Helical carbon nanotubes (HCNTs) were functionalized to fabricate HCNT/epoxy composites. Acid oxidation and a silane coupling agent, glycidoxypropyltrimethoxysilane (KH560), were used to modify the HCNTs. Remarkably, the flexural strength and the flexural strain were enhanced by 72.0% and 325.0%, respectively, compared to pure epoxy after adding a small amount of the KH560 modified HCNTs (K-HCNTs). Simultaneously, the tensile strength and Young’s modulus of K-HCNTs/epoxy composites were 51.3% and 270.9% higher than those of pure epoxy. It is found that the presence of silane molecules improved the dispersion of HCNTs in epoxy and the interfacial interaction. Moreover, it has been found that the mechanically interlocking effect from the helical shape of HCNTs also contributes to the improved mechanical properties of epoxy composites, compared to their straight multi-walled carbon nanotube (MWCNT) counterparts. This work provides a low-cost and efficient approach to strengthen and toughen epoxy composites. Epoxy resins have attracted great attention for wide applications in the aerospace and automobile industry, owing to their excellent mechanical properties, strong bonding strength and good thermal stabilities ,2. Howev2 bonding of CNTs. It has been found that the flexural strength and the flexural modulus of epoxy composites are increased by the addition of Triton-treated CNTs, owing to the fact that the surfactant serves as an interfacial coupling agent and a dispersant [To achieve a remarkable enhancement in the mechanical properties of CNT/epoxy composites, two main issues should be deliberately considered. One is that CNTs tend to aggregate in a polymer matrix due to the strong π–π interactions and van de Waal’s forces between them, resulting in the uneven dispersion of CNTs. In addition, the dispersion of filler become worse with increasing filler content owing to the presence of filler agglomerate . Furtherspersant .However, the mechanical properties of CNTs/epoxy composites are still far from expectations despite the extensive studies on surface modification of straight CNTs. The root may be the fact that it is difficult to form the mechanical interlocking at the straight CNT/epoxy interface. However, the curved surface morphology of helical carbon nanotubes (HCNTs) offers an advantage in forming the mechanical interlocking at the interface, which further enhances the interfacial interaction between HCNTs and epoxy. Lau et al.  comparedIn this work, the effect of surface-treated HCNTs on the flexural and tensile properties of HCNT/epoxy composites is systematically investigated. Two different surface modification methods, namely acid oxidation and silanization, were employed to promote the dispersion of HCNTs in epoxy and enhance the interfacial interaction between the HCNTs and epoxy. In addition, the contributions of straight CNTs and HCNTs on the mechanical properties of epoxy composites were compared to further reveal the strengthening and toughening mechanisms of HCNTs in epoxy.3, 63%), concentrated sulfuric acid  and glycidoxypropyltrimethoxysilane (KH560) were supplied by Chengdu Kelong Chemical Reagent Factory, Chengdu, China. All the reagents were used without further purification. A standard bisphenol A epoxy resin (E44) and a common aromatic diamine hardener (curing agent) 4,4′-diaminodiphenylsulfone (DDS) were supplied by Nantong synthetic Materials Co., Ltd., Nantong, China. HCNTs with an average diameter of 80–120 nm and length of 3–5 μm were prepared by chemical vapour deposition (CVD) in the laboratory . The mulv/v) mixture of H2SO4/HNO3 using an ultrasonic bath at 40 °C for 1 h. Then, the acid treated HCNTs were collected by filtration and washed with deionized water until the pH of the filtrate reached neutral. The resultant HCNTs are defined as A-HCNTs. In order to remove the amorphous carbon and organic impurities, the HCNTs were annealed at 700 °C for 2 h in Ar atmosphere. For the silanization of the A-HCNTs, the silane coupling agent (KH560) solution was prepared by mixing 7.5 mL KH560, 5 mL deionized water and 95 mL anhydrous ethanol and stirred continuously for 1 h to fully hydrolyse at pH = 5. Secondly, 0.2 g of A-HCNTs were treated with the KH560 solution for 3 h. Finally, the KH560-treated HCNTs were washed and dried at 60 °C for 24 h. The resultant materials are defined as K-HCNTs. It should be noted that the surface treatments of MWCNTs were exactly the same as those of HCNTs. The K-MWCNTs represent the silane treated MWCNTs.The HCNTs or MWCNTs were homogeneously dispersed into tetrahydrofuran (THF) by an ultrasonicator at high amplitude for 30 min. The dispersion was subsequently mixed with a certain amount of epoxy by using high shear mixing for 3 min at room temperature. The solvent (THF) was completely evaporated in an oven at 120 °C for 2 h. After that, the air bubbles and the residual THF were further removed at 120 °C for 2 h under vacuum. Then, the curing agent (DDS) was added into the epoxy/CNTs suspension and mixed on a magnetic stirrer at 150 °C for 30 min. Finally, the mixture was poured into Teflon molds and cured at 120 °C for 2 h, 160 °C 2 h and 180 °C for 2 h.−1.The surface chemical components of untreated and surface-treated HCNTs were analyzed by a Fourier transform infrared spectrometer (FTIR)  with a scanning range of 4000–400 cmX-ray photoelectron spectroscopy (XPS) was also used to study the chemical structure of HCNTs surface further. All XPS spectra were obtained using an Escalab 250Xi spectrometer  with a standard AlK X-ray source (200 W) and the pass analyzer pass energy of 30 eV.In order to investigate the structural integrity of HCNTs before and after the surface modification, Raman spectra were recorded by a high-solution Raman spectrometer  with laser excitation of 532 nm.The morphology of HCNTs and the fracture surface of HCNT/epoxy composites were observed by using field-emission scanning electron microscopy (FE-SEM)   at accelerating voltage of 20 kV. The dispersion of HCNTs or CNTs in uncured epoxy was observed by a DM 2700 P optical microscope .2 atmosphere.Thermogravimetry (TGA) measurements were performed on a thermo analysis system . The temperature was increased from room temperature to 800 °C at 10 °C/min in the NTensile and flexural tests were conducted on a universal testing machine  at a crosshead speed of 2 mm/min, following the GB1040-92 and GB1449-2005, respectively. All the specimens were tested at room temperature. Six separate measurements were performed to provide the average value and the deviation.The morphology of P-HCNTs, A-HCNTs and K-HCNTs was observed by using scanning electron microscopy (SEM) . As show−1 represent the bending and stretching vibrations of hydroxyl groups (–OH), respectively. The presence of –OH is probably induced by the ambient atmospheric moisture or the oxidation during the purification of HCNTs [−1 . It suggests that the surface of P-HCNTs is activated by the acid treatment. The peaks of C–O appear at 1049 and 1096 cm−1, resulting from the different alcohol structures on the HCNT surface. For the K-HCNTs, the new peaks of Si–O, Si–O–C and Si–C appear at 805, 1023 and 1260 cm−1, respectively. The peak of C–O–C and C–H from the KH560 molecules are also observed at 1184 and 1450 cm−1. Therefore, the presence of the KH560 molecules on the HCNT surface can be confirmed.In order to detect the chemical groups of HCNTs induced by surface treatments, the Fourier-transfrom infrared spectra (FTIR) of P-HCNTs, A-HCNTs and K-HCNTs are shown in of HCNTs ,25. The CNTs [−1 a and 163 [−1  can be used to represent the degree of CNT defects [ID/IG ratio is maintained after the surface treatments, which means that the graphitic integrity of HCNTs is not severely damaged by the acid oxidation or the silanization process. This is in full agreement with the SEM images (The defect degree of HCNTs before and after surface treatments was evaluated by Raman spectra e. It can defects ,27. FiguM images  in whichIn order to explain the contribution of HCNTs to the flexural and tensile properties of HCNT/epoxy composites, the dispersion state of HCNTs in epoxy and the interfacial interaction between the HCNTs and epoxy were investigated by optical microscope and SEM, respectively. The interfacial interaction between the HCNTs and epoxy is investigated by SEM. To gain a deeper insight into the strengthening and toughening mechanisms of HCNTs, the effect of straight MWCNTs on the mechanical properties of epoxy composites was comparatively investigated. To further explain the different effects of HCNTs and MWCNTs, the comparison of the dispersion of K-HCNTs and K-MWCNTs in epoxy and the interfacial interaction between them are presented in The effect of surface-treated HCNTs on the mechanical properties of epoxy composites was systematically investigated. The acid oxidation and the silanzation treatments were conducted to modify the HCNTs. The dispersion of HCNTs in epoxy and the interfacial interaction between the HCNTs and epoxy were significantly improved by these treatments. The flexural strength and the flexural strain of K-HCNT/epoxy composites are 72% and 325% higher than those of pure epoxy. In addition, the tensile strength and Young’s modulus of epoxy composites were simultaneously enhanced by 51% and 270.9% after combining with the K-HCNTs. This is because the presence of KH560 molecules promotes the dispersion of HCNTs in epoxy and enhances the interfacial interaction. Compared with the straight MWCNTs, the coiled HCNTs exhibit superior strengthening and toughening contributions to the epoxy composites, resulting from the mechanical interlocking between the coils of HCNTs and epoxy.


RelA/p65 a crucial member of NF-κB signaling pathway plays diverse role in mediating oncogenesis. Limited knowledge prevails on the mechanistic insights of RelA gene regulation. RNA helicase p68 apart from being a vital player of RNA metabolism acts as a transcriptional coactivator of several oncogenic transcription factors including β-catenin and is highly implicated in cancer progression. In this study, we aim to discern the molecular mechanism of how an RNA helicase, p68 deploys a major oncogenic signaling pathway, Wnt/ β-catenin to regulate the expression of RelA, an indispensable component of NF-κB signaling pathway towards driving colon carcinogenesis.in vitro findings.Immunoblotting and quantitative RT-PCR was performed for determining the protein and mRNA expressions of the concerned genes respectively. Luciferase assay was employed for studying the promoter activity of RelA. Chromatin immunoprecipitation was used to evaluate the occupancy of transcription factors on the RelA promoter. Immunohistochemical analysis was conducted using FFPE sections derived from normal human colon and colon cancer patient samples. Finally, a syngeneic colorectal allograft mouse model was used to assess physiological significance of the in vitro findings.p68, β-catenin and RelA proteins were found to bear strong positive correlation in normal and colon carcinoma patient samples. Both p68 and β-catenin increased RelA mRNA and protein expression. p68, β-catenin and Wnt3a elevated RelA promoter activity. Conversely, p68 and β-catenin knockdown diminished RelA promoter activity and led to reduced RelA mRNA and protein expression. p68 was perceived to occupy RelA promoter with β-catenin at the TCF4/LEF (TBE) sites thereby potentiating RelA transcription. p68 and β-catenin alliance positively modulated the expression of signature NF-κB target genes. Enhanced NF-κB target gene expression by p68 was corroborated by findings in clinical samples. Tumors generated in mice colorectal allograft model, stably expressing p68 further reinforced our We report for the first time a novel mechanism of alliance between p68 and β-catenin in regulating the expression of RelA and stimulating the NF-κB signaling axis towards driving colon carcinogenesis. This study unravels novel modes of p68-mediated colon carcinogenesis, marking it a potential target for therapy.The online version of this article (10.1186/s13046-019-1304-y) contains supplementary material, which is available to authorized users. Mounting evidences suggest p68 to play a crucial role in cancer development and progression. It is reported to be overexpressed in various cancers and acts as a transcriptional co-activator of several transcription factors like β-catenin, NF-κB p50, MAML1 and Androgen Receptor .In terms of both morbidity and mortality, colorectal cancer (CRC) is one of the most common adult malignant tumors worldwide. Aberrant regulation of Wnt/β-catenin signaling cascade is the prevalent theme in colon carcinogenesis, β-catenin being the central oncogenic driver. In CRC, mutations in APC, β-catenin gene or up-regulation of Wnt pathway disrupts the cytoplasmic destruction complex mediated regulation of β-catenin. This enables β-catenin to accumulate in the cytoplasm, translocate to the nucleus and form complex with TCF/LEF HMG box transcription factors including TCF4. This interaction ensures efficient activation of specific target genes that drives initial as well as later stages of neoplastic transformation and progression .Abnormal inflammatory response plays a critical role in colon carcinogenesis. Nuclear factor kappa B (NF-κB) activation, a cardinal feature of tumorigenesis has been proposed to act as a crucial link between inflammation and cancer. Mammalian NF-κB is a family of ubiquitous transcription factors and consists of five members, sharing a highly conserved Rel homology domain (RHD): Rel A p65), c-Rel, Rel B, NF-κB1 (p50) and NF-κB2 (p52). These factors exist in heterodimeric or homodimeric complexes of which heterodimer p65/p50 is the most predominant. In unstimulated cells, NF-κB complexes are sequestered in the cytoplasm through their interaction with members of the inhibitor of κB (IκB) family. Specific cellular stimulation activates IκB kinase (IKK) complex which phosphorylates IκB leading to its ubiquitination and proteosome-mediated degradation. Following IκB degradation, NF-κB translocates to the nucleus to promote the transcription of various target genes bearing consensus κB sequences in the promoter or enhancer regions 5, c-Rel,.The pivotal role played by NF-κB in mediating colon carcinogenesis is illustrated by its regulation of a great diversity of genes positioned at the interface between cell proliferation, survival and evasion from apoptosis. Several evidences support the role of NF-κB in regulating the expression of antiapoptotic members of the Bcl-2 family like Bcl-2  and Bcl-Regulation of NF-κB activity occurs at multiple levels ; hence iA lot remains to be explored regarding the mechanisms of RelA gene regulation, an indispensable member of NF-κB signaling pathway. p68 serves as a coactivator of β-catenin  and β-can = 22) colon and colon carcinoma (n = 45) samples were collected from Indian patients. All clinical and ethical regulations were followed for collecting the samples, including patient consent and with formal approval from the ethical committee of both CSIR-IICB and Park Clinic (source) ; human embryonic kidney cell line [HEK-293] and murine adenocarcinoma cells [CT26] were procured from ATCC . The cell lines were cultured in McCoy’s 5A, DMEM or RPMI-1640, as required, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and maintained at 5% CO2, 37 °C in a humid incubator. Penicillin/Streptomycin cocktail and Gentamycin  were added as prescribed antibiotic at the recommended dose. Transfections of different DNA constructs were performed using Lipofectamine 2000 (Invitrogen). Lipofectamine RNAi MAX (Invitrogen) was used for siRNA-mediated knockdown experiments. Small interfering RNAs (siRNAs) against β-catenin, p68 and TCF4 were purchased from Santa Cruz Biotechnology . CT26 cells were transfected using Lipofectamine LTX (Life Technologies), G418  (500 μg/ml) was used for selection of stable cell line.http://alggen.lsi.upc.es/) was used for putative analysis of the transcription factors binding to this region. All the constructs were verified by restriction digestion and confirmed by sequencing. All primers were synthesized from IDT . Sequences of the primers are given in Additional file p68 was sub-cloned from pSG5-Myc vector into pGZ21dx, pEGFP-c3 and pIRES-hrGFP-1a. pBI-β-catenin was sub-cloned into pGZ21dx vector and human Wnt3a gene was cloned in pcDNA3.1-myc-his, respectively. RelA promoter region (1746 bp fragment) was PCR amplified using genomic DNA from HEK-293 cells and following standard protocol cloned into pGL3 basic vector. ALGGEN-PROMO  in the RelA promoter construct were performed using the QuickChange XL Site-Directed Mutagenesis kit , following manufacturer’s instructions. All deletion constructs were verified by DNA sequencing. Sequences of the primers are given in Additional file Two different shRNAs targeting p68 and a control (scrambled) shRNA were subcloned in pMKO.1-Puro according to Addgene’s protocol. shRNA targeting β-catenin was purchased from Addgene (pLKO.1 puro shRNA β-catenin) (Addgene Cat no 18803). Scramble siRNA, p68, β-catenin and TCF4 siRNA  were used at a final concentration of 30 nM.http://biochemlabsolutions.com/GelQuantNET.html) was used for densitometric scanning of immunoblots. Blots were evaluated by using Luminata Classico Western HRP Substrate (Millipore) following manufacturer’s protocol.Preparation of whole cell lysates (WCL), cytoplasmic and nuclear extracts and immunoblot analyses were performed as described previously , 21. TheTotal RNA was extracted using the Trizol reagent , converted to cDNA (1 μg of RNA for each sample) using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific™) which was subsequently used for qRT-PCR analysis using FastStart Universal SYBR Green Master (Roche) on a ViiA 7 Real-Time PCR system (Applied Biosystems). In all the experiments, 18S rRNA served as the internal control. Quantification and Standard deviation (SD) calculations were based on three independent experiments in triplicates. Sequences of the primers are given in Additional file Cells were transiently transfected with pGL3-RelA promoter luciferase reporter construct along with Renilla luciferase plasmid (pRL-TK) and respective gene constructs as per experimental interest and indicated in the relevant figure(s). Luciferase Assay was performed with the cell lysates 36 or 48 h post-transfection following the manufacturer’s protocol. The efficiency of transfection was normalized with Renilla luciferase expression. Quantification was based on three independent experiments. Luciferase activity was determined luminometrically in the Varioskan Flash Multimode Reader  by the dual luciferase assay system  following manufacturer’s instructions and as described previously .The preparation of chromatin and the immunoprecipitation of such for ChIP assay were performed as described earlier , 24. ChIFor histology and IHC analysis, 5 μm thick sections from both human patient samples and murine allograft tumors were prepared from paraffin-embedded blocks. For histological analysis, the tissue sections were subjected to hematoxylin and eosin (H&E) staining following standard protocol. IHC was performed using SuperSensitive™ Polymer-HRP IHC Detection System (BioGenex), following manufacturer’s instructions and as described previously . Images All animal care and experimentation were conducted according to the CPCSEA guidelines; with approval of the animal ethics committee at CSIR-Indian Institute of Chemical Biology. Murine adenocarcinoma cell line CT26 was used for generation of syngeneic tumor model.n = 6; 4–5 weeks old; 18–20 g) were injected with 106 CT26 cells stably expressing EGFP or EGFP-p68, subcutaneously in the right flank region. The onset of tumor development in the animals was monitored daily by palpating the injection area. Growth of the tumor was observed for a period of 30 days after which the mice were sacrificed and the tumor was excised. The tumor volume was measured by vernier calipers. The tumor volume was determined as [length × width2] × 0.5. Tumors were fixed in 10% buffered formalin and embedded in paraffin for histology and immunohistochemical (IHC) analysis.To generate the primary tumor model, BALB/c mice  to generate metastatic growth in lung. 30 days post injection, animals were sacrificed, the lungs were removed, metastatic nodules were counted and the diameter of the nodules was measured. Lungs were fixed in 10% buffered formalin and embedded in paraffin for further processing.5 × 10http://www.graphpad.com/quickcalcs/index.cfm). H-scores were calculated manually. Mann–Whitney U-test was used to evaluate the differences in H-score values of all the concerned proteins between human normal colon and colon carcinoma samples, using R module Wilcoxon-Mann-Whitney Test calculator (https://www.wessa.net/rwasp_Reddy-Moores%20Wilcoxon%20Mann-Witney%20Test.wasp). Distribution of H-scores are represented by Box plots, generated using web tool BoxPlotR (http://boxplot.tyerslab.com/). Spearman’s rank correlation coefficient (rs) was used to determine the correlation between the expression levels of two different proteins  in both human normal colon and colon carcinoma samples, using R module Spearman Rank Correlation calculator (http://www.wessa.net/rwasp_spearman.wasp/). Throughout, the value of P < 0.05 was considered to be statistically significant (*), P < 0.01, P < 0.001 and P < 0.0001 were considered to be very significant (**), highly significant (***) and extremely significant (****), respectively.At least three independent experiments were performed in triplicates to calculate means and standard deviations (SD). To determine the significance values in all the experiments, Student’s t-test (unpaired) was performed, using GraphPad QuickCals  cell lines. In all the cases, p68 enhanced RelA protein expression. p68 co-activates β-catenin in regulating its target gene Cyclin D1 . Hence, To study whether the effect of p68 on RelA expression at the protein level is a consequence of its transcriptional regulation, we quantified its mRNA expression. In multiple CRC cell lines, p68 overexpression led to increase in mRNA expression of RelA Fig. a. ConverWe next speculated that RelA promoter activity might be regulated by p68, as p68 is a co-activator of several oncogenic transcription factors that might bind on the RelA promoter. We studied the promoter region of RelA 2000 bp upstream to the transcriptional start site and found that this region contains seven putative β-catenin/TCF-LEF binding elements (TBEs). Wnt/β-catenin signaling is highly implicated in colon cancer progression and p68 is reported to interact with β-catenin and thereby, regulate the expression of its target genes , 5. HencNext, we conducted Chromatin Immunoprecipitation (ChIP) assay to gain deeper mechanistic insights into the modulation of RelA promoter activity mediated by p68 and β-catenin cooperation. Immunoprecipitations (IPs) were carried out using anti-p68, anti- β-catenin and anti-TCF4 followed by amplification of different TBEs sites on the RelA promoter region, as shown in the schematic diagram Fig. e. The ChThe RelA promoter possesses seven TBEs namely: TBE1 , TBE2 , TBE3 , TBE4 , TBE5 , TBE6  and TBE7 . The positions are relative to the transcription start site. We generated RelA promoter constructs containing deletions (5 nucleotides deleted by SDM) of the seven TBEs, as shown in the schematic diagram  or the empty vector encoded EGFP (CT26EV). The murine CT26 is a grade IV carcinoma cell line developed in 1975 by exposing BALB/c mice to N-nitroso-N-methylurethane (NMU) and is extensively used as a model system in investigation of colorectal carcinomas. The expression of RelA and NF-κB target genes were significantly elevated in CT26p68 cells compared with CT26EV  with enhanced vasculature than that produced from CT26EV cells -p68  and elevated in size (approximately four times) and possessed compact tissue morphology with high cellular density as observed by the histological analysis (H&E staining) of the lung tissue sections in comparison to that formed by CT26EV cells  HCT 116 (left) and SW-480 (right) cells were seeded in 35 mm plates and transfected with p68 siRNA (1 μl) or scramble siRNA  along with pGL3-RelA-prom (1 μg) and Renilla luciferase plasmid (50 ng). The luciferase activity was measured 48 h post transfection. Renilla luciferase activity was used for normalization and the data is represented as fold activity with respect to control (scr). Error bars represent mean (+) s.d. Indicated P-values were determined using Student’s t-test. Scramble and p68 siRNA were used at a final concentration of 30 nM. (B) SW-480 cells were grown in 100 mm cell culture dishes followed by Chromatin immunoprecipitation (ChIP) using the indicated antibodies. ChIP using antibody against RNA Polymerase II (Pol II) and IgG served as positive and negative controls, respectively. Amplification of Cyclin D1 promoter containing TCF-binding elements (TBE) served as positive control for both p68 and β-catenin. Actin promoter served as positive control for Pol II. DNA extract (10% without ChIP) was used as input. PCR amplification of the immunoprecipitated DNA was performed using primers designed from the RelA promoter region – TBE [1–3]-(349 bp), flanking TBE1, TBE2 and TBE3; TBE4-(296 bp), flanking TBE4; TBE5-(140 bp), flanking TBE5 and TBE [6–7]-(213 bp), flanking TBE6 & TBE7. (C) Schematic representation of the RelA promoter reporter constructs (WT and mutated). White boxes represent TBEs and black boxes represent mutated TBE (5 nucleotides deleted by SDM). The numbers indicate the position of each TBE with respect to transcription start site. (TIF 385 kb)Additional file 2:Figure S2. β-catenin and TCF4 expression is elevated in CRC samples. (A) Notched box plots showing the distribution of H-scores of β-catenin and TCF4 in normal (n = 22) and colon carcinoma samples (n = 45); Mann–Whitney U-values were calculated from H-scores. (B) Comparison of the combined average H-scores of β-catenin and TCF4. Error bars represent the mean (+) s.d.; P < 0.0001 is represented as ****; calculated using Student’s t-test. (TIF 106 kb)Additional file 3:Figure S3. Amalgamation between p68 and β-catenin regulates RelA expression. (A) SW-480 cells seeded in 35 mm plates were transfected with pGZ-β-catenin or EV (2 μg). (B) HCT-15 cells seeded in 35 mm plates were transfected with scr or β-catenin shRNA (2 μg). HCT 116 cells seeded in 35 mm plates were transfected with either (C) scr or p68 shRNA-1 (1 μg), in combination with pGZ-β-catenin or EV (1 μg) or (D) scr or β-catenin shRNA (1 μg), in combination with pGZ-p68 or EV (1 μg). Total RNA was extracted; 36 h post transfection for (A) and 48 h post transfection for (B), (C) and (D), followed by analysis of RelA mRNA by qRT–PCR. 18S rRNA was used for normalization in all the above experiments. HCT 116 (left) and SW-480 (right) cells seeded in 35 mm plates were transfected with (E) scr or β-catenin shRNA plasmid (1 μg) along with pGL3-RelA-prom (1 μg) and Renilla luciferase plasmid (50 ng). The luciferase activity was measured, 48 h post transfection. (F) EV or pGZ-β-catenin (1 μg) along with pGL3-RelA-prom (1 μg) and Renilla luciferase construct (50 ng). (G) pGZ-β-catenin or pGZ EV (1 μg) along with pGL3-WT-RelA-prom or its deletion constructs (1 μg) and Renilla luciferase plasmid (50 ng). For both (F) and (G) the luciferase activity was measured, 36 h post transfection. For (E), (F) and (G) cells co-transfected with (1 μg) of EV, pGL3-WT-RelA-prom and Renilla luciferase plasmid (50 ng). Renilla luciferase activity was used for normalization and data is represented as fold activity with respect to control cells. For all the sub-figures results are presented as mean (+) s.d. from three independent experiments. Indicated P-values were determined using Student’s t-test and P < 0.0001 is represented as ****. (TIF 407 kb)Additional file 4:Figure S4. Abundance of β-catenin with p68 and NF-κB target genes maintain strong positive correlation in human colon cancer tissues. (A) Scatter plots representing the mean H-scores of β-catenin and p68, Bcl-2, Bcl-xL, Survivin and XIAP in normal (n = 22) and colon carcinoma tissue (n = 45) samples, respectively. (B) Spearman’s rank correlation coefficient (rs) between the mean H-scores of β-catenin and p68, Bcl-2, Bcl-xL, Survivin and XIAP was determined from both normal and colon carcinoma tissues in combination. (TIF 628 kb)Additional file 5:Table S1. List of primers used in the study. (DOCX 19 kb)


Carbamazepine is a drug that is widely used in the treatment of epilepsy and bipolar disorder. The prevalence of obesity in patients treated with carbamazepine has been frequently reported. However, whether carbamazepine affects adipogenesis, one of the critical steps in the development of obesity, remains unclear. Here, we show that carbamazepine increased the expression levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and fatty acid synthase (FASN) in 3T3-L1 cells. Notably, carbamazepine inhibited the expression levels of β-catenin, a negative regulator of adipogenesis, leading to enhanced adipogenesis. Conversely, β-catenin overexpression abolished the effect of carbamazepine on adipogenic gene expression. However, depletion of β-catenin further enhanced PPARγ expression. In addition, carbamazepine reduced β-catenin expression by lowering the levels of phospho-low density lipoprotein receptor-related protein 6 (p-LRP6) and phospho-glycogen synthase kinase 3β (p-GSK3β) in Wnt/β-catenin signaling. Moreover, carbamazepine reduced Wnt mRNA expression and decreased the promoter activities of TCF, the target of β-catenin during adipogenesis. These results suggest that carbamazepine enhances adipogenesis by suppressing Wnt/β-catenin expression, indicating its potential effects on obesity-related metabolism. Carbamazepine, a dibenzazepine compound with tricyclic structure, has been used as a prophylactic agent for the treatment of bipolar disorder . CarbamaRecent studies have revealed that treatment with carbamazepine increases the levels of total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride concentrations in serum without significantly affecting circulating insulin or leptin levels, suggesting the effect of carbamazepine in cholesterol metabolism ,10,11. AObesity represents an imbalance between energy intake and consumption, leading to increased sizes and numbers of adipose tissues. It is linked to the development of diabetes and cardiovascular diseases ,15,16. PAdipogenesis is a cellular differentiation process that includes the formation of pre-adipocytes from mesenchymal stem cells and their differentiation into adipocytes . The molIn this study, we found that treatment with carbamazepine promoted the differentiation of 3T3-L1 pre-adipocytes and inhibited the expression levels of Wnt/β-catenin, a negative regulator of adipogenesis, suggesting that carbamazepine might have novel pro-adipogenic roles in the development of obesity. 2 at 37 °C, and used for luciferase assay.The 3T3-L1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum, seeded onto 6-well-plates and cultured for 2 days. After that, pre-adipocytes were maintained in DMEM with 10% FBS. For adipocyte differentiation, pre-adipocytes were treated with DMI  cocktail containing 1 μM dexamethasone (DEX), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 5.0 μg/mL insulin for 2 days. Cells were further incubated with 5.0 μg/mL insulin and treated with fresh media every 2 days. The differentiation process to mature adipocyte was completed after 6 days of differentiation. Human embryonic kidney (HEK) 293T cells were maintained in DMEM supplemented with 10% FBS in 5% COThe carbamazepine was purchased from Sigma-Aldrich . The stock concentration of carbamazepine was 100 mM, dissolved in 100% ethanol. The stock solution was diluted to a concentration of 80 μM with culture medium, and serially diluted to concentration of 40, 20, and 10 μM before use. We used an ethanol solution for vehicle (Veh) treatment, and carried out serial dilution in the same way as carbamazepine solution. Ethanol levels were kept below 0.5% in all cells, which did not affect differentiation. 3T3-L1 cells were treated with carbamazepine on day 0 of differentiation process. When the differentiation medium was replaced at 2-day intervals, carbamazepine was added together at a similar concentration. The Oil Red O powder was purchased from Sigma-Aldrich . Fully differentiated cells were fixed with 10% formalin for 1 h at room temperature (RT), rinsed with 60% isopropanol, and stained with filtered Oil Red O for 20 min. These cells were then visualized under a light microscope. The absorbance of the Oil Red O eluted by adding 100% isopropanol was measured at 500 nm wavelength by spectrophotometry.Whole cell extracts were fractionated by SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer apparatus, according to the manufacturer’s protocol . The membranes were incubated with primary antibodies in Tris-Buffered Saline, and 0.1% Tween 20 detergent (TBS-T) containing 3% bovine serum albumin for overnight at 4 °C. After washing three times for 10 min with TBS-T, the membranes were incubated with secondary antibodies for 1 h at RT, followed by washing thrice with TBS-T, and developed with ECL system , according to the manufacture’s protocol. Antibodies against active β-catenin (cat. 05-665) from Merck Millipore, β-catenin (cat. 610154) and FASN (cat. 610963) from BD Bioscience, IRS1 (cat. 06-248) from Upstate Biotechnology , Glut4 (cat. ab652) from Abcam , Flag (cat. F1804) from Sigma , p-AKT (cat. 9271), AKT (cat. 9272), p-LRP6 (cat. 2567), AXIN (cat. 2087), p-GSK3β (cat. 9336), GSK3β (cat. 12456), and IR (cat. 3025) from Cell Signaling Technology . C/EBPβ (cat. sc-7962), PPARγ (cat. sc-7273), α-tubulin (cat. sc-5286), anti-rabbit IgG HRP (cat. sc-2030), anti-mouse IgG HRP (cat. sc-2005), and anti-goat IgG HRP (cat. sc-2020) from Santa Cruz Biotechnology  were used. Signals were detected using chemiluminescent substrates .The 3T3-L1 cells were seeded onto 96-well-plates and maintained in growth media for 24 h. The medium was replaced and cells were incubated with various concentrations of carbamazepine for 24 h. Cells were further incubated at 37 °C for 1 h with 1 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT). Precipitates were dissolved with 200 μL DMSO. Cell viability was then measured at 570 nm wavelength using a microplate reader.Total RNA was extracted using Trizol reagent , according to the manufacturer’s instructions. The cDNA synthesis was carried out with 1 μg of total RNA using the RevertAid First cDNA Synthesis kit (Thermo). Gene expression was determined by RT-PCR  using gene specific primers. The quantitative PCR analysis was performed with QuantStudio 6 Flex System Applied Biosystem . The β-catenin siRNA (si-β-catenin) and non-silencing siRNA (si-NS) were purchased from Macrogen . For siRNA transfection, pre-adipocytes were seeded onto 6-well-plate for 24 h. Cells were transfected with either si-β-catenin or si-NS at a concentration of 50 nM using Lipofectamine RNAi max , according to the manufacturer’s protocol. The plasmid expressing β-catenin was purchased from Addgene . For gene overexpression, cells were transfected with plasmids expressing either β-catenin or pcDNA3 (vector) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.The HEK293T cells were transfected with reporter vector and expression plasmid using TransIT-LT1 . Cells were lysed with cell lysis buffer  at two days after addition of differentiation stimulus. Luciferase activity was determined by luminometer (Promega) according to the manufacturer’s instruction.t-test . Differences between individual group means were analyzed using two-tailed t-tests. P value of less than 0.05 was considered significant.Data are presented as mean ± standard error of the mean (SEM). The main and interactive effects were analyzed using Student’s The structure of carbamazepine has previously been described A 22]. T. T22]. TPPARγ, C/EBPβ, ACC, and FASN were increased by treatment with carbamazepine in a time-dependent manner (We next investigated how carbamazepine affected adipocyte differentiation in 3T3-L1 cells. Adipogenic transcription factors such as adipocyte protein 2 (aP2), C/EBPα, C/EBPβ, PPARγ, and lipogenic enzymes, including acetyl-CoA carboxylase (ACC) and FASN, are critical for adipocyte differentiation ,21. Thust manner D. Proteit manner . After 3t manner F. When tt manner . Taken tWe next determined the critical time point of carbamazepine action. Confluent 3T3-L1 cells were exposed to DMI with or without carbamazepine for various time periods during adipogenesis. PPARγ were markedly increased (PPARγ expression. We divided the adipocyte differentiation process into four different phases: Early (days 0–2), intermediate (days 2–4), late (days 4–7), and throughout the 7-day period (complete) phase A,B. Aftencreased C. Carbamncreased C. These SREBP1), lipid synthesis-related genes , acyl-CoA:monoacylglycerol acyltransferase (MGAT1), and acyl-CoA:diacylglycerol acyltransferase (DGAT1)), and adipokine related genes (such as adiponectin (ADIPOQ), fatty acid binding protein 4 (FABP4), and Leptin) in carbamazepine treated adipocytes were increased, compared to those in vehicle-treated cells  co-receptors . Levels Lrp5/6 and Fizzled (FZD) act as receptors for Wnt signaling . FZD binβ-catenin in 3T3-L1 cells. Overexpression of β-catenin abolished the positive effect of carbamazepine on adipocyte differentiation  was used as a control for the si-RNA against β-catenin (si-β-catenin). si-NS and si-β-catenin transfected cells were treated with vehicle or carbamazepine. The positive effect of carbamazepine on adipocyte differentiation was further increased in β-catenin knockdown cells A. After mazepine B. The prmazepine . Depletimazepine . Decreasmazepine C. In addmazepine D.C/EBPα and PPARγ [We next examined whether carbamazepine affects the transcriptional activity of β-catenin. Considering that β-catenin is associated with DNA-bound TCF to serve as its co-activator for regulating Wnt-responsive genes  and thatnd PPARγ , we measWnt is an upstream regulator of β-catenin ,32, thatPPARγ and eventually resulting in the inhibition of adipogenesis [Wnt10a and Wnt10b mRNA expression levels at day 7, compared with those at day 2 after the initiation of differentiation , genes related to lipid synthesis , and adipokine genes  were increased after carbamazepine treatment. In contrast, Turpin et al. [PPARγ, C/EBPα, and C/EBPβ) and lipogenic enzymes  in 3T3-L1, 3T3-F442A, and T37i cells. However, the concentration of carbamazepine used in their study was very high (500 μM). The high dose of carbamazepine (500 μM) might affect cell viability. Our analysis revealed that 40 μM of carbamazepine did not affect cell viability. However, carbamazepine at a concentration greater than 100 μM significantly reduced cell viability. These results suggest that depending on its dosage, carbamazepine has a distinct effect on adipocyte differentiation and cell viability.Adipocyte differentiation is divided into three stages: Early, middle, and late stages . Specifin et al.  have repExpression of insulin signaling is known to increase as adipocyte differentiation progresses . Our resThe mechanism by which carbamazepine enhances adipogenesis is mostly unknown. Our results showed that carbamazepine treatment decreased β-catenin expression levels in 3T3-L1 cells. Among pathways that regulate the expression of β-catenin, the canonical pathway is well known . This paRecent studies have reported that Wnt ligand binds to LRP5/6 and FZD receptor to activate the Wnt/β-catenin signaling pathway . Our resWnt. It would be of interest in the future to determine whether carbamazepine suppresses Wnt expression via the inhibition of LRP6 phosphorylation.Our results showed that Wnt expression was decreased in mature adipocytes and further reduced by carbamazepine treatment. Suppression of Wnt expression by carbamazepine may involve various mechanisms. When pre-adipocytes differentiate into mature adipocytes, carbamazepine treatment might have further promoted adipogenesis and increased lipid accumulation of mature adipocytes. One hypothesis is that the expression of Wnt itself might be decreased due to elevated lipid accumulation in mature adipocytes . Our resWnt10a, Wnt10b, and β-catenin in 3T3-L1 cells. Consistent with our results, a study using mice overexpressing Wnt10b has revealed that Wnt10b inhibits adipocyte formation both in vitro and in vivo [Wnt factors are a group of extracellular signaling molecules. Among them, Wnt10b is expressed in dividing and confluent pre-adipocytes . Its exp in vivo ,27. In tIn conclusion, our results indicate that carbamazepine increases adipocyte differentiation by inhibiting Wnt/β-catenin expression. Based on our results, potential therapeutic strategies could be developed by targeting β-catenin signaling and its expression to control obesity in patients treated with carbamazepine.


TNBC is a highly heterogeneous and aggressive breast cancer subtype associated with high relapse rates, and for which no targeted therapy yet exists. Protein arginine methyltransferase 5 (PRMT5), an enzyme which catalyzes the methylation of arginines on histone and non‐histone proteins, has recently emerged as a putative target for cancer therapy. Potent and specific PRMT5 inhibitors have been developed, but the therapeutic efficacy of PRMT5 targeting in TNBC has not yet been demonstrated. Here, we examine the expression of PRMT5 in a human breast cancer cohort obtained from the Institut Curie, and evaluate the therapeutic potential of pharmacological inhibition of PRMT5 in TNBC. We find that PRMT5 mRNA and protein are expressed at comparable levels in TNBC, luminal breast tumors, and healthy mammary tissues. However, immunohistochemistry analyses reveal that PRMT5 is differentially localized in TNBC compared to other breast cancer subtypes and to normal breast tissues. PRMT5 is heterogeneously expressed in TNBC and high PRMT5 expression correlates with poor prognosis within this breast cancer subtype. Using the small‐molecule inhibitor EPZ015666, we show that PRMT5 inhibition impairs cell proliferation in a subset of TNBC cell lines. PRMT5 inhibition triggers apoptosis, regulates cell cycle progression and decreases mammosphere formation. Furthermore, EPZ015666 administration to a patient‐derived xenograft model of TNBC significantly deters tumor progression. Finally, we reveal potentiation between EGFR and PRMT5 targeting, suggestive of a beneficial combination therapy. Our findings highlight a distinctive subcellular localization of PRMT5 in TNBC, and uphold PRMT5 targeting, alone or in combination, as a relevant treatment strategy for a subset of TNBC. Subtype classification was based on immunohistochemical status for the estrogen receptor (ER), progesterone receptor (PR) and HER2, as follows. TNBC: ER−, PR− and HER2‐negative (n = 157); HER2+/ ER−: ER− and PR‐negative, HER2‐positive (n = 41); luminal B: ER− and/or PR‐positive, HER2‐positive (n = 153); luminal A: ER− and/or PR‐positive, HER2‐negative (n = 663). The TCGA database includes 113 referenced normal breast tissue samples.The TCGA breast invasive carcinoma (TCGA‐BRCA) cohort is publicly available.2.2Cell lines were purchased between 2005 and 2009 from the American Type Culture Collection  and authenticated by short tandem repeat profiling in 2018, using the Powerplex 16 system (Promega). All cell lines were cultured as described.2.3PRMT5 inhibitor EPZ015666 was purchased from Clinisciences and DC Chemicals. EPZ015938 was purchased from Selleckchem. Antibodies used are listed in Table 2.4Cell assays were performed as already described.2.5MDA‐MB‐453 cells were seeded into 384‐well plates (ViewPlate‐384 Black Perkin Elmer) in 40 µL of media, using a MultiDrop combi (Thermo Fisher Scientific). Twenty‐four hours later, cells were incubated with 1200 clinically licensed compounds from the Prestwick Chemical Library  , or one of six additional compounds were added (Table ΔRZscore=RZscorePrestwick+EPZ015666−RZscore(Prestwick) was calculated for each ‘hit' to quantify the effect of the Prestwick + EPZ015666 drug combination.A 2.6http://chalice.horizondiscovery.com/analyzer-server/cwr/analyze.jsp) was used to calculate the Loewe excess. Synerdrug Analyzer (https://github.com/bioinfo-pf-curie/synerdrug) was used to calculate Chou‐Talalay Combination Indexes. Experiments were repeated at least three times.Cells were seeded into 96‐well plates and treated with various concentrations ranging from 0 to 5 or 10 µmol/L of EPZ015666  and/or Erlotinib  or with DMSO alone after 24 hours. Cell viability was determined after 3 days (MDA‐MB‐453), as in the screen for experimental validation, or 7 days  by CellTiter‐Glo  assay. Luminescent signals were measured using an Infinite 200 spectrophotometer (Tecan). Chalice Analyzer (2.73) were grafted into the inter‐scapular fat pad of nude mice. When tumors reached 60‐100 mm3 , mice were randomly assigned to control or treatment groups (n = 7/group). Tumor volume was evaluated by measuring two perpendicular tumor diameters with a caliper, twice a week, as described.Six‐week‐old Female Swiss nude mice were purchased from Charles River  and maintained in specific pathogen‐free conditions. Their care and housing were in accordance with institutional guidelines as put forth by the French Ethical Committee. EPZ015666  was formulated at 1 mg/mL in 0.5% Methylcellulose (Sigma Aldrich) + 0.5% Tween 20 (Sigma Aldrich). EPZ015666 toxicity studies were performed by administration of 100 mg/kg, per‐os (po), twice daily, 5 days per week, to nude mice. Treatment was not associated with any mortality or body weight loss Figure . The pat2.8t tests and were considered significant if the P value was below 0.05. For the cell cycle experiment, we used cell counts to evaluate the difference between DMSO‐treated cells and EPZ015666‐treated cells for each population , in a Fisher‐exact test. We adjusted for multiple testing using the Benjamini‐Hochberg method. Differences were considered significant if the adjusted P value was below 0.05. For the in vivo experiment, differences observed between treated mice RTV and control group RTV were calculated using a two‐tailed Mann‐Whitney test. Differences were considered significant if the P value was below 0.05.For caspase activity assay, sub‐G1 cell cycle analysis, colony formation, and mammosphere formation assays, differences between groups were assessed using Student 33.1+ breast cancers .As mRNA and protein expression do not always concur, we next evaluated the expression of PRMT5 at the protein level in the samples from the Curie cohort by IHC using TMA, after validating an anti‐PRMT5 antibody for IHC staining Figure . We obse3.2www.kmplot.com)  and overall survival   Figure . Kaplan‐3.3To explore the potential of PRMT5 targeting in TNBC, we first examined the effect of PRMT5 depletion on six TNBC cell lines using two PRMT5 siRNAs. PRMT5 silencing significantly decreases viability Figure A, and coTo better assess the therapeutic relevance of PRMT5 targeting in TNBC, we next examined the effect of PRMT5 inhibition on a panel of breast cell lines using the PRMT5‐specific inhibitor EPZ015666.A newer more potent PRMT5 inhibitor , and show dose‐dependent increases in PARP, caspase‐7, and caspase‐8 cleavage following treatment Figure A. These Next, we examined the effect of PRMT5 inhibition on breast cancer cell cycle progression using flow cytometry. EPZ015666‐treated MDA‐MB‐453, MDA‐MB‐468, and HCC38 cells display a higher proportion of cells in the sub‐G1 phase 96 hours post‐treatment compared to untreated cells  are enriched in TNBC3.7per‐os (po). Treatment significantly slows tumor growth, with 39% tumor growth inhibition  after 4 weeks , including breast cancers.We further show here that PRMT5 is differentially localized in breast cancers and healthy mammary tissue, and that some important subcellular localization differences can be noted between breast cancer subtypes. Indeed, PRMT5 is expressed at lower levels in the nucleus of TNBC than in those of healthy breast tissues, HER2+, and luminal breast cancers. We thus posit that PRMT5 activity has different biological outcomes depending on PRMT5 subcellular localization and/or substrate specificity.Several studies have already pointed out the importance of PRMT5 localization in determining substrate specificity and resultant cell fate. During mouse embryogenesis, Prmt5 is predominantly found in the cytoplasm, where it maintains cell pluripotency via methylation of predeposited H2AR3.What controls the subcellular localization of PRMT5? A 2007 study by Teng et al may give a first element of response.Which additional signals and/or protein‐protein interactions are involved? These interrogations must be addressed to better understand PRMT5 substrate specificity and to further decipher the role of PRMT5 in cancers. Such knowledge could, in turn, allow the design of efficient therapeutic strategies targeting PRMT5 in a substrate‐specific manner. In conclusion, the present study highlights the importance of the subcellular localization of PRMT5 in determining TNBC prognosis and upholds continued attention for PRMT5 targeting, alone or in combination, as a potential treatment option in a subset of TNBC. Click here for additional data file.


In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation. Macro-autophagy (more commonly referred to as autophagy) is a highly conserved process present in all eukaryotes, which allows the degradation of proteins and organelles by lysosomes . It is cThe UNC51-like kinase 1  is a master regulator of autophagy . SpecifiThe coat protein complex II (COPII) is a multi-subunit protein complex essential for the transport of cellular cargos from the ER to the Golgi apparatus . A key cetc. (SKP1-CUL1-F-box protein (SCF) complexes form a family of multi-subunit ubiquitin ligases, which, in turn, is part of the super-family of Cullin-Ring Ligase (CRL) complexes . In humaetc. . We had etc. . A targeFBXW5 substrates, Streptag-FLAG (SF)-tagged FBXW5 was transiently expressed in HEK293T cells and affinity purified for analysis by Multidimensional Protein Identification Technology (MudPIT) (http://www.stowers.org/research/publications/libpb-1118). To confirm the binding between SEC23B and FBXW5 and its specificity, we screened a panel of nine human F-box proteins. SF-tagged F-box proteins were expressed in HEK293T cells and affinity precipitated to evaluate their interaction with SEC23B. We found that FBXW5 was the only F-box protein capable of co-precipitating with endogenous SEC23B (To identify SCF(MudPIT) . MudPIT s SEC23B .FBXW5 complex  induced an increase in both the steady state-levels and the stability of endogenous SEC23B in two different cell types . MoreoveFBXW5.Altogether, these results indicate that FBXW5 controls the ubiquitin- and proteasome-mediated degradation of SEC23B. Intriguingly, SEC23A did not bind FBXW5, and its levels were not affected by FBXW5 silencing , indicathttp://www.stowers.org/research/publications/libpb-1118), suggesting that the subpopulation of SEC23B interacting with FBXW5 is not integrated within COPII vesicles. SEC23B and SEC24 proteins form tight heterodimers  . TherefoNext, to test whether Ser186 on SEC23B can serve as a phospho-acceptor for ULK1, we co-expressed FLAG-tagged, wild-type SEC23B together with either wild type ULK1 or a kinase-dead (KD) mutant. Only wild-type ULK1 induced the phosphorylation of both endogenous and exogenous SEC23B on S186, as detected with our phospho-specific antibody  endogen. AdditioNext, we studied how nutrient deprivation, a condition that activates ULK1 and autophagy, modulates the phosphorylation of SEC23B on S186. After substituting the growth medium, which contains 10% fetal bovine serum, with Earle's balanced salt solution (EBSS), we observed de-phosphorylation of ULK1 and phosphorylation of ATG13 and BECLIN-1, two known substrates of ULK1 , indicatWe concluded that, in response to starvation, ULK1 phosphorylates SEC23B on S186.To investigate whether phosphorylation on S186 affects the SEC23B-FBXW5 interaction, we used a phosphorylated version of the synthetic peptide employed for the binding experiments. While the non-phosphorylated peptide efficiently bound FBXW5, as previously observed , a correConsistent with the observed impaired interaction with FBXW5, the levels of SEC23B(S186D) did not decrease in the presence of FBXW5  and it eSignificantly, although Ser186 is located at the interface between the SEC23 and SEC24 heterodimer binding region  Figure  and its Altogether, these results indicate that the ULK1-dependent phosphorylation of SEC23B blocks its FBXW5-dependent degradation but does not interfere with either the formation of the SEC23B-SEC24 heterodimer or its recruitment to the COPII vesicle coat by SAR1.Since ULK1 plays an essential role in the induction of autophagy, and since COPII proteins, in addition to their role in secretion, are also required for the proper execution of the autophagic program (see Introduction), we investigated whether the ULK1-dependent regulation of the interaction between FBXW5 and SEC23B regulates the autophagic flux. Automated quantification of both endogenous LC3  and exogNext, we used a tandem fluorescent-tagged LC3 construct (mCherry-GFP-LC3) that allows monitoring autophagosome maturation as a change from double-positive, green +red  fluorescent vesicles (autophagosomes), to single-positive, red fluorescent vesicles (autolysosomes) deprived of GFP fluorescence due to its quenching at low pH . Using tImportantly, co-depletion of SEC23B almost completely prevented the increase in autophagy mediated by FBXW5 silencing . MoreoveThe ERGIC compartment produces vesicles that are active for LC3 lipidation . We founAltogether, these results suggest that ULK1-mediated phosphorylation of SEC23B promotes both its localization to the ERGIC and its autophagic function, possibly at the expense of its secretory function.SEC23B were mutated to SEC23B(S186A)  . In contB(S186A) . SignifiB(S186A) . FinallyB(S186A) .The above results indicate that the presence of Ser186 in SEC23B and, presumably, its phosphorylation and consequent stabilization are required for the proper induction of autophagy in response to starvation.Vertebrates express four SEC24 paralogs , but it )-SEC24C . These oAltogether, these results suggest that upon serum starvation, the population of phosphorylated SEC23B preferentially associates with SEC24A and SEC24B.Notably, co-depletion of both SEC24A and SEC24B, but not SEC24C and SEC24D, inhibited the increase in autophagic flux induced by the silencing of FBXW5 , similarThus, our data demonstrate the existence of a high degree of specificity among SEC24 paralogs with respect to their ability to contribute to the autophagic flux.By searching for mutations of SEC23B in publicly available human cancer databases, we found a mutation in human melanoma that converts S186 of SEC23B to asparagine (ID#: COSM5391641). We thus generated SEC23B(S186N), a mutant that mimics the cancer associated mutation, and observed a reduced binding to FBXW5, SEC24C, and SEC24D  but not These results show that the SEC23B(S186N) mutation found in melanoma mimics SEC23B phosphorylated on Ser186 similarly to what observed for SEC23B(S186D). In fact, SEC23B(S186N) does not bind efficiently FBXW5, SEC24C, and SEC24D, and promotes autophagy, in agreement with a possible advantageous role resulting from increasing autophagic flux to ensure tumour cell homeostasis .SEC23B is an essential component of the COPII multi-subunit protein complex that is responsible for the transport of cargo proteins destined to be secreted . AlthougIt has been suggested that autophagic COPII vesicles can be distinguished from trafficking COPII vesicles by their different site of generation, that is autophagic COPII vesicles are generated from the ERGIC rather than the ERES, and they are potent stimulators of LC3 lipidation in response to starvation . PI3KC3,Recently, it has been shown that, in presence of nutrients, the basal activity of ULK1 mediates the phosphorylation of SEC16A (promoting the assembly of COPII complexes at the ERES) and that, in response to starvation, the activation of ULK1 induces the dissociation of SEC23A from SEC31A (inhibiting the secretory pathway) . Our stuAltogether, our results provide evidence for one of the molecular mechanisms by which ULK1 functions as a switch necessary to commit COPII to autophagy in response to starvation.Cell lines were purchased from ATCC and were routinely checked for mycoplasma contamination with the Universal Mycoplasma Detection Kit (ATCC 30–1012K). All cells were maintained in DMEM/GlutaMAX supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. For nutrient starvation, DMEM and FBS were removed and cells were grown in EBSS (Sigma) for the indicated times.For immunoprecipitation, cell extracts were prepared using lysis buffer , followed by incubation with Streptactin beads (IBA) or FLAG-M2 beads (Sigma) for 2 hr at 4°C. For immunoblotting, each sample was solubilized with lysis buffer . Cell extracts were quantified using BCA protein assay kit (Pierce) and solubilized with LDS-sample buffer (Life technology) followed by boiling at 95°C for five minutes.Immunofluorescence microscopy was performed as described previously . BrieflyAffinity purification and mass spectrometry were performed as described previously . BrieflyFBXW5 and SEC23B mutants were generated using KAPA HiFi polymerase (Kapabiosystems). All cDNAs were subsequently sequenced. ULK1 and ULK1(K46I) plasmids were purchased from Addgene. Sar1 plasmids were generously provided by Dr. Antonella De Matteis. SEC24A, B, C, and D plasmids were generously provided by Dr. Randy Schekman. ON-Target siRNAs targeting FBXW5, SEC23B, SEC24A, SEC24B, SEC24C, SEC24D were purchased from Dharmacon. The production of lentivirus was previously described . ULK1 anAn anti-FBXW5 antibody was generated by immunizing rabbits with FBXW5 peptides (Yenzyme) and affinity-purified using the same peptides immobilized on CNBr-sepharose. A rabbit polyclonal antibody against phospho-S186-SEC23B was generated and affinity purified by YenZym Antibodies. Anti- SEC23B, SEC24A, SEC24B, SEC24C, and SEC24D antibodies were provided by Dr. Randy Schekman. Mouse monoclonal antibodies were from Sigma-Aldrich (anti-FLAG M2), Covance (anti-HA), and Thermo Scientific (Sar1). Rabbit polyclonal antibodies were from Invitrogen (CUL1 and SKP1), Bethyl Laboratories, Inc. (Sec13 and SEC31A), Cell Signaling Technology (ULK1 and phospho-ULK1 (S757)), Novus Biological (LC3B), Rockland (phospho-ATG13 (S318)), and Abbiotec (phospho-Beclin-1 (S15).2, 25 mM β-glycerophosphate, 2 mM dithiothreitol and 100 μM sodium orthovanadate) were used to elute the purified proteins. Kinase and substrates were mixed and incubated at a final volume of 20 μL in KRB containing 20 μM ATP, 5 μg substrates at 30°C for 60 min. The reaction was stopped by the addition of sample buffer, boiled and analysed by immunoblot with phospho-SEC23B (Ser186) specific antibody.Kinase and substrates were purified from HEK293T cells that had been transfected with plasmids expressing individual kinase and substrates. Kinase reaction buffer  , while SEC23B was modeled by homology from the PDB structure of SEC23A (entry 5KYN), including the missing residues from the crystal structure. The structure of the mutant SEC23B(S186D) was obtained using PyMol Mutagenesis Wizard tool . MD simulations were performed with the GROMACS v5.1 package  using thermostat  with a cA scheme  providedA scheme  using thA scheme . The salTo generate SEC23B S186A and S186N knock-in cells, an optimal gRNA target sequence closest to the genomic target site and a 2.1 kb homologous recombination (HR) donor template were designed using the Benchling CRISPR Genome Engineering tool. The HR donor template was designed to introduce alanine or asparagine substitutions at position S186, and a silent mutation to introduce a KpnI restriction site for genotyping. SEC23B gRNA target sequence see;  was clonGaussia luciferase plasmid (ThermoFisher Scientific) was co-transfected to be used as a reporter by measuring luciferase activity in the conditioned medium , 3,000 × g (10 min), 25,000 × g (20 min) to collect the membranes sedimented at each speed. The 25,000 × g membrane pellet was suspended in 0.75 ml 1.25 M sucrose buffer and overlayed with 0.5 ml 1.1 M and 0.5 ml 0.25 M sucrose buffer . Centrifugation was performed at 120,000 × g for 2 hr , after which the interface between 0.25 M and 1.1 M sucrose (L fraction) was selected and suspended in 1 ml 19% OptiPrep for a step gradient containing 0.5 ml 22.5%, 1 ml 19% (sample), 0.9 ml 16%, 0.9 ml 12%, 1 ml 8%, 0.5 ml 5% and 0.2 ml 0% OptiPrep each. Each density of OptiPrep was prepared by diluting 50% OptiPrep  with a buffer containing 20 mM Tricine-KOH, pH 7.4, 250 mM sucrose and 1 mM EDTA. The OptiPrep gradient was centrifuged at 150,000 × g for 3 hr  and subsequently fractions of 0.5 ml each, were collected from the top. The fractions were then analyzed by immunoblot.Membrane fractionation was performed through a modified protocol based on e et al. . HeLa cee et al. . MembranImages were analyzed with an in-house developed python script. Cell counts were calculated by detecting nuclei labelled by DAPI in the blue channel of the image. First, a Gaussian smoothing was applied, and then an Otsu threshold to determine the nuclei mask. This was followed by watershed segmentation to separate touching nuclei, as well as filtering based on area to remove small spots. Partial nuclei touching image borders were included but were counted as a fraction based on the average nuclei size measured in the data set. The puncta (green channel) were detected using the Laplacian of Gaussian (LoG) blob detection algorithm as provided by the python package ‘scikit-image’ . Any blo In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ivan Dikic as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Catherine Rabouille (Reviewer #3).Thank you for submitting your article "The ULK1-FBXW5-SEC23B nexus controls autophagy" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The authors identified SEC23B as a substrate of the E3 ligase FBXW5. They found that FBXW5 leads to proteasomal degradation of SEC23B under normal conditions. In contrast, under starvation conditions, ULK1 phosphorylates SEC23B on Ser186, preventing recognition by FBXW5 and redirecting SEC23B to the ERGIC together with SEC24A/B. The data are clearly demonstrated, and the logic is easy to follow.Essential revisions:1) The authors propose that ULK1-mediated phosphorylation of SEC23B promotes its translocation to the ERGIC, but this is not directly shown. The authors should test whether the translocation of SEC23B to the ERGIC is indeed abolished in ULK1- or ULK1/2-knockdown cells.2) The authors conclude that FBXW5-dependent degradation of SEC23B downregulates basal autophagy based on the results of the LC3 puncta formation and mCherry-GFP-LC3 assays. However, the authors should rule out the possibility that SEC23B only increases the level of LC3-II on autophagosomes by enhancing LC3 lipidation at the ERGIC rather than the efficiency of autophagosome formation (the extent of autophagic degradation). To confirm that FBXW5 knockdown and the expression of the SEC23(SD) mutant indeed increase the autophagic flux even under nutrient-rich conditions, the authors should determine whether degradation of autophagic cargos such as p62 is also activated.3) Related to comment #2, if the expression of SEC23B(S186D) is indeed sufficient to induce autophagy, the data suggest that the primary function of the ULK1 complex is phosphorylation of SEC23B among many known substrates. To prove this hypothesis, it would be important to test whether the expression of SEC23B(S186D) can rescue autophagy in ULK1- or ULK1/2-double knockdown cells.4) If the regulation of the level of SEC23B by FBXW5 is important for the control of autophagy, is overexpression of even wild-type SEC23B sufficient to induce autophagy?5) The number of LC3 can be increased by not only autophagy activation but also inhibition of the lysosomal function. Given that overexpression of SEC23B(S186D) affects secretion , the authors should rule out the possibility that it also affects the lysosomal function and thereby causes accumulation of LC3. The lysosomal function can be tested by monitoring maturation of cathepsins or degradation of the EGF receptor.and fueling general secretion.6) Subsection “Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation”, first paragraph and Supplementary Figure 3D and E: The data on the secretory pathway by assaying secreted Gaussia Luciferase are critical and should be shown in the main figures rather than in the supplement. It could have been well the case that SEC23-P/SEC24A/B would lead to vesicles feeding autophagosomes 7) Perhaps one important point to clarify is what happens to the secretory pathway and ER to Golgi transport with SEC23B-P/SEC24A/B? Is there hard evidence to exclude the notion of SEC23B-P/SEC24A/B during starvation does not also fuel secretion (except for the luciferase secretion that is inhibited)? Any specific cargo? Why would this complex SEC23B-P/SEC24A/B not support full COPII coat formation? The authors seem in doubt exemplified with the question mark in their model. Does SEC23-P/SEC24A/B also bind the other COPII subunits to form a full coated vesicle? This should be experimentally tested. What about the other isoforms of SEC23 and SEC24C/D? Essential revisions:1) The authors propose that ULK1-mediated phosphorylation of SEC23B promotes its translocation to the ERGIC, but this is not directly shown. The authors should test whether the translocation of SEC23B to the ERGIC is indeed abolished in ULK1- or ULK1/2-knockdown cells.We now show that ULK1 knockdown significantly inhibits the translocation of SEC23B to the ERGIC .2) The authors conclude that FBXW5-dependent degradation of SEC23B downregulates basal autophagy based on the results of the LC3 puncta formation and mCherry-GFP-LC3 assays. However, the authors should rule out the possibility that SEC23B only increases the level of LC3-II on autophagosomes by enhancing LC3 lipidation at the ERGIC rather than the efficiency of autophagosome formation (the extent of autophagic degradation). To confirm that FBXW5 knockdown and the expression of the SEC23(SD) mutant indeed increase the autophagic flux even under nutrient-rich conditions, the authors should determine whether degradation of autophagic cargos such as p62 is also activated.We now show that FBXW5 knockdown and the expression of the SEC23(S186D) mutant activate the degradation of p62 under nutrient-rich conditions .3) Related to comment #2, if the expression of SEC23B(S186D) is indeed sufficient to induce autophagy, the data suggest that the primary function of the ULK1 complex is phosphorylation of SEC23B among many known substrates. To prove this hypothesis, it would be important to test whether the expression of SEC23B(S186D) can rescue autophagy in ULK1- or ULK1/2-double knockdown cells.We now show that the expression of SEC23B(S186D) is unable to rescue autophagy in ULK1 knockdown cells . This is likely because ULK1 is necessary to phosphorylate many pro-autophagic substrates. Moreover, in presence of nutrients, the basal activity of ULK1 is necessary to promote the assembly of COPII complexes . Therefore, in the absence of ULK1, it is expected that neither secretory nor autophagic COPII vesicles are efficiently formed.4) If the regulation of the level of SEC23B by FBXW5 is important for the control of autophagy, is overexpression of even wild-type SEC23B sufficient to induce autophagy?We now show that overexpression of wild-type SEC23B to levels similar to those of the stable SEC23B(S186D) mutant is sufficient to induce autophagy .5) The number of LC3 can be increased by not only autophagy activation but also inhibition of the lysosomal function. Given that overexpression of SEC23B(S186D) affects secretion , the authors should rule out the possibility that it also affects the lysosomal function and thereby causes accumulation of LC3. The lysosomal function can be tested by monitoring maturation of cathepsins or degradation of the EGF receptor.By monitoring the levels of p62 as well as pro-Cathepsin and active-Cathepsin, we now show that the overexpression of SEC23B(S186D) does not affect the lysosomal function .6) Subsection “Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation”, first paragraph and Supplementary Figure 3D and E: The data on the secretory pathway by assaying secreted Gaussia Luciferase are critical and should be shown in the main figures rather than in the supplement. It could have been well the case that SEC23-P/SEC24A/B would lead to vesicles feeding autophagosomes and fueling general secretion.We have moved these figures to the main figures .7) Perhaps one important point to clarify is what happens to the secretory pathway and ER to Golgi transport with SEC23B-P/SEC24A/B? Is there hard evidence to exclude the notion of SEC23B-P/SEC24A/B during starvation does not also fuel secretion (except for the luciferase secretion that is inhibited)? Any specific cargo? Why would this complex SEC23B-P/SEC24A/B not support full COPII coat formation? The authors seem in doubt exemplified with the question mark in their model. Does SEC23-P/SEC24A/B also bind the other COPII subunits to form a full coated vesicle? This should be experimentally tested. What about the other isoforms of SEC23 and SEC24C/D?The phosphorylation of Ser186 does not increase the affinity of SEC23B to SEC24A and SEC24B. Rather, it inhibits the binding of SEC23B to SEC24C and SEC24D. We never claimed in the text that the binding of phosphorylated SEC23B to SEC24A and SEC24B reduces secretion. The lack of binding between phosphorylated SEC23B and SEC24C/D is striking and we speculate that this inhibition in binding may explain the reduced secretion. However, we have shown that SEC23B(S186D) (which mimics the phosphorylated form of SEC23B) binds to SAR1(H79G), a GTP-bound SAR1 mutant that is constitutively associated with COPII vesicles, but not with the cytosolic GDP-bound SAR1(T39N) mutant . Moreover, SEC23B(S186D) also interacts with SEC13, SEC16, and SEC31 . In conclusion, we believe that, upon starvation, SEC23B-SEC24A/B promotes autophagy, but can possibly also sustain secretion since these proteins can bind other COPII subunits to form a full-coated vesicle. However, the decrease in the abundance of the SEC23B-SEC24C/D complexes likely contributes to a decrease in secretion. Concerning the other isoform of SEC23, Gan et al. (2017) have shown that ULK1 induces the dissociation of SEC23A from SEC31A in response to starvation, further inhibiting the secretory pathway. We now integrated better these points on the Discussion section. We also agree that the question mark going from the SEC23B-SEC24A/B to the “ER-to-Golgi trafficking”, which we drew in the old cartoon  is confusing and reductionist. In fact, we believe that a simple cartoon cannot easily summarize the effects of FBXW5, ULK1, SEC23B phosphorylation, and SEC23B accumulation on both secretion and autophagy. Therefore, we eliminated the cartoon and its shortcomings from the supplementary figures. Finally, we believe that the identification of specific cargos goes beyond the scope of the current study.


We evaluated the immunoexpression of LGR4 and β-catenin in primary gastric carcinomas, lymph node metastases and histologically normal gastric mucosa in the surgical margins of gastric primary tumours. We performed a cross-sectional, observational study, based on 75 gastric carcinoma specimens from gastrectomies conducted at the hospital of the Federal University of Ceará, Brazil. The samples were analysed by tissue microarray and immunohistochemistry. Chi-square, Fisher’s exact test and Pearson’s linear regression were used in this study. LGR4 expression was greater in the histologically normal gastric mucosa  of the tumour surgical resection margin than in the cases of primary carcinomas , and also in the number of cells stained in the normal mucosa (P<0.0001). Primary intestinal-type carcinomas showed greater positivity for LGR4 than diffuse tumours  and in these the positivity was higher in the metastases (P=0.0242). The membranous immunoexpression of β-catenin was ubiquitous in the normal mucosa and present in 2/3 of the positive carcinomas. In only one case, nuclear β-catenin expression was observed. Most LGR4-positive cases were stained for membranous β-catenin but not the opposite (P<0.01). LGR4 is a likely biomarker of stem cells in the normal gastric mucosa and carcinomas of the stomach, not specific to cancer cells and positively associated with cell proliferation. LGR4 immunoexpression is more frequent and found in a larger number of cells in normal tissues than in tumour samples. Expression of β-catenin in the junctional membrane-complex occurred predominantly, in positive cases of gastric carcinomas and very rarely in the nucleus. LGR4 apparently influenced the membranous expression of β-catenin. These findings suggest a controversial role for LGR4, related to proliferative status and inversely related to tumour progression, in contrast to most previous reports. Gastric cancer is the fifth most common neoplasm worldwide, preceded by lung, breast, colorectal and prostate cancer. In mortality, it ranks third after lung and liver cancer . According to Lauren’s classification, there are two main types of adenocarcinomas, with distinct histological, clinical and epidemiological characteristics: the intestinal type and the diffuse type .Cancer stem cells are associated with tumour initiation, development and metastatic dissemination . Molecules expressed in normal stem cells can be used as biomarkers of cancer stem cells . New candidates for stem cell biomarkers have been investigated recently. Among these, the G protein receptors rich in leucine residues (LGRs) monitor important physiological and pathological functions . LGR4, a member of this family of receptors, participates in cell signalling, activating transcription factors related to tumour formation, potentiating the Wnt signalling pathway and playing an important role in the development of multiple organs and cancer progression .β-catenin is a perimembranous cytoplasmic protein that interacts with the cadherins of the intercellular junctional complex in the adhesion zones. When excess β-catenin is present in the cytoplasm, it can migrate to the nucleus where it acts as a transcription factor for cell proliferation. In addition, β-catenin participates in cellular communication, mainly related to the Wnt signalling pathway . Mutations, cytoplasmic/nuclear overexpression of β-catenin and their membrane loss or decrease have been associated with several types of neoplasms including colorectal carcinoma and gastric carcinoma .In gastric cancer, LGR4 is a potential stem cell marker, and may be involved in the activation of β-catenin similar to LGR5 and LGR6 . Moreover, in cell signalling pathways, it activates transcription factors related to tumour formation , but these aspects are not yet clear. The aim of this study is to evaluate the immunoexpression of LGR4 and β-catenin in primary gastric carcinomas in both histotypes, lymph node metastases and histologically normal gastric mucosa at the tumour resection margins and to correlate the expression of these potential biomarkers with clinical variables such as age, sex and tumour staging.Patients and samplesThe study is a cross-sectional observational study of 75 paraffin-embedded gastrectomy specimens collected at the Department of Pathology and Legal Medicine of the Federal University of Ceará (DPML / UFC). Tissue samples of primary tumours (n=75), lymph node metastases (n=26) and histologically normal gastric mucosa at the tumour surgical margin (n=60) were selected, fixed in 10% formalin, processed and embedded in paraffin and then evaluated in histological sections stained with haematoxylin-eosin. In the evaluation of β-catenin, four cases of primary tumours and six lymph node metastases were not appropriate for analysis and were excluded. Similarly, eight cases of histologically normal mucosa were not suitable for LGR4 analysis and were also excluded. Specimens with inadequate fixation, hemorrhage and necrosis were not included in the original sampling.The cases were classified according to Lauren’s classification into two main types of adenocarcinomas, with distinct histological, clinical and epidemiological characteristics. The intestinal type, which is composed of cohesive neoplastic cells that form tubular structures that resemble the glands and the diffuse type, consisting of isolated cells that are dispersed throughout the stomach wall .A tissue microarray (TMA) technique was used in this study . Immunohistochemistry was performed using a Dako™ platform and Abcam™primary monoclonal antibodies anti-LGR4 (GR125979-19) and anti-β-catenin (GR205416-1) both diluted 1/500.As external positive controls for LGR4, normal bowel tissue  and normal kidney tissue  were utilized. For β-catenin, an intestine sample was used as positive control . For the negative control, immunohistochemistry on two samples was performed in the absence of primary antibody.Evaluation of immunostainingLGR4: based on previous studies, at least one cell with membranous or cytoplasmic labeling is required for the sample to be considered positive . Counts of stained cells  from 100 to 1,000 cells/case using an appropriate optical system (Carl Zeiss™) were performed. The fields were counted sequentially from a random point  at 400× magnification, until reaching the number of cells in the last field evaluated. Membranous or nuclear β-catenin expression was considered positive when the number of stained cells was ≥ 10% .Histological preparations for LGR4 and β-catenin immunostaining were double-blind investigated by different researchers . When the results for the same sample were different, a third investigation was performed for confirmation.Statistical analysisThe relationship between the differential expression of LGR4 and β-catenin and the clinical and pathological features was assessed using Fisher’s exact test or chi-square test, with a P value <0.05 considered statistically significant. The numbers of stained and unstained cells from the LGR4 positive cases of tumours and normal mucosa were analysed by the Kolmogorov-Smirnov normality test (P>0.05). The positive cell counts of the two researchers were compared by Pearson’s correlation, with a very strong (r=0.905) statistically significant correlation coefficient (P<0.001) validating its calibration. GraphPad Prism 6™ software was used for statistical analysis and plotting.Ethical AspectsThis project was approved by the Ethical Committee of the Federal University of Ceará, Fortaleza, CE, Brazil (Process number 2004326).The general clinicopathological variables data and the LGR4 and β-catenin immunoexpression obtained in this study are shown in Distribution of LGR4 immunoexpression in the normal gastric mucosa, primary gastric carcinomas and lymph node metastasesThe expression of LGR4 in the histologically normal mucosa in a total of 50 out of 52 cases (96%) was higher than in the primary tumours , with a significant difference (P<0.0001), as shown in Immunoexpression of LGR4 in the histologically normal mucosa, primary carcinomas and lymph node metastases, based on positive cell countsThe counts of LGR4-positive cells in the primary tumours, lymph node metastases and normal mucosa were evaluated, as shown in The immunoexpression for LGR4 in the primary tumour in relation to the number of positive cells was significantly higher in the intestinal type (7321/21001=35%) than in the diffuse type . In the histologically normal mucosa, at the margins of the diffuse subtype, the immunostaining was significantly higher (7024/15407=46%) than in the intestinal type . When evaluating the total cells immunostained in the metastases (3791/8996=42%), it was observed that it was well above the total in primary carcinomas  was greater than that in the primary tumours without distinguishing between histotypes  was observed compared to that in the primary tumour (19/35=54%)  were also β-catenin positive (22/26=85%), but the number of β-catenin positive cases that were also LGR4 positive was only 48%  in the histologically normal gastric mucosa, always in the basal third of the epithelial thickness. This site is involved with the cellular proliferation of the mucosal renewal, where cells are less differentiated as stem cells and progenitor cells. At the same time, higher LGR4 positivity was observed in the normal mucosa than in the tumour tissue, with a highly significant difference . These findings are indicative of LGR4 being involved in cell proliferation, whether of normal or tumour cells, without distinguishing them. LGR4 possibly represents a marker of stem/progenitor cells lato sensu, not being characteristic or specific of neoplastic cells.A few reports have compared the presence of LGR4 in normal and tumour mucosa. Wu et al., (2013) found significantly higher LGR4 expression in tumour cells than in normal tissues in histological sections of large intestinal mucosa and of colonic tumours. This finding is consistent with our data regarding the presence of this marker in the two types of tissue but discordant regarding the higher frequency in their cancer samples.Some recent studies have investigated LGR4 in stomach cancer. Steffen et al., (2012) observed a correlation between LGR4 expression and lymph node dissemination in human carcinoma samples. In human colorectal carcinomas, Wu et al., (2013 ) reported that LGR4 is a poor prognostic factor for increasing tumour invasion and metastasis when it is associated with an increase of nuclear β-catenin via Wnt. In an in vitro study with a culture of various types of cancer cells , Zhu et al., (2013) also observed that LGR4 overexpression was associated with increased expression of nuclear β-catenin. In addition, another in vitro and experimental study  reported that LGR4 was associated with a poor prognosis because its reduction decreased the number of cancer stem cells and of cells in epithelial-mesenchymal transition. All these reports point to a role of the pro-carcinogenic LGR4 in promoting tumour progression.Although in this study LGR4 was present in tumour samples, its expression was more common in those cases without lymph node metastases, with less local invasion and in the absence of perineural invasion, i.e., associated with clinical variables of better prognosis. Therefore, its expression was inversely related to tumour progression, the opposite of what has been generally reported .The cell count in the positive cases of LGR4 strengthens the information stated previously regarding the comparison between the number of normal and tumour positive cases: a much higher percentage of stained cells in the normal mucosa compared to the percentage of positive tumour cells. Among these cases, cell staining in primary gastric tumours was greater than that of the respective lymph node metastases. Moreover, in primary carcinomas of the intestinal histotype, the LGR4 immunoexpression was significantly higher than that of the diffuse type , a lesser differentiated histotype , finding consistent with that of Steffen et al., ( 2012). These findings suggest that LGR4 is a valuable biomarker of cells linked to proliferation (stem cells/progenitors) in normal and cancer contexts, but that it plays a role in opposing tumour progression.In recent studies, LGR4 has been shown to promote the proliferation of cancer cells through the activation of the Wnt/β-catenin signalling pathway that plays important roles in stem cell biology, tissue homeostasis and cancer . The key mediator of this signalling pathway is β-catenin that is found dynamically at multiple subcellular locations, including intercellular junctions, cytoplasm and nucleus . Mutations in β-catenin may cause activation of the Wnt pathway in gastric cancer , when they favor the nuclear localization of this protein. The cytoplasmic and/or nuclear localization of β-catenin can be used to estimate Wnt pathway activity. These relationships between LGR and β-catenin proteins during carcinogenesis and possibly during tumour progression prompted us to study this protein and LGR4 in the same samples of normal and tumour gastric mucosa. In this study, it was observed that immunostaining for β-catenin was universally present at membrane localization in histologically normal mucosal cells as expected. In the tumour samples, membranous immunostaining was present in 2/3 of the cases. And in only one case, there was nuclear staining in the lymph node metastasis whose primary tumour was of the intestinal histotype. This result was similar to that of Lins et al., (2016) who found only two cases (2/64=3.1%) of nuclear positivity in intestinal gastric cancer samples. The rarity of the nuclear expression of β-catenin in our case series drastically reduces the importance of the Wnt pathway during the carcinogenesis of these cases. However, Wang et al., (2015) found nuclear positivity in 31.6% of their cases, with predominance in the intestinal histotype. In a different way, Wu et al. (2013) observed high β-catenin expression in 52.9% of the colorectal cancer (CRC) cases, involving the cytoplasm (70.6%) and nucleus (26.5%) of the cells.The evaluation of β-catenin expression in relation to clinicopathological variables in this study revealed a predominance of positivity (membranous expression) in the absence of angiolymphatic and perineural invasion. These findings are consistent with the suppressor role of β-catenin during tumour progression, when localized in the junctional membrane complex, favouring adhesion between cells.Could there be a possible cause and effect association between LGR4 and β-catenin expression or vice versa based on the results of this study? When comparing the immunoexpression of LGR4 and β-catenin membranes, it was observed that when LGR4 was positive, the great majority of β-catenin (85%) was also positive. However, when β-catenin was positive, only half the cases stained by LGR4 were positive, the difference between the two comparisons being highly significant (P<0.01). These data suggest that LGR4 may influence the expression of β-catenin in the cell membrane but that the expression of the latter does not appear to significantly influence the positivity of LGR4. This observation suggests a possible mechanism of anti-neoplastic action of LGR4. No previous studies were found for comparison.In summary, LGR4 is a potential biomarker of stem cells in normal gastric mucosa and carcinomas of the stomach, not specific to cancer cells and positively associated with cell proliferation. Its immunoexpression is more frequent and occurs in a larger number of cells in normal tissues than in tumour samples. Expression of β-catenin in the junctional membrane complex occurred predominantly in positive cases of gastric carcinomas, and immunostaining of this protein in the nucleus was extremely rare. LGR4 apparently influenced β-catenin membranous expression but not the opposite. The findings from this study suggest a controversial role for LGR4, related to proliferative status and inversely related to tumour progression, in contrast to most previous reports.This project was partially funded from the Public Call MCTI/CNPq Nº 14/2013 - Universal/Universal, process Nº 483318/2013-2.The authors state that they have no conflicts of interest.