Musa sp.) is cultivated worldwide and is one of the most popular fruits. The soil-borne fungal disease Fusarium wilt of banana (FWB), commonly known as Panama disease, is caused by Fusarium oxysporum f. sp. cubense (Foc) and is a highly lethal vascular fungal disease in banana plants. Raman spectroscopy, an emerging laser-based technology based on Raman scattering, has been used for the qualitative characterization of biological tissues such as foodborne pathogens, cancer cells, and melamine. In this study, we describe a Raman spectroscopic technique that could potentially be used as a method for diagnosing FWB. To that end, the Raman fingerprints of Foc (including mycelia and conidia) and Foc-infected banana pseudostems with varying levels of symptoms were determined. Our results showed that eight, eleven, and eleven characteristic surface-enhanced Raman spectroscopy peaks were observed in the mycelia, microconidia, and macroconidia of Foc, respectively. In addition, we constructed the Raman spectroscopic fingerprints of banana pseudostem samples with varying levels of symptoms in order to be able to differentiate Foc-infected bananas from healthy bananas. The rate at which FWB was detected in asymptomatic Foc-infected samples by using the spectral method was 76.2%, which was comparable to the rates previously reported for other FWB detection methods based on real-time PCR assays, suggesting that the spectral method described herein could potentially serve as an alternative tool for detecting FWB in fields. As such, we hope that the developed spectral method will open up new possibilities for the on-site diagnosis of FWB.Banana ( Fusarium oxysporum f. sp. cubense (Foc), is a highly lethal vascular fungal disease in banana plants, and is thus reported to be one of the major limiting factors for banana production worldwide ,,in-plantTo evaluate the potential of the Raman spectroscopy assay for the rapid differentiation of banana pathogens, the SERS spectra databases of the fungal pathogens on banana in Taiwan were built, including those for Foc, Colm (which causes Anthracnose of banana), Dt (which causes Deightoniella leaf spot), Aa (which causes Alternaria speckle of banana), Bd (which causes Botryosphaeria crown rot of banana), and Corm (which causes Cordana leaf spot of banana).−1; −1; −1; The mean SERS spectra of the tested mycelia and conidia covering the spectral range of 400–1800 nm are presented in −1 peak, which corresponds to guanine [-1 . The vibration mode and major assignment of the peaks around 1590 cm−1, however, were not yet determined.It is notable that the 652 cm guanine , was onlnine [-1 , while mnine [-1  cm-1. Threathing , 33 and id chain , respect−1, as they were observed in all of the conidia samples  of Foc and Colm  of the field banana samples did not always perfectly match the Raman peaks of the Foc mycelia, microconidia, and macroconidia because it is easy for Raman peaks (300–1800 cm−1) to be interfered with by the Raman signals from endogenous biomolecules [A reliable method for the early monitoring of banana health is essential for formulating appropriate and timely disease management strategies to counteract the diseases affecting bananas. Traditional laboratory methods for diagnosing FWB involve lengthy assays including pathogen isolation, culturing, and morphological observation, as well as pathogenicity testing, which can take a few days to months . One sold chains  and prold chains , could bolecules .−1; mildly symptomatic: 896, 1044, and 1113 cm−1; moderately symptomatic: 614, 896, and 1044 cm−1; severely symptomatic: 830, 896, 1039, 1400, and 1572 cm−1; and 447 cm−1 as healthy control) were collected to differentiate field-infected bananas with varying levels of symptoms. A field detection evaluation was further performed to determine whether the spectroscopic method based on the reproducible characteristic Raman spectra was suitable for the diagnosis of FWB. For this purpose, we collected a total of 73 banana pseudostem samples  and 15 samples of banana infected with fungal pathogens other than Foc (3 samples for each disease) from 12 different fields that had been strongly affected by FWB for the Raman spectroscopic fingerprinting analysis and the three molecular detections. We chose three published methods for comparison, namely PCR-based pathogen-identification (MDIP), PCR-based in-planta detection (IPDP), and qPCR-based in-planta detection (IPDQP). As shown in Before this study, Raman spectroscopy had not been used for the diagnosis of banana diseases. In this study, the most reproducible characteristic Raman spectra  for differentiating conidia or mycelia of Foc from those of other fungal pathogens collected from bananas, such as Colm, Dt, Aa, Bd, and Corm, as well as for the diagnosis of FWB on pseudostem samples with varying levels of symptoms. An acceptable field-detection rate for FWB detection by the spectroscopic fingerprinting assay was observed among the results generated from the 73 random field-collected Foc-infected pseudostems with varying levels of symptoms, including asymptomatic pseudostems. In conclusion, to our knowledge, this is the first study to report the Raman spectroscopic fingerprinting databases of phytopathogenic fungi. The Raman spectroscopic fingerprinting assays developed in this study provide an alternative to conventional PCR and real-time PCR assays for the field-detection of FWB. The spectroscopic fingerprinting assays have the potential to serve as a rapid and simple tool for the routine diagnosis of FWB.S1 FigThe Raman spectra databases of banana samples with (A) pale yellowing, (B) black leaf streak, (C) black spot, and (D) necrosis on leaves, and (E) crown rot on fruit were built. Average Raman spectra were obtained from three independent replications of the surface-enhanced Raman spectroscopy measurements.(TIF)Click here for additional data file.


The influence of edges and ridges on the Wnt/β-catenin pathway in combination with the cells’ stress response has not been clear. Therefore, MG-63 osteoblasts were studied on defined titanium-coated micro-pillars (5 × 5 × 5 µm) in vitro and in silico. MG-63s on micro-pillars indicated an activated state of the Wnt/β-catenin pathway. The β-catenin protein accumulated in the cytosol and translocated into the nucleus. Gene profiling indicated an antagonism mechanism of the transcriptional activity of β-catenin due to an increased expression of inhibitors like ICAT (inhibitor of β-catenin and transcription factor-4). Cells on pillars produced a significant reactive oxygen species (ROS) amount after 1 and 24 h. In silico analyses provided a detailed view on how transcriptional activity of Wnt signaling is coordinated in response to the oxidative stress induced by the micro-topography. Based on a coordinated expression of regulatory elements of the Wnt/β-catenin pathway, MG-63s are able to cope with an increased accumulation of β-catenin on micro-pillars and suppress an unintended target gene expression. Further, β-catenin may be diverted into other signaling pathways to support defense mechanisms against ROS.The physico-chemical surface design of implants influences the surrounding cells. Osteoblasts on sharp-edged micro-topographies revealed an impaired cell phenotype, function and Ca Biofunctional materials which replace damaged tissue and stimulate cell regeneration are currently in great demand and will be in the future. Titanium (Ti) and titanium-based alloys are the most common metallic materials in implant applications due to their good biocompatibility, mechanical compliance, and high corrosion resistance ,2,3. The2+) was observed [The previous systematic analyses revealed that a defined topographical environment with cubic pillars reduced cell functions, such as proliferation and ECM-synthesis (collagen type I and fibronectin) ,12,13. Pobserved . These sThe Wnt signaling pathways are known to be one of the most important signal transduction systems in tissue homeostasis ,15,16 anThe key component of the canonical Wnt pathway is the protein β-catenin . In the Based on the available data, topography-induced osteogenic differentiation appears to be associated with an activated Wnt/β-catenin pathway ,17,18. BThe aim of this study was to understand the effects of sharp-edged geometrical micro-pillars on components of the Wnt/β-catenin pathway in human MG-63 osteoblasts. We hypothesize that a defined surface micro-topography modify the signal transduction and ultimately prevent the activation of context-specific target genes. The expression of regulator of Wnt signaling: β-catenin, inhibitors like ICAT, SOX17 (SRY-box transcription factor 17) and transcription factors (TCF4) was quantified in vitro and simulated in silico. The influence of cellular oxidative stress was evaluated as additional trigger of β-catenin. Finally, the complex interplay of ROS, β-catenin and specifically expressed inhibitors and transcription factors was evaluated in silico. The model illuminates the redox-sensitive pathway that activates β-catenin in a receptor and Wnt-independent manner as well as the corresponding cellular and transcriptional response mechanisms that suppress  Wnt/β-catenin target gene expression ,29.Silicon wafers with a size of 1 cm² were sputter-coated with 100 nm titanium . The unstructured bulk material served as reference (Ref). For the defined micro-pillars, the silicon wafers were structured by deep reactive-ion etching (DRIE) . The resulting geometrical sharp-edged pillars had the dimensions 5 × 5 × 5 µm (length × width × depth) (P5) ,30. The ® CRL-1427™), Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium  with 10% standardized fetal calf serum  and 1% gentamycin  (complete media) at 37 °C in 5% CO2 according to the protocols described previously [Osteoblast-like human cells MG-63 . In brief, protein degradation was prevented by a protease inhibitor cocktail and Benzonase® nuclease was used to remove contaminating nucleic acids. Then MG-63s were incubated with the respective extraction buffer to get the cytosolic-, membrane/organelle-, and nucleic protein extract fraction. Proof of subcellular fractionation were carried out by SDS-Page (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) and assign selected marker proteins were detected using immunoblot  were performed for mRNA (messenger ribonucleic acid) expression profile. After 24 h of cultivation (5 × 104 cells) on the surfaces, RNA extraction was done with the RNeasy Kit  according to the manufacturer’s protocol. The total RNA samples were quantified spectrophotometrically  and the integrity was controlled using the Agilent Bioanalyzer 2100 with the RNA Pico chip kit . RIN (RNA integrity number) values between 9.4 and 10 were achieved. A 100 ng whole RNA sample was used as the starting material in the GeneChipR Whole Transcript Sense Target Labeling protocol (Affymetrix). The microarray hybridization was done using the Affymetrix Clariom™ S Array Kit according to the manufacturer’s instructions (detailed described [The human Clariomescribed ). p-value < 0.05. Furthermore, TAC links directly to multiple public databases like WikiPathways to identify the molecular events associated with the Wnt signaling. A table containing an overview of genes related to Wnt signaling can be found as part of the The mRNA expression profiling and analysis of raw data was performed using the Transcriptome Analysis Console (TAC) version 4.0.2.1.5 . The subsequent evaluation of the datasets P5 versus Ref (triplicates from independent experiments) was summarized by Signal Space Transformation-Robust Multi-Chip Analysis (SST-RMA). Technical replicate groups were summarized by linear models for microarray data  statistics. Differentially expressed genes (DEG) were identified using filter parameters, the SST-RMA Algorithm with the settings fold change > 1.5 or <−1.5, LIMMA 4) were seeded on each sample for 24 h. Cells were fixed with 4% paraformaldehyde  for 10 min at room temperature (RT) followed by permeabilization with 0.1% Triton X-100 (Merck KgaG). MG-63s were incubated with the primary antibodies: β-catenin , inhibitor of β-catenin and TCF-4  and SOX17  at RT for 1 h and then fluorescence-conjugated secondary antibodies (dilution 1:200): Alexa Fluor® 488-labeled goat anti-rabbit or -mouse (Life Technologies), Goat F(ab’)2 anti-mouse IgG-Cy3  for 30 min at RT. Finally, the samples were fixed on microscope slides and mounted using Fluoroshield™ with DAPI . The location and expression of proteins were visualized by the inverted confocal laser scanning microscope LSM 780 . MG-63 osteoblasts  was used for image analysis. For MG-63s on micro-pillars, several images were acquired in the “Z-stack” function  and then generated a 3D-reconstruction (overlay). Confocal microscopy with 3D Z-stack generation was performed to visualize the protein localization on top of and between the micro-pillars to understand protein localization that was not limited to a single dimension. Using the “Profile” function, intensity profiles of the individual fluorescence’s of certain regions in the image can be created.4) cultivated for 1 h or 24 h on the Ti-samples were harvested by detachment with Trypsin-EDTA, fixed with 4% PFA (10 min) and permeabilized with 0.1% Triton X-100 (10 min). The cells were resuspended in the primary antibodies (dilution 1:50): β-catenin  and SOX17 (Acris) for 30 min each, followed by the secondary antibodies (dilution 1:200): Alexa Fluor® 488-labeled goat anti-rabbit or -mouse (Life technologies). After staining, the suspended cells were analyzed using a BD FACSCalibur™ . The fluorescence intensity of at least 10,000 events was measured using CellQuest Pro 4.0.1 (BD Biosciences) and calculated as the FL-1 mean channel by FlowJo_V.10.1r1 .The MG-63s (4 × 10® protein assay kit (Thermo Fisher Scientific Inc.). The cellular protein (25 µg per lysate) were loaded into Criterion™TGX Stain-Free™-gel  and separated using SDS-PAGE (Bio-Rad). Afterwards, the proteins were transferred onto PVDF membrane  by electro blotting (150 mA overnight). The PVDF membranes were blocked with 5% low fat milk solution  for 1 h, and incubated with the primary antibodies: β-catenin ; Apg1 , pan-cadherin ; TCF-1  bone sialo protein 2 , collagen type I ; fibronectin  and GAPDH  at 4 °C overnight. As secondary antibodies: horseradish peroxidase (HRP)-conjugated monoclonal anti rabbit or mouse IgG  was used at RT for 1 h. Protein expression was detected using enhanced chemiluminescence-substrate (Thermo Fisher Scientific Inc.). The band intensity was quantified by densitometry in the ChemiDoc™ MP imager (Bio-Rad) using the software Quantity One® and Image Lab™ (Bio-Rad). In order to control the equal protein amount or confirm the specificity of the purity of the fractions markers for nucleus, cytosol and membrane, Stain-Free-gel or PVDF membrane were visualized  and subcellular fraction lysates  of MG-63s on the Ti-samples. The protein content was measured using a Qubitsualized .®200 System (Bio-Rad) and the Bio-Plex Manager™ 4.1.1 software (Bio-Rad). The MFI values were normalized to the protein concentration determined by Bradford method.The secreted proteins osteocalcin (OCN) and osteopontin (OPN) from lysates of the cell culture supernatant after 96 h were analyzed using MILLIPLEX MAP Kit for Human Bone Magnetic Bead Panel (Merck KGAA) according to the manufacturer’s recommendations. Briefly, the bead mix was transferred to each well of the 96-well plate and mixed with lysates over night at 4 °C shaking with 300 rpm. Subsequently, incubation was performed with a detection antibody solution  and afterwards with a Streptavidin-Phycoerythrin solution . The protein quantification, mean fluorescence intensity (MFI), was measured with the Bio-PlexFor quantitative protein detection of ICAT in the cell fraction lysates of MG-63s within 24 h, the ICAT ELISA kit (human beta catenin interacting protein 1 (CTNNBIP1); MyBioSource, Inc., San Diego, CA, USA) was used according to the manufacturer’s instructions. Briefly, the 96-well pre-coated microtiter plate was loaded with equal amounts of the protein as well as the standards. Lysates were labeled with CTNNBIP1-HRP conjugates for 1 h and subsequently incubated with the substrate for HRP enzyme at 37 °C for 20 min. Immediately after stopping the reaction, the optical density  could be measured using Anthos Reader 2010  at 450 nm. For the calculation of ICAT protein expression level, a standard curve was plotted relating the O.D. to the standard concentration.5 osteoblasts were cultivated on the samples within 24 h. In the presence of an ROS, the DCF-DA is oxidized to the fluorescence compound DCF [®Green) or cytosol (CellROX®Orange) according to the manufacturer’s instructions. After 24 h adherence on the samples, cells were washed with PBS and loaded with 5 µM of one fluorogenic probe at 37 °C for 30 min. The production of intracellular ROS were detected using DCF-DA Cellular ROS detection assay kit . Therefore, a trypsinated MG-63 cell pellet was resuspended in a dye solution consisting of 2′,7′-dichlorofluorescein diacetate  and then shaken (300 rpmi) at 37 °C for 30 min in the dark. The stained MG-63s were resuspended in complete DMEM . Afterwards, 1 × 10ound DCF . The Cel® Lipid Peroxidation Kit . C11-BODIPY (581/591) fluorescence was measured using a filters for FITC and TexasRed. Upon oxidation (ROS production), fluorescence changes from red to green and its ratio indicates the degree of lipid peroxidation. As a positive control, cells were treated with 0.5% hydrogen peroxide (H2O2) or cumene hydroperoxide (CH)  . The flut-test (ROS and lipid peroxidation detection) or the multiple t-test . Herein, a probability value of p < 0.05 was accepted as indicating a significant difference. Data were expressed as mean ± s.e.m. (standard error of the mean).All in vitro experiments were repeated at least three times with independently passaged cells. To evaluate statistical differences and to prepare the graphs, the GraphPad Prism 7 software for Windows  was used. Data were normalized either to unstructured bulk material (Ref) or time point (1 h). After determining the normal distribution by Kolmogorov-Smirnov, subsequent statistical test was done: the Mann-Whitney U test , paired https://github.com/SFB-ELAINE/SI_Staehlke_MDPI_Cells. A table containing all parameter values employed in the (fitted) model as well as the model implementation in ML-Rules can be found as part of the The simulation model is defined in ML-Rules, a multi-level, rule-based modeling language . ML-RuleThe stabilization, accumulation, and translocation of the β-catenin into the nucleus is a marker for the activation of the Wnt/β-catenin pathway ,16,23 anIn flow cytometry a significantly increase in total protein expression of β-catenin in cells on P5 compared with Ref after 24 h was observed A. AdditiOn Ref β-catenin appears evenly distributed throughout the cytosol with an increased localization along the cell membrane in cell-cell contacts. On P5 a general higher occurrence was detected and β-catenin was distributed mainly in the cytosol C. Also, Also the quantification of β-catenin protein level in MG-63 subcellular fractions using Western Blot revealed a slightly higher level in the cytosol as well as a significantly increased expression in the nucleus on P5 versus Ref. No topographical influence could be observed in the protein expression of β-catenin in the membrane fraction E,F.Taken together, MG-63 cells on P5 indicated an activated state of the Wnt/β-catenin pathway due to the accumulation of the key effector β-catenin in the cytosol and their transduction in the nucleus after 24 h.In order to obtain information about the overall composition of the analyzed complex microarray datasets and to determine the direction of variability, the principal component analysis (PCA) was performed A. This ip < 0.05, and for the identification of differentially regulated genes (p < 0.05) differently regulated regarding the Wnt/β-catenin pathway  algorithm identified expression values for 21,448 annotated gene clusters, which were further filtered with the criteria fold change cutoff, fold change > 1.5 or <−1.5, linear models for microarray data (LIMMA) ed genes B. Of theed genes B. Under  pathway . AXIN2 or osteoblast-specific ones were not influenced on the mRNA expression level . In addition, the increased and abundant expression of WLS  was observed on P5. The datasets were evaluated according to the pathways tool WikiPathways. on level . All in SOST . Further significantly upregulated inhibitors were ICAT  as well as SOX17 , which finally antagonize the transcriptional activation of β-catenin. Another Wnt antagonist, the SFRP4 , showed a significantly lower expression on P5 versus Ref. Remarkably, osteoblasts on P5 exhibited an upregulation of antagonists of the Wnt/β-catenin pathway, such as The expression of β-catenin antagonists were also upregulated at protein level in MG-63s on P5 after 24 h . The fluIn previous studies a significant reduced expression of osteogenic markers could be observed for osteoblasts on micro-pillars after 24 h ,12,13. Ip < 0.05) just after 1 h cultivation (data not shown). Moreover, intracellular ROS was determined significantly in the nucleus, the mitochondria (both by CellROX®Green) as well as in the cytosol (CellROX®Orange) (The micro-pillars (P5) induced the reactive oxygen species (ROS) production and as consequence lipid peroxidation in MG-63s . To inve®Orange) B,C. Figup < 0.0345; Antioxidative enzymes like superoxide dismutase (SOD) eliminate ROS by catalyzing reactions. The protein expression of one of these ROS-scavenging enzymes—SOD3—was significantly increased  compared with unstructured samples (Ref). The model and the assumptions derived from the experimental results are briefly described below.According to the in vitro observations, P5 induce the production of ROS in MG-63s . SeveralIt is assumed that there is no extracellular Wnt stimulus; i.e., Wnt proteins are neither part of the medium nor being produced and secreted by the cells. This assumption is supported by the mRNA expression profile, which provides no significant difference between Ref and P5 in the expression of Wnt ligands. Therefore, the model disregards any membrane-associated signaling components, and considers solely the intracellular signaling components.Next, the intracellular components that are differentially expressed on P5, i.e., ICAT, SOX17 and TCF, were added to the model. The role of TCF was thoroughly described in the original Wnt model by Lee et al.  and can The surfaces of biomaterials play a central role in the modulation of cell behavior ,43 and i2+-release is decreased in cells on micro-pillars [Wnt signaling is one of the central signaling pathway that controls cell fate specification ,16,21,56-pillars ,12,13, iIn the present study, the key effector of the Wnt/β-catenin pathway, the β-catenin-protein, indicated a very pronounced and effective response of MG-63s to P5 after 24 h. β-catenin protein level is strongly enhanced on P5 in cells at time periods of 1 to 24 h. β-catenin in MG-63s on the Ref appeared evenly distributed throughout the cytosol, with a stronger signal along the cell border. This can be attributed to the presence of cell-to-cell contacts due to the increased spreading area, accompanied by increased concentration of cadherin to mediate cell-cell adherence ,16,65. SMany studies associate an activated state of the Wnt/β-catenin pathway with increased osteogenesis due to topography ,61,69,70In one study it was found that the topographical structure has an influence on the Wnt/β-catenin pathway: Schernthaner et al.  describep < 0.3533). Transcriptome analyses by microarray technology was used to identify the full array of genes whose expression changes under topographical features. In general, the regulation of mRNA (messenger ribonucleic acid) expression in MG-63s in Wnt signaling was only observed in Wnt/β-catenin pathway components. The most important components such as Wnt, receptors or target genes were topography independent after 24 h . InteresWLS) and abundancy in MG-63s on P5, which is implicated in the extracellular secretion of Wnt ligands [TCF4) was significantly enhanced on P5. The proteins T-cell factor/lymphoid enhancer factor (TcF/LEF) family of DNA-bound transcription factors is the major partner for β-catenin that triggers specific expression of Wnt target genes [TCF4 mRNA level, the expression of target genes (like AXIN2) was topographically unchanged. Otherwise, antagonists like extracellularly secreted antagonist Sclerostin (SOST) was upregulated due to micro-topography at mRNA level. SOST binds to the LRP5/6 receptor and finally inactivates the Wnt/β-catenin pathway [In the present study, six genes were identified as significantly regulated with respect to the Wnt/β-catenin pathway. The transmembrane protein Wntless displayed the highest gene expression  and inhibitor of β-catenin and TCF-4 (ICAT), were upregulated on P5 at mRNA level. Both antagonists are expressed and react only when β-catenin accumulates and can translocate into the nucleus, where β-catenin acts as a transcriptional co-activator [Two β-catenin signaling antagonists, SRY-box transcription factor 17 (ctivator . ICAT bictivator . In the ctivator ) for TCFctivator ,75,76, wctivator ,25,74,75ctivator , in theictivator  publishectivator . Sinner ctivator  demonstrctivator ,76,80. SFRP4), which interacts directly with Wnt, preventing binding to their receptor; in this way they antagonize the Wnt/β-catenin pathway [DVL3 showed slightly higher mRNA expression levels in MG-63s on P5  and SOX17 suggest that the β-catenin-dependent transcriptional activity (as co-activator) is affected due to micro-topography.The only significantly downregulated gene was secreted frizzled-related protein 4  and mitochondrial activity [p < 0.05) [SOD3  by inhibiting TCF-mediated transcription for activation of the FOXO/β-catenin pathway. The mRNA expression level of FOXO displayed only a very slight increase due to the topography  . The preWhile the connection between these experimental measurements is not immediately obvious, it becomes apparent when considering the in silico model. As shown in ,37, elevMyc, c-Jun and AXIN2. Notably, AXIN2 has a similar function as its homolog AXIN and induces the degradation of β-catenin. This means that nuclear β-catenin accumulation usually induces a negative feedback by increasing the expression of its own inhibitor  mobilization and induced a cellular stress reaction with low adenosine triphosphate (ATP) levels and increased ROS production [Defined sharp-edged micro-pillars caused a change in cell morphology , reducedoduction . In the oduction ,12,13 cooduction ,20,68. HOther antagonistic effects on Wnt signaling in MG-63 osteoblasts, such as hypoxia , treatme


The aberrant Wnt/β-catenin signaling pathway facilitates cancer stem cell renewal, cell proliferation and differentiation, thus exerting crucial roles in tumorigenesis and therapy response. Accumulated investigations highlight the therapeutic potential of agents targeting Wnt/β-catenin signaling in cancer. Wnt ligand/ receptor interface, β-catenin destruction complex and TCF/β-catenin transcription complex are key components of the cascade and have been targeted with interventions in preclinical and clinical evaluations. This scoping review aims at outlining the latest progress on the current approaches and perspectives of Wnt/β-catenin signaling pathway targeted therapy in various cancer types. Better understanding of the updates on the inhibitors, antagonists and activators of Wnt/β-catenin pathway rationalizes innovative strategies for personalized cancer treatment. Further investigations are warranted to confirm precise and secure targeted agents and achieve optimal use with clinical benefits in malignant diseases. The Wnt/β-catenin signaling pathway, also called the canonical Wnt signaling pathway, is a conserved signaling axis participating in diverse physiological processes such as proliferation, differentiation, apoptosis, migration, invasion and tissue homeostasis –3. IncreIn the Wnt/β-catenin pathway, abnormal regulation of the transcription factor β-catenin, which is the pivotal component of the Wnt signaling pathway, leads to early events in carcinogenesis –12. WithIn addition, multiple regulatory mechanisms have been identified on the phosphorylation and ubiquitination of β-catenin by the degradation complex. Notum, which removes palmitoleate from Wnt proteins, blocks their extracellular secretion. Dickkopf (DKK) negatively regulates the initiation of Wnt protein-mediated signaling by competitively binding to LRP5/6 receptors. Besides, secreted FZD-related proteins (sFRPs), which bind to FZD receptors also blocking the initiation of Wnt protein-mediated signaling. Moreover, Wnt inhibitory factor (WIF) inhibits signaling by binding directly to Wnt proteins . The traFurthermore, Wnt/β-catenin signaling orchestrates multiple cell signaling cascades, such as epidermal growth factor receptor (EGFR), Hippo/YAP, nuclear factor kappa-B (NF-κB), Notch, Sonic Hedgehog and PI3K/Akt pathway, which contribute to pivotal molecular mechanism in cancer development –24. EGFRCollectively, underscoring the physiological importance of Wnt/β-catenin signaling pathway in tumorigenesis, targeted agents are explored and presented promising therapeutic potential in preclinical studies and clinical trials of some cancer types. In the present review, we elaborated on the advances and challenges of Wnt/β-catenin signaling pathway targeted interventions in malignancies, aiming to provide rationales and insights on novel strategies in cancer therapy.The deregulation of Wnt/β-catenin signaling pathway is closely related to the initiation and progression of various types of cancers , 5, 29. Porcupine (PORCN), a family member of membrane-bound O-acyltransferases (MBOAT), is key for the secretion of Wnt ligands , 31. SevWith the antagonism of Wnt ligands and FZD receptors, canonical Wnt signaling pathway was suppressed and indicated potential strategy in cancer therapy. Ipafricept  is a recombinant fusion protein, including the cysteine-rich domain of FZD8 fused to a human IgG1 Fc fragment . This stOMP-18R5 (vantictumab) is a monoclonal antibody targeting FZD1, FZD2, FZD5, FZD7 and FZD8 –45. OMP-As the co-receptor of Wnt, the phosphorylation of LRP5/6 promotes the activation of Wnt/β-catenin signaling pathway. The molecular complex Wnt-FZD-LRP5/6-DVL forms a structural region for AXIN interaction that disrupts degradation of β-catenin. BMD4503-2, a quinoxaline moiety, was identified as a new small-molecule inhibitor of the LRP5/6-sclerostin interaction through pharmacophore-based virtual screening and in vitro assays. The compound BMD4503-2 could revert the down-regulated activity of the Wnt/β-catenin signaling pathway through competitively binding to the LRP5/6-sclerostin complex [DVL is important for Wnt signal transduction by recruiting components of the β-catenin destruction complex to the cell membrane , 52. DVLScaffolding protein AXIN is the rate-limiting component of the β-catenin destruction complex, which are constantly surveyed and regulated by tankyrases –58. TankStabilizing the β-catenin destruction complex can block the nuclear localization of β-catenin, suggesting as an attractive therapeutic target. Feasible strategy for the repositioning of existing FDA approved drugs is explored for the treatment of malignancies with deregulated Wnt signaling. For example, pyrvinium, an existing FDA approved drug, can bind all CK1 family members in vitro, selectively potentiating CK1α kinase activity . Colon cSeveral compounds targeting the downstream effectors, like transcription complex and co-activators, were identified by high through-put ELISA screening, such as PFK115-584 and CGP049090, which can block the β-catenin/TCF complex in a dose-dependent manner . LF3, a There are several co-activators of β-catenin-dependent transcription, including CREB binding protein (CBP). The CBPs are key transcriptional co-activators essential for a multitude of cellular processes and involved in human pathological conditions and cancer , 88. Sev50 values. To achieve high potency, formononetin was modified with a coumarin unit to design a derivate 10 via the molecular hybridization strategy. The analog 10 presented anti-proliferative effects through Wnt/β-catenin pathway in gastric cancer [It is notable that some of the natural agents exert anti-tumor activities via regulating canonical Wnt signaling pathway , 100. Cuc cancer . Besidesc cancer . Gigantoc cancer . Additioc cancer . Besidesc cancer . Moreovec cancer . It was c cancer . Moreovec cancer .In addition, researchers had found some old drugs performed new tricks, which play important roles in tumor growth, invasion and metastasis via regulating Wnt/β-catenin signaling pathway. Carbamazepine, an antiepileptic drug, was recently reported to bind the cysteine-rich domain of FZD8, which suggested to been explored as a promising therapy option in cancers . It was CSCs display many characteristics of embryonic or tissue stem cells and often show continuous activation of highly conserved signaling pathways related to development and tissue homeostasis , 127. ThAbnormal activation of the Wnt/β-catenin pathway promotes CSC progression and thus leads to the deterioration and metastasis of cancer . For insSeveral compounds have been identified to target CSCs via Wnt/β-catenin signaling pathway Table , Fig. 4.+ cells (liver CSCs) and the sphere-forming ability of hepatocellular carcinoma (HCC) cells, as well as in CRC and bladder cancer cells [Furthermore, many potential compounds targeting CSCs through inhibiting Wnt/β-catenin signaling pathway have been undertaken in preclinical evaluations. For example, XAV939 inhibited β-catenin signaling, thus attenuated CSC progression, thereby eliminating the CSC-mediated chemical resistance in head and neck squamous cell carcinoma (HNSCC) and colon cancer cells –147. IWRer cells . In addier cells . A similer cells , 155. Ther cells –158. Adder cells . The treer cells . Anti-prer cells .Aberrant activation of Wnt/β-catenin signaling drives oncogenic transformation in a wide range of cancers, indicating the key pathway modulators as attractive therapeutic targets in malignancies. Despite that Wnt/β-catenin targeted therapies are varied and clinical experience nascent, with the development of the targeted agents and combination strategies under investigation, the risk for off-targeting effectivity, side effects and toxicities are not allowed to be neglected. Of note, the critical role of Wnt/β-catenin signaling in stem cell maintenance raised concerns regarding the dose-limiting toxicity of targeted agents in bone, hair and gastrointestinal tract as well as in hematopoiesis, which limited of its clinical application –164. BesNovel strategies are imperative to improve the outcome of cancer patients. With great advances in the knowledge of molecular basis and the constant effort for improvement, preclinical investigations and clinical trials have been conducted on the Wnt/β-catenin signaling targeted interventions in malignancies. The Wnt/β-catenin signaling targeted regimens have been proved to represent promising candidates of individualized approaches in the treatment of cancer patients. Further investigations are expected on confirming the safety, efficacy, patient stratification and drug delivery of innovative Wnt/β-catenin targeted therapies in cancer.Additional file 1. Formulas and structures of agents targeted Wnt/β-catenin signaling pathway.


Existing rammed earth construction methods have disadvantages such as increased initial costs for manufacturing the large formwork and increased labor costs owing to the labor-intensive construction techniques involved. To address the limitations of the existing rammed earth construction methods, an autonomous rammed earth construction method was introduced herein. When constructing an autonomous rammed-earth construction method, an alternative means of assuring the performance at the initial age of the binder in terms of materials is needed. In this study, in order to satisfy the performance of the red clay binder, epoxy emulsion was added to analyze the compressive strength, water loosening, shrinkage, rate of mass change, and microstructure in the range of the initial age. As a result of the analysis, the applicability of the epoxy emulsion was confirmed as a new additive for application to an autonomous rammed-earth construction method. Earth buildings utilize soil as their primary material, allowing these buildings to reflect the characteristics of the soil itself. These buildings have excellent humidity control, deodorization rate, and energy efficiency, and are also eco-friendly owing to the influence of far-infrared radiation ,2,3,4. EIn existing rammed earth construction methods, a large integrated formwork is created and filled with soil, and then directly rammed with a vibrator to build the bearing wall ,12,13,14Prior to the experiments in this study, a preliminary experiment was performed to select the optimal formulation of the epoxy emulsion, which is composed of the epoxy and a hardener. The preliminary research results demonstrated that satisfactory performance could not be obtained with use of the epoxy and hardener alone. Thus, the total amount of epoxy emulsion was selected by considering the viscosity of the epoxy and hardener. Four epoxy emulsion concentrations were used for the red clay + polymer aqueous solution + epoxy emulsion (RPE) test specimens: 4.0, 5.6, 6.8, and 8.0%. In terms of the evaluation indices, the compressive strength was measured at 3, 6, 12, and 24 h of aging.2/g.The aqueous polymer solution was in the form of poly(AA-co-AM), in which the monomer acrylic acid (AA) and acrylamide (AM) were copolymerized. The intrinsic viscosity of the polymer aqueous solution was 2–3 dL/g.The experimental plan of this study is summarized in A mortar mixer was used to mechanically mix the formulation with the red clay binder. First, the sample was weighed, poured into the mixer container, and dry mixed for 30 s. The additives were then added uniformly, and the sample was mixed for an additional 1 min. The binder that had become attached to the bottom and wall surfaces of the mixer container was then removed and collected in the center of the container, after which it was mixed again for 1 min. In the epoxy emulsion, to prevent agglomeration due to the viscosity of the epoxy and hardener, the epoxy and hardener were each weighed using a 20 mL laboratory syringe in a beaker in which the polymer aqueous solution had previously been weighed. The epoxy emulsion was then mixed for at least 1 min using a reagent spoon and added to a mortar mixer container.To produce a specimen with a size of 50 mm × 50 mm × 50 mm, 240 g of the red clay binder was weighed. One-third of the weighed sample was then placed into each of three molds and consolidated to 2 MPa using a universal material tester to ensure uniform compaction. After reaching 2 MPa, the consolidation was maintained for 1 min to produce the specimen. The prepared specimens were cured at a constant temperature of 20 °C and relative humidity of 60%.To measure the compressive strength based on specification KS L 5105 , three sMeanwhile, to investigate the effect of curing temperature on the compressive strength, the compressive strength of the RPE specimens was measured at an age of 24 h after curing at temperatures of 5, 20, and 35 °C.Among the specimens cured for 7 d, the specimens coated with inorganic ceramic resin (CERAST red clay waterproofing coating agent) and uncoated specimens were separated, and the water loosening behavior was observed. First, an inorganic ceramic resin was applied to the specimens with sizes of 50 mm × 50 mm × 50 mm, and a primary coating was applied to permeate the coating agent. After 30 min, the coating was reapplied to complete the coating of the specimen. Then, each specimen was immersed and observed at 3 h intervals for 48 h.Specimens with sizes of 50 mm × 50 mm × 50 mm were prepared and cured for 1 d at a constant temperature of 20 °C and relative humidity of 60%, after which the shrinkage was measured. First, a pressure sensitive  adhesive was applied and reapplied to remove the surface voids of the specimen. Then, strain gauges (PFL-10-11-1LJC) were attached to both sides of the specimen with a cyanoacrylate  adhesive. The strain gauges were then connected to a data logger (TDS-303) and the strain was measured at 10 min intervals until 28 d of aging.Meanwhile, specimens with sizes of 50 mm × 50 mm × 50 mm were produced, the mass immediately after demolding was recorded, and the rate of mass change was measured at ages of 1, 3, 7, 14, 21, and 28 d at a constant temperature of 20 °C and relative humidity of 60%.Powder samples taken at 24 h of aging were coated with platinum, and the microstructure was observed at magnifications of ×3000 and ×5000 using a scanning electron microscope (SNE-3200M) at an acceleration voltage of 15 kV.Powder samples collected at 24 h of aging were completely dried in a drying furnace at a temperature of 60 °C. Non-wetting mercury was then pressurized at 0–60,000 psi, and the total cumulative porosity and pore diameter of the samples were measured based on the amount of intrusion using a porosimeter (AutoPore IV 9520).The RPE specimen reached a compressive strength of 2.4 MPa within just 12 h of aging, which satisfies the requirement for earthen walls specified in the ACP-EEC rammed earth construction regulations at 28 d of curing [In contrast, the coated red clay binders achieved water resistance at all formulation levels based on the water loosening results obtained up to 48 h after immersion. This indicates that when applying red clay binders in the field, a waterproof coating agent must be applied to ensure water resistance.Though the rates of mass change did not differ considerably overall, the RPE specimens had a smaller rate of mass change than the RP and RPC specimens at early ages. This is attributed to the increased attractive force between the red clay particles due to the curing mechanisms of the epoxy and hardener in the epoxy emulsion. Specifically, owing to the rapid setting of the red clay binder caused by the molecular bonds of the epoxy and hardener, the rate of mass change due to drying is small but the shrinkage is large.On the other hand, the RPE specimen c, which For the RPE formulation, which contains epoxy emulsion, to examine the curing properties of the red clay binder with varying temperature, the compressive strength was measured under different curing conditions. Adding an epoxy emulsion increased the attractive force between the red clay binder particles owing to the hardening mechanism of the epoxy and hardener in the epoxy emulsion, thereby causing the red clay particles to agglomerate in a wide plate shape and thus improving the strength of the red clay binder.In general, the use of cement as a solidifying material has been suggested to develop the strength of the binder in the rammed earth construction method ,26,27. OThe red clay binder without a coating agent could not achieve water resistance. On the other hand, applying a coating agent enabled the specimen to achieve water resistance, indicating that when applying red clay binders in the field, a coating agent must be applied to ensure water resistance.Owing to the molecular bonding between the epoxy and hardener, the red clay binder set rapidly. Accordingly, it was determined that shrinkage increases as the attractive force between the red clay particles increases with age. In addition, the red clay particles agglomerated in a wide plate shape and the pore diameter due to the gaps between the particles increased, while the total cumulative pore volume decreased.The epoxy emulsion applied in this study enhanced the strength and durability of the red clay binder as a result of the curing mechanisms of the epoxy and hardener. This suggests that an epoxy emulsion can be applied to satisfy the performance requirements for autonomous rammed earth construction, such as improving the durability and strength at an early age.This study evaluated the performance of a red clay binder with the addition of an epoxy emulsion by analyzing the compressive strength, water loosening, shrinkage, and microstructure of the red clay binder. The compressive strength characteristics were also evaluated under different curing conditions. In these evaluations, the formulation condition of the epoxy emulsion was varied. The following conclusions were obtained in this study.


MTAP gene is frequently codeleted with the adjacent cyclin‐dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock‐down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP‐deleted MM cells. We also observed that PRMT5 knock‐down in MTAP‐deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial‐to‐mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP‐deleted MMs.Malignant mesothelioma (MM) is an aggressive asbestos‐related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)‐deficient cancers, in which the accumulation of the substrate 5'‐methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the  MM is mainly associated with asbestos exposure and is the most common type of asbestos‐related cancer.Despite current intensive treatment regimens, including chemotherapy, radiation and surgery, MM patients’ overall survival (OS) range from 8 to 36 months, depending on the stage and the histological subtype.Therefore, a better understanding of the molecular machinery underlying MM development is required to move forward against this malignancy. In particular, the discovery of crucial pathways for MM cell proliferation and spreading and the identification of new druggable targets and predictive markers for the stratification of patients who could benefit from a specific therapy are fundamental goals of pre‐clinical research aimed to the improvement of patients’ outcome.CDKN2A) locus, inactivation of the retinoblastoma (RB) pathway, mutations in the BRCA1‐associated protein 1 (BAP1) and neurofibromatosis type 2 (NF2) genes, and aberrant regulation of phosphatidylinositol‐4,5‐ bisphosphate 3‐kinase (PI3K)/AKT pathway are all related to MM uncontrolled growth and resistance to treatment‐induced cell death.Among the most common genetic alterations or deregulated pathways identified in MM, deletions in the cyclin‐dependent kinase inhibitor 2A  gene, encoding a key enzyme in the adenosine and methionine salvage pathway from the substrate 5'‐methylthioadenosine (MTA), is frequently codeleted with CDKN2A in different cancer typesMTAP gene has been suggested to be a tumour suppressor, the loss of which results in a higher cell invasive potential and poor prognosis for patients with different cancer types.MTAP loss determines the accumulation of the MTA substrate, a natural inhibitor of protein arginine methyltransferase 5 (PRMT5), thus generating a hypomorphic PRMT5 state in MTAP‐deficient cancers that are, in this way, selectively sensitized to further PRMT5 inhibition. This vulnerability can be exploited therapeutically, and PRMT5 targeting in MTAP‐deficient cancers has indeed become the focus of recent research.Deletion on the short arm of chromosome 9, including the PRMT5 belongs to a family of ten protein arginine methyltransferases (PRMTs) ubiquitously expressed in mammalian cells, which methylate arginine residues on histones and other proteins, although their biological role is still underexplored. PRMT5 regulates a broad range of physiological and cancer‐associated processes, such as DNA damage response, apoptosis control, EMT and inflammation, and is involved in the inhibition of tumour suppressors, including RB proteins, p53, programmed cell death 4 (PDCD4) and activation of survival pathways such as PI3K/AKT axisOverall, these considerations prompted us to investigate whether PRMT5 could be a valuable MM therapeutic target, the inhibition of which could impact on pathways fundamental for MM biology.22.1Formalin‐fixed, paraffin‐embedded tumour specimens were used for tissue microarray (TMA) construction. Multi‐tissue pleural mesothelioma arrays were obtained from the Section of Pathology, Siena Hospital, Siena, Italy, and the Anatomy and Pathology Unit, Ospedale dei Colli, AORN, ‘Monaldi’, Naples, Italy, and consisted of 2‐mm representative areas of resected tumour and normal pleura controls. From each tissue microarray, 4‐μm‐thick paraffin sections were prepared for immunohistochemistry. Clinical information about mesothelioma specimens is summarized in Table Based on the expression patterns identified in the resection specimens, the tumour cell staining in TMA was evaluated in comparison with normal pleura. Two pathologists blinded to the clinical data evaluated the staining of each specimen. To avoid inter‐observer variability, the mean value of the scores was adapted for further analysis. The primary rabbit polyclonal anti‐PRMT5 antibody  at 1:70 dilutions was used according to the manufacturer's instructions.The assessment of PRMT5 expression levels included the staining intensity and the percentage of stained cells. PRMT5 was analysed for both nuclear and cytoplasmic staining. The staining intensity was scored as 0 = no staining, 1 = moderate expression and 2 = strong expression; the results were categorized according to the following distribution: 0 =< 10%, 1 = 10% – 50% and 2 ≥ 50% staining. The PRMT5 expression score was determined as a combined score of staining intensity and distribution. Samples with a final immunoscore ≥ 2 were considered as PRMT5‐positive.2.2NCI‐H2452  and MeT‐5A  cell lines were purchased from the American Type Culture Collection ; LP‐9 cells were from Coriell Institute ; IST‐Mes1 , IST‐Mes2  and MPP 89  were purchased from the ISTGE Cell Repository ; and MMB‐1 (RRID:CVCL_IW98) and REN (RRID:CVCL_M202) were a kind gift of Prof. Giovanni Gaudino . All the cell lines were cultured according to the manufacturer's protocols. Human mesothelial cells (HMC‐NEO) immortalized with a PSV3NEO plasmid were kindly provided by Prof. Paolo Pinton .MMP1, MMP2 and MMP4 mesothelioma cell lines were isolated from patients’ who underwent surgery at the Thoracic Surgery Unit  for decortication, without prior chemotherapy or radiotherapy. MMP6 cell line was derived from pleural effusion. All specimens were collected from patients diagnosed for pleural mesothelioma  selected for surgery based on the pre‐operative staging and with their written consent. Non‐immortalized HMC1 cells were obtained from pleural effusion of a patient with heart failure. Human investigations were performed after Research Ethics Committee (Comitato Etico Regione Toscana‐Area Vasta Sud Est) approval (#CCMESOLUNG). The study is conformed to the standards of the Declaration of Helsinki. The original pathologic materials were analysed by light microscopic analysis, followed by extensive immunocytochemical analysis using a battery of markers Table .Both solid tumours and pleural effusions were transported to the laboratory for primary cell culturing within 30 minutes of collection. Solid tissue was minced into small pieces, 1–3 mm, and then incubated in complete medium supplemented with collagenase type I from Clostridium histolyticum  at 200 U/mL concentration for 1 hour to digest collagen and release tumour cells. Macrophages, red blood cells and lymphocytes were the main contaminants; to avoid their interference in the analysis, all the primary cells were used after the 6th passage.g for 5 minutes and placed into culture flasks.Effusions were centrifuged at 400  l‐glutamine , 100 U/mL penicillin, 100 μg/mL streptomycin , 10% FBS , 20 ng/mL hEGF , 0.4 µg/ml hydrocortisone  at 37°C and 5% CO2. All cell lines were routinely passaged every 1–2 weeks.All primary cell lines were cultured in Medium 199 , supplemented with 2 mmol/L Mesothelial origin of primary cell cultures was assessed by haematoxylin/eosin, calretinin and WT‐1 staining.2.3CDKN2A gene  deletions and the classical satellite III region of chromosome 9 (CEN9)  were detected by fluorescence in situ hybridization (FISH), using SPEC CDKN2A/CEN 9 Dual Color Probe ZytoLight . Cell lines were fixed with 4% paraformaldehyde for 10 minutes at RT, washed with PBS and pre‐treated with heat pre‐treatment solution citric, following the manufacturer's protocol. FISH was performed with a hybridization automation . Probe was placed on the samples, covered with a glass slide and then sealed with rubber cement. After codenaturation at 78°C for 10 minutes, the probe and the target DNA were allowed to hybridize at 37˚C overnight in a humid and dark atmosphere. Next day, the excess of probe was washed in 25× wash buffer A at 37°C. Slides were air‐dried in the darkness and counterstained with 4',6‐diamidino‐2‐phenylindole (DAPI). Analysis was performed using a fluorescent microscope  and Leica LAS v3.8 Software (Leica) at ×630 magnification, equipped with SpectrumGreen™, SpectrumOrange™ filters. For each specimen, at last 100 intact non‐overlapping nuclei with good signals were required for valid scoring.Qualitative detection of 2.45 cells were scraped directly into 500 µL of pure methanol spiked with 2.48 nmol/L of stable isotope‐labelled internal standard. Scraped cells were centrifuged , and the supernatant was collected. The cell pellet was washed twice with 200 µL methanol and centrifuged, and all supernatants were combined, dried and reconstituted in 100 µL of water. All solvents for sample preparation and LC‐MS/MS were HPLC grade and purchased from Sigma‐Aldrich. MTA was obtained from Sigma‐Aldrich (Cat #D5011) and labelled. 15N5 8‐hydroxy‐2'‐deoxyguanosine was obtained from Cambridge Isotope Laboratories Inc. The water used was purified by means of a MilliQ (Millipore).The protocol used is based on the method published by Stevens and coworkersLC‐MS/MS was performed using an Agilent 1200 SL HPLC system and an ABSciex API 4000 triple quadrupole mass spectrometer (AB Sciex), which was equipped with a turbo ion spray source . A Luna C18  reversed phase column (Phenomenex) was used. LC separation was carried out using a mobile phase consisting of 0.1% acetic acid in water (Solvent A) and 0.1% acetic acid in acetonitrile (Solvent B). The gradient employed was as follows: 0‐2 minutes isocratic 5% solvent B, 2‐10 minutes linear increase from 5% to 100% solvent B, hold at 100% solvent B for 3 minutes, and 3 minutes post‐run equilibration. The flow rate was set to 500 µL/min. Sample volumes of 20 µL were injected. The API 4000 mass spectrometer was operated in positive mode using turbo ion spray with the following parameters: gas 1 as 55, gas 2 as 45 and the curtain gas as 17 .The turbo ion spray source was heated to 450°C. The declustering potential was set to 47 and the entrance potential to 9.4 V.The transition analysed was 296.3/136 (MTA Quantifier) 296.3/119.2  269.0/173.0 (IS).2.5MTA was obtained from Sigma‐Aldrich (Cat #D5011). Stock solutions of the drug were prepared in dimethyl sulfoxide cell culture grade (DMSO)  and stored at −20°C.http://www.graphpad.com/scientificsoftware/prism/ ).Cells were seeded in 96‐well plates  24 hours before treatment with MTA and incubated for further 72 hours. Control cells were treated with DMSO at the same amount used to deliver the molecule. Each experiment was conducted in triplicate. Cell viability was evaluated by sulforhodamine B (SRB) assay , as previously described.2.6PRMT5  or sham sequences  according to the manufacturer's instructions. Cell lines were selected with puromycin , and PRMT5 knock‐down was verified by real‐time qRT‐PCR and Western blotting.NCI‐H2452, IST‐Mes2, Met‐5A, MMB‐1 and MPP 89 cell lines were transduced with lentiviral vectors expressing short hairpins RNAs against 2.7MTAP, Assay ID qHsaCED0004212; PRMT5, Assay ID qHsaCED0042792; E2F1, Assay ID qHsaCED0042864; TS, Assay ID qHsaCED0047209; RBL2, Assay ID qHsaCED0042975; CCNA2, Assay ID qHsaCID0017452; CCNE, Assay ID qHsaCID0015131; EZH2, Assay ID qHsaCED0043150; MMP9, Assay ID qHsaCID0011597; GAPDH and Assay ID qHsaCED0038674. The housekeeping glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene was used to normalize the expression of genes of interest. Gene expression levels were calculated by the 2−ΔΔCt method.Total RNA was isolated from cell lines using the RNeasy Mini kit . RNA concentration was determined using a NanoDrop™ ND‐1000 (Thermo Fisher). Complementary DNA (cDNA) was synthesized from 500 ng of RNA using the iScript cDNA Synthesis Kit  and amplified in the LightCycler™ instrument (Roche Applied Sciences) using SsoAdvanced™ Universal SYBR® Green Supermix  according to the manufacturer's instruction. The primers used were from Bio‐Rad: 2.8For Western blotting analysis, cells were harvested on ice and lysed as previously describedMembranes were washed with TBS with 0.1% Tween‐20 and incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Membranes were washed before chemiluminescence detection using Clarity ECL reagents .2.9Single‐cell suspension cultures of log‐phase growing parental/transduced NCI‐H2452, IST‐Mes2, Met‐5A, MMB‐1 and MPP 89 cells were seeded in triplicate in Petri dishes (35 mm diameter). After 24, 48 and 72 hours, the cells from each well were harvested by trypsinization and viable cells were counted in a haemocytometer chamber. Cell viability was evaluated by the trypan blue dye exclusion test.2.10t test. Statistically significant differences among the growth curves were evaluated by two‐way repeated measures ANOVA P < .05 was considered statistically significant.Statistical analyses were performed using the GraphPad Prism Software, version 5.01 for Windows. Statistically significant differences between the means of multiple matched groups were evaluated by one‐way repeated measures ANOVA with Tukey's post‐test. To compare the means of 2 unmatched groups, we used the two‐sided unpaired Student's 33.1To explore the role of PRMT5 in MM, we first analysed its expression in 58 MM tissue specimens and 10 normal pleural samples through immunohistochemistry. Our analysis revealed that 43% of the MM samples expressed PRMT5 in both nuclei and cytoplasms Figure  or nucleThen, we analysed PRMT5 expression in a panel of previously established/commercial  and patient‐derived primary  MM cell lines and in normal mesothelial cells , by both real‐time quantitative reverse transcription‐PCR (qRT‐PCR) and Western blotting, and found that PRMT5 was expressed, although at different levels, in all the cells analysed . Consistent results were also obtained through Western blotting . Consistent with what previously demonstrated in other cancer types,MTAP‐negative MM cells showed a significantly higher mean intracellular MTA level compared with that of MTAP‐positive cells  assay. We observed that MTA had cytotoxic effects on all MTAP‐deleted cells, resulting in the indicated half maximal inhibitory concentration (IC50) values  or a non‐targeting (NT) control shRNA, the MTAP‐negative MM cells, NCI‐H2452, MMB‐1 and IST‐Mes2, the MTAP‐positive MM cells, MPP 89, and the non‐tumour mesothelial cells, MeT‐5A. Infected cells were selected for puromycin resistance, and PRMT5 silencing was verified by Western blotting  and MTAP‐negative  cells. As shown in Figure PRMT5 silencing only in MTAP‐negative MM cells.We then analysed the impact of 3.4MTAP‐deleted MM cells, we analysed the effect of PRMT5 knock‐down on the expression of genes involved in cancer cell growth. Among the broad range of factors regulated by PRMT5, we first investigated whether PRMT5 silencing could affect the E2F pathway in MTAP‐deleted MM cells. We observed that PRMT5 knock‐down inhibited the E2F1 growth‐promoting signalling, as suggested by the down‐regulation of the E2F1‐regulated genes CCNA2), cyclin E1 (CCNE1), enhancer of zeste homolog 2 (EZH2), thymidylate synthase (TS) and E2F1 itself  and matrix metallopeptidase 9 (MMP9).Therefore, we investigated the effect of 4MTAP, resulting from the deletion of the CDKN2A tumour suppressor gene, generates a selective vulnerability to PRMT5 inhibitionRecent advances in cancer metabolism research have identified molecular defects leading to vulnerabilities, which can be exploited therapeuticallyAlthough PRMT5 has been proposed as a possible therapeutic target also for MM,MTAP status and MTA intracellular content in a panel of MM and normal mesothelial cell lines and, in line with what previously reported for other cancer types,MTAP‐deleted MM cells and a greater cytotoxic effect of exogenous addition of MTA, the natural inhibitor of PRMT5, in these cells. Consistently, shRNA silencing of PRMT5 led to a decreased cell growth and clonogenic potential selectively in MTAP‐deleted MM cells, similar to a previous observation We then analysed the PRMT5 knock‐down led to a decreased expression of E2F1 target genes in MTAP‐negative cells. E2F1 has previously been found to exert controversial roles in different human cancers, promoting either proliferation or tumour suppression, depending on the context PRMT5‐silenced MM cells, it could be hypothesized that the E2F1 activity in MM is affected by PRMT5‐mediated arginine methylation. However, this needs further investigation.To analyse at the molecular level the growth‐promoting activity of PRMT5 in MM cells, we first focused on the E2F pathway. We observed that PRMT5 silencing and involved in cell cycle progression, the EZH2 gene encodes a factor implicated in the epigenetic regulation of gene expressionInterestingly, among the E2F1 target genes down‐regulated by Finally, we identified a subset of EMT markers regulated by PRMT5 in MM, including E‐cadherin, N‐cadherin, α‐smooth muscle actin and MMP9, indicating that targeting PRMT5 could hamper this crucial process for cancer progression.5MTAP‐deleted MM cells, also showing the effect of its silencing on the expression of genes implicated in MM growth and progression. In agreement with previous studies underlining the potential value of PRMT5 as a therapeutic approach,MTAP‐deleted mesothelioma.To the best of our knowledge, this is the first study extensively exploring the possible therapeutic potential of targeting PRMT5 in The authors have no conflicts of interest to declare.1 designed the research study and wrote the manuscript. MB1 and AG contributed to funding acquisition. AG, PI and LM reviewed the manuscript. DC, MB2, FMB and AN performed biological and molecular experiments. FP performed LC‐MS/MS measurement of MTA. PI provided suggestions on data presentation and performed statistical analysis. LL, LP and PP furnished patient‐derived biopsies for cell lines establishment. CB, MdS, RG, MM and PS provide IHC analysis and results.MB1: Marcella Barbarino; MB2: Maria Bottaro.MBFig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Metastatic urothelial cell carcinoma (UCC) is a significant public health burden with a median survival estimated at about 15 months. The use of immunotherapy with immune checkpoint inhibitors has greatly improved outcomes but only benefits a minority (~20%) of patients. In this review we discuss the evidence showing how a key molecular pathway known as Wnt/β-catenin signaling can be a driver of immunotherapy resistance and how these insights can serve as lessons for improving future treatment of urothelial carcinoma. Urothelial cell carcinoma (UCC) is a significant public health burden. It accounts for approximately 90 percent of all bladder cancers with an estimated 200,000 annual deaths globally. Platinum based cytotoxic chemotherapy combinations are the current standard of care in the frontline setting for metastatic UCC. Even with these treatments the median overall survival is estimated to be about 15 months. Recently, immune checkpoint inhibitors (ICIs) have demonstrated superior clinical benefits compared to second line chemotherapy in UCC treatment. However only a minority of patients (~20%) respond to ICIs, which highlights the need to better understand the mechanisms behind resistance. In this review, we (i) examine the pathophysiology of Wnt/β-catenin signaling, (ii) discuss pre-clinical evidence that supports the combination of Wnt/β-catenin inhibitors and ICI, and (iii) propose future combination treatments that could be investigated through clinical trials. Urothelial cell carcinoma (UCC) is the most common malignancy of the urinary system. It accounts for approximately 90 percent of all bladder cancers with an estimated 200,000 annual deaths globally ,2. UCC iCytotoxic chemotherapy is the current standard of care in the frontline setting for metastatic UCC. The median overall survival is estimated to be about 15 months with modern chemotherapy regimens containing platinum-based agents ,4. Once More recently, immune checkpoint inhibitors (ICIs) have demonstrated superior clinical benefits compared to second line chemotherapy in UCC treatment ,8. HowevSeveral mechanisms of ICI resistance in cancers have been reviewed extensively elsewhere ,12. PrevWnt signaling is a highly coordinated and conserved signaling cascade that occurs at the cell surface and within the cytoplasm. This pathway mediates an array of biological functions, including cell fate decisions during embryonic development, stem cell equipoise, and immune system homeostasis ,14,15,16Canonical, or β-catenin-dependent, Wnt signaling is one of the primary sources of dysregulated transcription in cancer. In the “on-state”, the signal cascade begins at the cell surface with Wnt ligands binding to the Frizzled:LRP5/LRP6 receptor complexes, and culminates in the nucleus with the formation of a transcription-activating complex . The priOnce bound to DNA by TCF/LEFs, β-catenin recruits other co-activators and regulatory components that collectively activate transcription of the downstream genes known as the Wnt target genes. These sets of Wnt target genes drive cells to proliferate, self-renew, differentiate and survive in a variety of tissues and contexts. In normal cells, feedback inhibition results in this activity occurring only transiently, which in turn prevents overactivation of Wnt target gene transcription. Signal transduction is thus “turned off” in cells with low or absent Wnt because β-catenin becomes unstable by being tagged in the cytoplasm for ubiquitination by the destruction complex, which then leads to proteasome degradation.APC, AXIN2 and FAM123B/WTX) or regulators of the receptors/ligand  components can lead to unchecked Wnt signaling. These mutations negate the cytoplasmic feedback controls and create cells with constitutive, high levels of β-catenin and aberrantly high levels of Wnt target gene transcription that can initiate carcinogenesis and immune suppression [However, in various cancers  mutations in the destruction complex components  promoter was found to lead to decreased transcription and increased Wnt signaling activity in human bladder cancer cell lines . KnockdoDue to the limited efficacy of ICI treatments, much effort is being dedicated to developing predictive biomarkers of response and understanding the biological mechanisms for resistance. One widely established predictive biomarker for ICI response is intratumoral enrichment of CD8+ T-cells prior to treatment ,43. TherA recent study by Sweis et al. used a bioinformatics approach to correlate CD8+ T-cell infiltration with various signaling pathways . The invOnce stratified into inflamed vs. non-inflamed phenotypes, the investigators uncovered that 730 genes were preferentially expressed in the non-T-cell-inflamed tumors. Ingenuity pathway analysis then showed that one of the top upstream regulators for these groups of differentially expressed genes were those that were regulated by β-catenin/Wnt signaling. The authors then went back to the 19 samples which they had initially performed CD8+ T-cell IHC staining and co-stained for nuclear β-catenin as a marker for active β-catenin dependent Wnt signaling. The investigators found a statistically significant inverse relationship between nuclear β-catenin and the density of CD8+ T cells infiltrating the tumor.CTNNB1 (gene for β-catenin) and other regulatory genes predicted to result in pathway activation, expression of downstream Wnt target genes, and β-catenin protein levels which were assessed through reverse phase protein array (RPPA). With respect to the 363 UCC samples included in this cohort, all three levels correlated with a non-T-cell inflamed tumor signature, the most pronounced of which was CTNNB1 protein level. Taken together, these findings suggest that there is a significant correlation between upregulation of Wnt signaling and a non-T-cell-inflamed microenvironment in UCC.To further validate the Wnt signaling pathway as a mediator of non-T-cell-inflamed tumor microenvironments, a follow up study done by Luke et al. employed a similar approach  and analyzed 9244 samples across 31 different types of cancers . The invAs previously discussed, translational studies have suggested that Wnt/β-catenin signaling may induce a non-T-cell-inflamed tumor phenotype thereby excluding immune cells from the tumor microenvironment and dampening the therapeutic effect of ICIs. To elucidate molecular mediators, the Gajewski group used a genetically engineered melanoma mouse model with active β-catenin signaling (BRAF/PTEN/CAT-STA) in the tumors .activated (BRAF/PTEN/Bcat-STA) mice but remained effective in Wnt-inactivated (BRAF/PTEN-STA) mice. These results suggested that upregulation of Wnt/β-catenin may indeed induce resistance to immune checkpoint inhibition.In their mouse model, the authors found that β-catenin signaling activation was associated with low levels of tumor infiltrating CD8+ T-cells. Conversely, mice in which β-catenin signaling was absent contained a high density of CD8+ T-cell infiltration. In order to discern if this was due to differences in neo-antigens, the authors introduced a neo-antigen (SIY) expressing construct genetically into the tumors and adoptively transferred T-cells with SIY T cell receptor. They found that the transferred T-cells accumulated in the BRAF/PTEN-STA tumors but not the β-catenin expressing BRAF/PTEN/Bcat-STA tumors despite both tumors now expressing the neo-antigen. Furthermore, anti-PD-1 and anti-CTLA-4 agents were rendered ineffective in the Wnt-The investigators then queried whether this blunted response to ICIs could be dependent on antigen presentation from CD103+ dendritic cells (DC). Within Wnt/β-catenin-activated T-cell-depleted tumors, they found that CD103+ DCs were nearly absent and IFN-β cytokine expression was reduced. The investigators then found that intratumoral injection of CD103+ DCs led to restoration of T-cells infiltration within the tumor. This supported the role of CD103+ dendritic cells as key mediators of an antitumor immune response. To characterize the mechanism of failed recruitment of the CD103+ DCs, the investigators analyzed the gene expression of these two tumor types and found that four chemokines  were lower in the non-T-cell inflamed BRAF/PTEN/Bcat-STA tumors. Of these four chemokines, only CCL4 was found on an in vitro DC migration assay to possess the ability to effectively modulate cell migration.Furthermore, the investigators found that the Wnt signaling target gene ATF3–which also binds at the promoter region of the CCL4 gene—was expressed at higher levels in the β-catenin activated melanoma tumors. This negative feedback was substantiated by then demonstrating that gene knockdown of ATF3 and CTNNB1 in melanoma cell lines led to upregulation of CCL4 expression .Tumor-associated macrophages (TAMs) are amongst the most common tumor immune infiltrating cells in the tumor microenvironment (TME) . TAMs arIt has been shown that Wnt/β-catenin signaling can modulate the TAMs population in the TME leading to a protumoral phenotype which may be ICI resistant ,51. In aAnother potential mechanism for ICI resistance is through the tumor’s ability to create a hostile TME that is acidic from increased lactic acid production which can lead to impaired cytotoxic T-cell function ,54,55. AAs discussed above, the Wnt/β-catenin signaling pathway was found to initially play a central role in carcinogenesis by driving cell proliferation . More relow murine melanoma and pancreatic cancer cell lines [high or LDHlow into syngeneic mice [low had impaired tumor growth and higher T-cell and NK cell infiltration compared to the LDHhigh tumors [It has also been shown that the lactic acid in the TME can play a role in immunosuppression and drive further tumor growth ,54,64. IThe above studies provide evidence supporting the hypothesis that the presence of lactic acid in the TME can be immunosuppressive by inhibiting the needed oxidative phosphorylation of cytotoxic T-cells. As a result, this has led to the proposal that targeting lactic acid production could be a potential way to overcome ICI resistance . In summThe above mentioned studies provide strong evidence that the Wnt/β-catenin signaling pathway drives immune cell exclusion which can then lead to immune checkpoint inhibitor resistance in cancer treatment ,45,65. AEarly therapeutic efforts primarily centered on finding targets for Wnt inhibition ,66,67,68More recently, several studies have shifted focus toward downstream inhibition of the intranuclear transcriptional β-catenin complex to enhance immune cell infiltration within the tumor microenvironment. For instance, Ganesh et al. designed a β-catenin inhibitor (DCR-BCAT) that selectively silenced CTNNB1 (the gene which transcribes/β-catenin) in tumors using an RNAi oligonucleotide . Using aEncouraged by these results, the investigators subsequently examined if their β-catenin inhibitor could reconstitute an immune response within their T-cell-excluded tumor model. Although monotherapy with either the DCR-BCAT or an ICI was minimally effective, the combination of DCR-BCAT plus ICI elicited a synergistic effect with reductions in tumor size by as much as 87% . MoreoveIn recent years, several novel agents with varied mechanisms of action have attempted to mitigate the immunosuppressive tumor microenvironment through WNT/β-catenin inhibition , Table 1Another class of WNT/β-catenin inhibitors disrupt PORCN, an enzyme that facilitates WNT secretion . A recenIn summary, WNT/β-catenin signaling can drive immune cell exclusion and may be a resistance mechanism for immune checkpoint inhibitors. Several preclinical studies have shown that inhibition of the WNT/β-catenin pathway in conjunction with an ICI can effectively overcome this resistance mechanism. With respect to UCC, this combination is particularly promising given the high frequency of WNT/β-catenin aberrations in correlative studies as well as its potential role in upregulating urothelial oncogenesis. Thus, to complement ongoing clinical trials across other solid tumors, additional studies that validate the synergistic relationship of ICIs and WNT/β-catenin inhibitors in UCC are urgently needed.


Emerging evidence shows that type II protein arginine methyltransferase 5 (PRMT5) serves as an oncoprotein and plays a critical role in many types of human cancer. However, the precise role and function of PRMT5 in human colorectal cancer (CRC) growth and epithelial-mesenchymal transition (EMT) are still unclear, and the related molecular mechanism and signaling axis remains largely obscure. Here, we show that PRMT5 is highly expressed in CRC cell lines and tissues. Using PRMT5 stable depletion cell lines and specific inhibitor, we discover that down-regulation of PRMT5 by shRNA or inhibition of PRMT5 activity by specific inhibitor GSK591 markedly suppresses CRC cell proliferation and cell cycle progression, which is closely associated with PRMT5 enzyme activity. Moreover, PRMT5 regulates CRC cell growth and cycle progression via activation of Akt, but not through ERK1/2, PTEN, and mTOR signaling pathway. Further study shows that PRMT5 controls EMT of CRC cells by activation of EGFR/Akt/GSK3β signaling cascades. Collectively, our results reveal that PRMT5 promotes CRC cell proliferation, cell cycle progression, and EMT via regulation of EGFR/Akt/GSK3β signaling cascades. Most importantly, our findings also suggest that PRMT5 may be a potential therapeutic target for the treatment of human colorectal cancer. APC, KRAS, and TP53, was involved in the CRC development and progression [BRAF activation and CpG methylation [Human colorectal cancer (CRC) is the most common tumor globally, which affects human life and results in death. CRC is a chronic disease, and the clinical manifestations are sporadic and develop very slowly in most humans over many years. The accumulating evidence suggested that in most cases, several genes mutation, including gression . Neverthhylation . Thus, aProtein arginine methyltransferases (PRMTs) have been involved in and play critical roles in multiple cellular physiological processes, including chromatin remodeling, gene expression, RNA splicing, cell cycle, cellular signaling crosstalk, proliferation, apoptosis, and protein functions , 4. ProtIn the current study, we uncovered the novel role of PRMT5 in human colorectal cancer and revealed the underlying molecular mechanism by which PRMT5 regulated CRC cell proliferation and EMT. Our findings showed that PRMT5 was highly expressed in CRC cell lines and tissues. Moreover, PRMT5 controls cell growth and cell cycle progression via activation of Akt, which is closely related to the enzyme activity of PRMT5. Most importantly, PRMT5 gated EMT probably through EGFR/Akt/GSK3β signaling cascades.A previous study has shown that PRMT5 is involved in many types of human cancer and tumor progression, including colorectal cancer . In ordeArginine methylation plays a key role in the regulation of gene transcription and post-translational protein modification . MoreoveIn order to explore the role of PRMT5 in human colorectal cancer, we generated the PRMT5 stable knockdown cell lines using lentivirus containing PRMT5 shRNAs and Scramble (Scr). As shown in PRMT5 regulated gene expression and protein post-translational modification through methylation . TherefoAlthough PRMT5 is participated in human colorectal cancer, how PRMT5 gates the tumor cell growth and epithelial-mesenchymal transition (EMT) and the related molecular mechanisms are still entirely unknown. To this end, we assessed the protein kinase B  activity, which is a master regulator for tumor cell proliferation and cell cycle progression . As showNext, we asked if the enzyme activity of PRMT5 is associated with Akt activation. In order to test this hypothesis, HCT116 cells were treated with PRMT5 inhibitor GSK591, and the phospho-Akt was detected by Western blotting. As shown in The emerging evidence has been shown that Akt/EMT signaling axis plays a vital role in human colorectal cancer progression . NeverthSo far, it has been reported that PRMT5 acts as an oncoprotein and plays a crucial role in different types of human cancer via regulation of specific downstream targets or signaling cascades. Nevertheless, it is completely unknown whether PRMT5 is engaged in human colorectal cancer cell proliferation and EMT. Besides, the related molecular mechanism is also unclear. In the current study, we confirm that PRMT5 is frequently highly expressed in human colorectal cancer cells and tissues in both mRNA and protein levels . FurtherThe oncoprotein PRMT5 promotes cancer cell proliferation, tumorigenesis, tumor invasion, and metastasis, which has been well elucidated in many human cancers. For instance, PRMT5 epigenetically activates the transcription of the androgen receptor and facilitates prostate cancer cell growth . PRMT5 aThe PI3K-Akt signaling pathway is responsible for cell growth, cell survival, cell metabolism, cell cycle, gene expression, cell development, and anti-apoptosis in various cell types . It has In summary, our study provides evidence that PRMT5 is overexpressed in human colorectal cancer cells and tissues. Our study also proves that PRMT5 promotes cell proliferation and EMT in human colorectal cancer via activation of the EGFR/Akt/GSK3β signaling axis, but not ERK1/2 or PTEN/mTOR signaling pathways, which is strongly linked to the PRMT5 enzyme activity. Our results further imply that PRMT5 may act as a novel therapeutic candidate for the treatment of human colorectal cancer. More importantly, the new insights into the activation of EGFR/Akt/GSK3β signaling axis medicated by PRMT5 provide much new information for the mechanisms of the carcinogenic effect of PRMT5 in human colorectal cancer.2. The fresh human CRC samples and matched adjacent normal tissues were obtained between 2017 and 2019 from the Department of Pathology, Southern Medical University. The human samples were obtained from the patients with informed consent, and the Ethics Committee approved the current project of Southern Medical University.All human colorectal cancer cell lines, including HCT116, SW480, CX-1, SW620, LoVo, COLO 205, LS-174T, and normal colonic mucosal FHC cells, were purchased from American Type Culture Collection . The HCT116 cells were grown in DMEM/F12 containing 10% fetal bovine serum (FBS); the SW480, SW620, and LoVo cells were cultured in DMEM supplemented with 10% FBS; the CX-1 and COLO 205 cells were grown in RPMI-1640 medium containing 10% FBS; the LS-174T were cultured in Eagle’s MEM containing 1% non-essential amino acids solution, 1 mM sodium pyruvate, and 10% FBS. The cells were maintained at 37° C under 5% COPRMT5 cDNA subcloned into Flag vector was purchased from Addgene and verified by sequencing.Human PRMT5 knockdown lentiviral plasmids were as described previously . The pacTotal RNA was isolated from HCT116, SW480, CX-1, SW620, LoVo, COLO 205, LS-174T, and FHC cells and the human CRC and normal tissues using TRIzol reagent  according to the manufacturer’s protocol. Subsequently, an equal amount of RNA was applied to reverse transcription using Bio-Red PCR thermal cycler (C1000). For gene expression analysis, the SYBR green fluorescent Dye  was subjected to the quantitative real-time PCR (qRT-PCR) with an ABI7500 PCR machine (Applied Biosystems). The following primers were used: human PRMT5 forward: 5’-CCTGTGGAGGTGAACACAGT-3’ and revise: 5’-AGAGGATGGGAAACCATGAG-3’; GAPDH, forward: 5’-GAAGGTGAAGGTCGGAGTCAACG-3’ and revise: 5’-TGCCATGGGTGGAATCATATTGG-3’; β-catenin forward: 5’-AAAGCGGCTGTTAGTCACTGG-3’ and revise: 5’-CGAGTCATTGCATACTGTCCAT-3’; Vimentin forward: 5’-AGTCCACTGAGTACCGGAGAC-3’ and revise: 5’-CATTTCACGCATCTGGCGTTC-3’;Collagen I forward: 5’-GAGGGCCAAGACGAAGACATC-3’ and revise: 5’-CAGATCACGTCATCGCACAAC-3. GAPGH was served as an internal control. Relative mRNA expression was determined by the method of ΔΔ-Ct.Lentivirus containing PRMT5-shRNAs was prepared by co-transfection along with the packaging plasmid MD2G and helper plasmid PAX2 into 293T cells. The medium was harvested after 48h, and the viral titer was pre-determined. The equal amount of virus particles was used in the indicated experiments. In order to generate the PRMT5 stable knockdown cells, the HCT116 and SW480 cells were infected with the lentivirus containing PRMT5-shRNAs or scramble shRNA. Those cells were then selected with puromycin  for 48 h, and the non-infected cells were killed. Finally, the PRMT5-depletion stable cells were used for the indicated experiments.TM 3000  was used. Before transfection, the HCT116 and SW480 cells were seeded into 6-well plates. When the cells were grown around 70%, the plasmids of flag-vector and flag-PRMT5 was delivered into those cells. After 48h, the cells were harvested for Western blotting.In order to deliver the exogenous genes into cells, the LipofectamineWestern blotting was carried out as described previously . BrieflyHCT116 and SW480 cells were transduced with lentivirus containing scramble shRNA and PRMT5 shRNAs and seeded into 24-well plates (5000 cells/plate). Cell viability was detected by cell counting kit-8  during the different time points according to the manufacturer’s recommendation. The Infinite 200 plate reader  was applied to evaluate cell viability. In order to assess the effect of PRMT5 inhibitor GSK591 on cell viability, HCT116 and SW480 cells were seeded into 96-well plates (5000 cells/plate) and treated with DMSO and different concentrations of GSK591.o C. Subsequently, the cells were incubated with blocking buffer  for 1 hour at room temperature, followed by incubation with Ki67  and β-actin  antibody (diluted 1:500 in blocking buffer) at 4° C overnight. Next, the cells were washed three with PBS for 10 minutes, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody  for Ki67 and Alexa Fluor 594-conjugated goat anti-mouse secondary antibody  for β-actin (diluted 1:500 in blocking buffer) and at room temperature. Nuclei were stained with DAPI  for 30 minutes at room temperature before observation. The Nikon Eclipse E800 fluorescence microscope was used to capture the images.HCT116 cells were seeded into 6-well cell culture plates and incubated overnight to establish adherence. The cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization in ice-cold methanol for 15 minutes at -20t-test was used for comparisons with the indicated group. The difference with P<0.05 was considered statistically significant. For all Figures, *P< 0.05 and **P < 0.01 vs. indicated group.Unless stated otherwise, the data were represented as means ± SEM. Paired and unpaired two-tailed Student’s


Notably, ectopic survivin expression curtailed Obatoclax-induced apoptosis and cytotoxicity, confirming an essential role of survivin downregulation in Obatoclax-elicited anti-CRC effect. Moreover, Obatoclax was found to repress hyperactive WNT/β-catenin signaling activity commonly present in human CRC cells, and, markedly, ectopic expression of dominant-active β-catenin mutant rescued the levels of survivin along with elevated cell viability. We further revealed that, depending on the cell context, Obatoclax suppresses WNT/β-catenin signaling in HCT 116 cells likely via inducing β-catenin destabilization, or by downregulating LEF1 in DLD-1 cells. Collectively, we for the first time define survivin downregulation as a novel, pro-apoptotic mechanism of Obatoclax as a consequence of Obatocalx acting as an antagonist to WNT/β-catenin signaling.Colorectal cancer (CRC) is a highly prevailing cancer and the fourth leading cause of cancer mortality worldwide. Aberrant expression of antiapoptotic BCL-2 family proteins is closely linked to neoplastic progression and chemoresistance. Obatoclax is a clinically developed drug, which binds antiapoptotic BCL-2, BCL-xL, and MCL-1 for inhibition to elicit apoptosis. Survivin is an antiapoptotic protein, whose upregulation correlates with pathogenesis, therapeutic resistance, and poor prognosis in CRC. Herein, we provide the first evidence delineating the functional linkage between Obatoclax and survivin in the context of human CRC cells. In detail, Obatoclax was found to markedly downregulate survivin. This downregulation was mainly achieved via transcriptional repression, as Obatoclax lowered the levels of both  Colorectal cancer (CRC) is the second and third most common cancer diagnosed in women and men, respectively, and is the fourth leading cause of cancer death worldwide . ApproxiAPC gene or activating mutations in the β-catenin-encoding gene CTNNB1 together account for the majority of genetic lesions in CRC cells, which lead to stabilization and ensuing nuclear translocation of β-catenin to facilitate TCF/LEF-dependent transcription of WNT/β-catenin signaling target genes to drive cell proliferation, metastasis, and cancer stemness [Dysregulation of signaling pathways including EGFR/MAPK, Notch, PI3K/AKT, TGF-β, or WNT/β-catenin has been linked to CRC genesis and progression . In partstemness ,7,8. It stemness ,9.Survivin, a well-defined WNT/β-catenin target gene , is the Obatoclax, a synthetic derivative of bacterial prodiginines , is a clHerein, we reported the first evidence establishing the molecular connection among Obatoclax, survivin, and WNT/β-catenin signaling in the context of CRC cell lines. We proved that, aside from acting as a pan-BCL-2 inhibitor, Obatoclax’s proapoptotic action involves survivin downregulation via suppressing WNT/β-catenin signaling. Our novel discovery thus reveals the multiple modes of Obatoclax’s pharmacological action but also the potential application of Obatoclax to CRC therapy.50 values of 257.19 ± 1.46, 89.96 ± 1.68, 283.82 ± 3.46, and 231.04 ± 2.01 nM for DLD-1, HCT 116, LoVo, and WiDr cells, respectively (p < 0.001)  B. Thus, < 0.001) C. Taken survivin mRNA in Obatoclax-treated cells. It is clearly noted that Obatoclax treatment caused a dose-dependent reduction in survivin mRNA expression  to withstand Obatoclax-induced survivin downregulation. It is noticeable that in both DLD-1 and HCT 116 stable clones, HA-survivin ectopic expression allayed Obatoclax-induced PARP cleavage A. Likewisurvivin transcription but also the prerequisite role of its aberrant activation in CRC tumorigenesis. To this end, we evaluated the effect of Obatoclax on the activity of WNT/β-catenin signaling revealed by the cell-based TOPFlash reporter system [p < 0.001)  A. To furWe next delineated the functional significance of WNT/β-catenin signaling in Obatoclax-induced survivin downregulation and consequent CRC cell death. To this end, we generated DLD-1 and HCT 116 clones with stable expression of a dominant-active β-catenin mutant  to cope with Obatoclax-evoked inhibition of β-catenin-mediated transcriptional activity. It is noteworthy that, in contrast to the vector controls where survivin was markedly downregulated by Obatoclax, ectopic ∆N90-β-catenin expression sustained the levels of survivin, along with attenuation in PARP cleavage A. MoreovLastly, the mechanisms by which Obatoclax inhibits WNT/β-catenin signaling were investigated. With respect to β-catenin itself, earlier immunoblotting revealed that β-catenin levels were dose-dependently lessened in Obatoclax-treated HCT 116 cells B; notablGiven activation of WNT/β-catenin signaling culminated at β-catenin nuclear translocation to facilitate transcription factors of the TCF/LEF family, such as TCF4 and LEF1, to initiate transcription of downstream target genes, we probed the effect of Obatoclax on TCF4 and LEF-1 in DLD-1 and HCT 116 cells. It was noticed that, in DLD-1 cells, Obatoclax barely altered TCF4 expression while evidently inducing a dose-dependent downregulation of LEF-1 , resulting in constitutively active β-catenin to promote TCF/LEF-dependent transcription of WNT target genes irrespective of upstream WNT signals [CTNB1 but wild-type APC [Contrary to other human cancers, in CRC almost all mutations that activate WNT/β-catenin signaling are present in genes such as  signals . To thistype APC , or by ptype APC  , prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO) , and stored at 4°C until use. MG132 was obtained from AdooQ BioScience . Polybrene was obtained from Sigma-Aldrich .® CCL-221™), HCT 116 (ATCC® CCL-247™), LoVo (ATCC® CCL-229™), and WiDr (ATCC® CCL-218™) were purchased form Bioresource Collection and Research Center (BCRC)  and were cultured in RPMI-1640, McCoy’s 5a, F-12K, and MEM medium , respectively. All culture media were supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% sodium pyruvate (Gibco Life Technologies). Cells were grown at 37 °C in a humidified environment with 5% CO2.Human colorectal carcinoma (CRC) cell lines DLD-1 (ATCC® AQueous One Solution Cell Proliferation Assay (MTS) assay , as well as evaluating colony-formation capacity (clonogenicity) of Obatoclax-treated cells. Both cell viability and clonogenicity assays were performed in accordance to our established protocol [The cytotoxic effect of Obatoclax on CRC cell lines was evaluated by measuring the viability of cells after 24 and 48 h treatment with Obatoclax using CellTiter 96protocol ,46.survivin mRNA in Obatoclax-treated CRC cells were determined using quantitative real-time reverse transcription polymerase chain reaction (PCR). Total RNA extraction, reverse transcription, and real-time PCR analysis for survivin mRNA expression levels were conducted according to our laboratory protocol as previously described [The levels of escribed .survivin promoter covering the region between nucleotides 1824 and 2800 of the human survivin gene (GenBank accession number U75285), has been described previously [http://n2t.net/addgene:12456; RRID:Addgene_12456), the luciferase reporter vector for TCF/LEF-mediated transcriptional activity, and its negative control M51 Super 8x FOPFlash (TOPFlash mutant)  were gifts from Professor Randall Moon.pSRVN-Luc, the luciferase reporter vector for the activity of the human eviously . M50 Supsurvivin promoter activity as well as the WNT/β-catenin signaling activity revealed by the TOPFlash reporter system were determined by the activity of the respective luciferase reporter. CRC cells (2 × 105) seeded onto 6-well plates were transiently transfected by jetPEI™  for 24 h with pSRVN-Luc, M50 Super 8x TOPFlash, or M51 Super 8x FOPFlash, together with a plasmid expressing Renilla luciferase  for normalization of transfection efficiency. CRC cells transfected with luciferase reporter vectors were then treated with Obatoclax for 24 h, followed by dual luciferase activity assay in accordance to our experimental protocol reported previously [The levels of the human eviously .The open reading frames (ORFs) of survivin and the dominant-active β-catenin mutant  were PCR-amplified from the cDNA pools of the DLD-1 cells as the template using the following primers: survivin—forward: 5′-GGTGC-CCCGACGTTGCCCCCTGCCTG-3′, survivin—reverse: 5′-TCAATCCATGGCAGCCAGCT-GCTCG-3′, ∆N90-β-catenin—forward: 5′-GCTCAGAGGGTACGAGCTGCTATG-3′, and ∆N90-β-catenin—reverse: 5′-CAGCTGCACAGGTGACCAC-3′. All PCR products were first TA-cloned into the pGEM-Teasy vector  to verify sequence, followed by directional subcloning to the pBabe-HA vector, a pBabe.puro vector engineered to encode an in-frame N-terminal hemagglutinin (HA) epitope. These pBabe-based expression plasmids were then subjected to production of retroviral particles and subsequent infection to target cells according the established protocols in our laboratory .5) were treated with Obatoclax  for 24 h, or treated with Obatoclax for 22 h and then combined with MG132 (20 μM) for an additional 2 h. After drug treatment, cells were lysed, subjected to SDS-PAGE, blot transfer, incubation with primary and secondary antibodies, exposure to enhanced chemiluminescence (ECL) reagent, and final visualization of the antigen-of-interest using the LAS3000 system  as stated previously [CRC cells (4 × 10eviously ,46. Denst tests. p < 0.05 was recognized as statistically significant.All data were derived from three individual experiments and were expressed as means ± SD. Statistical differences between two independent experimental groups were analyzed using unpaired two-tailed