP and CRC patients was significantly higher than that in healthy controls. The serum β-catenin level of the CRC patients was also significantly increased compared with that of the CRP group. These results indicated that the serum β-catenin level tends to increase gradually in the process of colorectal polyps or colorectal cancer. To explore the relationship between serum β-catenin and CRC disease progression, we grouped these cases according to the factors, e.g., gender, age, tumor size, tumor location, metastasis, and Dukes staging. We found that the level of serum β-catenin in CRC patients was not associated with these pathological parameters. Therefore, when the serum β-catenin level of CRC patients is elevated, we can only reasonably suspect the possibility of early development for CRC, but could not estimate the stage of the disease.This study showed that the serum β-catenin level and the CEA level. The Spearman rank correlation coefficient analysis showed no correlation between the two factors. This phenomenon might be due to the limitations of CEA, which had lower specificity and sensitivity. It is difficult to capture the changes in the early stages of the tumor, and β-catenin may change at an earlier stage.Furthermore, carcinoembryonic antigen (CEA) is a common cancer biomarker. It is a high molecular weight glycoprotein found in embryonic tissues and colorectal malignancies. High CEA levels are associated with tumorigenesis. Many tumorigeneses in human were found in the increase of serum CEA level, and it is better for efficacy judgment, disease development, detection, and pretreatment for colorectal cancer, breast cancer, and lung cancer –43. Inveβ-catenin in the process of CRP to CRC disease. The results showed that serum β-catenin had the highest diagnostic efficacy in the diagnosis of CRP (AUC = 0.74). In the evaluation of CRC, β-catenin combined with CEA had the highest diagnostic efficacy (AUC = 0.88). Also, we evaluated the detection efficiency of CRC in CRP, which determined by β-catenin combined with CEA. The AUC was 0.70. These results demonstrated that serum β-catenin level could detect changes in early colorectal disease and combined with CEA can improve the diagnostic accuracy of CRC.Finally, we used the ROC curves to assess the diagnostic value of serum β-catenin. It has little correlation with the disease progression of CRC. While combining the value of CEA, it may improve the accuracy of the early diagnosis of colorectal diseases. The diagnostic performance of CRC is fast and straightforward. Therefore, these studies not only have a role in screening and early detection of disease but are also important for prognosis and to predict response to therapy in CRC, reducing its morbidity and mortality.In sum, CRP and CRC have a significant increase in the level of serum


Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/β-TrCP SCF E3 ligase complex. Here, we show that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/β-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in  The maintenance of epithelial tissue architecture through cell-cell and cell-extracellular matrix contacts, as well as cell polarity, is essential for organ function and size control . The evoex is a Yki target gene and, therefore, it is a prime candidate to mediate the Hpo pathway feedback regulation that controls tissue homeostasis  family of protein kinases as regulators of Ex stability that function downstream of the polarity protein Crb. Depletion of CKI kinases suppresses Crb-induced Ex phosphorylation, ubiquitylation and degradation. Interestingly, CKI kinases regulate Ex in a partially redundant manner, which suggests that regulation of Ex stability is a key step in the regulation of Yki function and the maintenance of tissue homeostasis.Drosophila β-TrCP) interaction is mediated by a β-TrCP consensus sequence immediately following the Ex N-terminal FERM domain (452TSGIVS457). In agreement with the fact that β-TrCP targets substrates for ubiquitylation and degradation through the recognition of a phosphodegron, Ex is phosphorylated in Drosophila S2 cells in the presence of ectopic Crb   (1-468 AP-MS only upon co-expression with wild-type (wt) Crb but not with a FERM-binding motif mutant version of Crb (ΔFBM) that cannot bind Ex or promote its depletion (We have previously shown that Crb regulates Ex protein stability in a β-TrCP-dependent manner . The Ex:rbintra) . Howeverrbintra) . Upon reEx1-468)  (RibeiroEx1-468) . Importaepletion   locus produces multiple protein isoforms, one of which, GishisoF, lacks a conserved C-terminal palmitoylation motif (isoI) or lack (GishisoF) this palmitoylation sequence. Interestingly, only GishisoI, but not GishisoF, was able to interact with Ex  analyses in S2 cell lysates. In reciprocal co-IP experiments, an interaction between Gish and Exf wt Crb . The Casf wt Crb , with hif wt Crb , which af wt Crb . The Droon motif , which won motif . Therefo with Ex  suggesti1-468 and GishisoI in the absence of Crb resulted in a mild depletion of Ex1-468 protein levels . Importantly, this variant remains responsive to Crb  (Drosophila CkIδ/ε orthologue), had a similar effect on Ex and whether this effect was dependent on their kinase activity. Indeed, co-expression of Ex1-468 CAAX or full-length (FL) Ex with wt GishisoI, CkIα or Dco led to Ex mobility shift and depletion, although their kinase-deficient (KD) versions did not  . Next, w did not . TogetheisoI in S2 cells with either wt Ex1-468 CAAX or a variant carrying a mutation at S453 (Ex1-468 S453A CAAX), one of the crucial residues that mediates the interaction between Ex and Slmb  was refractory to degradation, and did not display a mobility shift following GishisoI expression  was involved in the regulation of Ex phosphorylation  . Thus, wrylation . Howeverrylation . Moreoveradation . TogetheDrosophila tissues. However, assessing Ex protein stability in vivo is complicated by the fact that ex is a Yki target gene . We also generated a mutant version that is refractory to Crb-induced degradation by mutating Ser 453, which mediates binding to Slmb , ubi-Ex1-468::GFP was lost from the apical surface and degraded  or all CKIs . Similar to wt Crb, Crb4A mut efficiently promoted Ex degradation  or dco (le88dco) alleles, we observed no differences from control cells, suggesting that these kinases are not essential for maintaining Ex levels at steady state in vivo , as well as a mutant for the poorly characterised CKI family member CG7094, which is also expressed in imaginal discs  using tstub-Gal80   . In contapically . TogetheckIα loss  inhibited Ex degradation (452TSGIVS457), which is phosphorylated to allow the interaction to occur (1-450) fails to undergo a mobility shift in the presence of Crbintra or Gish  depletion has a strong effect on Ex stability , while Cocalised . This isocalised . Howeverocalised . It is tocalised .ckIα depletion and increased by CkIα overexpression, while Gish manipulations had no such effect   . Thus, ickIα or slmb function results in an increase in the apical levels of Crb as well as Ex , and FRMD6, in a density-dependent manner . Cells were obtained from the ATCC and mycoplasma testing revealed no contaminations. Drosophila S2 cells were grown in Drosophila Schneider’s medium (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 50 μg/mL penicillin and 50 μg/mL streptomycin. Expression plasmids were transfected using Effectene transfection reagent (QIAGEN). Expression plasmids were generated using Gateway technology (Thermo Fisher Scientific). ORFs were PCR amplified from cDNA clones obtained from the Drosophila Genomics Resource Center  and cloned into Entry vectors . Vectors from the Drosophila Gateway Vector Collection and an in-house V5 tag expression vector were used as destination vectors (1-468 and Crbintra plasmids were previously described (10P12E16 to A10A12A16) (Drosophila S2 cells), HA-ubiquitin and Crb4A mut plasmids were kind gifts from M. Ditzel , P. Meier  and B. Thompson , respectively. mScarlet  and pCFD3  plasmids were obtained from Addgene. Where indicated, proteasome inhibition was achieved by treating cells with 50 μM MG132 (Cambridge Bioscience) and 50 μM calpain inhibitor I (Ac-LLnL-CHO or LLnL) (Sigma) for 4 hr before cell lysis or with 5 μM MG132 overnight.Work involved the use of the  vectors . All Entescribed . The WT 0A12A16) . The NTAhttp://www.dkfz.de/signaling2/e-rnai/). The following primers were used: lacZ (Fwd –TTGCCGGGAAGCTAGAGTAA and Rev – GCCTTCCTGTTTTTGCTCAC); gish (Fwd – TGGCCAAAGAATACATTGATTTAGA and Rev – GGCAGTGAACCCCTTAAGAAATAC); ckIα (Fwd – GGAGTGCATCAGAGAAGGAGAAC and Rev – GTGGGTGTGTTATGCAAGTATGTT); panckI (Fwd – GAATTAATACGACTCACTATAGGGAGAGGCCATCAAGATGGAGAGC and Rev – GAATTAATAC GACTCACTATAGGGAGACATGTAATCTGGCTGCTCC) (dco (Fwd – ACGCAGGCATTTAATTCTGTTT and Rev – GGTGTCCTTTGTTTCTTTTACACA); slmb (Fwd – TGTACTGTAGGCAGGCGATG and Rev – AGGTGATCATCAGTGGCTCC); 24sgg (Fwd – AGCTCTCAATACAGCCCAGC and Rev – CGGTTCCTGCTGTTGCTC); 25sgg (Fwd – CCGCAATTTCAAAAGAACTC and Rev – AAAATGACAATCGATCAGCG). After cell seeding, S2 cells were incubated with 15–20 μg dsRNA for 1 hr in serum-free medium, before complete medium was added. 72 hr after dsRNA treatment, cells were lysed and processed as detailed below.RNAi production and treatment dsRNAs were synthesised using the Megascript T7 kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. DNA templates for dsRNA synthesis were PCR amplified from genomic DNA or plasmids encoding the respective genes using primers containing the 5’ T7 RNA polymerase-binding site sequence. dsRNA primers were designed using the DKFZ RNAi design tool (CTGCTCC) ; dco . Western blots were probed with mouse anti-FLAG , mouse anti-Myc , rat anti-HA , mouse anti-V5 , mouse anti-Crb , or mouse anti-tubulin . For densitometry analysis of immunoblots, X-ray blots were scanned using an Epson Perfection V700 flatbed scanner and further analysed with the Gel Analyzer function on ImageJ (RRID:SCR_003070). Relative Ex protein levels  were represented as a ratio to the average of the Ex levels in the CrbΔFBM negative control samples.For purification of FLAG-tagged proteins, cells were lysed in lysis buffer  glycerol, and 1 mM EDTA), to which 0.1M NaF, phosphatase inhibitors 2 and 3 (Sigma) and protease inhibitor cocktail  were added. Cell extracts were spun at 17,000 g for 10 min at 4°C. FLAG-tagged proteins were purified using anti-FLAG M2 Affinity agarose gel (Sigma) for >1 hr at 4°C. FLAG immunoprecipitates were then washed four times with lysis buffer before elution using 150 ng/μl 3x FLAG peptide for 15–30 min at 4°C. For isolation of ubiquitylated proteins, cells were collected by centrifugation and washed with cold PBS. 10% of cell material was lysed as described above. The remaining 90% was lysed with boiling 1% SDS-PBS for 5 min. Following quick vortexing, samples were incubated for 5 min at 100°C before 5-fold dilution using 0.5% BSA-1% Triton X-100-PBS. DNA was sheared by sonication and cell extracts were cleared by centrifugation at 17,000 g for 10 min at 4°C. Samples were diluted 2-fold with 0.5% BSA-1% Triton-X-100-PBS and incubated overnight at 4°C with monoclonal anti-HA agarose beads (Sigma) using Bio-Spin Columns (Bio-Rad). Following incubation, samples were washed with 0.5% BSA-1% Triton-X-100-PBS and 1% Triton X-100-PBS before elution. HA immunoprecipitates were eluted from HA beads using 0.2 M glycine pH 2.5 for 30 min at room temperature and eluted samples were equilibrated with 1 M NHAB_2109711) was used at 1:50 or 1:100, chicken anti-GFP  was used at 1:1000, rat anti-Crb-ICD (a kind gift from F. Pichaud) was used at 1:200, rabbit anti-aPKC  was used at 1:500, and mouse anti-β-galactosidase  was used at 1:500. Anti-mouse, anti-rat and anti-rabbit Rhodamine Red-X-, FITC-, or Cy5-conjugated (Jackson ImmunoResearch) secondary antibodies were used at 1:400 or 1:500. Anti-rat Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher) was used at 1:500. Anti-rat Alexa Fluor 568-conjugated (Abcam), anti-chicken Alexa Fluor 488-conjugated (Abcam) or anti-mouse Alexa Fluor 647-conjugated (Jackson ImmunoResearch) secondary antibodies were used at 1:1000. Secondary antibodies were incubated for at least 2 hr at room temperature. After washes, tissues were mounted in Vectashield (with or without DAPI) , or stained with DAPI (1 μg/mL) for 10 min before mounting with Mowiol 40–88 (Sigma). Fluorescence images were acquired on Zeiss LSM510 Meta, Zeiss LSM710 or Zeiss LSM880 confocal laser scanning microscopes (40x objective lens).Larval tissues were processed as previously described . PrimarySCR_004870) using the full-length Drosophila CkIα sequence as query. Sequence alignments were performed using MUSCLE (RRID:SCR_011812) (SCR_014629) and DrawTree tools in the Phylogeny online tool (http://www.phylogeny.fr/simple_phylogeny.cgi) (D. melanogaster CkIα); NP_001020276.1 (H. sapiens CKIα); NP_733414 (D. melanogaster Dco); NP_001884.2 (H. sapiens CKIδ); NP_001885.1 (H. sapiens CKIε); NP_001163628 (D. melanogaster Gish isoform I); NP_001014628 (D. melanogaster Gish isoform F); NP_071331.2 (H. sapiens CKIγ isoform 1); NP_001310.3 (H. sapiens CKIγ isoform 2); NP_004375.2 (H. sapiens CKIγ isoform 3); NP_572794 (D. melanogaster CG2577); NP_609851 (D. melanogaster CG7094); NP_608697 (D. melanogaster CG9962); NP_649536 (D. melanogaster CG12147).CKI kinases were compiled with BLAST   and CKI eny.cgi) . ProteinDrosophila Resource Center  . 1-468::GFPubi-Ex (wt or S453A) transgenes were cloned using Gateway technology into a modified pKC26-pUbiq plasmid (UAS-CKI transgenes were cloned using Gateway technology into the pUASg-HA(N)-attB vector. FLUAS-Ex (wt or S453A) transgenes were cloned into the pUAST-attB plasmid (SCR_012605). 1-468UAS-Ex (wt or S453A) transgenes were cloned into the pUASg-attB plasmid . 9H4-17slmb was obtained from Daniel St Johnston . 1slmb was obtained from Daniel Kalderon (Columbia University). le88dco was obtained from Tomas Dolezal (University of South Bohemia). 8B12ckIα was obtained from Jessica Treisman . K03891gish was obtained from Bloomington (BL-13263).Transgenic RNAi stocks were obtained from the Vienna  plasmid . UAS-CKI plasmid  and gene plasmid  and tranAll crosses were raised at 25°C unless otherwise stated. Genotypes were as follows:1-468::GFP/FRT82B crb82-04hsFLP;; FRT82B ubi-RFP, ubi-Ex1-468::GFP/FRT82B slmb9H4-17hsFLP;; FRT82B ubi-RFP, ubi-Ex9H4-17, crb82-04/ubi-Ex1-468::GFP, FRT82B ubi-RFPhsFLP/+; ; FRT82B slmb1-468::GFP / +w;; hh-Gal4, ubi-Ex1-468::GFP/UAS-crbintraw;; hh-Gal4, ubi-Ex1-468 S453A::GFP / +w;; hh-Gal4, ubi-Ex1-468 S453A::GFP/UAS-crbintraw;; hh-Gal4, ubi-Ex1-468::GFP/UAS-HA::ckIαw;; hh-Gal4, ubi-Ex1-468::GFP/UAS-HA::gishisoIw;; hh-Gal4, ubi-Ex+>Gal4 / +; hh-Gal4, ubi-Ex1-468::GFP/UAS-crbintraw; Act > yRNAi / +; hh-Gal4, UAS-CD8::GFP / +w; UAS-lacZRNAi / +; hh-Gal4, ubi-Ex1-468::GFP / +w; UAS-lacZRNAi / +; hh-Gal4, ubi-Ex1-468 S453A::GFP / +w; UAS-lacZw; en-Gal4, UAS-CD8::GFP/UAS-CD8::GFP (18°C cross)intra / +w; en-Gal4, UAS-CD8::GFP / +; UAS-crb (18°C cross)WTw; en-Gal4, UAS-CD8::GFP/UAS Ex (18°C cross)WT / +; UAS-crbintra / +w; en-Gal4, UAS-CD8::GFP, UAS-Ex (18°C cross)S453Aw; en-Gal4, UAS-CD8::GFP/UAS-Ex (18°C cross)S453A / +; UAS-crbintra / +w; en-Gal4, UAS-CD8::GFP, UAS-Ex (18°C cross)1-468::GFP/FRT82B dcole88hsFLP;; FRT82B ubi-RFP, ubi-Ex1-468::GFP/FRT82B gish17hsFLP;; FRT82B ubi-RFP, ubi-Ex8B12;; ubi-Ex1-468::GFP / +hsFLP, FRT19A ubi-RFP/FRT19A ckIα1-468w;; hh-Gal4, UAS-CD8::GFP/UAS-Ex1hsFLP;; FRT82B ubi-GFP/FRT82B slmb9H4-17hsFLP;; FRT82B ubi-GFP/FRT82B slmb1-468::GFP/FRT82B gishKG03891hsFLP;; FRT82B ubi-RFP, ubi-ExF2; ubi-Ex1-468::GFP / +hsFLP; FRT40A ubi-RFP/FRT40A CG70948B12hsFLP, FRT19A ubi-RFP/FRT19A ckIα8B12;; ubi-Ex1-468::GFP / +hsFLP, FRT19A ubi-RFP/FRT19A ckIαF2; FRT82B gish17, dcole88/FRT82B ubi-RFP, ubi-Ex1-468::GFPhsFLP; FRT40A CG70948B12;; FRT82B gish17, dcole88/FRT82B ubi-RFP, ubi-Ex1-468::GFPhsFLP, FRT19A ubi-RFP/FRT19A ckIαRNAi (107574KK) / +; hh-Gal4, ubi-Ex1-468::GFP/tub-Gal80tsw; UAS-ckIαRNAi (107574KK) /+; hh-Gal4, ubi-Ex1-468::GFP/UAS crbintra, tub-Gal80tsw; UAS-ckIαRNAi (108680KK) / +; hh-Gal4, ubi-Ex1-468::GFP/tub-Gal80tsw; UAS-gishRNAi (108680KK) / +; hh-Gal4, ubi-Ex1-468::GFP/UAS crbintra, tub-Gal80tsw; UAS-gish1-468::GFP/UAS crbintra, tub-Gal80tsw;; hh-Gal4, ubi-ExRNAi (330069shRNA) / +; hh-Gal4, ubi-Ex1-468::GFP / +UAS-dcoRNAi (330069shRNA) / +; hh-Gal4, ubi-Ex1-468::GFP/UAS-crbintraUAS-dcotub-Gal4, hs-FLP, UAS-nucGFP::myc / + or intra / +; FRT82B blank/ubi-Ex1-468::mScarlet, FRT82B tub-Gal80Y; UAS-Crbtub-Gal4, hs-FLP, UAS-nucGFP::myc / + or intra / +; FRT82B gish17/ubi-Ex1-468::mScarlet, FRT82B tub-Gal80Y; UAS-Crb1-468::mScarlet, UAS-Crbintra/tubFRT19A blank/tub-Gal80, hs-FLP, FRT19A; UAS-nls-lacZ, UAS-CD8::GFP / +; ubi-Ex Gal48B12 FRT19A/tub-Gal80, hs-FLP, FRT19A; UAS-nls-lacZ, UAS-CD8::GFP / +; ubi-Ex1-468::mScarlet, UAS-Crbintra/tubckIα Gal48B12 FRT19A/hsFLP ubi-RFP FRT19A; ex697 (ex-lacZ) / +ckIα697 (ex-lacZ) / +; FRT82B gish17/FRT82B ubi-RFPhsFLP / +; ex8B12 FRT19A/hsFLP ubi-RFP FRT19A; ; diap1-GFP3.5 / +ckIα8B12 FRT19A/hsFLP ubi-RFP FRT19A; yki::GFP / +ckIαw;; hh-Gal4, UAS-CD8::GFP / +w;; hh-Gal4, UAS-CD8::GFP/UAS-ckIαisoIw;; hh-Gal4, UAS-CD8::GFP/UAS-gish1-468::GFP (wt or S453A) (SCR_002285). n ≥ 14 for all genotypes; one-way ANOVA with Dunnett’s multiple comparisons test. Quantification of aPKC levels  with Dunnett’s multiple comparisons test. For ex-lacZ intensity was calculated by manually drawing around each compartment of the pouch in maximum intensity projections, then measuring the mean grey pixel value in Fiji. Significance was calculated using a one-way ANOVA comparing all means to hh >control, with correction for multiple comparisons; n ≥ 8 for all genotypes.For quantification of Exr S453A)  or Crb (r S453A)  ratios iC levels  were perC levels . Signifigish AATGGAGCCTATGAAGTCAA; CG7094-up ATATCTCGGACTAAGCATCA; CG7094-down ACGGGGTTGTGAGCCTCAGC. gRNA expression plasmids were created by ligation of annealed oligos into pCFD3 (Addgene 49410) . Progeny of injected animals were screened for homozygous lethality (gish) or PCR screened to identify a deletion . The 17gish mutation was genotyped using primers Gish_PCR_F1 GCGAATGTGTTGCTTTGGTG and Gish_M17_mut2 GTGTAGTTGCGGAGCCTTTC .gRNA sequences were as follows: e 49410) , dilutedDrosophila wing, flies with the genotypes of interest were collected and preserved in 70% EtOH for at least 24 hr. Wings were removed in 100% isopropanol, mounted in microscope slides using Euparal (Anglian Lepidopterist Supplies) as mounting medium and baked at 65°C for at least 5 hr. Adult wing images were captured using a Pannoramic 250 Flash High Throughput Scanner (3DHISTECH) and extracted using the Pannoramic Viewer software (3DHISTECH). Wing area was quantified using ImageJ . Images were processed using Adobe Photoshop (RRID:SCR_014199).For analysis of genetic interactions in the SCR_014322) against the Drosophila melanogaster Uniprot reference proteome. Methionine oxidation was entered as a variable modification and search tolerances were 5 ppm and 0.8 Da for peptides and fragments, respectively. Individual lane searches were combined and results compiled in Scaffold 4.0.3 (RRID:SCR_014345).AP-MS experiments followed a GeLC MS/MS approach. Gel lanes were fragmented into eight equally sized pieces and subjected to in-gel trypsin digestion using a Perkin Elmer Janus Automated Workstation. Peptide mixtures were acidified to 0.1% TFA and injected onto a nanoACQUITY UPLC (Waters Corporation) coupled to an LTQ-Orbitap XL (Thermo Fisher Scientific) via an Advion Biosciences Nanomate. Peptides were eluted over a 30 min gradient (5–40% ACN). Mascot distiller was used to extract peak lists, which were searched with Mascot v.2.4.1  require clear conclusions.The reviewers have discussed the reviews with one another and the Reviewing Editor. They have agreed that, in principle your manuscript is appropriate for Reviewer #1:eLife after revisions.The work of Fulford et al. fills the gap in knowledge of the kinase that phosphorylates Ex and targets it for degradation, downstream of Crb overexpression. The authors provide sufficient in vivo and biochemical data to support their conclusions. Overall, the data are of high quality and well controlled; I recommend publication in 1) My major concern/confusion is the lack of any discussion about the seeming contradiction that both loss of Crb and excess Crb leads to Ex degradation. How can Crb be necessary for Ex stabilization at the membrane and at the same time target it for degradation? What is it about suboptimal Crb levels that triggers Ex degradation? Are Slimb and CK1 proteins required for Ex degradation upon Crb loss as well? The authors could check this by generating double loss-of-function clones for Crb and Slimb or CK1 proteins, and assaying Ex levels.slimb and CK1α mutant clones with and without Crb overexpression.2) in vivo, at least in wing discs, CK1α seems to be the major kinase. Is this also true in other imaginal discs? They could extend their results to other discs by analyzing hedgehog-Gal4 driver , ubi-Ex1-468::GFP was lost from the apical surface and degraded . In contrast, the S453A mutant reporter accumulated all over the cell upon Crbintra expression, presumably due to the fact that it cannot be recognised by Slmb and degraded . "3) The authors state: "When a Crbintra transgene was expressed in the posterior compartment using the S453A mutant accumulates all over the cell, it does not accumulate at the apical membrane. Therefore, it seems to be still degraded at the membrane suggesting involvement of other residues. Alternatively, it is mislocalized to the basolateral membrane by ectopic Crb and cannot be degraded there.I disagree with the conclusion that ExReviewer #2:It has been demonstrated in previous papers that Crb triggers Ex turnover in a Slmb/β-TrCP-dependent manner. However, recognition by Slmb/β-TrCP requires substrate phosphorylation, which had remained an unexplored aspect of Ex regulation. In this paper, Fulford et al. provide evidence that the CKI family kinases, CkIα and Gish, act downstream of Crb to promote phosphorylation of Ex, in turn promoting Ex degradation. The paper comprises a large amount of supporting data, which is generally of very good quality and consistent with the authors' interpretations. The strong points of this manuscript are the thoroughness of the biochemistry assays although it is still unclear about the phosphorylation site(s) of CKI on Ex. Overall, this paper gave me a feeling that the authors described a quite simple biological context using a very complicated way. I would suggest the authors simplify this paper. In addition, the major weakness of this paper is that it doesn't show any biological function of CKI in regulating organ size and Yki activity, which dramatically reduced the significance of this work. My concerns listed below should be addressed in revision.1) To assess the functional significance of CKI kinases in controlling Hippo pathway activation, it will be important to examine whether CKI controls organ growth. CkIα (and/or Gish) mutant- or overexpression- MARCM clone assays would be required.ex-lacZ reporter.2) Following the above question, the authors need to examine Yki activity in CkIα (and/or Gish) mutant/overexpression background using Diap1-lacZ or 3) The authors showed that CKI phosphorylates Ex in vitro, however, it is important to identify the phosphorylation site(s) of Ex and generate the phosphor-specific antibody to examine the endogenous Ex phosphorylation. Alternatively, the phosphor-tag gel or pan-phosphor S/T antibody could be used for the phosphorylation detection.gish-mediated transcriptional regulation of endogenous Ex expression.4) All biochemistry assays were using overexpression system. They should detect endogenous Ex expression and degradation. For example, CHX treatment could be used to avoid hh> Crbintra vs. hh>Crbintra + CkIαRNAi", then compare the Yki reporter induction (e.g. Diap1-LacZ) and P-compartment size.5) Although the biochemistry assays have shown that CKI kinase Gish is required for Crb-mediated Ex turnover, the genetic epistasis between Crb and CkIα in controlling Yki activation and organ growth is still needed to be tested. I would suggest the authors perform the following experiments " Reviewer #1:[…] 1) My major concern/confusion is the lack of any discussion about the seeming contradiction that both loss of Crb and excess Crb leads to Ex degradation. How can Crb be necessary for Ex stabilization at the membrane and at the same time target it for degradation? What is it about suboptimal Crb levels that triggers Ex degradation? Are Slimb and CK1 proteins required for Ex degradation upon Crb loss as well? The authors could check this by generating double loss-of-function clones for Crb and Slimb or CK1 proteins, and assaying Ex levels.Slimb complex, as shown by our Affinity Purification/Mass Spectrometry analysis . Thus, we believe Crb is both responsible for Ex recruitment to its site of activity and also for ensuring its fast turnover through degradation in order to fine-tune Yki regulation. We have clarified this issue in the first paragraph of the Discussion.We apologise for not being sufficiently clear in our description of previous data regarding the effect of Crb on Ex protein levels. Previous work from ourselves and others had indeed shown that Crb is required to recruit Ex to the apical plasma membrane . However, as well as promoting Ex activation by membrane recruitment, Crb also brings Ex into proximity with CKI family members and the SCFex transcriptional upregulation in crb mutant clones. Indeed, work published by the Jiao lab suggests SCFSlimb can also degrade Ex in the cytoplasm . To directly test this possibility and as requested by the reviewer, we examined the Ex::GFP sensor in crb, slmb double mutant clones . We observed that, as anticipated, loss of slmb leads to Ex sensor cytoplasmic accumulation in crb mutant clones, confirming the finding from the Jiao lab that Ex can be degraded in the cytoplasm and supporting the idea that transcriptional elevation of ex plays a role in cytoplasmic accumulation of Ex protein in Crb clones. Thus, although Crb promotes Ex degradation by SCFSlimb at the plasma membrane, there is also a Crb-independent Slimb-dependent cytoplasmic Ex degradation pathway, though the relative turnover rate of these mechanisms is not clear. Since Ex activity in growth control is thought to occur at the apical plasma membrane , it is likely that the Crb-dependent mechanism is key in determining Ex functional levels. We discuss this new result in the first paragraph of the Discussion.The reviewer raises in interesting point in enquiring about the fate of Ex in the cytoplasm upon loss of Crb. Previously published data indicated that endogenous Ex accumulates in the cytoplasm upon Crb loss , which had been interpreted as meaning that Ex is mainly degraded at the apical plasma membrane, and failure of apical recruitment leads to stabilisation and cytoplasmic accumulation. However, our Ex::GFP reporter does not accumulate in the cytoplasm in Crb clones , suggesting a different possibility: that endogenous Ex cytoplasmic accumulation is primarily due to 2) in vivo, at least in wing discs, CK1α seems to be the major kinase. Is this also true in other imaginal discs? They could extend their results to other discs by analyzing slimb and CK1α mutant clones with and without Crb overexpression.ckIα and slmb clones, suggesting a similar mechanism operates as in the wing disc. Interestingly, we did not see a clear increase in the eye disc. However, ckIα and slmb loss in these tissues elicited a very strong clone extrusion phenotype, and a complete loss of ommatidial differentiation, making the analysis difficult, especially since the most prominent Ex localisation in the eye disc is at the apical side of the emerging ommatidial clusters. These new data are presented in Figure 5—figure supplement 2.As requested by the reviewer, we examined the levels of the Ex::GFP reporter in haltere, leg and eye imaginal discs. In the haltere and leg disc, the levels of the Ex reporter were elevated in both 1-468::GFP was lost from the apical surface and degraded . In contrast, the S453A mutant reporter accumulated all over the cell upon Crbintra expression, presumably due to the fact that it cannot be recognised by Slmb and degraded . "3) The authors state: "When a Crbintra transgene was expressed in the posterior compartment using the hedgehog-Gal4 driver , ubi-ExI disagree with the conclusion that Ex-S453A mutant accumulates all over the cell, it does not accumulate at the apical membrane. Therefore, it seems to be still degraded at the membrane suggesting involvement of other residues. Alternatively, it is mislocalized to the basolateral membrane by ectopic Crb and cannot be degraded there.S453 reporter is mislocalised to the basolateral membrane by ectopic Crb and is refractory to degradation there. We have changed the manuscript text to reflect this possibility. “It is therefore possible that Ex1-468 S453A is mislocalised to the basolateral membrane by ectopic Crb, where it cannot be degraded”.We agree with the reviewer that it is possible that the ExReviewer #2:[…] Overall, this paper gave me a feeling that the authors described a quite simple biological context using a very complicated way. I would suggest the authors simplify this paper. In addition, the major weakness of this paper is that it doesn't show any biological function of CKI in regulating organ size and Yki activity, which dramatically reduced the significance of this work. My concerns listed below should be addressed in revision.ex make these studies challenging to perform and to explain in a simple manner. Nevertheless, based on the reviewer’s comments, we have endeavoured to highlight the relevance of the biological phenomenon studied and to clarify our arguments in the manuscript accordingly. We believe that our revised manuscript and new data significantly improve the quality and strength of our findings.We thank the reviewer for the insightful comments and suggestions, which have helped us improve the manuscript. We believe that whilst it is tempting to consider that Expanded regulation by Crb is a simple biological process, the reality is that the existence of multiple layers of regulation and the fact that there is Yki-dependent feedback regulation of transcriptional levels of 1) To assess the functional significance of CKI kinases in controlling Hippo pathway activation, it will be important to examine whether CKI controls organ growth. CkIα (and/or Gish) mutant- or overexpression- MARCM clone assays would be required.2) Following the above question, the authors need to examine Yki activity in CkIα (and/or Gish) mutant/overexpression background using Diap1-lacZ or ex-lacZ reporter.ckIα mutant clones with Yki activity. To address this, we examined two transcriptional readouts of Yki activity: ex-lacZ, an enhancer trap in the ex locus , and diap1-GFP3.5, a fragment of the diap1 promoter driving a GFP reporter , both of which are widely used in the field. Results of this analysis are shown in the new Figure 7. In accordance with our model, while gish mutant clones had wild type levels of ex-lacZ , both diap1-GFP3.5 and ex-lacZ levels were markedly reduced in ckIα clones . In addition, we used a Yki-GFP knock-in fly line  to show that Yki is excluded from the nucleus in ckIα mutant cells compared with control . Thus, decreased Yki activity is correlated with increased Ex stability upon CkIα disruption, while Gish loss has no effect. We then examined the effect of CKI overexpression on ex-lacZ. In further support of our model, CkIα overexpression increased ex-lacZ expression, while Gish expression had no effect .We agree with the reviewer that it is important to correlate the stabilisation of Ex in ckIα clones are larger than control clones and are often excluded from the disc epithelium once they reach a certain size. This overgrowth phenotype is due to excess Wingless (Wg) signalling, and can be suppressed by expression of dominant negative TCF . In fact, the clonal overgrowth and exclusion phenotype of ckIα mutants closely resembles that of the Wg signalling negative regulators axin and APC1/2 . Thus, it is likely that the undergrowth induced by Ex stability and consequent Yki downregulation is masked by the very strong overgrowth due to Wg signalling upregulation upon ckIα loss. We now consider these points in the Discussion. In the case of CkIα overexpression, we do observe a mild overgrowth phenotype . As expected, Gish overexpression causes neither ex-lacZ expression  nor overgrowth  The authors showed that CKI phosphorylates Ex in vitro, however, it is important to identify the phosphorylation site(s) of Ex and generate the phosphor-specific antibody to examine the endogenous Ex phosphorylation. Alternatively, the phosphor-tag gel or pan-phosphor S/T antibody could be used for the phosphorylation detection.intra expression, potentially directly by CKIs. Unfortunately, we were unable to validate the p-S453 antibodies we generated in both rabbits and sheep, in any system we tested  and the antibody failed to recognise Ex. Even in conditions where Ex should be extensively phosphorylated, we failed to detect a band recognising either endogenous or exogenous Ex.We performed several experiments to address this point but, unfortunately, we were unable to unequivocally identify the CKI phosphorylation site(s). As suggested by the reviewer, we attempted to raise antibodies that recognise a peptide phosphorylated at S453. We reasoned that since S453 is essential for the Ex:Slmb interaction, it is likely to be phosphorylated in response to CrbDrosophila S2 cells. However, we found that the widely used anti-Ex antibody was not sufficiently specific . Shown in 1-468 was coexpressed with either WT or mutant Crbintra and the lysates were assessed by Phos-tag gel electrophoresis followed by Western blot analysis.As suggested by the reviewer, we also used Phos-tag gels to examine point mutants of Ex1-468 with Crbintra WT, but not DFBM, resulted in a shift in Ex mobility in Phos-tag gels, consistent with our previous results on standard PAGE gels. Our analysis of the Ex mutants confirmed our previous observations regarding their effect on the stability of Ex and their ability to interact with Slmb/b-TrCP. We have previously shown that S453 is crucial for Slmb/b-TrCP recognition and Ex degradation. When S453A is mutated, Ex fails to bind to Slmb/b-TrCP and is therefore more stable. We had previously observed that S453A mutation does not abrogate the Ex mobility shift and this is confirmed with the Phos-tag analysis. Ex1-468 is still shifted and it is significantly more stable. Interestingly, unmodified Ex S453A is also seen in the Phos-tag gel, perhaps indicating that, indeed, this residue is phosphorylated in response to expression of Crb. However, it is clear that even if that is the case, it is not the only phosphorylation event triggered by Crb expression.Co-expression of ExThe other mutations tested (S457A and S462A) behaved very similarly to WT Ex, showing only a slight increase in Ex stability. In the case of S457A, the Ex detected is nearly completely modified, suggesting that this residue may not be phosphorylated in response to Crb expression. In the case of S462A, we could detect some unmodified Ex, again perhaps suggesting that it may be phosphorylated but, like S453, it is not the only residue involved. What is clear from the modest band shifts we observed is that Phos-tag gels do not allow us to readily separate different individual phosphorylated species of Ex any better than standard PAGE gels. Therefore, given the fact that the sequence surrounding the Slmb/b-TrCP consensus site is extremely rich in Ser and Thr residues, this approach is very unlikely to allow us to identify the  CKI phosphorylation sites.Finally, we attempted to immunoprecipitate CkIα from S2 cell lysates to perform in vitro kinase assays on bacterially expressed Ex followed by Mass Spectrometry analysis. Unfortunately, we could not find conditions where the purified CkIα was active in the in vitro setting and had no time to extensively troubleshoot the experiment given the time constraints on the revision.4) All biochemistry assays were using overexpression system. They should detect endogenous Ex expression and degradation. For example, CHX treatment could be used to avoid gish-mediated transcriptional regulation of endogenous Ex expression.Drosophila S2 cells. However, we found that the Ex antibody was not sufficiently specific  Although the biochemistry assays have shown that CKI kinase Gish is required for Crb-mediated Ex turnover, the genetic epistasis between Crb and CkIα in controlling Yki activation and organ growth is still needed to be tested. I would suggest the authors perform the following experiments "hh> Crbintra vs. hh>Crbintra + CkIαRNAi", then compare the Yki reporter induction (e.g. Diap1-LacZ) and P-compartment size.intra phenotype (overgrowth and Yki activity using ex-lacZ) in the posterior compartment of the wing disc (ex-lacZ increase elicited by Crbintra expression are suppressed by CkIα depletion. However, the discs combining Crbintra and CkIα depletion appear heavily disrupted and, therefore, we want to be cautious about the interpretation of this experiment. In particular, it is possible that the suppressed growth is due to toxicity of combining Crbintra with CkIα depletion rather than specifically lowering Yki activity. Nevertheless, we are happy to include the data as supplementary material if required by the reviewer.As requested by the reviewer, we examined the effect of CkIα depletion on the Crbing disc . We obse


The present study aimed to evaluate the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1) on the proliferation and osteogenic differentiation potential of human bone marrow-derived MSCs (hBMSCs). hBMSCs with stable TIMP-1 overexpression or TIMP-1 knockdown were generated. Osteogenic differentiation was assessed by Alizarin Red S staining, alkaline phosphatase (ALP) activity and expression of specific markers. Compared with the vehicle controls, TIMP-1 knockdown significantly promoted the growth of hBMSCs. TIMP-1 knockdown up-regulated β-catenin and cyclin D1 proteins. During osteogenic differentiation, TIMP-1 knockdown elevated the deposition of calcium nodules, ALP activity and the mRNA levels of the osteogenic markers sex determining region Y-box 9 (Sox9), CCAAT-enhancer-binding protein and peroxisome proliferator-activated receptor γ. During osteogenic differentiation, TIMP-1 knockdown significantly enhanced the up-regulation of osteocalcin proteins. Meanwhile, TIMP-1 overexpression attenuated the Wnt/activator Wnt3a-induced up-regulation cyclin D1 and Runt-related transcription factor 2 (RUNX-2) (during osteogenic differentiation) proteins, while TIMP-1 knockdown restored the inhibitor Dickkopf 1-induced inhibition effect on the expression of β-catenin, cyclin D1 and RUNX-2. TIMP-1 plays a negative regulatory role in the proliferation and osteogenesis of hBMSCs, at least partially, through Wnt/β-catenin signaling. Bone marrow-derived mesenchymal stem cells (BMSCs) are commonly adopted in various cell-based therapy for tissue repair and regeneration . It has During the early stage of osteogenic differentiation of MSCs, accumulated extracellular matrix (ECM) forms 3D nodules at the sites of mineral deposition . Matrix In addition to regulating MMPs, TIMP-1 has been shown to participate in the regulation of various MMP-independent biological activities, including cell growth and survival , cell miIt has been demonstrated that TIMP-1 plays an important role in the osteogenesis of osteoblasts and MSCs . GrässelTo further elucidate the effect of TIMP-1 on MSCs, the present study aimed to investigate the regulatory effect of TIMP-1 on the proliferation and osteogenic differentiation of human BMSCs (hBMSCs).Bone marrow sample was collected from the drill holes of the pedicle during the operation of spine internal fixation of ten patients with traumatic lumbar spondylolisthesis at our center from March 2014 to June 2014. The present study was approved by the Institutional Review Board (IRB) of the First Affiliated Hospital of Sun Yat-sen University, and all the procedures were performed in accordance with the IRB guidance. Written informed consent was obtained from each patient.hBMSCs were isolated and cultured as described in our previous studies ,25. The puro vector, the puromycin resistance encoding sequence was used to replace both the blasticidin resistance encoding sequence and the bacterial EM7 promoter of lentiviral 2k7bsd vector. In order to generate entry vectors, human TIMP-1 cDNA (648 bp) flanked by attB sites was PCR-amplified from BAC clone GM090230  and cloned into Pdonr™ 221  by utilizing the Gateway BP recombination reaction according to the manufacturer’s protocol. The EF1a promoter and heGFP cDNA were cloned into Pdonr™ P4-P1R (Invitrogen) and Pdonr™ P2r-P3 (Invitrogen) by the same method. The resulting vectors, pDown-TIMP-1, pUp-EF1a and pTail-IRES-heGFP, were then recombined into the Destpuro vector according to the protocol for LR recombination reaction using the Gateway LR plus clonase enzyme mix (Invitrogen). The constructed lentiviral vector was designated as pLV/Final-puro-EF1a-TIMP-1-hrGFP. In the present study, we used the pDestpuro vector with heGFP gene inserted as the vehicle control.To generate a pDest™ Lentiviral packaging mix (Invitrogen) using Lipofectamine 2000 (Invitrogen). Three days after transfection, viral particles were harvested from the medium, filtered through 0.45 µm pore-sized polyethersulfone membrane and concentrated by ultracentrifugation at 50000×g, 4°C for 120 min. For lentiviral transduction, hMSCs were washed with PBS and dissociated into single cells by 0.25% trypsin and were replated with lentiviral particles and 8 mg/ml polybrene . The medium was changed to fresh culture medium after infection for 12 h. After twice transduction, puromycin (Sigma) was added to the culture medium at a concentration of 1 to 5 μg/ml and maintained for 5 days.The lentiviral particles were prepared by transient cotransfection of 293FT cells with the lentiviral vectors and ViraPower4 MSCs were seeded in a well of 24-well plate (500 µl) and was transfected with 50 ng plasmid DNA ) of Wnt/β-catenin signaling were used. As shown in P<0.001 P<0.01, At 7 days after osteogenic differentiation, TIMP-1 overexpression suppressed the Wnt3a-induced up-regulation of RUNX-2 proteins P<0.001 B, while It has been shown that TCF4 play a crucial role in the Wnt/β-catenin signaling and cellular development ,30. We ihttp://jaspar.genereg.net) [Previous study has shown that TCF4 is a conserved bHLH transcription factor that binds E-box sequences in the promoters and enhancers of certain genes . We, thereg.net) . As showreg.net) . Two putTCF4 knockdown significantly down-regulated mRNA expression of osteogenic gene RUNX-2 at 72 h after osteogenic induction C, indicaIn the present study, we investigated the effect of TIMP-1 on the proliferation and osteogenic differentiation potential of hBMSCs by using lentiviral-mediated overexpression and shRNA interference technique. The results showed that TIMP-1 knockdown significantly increased the growth of hBMSCs as compared with the vehicle control. TIMP-1 overexpression significantly down-regulated the protein levels of β-catenin and cyclin D1, but TIMP-1 knockdown up-regulated these two proteins. In addition, during osteogenic differentiation, TIMP-1 overexpression reduced the deposition of calcium nodules and ALP activity, and the mRNA levels of SOX-9, C/EBP and PPARγ, whereas TIMP-1 knockdown exhibited the opposite effects. During osteogenic differentiation, TIMP-1 overexpression significantly attenuated the up-regulation of OC, whereas TIMP-1 knockdown displayed the opposite trends. Meanwhile, TIMP-1 overexpression attenuated the Wnt3a-induced up-regulation of cyclin D1 and RUNX-2, while TIMP-1 knockdown restored the DKK1-induced inhibition on the expression of β-catenin, cyclin D1 and RUNX-2 (during osteogenic differentiation). Taken together, these results suggested that TIMP-1 has a negative regulatory role in the proliferation and osteogenesis of hBMSC through Wnt/β-catenin signaling.The growth-stimulating effect of TIMP-1 has been described in a variety of cell types . ContrarIt has been reported that the Wnt/β-catenin signaling plays a positive role in the proliferation and osteogenic differentiation of MSCs ,34. Our It is known that activation of the Wnt/β-catenin pathway can suppress the degradation of cytosolic β-catenin by inducing saturation on the APC/Axin1 destruction complex ,39. Subsin vitro findings should be validated in an in vivo model. In addition, the biological meaning of the inhibitory effect of TIMP-1 on differentiation markers requires further study. Moreover, the detailed mechanism underlying Wnt/β-catenin signaling involved in TIMP-1 effect on the proliferation and osteogenic differentiation remains to be further investigated. All these limitations should be addressed in the following study.There are still some limitations of the present study. First, the Overall, our findings demonstrate that TIMP-1 is a negative regulator of the growth and osteogenic differentiation of hBMSCs, at least partially, through inhibiting Wnt/β-catenin signaling. Our findings should be helpful for better understanding the mechanism of growth and osteogenic differentiation of hBMSCs.