In conclusion, the present study suggests that cisplatin-based adjuvant chemotherapy needs to be carefully applied to early stage NSCLC patients with overexpressed β-catenin in combination with reduced NME1 expression.This study aimed to understand whether the effect of non-metastatic cells 1 (NME1) on recurrence-free survival (RFS) in early stage non-small cell lung cancer (NSCLC) can be modified by β-catenin overexpression and cisplatin-based adjuvant chemotherapy. Expression levels of NME1 and β-catenin were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 425 early stage NSCLC patients. Reduced NME1 expression was found in 39% of samples. The median duration of follow-up was 56 months, and recurrence was found in 186 (44%) of 425 patients. The negative effect of reduced NME1 expression on RFS was worsened by cisplatin-based adjuvant chemotherapy (adjusted hazard ratio = 3.26, 95% CI = 1.16–9.17,  Lung cancer is one of the most common causes of cancer-related deaths in the world. Despite recent advances in the early detection and treatment of lung cancer, the prognosis is very poor, partly because of a high rate of recurrence even after curative resection. Approximately half of the patients diagnosed with non-small cell lung cancer (NSCLC) develop recurrence and die of the disease even after curative resection. Adjuvant chemotherapy plays an important role in preventing recurrence following curative resection of lung cancer. A survival benefit of platinum-based adjuvant chemotherapy in NSCLC was confirmed by phase III trials and the Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis ,2. HowevNME1 gene into different types of cancer cells has resulted in the inhibition of metastatic properties, including migration, invasion, and colonization [NME1 silencing is known to upregulate β-catenin-dependent TCF/LEF-1 (T-cell factor/lymphoid enhancer-binding factor) transactivation through glycogen synthase kinase (GSK)-3β-independent mechanisms by promoting nuclear translocation of β-catenin [Non-metastatic cells 1 (NME1), also known as NM23-H1, was the first metastasis suppressor discovered by its reduced mRNA transcript levels in a murine melanoma cell line exhibiting high metastatic activity . An invenization ,14,15,16-catenin .c-MYC and CCND1, and influences the metastatic cascade by regulating the expression of genes such as AXIN2, SNAIL, ZEB1, COX2, and S100A4 [NME1 silencing, Wnt/β-catenin-mediated resistance to cisplatin has been demonstrated in human cancers [Activation of the canonical Wnt signaling pathway inhibits axin-mediated β-catenin phosphorylation and degradation and allows β-catenin to accumulate in the cytoplasm and then translocate into the nucleus. Nuclear β-catenin forms a stable complex with members of the TCF/LEF transcription factor family and induces the expression of target genes such as d S100A4 . It has d S100A4 ,20,21,22 cancers ,24. BaseIn this study, we analyzed whether the effect of NME1 on recurrence-free survival (RFS) can be modified by cisplatin-based adjuvant chemotherapy and β-catenin overexpression in early stage NSCLC.This was a retrospective study. Formalin-fixed paraffin-embedded (FFPE) tissue specimens stored at room temperature were obtained from 425 patients with pathologic stage I–IIIA NSCLC who had undergone anatomical lung resection with mediastinal lymph node dissection between November 1994 and April 2004 at Samsung Medical Center in Seoul, Korea. Patients with incomplete resection of lung tissue  or history of neoadjuvant therapy were excluded from this study. Postoperative follow-up was performed according to a previously described protocol . SupportTissue microarrays (TMAs) were constructed from paraffin blocks prepared from 425 NSCLC samples. Expression levels of β-catenin and NME1 proteins were analyzed using immunohistochemistry. In brief, serial sections of 4 μM in thickness were cut from TMA blocks, deparaffinized in xylene, and rehydrated through a series of decreasing concentrations of alcohols. Antigens were recovered by heating these sections in 10 mM (pH 6) citrate buffer for 10 min using a pressure cooker. These sections were then incubated with primary antibody β-catenin clone 17C2  or NME1 clone 4B2  at 4 °C overnight. Immunoreactivity of each primary antibody was detected with Envision™ + peroxidase . Antibody-bound peroxidase activity was visualized after incubating with chromogen 3,3′-diaminobenzidine (DAB) at room temperature for 1–5 min. Normal bronchial epithelial cells were used for positive control of staining, and primary antibody was replaced by immunoglobin for negative control. All sections were counterstained with Mayer’s hematoxylin.Immunohistochemical staining was interpreted by consensus between two authors (O.-J.L. and D.-H.K.) in a double-blinded fashion to minimize inter-rater variability. Samples with a Cohen’s kappa coefficient of less than 0.20 were removed from further analysis. Although immunoreactivity for β-catenin was found in the membrane, cytoplasm, and nucleus, only cytoplasmic staining was assessed for scoring. The expression of NME1 protein in tumor cells was evaluated based on cytoplasmic staining. Cytoplasmic staining of both proteins was semi-quantitatively evaluated using a score calculated by multiplying the intensity score  with the proportion score of positive cells . For NME1, its expression was defined as reduced if a composite score was less than two in a tumor. β-catenin expression was considered to be overexpressed in a tumor with a composite score greater than or equal to two. Staining was performed in triplicate and average values of scores were used to determine the expression levels. Cutoff values for the abnormal expression of NME1 and β-catenin were determined considering an internal control consisting of 23 normal lung cores. Representative positive stainings for β-catenin and NME1 expression are shown in adenocarcinoma and squamous cell carcinoma A. DetailPatients were randomly selected without stratification or matching by age. The median duration of follow-up was 56 months. The clinical endpoint of the study was recurrence-free survival (RFS), which was defined as the time from the date of the diagnosis to the first recurrence. Variables such as age, sex, histology, pathologic stage, NME1 expression, and adjuvant chemotherapy were initially considered for the analysis of RFS. FFPE tissue samples were obtained from 425 patients because at least 365 patients were needed for analysis of the effect of NME1 expression on RFS under 2-sided α = 0.05 and β = 0.1 .t-test (or one-way ANOVA) were used for univariate analyses of continuous and categorical variables, respectively. A linear relationship between two continuous variables was analyzed using Pearson’s correlation coefficient. The prognostic significance of NME1 on RFS was evaluated by Kaplan–Meier survival curves. The difference between two survival curves was assessed using the log-rank test. Variables with P ≤ 0.25 in the univariate analysis were included in the multivariate model. Hazard ratios of predictor variables for survival were estimated using the Cox proportional hazards model after controlling for potential confounding factors. The effect of β-catenin expression and adjuvant chemotherapy on NME1 function was analyzed using a stratified Cox proportional hazards model. No replacement was made for missing values. All statistical analyses were two-sided with a type I error of 5%.To find factors associated with NME1 reduction in NSCLC, chi-square test (or Fisher’s exact test) and Student’s p = 0.01; A total of 425 patients with early stage NSCLC were included in the data analysis without dropout. The mean age of the patients at diagnosis was 61 years , and men accounted for 74% of the cases. Adenocarcinoma and squamous cell carcinoma comprised 46% and 47% of the cases, respectively. Patients at I, II, and IIIA stages accounted for 56%, 43%, and 5%, respectively. The relationship between NME1 expression and clinicopathological characteristics is summarized in p < 0.0001). β-catenin was overexpressed in 55% of patients, with a higher prevalence in squamous cell carcinoma than that in adenocarcinoma  of 425 patients. Patients with reduced NME1 expression had a higher recurrence rate than those without  times worse in patients with reduced NME1 expression than in those without (p = 0.18), sex , adjuvant chemotherapy , and β-catenin expression .Multivariate Cox proportional hazards analysis was conducted to measure the effect of reduced NME1 expression on RFS in early stage NSCLC, after adjusting for potential confounding effects of variables. Considering the pathological stage, hazard ratios for RFS ranged from 1.64 to 3.93, after adjusting for patient age, sex, β-catenin expression, adjuvant chemotherapy, and histology . The haz without . Howeverp = 0.03; p = 0.005) to 2.54  plays a crucial role in promoting metastasis of carcinoma derived from epithelial cells. Tumor cells lose their epithelial characteristics such as cell polarity and gain mesenchymal features such as increased migratory and invasive potentials during EMT. Our data did not show an association of NME1 expression with tumor growth , consistdvantage . A numbedvantage ,29. In cdvantage . Ohta etdvantage  have alsdvantage ,33. The dvantage ,31,32,33NME1 inhibits the metastasis of lung cancer cells by interacting with Inhibitor of nuclear factor Kappa-B Kinase subunit beta (IKKβ) in an isotype-specific fashion and regulating tumor necrosis factor alpha (TNFα)-stimulated Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling negatively [NME1 silencing induces the nuclear translocation of β-catenin by disrupting adherence junction complexes mediated by E-cadherin and promotes extracellular matrix invasion by increasing invadopodia formation and pericellular matrix metalloproteinase (MMP) activity [The negative effect of reduced NME1 expression on RFS in this study was worse in patients with β-catenin overexpression than in those without. How does β-catenin overexpression influence the effect of reduced NME1 expression on RFS? Mechanisms underlying the metastasis suppression of NME1 have been addressed in multiple types of cancer cells. Early efforts have revealed that NME1 may mediate its inhibitory effects on cellular motility and invasion through interactions with signaling cascades ,34,35,36gatively . In addiactivity .ITGβ3) gene through direct physical interaction with the promoter [In addition to its effect on signaling pathways, NME1 is also known to regulate gene transcription by binding to single-stranded DNA. A promoter region between –922 to –846 of Kangai 1 (KAI1) is known to suppress metastasis through the inhibition of cell movement. It responds to NME1 in high-metastatic lung cancer cell line L9981 . NME1 supromoter . NME1 alpromoter ,40,41,42NME1-transfected breast cancer cells [NME1 by shRNA transfection in head and neck squamous carcinoma cells attenuates the chemosensitivity of cells to cisplatin by downregulating cyclins E and A and reducing cisplatin-induced S-phase accumulation [Platinum derivatives such as cisplatin are widely used chemotherapeutic agents for NSCLC. However, cisplatin resistance is a major challenge in the use of these drugs. The molecular mechanism of cisplatin resistance in lung cancer cells is not fully understood. Therefore, there are few efficient strategies to overcome such resistance. Cisplatin-based adjuvant chemotherapy in the present study did not affect RFS in univariate analysis. However, it worsened the RFS in patients with reduced NME1 expression . A functer cells . NME1 haer cells . Thus, rmulation . These lBcl-xl and increase cisplatin sensitivity in A549 wild-type cells. Despite these associations of β-catenin overexpression and cisplatin resistance in various types of cancer cells, β-catenin overexpression alone was not associated with cisplatin resistance in the present study. However, β-catenin overexpression aggravated RFS when patients with reduced NME1 received cisplatin-based adjuvant chemotherapy  polymorphism in NME1 gene is associated with increased susceptibility to NSCLC [This study was limited by several factors. First, this was a retrospective study that was prone to selection and surveillance biases. Second, it is necessary to investigate combined effects of β-catenin and NME1 on apoptosis, migration, invasion, or metastasis in different cell types of lung cancer to clearly understand the molecular mechanisms underlying the effect of β-catenin and NME1 on poor RFS. Third, the lack of a negative effect of β-catenin in the univariate analysis  in this to NSCLC  and coulIn conclusion, the present study suggests that the adverse effect of reduced NME1 expression on RFS may be exacerbated by cisplatin-based adjuvant chemotherapy and β-catenin overexpression through other mechanisms rather than through the nuclear translocation of β-catenin in early stage NSCLC. Accordingly, it is recommended that cisplatin-based adjuvant chemotherapy in patients with completely resected stage I–IIIA NSCLC be carefully applied after examining the expression levels of β-catenin and NME1.


Jumonji AT-rich interactive domain 1B(JARID1B) has been shown to be upregulated in many human cancers and plays a critical role in the development of cancers cells. Nevertheless, its functional role in colorectal cancer (CRC) progression is not fully understood.Herein, JARID1B expression levels were detected in clinical CRC samples by western blotting and qRT-PCR. DLD-1 cells with JARID1B knockdown or overexpression by stably transfected plasmids were used in vitro and in vivo study. Colony formation, 5-ethynyl-20-deoxyuridine (EdU) and Real Time Cellular Analysis (RTCA) assays were used to detect cell proliferation and growth. Transcriptome and CHIP assays were used to examine the molecular biology changes and molecular interaction in these cells. Nude mice was utilized to study the correlation of JARID1B and tumor growth in vivo.Here, we first observed that JARID1B was significantly upregulated in CRC tissue compared to adjacent normal tissues. In CRC patients, JARID1B high expression was positively relation with poor overall survival. Multivariate analyses revealed that high JARID1B expression was an independent predictive marker for the poor prognosis of CRC. In addition, we found that JARID1B promoted CRC cells proliferation by Wnt/β-catenin signaling pathway. Further studies demonstrated CDX2 as a downstream target of JARID1B, and our data demonstrated that CDX2 is crucial for JARID1B -mediated Wnt/β-catenin signaling pathway. Mechanistically, we demonstrated that JARID1B regulated CDX2 expression through demethylation of H3K4me3.CDX2 inhibited by JARID1B-derived H3K4me3 methylation promoted cells proliferation of CRC via Wnt/β-catenin signaling pathway. Therefore, our studies provided a novel insight into the role of JARID1B in CRC cells proliferation and potential new molecular target for treating CRC.Video abstractThe online version contains supplementary material available at 10.1186/s12964-020-00660-4. Colorectal cancer (CRC) is the most common cancer with high morbidity worldwide and is the third leading cause of cancer-related death in men and the second leading cause in women , 2. In rJARID1 proteins are histone demethylases that regulate the fate of normal cells and contribute to malignant transformation . The JARThe Wnt family consists of 19 secretory cysteine-rich glycoproteins that all play principal regulatory roles in many developmental and biological processes, such as cell fate specification, proliferation, migration and asymmetric cell division . FurtherIn the present study, we found that JARID1B was elevated in CRC tissues compared with normal colonic tissues using quantitative reverse transcription (qRT)-PCR, western blot and immunohistochemistry. JARID1B expression was correlated with patient survival time. Furthermore, JARID1B promoted CRC cell proliferation in vivo and in vitro. In addition, our results further show that JARID1B regulates Wnt/β-catenin signalling to influence CRC cell proliferation. Additionally, we demonstrated that JARID1B significantly inhibited CDX2 expression in CRC, and CDX2 is crucial for JARID1B-mediated Wnt/β-catenin signalling in CRC. Finally, our study verified that JARID1B decreases CDX2 expression by demethylating H3K4me3. Therefore, our findings indicate that JARID1B is believed to be a promising target in the treatment of CRC.All the specimens of 54 patients from the Second Affiliated Hospital of Nanchang University were diagnosed as CRC by pathological test from June 2018 to December 2018. Total proteins and mRNAs from these patients’ clinical tissues were immediately obtained when tissues was still fresh. Moreover, formalin-fixed paraffin-embedded 130 CRC patients’ tissues from December 2012 to December 2017 were randomly selected to observe the expression levels by immunohistochemistry and their corresponding follow-up data about survival was obtained from the hospital database. Informed consent was obtained from all patients, those patients’ clinical data was recorded during hospitalization and the research program was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.According to what the manufacturer’s protocol described, 20 μM BrdU was put into CRC cells for 4 h at 37 °C. Then washed with PBS for three times, the cells were mixed with apollo reaction for 1 h. The cells were stained with 100 ul of Hoechst 33342 (5 μg/ml) for 30 min to visualize the nuclei and observed under a fluorescence microscope  .The xCELLigence real-time cell analysis (RTCA) system (ACEA Bioscience) was used to analyze cell proliferation. Cells were seeded on a 96-well plate . After treated with different approaches for 12 h, the growth rates were recorded every 10 min by the instrument. Under the same xCELLigence RTCA program, continue to monitor the changes of cell index for 36 h. We used mean cell index values in real-time to present cell proliferation changes. For each experiment, we tested three biological replicates.6 cells in 200 ul of PBS were injected subcutaneously into the flanks of nude mice . We used the random number table as a random method to determine the experimental animals, making sure more than 40 mices. Five mice were randomly selected every 5 days to get tumor tissues, and then tumor volumes were measured according to the protocol: V = 1/2 (largest diameter) × 2. In vivo imaging showed tumor growth on day 30, and fluorescent pictures were taken. After 40 days, tumors tissues from 5 mices were harvested and individually weighed after the mice were anesthetized. The data was presented as tumor weight (mean ± SD).After construction of a DLD-1 cell line that stably interfered with JARID1B expression, 5 × 102 condition. Moreover, 10 uM XVA-939 from American AbMole was used to inhibite β-catenin degradation. Over-expression plasmids and shRNA interference fragments are transfected into cells through lipofectamine 2000 and lipofectamine LTX .Commercialized CRC cell lines SW620, HCT116, LOVO, SW480 and DLD-1 were obtained from American Type Culture Collection . SW620 and SW480 were cultured in L-15 medium (Gibco). LOVO was cultured in F12K medium (Gibco). DLD-1 and HCT116 were cultured in DMEM medium (Gibco). All cell lines were cultured at 37 °C, 5% COSiRNA of JARID1B and CDX2 was synthesized by InvivoGen. The target sites of shRNA are detailed in Supplementary Table SqRT-PCR and western blot analysis were made as previously reported . Whetherhttp://www.kegg.jp) assessment. Results from the GSEA software were visualized by the GOplot R software package. The GSEA software used a genome false discovery rate (FDR) of less than 25%, suggesting that FDR < 25% indicated significantly enriched gene sets.GSEA was a statistical analysis method for assessing whether a particular gene set showed statistically significant and consistent differences between two biological states, such as tumor and non-tumor or low-expression and high-expression groups . SubsequFragments of the CDX2 were amplified PCR using primers . Briefly, the cells were fixed with 1% formaldehyde for 10 min at room temperature, then washed with PBS for three times. After a series of processing according to the protocol, Digested DNA fragments was sonicated in the range of 150–300 bp. The immunoprecipitations were mixed with H3K4me3 antibody and agarose. The input and DNA were then subjected to qPCR.p-values of < 0.05 indicated statistically significant changes. Each experimental design included more than three biological repeats.Paired t test for the two groups and one-way ANOVA for more than two groups were used to analyze JARID1B expression data and its relationship with various clinicopathological factors. When data did not follow normal distribution, Mann-Whitney U test between two groups and Kruskal-Wallis H test for three or more groups should be used. Kaplan-Meier analysis and the logrank test were used for survival analysis. Correlation between two continuous values was analyzed by pearson’s correlation. Furthermore, univariate and multivariate analyses were performed using the logistic regression model. All of the data were analyzed using GraphPad Prism and SPSS 22.0. To investigate the role of JARID1B in the development of colorectal cancer, we investigated JARID1B expression levels in colorectal cancer, and 54 paired fresh CRC tissue samples and matched adjacent non-tumour tissues were used. qRT-PCR revealed that the mRNA expression level of JARID1B was notably elevated in CRC tissue samples compared with the corresponding adjacent tissues Fig. . AltogetTo investigate the potential biological function of JARID1B in CRC development, we initially examined JARID1B expression levels in CRC cells  and the non-malignant cell line (HCoEpic) by qRT-PCR and western blotting. The results showed that JARID1B expression was notably elevated in CRC cells  revealed that JARID1B expression was positively correlated with Wnt signalling pathway Fig. a. SecondNext, we would like to validate that cells proliferation mediated by JARID1B depended on the Wnt/β-catenin signalling pathway. Thus, we increased β-catenin expression in JARID1B-knockdown DLD-1 cells, showing that β-catenin over-expression significantly increased β-catenin, c-MYC and cyclin D1 in JARID1B–knockdown DLD-1 cells, demonstrating β-catenin could rescue the decreased Wnt/β-catenin pathway activity induced by knockdown of JARID1B Fig. e, f. MorWe further explored how JARID1B regulated Wnt/β-catenin signalling in CRC cells. It has been reported that CDX2 knockdown promotes the proliferation of colorectal cancer cells via Wnt/β-catenin signalling. Thus, we speculated that JARID1B might regulate the expression of CDX2 to enhance the activity of Wnt/β-catenin signalling in CRC cells. Given the role of JARID1B in the epigenetic regulation of transcription, we first performed RNA-seq to identify potential JARID1B target genes involved in cell proliferation. The results showed that CDX2 was one of the most notably upregulated transcripts when JARID1B was knocked down Fig. a. Next, To further validate whether JARID1B activated the Wnt/β-catenin signalling pathway by regulating CDX2 expression. After we first decreased the expression of CDX2 in JARID1B–knockdown DLD-1 cells, CDX2, GSK-3β, Axin2 and p-β-catenin, β-catenin, c-MYC and cyclin D1 expression levels were observed. The qRT-PCR results showed that knockdown of CDX2 inhibited the increase in CDX2 mRNA expression induced by JARID1B knockdown Fig. a. WesterWe then explored how JARID1B regulated CDX2 expression at the transcriptional level. Studies have shown that JARID1B affects histone H3 lysine 4 (H3K4) demethylase and exhibits a strong transcriptional repression function. We explored whether JARID1B demethylates H3K4me3 at the promoter of CDX2 in CRC cells. We first decreased the expression of JARID1B and then observed the H3K4me3 protein expression levels and CDX2 promoter activity. Western blotting data showed that JARID1B knockdown markedly increased H3K4me3, CDX2, GSK-3β, Axin2 and p-β-catenin expression and decreased β-catenin, c-MYC and cyclin D1 expression Fig. a. SimultTumour proliferation plays a crucial role in the development of CRC. Studies have confirmed that the proliferation of CRC cells is significantly correlated with the abnormal expression of histone demethylase. JARID1B plays an important role in cell fate decisions, cancer progression, and stem cell self-renewal , 26. EviNext, we further explored the underlying mechanism by which JARID1B regulates CRC proliferation. The present studies have demonstrated that abnormal activation of the Wnt/β-catenin signalling pathway is an important cause of CRC . StudiesOur current study also highlights the detailed mechanism by which the Wnt/β-catenin pathway is regulated by JARID1B in CRC cells. Caudal-type homeobox transcription factor 2 (CDX2), an essential intestine-specific regulator, is involved in the development and differentiation of intestinal epithelial cells and regulates the balance between cell proliferation and differentiation . In the Therefore, we proposed the underlying mechanism: JARID1B regulates CDX2 expression through H3K4me3. This conclusion was based on the following observations: on the one hand, JARID1B knockdown markedly increased H3K4me3 protein expression and CDX2 promoter activity; on the other hand, JARID1B overexpression decreased H3K4me3 levels and CDX2 promoter activity. However, further studies are needed to clarify whether there were some proteins as a complex participating in the mechanism between JARID1B and H3K4me3, and further study on the mechanism of how JARID1B affects the activation of CDX2 promoter, providing new ideas for further understanding the pathogenesis and development of CRC.In summary, our study demonstrated that JARID1B acted as an oncogene to promote the CRC progression. JARID1B inhibited CDX2 transcription level by demethylation of H3K4me3, which was effected at the CDX2 promoter region. Following CDX2 low expression, GSK-3β and Axin2 expression levels were decreased, and further phosphorylated β-catenin level was inhibited. So that less β-catenin was bound by ubiquitination to degrade, and more β-catenin was transported into the nucleus, which caused downstream target genes (c-MYC and cyclinD1) to be activated Fig. e. FinallAdditional file 1:Table S1. Primers and shRNA target sequences. (DOC 33 kb)Additional file 2:Figure S1. JARID1B knockdown in HCT116 resulted in decreased CRC proliferation. a, b Western blot showed the knockdown efficiency of JARID1B in DLD-1 and HCT116. c, d, e Cells proliferation capacities were detected by EdU, colony formation assay and RTCA assays in CRC HCT116 cells transfected with the shJARID1B#1/#2 plasmid. *p < 0.05, **p < 0.01. (TIF 15203 kb)Additional file 3:Figure S2. JARID1B overexpression promoted CRC cells proliferation. a, b Cells proliferation capacities as detected by Colony formation and EdU in LOVO cells transfected with Vector or Fla-JARID1B.**p < 0.01. (TIF 4842 kb)Additional file 4:Figure S3. JARID1B overexpression promoted CRC cells proliferation by inhibiting CDX2 expression. a Western blot detected JARID1B, β-catenin, c-MYC and cyclinD1expression in LOVO cells transfected with Vector or Flag-JARID1B. b The total and nuclear protein levels of β-catenin were assessed by western blotting in LOVO cells transfected with Vector or Flag-JARID1B. c The effect of JARID1B overexpression on Wnt/β-catenin pathway was detected by TOP-Flash luciferase reporter assay. d Western blotting analysis showed downregulation of β-catenin attenuated the increased expression of β-catenin, c-MYC and cyclinD1 in LOVO-JARID1B cells. e TOP-Flash luciferase reporter assay showing that β-catenin knockdown rescued the increased Wnt/β-catenin pathway activity of LOVO-JARID1B cells. g, h Edu assay results showed that β-catenin knockdown significantly inhibited the increase of cells proliferation in LOVO-JARID1B cells. *p < 0.05. (TIF 12820 kb)Additional file 5:Figure S4. β-catenin IB in β-catenin IP ensured that IP worked and the differences between samples were real. a In DLD-1, β-catenin IP while there were different groups including shJARID1B, shCDX2 and shJARID1B/shCDX2. b In LOVO, β-catenin IP while there were different groups including Flag-JARID1B, HA-CDX2 and Flag-JARID1B/HA-CDX2. (TIF 5295 kb)


Cancer is one of the greatest public health challenges. According to the World Health Organization (WHO), 9.6 million cancer deaths have been reported in 2018. The most common cancers include lung, breast, colorectal, prostate, skin (non-melanoma) and stomach cancer. The unbalance of physiological signalling pathways due to the acquisition of mutations in tumour cells is considered the most common cancer driver. The Wingless-related integration site (Wnt)/β-catenin pathway is crucial for tissue development and homeostasis in all animal species and its dysregulation is one of the most relevant events linked to cancer development and dissemination. The canonical and the non-canonical Wnt/β-catenin pathways are known to control both physiological and pathological processes, including cancer. Herein, the impact of the Wnt/β-catenin cascade in driving cancers from different origin has been examined. Finally, based on the impact of Extracellular Vesicles (EVs) on tumour growth, invasion and chemoresistance, and their role as tumour diagnostic and prognostic tools, an overview of the current knowledge linking EVs to the Wnt/β-catenin pathway is also discussed.  FZD) receptors. This, in turn, drives the biological response [The human wingless-related integration site (Wnt) genes encode 19 evolutionarily conserved glycoproteins with 22-24 Cys residues. In the endoplasmic reticulum (ER), the Wnt ligands are post-translationally acetylated by porcupine, a membrane associated O-acyl transferase. Acetylation leads to palmitoylation, which is required for the release and binding of Wnt to the frizzled , glycogen synthase kinase 3 β (GSK-3β) and two scaffolds: axis inhibition (Axin), and adenomatous polyposis coli (APC). Firstly, β-catenin undergoes phosphorylation by CK1α at serine 45 (Ser45), Ser33, Ser37 and threonine 41 (Thr41) by GSK-3β. Then, the E3 ubiquitin ligase, denoted as β-transducin repeat-containing protein (βTrCP), marks β-catenin ubiquitination and degradation [The canonical pathway turns around the β-catenin intracellular level . In the radation   co-receptors, phosphorylation of which is essential for receptor activation. Wnt binding to its receptor results in dishevelled (DVL) phosphorylation, leading to Axin de-phosphorylation and decline of its cytoplasmic content [The activation of the canonical Wnt signal requires both the  content . Thereby content .TCF/LEF) family of transcription factors [Although several nuclear β-catenin binding partners have been involved in the control of gene transcription, the most relevant β-catenin partners are the members of the T-cell factor/lymphoid enhancer factor , RAR-related orphan receptor (ROR) or the receptor like tyrosine kinase (RYK) and convey the signal to DVL. On the one side, DVL forms the disheveled associated activator of morphogenesis 1 (DVL-Daam-1) complex, which triggers a small guanosine-5’-triphosphate (GTP) GTPase, such as ras homolog gene family member A (RhoA), RHO and RHO-associated kinase (ROCK). DVL also triggers ras-related C3 botulinum toxin substrate (RAC), JUN-N-terminal kinase (JNK) and the activator protein-1 (AP-1). [In the non-canonical PCP pathway, Wnt ligands bind to  (AP-1).  , which hydrolyses the phosphatidylinositol -biphosphates (PIP2) to inositol -triphosphates (IP3) and diacylglycerol (DAG). This translates into the release of the intracellular calcium and the activation of both calcineurin (CaN) and calcium/calmodulin-dependent kinase II (CamKII). Moreover, the activation of calmodulin promotes the activation of the TGF-β-Activated kinase 1 (TAK-1) and nemo-like kinase (NLK), thereby antagonizing and neutralizing the canonical Wnt/β-catenin cascade. CaN activates the nuclear factor of activated T-cells (NFAT), which moves to the nucleus and regulates the expression of target genes [In the calcium-dependent pathway, Wnt ligands bind to et genes  . Among them CMS2, CMS3, and CMS4 have a higher rate of APC mutations (over 50%) compared to CMS1. Each CMS has unique features: CMS1 : hyper- mutated, microsatellite instability, strong immune activation; CMS2 : epithelial, chromosomally unstable, marked Wnt and myc signalling activation; CMS3 : epithelial, metabolic dysregulation; and CMS4 : a prominent transforming growth factor β (TGFβ) activation, stromal invasion, and angiogenesis. Samples with combined features (13%) represent transition phenotypes or are supposed to reflect the intra-tumour heterogeneity [Colorectal cancer (CRC) is one of most common cancers worldwide and represents a deep cause of cancer mortality  with a rThe heterogeneous genetic ground underlying CRC initiation and progression mainly involves gene fusion, deletion or amplification, somatic gene mutations and epigenetic alterations. Wnt/β-catenin signalling has emerged as one of the most significant biological pathways in both the physiological setting and in CRC development. Almost all CRCs are characterized by a hyper-active Wnt/β-catenin pathway, which, in many cases, is considered the most critical cancer initiating and driving event. Proteins and miRNAs guiding the Wnt/β-catenin pathway and proposed as potential CRC therapeutic targets are discussed.RNF6) is an oncogene frequently upregulated by gene amplification in primary CRC. Moreover, APC mutation and RNF6 copy number amplification were commonly found in CRC patients. RNF6 is a RING-domain E3 ubiquitin ligase and exerts its pro-metastatic effects by promoting CRC cell growth, cell-cycle progression, and epithelial to mesenchymal transition (EMT). Furthermore, RNF6 expression and its gene amplification have been considered independent patients‘ prognostic factors. RNF6 mediates the polyubiquitination of the transducin-like enhancer of split 3 (TLE3), a transcriptional repressor of the β-catenin/TCF4 complex, and its proteasome degradation. The lack of TLE3/TCF4/LEF interaction enhances the Wnt/β-catenin transcriptional activity, and the expression of its downstream target genes [Ring finger protein 6 (et genes  Table 1RNF6 is aLGR5) is a Wnt/β-catenin target gene implicated in cancer cell proliferation and migration. It has been reported that LGR5 is highly expressed in CRC tissues compared to the healthy ones. A decline in β-catenin and c-myc mRNA expression were detected by knocking-down LGR5 expression, suggesting that it may regulate the Wnt/β-catenin activity by modulating the expression of β-catenin. Furthermore, since targeting LGR5 improves the response to chemotherapy, LGR5 has been proposed as a novel therapeutic target in CRC [The leucine-rich repeat-containing G-protein coupled receptor 5  expansion. High levels of β-catenin and RAS proteins are considered the major drivers of CSC expansion and cancer dissemination and are associated with poor patient’s outcome . CBP). Moreover, ICG-001 effectively and without side effects abrogated drug-resistant cells. On the same line, PRI-724, a second generation of CBP/β-catenin antagonist, was found safe in pre-clinical studies and displayed an acceptable toxicity profile.Targeting the CSC pool without affecting the somatic stem cell (SSC) niche is one of the major goals of recent decades. As reported by Lenz et al. , the β-cTNIK) amplification and its role in tumor progression by applying siRNA technology, while Masuda et al. [TNIK inhibitor. TNIK selectively binds both to TCF4 and β-catenin in order to promote cancer cell growth via Wnt/β-catenin cascade and drives colorectal CSC expansion. The NCB-0846 inhibitor was effective in interfering with TNIK activity tumour growth. Yu et al.  investiga et al.  have genGSK-3β via Axin binding and β-catenin/RAS destabilization. In line with this observation, treatment with KYA1797K abrogated CRC stem cell features both in vitro and in vivo. Mechanistically, KYA1797K pushes β-catenin and RAS towards the Axin binding [KYA1797K, a small molecule identified by Cha et al. , was fou binding  , a nuclear protein belonging to the BTB/POZ/zinc finger (ZF) family of transcription factors, was found upregulated and surprisingly post-transcriptionally regulated by miR-144-3p. Previous studies revealed that BCL6 is involved in the control of cell cycle progression and differentiation [BCL6 co-operate to inhibit cellular proliferation, development, and progression of CRC by interfering with c-myc and cyclin D1 expression [Sun and co-workers  identifintiation ,23. Indepression  Table 1BCL6, a nGSK-3β, a direct miR-377-3p target, was found upregulated upon miR-377-3p overexpression. These data suggest that a complex regulatory network boosting tumour progression is associated with the expression of miR-377-3p in CRC. miR-377-3p displays an ambiguous role in CRC. Liu and colleagues  uncovereZEB2) and the X-linked inhibitor of apoptosis protein (XIAP) are two positive regulators of the Wnt/β-catenin cascade [ZEB2 enables tumour progression and invasion, whereas XIAP promotes cell proliferation and chemoresistance. De facto, miR-377-3p overexpression was found to suppress the malignant CRC phenotype, as well as cell proliferation, invasion and drug resistance by directly targeting the 3’ UTR sequence of both ZEB2 and XIAP mRNAs. Since miR-377-3p/ZEB2-XIAP inhibited CRC progression by reducing Wnt/β-catenin-associated gene expression , MMP-9, CD44, vascular endothelial growth factor (VEGF), and Twist), approaches involving increasing its expression have been proposed for novel therapeutic options , which acts as an oncogene [GSK-3β phosphorylation and β-catenin expression. Mechanistically, it was found that miR-520e regulates cancer cell behaviour by targeting AEG-1, which in turn inactivates the Wnt/β-catenin signalling and the transcription of its downstream genes. Hence, miR-520e overexpression could represent a promising therapeutic target in CRC by AEG-1 suppression.Functional experiments showed that miR-520e plays a pivotal role in regulating CRC cell proliferation, colony formation and invasion . Moreoveoncogene , is a diRHBDD1) plays a crucial role in driving metastasis formation in CRC patients, via the Wnt/β-catenin pathway. It has been shown that RHBDD1 is able to influence the Wnt/β-catenin cascade by increasing the phosphorylation of β-catenin at the Ser552 and Ser675 residue without affecting its nuclear translocation. Moreover, it promotes EMT, stemness, migration and invasiveness. RHBDD1 also improves the expression of the β-catenin target gene, ZEB1. Furthermore, the protein level of RHBDD1 positively correlated with ZEB1. Thereby, RHBDD1 has been proposed as a novel therapeutic target and/or a clinically useful biomarker for metastatic CRC  superfamily of solute carriers. Deng’s group [SLC35C1 that regulates the canonical Wnt/β-catenin pathway in CRC. They demonstrated a reduction in SLC35C1 and an increase in β-catenin at all tumour stages. Indeed, silencing SLC35C1 resulted in the increased release of Wnt3a and c-myc, Axin2 and cyclin-D1 expression. This suggests that SLC35C1 is involved in the control of the canonical Wnt/β-catenin pathway, and thereby in tumour cell proliferation and tumour progression (’s group  exploredgression . NPTX2) is a member of the neuronal pentraxin family and is essential for the formation of synapsis. NPTX2 was found overexpressed at both mRNA and protein level in CRC, particularly in metastatic lesions [NPTX2, which was found to positively correlate with tumour stages, lymphatic invasion, distant metastasis, and poor patients’ outcome, promotes β-catenin nuclear translocation and the expression of c-myc, cyclin D1, Snail, and N-cadherin. No NPTX2 receptors have been identified in CRC; however, its cellular internalization was found mediated by the Wnt/β-catenin receptor, FZD6. Additionally, it has been reported that NPTX2/FZD6 interaction translates in cancer cell proliferation and metastasis formation by triggering the Wnt/β-catenin pathway [Neuronal pentraxin 2  cells. The authors have found that APC disruption rapidly induces changes in DNA methylation, indicating that focal remodelling of the DNA methylation profile occurs early and concomitantly with the first oncogenic event. Moreover, it has been demonstrated that the hyper-activation of the Wnt/β-catenin pathway associated with the APC loss-of-function turns out in a rapid increase in intestinal stem cell commitment towards differentiation. Again, it was correlated with the remodelling of the DNA methylation profile. This study unveils that early changes in DNA methylation are crucial for the impaired fate decision program associated with APC loss-of-function.Aberrant gene expression and DNA methylation profiles are considered hallmarks of CRC initiation and progression . Due to c spread . Bruschic spread  investigKLHL22) is a tumour suppressor protein involved in the development/progression of several cancers [KLHL22 was found in CRC tissues. KLHL22 overexpression was associated with decreased migration, invasion and reduced expression of the EMT markers, vimentin, N-cadherin, Twist1 and Snail1. Intriguingly, KLHL22 knockdown led to increased expression of β-catenin and LEF, while KLHL22 overexpression translates into GSK-3β upregulation and β-catenin downregulation [The kelch-like family member 22 , the hydroxyacyl-CoA dehydrogenase (HADH), ligand-dependent corepressor (LCOR) and the receptor expression-enhancing protein 1 (REEP1) as candidate genes regulated by the non-canonical Wnt/β-catenin pathway. Second, these genes were found regulated by Wnt5a/b, as well as by ROR2, the DVL2, the activating transcription factor 2 (ATF2) and ATF4 in a non-canonical Wnt/β-catenin independent manner. Lastly, Wnt5a/b silencing was found to impair cancer cell proliferation.Three relevant goals have been recently achieved by Voloshanenko et al.  supportiFZD6, RYK, PTK7 receptors, positively correlate with poor prognosis. Additionally, Wnt11 downregulated β-catenin transcriptional activity and increased ATF2 via the non-canonical Wnt signalling pathway. Thereby, Wnt11 has been proposed as a prognostic biomarker and therapeutic target in CRC patients.Among several soluble Wnt proteins, Wnt11 was found to be upregulated in CRC patients . RecentlCCL2) in Wnt5a-treated macrophages. Consistently, Wnt5a knockdown reduced CCL2 expression in TAMs and their cancer-promoting activity. In Wnt5a-treated macrophages, both CaMKII and ERK1/2 undergo phosphorylation and lead to CCL2 secretion. This study provided evidence for a new role of Wnt5a in CRC and describes a potential novel therapeutic target (Tumour micro environment (TME) has a pivotal role in cancer development . Liu et c target .Xfz7) [Breast cancer (BC) is the most diagnosed cancer in women , the firXfz7)  have beesFRP1) is an early event in BC patients and is associated with poor prognosis [Dysregulation of the Wnt/β-catenin cascade has been associated with cancer initiation and metastasis formation . Moreoverognosis . Furtherrognosis . cyclin D1, c-myc and Ki67, impaired tumour growth and induced apoptosis [A recent study demonstrated that high β-catenin level is associated with miR106a overexpression and involved in BC cell growth. Additionally, high levels of miR106a were reported to reduce cisplatin sensitivity. Major results were obtained exploiting the Wnt inhibitor, FH535. In fact, FH535 treatment reduced the expression of β-catenin, poptosis .In a different study , the impFOXO1). In the physiological setting, FOXO1 binds β-catenin and induces its degradation. This implies that miR-5188 overexpression leads to β-catenin nuclear accumulation and transcription of its downstream target genes, mainly involved in EMT, tumour cell proliferation, metastasis formation and chemo resistance. Moreover, the authors elegantly showed that miR-5188 expression is under the control of c-Jun, which directly binds to its promoter region. This in turn generates a positive loop, accelerating tumour progression. Clinically, miR-5188 has been proposed as a diagnostic or prognostic factor and/or a direct target for anti-cancer therapy [A recent study demonstrated that miR-5188, aberrantly expressed in breast cancer patients, positively correlates with poor prognosis. The molecular analyses revealed that miR-5188 directly targets the forkhead box protein O1 ( therapy .HOTTIP) has also been linked to poor prognosis in BC patients. Overexpression of HOTTIP correlates with the expansion of breast CSCs (BCSCs) and the expression of the stem cell markers, OCT4 and SOX2. Han et al. [CK18 and CK14 and that miR-148a inhibits BC cell migration and invasion by directly targeting Wnt1. Moreover, it has been reported that HOTTIP controls miR-148a-3p by acting as a competing endogenous RNA (ceRNA). Thereby, HOTTIP promotes expansion of CSCs in vitro and tumorigenesis in vivo by regulating the miR-148a-3p/Wnt1/β-catenin axis [The upregulation of the lncRNA hoxa transcript at the distal tip , increased EMT [LGR4 knockdown leads to SLUG and ZEB1 downregulation, thereby impairing invasion and metastasis [LGR4 homolog LGR5 was also reported. LGR5 maintains the pool of BCSCs and promotes tumour progression and invasiveness by activating the Wnt/β-catenin canonical pathway [The rognosis . A tight cascade . Previouased EMT . As a matastasis . A corre pathway  (Table 1ST7L) is downregulated in BC cells, and more importantly, that ST7L acts as an antitumor supervisor by reducing GSK-3β phosphorylation and inducing β-catenin degradation. However, the mechanisms through which ST7L controls GSK-3β phosphorylation are still missing  in BC and its correlation to poor prognosis. An aberrant level of TMED boosts cell cycle progression, colony formation, migration and invasion and the expression of CDK2, CDK4, CDK6, cyclin E, β-catenin, cyclin D1, c-myc, MMP-7 and TCF4. Conversely, silencing TMED3 drastically reduced migration and invasion. Moreover, the observation that β-catenin knockdown translates in the reduction of its regulated genes supports the notion that the oncogenic effect of TMED goes through the Wnt/β-catenin pathway (A recent study  reported pathway .PKM2), a protein involved in glycolysis, and more importantly in β-catenin activation [Cryptotanshinone (CTS) is an herbal medicine derived from roots of salvia miltiorrhiza, which displays anti-tumour properties. It has been shown that in vitro CTS reduces tumour cell growth, migration and invasion by downregulating the pyruvate kinase muscle isozyme M2 . They have shown that low PFKP level correlates to cancer cell migration and poor patients’ survival. The growth and expansion of tumour cells also rely on glucose consumption, resulting in the accumulation of lactate. Cancer cell metabolism was also associated with β-catenin activation [PFKP.Foxy5 is a Wnt5a mimicking hexapeptide able to decrease BC cell migration and invasion . More retivation . In thiscyclin D1. The authors have shown that Wnt5a supports TGF-β-mediated tumour suppressive functions by antagonising Wnt/β-catenin signalling and limiting tumour cell proliferation.According to the study of Borcherding et al. , Roarty Moreover, Leris and colleagues  proved tALCAM), a protein involved in migration and invasion. Knockdown of either Wnt5a or ALCAM inhibited tumour cell migration, confirming the role of the Wnt5a/ALCAM axis in the migratory phenotype of ER-positive BC  than in normal monocytes and macrophages. Wnt5a was found to inhibit the generation of Mo-mDCs by stimulating BC cells to produce IL-6. In addition, the presence of IL-6 in the conditioned media of Wnt5a stimulated BC cells was found to be involved in the inhibition of Mo-mDC differentiation . ConsistWnt5a signalling is also able to modify the CD44-AKT signalling pathway, leading to a reduced BC cell migration and invasion. In epithelial BC cells, silencing of Wnt5a drives EMT-like changes without altering the expression of common EMT markers. On the contrary, it interferes with CD44 expression and induces pAKT downregulation, thereby acting via a EMT-independent mechanism . Wnt5a-L) and Wnt5a-short (Wnt5a-S) isoform. When analysed in several cell lines, Wnt5a-L reduced tumour progression, while Wnt5a-S promoted tumour growth. The dual activity of Wnt5a has also been ascribed to the Wnt5a isoforms. Bauer et al.  have shoOverall, Wnt5a may play multiple roles. Whether it acts as a tumour suppressor or a tumour promoter remains elusive and depends on the availability of essential receptors, the TME, and the activation of discrete signalling pathways.Triple-Negative Breast Cancer (TNBC) is an invasive type of breast carcinoma that lacks the expression of estrogen and progesteron receptor as well of the human epidermal growth factor receptor 2 (HER2)  and accoTNBC patients have poor outcome due to the high grade of proliferation, early tumour dissemination, and the lack of targeting approaches ,114. TheNek2) and β-catenin correlated with patients’ poor prognosis. β-catenin binds to and is phosphorylates by the Nek2B isomer. Thereby, in TNBC, Nek2B functions as a β-catenin regulator by activating the Wnt signalling pathway and its downstream target genes. In addition, it has been suggested that Nek2B and β-catenin may synergize to promote resistance to chemotherapy. However, further studies are required to better elucidate the relationship between β-catenin and Nek2 and its possible implications in cancer development (Gene expression omnibus (GEO) databases were applied by Shen et al.  to gatheelopment .VANGL2), a core Wnt/PCP component, plays a crucial role in cancer cell migration, anchorage-dependent and independent cell proliferation, as well as in tumour growth. Since the scaffold p62/SQSTM1 protein, a VANGL2-binding partner, has a key role in the VANGL2–p62/SQSTM1–JNK pathway, the possibility to exploit p62/SQSTM1 as a potential therapeutic target has been proposed. This would be of particular relevance since the JNK targeting approaches are associated with major side effects in the clinical setting  stabilizes β-catenin in the cytoplasm and allows its nuclear translocation by regulating the phosphorylation of GSK-3β. This results in the transcriptional activation of target genes, leading to cell migration and invasion. Since knockdown of HePTP significantly suppresses metastases formed by TNBC cells, HePTP has also been proposed for therapeutic approaches in TNBC (Yu and colleagues  demonstr in TNBC .DLC-3), is downregulated in TNBC and its expression is linked to lymphatic metastases. DLC-3 overexpression leads to β-catenin and c-myc downregulation as well as in reduced in vitro cell proliferation, colony formation, migration, and invasion. Hence, a tumour-suppressor role related to the inhibition of the Wnt/β-catenin signalling pathway has been postulated  is a global issue representing the fourth most common cancer in the female population, particularly in developed countries . The pooThe Wnt/β-catenin signalling pathways play a crucial role in carcinogenesis of all OC subtypes . In partDKK1). They showed that DKK1 is involved in the control of OC stemness. Mechanistically, it has been shown that STAT3 directly activates the transcription of miRNA-92a, translating in DKK1 downregulation [SFRP1-AXIN2 and the inhibitor of β-catenin and T cell factor 4 (ICAT). They found that miR-1207 overexpression was associated with a reduced SFRP1-AXIN2 and ICAT expression and the appearance of a stem-like phenotype  has also been reported to display several repressive actions on EOC-derived tumour cell lines. It inhibits cell proliferation, migration, invasion and EMT. Thereby, since miR-590-3p post-transcriptionally regulates FOXA2, FOXO3, CCNG2 and DDK1 expression, miR-590-3p has been proposed as a potential target in EOC patients [SFRP1 in OC growth has also been proposed. Since miR-1180 is highly expressed in neoplastic tissues, Hu et al. [SFRP1/Wnt/β-catenin signalling pathway in this context, demonstrating that miR-1180 triggers the activation of the Wnt/β-catenin cascade by targeting SFRP1. Salem et al.  proved t in vivo . Similarp target . The cycpatients . A cruciu et al.  exploredLGR4-6 plays crucial roles in the activation of the Wnt/β-catenin cascade [LGR6 silencing  was found to be highly overexpressed in OC [ITGBL1 promotes cell migration and adhesion via Wnt/PCP, RhoA, the focal adhesion kinase, and the steroid receptor coactivator (FAK/src) pathway  in OC. They demonstrated that ALPL overexpression inhibits EMT, migration and invasion of high grade serous OCs (HGSOC) and FZD2 correlates with a poor survival rate [FZD2–mediated EMT activation, possibly by interfering with STAT3 activation [Luo and colleagues  have invval rate . Mechanitivation  Table 1ALPL in Ocyclin D1 is associated with high level of the long noncoding RNA, FGD5 antisense RNA 1 (lncRNA FGD5-AS1). A close relationship between them was straitened by the observations that inhibition of FGD5-AS1 reduced β-catenin and cyclin D1 expression while β-catenin downregulation decrease lncRNA FGD5-AS1 expression. This results in the impaired tumour cell migration and invasion.Glioma is an aggressive tumour of the nervous system displaying rapid progression and poor prognosis. Zhao et al.  have fouHDGF).Prostate cancer (PCa) is among the most common tumour in male. A recent study by Situ et al.  providedOsteosarcoma (OS) is a common bone paediatric tumour displaying high rates of lung metastasis. The inhibition of β-catenin activation, metastasis formation and chemo-resistance were found modulated by tegavivint (a Wnt/β-catenin inhibitor), which has been proposed as an alternative therapeutic option in OS .VDR) level correlates to increased cancer incidence and melanoma progression, respectively. Recently, it has been shown that high VDR expression correlated with the inhibition of tumour growth, low Wnt/β-catenin activation and the induction of the immune response [Melanoma is among the most immunogenic tumours displaying increased lymphocytic infiltration. Low 1α,25-dihydroxyvitamin D3 and vitamin D receptor (response  Table 1VDR levelTCF4 cascade and the metastatic spread by regulating the miR-552 5p/FOXO3 axis [The long non-coding RNA00261 (Linc00261) has been shown to display onco-suppressor properties in Pancreatic Cancer (PC). Linc00261 overexpression inhibits PC cell proliferation, invasion, EMT and metastasis. Bioinformatics analysis revealed that Linc0026 inhibits the activation of the β-catenin/XO3 axis .EVs are heterogeneous small membrane-bound carriers with complex cargoes released under both physiological and pathological conditions. Almost any cell can release EVs, which act as inter-cellular mediators modifying target cell fate at closed or distant sites .Based on the biogenesis, size, content, mechanisms of release and function, three discrete EV subtypes are recognized: microvesicles (MVs), exosomes, and apoptotic bodies . EVs-mediated transfers of specific molecules are known to dictate the phenotype of the recipient cell. They can act on proliferation, motility, EMT, migration, invasion, immune evasion, chemo-resistance, and TME reprogramming .Moreover, EVs derived from serum or other biological fluids have been proposed as tumour biomarkers. More importantly, EVs have gained attention as anti-cancer tools. Indeed, EVs can be used as drug delivery systems or potential cancer vaccines. Moreover, the transfer of Wnt ligands or β-catenin via EVs has been proposed as a Wnt signalling activation mechanism.c-myc and cyclin D1 when transferred to recipient cells  is a negative regulator of CRC. Low levels of lncRNA-APC1 correlate with metastasis, advanced clinical stage and poor prognosis in CRC patients. APC, via lncRNA-APC1, promotes cell-cycle arrest and suppresses angiogenesis by lowering the release of CRC cell-derived EVs. Finally, it has been shown that EV-derived from CRC are enriched in Wnt1 and enhance CRC cell proliferation and migration via non-canonical Wnt/PCP signalling [Long non-coding RNA-gnalling .EpCAM) [EpCAM positive liver cancer stem cells (LCSC) targeted by EVs engineered with a β-catenin specific siRNA (Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide. Constitutive activation of the Wnt/β-catenin pathway turns into the expression of the epithelial cell adhesion molecule (EpCAM) . IshigurEpCAM)  providedic siRNA .DKK-1 were found to boost the Wnt/β-catenin signalling and contribute to the abnormal osteogenesis. The inhibition of EV shedding, combined to chemotherapy, was found to impair tumour load, angiogenesis and osteolysis [Multiple myeloma (MM) is a hematopoietic malignancy associated with an altered homeostasis of bone formation/resorption. MM-derived EVs enriched in teolysis  (Table 3Furthermore, a recent study noticed that the release of EVs from HCC cells is increased in hypoxic conditions and linked to cancer cell proliferation, migration, invasiveness and EMT. Mechanistically, they have shown that miR-1273f enriched in EVs activates the Wnt/β-catenin signalling cascade by targeting the Wnt/β-catenin inhibitor LHX6 .Chen et al.  proved tXia et al.  have demHarada et al.  purifiedLuga et al.  demonstrAPC, GSK-3β and drive β-catenin nuclear accumulation via miR-214-3p and miR-24-3p (Lombardo et al.  providediR-24-3p b. OveraliR-24-3p .Overall, these data indicate a crucial contribution of EVs released by different cell sources in driving tumor development and dissemination. Several data suggest that these effects mainly rely on the transfer of their specific cargo into target cells. Therefore, approaches able to modify their cargo, particularly miRs and proteins involved in their tumor promoting action, have been proposed as useful therapeutic options. EV engineering by using siRNA for mutated protein has been tested and its effectiveness demonstrated in pancreatic cancer . This suCell-to cell communication is part of the evolutional processes. Wnt ligands are essential for homeostasis and, in the last 30 years, genetic, biochemical, and molecular investigations have uncovered several Wnt signalling components ,3. DriviAPC mutations [The identification of specific tools able to interfere with the Wnt/β-catenin cascade has been a hotspot for many years. This is particularly true for CRC, in which almost 70% of CRC patients display utations . Apart futations ,124,125.utations ,114. Likutations . Severalutations .In the TME, intercellular communication has been recently reported as mediated by the transfer of EV molecular cargo and revised in . Their cIn recent decades, several efforts have been directed to the development of Wnt/β catenin targeting approaches in order to interfere with tumour progression. However, these efforts have been limited by the crucial role of the Wnt/β catenin pathway in preserving tissue homeostasis. Therefore, future energies should be directed to clearly dissect the mechanisms driving the unbalanced Wnt/β catenin pathway in cancer, and the EV mechanism of action should be considered amid them. Should they be identified, targeting approaches would become a suitable anti-cancer option.


Reactive astrogliosis is prominent in most neurodegenerative disorders and is often associated with neuroinflammation. The molecular mechanisms regulating astrocyte-linked neuropathogenesis during injury, aging and human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are not fully understood. In this study, we investigated the implications of the wingless type (Wnt)/β-catenin signaling pathway in regulating astrocyte function during gliosis. First, we identified that HIV-associated inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced mediators of the Wnt/β-catenin pathway including β-catenin and lymphoid enhancer-binding factor (LEF)-1 expression in astrocytes. Next, we investigated the regulatory role of β-catenin on primary aspects of reactive astrogliosis, including proliferation, migration and proinflammatory responses, such as IL-6. Knockdown of β-catenin impaired astrocyte proliferation and migration as shown by reduced cyclin-D1 levels, bromodeoxyuridine incorporation and wound healing. HIV-associated cytokines, IL-1β alone and in combination with TNF-α, strongly induced the expression of proinflammatory cytokines including C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)8 and IL-6; however, only IL-6 levels were regulated by β-catenin as demonstrated by knockdown and pharmacological stabilization. In this context, IL-6 levels were negatively regulated by β-catenin. To better understand this relationship, we examined the crossroads between β-catenin and nuclear factor (NF)-κB pathways. While NF-κB expression was significantly increased by IL-1β and TNF-α, NF-κB levels were not affected by β-catenin knockdown. IL-1β treatment significantly increased glycogen synthase kinase (GSK)-3β phosphorylation, which inhibits β-catenin degradation. Further, pharmacological inhibition of GSK-3β increased nuclear translocation of both β-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. HIV+ human astrocytes show increased IL-6, β-catenin and NF-κB expression levels and are interconnected by regulatory associations during HAND. In summary, our study demonstrates that HIV-associated inflammation increases β-catenin pathway mediators to augment activated astrocyte responses including migration and proliferation, while mitigating IL-6 expression. These findings suggest that β-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies, such as HAND. More than 30% of human immunodeficiency virus-1 (HIV-1) positive patients develop disorders associated with the central nervous system (CNS) . DespiteAstrocytes, the most abundant glial cell in the brain, maintain CNS homeostasis and blood-brain barrier (BBB) integrity ,6. ReactThe canonical wingless type Wnt (Wnt/β-catenin) signaling pathway is an important intracellular signal transduction pathway that promotes cell survival, differentiation, proliferation and communication. It is implicated in several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and neurological complications of acquired immunodeficiency syndrome ,24,25,26The nuclear factor (NF)-κB signaling cascade is a pivotal modulator of inflammation and regulates innate and adaptive immune responses, cell proliferation, and apoptosis in various cell types ,36. Whil2 flasks at 2 × 106 cells/well and 20 × 106 cells/flask, respectively, and treated with IL-1β , TNF-α  and 6-bromoindirubin-3′-oxime (BIO)  for 8 or 24 h at 37 °C and 5% CO2.Human astrocytes were isolated from brain tissues recovered from first- and early second-trimester conceptual tissue as described previously ,43,44. TTM P3 primary cell 96-well nucleofector kit . Briefly, 1.6 × 106 astrocytes were suspended in 20 μL nucleofector solution containing mock, siCon or siβ-catenin (200 nM) and transfected using a Nucleofector/Shuttle (Lonza) device. Transfected cells were supplemented with media and incubated for 20 min at 37 °C prior to plating. Cells were then cultured and allowed to recover for 48 h prior to experimental use. The knockdown efficiency of siβ-catenin was verified in all donors and averaged −88.5 ± 3.6% (±SD) as assayed by real-time polymerase chain reaction (RT-PCR) and −46.6 ± 3.9% by Western blot as described below.Human astrocytes were transfected either with no siRNA (mock), scrambled non-targeting siRNA , or siRNA specific to β-catenin (siβ-catenin) using the Amaxa6 cells per 6-well tissue culture plate well and grown to confluence for 48 h. The media was then aspirated, and a thin stretch of the confluent monolayer was scraped to create a “scratch or injury” using a sterile 10 μL pipette tip. Fresh astrocyte medium was added, and the wound was allowed to heal. The culture plates were examined periodically at 8, 24, 36 and 48 h and then returned to resume incubation. Live images of migrating cells were obtained using a phase contrast microscope (Zeiss Invertoskop 40C) .A well-established in vitro wound-healing model to study the migration of astrocytes was used . BrieflyMetabolic activity of astrocytes was measured by 3--2,5-diphenyl tetrazolium bromide  assay as previously described . Briefly4 cells/well and allowed to recover overnight. BrdU was added 12 h prior to the measurement of incorporation using BrdU Cell Proliferation Assay Kit  as per the manufacturer’s instructions. Data were normalized to mock transfected astrocytes.Transfected astrocytes were plated in triplicates in 96-well tissue culture plates at 5 × 10Cell culture supernatants were collected at specified timepoints after treatment and used to quantify proinflammatory cytokines CXCL8, CCL2, and IL-6 . Assays were performed as per the manufacturer’s instructions and normalized to MTT levels.® gene expression assays  as previously described  followedn assays  and the 2 flasks at a density of 4 × 106 cells/flask and allowed to recover for 24 h. Following recovery, cells were treated with IL-1β (20 ng/mL) for 5, 15, 30, and 45 min. Cells were harvested by scraping in sterile ice-cold PBS to avoid alteration of protein expression on the surface of cell membranes. Cell lysate proteins (15 μg) were boiled with 4X NuPAGE lithium dodecyl sulfate loading sample buffer at 100 °C for 5–10 min, resolved by Bolt 4–12% bis tris gel, and subsequently transferred to nitrocellulose membranes using i-Blot . The membranes were incubated with antibodies against β-catenin , GSK-3β , p-GSK-3β , or GAPDH  overnight at 4 °C, washed and then incubated with anti-mouse IgG or anti-rabbit IgG conjugated to horseradish peroxidase  for 2 h at room temperature. The membrane was then developed with SuperSignal West Femto Substrate (Thermo Fisher Scientific) and imaged in a Flourochem HD2 Imager . Densitometric analyses were performed using AlphaView software .Astrocytes were cultured as adherent monolayers in 25 cmCytoplasmic and nuclear lysates were isolated using a nuclear and cytoplasmic extraction kit according to the manufacturer’s methods . Protein concentrations were determined by the BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein (0.4 mg/mL) was loaded into WES capillaries, and columns were probed with antibodies for lamin A/C , β-catenin, NF-κB, and GAPDH. The levels of specific proteins were determined by the WES capillary protein detection system (ProteinSimple) according to the manufacturer’s directions.p ≤ 0.05.Statistical analyses were performed using GraphPad Prism 8.3.1 . All numerical data were taken as mean ± standard error of the mean (SEM). All data were analyzed using student t-test, one-way, or two-way analysis of variance (ANOVA) with Tukey or Fisher’s least significant difference post-hoc tests for pair-wise comparisons. Differences were considered statistically significant at The R/G-HIV-1 transcriptome data used for the Ingenuity Pathway Analysis (IPA)  in this publication have been deposited in NCBI’s gene expression omnibus (GEO) and are accessible through GEO series accession number GSE141756 .HIV-1 infection of macrophages and microglia in the CNS leads to cellular activation and secretion of neurotoxic cytokines such as IL-1β and TNF-α ,11. ThesCrosstalk between Wnt/β-catenin and NF-κB signaling during inflammation in various peripheral cell types has been well summarized by Ma et al. . HoweverWnt/β-catenin canonical pathway regulates cell proliferation, cell mobility and differentiation. To investigate the role of β-catenin signaling in astrocyte proliferation and migration following injury, either no siRNA (Mock), non-targeting siRNA (siCon) or siRNA specific for β-catenin (siβ-catenin) transfected astrocytes were plated, and a scratch was made following recovery. Wound sites were observed at defined time points to measure astrocyte migration. No difference in astrocyte migration was seen at 8 h in all conditions C4.Canonical Wnt/β-catenin pathway regulates the expression of several transcription factors such as c-myc, cyclin D1 and survivin that mediate cell proliferation . To studTo determine if β-catenin regulates neuroinflammation, siRNA transfected astrocytes were treated with IL-1β and TNF-α, and levels of CXCL8, CCL2 and IL-6 were measured at both mRNA and protein levels. IL-1β and TNF-α treatment significantly increased CXCL8 and CCL2 expression both at mRNA (data not shown) and protein levels C. While Wnt/β-catenin signaling involves inhibition of GSK-3β kinase activity by phosphorylation at serine 9, permitting β-catenin accumulation and subsequent expression of Wnt target genes. Since GSK-3β activity is inhibited by phosphorylation at serine 9, we investigated GSK-3β phosphorylation following IL-1β stimulation. Astrocytes were treated with 20 ng/mL of IL-1β for various time points up to 45 min. IL-1β treatment significantly increased GSK-3β phosphorylation at 15 min through 45 min A,B. DataTo determine if activation of the Wnt/β-catenin signaling pathway regulates the nuclear localization of NF-κB, cells were treated with BIO, cytosolic and nuclear fractions of cell lysates were analyzed for β-catenin and NF-κB levels using simple WES. β-catenin stabilization slightly decreased cytosolic levels of both β-catenin and NF-κB E,F. TogeTo examine the interrelationships in the context of HIV-infected astrocytes, we accessed a previously published dataset that quantified mRNA expression levels in Red/Green HIV + astrocytes . ExpressIn this study we investigated the implications of the Wnt/β-catenin signaling pathway in regulating astrocyte function in the context of HIV-associated neuroinflammation and gliosis. HIV-associated inflammation, IL-1β and TNF-α alone and in combination, strongly induced the expression of proinflammatory cytokines including CCL2, CXCL8 and IL-6 and showed significant activation of both the Wnt/β-catenin and NF-κB signaling pathways in primary human astrocytes. Knockdown of β-catenin impaired astrocyte proliferation and migration but only affected one of the three proinflammatory responses of astrocytes that we examined. IL-1β-induced IL-6 levels were increased by β-catenin knockdown and decreased with BIO, a β-catenin stabilizer, while CCL2 and CXCL8 remained unaffected by β-catenin knockdown. To better understand this relationship, we examined the crossroads between β-catenin and NF-κB pathways. While NF-κB levels were not affected by β-catenin knockdown, β-catenin stabilization increased translocation of both β-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. Together, these data strongly indicate crosstalk between these two pathways in astrocytes during injury and neuroinflammation, wherein reactive astrocyte responses including migration and proliferation were augmented by β-catenin signaling, while the IL-6 response was mitigated. These findings suggest that β-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies such as HAND .At early stages of CNS insults, astrocytes migrate and form barriers at the injured site to restrict the spread of infectious agents or inflammatory cells ,50. The While HIV-1 predominantly replicates in microglia and macrophages in the CNS, restrictively infected astrocytes were recently shown to be a reservoir that is capable of reinfecting the periphery . InfecteReactive astrocytes release a wide variety of inflammatory mediators such as cytokines and chemokines that are both neuroprotective (cytokines such as IL-10 and transforming growth factor-β) and neurotoxic  in nature . IL-1β iCrosstalk between NF-κB signaling and canonical Wnt/β-catenin signaling during inflammation has been extensively studied in the periphery, and has shown both positive and negative associations. Studies have demonstrated modulation of inflammatory responses via interaction of Wnt/β-catenin signaling with NF-κB in murine hepatocytes  and posiRobinson et al. recently demonstrated how the Wnt/β-catenin pathway discordantly regulates IL-6 expression in naïve astrocytes . Robust , making it a hallmark for HIV-associated inflammation. Early in the epidemic, IL-6 reportedly increased HIV-1 replication in several cell types such as PBMC, monocyte-derived macrophages, and U1 latently infected cells derived from U937 cells [Elevated plasma IL-6 levels have been associated with increased mortality and morbidity in people living with HIV even with effective retroviral suppression ,64,65, m37 cells ,67. This37 cells . In fact37 cells ,70,71, w37 cells . NF-κB-i37 cells . HIV-1 v37 cells ,73. Thus


Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by the selective death of the upper and lower motor neurons (MNs). MN loss causes a progressive paralysis that affects a patient’s ability to move, speak, and breathe; ALS typically leads to death within 3 to 5 years of diagnosis. The vast majority of cases (approximately 90%) occur in the absence of a family history, denoted as sporadic ALS (sALS) . HoweverProtein aggregates can be found in virtually all ALS patients ,4,5,6. MInduced pluripotent stem cells (iPSCs) represent a powerful tool to model a wide array of diseases as iPSCs can theoretically be generated from any individual regardless of health status or genetics. Like embryonic stem cells, iPSCs can be differentiated into virtually every cell type of the body , therebyIn this study, we generated iPSCs from discordant identical twin brothers to model sALS in an isogenic manner. We found that the brothers have no pathogenic mutations in known ALS-causing genes, and that MNs generated from both twins exhibit high levels of insoluble proteins. Additionally, whereas MNs derived from iPSCs expressing pathogenic SOD1 or C9orf72 mutations alter protein burden in response to excess glutamate in a protein- and genotype-dependent manner, the MNs from the identical twins do not robustly change their aggregate levels after the same stress. They are, however, capable of inducing autophagic flux at levels seen in healthy control cells in response to excess glutamate, which is a mechanism that the SOD1 and C9orf72 MNs do not activate. Together, these data indicate that these novel iPSC lines may provide key insights into disease pathology and identify convergent and divergent mechanisms between fALS and sALS.Identical twin males discordant for ALS were identified from the ALS Multi-Disciplinary Clinic at Froedtert Hospital and consented to sample collection in accordance to the procedures outlined by IRB PRO00024167 (approved 29 September 2015). The affected individual was diagnosed with ALS with left leg onset at the age of 59 and collection occurred at age 61. The disease duration was 64 months from symptom onset.Peripheral blood mononuclear cells (PBMCs) were isolated and reprogrammed as described previously  using thWhole genome libraries were constructed by standard automated methods using Illumina TruSeq technology. Quantitative Real-time PCR was used to assess the overall amount of DNA library. Samples were sequenced on the Illumina HiSeq 2500  with 125 bp paired-end reads with an average depth of coverage of 43X. Raw sequencing data were converted to the fastq format by bcl2fastq conversion software v 1.8.4  and were aligned to Human reference GRCh37 by the Burrows–Wheeler Alignment Minimal Exact Matches (BWA-MEM) algorithm v0.7.15 . VariantInduced pluripotent stem cells (iPSCs) were maintained in Essential 8  on Matrigel  coated 6-well plates  and passaged every 4–6 days with Versene . Cultures were routinely tested for mycoplasm using the Mycoalert Detection Kit . Two independent control lines ,20, one Motor neurons (MNs) were generated as described previously  and wereMotor neurons (MNs) were fixed after four weeks of differentiation and stained as previously described . Images Protein was isolated using the Triton-X 100 fractionation method. Briefly, cell pellets were resuspended in a 1% Triton X-100 lysis buffer and briefly sonicated. Protein concentration was determined through a bicinchoninic acid (BCA) assay  according to manufacturer’s instructions. Insoluble protein was isolated via ultra-centrifugation at 45,000 rpm for 30 min and the resulting pellet was resuspended with 2% SDS. Insoluble protein was quantified using a BCA assay. 10 µg of soluble protein or 30–40 µL of insoluble protein were used for Western Blot analysis . Blots wt-test or a one-way ANOVA with Tukey’s post test, as appropriate. p < 0.05 was considered significant.A minimum of three independent experiments each with a minimum of three biological replicates were analyzed. The data were analyzed with a Student’s We identified identical twin males who are discordant for ALS. The affected twin was diagnosed at age 59 with no family history of ALS and died at age 63. The unaffected individual is still ALS-free at the time of writing. Whole blood samples from both individuals were collected when the twins were 61 years old. Peripheral blood mononuclear cells (PBMCs) were then isolated and reprogrammed using Sendai virus expressing the Yamanaka factors . IndividOPTN). However, this particular variant has been linked to open-angle glaucoma [SIGMAR1. However, mutations in SIGMAR1 are linked to juvenile-onset ALS, so we speculate that this mutation is likely not disease-causing in these patients. Additionally, we found two missense mutations in chromogranin B (CHGB) that have been linked to Schizophrenia [NIPA1, a known risk allele for ALS [Sporadic ALS (sALS) generally lacks known genetic mutations; however, de novo mutations in known ALS-causing genes have occurred in sporadic patients . We therglaucoma  and is fophrenia ,27. Fina for ALS . HoweverWe next differentiated these iPSCs into MNs to determine whether any phenotypic alterations existed between the affected and unaffected samples. Using an established MN protocol , we founSince there were no overt phenotypes in the affected MN cultures compared to the unaffected control and baseline, we tested how the MNs reacted to exogenous stress. There are several non-cell autonomous contributors to ALS pathogenesis, and glutamate excitotoxicity is considered one of the prominent features . We therConsistent with our previous results , SOD1 anWe next tested the impact of excess glutamate on fALS iPSC-derived motor neurons. Like the MNs derived from the identical twins, a 96-h treatment with 100 µM glutamate did not alter overall morphology or viability in mutant SOD1, C9orf72, and control iPSC-derived MNs (data not shown). Yet, this treatment was sufficient to induce a consistent change in insoluble TDP-43 protein levels C. Both tSince autophagy is a common mechanism by which a cell degrades misfolded and aggregated proteins, we next tested whether autophagic flux was disrupted in ALS iPSC-derived MNs. We found that the ratio of LC3-II/LC3-I is not changed in the affected MNs compared to the unaffected twin at baseline but does increase after glutamate treatment A. This sInduced pluripotent stem cells (iPSCs) are a powerful modeling tool to study both sporadic and genetically linked disease. Still, few studies have generated iPSC lines from sALS patients ,36,37,38While sALS is generally believed to occur in the absence of genetic mutations, previous data have found de novo mutations in sporadic patients . Still, Like most MNs derived from ALS iPSC lines , the affConsistent with our previous work , we founInterestingly, we also observed a 35 kDa fragment of TDP-43 within the soluble fraction in the C9orf72 MNs A. EvidenWhile the field has largely taken a MN-centric view of ALS, there are undoubtably multifaceted interactions between additional cell types within the brain and spinal cord of ALS patients. Indeed, it is well established that astrocytes can drive disease pathogenesis . AlthougThe iPSC-derived MNs from discordant identical twins seem to exist in a phenotypic space between “ALS-like” and “control-like”. MNs from both twins exhibit evidence of protein aggregation that are not significantly different from each other but do appear higher than independent controls . AdditioTogether, these data suggest that glutamate may contribute to protein aggregation in a protein- and genotype-dependent manner. Moreover, sALS and fALS may follow different pathological mechanisms and should be separated into distinct subgroups when developing and validating new therapies. As such, the iPSC lines from discordant identical twins generated in this study will be a valuable tool for studying sALS in an isogenic manner in order to better understand the phenotypes and pathological mechanisms in ALS.


In this study, an analysis of the influence of polymer modification on the mechanical behavior, porosity, and microstructure of mortar is carried out. Epoxy latexes contents of 5%, 10%, 15%, and 20% of cement were employed in the preparation of cement mortars based on the same workability. The specimens were subjected to dry, wet, and wet–dry curing regimes. Compressive strength, flexural strength, Mercury intrusion porosimetry (MIP), and scanning electronic microscope (SEM) tests were conducted to analyze the effect of epoxy latexes on the mechanical property and porosity of modified mortars. Based on the compressive strength test results, a quantitative method was established to calculated the degree of hydration and polymerization. The results show that the mechanical behavior and porosity property of epoxy latexes modified mortar are influenced by the degree of hydration, the degree of polymerization, and the volume changing effect of mortar. The polymerization of epoxy latexes could improve the flexural strength of the mortar. The macropores of specimens tended to decrease with the increase of the degree of epoxy latexes polymerization and cement hydration. In practical engineering, it is necessary to ensure the degree of hydration and increase the polymerization rate. Thus, the wet–dry curing regime is recommended. Concrete is the most widely used material in China’s hydropower construction thanks to its low cost and relatively good strength in compression. Concrete structures often suffer various deteriorations caused by the ambient environment, such as frost damage, carbonation, corrosion, alkali–aggregate reaction, and so on . In the With the recent development in the polymer material technologies fields, applications of polymer are more and more extensive. Innovative strengthening strategies have been developed, based on externally bonded fiber-reinforced polymer and fiber-reinforced cementitious matrix systems, to improve the capacity of existing structures in order to guarantee the safety of masonry structures ,5,6. FurIn general, polymer latexes were used as a cement modifier and will form flexible polymer films in polymer-modified cement-based material ,17. The Numerous test programs have been conducted to study the effect of epoxy latexes on the properties of cement-based materials. The test results show that the flexural strengths and ductility of PMM and PMC are increasing significantly, while the compressive strengths are quite discrete. Aggarwal et al.  pointed In general, the tensile strength, flexural strength, and deformation properties of PMM and PMC are relatively higher compared with the conventional cement-based materials ,39. It hIn this research, polymer-modified mortars using epoxy latexes with hardener were prepared with various epoxy latexes contents and curing conditions. Compressive and flexural strength were tested to evaluate the effectiveness of modification. Mercury intrusion porosimetry (MIP) tests were conducted to analyze the effect of epoxy latexes on the porosity of modified mortar. Scanning electronic microscope (SEM) tests were used to evaluate the influence of epoxy latexes on the morphologies of the co-matrix. Based on the experimental results, a quantitative method was established to clarify the degree of cement hydration and epoxy resin polymerization. The effects of these two parameters on the properties of modified mortars were analyzed and discussed.3 was adopted in this research. The specific area of the cement was 390 m2/kg. Well-graded manufactured quartz sand with a specific gravity of 2700 kg/m3 was employed as fine aggregates. The particle sizes of manufactured sand range from 0.075 mm to 4.75 mm. The particle size distribution curves of manufactured sand are shown in Type PO42.5 ordinary Portland cement with a relative density of 3080 kg/m3, epoxide equivalent value of 200–300 g eq, and total solids of 99.9%. The epoxy resin hardener had a density of 1.00–1.05 g/cm3 and total solids of 50%.Epoxy latexes were prepared by emulsifying epoxy resin, based on bisphenol A-type epoxy resin and curing agent in water using an emulsifier. As shown in In this investigation, a high-speed shear dispersing emulsification tool was used for mixing the epoxy latexes, as shown in 3 based on Chinese standard CECS 203:2006 [The mix proportions of epoxy latexes modified mortar were designed based on that of a self-compacting mortar. The mix proportions of self-compacting mortar were selected based on our previous research ,48,49,50203:2006 , which c203:2006 ,48,49,50203:2006 , the supWith reference to ISO 679:2009 , a standMortar cube specimens with the size of 70.7 mm × 70.7 mm × 70.7 mm were cast for the compressive strength test and mortar prism specimens with the size of 40 mm × 40 mm × 160 mm were cast for the flexural test. After casting, the mixtures were covered with plastic sheets to avoid water evaporation. After 2 days, all specimens were demoulded and cured according to three different curing regimes.To investigate the coupled effect of cement hydration and epoxy resin polymerization on the properties of epoxy latexes modified mortar, dry curing, wet curing, and wet–dry curing regimes were applied for all the mortar specimens.Dry curing: after de-moulding, the mortar specimens were transferred into a standard curing chamber and cured under the standard environmental condition at a temperature of 20 ± 1 °C and relative humidity of 60 ± 5% until the test age;Wet curing: after de-moulding, the mortar specimens were immediately immersed in a water chamber with a temperature of 20 ± 1 °C until the test age;Wet–dry curing: after de-moulding, the mortar specimens were immediately immersed in a water chamber with a temperature of 20 ± 1 °C for 5 d. Then, the mortar specimens were transferred into a standard curing chamber and cured under the standard environmental condition at a temperature of 20 ± 1 °C and relative humidity of 60 ± 5% until the test age.cmf represents compressive strength, cF is the total maximum load, and A is the area of the loaded surface (70.7 mm × 70.7 mm = 4998.49 mm2).The compressive strength tests of mortar cube specimens were carried out at the ages of 7 d, 14 d, 28 d, 45 d, 60 d, and 90 d with a standard compression test machine, according to BS EN 12390 . All thefmf represents flexural strength; fF is the peak load; L (100 mm) is the distance between the support points; and b (40 mm) and t (40 mm) are the width and height of the cross-section of the specimens, respectively.The flexural strength tests of mortar prism specimens were carried out using a universal test machine at the ages of 7 d, 14 d, 28 d, 45 d, 60 d, and 90 d, according to standard ISO 679:2009 . The tesThe porosity and pore size distribution of mortar specimens were measured by the mercury intrusion test using a Micromeritics Poresizer model AutoPore IV 9500 . The mortar specimens were collected from the crushed specimens in compressive tests at the ages of 7 d, 14 d, 28 d, 45 d, 60 d, and 90 d.SEM tests were carried out to clarify the morphologies of epoxy latexes modified mortars. Test specimens were also collected from the crushed specimens in compressive tests at the ages of 7 d, 14 d, 28 d, 45 d, 60 d, and 90 d. All the specimens were etched with ethanol for 24 h and coated with a plastic film before the test. Magnifications ranged between 5000× and 20,000×.The P/C ratios varied from 0 to 20%. The relationship between the epoxy latexes content and the compressive strength of mortar specimens with different curing regimes at the ages of 7 d, 14 d, 28 d, 45 d, 60 d, and 90 d is shown in As mentioned, the properties of epoxy latexes modified mortar were significantly governed by both the process of cement hydration and the epoxy resin polymerization ,32. It icf is the compressive strength in MPa, 234 MPa represents the intrinsic strength of the hydrates gel for the type of cement, and the gel/space ratio r is defined as the ratio of the volume of gel and the total space available to the gel:hk is the hydrate volume expansion coefficient of ordinary Portland cement, which indicates that the products of complete hydration of 1 mL of cement will be assumed to occupy 2.06 mL gel. cv is the specific volume of cement, which denotes the volume of unit mass with a value of 0.317 cm3/g. α is the degree of hydration of cement. 0w is the volume of mixing water. Finally, c is the mass of cement.Neville  developeThe validity of the model was verified by plenty of tests in the cement paste and mortar scale without polymer. In this research, new parameters will be introduced to represent the interaction between cement hydration and epoxy resin polymerization based on the model.The degree of cement hydration is calculated by Equations (3) and (4), based on the experimentally determined compressive strengths of the control mortars. The hydration degree of cement under dry curing, wet curing, and wet–dry curing regimes was calculated by fitting the experimental results with Equations (3) and (4). Moreover, the calculated results of the degree of hydration for all specimens are listed in wV/cV). As shown in i was first introduced as shown in Equation (5):i is the volume changing index and can be calculated according to It should be noted that, in this research, the mixture proportions of epoxy modified mortar were designed with a constant volume water/cement ratio (ξ. The gel/space ratio in epoxy latexes modified mortar could be modified as Equations (6) and (7):α’ a is the actual hydration degree of epoxy latexes modified cement mortars. ξ is the retardation coefficient, which indicates the retardation effect of epoxy latexes on the degree of cement hydration. For the series of control cement mortars, ξ = 1.It has been reported that the addition of polymer latexes has a significant retardation effect on the cement hydration process . The degAs reported before, Ohama proposed a simplified model of polymer–cement co-matrix formation wherein, with water withdrawal by cement hydration, the close-packed polymer particles on the cement hydrates coalesce into continuous films or membranes ,58. In tφ is defined as the beneficial effect of epoxy resin polymerization on the compressive strength; η is the degree of polymerization; and pV and cV are the volume of polymer and cement, respectively.It can be seen from the test results that the polymerization of epoxy resin has a beneficial effect on the compressive strength of mortar specimens. To represent the beneficial effect, the degree of epoxy resin polymerization is introduced into the model, as shown in Equation (8):ξ, the degree of polymerization (η) can be calculated by fitting the experimental results through Equations (6) and (8).It has been reported that the degree of polymerization of epoxy resin has little influence on the hydration kinetics of epoxy resin-modified cement at normal temperatures . Thus, tThe calculated results of polymerization for all of the specimens are listed in An investigation of the material’s ability to resist deformation under load was done through the flexural test. 1λ is the influence factor representing both the effect of cement hydration and the volume of mortar. 2λ is used to represent the coupled effect of the degree of hydration, the degree of polymerization, and the volume changing effect of mortar.It has been reported that the polymer–cement co-matrix formed in PMM can significantly improve the flexural strength of mortar and concrete ,39. It i1λ is shown in 2λ is shown in λIt can be concluded that the flexural strength of epoxy latexes modified mortar was also influenced by the degree of hydration, the degree of polymerization, and the volume changing effect of mortar. Meanwhile, the improvement of the flexural strength depends mainly on the degree of epoxy resin polymerization. It can be speculated that, for the engineering application of epoxy latexes modified mortar, the dry and wet–dry curing regimes are suitable for the development of the mechanical properties.The relationship between the flexural strength of specimens under the wet curing regime and The effects of epoxy latexes modifications on pore size distributions of cement mortars determined from MIP are discussed in this section. In general, the pores in concrete can be classified as gel (d < 10 nm), transition (10 nm ≤ d < 100 nm), capillary (100 nm ≤ d < 1000 nm), and large (d ≥ 1000 nm) pores . Capilla3λ is the influence factor to represent both the coupled effect of the degree of hydration and polymerization. 3λ and the macropores under the dry and wet–dry curing regimes. As seen in Similarly, an influencing factor was defined:2 crystals and ettringite needles produced by cement hydration could be found. However, contrary to the mortar specimens under dry curing regimes, the polymer films could not be observed clearly from the mortar specimens under wet curing regimes. It is interesting to note a large amount of unhardened epoxy resin in the mortar aggregates, which proves that no polymerization reaction was produced in the case of the wet curing regime. Meanwhile, as seen in To understand the modification mechanism of epoxy latexes more clearly, the microstructure of the fracture surfaces of the 45 d cured modified mortar specimens under different curing regimes with the addition of 15% epoxy resin was observed by SEM. As seen in The compressive strength of epoxy modified mortars tends to decrease with the increase of the P/C ratios. The compressive strength of specimens under wet–dry and dry curing regimes is higher than that of specimens under wet curing.Based on the proposed model, the degree of hydration and polymerization of mortar specimens under different curing regimes can be calculated. The degree of hydration decrease with the increase of the P/C ratio, and the hydration rate tends to decrease as the curing ages increase. The compressive strength of epoxy latexes modified mortar was mainly governed by the cement hydration.Epoxy latexes provide a considerable increase in the flexural strength under the dry and wet–dry curing regimes. Meanwhile, for the case of wet curing, the flexural strength of epoxy latexes modified mortars is less than that of than control mortars. The reduction of the flexural strength is caused by the low polymerization rate under the wet curing regime.The flexural strength of epoxy latexes modified mortar is influenced by the degree of hydration, the degree of polymerization, and the volume changing effect of mortar. Meanwhile, the improvement of the flexural strength depends mainly on the degree of epoxy resin polymerization.According to the findings of the study, the strength of epoxy latexes modified concrete is related to hydration and polymerization. In practical engineering, it is necessary to ensure the degree of hydration and increase the polymerization rate, thus the wet–dry curing regime is recommended.The micropores of epoxy latexes modified mortars tend to increase with the increase of the P/C ratios. The macropores of epoxy latexes modified mortar specimens tended to decrease with the increase of the degree of epoxy latexes polymerization and cement hydration.The addition of epoxy latexes is helpful to improve microstructures by forming the continuous films in cement mortar under dry and wet–dry curing regimes. Meanwhile, little continuous films can be founded under the wet curing regime owing to no polymerization of epoxy resin.In this research, polymer-modified mortars using epoxy latexes were prepared with various epoxy latexes contents and curing conditions. Compressive, flexural strength, MIP, and SEM were conducted to analyze the effect of epoxy latexes on the mechanical behavior and porosity property of cement mortar. Based on the experimental results, an integrated model for the degree of cement hydration and epoxy latexes polymerization was established. The following conclusions can be drawn from the present study: