This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines  and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes  confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.All colorectal cancer cell lines except RKO displayed active  APC mutations in colorectal carcinogenesis showed that the majority of colorectal cancer cell lines have acquired activated β-catenin/TCF regulated transcription and hence active canonical Wnt signaling pathway, as a result of inactivation of APC or activating mutations of β-catenin gene CTNNB1 [ APC tumor suppressor gene [ APC or CTNNB1 and did not show any evidence of activated β-catenin/TCF regulated transcription [β-catenin subcellular localization was established as a surrogate marker for its activity, where nuclear β-catenin localization indicated activated β-catenin/TCF regulated transcription and membranous localization of β-catenin indicated inactive β-catenin/TCF regulated transcription. Using this approach, the majority of primary colon tumors showed nuclear β-catenin with the exception of few subsets including familial colon cancer [Early research into the role ofe CTNNB1 , 3. The sor gene . Excepticription , 5. Thisn cancer . Hence, β-catenin/TCF regulated transcription, it is important to address the interaction of these mechanisms in such cells.Extensive studies showed that genetic instability and epigenetic changes play a significant role in colorectal carcinogenesis. Different patterns of genetic instability were described in colorectal cancers including chromosomal instability which may have diverse underlying causes and microsatellite instability (MSI) due to deficiency in the mismatch repair (MMR) system . ChromosWe compared the RKO cell lines to seven classical colorectal cancer cell lines  and three normal colon mucosa specimens. The seven cell lines were chosen to be MSI similar to RKO, to eliminate the influence of this mechanism. APC or CTNNB1. To maintain this as the differentiating factor, cell lines for microarray analysis were selected to be MSI similar to RKO and, moreover, the majority of these cells were CpG island methylator phenotype- (CIMP-) positive similar to RKO. Many of these features were confirmed in our lab (see β-catenin). Fresh frozen and/or paraffin derived specimens of tumor and matching normal tissues were collected from pathology departments of different hospitals and used for immunohistochemical analysis and DNA extraction according to standard protocols. The study was approved by the institutional review board of the Helsinki University Central Hospital (Dnro 466/E6/01) and the National Authority for Medicolegal Affairs (Dnro 1272/04/044/07).The colon cancer cell lines were RKO, HCA7, KM12, LoVo, HCT15, HCT116, SW48, and LIM1215. RKO was the only cell line with no mutation in lab see . We incl CTNNB1 exon 3 was directly sequenced as described [ BRAF mutation analysis focused on codon 600 and was conducted as described in Niskakoski et al. [APC mutation analysis was as described . CTNNB1 escribed . BRAF mui et al. .Multiplex fluorescence in situ hybridization (M-FISH) was performed using the Xcyting colors  according to the manufacturer's protocol. Data were analysed using ISIS 4.4.21 software (MetaSystems GmbH). Comparative genomic hybridization (CGH) was performed and results were analyzed according standard protocols. Briefly, tumor DNA and reference DNA  were labelled by nick translation with fluorescein-iso-thiocyanate (FITC)-conjugated dCTP and dUTP , and Texas Red-conjugated dCTP and dUTP (Dupont), respectively, to obtain fragments ranging from 600 to 2000 bp. The results were analyzed using the ISIS digital image analysis system , based on an integrated high-sensitivity monochrome charge-coupled device (CCD) camera and automated CGH analysis software.Gene expression was analyzed using Affymetrix Human Genome U133 Plus 2.0 GeneChip® microarrays , containing over 54 000 probe sets covering 47 000 transcripts. The samples for microarray were biological duplicates of all the 8 cell lines plus the three normal mucosal specimens.Total RNA was extracted and purified using Qiagen RNeasy kit . RNA integrity and yield were assessed by Agilent Bioanalyzer 2100 . Only samples with an RNA integrity number (RIN) higher than 7.0 were included in the analysis.Samples were amplified, labelled and hybridized according to manufacturer's protocol and as described in Nymark et al. . The arrt-test analysis with Benjamini-Hochberg multiple testing correction was utilized to obtain genes whose fold change between Wnt inactive and Wnt active colorectal cancer cell lines was ≥2.0 . Unsupervised hierarchical clustering analysis was performed in GeneSpring software on normalised data for differentially expressed genes for all samples. The euclidean distance metric was used, this calculates the standard sum of squared distance between two entities, with centroid linkage . The raw microarray data are deposited at GEO with the accession number GSE58058.Microarray data were analyzed using GeneSpring GX12.0 software and processed using robust multiarray analysis (RMA) algorithm for background correction, normalization and log2-transformation. Unpaired http://david.abcc.ncifcrf.gov/home.jsp using KEGG and Biocarta annotation.Pathway analysis was performed at the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7, NIAID, NIH at http://dx.doi.org/10.1155/2016/6089658) had high allele number and heterozygosity frequency. Primer sequences and PCR amplification conditions were retrieved through NCBI website (http://www.ncbi.nlm.nih.gov/). The experimental procedure and data analysis were as described [β-catenin).MSI status was determined using the Bethesda panel . The 31β-catenin and 10 with membranous β-catenin) and then was extended to include all available sections.The primary antibodies are listed in Supplementary Table S2. Immunohistochemistry was performed as described before  on a pilDm values) were calculated as described [Methylation analysis was conducted by Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). In brief, promoter-associated CpG islands were examined for methylation by bisulphite sequencing with primers indicated in Supplementary Table S3-A and representative HhaI sites used for MS-MLPA probe design according to the protocol of MRC-Holland (Supplementary Table S3-B). Upon digestion of the sample DNA/probe hybrids with the methylation sensitive restriction enzyme HhaI, only samples with the HhaI site methylated will generate an amplification product. Synthetic MS-MLPA probes for the chosen genes were added to the P300 commercial test  for control fragments. The DLC1 gene had two separate MS-MLPA probes, with the probe for DLC1 isoform 4 (DLC1-i4) designed based on a previous publication . Methylaescribed .Dm <0.15 by MS-MLPA correlated with the lack of methylation (T/T) by bisulphite sequencing, whereas Dm >0.15 correlated with methylation (C/T or C/C) by bisulphite sequencing. To evaluate increased methylation in tumor DNA versus normal DNA , a normal tissue-based hypermethylation threshold was determined individually for each locus. The hypermethylation threshold was defined as the average Dm +1 standard deviation and derived from the combined set of normal mucosa specimens (n = 110) from all patient groups (Supplementary Table S4).Custom MS-MLPA assays were validated against bisulfite sequencing by using normal tissue and cancer cell lines. The technical threshold for methylation detection by MS-MLPA was 0.15 for all loci since http://www.vassarstats.net/tab2x2.html). Independent samples t-test was used for comparisons of two normally distributed tumor groups. One-way ANOVA was used for statistical analyses of normally distributed data. When methylation patterns between normal and tumor samples were examined parametric t-test for paired samples or nonparametric paired-samples Wilcoxon signed rank test was used. P values <0.05 (two-tailed) were considered significant.Statistical analyses were performed using SPSS Statistics Software . The percentages of tumors with hypermethylation were compared by Fisher Exact Probability Test from VassarStats Web site  positive similar to RKO as well. We considered it important that these cell lines used for comparison shared these characteristics. The MSI status and the β-catenin gene (CTNNB1) mutation status were confirmed in our lab during previous studies [ APC gene was fully sequenced in the 3 cell lines HCA7, KM12, and RKO in our laboratory [RKO was the only cell line with no mutation in studies , 24. The BRAF mutation; we also extended the BRAF mutation analysis to the primary tumors. We found that BRAF V600E mutation was present as follows: sporadic MSI-colorectal cancers 17/33 (51%) (result was not available for 7), sporadic MSS- colorectal cancers 0/40 (0%) (result was not available for 3), and Lynch syndrome-colorectal cancers 0/15 (0%) (result was not available for 12).Since RKO was the among the only in vitro colorectal cancer model holdingThe RKO karyotype by M-FISH  showed mKaryotypes of other cell lines used here were available in previous publication by our group  or otherβ-catenin/TCF regulated transcription, the RKO cell line cDNA expression profile was compared to 10 specimens: 7 MSI colon cancer lines and three normal colon mucosa samples as described above. Taking a twofold change as threshold limit, there were 815 differentially expressed Affymetrix probes (272 downregulated and 543 upregulated) in RKO compared to Wnt-active colon cancer cell lines. The probes corresponded to 611 unique genes . Cluster analysis showed that samples clustered into 3 distinct groups with RKO duplicate samples in one group, the normal samples in one group, and all other cancer cell lines in one group   showed variation of protein expression among the 22 tumors of the pilot study. For RGS4, DGKI, OBCAM/OPCML, and GLIPR1 proteins, staining was extended to include all tumors available. Expression of DGKI and GLIPR1 showed significant relationship with the β-catenin subcellular localization in a pattern similar to that observed in the microarray analysis of the cell lines  whereas two genes had low gene expression in RKO (EPCAM and KLK10). DLC1 showed a fold change <2 in the microarray analysis but it was included because of its potential significance as a tumor suppressor in colon and liver cancers [As promoter methylation can underlie expression changes, six genes from the cell line experiments, all with verified or putative roles in growth regulation and with CpG islands in the promoter region, were selected for methylation analyses by MS-MLPA. Four genes exhibited high expression in RKO compared to the other MSI colon cancer cell lines  revealed high frequencies of hypermethylation (78–98%) and no apparent subgroup-specificity. KLK10 and EPCAM showed Lynch syndrome-specific methylation patterns. For KLK10, 48% of Lynch colorectal tumors showed hypermethylation compared to 8% of sporadic MSI (P < 0.001) and 12% of sporadic MSS tumors (P < 0.001). Hypermethylation of EPCAM was restricted to Lynch syndrome colorectal cancers . DLC1 hypermethylation was characteristic of sporadic MSI colorectal cancer as compared to Lynch  or sporadic MSS colorectal cancer .Since the baseline levels of methylation may show locus and patient group-specific variation, thresholds for hypermethylation were determined as described in β-catenin subcellular localization, the number of gene promoters showing hypermethylation out of 7  was calculated for each tumor and compared with nuclear versus membranous localization of β-catenin. In all three patient groups , membranous β-catenin was associated with a higher number of hypermethylated loci , compared with tumors showing nuclear β-catenin . As evident, the association of hypermethylation with β-catenin status was stronger among sporadic (MSI plus MSS combined) than Lynch-associated colorectal cancers, and reached statistical significance among the former tumors .To investigate the relationship between promoter hypermethylation and  APC mutations which almost always cause deletion of both central domains that interact with β-catenin and C-terminal domains that interact with the cytoskeleton and play a role in microtubule binding, cell polarity, and chromosome segregation. Hence, deletion of APC C-terminal domains is expected to contribute to the chromosomal instability observed in many colorectal cancers. We have analyzed the chromosomal instability in RKO by M-FISH and CGH which revealed almost stable chromosomal number as evidenced by low aneuploid index and low variability of centromere number in RKO similar to the chromosomally stable MSI lines. This finding is consistent with having wild type APC protein. Interestingly, chromosome structure was not stable in RKO as evidenced by high number of rearranged chromosomes which was not expected for MSI cell line. Previously, we have described MSI cell line with specific tendency to acquire structural rearrangements most of which were balanced translocations [Colorectal tumors are dominated byocations  but the ocations . The posβ-catenin at the majority of the tested loci comparable to the LOH frequency in tumors with nuclear β-catenin. The LOH analysis also showed that some specific events are selected for in tumors with membranous β-catenin  consistent with the down regulation of nearby genes  observed by expression microarray analysis. The loss of EPCAM was reported in Lynch syndrome cancers and germline deletion of this gene was associated with predisposition to colorectal cancer in Lynch syndrome [β-catenin were overrepresented among familial colon cancer [ ERCC1 and RECQL which were rather upregulated in RKO. Overall, the tendency for structural chromosomal instability in RKO justifies more detailed studies to analyze its extent, mechanism, and consequences in the development of this subset of colorectal cancers.Consistent with the structural instability in RKO, we observed high frequencies of LOH in primary tumors with membranous syndrome –34. This 2p25.1, p25.1 conβ-catenin activation status. We cannot exclude the effect of other possible molecular differences  on the expression pattern of the cell lines. BRAF mutation is known to correlate with MSI and CIMP, but RKO and most of other cell lines were MSI-positive and CIMP-positive, regardless of BRAF mutation status; hence this effect remains to be a confounding factor. We characterized our primary tumors for BRAF mutation; the frequencies we obtained were in agreement with published reports on the occurrence of BRAF V600E mutation in sporadic and Lynch associated colorectal cancers [The first observation from the expression microarray was that cluster analysis based upon global gene expression profiling corroborated our hypothesis that RKO cell line is unique as it formed a distinct cluster separate from all other cell lines. The high number of the differentially expressed genes (611) exceeding the twofold change threshold and the large number among these with a very high (>10) fold change  also supports that the differences in the carcinogenic mechanisms between these clusters are substantial. Considering known and common pathways in colorectal carcinogenesis, a major difference between RKO and other cell lines is the  cancers –37.β-catenin. Some loci showed high frequency of LOH consistent with common areas of gains and losses reported in colorectal cancers such as 7p15.2, 7q22.1, 8p22, 9p21.1, 12p12.3, 12q14.1, 17p13.1, 20q1.21, and 20q13.32 [ MUC12 [ MTUS1 [ ARFGAP1/SMAP1 [NFE2L3, DGKI, ADAMTSL1, RASSF8, DCTN2, GLIPR1, and ASGR1). Loci that were not commonly reported as targets of gains, losses, or allelic imbalances in colorectal cancers included 1q23.2, 2q11.2, 5q35.2, 11q22.3, and 11q24.1 and their associated target genes also included known tumor suppressors/immune killers such as IL18 [ OPCML/OBCAM [IFI16, RGS4, DEDD, AFF3, SORL1, and PDLIM7).We then proceeded to verify whether or not selected target genes have a role in well characterized categories of primary colorectal cancers. We started by LOH analysis as it was recently reported that LOH is an alternative mechanism that plays a significant role in the pathogenesis of colorectal cancers with chromosomal and microsatellite instability . Overall20q13.32 . Some ta [ MUC12 , MTUS1 [ [ MTUS1 , and ARFP1/SMAP1 , while o as IL18 , OPCML/OML/OBCAM , or noveIFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) in primary tumors confirmed that they were differentially expressed in clinical specimens. DGKI and GLIPR1 showed significant relationship with the β-catenin subcellular localization in a pattern similar to that observed by the cDNA expression microarray analysis of the cell lines. GLIPR1 (glioma pathogenesis-related protein 1) is a p53 target gene that has p53-dependent and p53-independent proapoptotic activities in tumor cells [β-catenin activity. DGKI  is a member of type IV diacylglycerol kinase subfamily which regulates the intracellular concentration of diacylglycerol through its phosphorylation. The specific role of this particular enzyme is undetermined and very few publications are available on this gene in general. Interestingly, DGKI was listed among genes mutated in melanoma as identified by next generation sequencing and also among hypermethylated gene list identified by whole-genome methylation analysis of glioblastoma [ DGKI is a novel potential tumor suppressor in colorectal cancers and its expression is downregulated subsequent to β-catenin activation too.The immunohistochemistry of selected target genes  and the most significant methylation degree compared to the matching normal tissues  β-catenin being associated with a higher frequency of hypermethylated genes compared to nuclear (active) β-catenin. This is an intriguing observation and supports previous evidence implicating epigenetic modifications as one important layer in the multilayered control system responsible for cell-type specific regulation of Wnt target genes [Interestingly, et genes .β-catenin/TCF regulated transcription using a model cell line and groups of primary sporadic and hereditary colon cancers. The data illustrate that these cancers might evolve through distinct cytogenetic structural chromosomal instability and also involves distinct groups of cancer-related genes which are susceptible to LOH and methylation. The essential regulatory systems important in colon cancer development  appeared to be closely connected. The differentially expressed genes validated here could be used to develop personalized tumor profiling.This work analyzed the carcinogenic mechanisms of colorectal cancers that lacks active The Supplementary Material consists of 9 components. Supplementary Figure SF1 shows representative examples from the immunohistochemistry of colon cancers. Supplementary Tables S1 to S7 (supplementary Table S3 has 2 parts S3A and S3B) show some detailed methods and results as referred to in the main article and indicated in each table title.


Epithelial–mesenchymal transition (EMT) contributes to the progression and metastasis of cancer cells and is associated with a more invasive phenotype of cancer. The Wnt/β-catenin signaling pathway is one of the major pathways involved in EMT regulation. Many studies provide evidence that β-catenin, the key regulator of the canonical Wnt signaling pathway, is important in regulating EMT in cancer. However, the roles of Wnt3a, the representative canonical Wnt ligand, in EMT and colon cancer progression have not yet been fully explored.The expression levels of Wnt3a and EMT-associated proteins  were assessed by immunohistochemistry in human colon cancer tissues to evaluate the clinicopathological significance of Wnt3a, as well as the correlation between Wnt3a and EMT. We then upregulated Wnt3a expression in HCT116 colon cancer cells, established a nude mouse xenograft model, detected the expression of EMT and Wnt/β-catenin signaling-associated proteins, and observed invasion and clone-initiating abilities.P = 0.004), clinical stage (P = 0.008), presence of metastasis and recurrence (P = 0.036), and survival time (P = 0.007) of colon cancer patients. Wnt3a expression was notably concomitant with EMT immunohistochemical features, such as reduced expression of the epithelial marker E-cadherin (P = 0.012), increased expression of the mesenchymal marker vimentin (P = 0.002), and cytoplasmic distribution of β-catenin (P = 0.021). Results of in vitro and in vivo experiments showed that Wnt3a overexpression could alter cell morphology, regulate EMT-associated protein expression, and enhance clone-initiation and invasion. Dkk1  could also partially reverse the expression of EMT-associated proteins in Wnt3a-overexpressing cells.In 203 human colon cancer tissue samples, Wnt3a protein overexpression was related to colon cancer histological differentiation (Wnt3a expression was associated with EMT and promoted colon cancer progression. The EMT-inducing effect was partially due to the stimulative effect of Wnt3a on the Wnt/β-catenin pathway. Colorectal carcinoma is the third most common cause of cancer-related deaths worldwide . AlthougEpithelial–mesenchymal transition (EMT) is an early phase of the malignant transformation of epithelial cells. In EMT, cells lose their polarities and contacts with neighboring cells and then acquire mesenchymal-like and motile phenotypes . Tumor-cThe Wnt signaling pathway is pivotal in embryogenesis and development and is also widely implicated in a number of human diseases -6. DeregThe canonical Wnt/β-catenin signaling pathway is well established in colorectal oncogenesis, with >85% of these malignancies harboring mutations along the pathway that lead to constitutive activation. Many studies provide evidence that the Wnt/β-catenin signaling pathway plays an important role in EMT regulation ,15. HoweIn this study, we evaluated the clinicopathological significance of Wnt3a and analyzed the correlation between Wnt3a expression and EMT immunohistochemical features in tissue specimens from 203 colon cancer patients. The effects of Wnt3a ectopic expression in the colon cancer cell line HCT116 on the expression of epithelial and mesenchymal markers and EMT transcription factors were studied. We also investigated cell proliferation and invasion in cell cultures with Wnt3a overexpression, as well as tumor growth and metastasis in a colon cancer xenograft model. We further treated Wnt3a-overexpressing cells with a Wnt/β-catenin pathway antagonist Dkk1-conditioned medium and detected the expression of EMT-related proteins to verify whether Wnt3a promoted EMT by activating the Wnt/β-catenin signaling pathway.Tissue samples of colon cancer were harvested from 203 patients who had undergone surgery for colon cancer in Tianjin Medical University Cancer Institute and Hospital  between January 2002 and December 2004. None of the patients had received any chemotherapy or radiotherapy before their operation. Data of clinicopathological parameters were obtained from patients’ clinical records and pathological reports.The human colon cancer cell line HCT116 was obtained from the Cell Resource Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College . Cells were cultured in Iscove modified Dulbecco medium with 10% FBS. Dkk1 recombinant protein was obtained from R&D Systems. For Dkk1 administration in vitro, recombinant Dkk1 (1 μg/mL) was added to culture medium. Typically, 50% of the medium was replaced every 24 h with fresh conditioned medium containing Dkk1 at the original concentrations. Cells were harvested 48 h post-treatment, and total cell lysates were collected for measurement by Western blot. The micro-Boyden chambers used were from NeuroProbe . Antibodies to β-catenin, goat anti-rabbit, and goat anti-mouse IgG-FITC were from Santa Cruz Biotechnology . Antibodies to Wnt3a, Snail, Slug, and Twist were from Abcam . Antibody to E-cadherin was from BD Biosciences . Antibody to vimentin was from Epitomics . Phalloidin was from Invitrogen . Alexa Fluor 488 and 546 were from Molecular Probes . BALB/C nude mice (4–5 weeks old) were obtained from Wei Tong Li Hua Experimental Animal Company .Streptavidin–biotin–peroxidase staining was performed as previously described . In a tyThe expression of Wnt3a, E-cadherin, vimentin, and β-catenin was analyzed only histologically in normal and neoplastic epithelial cells and not in stromal tissues. Wnt3a staining was considered immunoreactive when brown granules were identified in the cytoplasm. The staining intensity of Wnt3a was graded on a scale from 0 to 2 . Percentage immunoreactivity was scored on a scale from 0 to 3 . We multiplied the two scores to obtain a composite Wnt3a expression score. Wnt3a expression was classified as negative (score = 0), weakly positive , or strongly positive . E-cadherin expression was considered to be positive if >90% of cancer cells exhibited a staining pattern similar to that in normal epithelial cells. Vimentin expression was classified as positive when >10% cells were stained. β-Catenin nuclear staining was considered positive if >10% of cells showed brown granules in nuclei.Transfection with plasmid carrying Wnt3a and controlled scrambled plasmid  was performed with Lipofectamine 2000  according to the manufacturer’s instructions. To establish stable HCT116 cells that overexpressed Wnt3a, G418-resistant cells were screened.Protein (30–50 μg/lane) was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were blocked and incubated with primary antibodies overnight at 4°C, incubated with secondary antibody, and detected with ECL Western blot substrate (Millipore) according to the manufacturer’s instructions.4 cells were mixed gently at 37°C and plated onto the bottom agar. Dishes were incubated at 37°C and 5% CO2. Six days later, colonies (>50 μm) were counted from 10 random fields per dish.To form bottom agar, 1.5 mL of culture medium containing 0.6% agarose was added to each 35 mm dish. Then, 1 mL of culture medium containing 0.6% agarose and 1 × 105 cells/mL) contained in serum-free medium was added to the upper chamber, and 300 μL of culture medium supplemented with 20% FBS was added to the lower chamber. After incubation for 20 h, the passed cells were fixed and stained.Matrigel (BD Biosciences) with a final concentration of 1.5 mg/mL was added to the upper surface of the chamber filter (8 μm pore). Then, 200 μL of cell suspension .Cells were cultured on sterile glass cover slips on the day before staining. Cells were fixed with 4% paraformaldehyde, quenched with 50 mmol/L NH6 control or HCT116 cells overexpressing Wnt3a (clone7) by subcutaneous injection in right groin. Tumor size was measured every 3 days for 21 days. Tumor volumes were calculated using the following formula: volume = [length(in millimeters) × width2[in square millimeters])/2. Tumor samples were formalin fixed and paraffin embedded.Twenty mice were randomly and evenly divided into two groups and given either 3 × 10P < 0.05.SPSS v.16.0 software  was used for data analysis. The associations between Wnt3a and clinicopathologic parameters and the differential expression of E-cadherin, vimentin and β-catenin between different Wnt3a expression level groups were assessed with Fisher’s exact test and chi-square test. Differences or correlations between groups were assessed by the Mann–Whitney U-test, Student’s t-test and Pearson’s correlation test. Survival analysis was carried out according to Kaplan–Meier. Differences in survival curves were assessed using the log rank test. Significance was set at Among 203 samples, 179 (88.2%) showed positive Wnt3a expression, whereas the remaining 24 (11.8%) were negative. Tumors were categorized as strong expression, weak expression, or negative for Wnt3a . The average survival time for Wnt3a-negative patients was 76.4 months, whereas the average survival time for Wnt3a-weak expression and Wnt3a-strong expression group patients were 57.6 and 41.7 months respectively , vimentin , and β-catenin (nuclear) . These data provided proof about the role of Wnt3a as a potent activator of Wnt/β-catenin signaling and as a regulator involved in tumor progression in colon cancer.To assess the relationship between Wnt3a and EMT in colon cancer, we investigated the expression of the EMT-associated markers E-cadherin, vimentin, and β-catenin . As shown in Table We established stable Wnt3a-overexpressed colon cancer cells to study the EMT-promoting effect of Wnt3a on colorectal cancer cells. To rule out clone-to-clone variations, we selected two clones (clone7 and clone15). HCT116 cells with Wnt3a overexpression had increased expression of c-myc and cyclin D1 , a representative of cytoskeleton, and observed that Wnt3a overexpression caused HCT116 cells to form structures with irregular shape and non-uniform composition or density  and preventing the formation of Wnt-Fz-LRP ternary complexes and the downstream signaling transduction. After Dkk1 treatment, Wnt3a-overexpressing cells showed decreased β-catenin expression, indicating the effectiveness of Dkk1 as an inhibitor of Wnt/β-catenin pathway. Western blot assays also demonstrated that Wnt3a-overexpressing cells treated with Dkk1 had higher expression of E-cadherin and lower expression of vimentin, Snail, and Twist compared with untreated cells  promoter and also a cancer stem cell marker. We further demonstrated that Wnt3a could promote the expression of Snail, which is a key transcription EMT regulator. We also found that Wnt3a has significant stimulative effects on tumor growth and metastasis in nude mice. These findings demonstrated that ectopic Wnt3a expression exerted EMT-inducing effects that promote the progression of colon cancer. Meanwhile, another study on hepatocellular carcinoma showed that recombinant Wnt3a could not induce Huh-7 or HepG2 cells to undergo EMT in normoxia. Yoshida also indicated that oxidative stress could induce canonical Wnt activation. All these observations emphasize the importance of the microenvironment for Wnt signaling and EMT ,33. We sβ-Catenin has a dual cellular function and is involved in both transcription regulation and cell adhesion. When Wnt ligands such as Wnt1, Wnt3a, and Wnt7a act on their cell-surface receptors, cytoplasmic β-catenin is stabilized by its release from the axin complex and accumulation in the nucleus, where it binds to TCF/LEF and stimulates the expression of various genes involved in EMT . β-CatenAfter treating with Dkk1, the expression of EMT-associated proteins in Wnt3a-overexpressing cells did not recover to the same level as that of control cells. Thus, a Wnt3a-induced EMT mechanism that is independent of the Wnt/β-catenin pathway may exist. Another possibility was that the cancer cells may be responsive to Dkk1 differently in 2-D versus 3-D cultures, which more closely mimic the in vivo environment ,37.Multiple interactions were observed between Wnt3a and other families of signaling molecules, such as FAK, TGF-β, and EGF -41. NaleIn conclusion, we showed that Wnt3a expression activated the Wnt/β-catenin signaling pathway, and this activation was a mechanism underlying EMT in colon cancer. The results provide better understanding of the importance of EMT in tumor development and may enable the establishment of clinically useful therapy targets.


VP16. Ivermectin inhibits the proliferation and increases apoptosis of various human cancer types. It represses the levels of C-terminal β-CATENIN phosphoforms and of CYCLIN D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases.In vivo, Ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without obvious side effects. Analysis of single semi-synthetic derivatives highlights Selamectin, urging its clinical testing and the exploration of the macrocyclic lactone chemical space. Given that Ivermectin is a safe anti-parasitic agent used by > 200 million people against river blindness, our results suggest its additional use as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.Constitutive activation of canonical WNT-TCF signaling is implicated in multiple diseases, including intestine and lung cancers, but there are no WNT-TCF antagonists in clinical use. We have performed a repositioning screen for WNT-TCF response blockers aiming to recapitulate the genetic blockade afforded by dominant-negative TCF. We report that Ivermectin inhibits the expression of WNT-TCF targets, mimicking dnTCF, and that its low concentration effects are rescued by direct activation by TCF For example, a variety of sporadic human cancers have been found to harbor hyperactive canonical WNT signaling, leading to the constitutive activation of β-CATENIN, a key polyvalent protein that is modified by phosphorylation at multiple sites. In particular, early colon cancers commonly display loss of function of the tumor suppressor Adenomatous polyposis coli (APC), a key component of the β-CATENIN destruction complex, or harbor N-terminal gain of function mutations in β-CATENIN that render it APC-resistant. In both of these cases, this leads to canonical WNT pathway activation and the resulting regulation of target genes  targeting it for proteasomal degradation. Without active nuclear β-CATENIN, there is no activation of positive WNT-TCF targets. In contrast, under active WNT signaling, β-CATENIN escapes cytoplasmic degradation and is phosphorylated at its C-terminus, for instance at positions Ser552 and Ser675, by various kinases including AKT, PAK, PKA, and possibly AMPK. Such C-terminal phosphorylation has been found to be essential for the transcriptional function of β-CATENIN with TCF factors . These include inhibitors of Porcupine and Tankyrases, which promote normal WNT ligand secretion or cytoplasmic transduction, respectively  is a specific WNT-TCF response blocker at low micromolar concentrations.et al, et al, et al, in vivo efficacy against human colon cancer xenografts sensitive to TCF inhibition with no discernable side effects. The ability of systemic Ivermectin to also block lung cancer growthin vivo supports the possible use of Ivermectin in particular, and other macrocyclic lactones such as Selamectin in general, as WNT-TCF pathway response blockers to combat WNT-TCF-dependent human diseases including cancers of the intestine, breast, skin, and lung.Ivermectin and other macrocyclic lactones are well-tolerated agents that have been used to treat millions of people for river blindness  . The expression of these genes was unaffected by the candidate compounds, providing an additional control against general transcriptional effects.Tertiary analyses measured the mRNA levels of an established cohort of canonical WNT-TCF target genes in Ls174T cells  and in primary colon cancer liver metastasis mCC11 cells , perfectly tracked the gene expression profile induced by dnTCF4 Fig, arrow. Streptomyces avermitilis. It is a fermentation mixture of > 80% Avermectin B1a (5-O-demethyl-Avermectin A) and < 20% B1b (5-O-demethyl-25-de(1-methylpropyl)-25-(1-methylethyl-Avermectin A1) (S3). Ivermectin is a clinically approved Avermectin B1 derivative   as Abamectin—the commercial name of Avermectin B1 (IC50: 0.8–2 μM)  Avermectin A1a) and Selamectin - 5-demethoxy-25-de(1-methylpropyl)- 22,23-dihydro-5-(hydroxyimino)- Avermectin A1a) Fig. Doramecin vitro and widespread veterinarian use of the second. Activated Caspase3 was used as a marker of apoptosis by immunohistochemistry 48 h after drug treatment. Selamectin and Ivermectin induced up to a sevenfold increase in the number of activated Caspase3+ cells in two primary (CC14 and CC36) and two cell line (DLD1 and Ls174T) colon cancer cell types  with the exception of 0.1 μM Selamectin in DLD1, CC14 and CC36, and were concentration and cell-type-dependent, with strongest effects detected in DLD1 cells with 2.5 μM Ivermectin  begged the question of their relative abilities to block TCF responses in colon cancer cells. Analysis of the TCF targetsation FigD and E tASCL2 andLGR5  or Selamectin (0.01–0.1 μM)  FigA diminis1 μM) FigB–E. Wherin vitro as well as the growth of resulting clonal spheroids. These results suggest an action on both the bulk of the tumor and its cancer stem cells. Indeed, analyses of primary vs. secondary clonogenic events in Ls174T revealed that Ivermectin pre-treatment only affected primary cloning, without effects on secondary events far removed from the time of drug action . Taken the results of the two distinct assays together, the data indicate that Ivermectin and Selamectin affect the frequency of clonogenic eventset al, + cells by magnetic activated cell sorting after dissociation of 2D cultures with limited Trypsin or StemProAccutase treatments revealed no differences between control vs. (2.5 and 5 μM) Ivermectin-treated cells . Ivermectin treatment thus separates CD133 expression and spheroid clonogenicity.Colon cancer cells with clonogenic spheroid capacity often express high levels of the CD133 (the AC133) epitope as compared with non-clonogenic cells  does not generally lead to arrested growth or cell death, with the exception of DLD1 cells  yielded a poor response (8/9) to Ivermectin treatment via IP (not shown), possibly due to ineffective drug penetration.Intraperitoneal injections of cyclodextrin-conjugated Ivermectin given daily at 10 mg/kg after tumor establishment inhibited DLD1 tumor growth as compared with cyclodextrin carrier-only injected mice xenografted with sibling cells Fig. Criticain vivo data to a second WNT-dependent colon cancer type, we used HT29, which harbors a similar truncation in APC as DLD1 cells. Subcutaneous injection of HT29 cells into the flanks of Nude mice yielded tumors, the growth of which was repressed by Ivermectin treatment  and enhancedp21 levels . Ivermectin also diminished the protein levels of CYCLIN D1, a direct TCF target and oncogene, in both HT29 and H358 tumor cells  , which blocks the activity of the serine protein phosphatases PP2A and PP1. As expected, treatment with OA led to elevated levels of β-CATENIN C-terminal phosphoforms and of the TCF target CYCLIN D1 . However, Ivermectin was ineffective in reducing OA-enhanced expression  and Ivermectin (5 μM)-treated CC14 cells 6 h after treatment  than those required to block TCF responses and induce apoptosis. General toxicity related to chloride channel deregulation has been suggested to underlie the high micromolar toxicity of Ivermectin for liquid tumor cells-CATENIN/TCF transcriptional responses by Ivermectin, Selamectin and related macrocylic lactones. This conclusion is based on (i) The ability of Avermectin B1 to inhibit the activation of WNT-TCF reporter activity by N-terminal mutant (APC-insensitive) β-CATENIN as detected in our screen; (ii) The ability of Avermectin B1, Ivermectin, Doramectin, Moxidectin and Selamectin to parallel the modulation of WNT-TCF targets by dnTCF; (iii) The finding that the specific WNT-TCF response blockade by low doses of Ivermectin and Selamectin is reversed by constitutively active TCF; (iv) The repression of key C-terminal phospho-isoforms of β-CATENIN resulting in the repression of the TCF target and positive cell cycle regulator CYCLIN D1 by Ivermectin and Selamectin; (v) The specific inhibition ofin-vivo-TCF-dependent, but notin-vivo-TCF-independent cancer cells by Ivermectin in xenografts.Our data point to a repression of WNT-β-CATENIN after treatment with Ivermectin or Selamectin under PP2A/PP1 protein phosphatase-blocked conditions suggest that these drugs may act by enhancing, directly or indirectly, phosphatase activity involved in dephosphorylating P-Ser552/P-Ser675. This effect can help explain the phenotype of Ivermectin-treated cells since P-Ser552- and P-Ser675-β-CATENIN show enhanced transcriptional activity in cooperation with TCF factors and are essential for WNT signaling in colon cancer cells   alleles leading to a broken blood–brain barrier show Ivermectin neurotoxicity  was used at 1–15 nM.Abamectin , Ivermectin , Doramectin , Selamectin , Stromectol™ (Merck) and Moxidectin  were diluted in 100% DMSO or ethanol and used at 0.01–10 μM forColon primary cell culture and cell lines were plated in medium containing 2% FBS and treated with different drug concentrations for 48 h. After a 15′ pulse with BrdU (10 mg/ml), cells were fixed in 4% PFA, incubated in 2N HCl for 15′, neutralized with 0.1 M Boric acid for 10′, blocked in PBS-10% HINGS for 10′, and labeled with anti-BrdU antibodies . Signal was revealed with a rhodamine-coupled anti-mouse secondary antibody  and counterstained with DAPI (Sigma). At least 10 fields were quantified for each condition.Colon cancer cells were plated at 30% confluency and treated with different drug concentrations for 48 h. After treatment, cells were fixed in 4% PFA for 2 min and blocked in 10% HINGS for 1 h. Apoptosis was evaluated by immunofluorescence using a rabbit anti-cleaved Caspase3 antibody  and Cy3-labeled secondary antibody . Cells were counterstained with DAPI . At least 10 fields were quantified per plate for each condition.et al, RNA extraction, reverse transcription and qPCR were performed as previously described (VarnatqPCR primer sequences were as follows (5′->3′):et al, Adherent colon cancer cells were treated with Ivermectin or Selamectin at different concentrations as described, washed, lifted, and plated at 1 cell/well in 96-well non-adherent plates in colon spheroid medium (DMEM-F12 supplemented with B27 and 10 ng/ml EGF) without drugs and grown for 14 days. Spheres were then counted visually using an inverted Zeiss optical microscope. For secondary clonogenic assays, primary spheres were dissociated and replated at 1 cell/well in 96-well plates and cultured as described above. Each experiment was repeated at least thrice. Alternatively, untreated cells were plated and treated in 96-well plates with drugs and subsequently scored for spheroid formation. CD133 MACS (Miltenyi Biotech) was performed as described previously (VarnatCells were plated in media containing 2% FBS and treated with Ivermectin or Selamectin at different concentrations for 12 h. Protein lysates were in RIPA buffer. 10–20 μg of protein were loaded onto 12% polyacrylamide gels and the proteins transferred onto nitrocellulose membranes (Hybond), which were probed with primary and secondary antibodies as noted. Signal was revealed using the ECL system  and film. ImageJ was used for quantification. Membranes were sequentially reprobed after washing in TBS-1% Tween-20 and controlling for loss of signal. The following primary antibodies were used: anti-GAPDH , anti-cyclinD1 , anti-Pser552 β-CATENIN , anti-Pser675 β-CATENIN , and anti-total β-CATENIN . As secondary antibodies, HRP-coupled anti-rabbit or anti-mouse  were incubated for 1 h at RT.5 control or lentivector-infected cancer cells were injected subcutaneously into the flanks of 6- to 8-week-old female NMRI Nude mice. After tumor establishment, mice were treated with cyclodextrin carrier alone or Ivermectin (or Stromectol™) conjugated with cyclodextrin (45%) via daily intraperitoneal injections at 10 mg/kg. Tumor volumes were measured every 2–3 days.5 × 10APC andCTNNB1 (β-CATENIN) genes. The Mutation Cluster Region (MCR) ofAPC was amplified with two pairs of primers: (1) MCR1FWD 5′-GATACTCCAATATGTTTTTC-3′ and MCR1REV 5′-GGAAGATCACTGGGGCTTAT-3′, (2) MCR2FWD 5′-GTGAACCATGCAGTGGAATG-3′ and MCR2REV 5′-TCTGAATCATCTAATAGGTC-3′. The primers forCTNNB1 were as follows:CTNNB1FWD 5′-CAATGGGTCATATCACAGATTCTT-3′ andCTNNB1REV 5′-TCTCTTTTCTTCACCACAACATTT-3′.One-hundred nanograms of genomic DNA from primary colon cancer cells (CC14 and CC36) or cell lines (DLD1 and Ls174T) was used for PCR using Phusion HF DNA Polymerase (BioLabs). Specific primers were designed to amplify commonly mutated regions inThe WNT-TCF intercellular signaling pathway is constitutively activated in a large number of diseases, including cancers of the intestine, skin, lung, endometrium, and brain. Finding small molecules that would act as pathway blockers has attracted much therapeutic interest but so far there are no available WNT-TCF blockers in the clinics, partly due to the time and cost of developing new drugs.in vitro andin vivo. Selamectin is 10 times more potent than Ivermectin, acting in the nanomolar range. Low doses of these drugs mimic the effect of dominant-negative TCF and their actions are rescued by activated TCF (TCFVP16). Ivermectin shows exquisite selectivityin vivo, repressing TCF-sensitive human cancer xenograft growth with no discernable side effects at the doses used. WNT-TCF blocking activity is detected at doses below those reported for chloride channel deregulation and general cell toxicity, suggesting a therapeutic window to target hyperactivated WNT-TCF-dependent diseases.Here, we have performed a repositioning small molecule screen using a collection of clinically approved drugs. We describe the identification of Ivermectin and related macrocyclic lactones, including the single-component semi-synthetic drug Selamectin, as WNT-TCF blockers in human cancer cellsGiven that Ivermectin is an approved drug widely used to combat multiple parasitic infections, our data suggest its additional use as a therapeutic agent against WNT-TCF-dependent diseases, notably including several cancer types, and the further investigation of the macrocyclic lactone chemical space.Colon cancer cells were plated at 40% confluency and treated with 5 μM Ivermectin or 2% DMSO for 6 h. After treatment, cells were fixed in fresh 4% PFA for 2 min. Antigen retrieval was conducted by boiling for 7 min in 10 mM citrate buffer . Cells were then incubated at RT for 30 min, washed with PBS and permeabilized in PBS with 1% Tween-20 for 15 min at RT. Anti-β-CATENIN antibodies  were applied overnight at 4°C after blocking with 3% BSA in PBS. Signal was revealed with a rhodamine-coupled anti-rabbit secondary antibody  and counterstained with DAPI . Samples were imaged with Zeiss LSM 510META confocal microscope. Five independent slides were analyzed per each condition.


The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin. Streptococcus pneumoniae is normally found as a commensal in the nasopharynx of humans, colonising 10% to 40% of adults and children respectively .,17.13,17stkP (T4ΔstkP∇ST) this reverted the effects and there was a decrease in the expression of the pilus islet genes (stkP (T4ΔstkP∇XST) there was also a drop in expression of the pilus islet genes but not to the same level as that seen in T4ΔstkP∇ST.In the StkP knockout RT-PCR analysis showed an up-regulation of all genes present on the pilus islet compared to TIGR4, suggesting that StkP normally functions to repress expression of the pilus islet genes. When the full length StkP was expressed in T4Δet genes . When thstkP, T4ΔstkP∇ST, T4ΔstkP∇XST, T4ΔrrgB and TIGR4 for the pilus adhesin RrgA (stkP compared to TIGR4 was observed. When StkP (T4ΔstkP∇ST) was complemented there was a large decrease in RrgA protein levels back to levels similar to those in TIGR4. This was not observed when the allelic variant of StkP was expressed in T4ΔstkP (T4ΔstkP∇XST) and only a small drop in RrgA protein levels was observed compared to T4ΔstkP.Western blot analysis was also performed to evaluate if the change in gene expression observed correlated to changes in total protein level. Changes in protein levels were evaluated in T4Δsin RrgA  and the sin RrgA , which astkP∇ST) there was a clear drop in RrgB protein levels compared to TIGR4 and T4ΔstkP suggesting that overexpression of the kinase suppresses RrgB expression. When complementing this mutant with the allelic variant of StkP (T4ΔstkP∇XST) the drop in RrgB levels was not as prominent but there was still a decrease compared to TIGR4 and T4ΔstkP.RrgB protein levels were not clearly different in the kinase knockout compared to TIGR4. However when complementing this mutant  at an unlinked part of the chromosome [stkP in TIGR4. Rather it is a consequence of altered StkP levels, which are closely regulated to levels of PhpP [y linked ,39,55. Fy linked . Thereforomosome . This woin vitro adherence of pneumococci to host cells during meningitis and pneumonia respectively.To evaluate the effect of altered pilus levels on adherence in the StkP mutant and complemented strains, their ability to adhere to Human Brain Microvascular Endothelial cells (HBMEC) and A549  were assessed . These cstkP (T4ΔstkPΔrrgB) abolished this increase in adherence showing the effect is solely due to an increase in pilus expression and not other adhesins. T4ΔstkPΔrrgB adherence capabilities were not different to that of T4ΔrrgB and TIGR4.Deletion of the kinase gene caused an increase in adherence to both cell lines. Deletion of RrgB in T4ΔstkP the adherence to both cell lines reverted to the levels seen with TIGR4. When the allelic variant of StkP was expressed in T4ΔstkP there was a trend towards a drop in adherence to both cells lines but this was not significant. Similar adherence patterns of T4ΔstkP were observed also to Detroit 562 cells  (When the wild type kinase was expressed in T4Δll line) ..rlrA and the regulator protein RlrA is part of a positive feedback mechanism for pilus expression in individual bacterial cells [StkP modulates pilus levels through expression changes, which in turn modulates adherence. It is not known if this is due to varying the number of pili on a single cell or the number of cells that have pili. Recent findings have shown that the number of pili-positive cells within a population can vary, although currently no genes have been shown to modulate this outside the pilus islet genes ,27. Thisal cells ,27.Flow cytometry was used to evaluate the biphasic expression of pili. To deduce the number of cells in a growing population that had RrgB present on the cell surface the whole bacterial population was stained with anti-capsular antibody and the proportion of those expressing pilli were measured using an RrgB specific antibody .stkP the proportion increased to 99% of cells being pili positive. When T4ΔstkP was complemented with StkP (T4ΔstkP∇ST) the proportion of pili-positive cells decreased, with 26% of bacteria in the capsule positive population also being RrgB positive. When the allelic variant of StkP (T4ΔstkP∇XST) was expressed 51% of cells were RrgB positive. These data suggest that StkP modulates the pilus at the population level.TIGR4 bacteria were gated in the FACS using the anti-capsular antibody. Gating using the anti-RrgB fluorescence channel showed that 88% of the cells within this population were RrgB positive. In T4ΔIn this study we show that StkP regulates expression of the pneumococcal pilus in TIGR4. We have shown that in strain TIGR4 the pilus modulates most of the adherence to human cells grown in culture and that the level of adhesion is mediated through the kinase StkP.Streptococcus agalactiae (GBS) where the pilus plays a role in adherence to HBMEC cells and has also been shown to aid penetration of the blood brain barrier [stkP knockout on virulence as it causes other deleterious phenotypes, such as defects in cell division [The pneumococcal pilus has previously been shown to promote adherence to A549 cells. Here we show it is also important for adherence to HBMEC cells suggesting a broad role of the pilus in adherence to different host cells ,25,53. S barrier ,57. Howedivision .Staphylococcus aureus [The role of the pilus in disease is still debated as presence of the pilus genes has not been linked to invasive disease or outbreak strains . Howevers aureus . Here wes aureus . This suRecent studies have shown that the pilus displays a biphasic pattern of expression. In TIGR4 30% of cells express the pilus on the cell surface however this varies between serotypes ,27. In oStreptococcus pyogenes modulating pilus expression at the population level based on temperature [It has been shown that there are no gene expression differences between pili positive and negative bacteria other than the genes present on the pilus islet itself . This biperature . This phperature .Little is currently known about pilus biogenesis and assembly kinetics, which may explain why in some instances we observe differences in gene expression and protein levels of the pilins in the kinase knockout and complemented strains.A large increase in adherence of TIGR4 to both HBMEC (7.5 fold) and A549 (15 fold) was observed when StkP was deleted. Due to the fact there are only 11% more cells in the population that are pili positive this increase seems disproportionate. However the recent structure of the pilus shows that RrgA can also multimerise forming chains of the adhesin reaching beyond the RrgB backbone . The facrd PASTA domain is not present. Whether this deletion alters StkP function due to reduced activation of the kinase by a reduced ability to bind peptidoglycan precursors or by physically reducing the size of the extracellular domain that may reduce the proximity to its substrate would require further study.The kinase is highly conserved within the pneumococcus and shows 99.5% amino acid sequence homology between strains . Here werlrA [The mechanism by which the kinase modulates pilus expression is still unclear but a hypothesis can be formed based on previous literature. The kinase has previously been shown to phosphorylate two pneumococcal response regulators, one being the orphan response regulator (SP_0376) and the other being the response regulator of TCS06 (SP_2193) ,42. RR06rlrA . ThereforlrA –65.In summary this study has shown that the single serine threonine protein kinase regulates the pneumococcal pilus in a serotype 4 and modulates adherence to HBMEC cells. This suggests that the pneumococcal type 1 pilus may play a role in meningitis as well as its already defined role in pneumonia and colonisation ,23,53. TS1 Fignd and 3rd PASTA domain, where the recombination event occurred in a serotype 4 strain (Xen35) containing the stkP allelic variant. Amino acids in red represent the amino acids deleted in the StkP allelic variant. B- shows the StkP amino acid sequence in Xen 35, with the 3rd PASTA domain not present, and in dark blue the remaining 10 amino acid repeat.Diagram shows the amino acid sequence of the extracellular PASTA domains present at the C-terminal end of StkP. A- shows the four extracellular PASTA domains present in TIGR4 StkP. In dark blue are the two 10 amino acid repeats present at the end of the 2(TIF)Click here for additional data file.S2 FigphpP in T4ΔstkP, T4ΔstkP∇ST and T4ΔstkP∇XST compared to TIGR4. Fold change represents that of the mutant strain compared to TIGR4. Each bar represents the average of three replicas and errors bars the standard deviation. B- Shows RT-PCR expression of stkP in T4ΔstkP∇XST compared to T4ΔstkP∇ST. Fold change represents that of T4ΔstkP∇XST compared to T4ΔstkP∇ST. Each bar represents the average of three replicas and errors bars the standard deviation.A- Graph shows RT-PCR expression of (TIF)Click here for additional data file.S3 FigstkP, T4ΔstkPΔrrgB and T4ΔrrgB was assessed to Detroit 562 cell lines. Data is represented as percentage adherence relative to that of TIGR4 . Each bar is an average of at least two replicates and the error bars represent the standard error of the mean. Statistical analysis was performed using a 1-way ANOVA with a Tukeys testing correction, * P<0.01.Adherence of strains TIGR4, T4Δ(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Objective. To investigate the role and possible mechanism of α-Klotho in the calcification and the osteogenic transition of cultured VSMCs. Methods. VSMCs were cultured in vitro and divided into 5 groups, each using a different medium: (1) control; (2) β-GP; (3) β-GP + Klotho; (4) β-GP + LiCl; (5) β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 and β-catenin levels were estimated by western blotting, and the level of α-SMA was determined by using immunofluorescence at day 12. Results. β-GP induced an increase in the expression of BMP2, Runx2, and β-catenin. The calcium content increased, and the expression of α-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl. Conclusions. Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway. Klotho gene knockdown mice show obvious vascular calcification and premature aging [β-glycerophosphate (β-GP) to investigate the role and possible mechanism of Klotho in the calcification and osteogenic transition of cultured rat aortic VSMCs.The Klotho protein was originally identified as an aging suppressor , and it re aging , and there aging . The Klore aging . Altogetre aging , 7. The μg/mL streptomycin and maintained in an incubator at 37°C in a humidified 5% CO2 atmosphere.The A7r5 cell line, originally derived from embryonic rat aorta, was purchased from the American Type Culture Collection . The cells were cultured in Dulbecco modified Eagle's medium (DMEM) containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 β-GP , 50 μg/mL vitamin C, and 1 × 10−7 mol/L insulin for 12 d. The VSMCs were divided into 5 groups: (1) the control group, which was cultured in ordinary medium; (2) the calcification group (β-GP group), which was cultured in medium with 10 mM β-GP added; (3) the Klotho intervention group (β-GP + rmKlotho group), which was cultured in medium with β-GP as in group (2) and different concentrations  of rmKlotho  to choose an appropriate treatment concentration for rmKlotho; (4) the LiCl intervention group (β-GP + LiCl group), which was cultured in medium with β-GP as in group (2) and the Wnt signaling agonist LiCl ; and (5) the Klotho + LiCl intervention group (β-GP + rmKlotho + LiCl group), which was cultured in medium that contained β-GP as in group (2), rmKlotho (50 ng/mL), and LiCl (5 mM). rmKlotho was added at each culture medium replacement.At 70 to 80% confluence, the culture medium was replaced with serum-free medium for 24 h to render the cells quiescent. Calcification was induced by adding medium with 10 Mm After VSMCs were cultured for 12 d, the calcium concentrations of the cell layers were determined by the o-cresolphthalein complexone method and normalized to protein content as previously described . Alizariα-SMA antibody , followed with three PBS washes before using the FITC-conjugated AffiniPure goat anti-rabbit IgG(H+L) antibody  for detection. Labeled cells were examined under a Zeiss confocal microscope, and images were obtained with the laser scanning confocal microscope LEICA TCS SP2 . The images were then quantified by Image J.The cells were washed and fixed with ice-cold paraformaldehyde before blocking with 3% bovine serum albumin (BSA). Samples were then treated at 37°C for 1 h with the rabbit monoclonal anti β-catenin  polyclonal antibodies. After the membranes were washed thrice with Tris buffer containing 0.1% Tween-20 (TBST), they were incubated with goat anti-rabbit IgG-HRP . Bands were visualized with a chemiluminescence system . The bands on the films were quantified using the Quantity One software . The BMP2, Runx2, and β-catenin levels were normalized to the levels of GAPDH as an internal control.After the proteins were isolated from each group of cells, the proteins were transferred to membranes, and the membranes were incubated overnight at 4°C with rabbit anti-rat BMP2 , Runx2 , and t-tests. P < 0.05 indicated a significant difference.Statistical analyses were performed using SPSS software v19.0 . The data were expressed as the means ± standard deviations, and the comparison among groups was performed using β-GP induced VSMC calcification. In the β-GP-treated group, the extracellular matrix contained significant calcification crystallization, and the Alizarin Red S staining was positive for calcium deposits . Furthermore, the levels of β-catenin, BMP2, and Runx2 as well as the calcium content were all increased in the Klotho + LiCl treatment group compared with the Klotho only-treated group (β-GP + Klotho + LiCl group versus β-GP + Klotho group)  was used to treat VSMCs in subsequent experiments. As a result, the expression of α-SMA in the β-GP + Klotho-treated group was significantly increased compared with the β-GP-treated group; however, BMP2 and Runx2 exhibited downregulated expression patterns. These data demonstrate that Klotho inhibits the differentiation of VSMCs into osteoblasts and ameliorates VSMC calcification.In our research, VSMCs were cultured in medium containing 10 mM β-glucuronidase, hydrolyzing steroid β-glucuronides, or represents a novel mechanism for the regulation of the activity of cell-surface glycoproteins [At present, the mechanism of the influence of Klotho on the calcification of VSMCs is unclear. Various studies , 16 haveproteins .β-catenin signaling pathway may increase the expression of BMP2 and stimulate the osteoblast differentiation of VSMCs. Vascular calcification is part of the active process of the transformation of VSMCs into the osteoblasts. In this study, we attempted to investigate the relationship between Klotho and the Wnt/β-catenin signaling pathways. Wnt signaling pathways are divided into classical and nonclassical pathways. Wnt signaling can be highly activated by various mechanisms, and its main function is to inhibit the proteolysis of β-catenin, which is controlled by phosphorylation. Free β-catenin can enter the nucleus and activate the target genes of Wnt. Steady-state levels of Axin are very important, as this scaffolding protein initiates the formation of the β-catenin degradation complex [ in vivo [β-catenin signaling induces BMP expression, whereas BMPs induce Wnt expression [β-GP exposure resulted in calcification and a marked increase in β-catenin (β-GP group), whereas the level of β-catenin significantly decreased and calcification was alleviated following Klotho treatment (β-GP + rmKlotho group). However, the expression of β-catenin was obviously upregulated again after treatment with 5 mM LiCl (β-GP + LiCl group). In addition, our results showed that the effects of Klotho in calcification protection could be reversed after treatment with a Wnt signaling agonist (β-GP + rmKlotho + LiCl group). These data demonstrated that Klotho may attenuate osteoblastic differentiation and the calcification of VSMCs by inhibiting the Wnt/β-catenin signaling pathway.Wnt signaling plays a key role in vascular calcification . The act complex . As a GS complex . Moreove in vivo . Thus, Bpression , suggestpression  involvinβ-catenin-BMP axis of calcification regulation by Klotho. β-GP exposure triggered the Wnt/β-catenin pathway and inhibited the degradation of intracytoplasmic β-catenin. Thus, more free β-catenin can enter the nucleus and activate the BMP genes, which subsequently induces the high expression of BMP2. BMP2 stimulates the transcriptional activation of Runx2. In addition, Klotho downregulates the expression of β-catenin (We proposed a possible Wnt/-catenin  and alsoβ-catenin pathway.Klotho attenuates osteoblastic differentiation and the calcification of VSMCs induced by a high content of phosphorus. The effect of Klotho on calcification may be associated with the classic Wnt/Unfortunately, in this study, we cannot determine the relationship between Klotho and the Wnt receptor. Whether this process relates to the glucuronic acid activity of Klotho requires further study. Moreover, the role of the Wnt signaling pathways is broad and complex. Further research is required to confirm whether Klotho affects other pathological processes through this pathway. The Klotho/Wnt pathway may be a promising new therapeutic target to prevent the calcification of vasculature.


We show that the population of induced mesenchyme cells is significantly reduced in s-/-β-cat mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. Wnt9b, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in s-/-β-cat. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and Wnt9b was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cat Development of the mammalian kidney is dependent upon inductive interactions between epithelial and mesenchymal cells . DevelopThe renal stroma is a population of matrix-producing fibroblast cells , 5]. Du. Du5]. Dβ-catenin is a multi-functional protein involved in cell adhesion and the regulation of gene transcription. In the cell, β-catenin localizes to the cell membrane, cytoplasm, and nucleus where it performs different cellular functions. At the cell membrane β-catenin is involved in the formation, maintenance, and function of adherens junctions by linking cadherins to the actin cytoskeleton . In the β-cats-/-). These mutant mice demonstrate marked kidney abnormalities, most notably reductions in the condensing mesenchyme. Using in situ hybridization and real-time quantitative PCR (qRT-PCR), we demonstrated Wnt9b, the key signal for the induction of nephron progenitors, was markedly reduced in ureteric epithelial cells in β-cats-/-. Consistent with reduced levels of Wnt9b, we demonstrated a significant down regulation of Wnt9b-dependent genes, while Wnt9b-independent genes remained unchanged in nephron progenitors. Moreover mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in induced nephron progenitors and Wnt9b. Together these data support a model in which β-catenin in the renal stroma communicates with the ureteric epithelium to modulate a genetic program that is required for the induction of nephron progenitors during mammalian kidney development.In this study, we investigated the role of stromally expressed β-catenin in kidney development. We show β-catenin expression in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. To understand the significance of stromally expressed β-catenin we generated a conditional knockout mouse in which β-catenin is specifically deleted in the renal stroma cell lineage , and localizes to the membrane, cytoplasm and nucleus . The renAt E13.5, the Pbx1 positive renal stroma cells are located between adjacent condensed mesenchymal populations and also form the renal capsule  Fig. 1DFig. 1D. During our analysis of β-catenin expression, we observed that the intracellular distribution of β-catenin varied between the capsular, cortical, and medullary stromal compartments. In the capsular stroma, the intracellular distribution of β-catenin is primarily in the cytoplasm and membrane and virtually absent from the nucleus , indicatFoxd1-Cre transgenic mouse line [Foxd1-Cre mice with transgenic mice containing LoxP sites flanking exons 2–6 of the β-catenin allele [β-catS-/-). We performed immunofluorescence using Pbx1 and β-catenin antibodies to confirm the absence of β-catenin within all stromal cells in β-catS-/- mutant kidneys  (WT littermates (data not shown). We then performed a histological analysis of β-catS-/- kidneys. In contrast to WT  . One mutst to WT , β-catSed cysts  in the med cysts . In addied cysts  and tubued cysts . β-catSYu et al . Yu et aYu et al . Their sYu et al .β-catS-/- kidneys revealed a paucity of renal capsule and sporadic non-adherent capsular cells  and at E15.5 (WT: 34.09% versus β-catS-/-: 26.73%)  . In conttermates . This daWnt9b expression by section in situ hybridization. We observed a marked reduction in Wnt9b mRNA expression in the majority of ureteric epithelial cells in β-catS-/- kidneys . The histological analysis of β-catGOF-S kidneys revealed a marked increase in the progenitor population at E14.5  [Foxd1-Cre;β-cat+/- males were then crossed with Δ2–6/Δ2–6β-cat to generate homozygous β-catenin loss-of-function mutants in the renal stroma (termed β-catS-/-). β-catenin gain-of-function mice were generated by crossing Foxd1EGFPCre mice with mice containing LoxP site flanking exon 3 of the β-catenin allele (Δ3/Δ3β-cat). Tail genomic DNA was isolated and PCR was used to detect the mutants using Foxd1EGFPCre primers 5’-GCGGCATGGTGCAAGTTGAAT-3’ and 5’-CGTTCACCGGCATCAACGTTT-3’. Primers used to identify the β-catenin mutants are previously described [Foxd1EGFPCre recombinase activity the Foxd1EGFPCre mice were crossed with Gt(ROSA)26Sor(ROSA) mice. All animal studies were performed in accordance with animal care and guidelines put forth by the Canadian Council for Animal Care and McMaster’s Animal Research Ethics Board (AREB)  and approved the project described in this manuscript.–6/Δ2–6)  of the βescribed . To vali20) and stained with hematoxylin and eosin . For immunofluorescence, tissue was prepared as described above and antigen retrieval was performed for 5 minutes in 10mM sodium citrate solution pH 6.0 in a pressure cooker, followed by blocking with serum-free protein block . Sections were incubated with primary antibodies to β-catenin , Pbx1 , Pax2 , Six2 , Cited1 , Foxd1 , Meis1/2 , TN-C , Amph , Ncam , Jag1 , Wt-1 , Cytokeratin , Aquaporin-3 , overnight at 4°C. Tissue sections were washed in PBS pH 7.4, incubated with secondary antibodies Alexafluor 488 or 568  for 1 hour at room temperature and stained with Dapi  for 5 minutes and cover-slipped using Fluoromount  and photographed on a Nikon 90i-eclipse upright microscope.Whole kidney tissue was fixed in 4% paraformaldehyde for 24 hours at 4°C. Kidneys were paraffin-embedded, sectioned to 5μm, and mounted on Superfrost Plus slides  and incubated overnight at 37°C. Sections were deparaffinized using xylene washes and rehydrated using graded ethanol washes , as previously described . PregnanCited1, Six2, Pax2, Wnt9b, Amph, Tafa5, Eya1, Ret, Wnt11 were designed using the Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/) and verified using the UCSC genome bioinformatics website (genome.ucsc.edu). Relative levels of mRNA expression were determined using the 2(-ΔΔCt) method. Individual expression values were normalized by comparison to β-2-microglobin.Real-time PCR was performed using the Applied Biosystems 7900HT fast RT-PCR system . cDNA was generated using first strand cDNA synthesis  from total RNA. Real-time PCR reaction mix contained 2.5ng of each cDNA sample, SYBR green PCR Master Mix  and 300nM of each primer to a total volume of 25 μl. Primers for Wnt9b, Ret, or Wnt11 digoxigenin-labeled (DIG) riboprobes overnight at 68°C. Sections were treated with 2μg/ml RNase for 15 minutes at 37°C and incubated in anti-DIG-AP antibody  at 4°C overnight and incubated with BM purple at room temperature to visualize signals. Sections were fixed in 4% PFA, mounted in glycergel mounting media . In Situ Hybridization for Wnt4 was performed using the Affymetrix QuantiGene ViewRNA assay. Briefly, paraffin blocks were sectioned at a thickness of 5 μm, deparaffinized, boiled in pre-treatment solution  and digested with proteinase K. Sections were incubated with a custom designed QuantiGene ViewRNA Wnt4 probe for 2 hrs at 40°C. Signal was amplified with Pre-Amp and Amp solutions and then developed Fast-Red Substrate. Slides were counterstained with DAPI, mounted with Fluoromount  and photographed on a Nikon 90i-eclipse upright microscope.Frozen blocks were sectioned at a thickness of 20 μm. Non-radioactive In situ hybridization was performed as described  using 1.The qRT-PCR, apoptosis, and proliferation data was analyzed using a two-tailed Student’s t-test using GraphPad Prism software, version 5.0c . P < 0.05 indicates statistical significance.All animal studies were performed in accordance with animal care and institutional guidelines at McMaster University .S1 FigWT kidneys at E15.5. Pbx1 (A) and Meis1/2 (B) mark the capsular (rc), cortical (cs), and medullary storma (ms), whereas TN-C (C) and Foxd1 (D) expression is restricted to the capsular and cortical stroma between the developing nephrons. The insets demonstrate the expression pattern of each marker in the whole kidney .(A-D) Immunofluorescence analysis of Pbx1, Meis1/2, TN-C, and Foxd1 in (TIF)Click here for additional data file.S2 Figβ-catS-/- kidneys. (A-F) At E13.5 in β-catS-/- kidneys, β-catenin is not expressed in Pbx1 positive stromal cells. β-catenin expression is maintained in the ureteric epithelium and mesenchyme populations. (G-R) In β-catS-/- kidneys at PN0 β-catenin is not expressed in capsular, cortical or medullary stroma. β-catenin expression persists in the ureteric epithelium and mesenchyme populations. .(A-R) Pbx1 and β-catenin immunofluorescence demonstrating the loss of β-catenin from stromal cells in (TIF)Click here for additional data file.S3 FigWT and β-catS-/- kidneys at PN0. All cysts observed in β-catS-/- kidneys originated from the ureteric epithelium. No cysts were found in the tubules or loops of Henle.(A-H) Expression analysis of collecting duct markers Aquaporin-3 (A-D) and Cytokeratin (E-H) in (TIF)Click here for additional data file.S4 FigWT and β-catS-/- kidneys. (G-L) Immunofluorescence analysis of nephrogenic markers Ncam , Jag1 , and Wt-1  in WT and β-catS-/- kidneys at E15.5. Despite the reduction in condensing mesenchyme, β-catS-/- kidneys undergo nephrogenesis and form mature nephrons (glomerulus in L).  .(A-F) Whole-mount branching analysis using the ureteric epithelium marker cytokeratin at E13.5 , E15.5 , and PN0 . No overt changes were observed between (TIF)Click here for additional data file.


Vav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein β-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity. The Vav1 proto-oncogene has a restricted hematopoietic expression and exhibits both GTP/GDP exchange activities (GEF) for Rho family GTPases and adaptor functions within signalling complexes . Two othThe initial characterization of Vav1 consisted in a truncated version of the proto-oncogene (wt-Vav1) with deletion of the first 66 residues within the CH domain that resulted in a transforming protein (onco-Vav) when expressed in fibroblasts . FurtherThe structural complexity and high similarities of Vav proteins have indicated both GTPase-dependent and GTPase-independent roles. Truncations of the inhibitory CH or AcR domains resulted in deregulated GEF activity, constitutive activation of Rho proteins and transformation , 20. HowIn this study, we investigated whether alterations within the protein-protein interaction CSH3 domain might activate the transforming properties of the Vav1 proto-oncogene. We identified D797 as one novel key residue controlling Vav1 oncogenicity, since its unique mutation confers transforming properties to Vav1 proto-oncogene. We also identified β-catenin as a new Vav1 interacting partner in fibroblasts and epithelial lung cancer cells. The transformation, which is dependent on Rac activation, is associated with the phosphorylation and the stabilization of β-catenin altogether with its relocation from junctional complexes to random membrane localization in fibroblasts. Our data provide novel insights on the mechanism of Vav1-regulated oncogenesis that involves cell-cell contact dysregulation.The C-terminal SH3 adaptor region of Vav1 contains several conserved residues present in Src, Grb2 or Abl SH3 domains but also unique residues specific for Vav family members Figure . We analNext, we compared the proliferative properties of stable cell lines expressing wt-Vav1, onco-Vav or D797N mutant. Higher proliferation rates and saturating densities were observed for transforming proteins-expressing cells while wt-Vav1-expressing cells exhibited an intermediate growth rate as compared to control with empty vector . Unlike Altogether these data demonstrate that one unique CSH3 specific substitution into Vav1 proto-oncogene confers activation to the protein leading to transformation and tumorigenesis.The transforming activity of all N-terminal deleted Vav oncoproteins has been ascribed to uncontrolled GEF activity on GTPases of the Rho family , 20, 32.To explore downstream events mediating the transforming activity, we investigated the activation of c-jun NH2-terminal (JNK) and of stress-activated protein p38 (SAPK) kinases, two direct effectors of Vav and Rac/Rho GTPases . ImmunobImplication of JNK kinases in onco-Vav- and D797N-mediated transformation was further confirmed using the SP600125 specific inhibitor. As shown in Figure Altogether these results indicate that, similarly to onco-Vav-, D797N-mediated transformation involves the GEF activity toward Rac/Rho GTPases leading to typical cell morphology changes and to the subsequent activation, albeit to a lesser extent, of JNK-dependent pathways.Next, we analysed downstream targets of the Rac/JNK-mediated pathway that has proven relevant for cell-cell contacts and adherens junctions. Both Rac1 and JNK2 are involved in the dynamic regulation between the signalling activity and the structural function of the stable pools of β-catenin , 35-37. Since subcellular localization and phosphorylation on particular residues of β-catenin are crucial events that modulate its different functions, we further investigated whether Vav1 proteins could have an impact on these modifications. Western blot analysis with anti pS675 β-catenin, a residue phosphorylated by the JNK kinase , 40 confSince phosphorylation of S675 and the non-phosphorylated β-catenin have been implicated in the balance between membrane function and nuclear translocation of β-catenin, we sought to verify whether expression of Vav1 mutants could impact β-catenin transcriptional activity. Upon transient transfection of a luciferase reporter construct based on β-catenin-TCF/LEF transcriptional function we did not observe significant differences between the various Vav1 constructs in 293T cells. On the other hand, treatment with LiCl, a potent inhibitor of GSK3β−dependent degradation of β-catenin resulted in a substantial and comparable induction of the transcriptional response in all cases Figure .Next, we evaluated β-catenin distribution at the cell membrane. As expected, endogenous β-catenin was abundantly localized at the cell membrane in control cells with a clustered accumulation at the sites of adhesion and cell-cell contacts. Importantly, expression of Vav1 proteins resulted in a redistribution of β-catenin all along the membrane with a decreased clustering. This peculiar reduction of the clustering of β-catenin was even more pronounced with the two oncogenic Vav1 proteins. Also, we noticed in several cells a significant cytoplasmic accumulation in association with perinuclear labelling Figure . These rThus, these results support an impact of Vav1 expression on certain phosphorylations and on structural function of β-catenin at the cell-cell contacts and adhesive complexes. Notably, a differential phosphorylation by wt-Vav1, and more importantly by the two oncogenic proteins, was observed on residues that are targets of Vav1-dependent pathways.The ectopic expression of Vav1 is observed in numerous non-hematopoietic cancer cells , 13, 41.Next, the impact of these overexpressions on the proliferation rate and the cell morphology were evaluated. As shown in Figure These results demonstrate that the expression of Vav1 contributes in cancer cells to the phosphorylation at specific residues of β-catenin with an advantage for the D797N mutant that demonstrated in fibroblasts higher GEF activity. These modifications of β-catenin participate to the modulation of its function at cell-cell junctions and to the subsequent alteration of cell shape and growth. Moreover, Vav1 interacts also with β-catenin partners in adherens junctions strongly suggesting a key role in cell-cell adhesion.This study highlights the essential role of the adaptor region of Vav1 not only as a scaffold domain for immune receptors signalosomes but also as a tranformation potentiator. Using, in Vav1 proto-oncogene, the unique mutation of an aspartic acid residue, specific for the Vav family of proteins, we show that alteration of the C-terminal SH3 domain activates the transforming capacity of the proto-oncogene. A previous study, addressing the role of the CSH3 domain in the transforming capacity of onco-Vav, showed that the D797N substitution did not abrogate the oncogenicity and was considered as unsignificant compared to mutations on conserved residues among SH3 domains . ImportaThis gain of function mutation occurs on a charged residue that does not belong to the consensus residues required for the structural integrity of the SH3 domains , 42. It One key finding of this work is the identification of a new partner for Vav1, β-catenin. The SH2-domain of Vav1 mediates this interaction likely by recognition of phosphorylated tyrosine residues of β-catenin. Multiple phosphorylated Ser/Thr or Tyr residues of β-catenin are involved in the regulation of the protein. In particular, several sequence motives contribute to conformational changes that lead to signalling functions or recognition by the proteasomal machinery for its degradation . Our resIn other cellular processes, such as osteoblastogenesis, or in colon cancer cells, a Rac1/JNK2-dependent phosphorylation of β-catenin was described at sites that regulate its nuclear accumulation and signalling function . The traIn light with the recent findings on Vav1 as a central regulator of invadopodia , our stuThe pEF-neo expression plasmids encoding Myc-tag human wt-Vav1 has been previously described . PlasmidAntibodies for Myc-Tag, Vav1, JNK, p-JNK, p38, p-p38, lamin A/C, p-βcatenin S33/37/T41, Hsp70, P-c-Jun, c-jun, pY654-β-catenin, pS675- β-catenin, no-P- β-catenin and Alexa 647-conjugated anti-β-catenin were obtained from Cell Signalling Technology, Anti-β-catenin, anti-E-cadherin, EEA1 Abs and anti-IgG control isotype were purchased from Becton Dickinson (BD), anti-α-catenin and anti-Tyr174 Vav1 from Santa-Cruz, and anti-β actin or anti-tubulin from Sigma.NIH3T3 cells were cultured in DMEM supplemented with 10% Newborn Calf Serum . For the focus formation assays, cells were cultured for 12-14 days after transfection with Lipofectamine and Reagent Plus (Invitrogen); medium was replaced every 2 or 3 days and was supplemented or not with JNK (SP600125) or P38 (SB203580) kinases inhibitors . Foci were scored after Giemsa staining. Stable NIH3T3 cell lines expressing the various Vav proteins and vector control were selected in the presence of 1 mg/ml neomycin after transfection. Neomycin-resistant colonies were scored after 10-12 days of selection.H358, H441 and A549 cell lines were maintained in RPMI and DMEM respectively, supplemented with 10% Foetal Bovine Serum . H358 and A549 cell lines were transfected with pcDNA constructs by using the Amaxa or JetPrime (Polyplus) following recommendation.SiRNA control or targeting Vav1 were purchased from Dharmacon and transfected using JetPRIME reagent (Polyplus).NIH3T3 cells were cultured in DMEM-10% NCS containing 0,4% agar over a layer of DMEM-10% NCS containing 0.8% agar during 21 days. Growth medium was added every other week.6 cells). Development of tumours was monitored weekly.Stable NIH3T3 cells lines were injected subcutaneously in Nu/Nu mice (1.105 cells were seeded in 6-well dishes 24h prior to treatment with 100 μM cycloheximide. Cells were harvested at different time points after drug addition (2-8 hours). Stability of the Vav mutant proteins was evaluated by western blot.4.103VO4). Proteins were separated by SDS-PAGE transferred to Hybond membrane (Amersham) and probed with appropriate primary and HRP-conjugated secondary (Sigma) Abs. Quantitation was performed using Image Lab 4.10 (Bio-Rad). For immunoprecipitation, Triton-X100 was replaced by 1% NP-40. After preclearing, cell extracts were incubated overnight with the indicated Abs or agarose beads-conjugated anti-Myc-tag Ab (Sigma). Beads were further washed with the lysis buffer and precipitates were eluted in 4X Laemmli buffer prior to immunoblotting.Protein extracts were prepared in Triton lysis buffer  containing protease inhibitors , 1mM DTT, 25mM β-glycerophosphate, 1mM sodium fluoride and 1mM NaNuclear, membrane and cytoplasmic fractions were prepared using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) as per manufacturer's instruction. Protein concentration was quantitated using the BCA protein assay kit (Pierce).Expression of the Glutathione S-transferase (GST) fusion proteins was induced with 500μM isopropyl-β-thiogalactopyranoside (IPTG). Fusion proteins were purified from bacterial lysates using gluthatione-Sepharose 4B beads . Proteins extracts from NIH3T3 stable cell lines were incubated 2 hours with fusion proteins bound to gluthatione-Sepharose 4B beads. Precipitates were recovered as described for immunoprecipitation assay and analysed by immunoblotting.293T cells were seeded in 12-well dishes 24h prior to transfection using JetPrime (Polyplus) with Vav constructs, Top-firefly or Fop-firefly negative control luciferase plasmids (Millipore) and Renilla luciferase plasmid under the control of SV40 promoter, pRL(Promega). 48h after transfection, cell lysis was performed using the Passive Lysis buffer followed by the Dual luciferase assay (Promega). Firefly and Renilla luciferase activities were monitored following manufacturer's instructions. LiCl (30mM) treatment was applied 3 hours before lysis. Top-firefly luciferase values were normalized to the respective Renilla and Fop-luciferase values for each treatment.Cells were fixed in paraformaldehyde (4%) and permeabilised with 0.25% Triton-X100 in PBS. Cells were stained with the indicated Abs. Actin filaments were visualized with Alexa 546- or Alexa 647-conjugated-phalloidin. Confocal images were acquired under a BD Pathway 855 Bioimaging System with a 40x objectif and further analysed using AttoVision and Image J softwares.Rac1 activation assays were performed using the Active Rac1 Pull-Down and Detection Kit. The assay consists on a GST-pull down experiment with the p21-binding domain of PAK1 that selectively precipitates active Rac1 from whole cell extracts. Active Rac1 is quantified by immunoblot analysis using anti-Rac1 monoclonal Ab (Thermo Scientific Pierce).Statistical significance was assessed using impaired Student's t test . P values <0.05 were considered significant with the following degrees: *p<0.05; **p<0.01; ***p<0.001.