HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the β-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of β-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting β-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase , is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that  Lung cancer is not only the most commonly diagnosed cancer but also the leading cause of cancer-associated death, due to its poor prognosis and active metastasis to other organs after diagnosis . SuppresHOXB9, a class 1 homeobox gene associated with tumorigenesis, metastasis, and poor prognosis [Wnt signaling, one of the most well-characterized signaling pathways in cancer metastasis , is actirognosis , 7, is urognosis . Thereforognosis .The N-acetyl-galactosaminyl-transferases  are responsible for synthesizing the Thomsen-nouvelle antigen (Tn), one of cancer-associated O-glycans , 12, whoHOXB9, the expression of which is critical for the invasive properties of lung adenocarcinoma. In addition, pharmacological inhibition of β-catenin significantly suppressed invasive features, suggesting that treatment of GalNAc-T14-expressing lung cancers with a β-catenin inhibitor would be a valid therapeutic approach to reduce their metastatic potential.Previously, we reported that expression of GalNAc-T14 (or GALNT14) was highly associated with poor recurrence-free survival in a clinicogenomics study with 138 non-small cell lung cancer (NSCLC) patients . HoweverTo investigate the role of GalNAc-T14 in lung cancer metastasis, the GalNAc-T14 expression levels in NSCLC cell lines was first compared. As shown previously , H460 exNext, to examine the molecular mechanism through which GalNAc-T14 controls metastatic potential, a microarray analysis was performed to compare gene expression in the two shGal-H460 cell lines compared to the parental control cells . The comTo identify a signaling pathway governing GalNAc-T14-dependent metastatic potential from among a number of signaling pathways underlying metastasis , 26, theFST), versican (VCAN), amphiregulin (AREG), and homeobox B9 (HOXB9), were found to satisfy these criteria  [HOXB9 expression was most significantly altered in patients with high-grade tumors (GSE31210) and in recurrent lung cancer (GSE8894), respectively. Moreover, HOXB9 was also found to be the most significantly increased gene in a microarray database of PC9-BrM3 cells (GSE14107), which are derived from the PC9 lung adenocarcinoma cell line and form brain metastases with close to 100% efficiency  for cancer cell line and cancer tissue analysis, respectively. Intriguingly, a close positive correlation between HOXB9 and GalNAc-T14 was observed both in 31 NSCLC cell lines (top panel) and in 23 lung adenocarcinoma patients (bottom panel) , and that are downstream of Wnt and involved in metastasis (including invasion and migration), as defined by either gene ontology, gene card analysis, or literature search, were further selected. Only four genes, follistatin  . Interesncy Figs  and S4B.ncy Figs . In a prdel Figs  and S4B,el) Fig. , implyinion Fig. .HOXB9 was the most promising Wnt downstream target to account for the induction of metastatic potential by GalNAc-T14, HOXB9 knockdown cells were next generated , and migration capability with or without GalNAc-T14 expression was monitored. Cell migration , none of the mice that received either the shGal#3 or the shHOXB9 cell line developed tumors . TCGA data was generated with about 500 lung adenocarcinoma samples using RNA-Seq. In the data set, a considerable number of the cancer samples showed highly increased expression of GalNAc-T14 or HOXB9 compared to the normal samples, but there were no differences between normal and tumor samples in the expression of the other three candidate genes, FST, VCAN, and AREG  and recurrence  . Taken together, expression level of GalNAc-T14 and HOXB9 are each associated with the risk of poor overall survival and high recurrence in lung adenocarcinoma patients.To investigate the prognostic significance of GalNAc-T14 and and AREG . For surles Figs  and 6B. les Figs  or HOXB9Pharmacological approaches targeting Wnt or β-catenin signaling are being extensively studied for the inhibition of tumor growth and metastasis –38. A feHOXB9 expression. Therefore, pharmacological inhibition of β-catenin, in order to block transcriptional factor complex formation, significantly attenuated the metastatic properties of GalNAc-T14-expressing cells -related apoptosis-inducing ligand (TRAIL) by increasing GalNAc modification of the death receptor (DR), which leads to an increase in death complex formation . As the HOXB9 in patients with lung adenocarcinoma. In a large data set (461 cases for the TCGA), GalNAc-T14 and HOXB9 expression was apparently associated with an increased risk of poor overall survival and high recurrence in lung adenocarcinoma patients, indicating a possible clinical relevance for their involvement in metastasis.We also assessed the prognostic significance of GalNAc-T14 and HOXB9 expression, which was critical for increased metastatic potential in lung adenocarcinoma. Thus, targeting β-catenin with a small molecule in GalNAc-T14- and HOXB9-expressing lung cancers may be an effective therapy to suppress metastatic properties.Taken together, we showed evidence that GalNAc-T14 expression is responsible for Wnt/β-catenin-dependent The primary antibody against GalNAcT-14 (16939–1-AP) was acquired from proteintech. The primary antibody against ERK2 (Sc-154), PARP (Sc-7150) and b-actin (Sc-47778) were obtained from Santa Cruz Biotechnology Inc. The primary antibody against total form of b-catenin (Cat.610153) and Active β-catenin (ABC) (Cat.05–665) was purchased from BD Biosciences pharmingen and millipore respectively. ICG-001  was purchased from Selleckchem.Total RNA was extracted with Trizol (Invitrogen) and the synthesis of target cRNA probes and hybridization were performed using Agilent's Low RNA Input Linear Amplification kit  according to the manufacturer's instructions. The fragmented cRNA directly pipetted onto assembled Agilent's Human Oligo Microarray (44K). The arrays hybridized as the manufacturer's protocol. The hybridized images were scanned using Agilent's DNA microarray scanner and quantified with Feature Extraction Software .http://imagej.nih.gov/ij/).For wound healing assay, after cells were packed in the 6well plate, media was changed without FBS for 24 hrs. A scratch was made on a uniform layer of cells using sterile micropipette tip. Cell migration was monitored daily using microscope and live image were taken by using Incucyte cell live image system . For migration assay, coverslip on which cells were plated compactly was transfer to empty 6well plate as previously described . After 4http://cancergenome.nih.gov/) database and on-line survival analysis , then the normalized read counts were converted to log2 RPKM (Reads Per Kilobase of transcript per Million mapped reads) values [survival package.Survival analyses were performed with TCGA . TCGA i) values , 46. Kap6) were injected into lateral tail vein of male BALB/C nude mice. 4 weeks after injection tumor mass were harvested, fixed with 4% paraformaldehyde for 1 week, embedded in paraffin and stained with hematoxylin and eosin (H&E).shCont, shGal#3 and shHOXB9 cells , p < 0.01 (**), p < 0.001 (***).Graphical data were presented as mean ± S.D. Statistical significance among three groups and between groups were determined using one- or two-way analysis of variance (ANOVA) following Bonferroni multiple comparison pos


The CAV1-β–catenin axis is mediated by a feedback loop in which β-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of β-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of β-catenin independent of the PI3K–AKT–GSK3β axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent β-catenin transcriptional modulation  The mechanism by which PTEN mutation is melanomagenic is complicated by different PTEN functions in different cellular locations. Here, the authors identify an alternative to membrane PI3K–AKT signalling, a caveolin-1-dependent PTEN pathway that induces nuclear localization and activation of β-catenin. INK4A is critical for this process and its expression is induced by the histone demethylase JMJD3 (ref. p16INK4A gene or suppression of its transcription by nuclear β-catenin235PTEN) loss is frequent in various cancers. Mutational inactivation and/or deletion of PTEN is found in about 20% of uncultured primary and metastatic melanomas7897PTEN loss in nevi is rare, that is, 2 out of 39  signalling promotes proliferation of melanocytes, but this is effectively blunted by the induction of cellular growth arrest known as oncogene-induced senescence (OIS)12PTEN may occur at different stages of melanomagenesis, but remains controversial for its role in senescence. On one hand, the acute loss of PTEN and APC/FZR1 induces senescence in mouse primary fibroblastsPTEN failed to induce a robust growth arrest in human IMR90 fibroblastsV600E-mutated melanocytes, reducing PTEN expression was sufficient to bypass senescenceBRAF mutation after birth induces nevi formation and melanomas arise harbouring deletion of p16INK4A or PTEN1in vivo. PTEN has different functions depending on its subcellular localizationEpigenetic inactivation or loss of INK4A. The co-occurrence of NRASG183T mutation and PTEN loss was detected in a fraction of human melanoma biopsies, suggesting a non-epistatic mechanism. Indeed, in a mouse melanoma model, hemizygous PTEN loss synergized with NRAS mutation and led to bypass of senescence. Thus, we have identified a novel CAV1-dependent pathway by which PTEN affects β-catenin activity and mediates melanomagenesis.In this study, we uncovered a signalling mechanism by which PTEN affects nuclear localization and transcriptional activity of β-catenin through a reciprocal interplay with CAV1. We discovered that the lack of PTEN, through CAV1, induces β-catenin transactivation, leading to the repression of p16null human cells (Hs944T)  . In non-(Hs944T) . In addi(Hs944T) . Conversanalysis , resulteanalysis . These ranalysis . One posanalysis . Re-expranalysis  and induanalysis . Furtheranalysis . Lastly,analysis . AltogetAccording to ingenuity pathway analysis, PTEN and β-catenin share 63 interactors , includi192223S-transferase-β (GST-β)–catenin fusion protein, we precipitated CAV1 on Hs944T cells transfected with GFP and, to a lesser extent, in cells expressing exogenous PTEN  PTEN-null cell line  β-catenin targets.We first assessed whether PTEN and CAV1 expression affects the transcriptional activity of β-catenin, using the TOP flash reporter assay in Hs944T cells. TOP flash activity is significantly reduced in the presence of PTEN and conversely induced with CAV1 (or β-catenin as positive control) . Co-exprell line . The traand BCAT . Owing tactivity . Overexpactivity . PTEN ovreporter . Howevere levels . We alsoe levels . We thening CAV1 . Finallye tumour . In concQ61K/R) and the lack of PTEN are found in ∼20% and 30% of human melanoma, respectively, and it has generally been assumed that they are mutually exclusivePTEN loss and for the presence or the absence of point mutations affecting NRAS. NRASG183T mutation, resulting in an amino-acid change Q61K, was found in 16 samples (15%), of which 2 also showed homozygous PTEN loss , Tyr::NRASQ61K/°;Tyr::Cre/° (NRAS) and Tyr::NRASQ61K/°;Tyr::Cre/°;PTENf/+ (NRAS-ΔPTEN). None of the ΔPTEN mice developed melanoma during 2 years of follow-up observation  and a shorter latency (27±13 weeks) than the NRAS mice. Most melanomas appeared in the hairy part of the skin for both genotypes  and pGSK3β (Ser9) was dramatically increased in NRAS-ΔPTEN compared with NRAS; however, most interestingly, the levels of pS6 remained unaffected. These results suggest that signalling via mammalian target of rapamycin-associated proteins was the same in NRAS and NRAS-ΔPTEN melanomas.To understand the molecular mechanisms underlying the differences between elanomas  and 5i,jenotypes . PTEN waINK4A. In agreement with our results with melanoma cell lines, the level of pSer33/37-Thr41 β-catenin, targeted for proteasomal degradation, is largely unaltered in NRAS-ΔPTEN compared with NRAS. Conversely, the amount of total and pSer675 β-catenin, corresponding to the transcriptionally active form of β-catenin, is significantly higher in NRAS-ΔPTEN compared with NRAS. Consequently, we reproducibly observed a reduction of p16INK4A protein in NRAS-ΔPTEN as compared with NRAS.We proceeded to evaluate the amount of β-catenin and p16Tyr::Cre/°; PTENf/f (Hom), Tyr::Cre/°; PTENf/+ (Het) and Tyr::Cre/°; PTEN+/+ (WT) mice. No obvious difference was observed between Het and WT melanocytes. The initial rates of growth of the Het and Hom melanocytes in vitro were indistinguishable, confirming that the absence of PTEN does not induce proliferation before transformation  of Hom newborn pup skins, but only from 28% (2 of 7) of their Het littermates, implying that the absence of PTEN from melanocytes increased the efficiency of immortalization. We confirmed that by modulating PTEN levels we affected p16INK4A expression in primary normal human epithelial melanocytes as well as in transformed Lyse human melanoma cells . ECAD can be internalized using caveolaeNRAS-ΔPTEN tumours compared with NRAS  and non-epithelial (melanocyte) cells, and appear to be independent of the RAS-BRAF context.In this study, we demonstrate the existence of a complex signalling network involving reciprocal interactions among PTEN, CAV1 and β-catenin; regulating molecular and cellular mechanisms that play a critical role in tumour initiation and progression. PTEN is classically known to inhibit the PI3K/AKT signalling axis, but here we show that it also remarkably controls the nuclear levels and transcriptional activity of β-catenin in an alternative PI3K/AKT way. β-Catenin transcriptional activity represses p16Q61K-induced senescence, was modelled in a mouse model, relevant for human melanomagenesis. In fact, based on a small melanoma series, it was generally assumed that NRAS mutations and PTEN loss are mutually exclusive events in human melanomagenesis; however, we showed that these two events co-exist in a fraction of human melanomas (5 out of 206 melanoma from 2 independent cohorts), as it was recently showed in one caseQ61K mutations. Our study was limited to the PTEN loss-mediated bypass of OIS on the NRASQ61K background, but the PTEN/CAV1/β-catenin/p16INK4A pathway may hold true in BRAFV600E melanomas as well (V600E (or HRASV12G)-induced senescence15Our work identifies a novel mechanism by which a subset of melanomas can escape OIS and result in aggressive tumours. This mechanism by which loss of PTEN induces bypass of senescence, allowing an earlier melanoma initiation with a higher penetrance after oncogenic NRAS as well . MoreoveINK4A proteins. In our models, oncogenic NRAS with or without PTEN loss did not affect neither P53, MDM2 nor MDM4 expression levels (data not shown). This supports a p53-independent model of senescence in melanoma cells in the NRASQ61K background, in which β-catenin-regulated expression of senescence-inducing p16INK4A is directly affected by the absence of PTEN6313334353637Bypassing senescence is classically associated with p53/MDM and Rb/p16Q61K PTEN-null cells, p16INK4A is repressed through CAV1/β-catenin; this interaction is ablated on PTEN re-expression. Thus, CAV1 would serve as a promoter of tumour initiation and progression by enhancing β-catenin-related transcription.The role of CAV1 in tumorigenesis is subject of debate393921NRAS-ΔPTEN murine melanoma tumours, western blot analysis revealed that the levels of CAV1, β-catenin and ECAD were higher than in NRAS tumours. Moreover, it appears that ECAD is more abundant in the cytoplasm of NRAS-ΔPTEN melanoma cells than in NRAS cells, and less at the cell–cell contact  of CAV1high PTENlow or the inverse CAV1low PTENhigh in 12 of 50 samples. Other combinations were found in the remaining samples, indicating a high level of molecular and clinico-pathological complexity that indicates that other mechanisms of OIS escape exist in human melanomagenesis.Whereas melanoma cell lines clearly demonstrated the causal relationship between PTEN, CAV1, β-catenin and p16of cases . In factINK4A repression is supported by our findings in cell culture, mouse models and human samples, the role of PTEN in senescence bypass is intricate and most likely context dependent. Be as it may, our studies indicate that CAV1 and CAV1-related pathways may be a potential therapeutic target for melanoma treatment. On the other hand, our findings predict that PI3K/AKT inhibitors will not block effectively the mechanism of senescence bypass caused by PTEN loss.Thus, although the validity of our model of PTEN/CAV1/β-catenin-regulated p162. Following this incubation, the dermis and epidermis were separated using forceps and the epidermis was washed in wash buffer . After washing, it was centrifuged for 5 min at 1,100 r.p.m. at room temperature. The resulting cell pellet was resuspended in dissociation buffer (GIBCO) and incubated at 37 °C and 5% CO2 for 10 min in a petri dish. Next, the cells were put through 18- and 20-g needles and washed in a 15-ml tube with wash buffer for 10 min, allowing for the removal of grease and hairs. The supernatant was centrifuged for 5 min at 1,100 r.p.m. at room temperature (RT) and the resulting cell pellet was resuspended in PBS and counted. The cells were then centrifuged again for 5 min at 1,100 r.p.m. (room temperature) and plated in tissue-culture dishes in F12 media supplemented with 10% FCS and 200 nM 12-O-tetradecanoylphorbol-13-acetate.Mouse primary melanocyte cell lines were established from mice 1–5 days after birth. Mice were rinsed with 70% ethanol and then in ice-cold PBS. The skin was removed and stored in PBS. Next, the skin was cut into small pieces and incubated with collagenases type 1 and 4 for 40 min at 37 °C and 5% COL-glutamine . Normal human epidermal melanocytes were obtained from Promocell, grown and transfected according to the manufacturer's instructions (OZ Biosciences).Human melanoma cell lines were obtained from Ruth Halaban , Sylvie Robine, Florence Faure and Alain Mauviel (Institut Curie), and were previously published by the laboratory4344454647PTEN-GFP WT, PTEN-C124S-GFP, PTEN-G129E-GFP and PTEN-Y138L-GFP expression vectors were obtainedWhole-cell lysate was prepared from human melanoma cell lines using RIPA buffer and whole-tissue lysate was prepared from mouse melanoma tumour using SDS lysis buffer5 cells were seeded in 18mm glass cover slips and allowed 24 h to recover prior immunofluorescence analysis. Similar procedure was followed for CSG, KPC1 and KCPTEN2 cells. Human Hs944T melanoma cells were transfected with CMV::PTEN-GFP (#1031) and allowed 48 h to recover before being fixed in 4% paraformaldehyde (PFA) for 20 min at RT. Human or mouse cells were permeabilized with 0.2% v/v PBS/Triton X-100 for 5 min at RT. Then, cells were washed twice with PBS and blocked with 1% BSA (w/v) and 10% fetal bovine serum in PBS for 20 min at RT. Cells were incubated with the primary antibody anti-β-catenin (dilution 1/100) at 4 °C overnight. Alexa 555 anti-rabbit (Sigma) secondary antibody was incubated for 1 h at RT in the dark. Cells were counterstained with 0.5 μg μl−1 4,6-diamidino-2-phenylindole to visualize the nucleus.Primary murine melanocytes were grown to near confluence upon which point were counted, 2.5 10f/f, Tyr::Cre/°; PTENf/+ and Tyr::Cre/°;PTEN+/+ mice were crossed with Dct::LacZ miceD-galactoside, as previously describedTyr::Cre/°;PTEND-glucopyranoside , 1 × protease and phosphatase inhibitors) and were incubated on ice for 30 min. Next, the lysates were centrifuged at 13,000 r.p.m. for 10 min at 4 °C. Following centrifugation, the supernatant (extract) was removed and used for IP and the pellet discarded. Fifty microlitres of the extract was kept for a total cell lysate (Input). For each IP, 1 ml extract (corresponding to ∼1.5 × 107 (Rosi) and 2 × 107 (HCT116) cells, respectively) was used. Extracts were pre-cleared for 2 h at 4 °C (with rotation) by incubation with 150 μl of 1:1 PBS:protein-G sepharose beads . After pre-clearing, the lysates were centrifuged at 10,000 r.p.m. for 10 min at 4 °C. The supernatants were transferred to new tubes and pre-incubated (with rotation) with anti-β-catenin , anti-CAV1 , anti-PTEN  or control IgG  antibodies at 4 °C for 3 h . Following pre-incubation, 150 μl PBS:protein G sepharose (2:1) was added to each antibody-extract mixture and left to rotate at 4 °C overnight.Co-IP experiments were performed as previously describedThe next morning, the immune complexes were collected by centrifugation at 14,000 r.p.m. for 10 min at 4 °C. The IP supernatants were removed and the beads were washed four times with IP buffer, with centrifugation at 10,000 r.p.m. for 10 s between each wash. The beads were then washed four times with EDTA buffer (50 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100). Following the last wash, the immunoprecipitated proteins were solubilized in SDS sample buffer and boiled for 10 min. Samples were then resolved by SDS–PAGE. Trueblot anti-rabbit IgG HRP  was used as a secondary antibody for western blotting in Quantification of all images was performed using the ImageJ imaging software. Briefly, using the Threshold function, all images were equally calibrated to either control GFP or si Scramble. Once a black and white image was obtained after processing through the Threshold function, a merge with 4,6-diamidino-2-phenylindole and other fluorescent channels  was generated, after which point we manually counted only the cells that were positive for GFP or red fluorescent protein (depending on the construct) and displayed a nuclear signal.2, 1 mM dithiothreitol, 0.2% Nonidet P-40, 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail), while rotating on a rocker rotator at 4 °C for 15 min. The cells were then centrifuged at 14,000 r.p.m. at 4 °C for 5 min and the resulting supernatant (cytoplasmic fraction) was collected. The pellet was resuspended in nuclear extraction buffer  and incubated at room temperature for 10 min, after which it was centrifuged at 14,000 r.p.m. at 4 °C for 5 min. The resultant supernatant (nuclear fraction) was removed from the pellet (cytoskeleton) and the purity of each fraction was assessed by immunoblotting with antibodies to α-tubulin and lamin B1, respectively.Hs944T cells transfected with complementary DNAs encoding either GFP or PTEN were separated into nuclear and cytoplasmic fractions. Cells were lysed with cytoplasmic extraction buffer . Samples were resolved in 12% SDS–PAGE gel.Five hundred micrograms of whole-cell lysates were incubated with 20 μl of β-catenin–GST beads in PBS supplemented with Protease inhibitors for 3 h at 4 °C under slight agitation. Beads were then collected by centrifugation at 10,000 r.p.m. for 10 min at 4 °C and washed 4 times with 10 mM Tris (pH8), 150 mM NaCl, 1%(v/v) Triton X-100, 60 nM Octyl β-Renilla luciferaseRenilla activity. Briefly, cells were transfected with 750 ng of either CMV::CAV1-GFP (#1040), CMV::PTEN-GFPCMV::β-cateninCMV::GFP (1085) and 350 ng of CDKN2A::Luciferase or MITF::LuciferaseRenilla luciferase construct as a control. Luciferase activity was determined 48 h post transfection and normalized to Renilla luciferase. Statistical analysis was performed using Prism v5.0.Cells were transiently transfected in 12-well plates with Magnetofectamine (OZ Biosciences) following the manufacturer's specifications. Briefly, cells were transfected with 1.25 μg of total plasmid DNA. As control cells were also co-transfected with thymidine kinase::Caveolin 1 (39) β-catenin (35) were purchased from Dharmacon as a SMART pool mix of four sequences and PTEN (38) from Santa Cruz Biotechnologies as a mix of three sequences. Caveolin-1: 5′-GCA AAU ACG UAG ACU CGG A-3′, 5′-AUU AAG AGC UUC CUG AUU G-3′, 5′-GCA GUU GUA CCA UGC AUU A-3′ and 5′-CUA AAC ACC UCA ACG AUG A-3′. β-Catenin: 5′-GAA CGC AGC AGC AGU UUG U-3′, 5′-CAG CUG GCC UGG UUU GAU A-3′, 5′-GCA AGU AGC UGA UAU UGA C-3′ and 5′-GAU CUU AGC UUA UGG CAA U-3′. si Scrambled, with no known human targets, was purchased from Invitrogen as a mix. Briefly, cells were transfected with 50 pmol of siPTEN, siCAV1, siβ-catenin or siScrambled (siScr) and were assayed for Luciferase activity or protein content 48 h post transfection.siRNA targeting human www.federa.org/codes-conduct) and approved by the institutional review board of the Radboud University Nijmegen Medical Center. Pathological and genomic data were obtained from paraffin-embedded tumour tissue.The first set of anonymized human melanoma samples was genomically profiledStaining for p16 and CAV1 expression was performed on a Discovery XT Autostainer . All solutions used for automated immunohistochemistry were from Ventana Medical System, unless otherwise specified. Tissue section (4 μm) underwent de-paraffinization with the EZ PREP solution, heat-induced epitope retrieval with Cell Conditioning solution CC1, pH 8.0, at standard condition (60 min at 95 °C). Pre-diluted mouse monoclonal anti-p16  or polyclonal anti-CAV1  diluted in antibody diluent were incubated for 32 min at 37 °C, then followed by the detection kit  and developed with 3,3′-Diaminobenzidine (DAB). A negative control was performed by the omission of the primary antibody. Slides were counterstained with haematoxylin for 4 min , blued with Bluing Reagent for 4 min , removed from the autostainer, washed in warm soapy water, dehydrated through graded alcohols, cleared in xylene and mounted with Permount. Immunostaining for PTEN was performed manually. Following de-waxing and rehydration through xylene and graded alcohol baths, quenching of endogenous peroxidase , a microwave antigen-retrieval step was performed . Each section was incubated with mouse monoclonal anti-PTEN antibody  overnight at 4 °C. Following three 10-min washes with PBS, tissues were incubated with powervision poly-HRP-GAM/R/R IgG (Immunologic) and immunoreactivity visualized with PowerDAB (Immunologic), counterstained with haematoxylin and mounted with Permount. Slides were scanned with a ScanScope AT Turbo scanner  at × 20 magnification. Vascular endothelium served as an internal positive control for PTEN expression and breast carcinoma as an external positive control. Cytoplasmic and nuclear PTEN and p16 expression was scored as positive immunostaining. CAV1 staining included membranous and/or cytoplasmic expression. Two events occur independently when the % PTEN negative (36/(101–3)) times the % NRAS mutant (11/(101–3)) equals to the % PTEN negative and % NRAS mutant (3/101).NRAS gene was performed using a 5′ WT PCR primer  and a mutated PCR primer corresponding to the NRASQ61K mutation . The reverse primer  was used for all the PCR reactionsNRAS gene in DNA extracted from tumours was also sequenced following PCR amplification. The primer sequences were 5′-GTT ATA GAT GGT GAA ACC TG-3′  and 5′-GAG GTT AAT ATC CGC AAA TGA CTT-3′ . The NRAS gene exon 3 sequences were analysed by direct DNA sequencing according to the Sanger technology.DNA was extracted from 20-μm-thick paraffin-embedded sections using NucleoSpin Extract II  according to the manufacturer's instructions and was amplified by PCR. Allele-specific PCR of the Tyr::N-RASQ61K/° mouse line was described previously61Q61K/° to generate Tyr::NRASQ61K/°;Tyr::Cre; PTENf/+ (NRAS-ΔPTEN mice), Tyr::NRASQ61K/°; PTENf/+ (NRAS mice) and Tyr::NRASQ61K/°;PTENf/f (NRAS mice). Mice were genotyped by PCR using DNA extracted from tails. The mice were evaluated weekly for tumour appearance and progression. Once tumours were 1 cm across, the mice were killed and autopsied. Some mice were also killed because of poor health. Tumour samples were fixed in 4% PFA and paraffin-embedded for histological analysis and immunostaining. When sufficient tumour tissue was available, samples were frozen for subsequent western blot analysis.The transgenic Mouse melanomas were collected, rinsed in cold PBS and fixed in 4% PFA at 4 °C O/N. Samples were dehydrated, embedded in paraffin wax and sectioned into 5-μm-thick transverse sections. Paraffin-embedded sections were stained with haematoxylin and eosin, and examined by light microscopy. For immunostaining, sections were deparaffinized, rinsed in Tween Buffer saline , boiled for 20 min in 10 mM sodium citrate and treated overnight at 4 °C in TBST (TBS/0.1% Tween-20) containing 5% normal goat serum with antibodies against S100 . AEC  was used to reveal bound antibody according to the manufacturer's instructions. All sections were counterstained with haematoxylin. Ki-67  and gp84 (ECAD antibody) antibodies were both produced in rabbit.Cells were transfected with 100 nM of miRNA mimics, hsa-miR-203  and hsa-miR-199a-5p . Forty-eight hours post transfection, cells were lysed for mRNA and protein contents, and analysed.How to cite this article: Conde-Perez, A. et al. A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity. Nat. Commun. 6:8093 doi: 10.1038/ncomms9093 (2015).Supplementary Figures 1-9 and Supplementary Table 1.CTNNB1 interactomePTEN interactomeCommon list for PTEN and CTNNB1NRAS and NRAS-ΔPTEN tumorsmiRnome of murine


Ctnnb1 mutant without the transactivation domain was unable to rescue presynaptic deficits of Ctnnb1 mutation, indicating the involvement of transcription regulation. On the other hand, the cell-adhesion function of Ctnnb1 is dispensable. We screened for proteins that may serve as a Ctnnb1-directed retrograde factor and identified Slit2. Transgenic expression of Slit2 specifically in the muscle was able to diminish presynaptic deficits by Ctnnb1 mutation in mice. Slit2 immobilized on beads was able to induce synaptophysin puncta in axons of spinal cord explants. Together, these observations suggest that Slit2 serves as a factor utilized by muscle Ctnnb1 to direct presynaptic differentiation.Neuromuscular junction formation requires proper interaction between motoneurons and muscle cells. β-Catenin (Ctnnb1) in muscle is critical for motoneuron differentiation; however, little is known about the relevant retrograde signal. In this paper, we dissected which functions of muscle Ctnnb1 are critical by an in vivo transgenic approach. We show that DOI:http://dx.doi.org/10.7554/eLife.07266.001 Motor nerves are like electrical wires that connect our spinal cord to the muscles in our body. These nerves communicate with muscles across a connection called the neuromuscular junction. To first form a neuromuscular junction, the motor nerves and muscles each produce molecular cues that tell each other to do their part to build a connection. Beta-catenin in the muscle is known to regulate motor nerve development. However, beta-catenin has two different roles: it helps to coordinate whether neighboring cells stick together, and it can regulate which genes are ‘transcribed’ to produce proteins. It was not known which of these roles is necessary for forming neuromuscular junctions.Wu, Barik et al. now investigate this question by creating mice with mutant forms of beta-catenin in their muscles. Some mice had muscle beta-catenin that could not help cells stick together, and others had beta-catenin that could not control gene transcription. Only mutations that affected the ability of beta-catenin to control transcription caused abnormalities in the neuromuscular junction. However, these problems could be fixed by adding either normal beta-catenin or the mutant form that cannot help cells stick together.Wu, Barik et al. then used molecular tools to explore which genes are turned on by beta-catenin. The experiments showed that beta-catenin causes muscle fibers to produce a protein called Slit2—a developmental cue that controls where neurons grow. Furthermore, the neuromuscular junction defects found in mice without beta-catenin in their muscles could be reduced by making the muscle fibers produce more Slit2. However, not all defects in beta-catenin mutant mice are rescued by Slit2. Future research is needed to identify other beta-catenin-controlled signals and to determine whether such a pathway is altered in neuromuscular disorders.DOI:http://dx.doi.org/10.7554/eLife.07266.002 The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells to control muscle contraction. It exhibits a high degree of subcellular specialization characteristic of chemical synapses and has served as a model to study synaptogenesis . AbnormaCaenorhabditis elegans, Drosophila as well as vertebrates  to impair the cell-adhesion function and TAD-deleted Ctnnb1 (LSL-Ctnnb1ΔTAD) to eliminate the transcriptional regulation, as well as wild-type LSL-Ctnnb1. Each transgene was placed downstream of a loxP-flanked transcriptional STOP (LSL) and contained the lacZ/neomycin fusion gene and triple repeats of the SV40 polyadenylation signal   gene , 2012b. nal cord  (data nob1fl/fl)  (data nob1fl/fl)  (data noCtnnb1fl/+ mice (ACTA1-Ctnnb1fl/+) to determine whether and which transgene was able to rescue NMJ deficits by Ctnnb1 mutation. Notice that both the expression of wildtype or mutant Ctnnb1 and the deletion of endogenous Ctnnb1 depend on the ACTA1 promoter. In control mice (Ctnnb1fl/fl), primary branches of the phrenic nerve were located in the middle of muscle fibers. The secondary branches were numerous and short, many of which end with AChR clusters that were confined to the central region of muscle fibers  mutants, the primary branches were mislocated to the tendon region close to the central cavity, the secondary branches were fewer and longer, and the region where AChR cluster distributed became wider , in agreement with previous report    . These r n = 17) , demonst-AA mice . As repo-AA mice , Ctnnb1 rameters . These rCtnnb1−/− mice, compared with control mice , but no change in mEPP amplitude  , events that are generated by spontaneous vesicle release. There was about 50% reduction in mEPP frequency in ACTA1-rol mice   . In contTAD mice . TogetheAxin2-nlacZ mice, where the nuclear-localized β-galactosidase (nlacZ) DNA was inserted in the Axin2 gene, a target of Wnt/Ctnnb1 signaling. Expression of β-galactosidase in Axin2-nlacZ mice is controlled, which has been used as a reporter of Wnt or Ctnnb1 activity  to screen for genes that are down-regulated in ACTA1-tal cues . C-Myc ay Ctnnb1 , were use method . In a pa/fl mice . Of partt family . Slit2 at family . Intereslocation , akin to muscles . The redogenates .10.7554/Ctnnb1 and resulting myotubes were subjected to Western blotting for endogenous Slit2. As shown in Muscle homogenates contain, in addition to muscle, nerve terminals, Schwann cells, and blood vessels and connective tissues where Slit2 may be expressed . To demoTcf4/Lef1 transcription factors , although the primers were able to detect a band in input, suggesting that the Tcf4 and Lef1 sites in this region may not be able to bind to Ctnnb1. Together, these results suggest that Ctnnb1 binds to the promoter region of the Slit2 gene to promote its expression in muscle cells.Ctnnb1 regulates gene expression by binding to the  factors . We idenP-MAPPER . There wion site . To deteion site . As showSlit2 is a target of Ctnnb1-regulated transcription in muscles, we wanted to determine if overexpressing Slit2 in muscles was able to rescue presynaptic deficits in ACTA1-Ctnnb1−/− mice. It should if the hypothesis is correct. To this end, we generated a transgenic mouse, ACTA1-Slit2, which expressed Flag-Slit2 under the control of the ACTA1 promoter   enlarges AChR clusters and increased the central band area where AChR clusters are distributed  , reducedtaining) , and redtaining) . While taffected .These remplitude    with ACTA1-Cre transgenic mice that were generously provided by Dr. Melki, to generate the ACTA1-Ctnnb1−/− mice which lacked Ctnnb1 in skeletal muscles. For the rescue experiments, three loxP-STOP-loxP (LSL)-Ctnnb1 transgenic mice  were generated. Ctnnb1TV-AA and Ctnnb1∆TAD (lacking amino acids 684–781) were generated using PCR. The HA tag at the C-terminus was added by subcloning the transgenes into the SalI and XbaI sites of pKH3. HA-tagged wild type and mutants were subcloned into the XhoI and NotI sites of pCCALL2 (loxP-STOP-loxP) vector to generate LSL-Ctnnb1 or mutant mice. LSL-transgenic mice were crossed with Ctnnb1fl/+ to generate Ctnnb1fl/+;LSL-Ctnnb1 or mutant mice. To generate ACTA1-Slit2 mice, Flag-Slit2 was subcloned into pBSX-ACTA1 as described previously (Axin2-nlacZ mice were obtained from Jackson laboratory (stock #:009120). DNA sequences of all transgenes were validated by sequencing. Linearized DNA was microinjected into FVB blastocysts, which were introduced into pseudo-pregnant recipient females. Mice were genotyped by PCR analysis of tail biopsy DNA. Tail DNA was digested with buffer A ) for 45 min at 100°C and neutralized with buffer B  for genotyping. Mice were housed in cages that were maintained in a room with 12-hr light/dark cycle with ad libitum access to water and rodent chow diet . P0 pups of either sex were analyzed, unless otherwise indicated. ‘Material and method’ have been approved by the Institutional Animal Care and Use Committee (IACUC) of Georgia Regents University.eviously . Axin2-nMuscles were lysed in modified RIPA buffer , 2% deoxycholate, 1 mM PMSF (phenylmethanesulfonylfluoride), 1 mM EDTA, 5 mM sodium fluoride, 2 mM sodium orthovanadate, and protease inhibitors including 1 mM phenylmethylsulfonyl fluoride, 1 μg/μl pepstatin, 1 μg/μl leupeptin, and 2 μg/ml aprotinin). After centrifuging at 10,000 RPM at 4°C, supernatant was designated as lysates. Protein concentration was measured using Pierce BCA kit. Samples (50 μg of protein) were resolved by SDS-PAGE and transferred to nitrocellulose membrane, which was incubated in 5% milk in phosphate buffer saline (PBS)-0.3%Tween20 overnight at 4°C and then with primary antibodies in 2% milk in PBS-Tween buffer: Ctnnb1  ; Slit2  ; HA  ; Flag ; GFP ; Cre ; β-actin . After washing, the membrane was incubated with PBS-Tween buffer containing HRP-conjugated goat anti-mouse and rabbit IgG from Pierce . Immunoreactive bands were visualized by using enhanced chemiluminescence . Quantitative densitometry of captured images was analyzed with Image J (NIH), as described before .Diaphragms were stained whole mount as previously described with modification , 2012b. Electron microscopy was performed as described previously . Entire 2/5% CO2) Ringer's solution  and pinned on Sylgard gel in a dish perfused with oxygenated Ringer's solution. To measure mEPP, microelectrodes, 20–50 MΩ when filled with 3 M KCl, were pierced into the center of muscle fibers with the resting potential between −45 and −55 mV. To evoke end-plate potentials, phrenic nerves were stimulated by a suction electrode with suprathreshold square pulses (0.1 ms) using Master-8 . Data were collected with axonpatch 200B amplifier , digitized with Digidata 1322A , and analyzed using pClamp 9.2 .Recording was performed as described previously . NeonataMouse muscle C2C12 myoblasts were propagated and induced to form myotubes as described previously . C2C12 mCtnnb1, were washed twice with PBS and then incubated in 1% formaldehyde in PBS at room temperature for 10 min. After washing twice with PBS, cells were scraped in PBS containing protease inhibitors, centrifuged at 2000 rpm at 4°C, and resuspended in SDS lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris–HCl (pH 8.0) plus protease inhibitors. Cells were sonicated by Sonic Dismembrator model 100 , strength 3, for 15 s 3 times, to shear chromosome DNA. After centrifugation at 13,000 at 4°C, the sonicated mixture was diluted 10-fold with ChIP dilution buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8.1), and 167 mM NaCl. The mixture was precleaned once with salmon sperm DNA-protein A-agarose beads at 4°C for 1 hr. The resulting supernatant was incubated with anti-Ctnnb1 antibody at 4°C overnight, when salmon sperm DNA-protein A-agarose beads (50 μg/μl) was added. The beads/mixture was incubated for another hour at 4°C and washed sequentially with a low-salt wash buffer containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), and 150 mM NaCl; a high-salt wash buffer containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), and 500 mM NaCl; an LiCl wash buffer containing 0.25 M LiCl, 1% NP-40, 1% deoxycholic acid, 1 mM EDTA, and 10 mM Tris–HCl (pH 8.1); and a wash with TE buffer. Beads were then incubated twice with 250 μl of the elution buffer  for 15 min at room temperature to elute bound DNA. Combined eluates were added to 20 μl of 5 M NaCl to reverse cross-links by heating at 65°C for 4 hr. After incubation with 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris–HCl (pH 6.5), and 2 μl of 10 mg/ml proteinase K (1 hr at 45°C), DNA was recovered by phenol-chloroform extraction and ethanol precipitation and used as template for PCR. Primers for the two TCF4/ LEF1-binding regions were as follows; Slit2 (primer1)- 5′-GTCCC CTTTA GGATC GCG-3′/5′-CAGCG GGAGA ACGAG G-3′ and Slit2 (primer2)-5′-AGAGA AGGTT GAAAA CACTA CTCCC-3′/5′-TGAAA TAGAT CTGCC TCCGT G-3′.ChIP assays were performed according to the protocol provided by Upstate Biotechnology . C2C12 mTotal RNA was isolated from muscles using Trizol (Invitrogen) and purified using the RNeasy mini kit (Qiagen). Quantitative RT-PCR was performed as described previously . Equal a−1), GDNF (2 ng ml−1), CTNF (2 ng ml−1), NGF (1 ng ml−1) (Sigma), and penicillin-streptomycin . Every alternate day, half of the medium was replaced by freshly prepared medium. 6 days after plating, the explants are subjected to incubating with Slit2-conjugated beads (see below).Spinal cord explant culture from E12.5 embryos was performed as previously described . In shorSlit2 or control empty Flag vector, to produce Flag-Slit2 and control conditioned media, respectively. Biotin-conjugated mouse monoclonal antibody (2 μl)  was conjugated with 50 μl of FluoSpheres NeutrAvidin-labeled Microspheres  by incubation in PBS for 4 hr at 4°C and then washed with ice-cold PBS, 3 times. Beads were re-suspended in 200 μl of ice-cold PBS with protease inhibitors and incubated with Flag-Slit2 or control conditioned media (200 μl) overnight on a rotating shaker at 4°C. Flag-Slit2-conjugated neutravidin microspheres were isolated by spinning at 5000 rpm at 4°C. Microspheres were washed 3 times with ice-cold PBS and diluted in explant culture media (1:100) and incubated with spinal cord explants for 24 hr before analysis.HEK293 cells were transfected with Flag-Dorsal horn explants were fixed 4% ice-cold PFA for 20 min at room temperature and washed twice (5 min each) with 1 ml PBS. Explants were then permeabilized by 1 ml blocking solution  for 5 min at room temperature and incubated with primary antibodies in blocking solution for 2 days at 4°C. Explants were rinsed 3 times with 1 ml PBS, 5 min each, and incubated with secondary antibodies in blocking solution for 90 min at room temperature. After washing thrice with 1 × PBS, 5 min each, coverslips were mounted on glass slides and covered with Vectashield mounting medium . Images were obtained by confocal scanning microscopy. FIJI (NIH) was used to measure and quantify synaptophysin puncta number and size.t-test was used to compare data between two groups. One-way ANOVA  was used to compare data between multiple groups, and Bonferroni's Multiple Comparison Test was used to determine the significance. Unless otherwise indicated, data were expressed as mean ± SEM, and considered statistically significant if p value was less than 0.05.Two-tailed student  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  a number of the conclusions are feasible.We would like to encourage you to resubmit a revised manuscript that addresses the specific issues raised in the reviews. We realize that several of the more ambitious suggestions are beyond the scope of the present work, but we have not removed them from the reviews since they convey the reaction to the work overall.Reviewer #1:In this manuscript, Wu et al. investigated the mechanism by which β-catenin controls presynaptic differentiation. The present work is an extension of their previous work (Nat Neurosci 11: 262-268), in which they showed that expression of β-catenin in muscle is critical for motoneuron differentiation. In this current work, they identified Slit2 as the retrograde factor activated by β-catenin. Specifically, 1) they showed that β-catenin TAD domain is required for rescuing presynaptic deficits of NMJ deficient in β-catenin; 2) they identified Slit2 as the retrograde factor activated by β-catenin; 3) transgenic expression of Slit2 rescued the presynaptic deficits of NMJs in β-catenin mutant mice.Overall this is a very interesting study which uncovers an unexpected retrograde factor Slit2 controlled by β-catenin. The manuscript is well written, the analyses of data were carefully performed and main conclusions are supported by the provided results. Thus the publication of the paper would be of interest to the general readership of the journal.Reviewer #2:This paper addresses how the β-catenin protein, earlier implicated in neuro-muscular junction (NMJ) formation, operates at the molecular level. The authors characterize transgenic mice that express wild-type or mutant forms of β-catenin, constructed such that the transcriptional activity and the cell adhesion functions of β-catenin are separated. By mutant rescue experiments, a role for the transcription by β-catenin is revealed. The target gene relevant for the activation of muscle-derived retrograde factors is identified as Slit2, a protein known to be cue for axon guidance but (to the knowledge of this reviewer) not yet implicated in NMJ formation. The Slit2 promoter may have essential binding sites for the β-catenin-TCF complex. There are some experimental issues that should be addressed.To start with a general question, if Slit2 is a transcription target of the β-catenin-TCF complex, it would be likely that β-catenin is activated by a Wnt signal, as the majority of cases of β-catenin acting as a transcription factor are due to Wnt signals. In the muscle, this would be interesting to know, in particular where the Wnts are expressed. In The survey of secreted molecules  shows thRegarding the experiments that show a role of β-catenin-TCF in the activation of Slit2, the text mentions binding sites in the 5'UTR (subsection “Identification of Slit2 as a retrograde target”). Do the authors mean that these sites are indeed in the transcript, or do they refer to the promoter area, upstream of the transcriptional start site?In the same section,  CHIP expA full analysis of the role of β-catenin-TCF on the Slit2 promoter would include mutating the binding sites and test for requirements. This is not unfeasible.Reviewer #3:Wu et al report an interesting study on a plausibly novel role of Slit2 in presynaptic differentiation in neuromuscular junction. The authors used a clever genetic trick to replace β-catenin in muscle cells with mutant versions that either cannot mediate cell adhesion or cannot activate downstream genes and showed that a transcription based mechanism in the muscle cells requiring β-catenin regulates the presynaptic differentiation in the motoneurons. From there, they screened for transcription targets of β-catenin in muscle cells. Slit2 was one of the many targets. Using a transgenic line expressing Slit2 in muscle cells in β-catenin mutant mice, the authors were able to rescue the phenotypes of overgrowth of the presynaptic terminals. Recombinant Slit2 can also induce synaptophysin puncta of spinal cord explants. Although the finding that Slit2 may be a novel retrograde signal for synapse formation is interesting, the current study does not provide sufficient evidence for this conclusion. There is also some concerns about the approach that is used, which involves overexpression of β-Catenin and Slit2.1) Genetic evidence that Slit2 is important for NMJ development is lacking. Slit2 is one of the many downstream factors of β-catenin. Overexpressing Slit2 in β-catenin mutant mice can rescue the overgrowth of presynaptic terminals. However, the authors did not show or discuss loss of function results. It is not clear whether other factors are more important in vivo.2) The phenotype of β-catenin knockout in muscle cells lead to overgrowth of motoneuron terminal arbors. Slit2 is an inhibitor of axon outgrowth. It is possible that overexpressing Slit2 reduces the arbor growth by enhancing growth inhibition. And the synaptic differentiation phenotypes may be due to a secondary effect to axon growth because the axons were not able to find the target area for efficient synapse formation. Therefore, Slit2 KO phenotype will be informative.3) The promoters for expressing both β-catenin and Slit2 are much stronger than their endogenous promoters. Therefore, β-catenin and Slit2 are overexpressed several fold more . This co Reviewer #2:[…] To start with a general question, if Slit2 is a transcription target of the β-catenin-TCF complex, it would be likely that β-catenin is activated by a Wnt signal, as the majority of cases of β-catenin acting as a transcription factor are due to Wnt signals. In the muscle, this would be interesting to know, in particular where the Wnts are expressed. In, some Wnts are tested for overall expression but this is a limited survey. As a locally acting signal, the Wnt protein could contribute, in a deterministic way, where on the muscle fiber β-catenin and subsequently Slit2 would be expressed, setting up the site of the NMJ. It could of course be that Wnt is coming from the incoming neuron. I realize that possible local signaling in a muscle fiber is complicated by the multinucleated nature of the muscle, but it still remains an important question whether β-catenin is locally activated. The latter point could be examined by a simple stain for β-catenin in the muscle while the use of Wnt/TCF reporter mice could indicated local transcriptional activation.Axin2-nlacZ mice, where the nuclear-localized β-galactosidase (nlacZ) DNA was inserted in the Axin2 gene, a target of Wnt/β-catenin signaling. Expression of β-galactosidase in Axin2-nlacZ mice is controlled by Axin2 promoter has been used to as a reporter of Wnt or β-catenin activity .Regarding the experiments that show a role of β-catenin-TCF in the activation of Slit2, the text mentions binding sites in the 5'UTR (subsection “Identification of Slit2 as a retrograde target”). Do the authors mean that these sites are indeed in the transcript, or do they refer to the promoter area, upstream of the transcriptional start site?Sorry for being unclear. Sites were identified upstream from the transcription initiation site. This is now described in revised Results.In the same section,  CHIP experiments are done using C2C12 cells. Are these cells over-expressing HA-tagged β-catenin, as was done in, or non-transfected cells?We apologize for being unclear. ChIP experiments were done in C2C12 cells transfected with HA-tagged β-catenin. This information is now provided in the revised Methods.A full analysis of the role of β-catenin-TCF on the Slit2 promoter would include mutating the binding sites and test for requirements. This is not unfeasible.We agree with the reviewer that a full analysis of the Slit2 promoter could be informative. This paper focuses on functional evidence that Slit2 serves as a β-catenin-regulated retrograde factor for presynaptic differentiation. We demonstrate, first, that Slit2 levels were reduced in β-catenin mutant muscles. Second, β-catenin associates with TCF cis-elements in the promoter region of the Slit2 gene. Third, Slit2 expression in muscle cells is promoted by elevating β-catenin. Slit2 was concentrated at the NMJ. Fourth, transgenic mice expressing Slit2 specifically in the muscle was able to diminish presynaptic deficits by β-catenin mutation, including poor innervation, reduced vesicle density and active zone, and depressed mEPP. Finally, Slit2 immobilized on beads was able to induce the synaptophysin puncta of axons of spinal cord explants.We hope that the reviewer agree that a full analysis of the Slit2 promoter would be beyond the scope of this paper.Reviewer #3:1) Genetic evidence that Slit2 is important for NMJ development is lacking. Slit2 is one of the many downstream factors of β-catenin. Overexpressing Slit2 in β-catenin mutant mice can rescue the overgrowth of presynaptic terminals. However, the authors did not show or discuss loss of function results. It is not clear whether other factors are more important in vivo.These points are well taken. First, as suggested, phenotypes of Slit2 mutant mice were discussed in the third paragraph of Discussion. Second, we agree with the reviewer that other factors may be involved. In fact, Slit2 expression in muscles is unable to rescue all defects in β-catenin mutant mice. This point is now indicated in the revised Discussion (fourth paragraph).2) The phenotype of β-catenin knockout in muscle cells lead to overgrowth of motoneuron terminal arbors. Slit2 is an inhibitor of axon outgrowth. It is possible that overexpressing Slit2 reduces the arbor growth by enhancing growth inhibition. And the synaptic differentiation phenotypes may be due to a secondary effect to axon growth because the axons were not able to find the target area for efficient synapse formation. Therefore, Slit2 KO phenotype will be informative.Our paper provides solid evidence that muscle Slit2 regulates presynaptic differentiation.Having demonstrated that the transactivation activity of β-catenin is critical, we reasoned that muscle β-catenin is likely to regulate presynaptic differentiation by controlling the expression of muscle-derived proteins. This notion was supported by results of additional experiments for the revision—that β-catenin and Wnt signaling reporter were higher in synaptic nuclei at the NMJ than those in extra synaptic areas. We reasoned that such a protein is secretable or has a transmembrane domain and its expression is down regulated in β-catenin mutant muscle. A screen for proteins that fulfill these criteria led to the identification of Slit2.Next, in a set of in vivo and in vitro experiments we demonstrated, first, that Slit2 levels were reduced in β-catenin mutant muscles. Second, β-catenin associates with TCF cis-elements in the promoter region of the Slit2 gene. Third, Slit2 expression in muscle cells is promoted by elevating β-catenin. Slit2 was concentrated at the NMJ. Fourth, transgenic mice expressing Slit2 specifically in the muscle was able to diminish presynaptic deficits by β-catenin mutation, including poor innervation, reduced vesicle density and active zone, and depressed mEPP. Finally, Slit2 immobilized on beads was able to induce the synaptophysin puncta of axons of spinal cord explants.Together, these observations make a reasonably compelling case for Slit2 as a β-catenin downstream factor for presynaptic differentiation.We agree with the reviewer that a full study of Slit2 mutant mice could be more informative. However, we hope that the reviewer would agree that such a study is beyond the scope of this paper, considering that the study has utilized multiple transgenic strains and muscle-specific mutant lines and the paper has 8 figures, each with multiple panels, plus several supplemental figures.3) The promoters for expressing both β-catenin and Slit2 are much stronger than their endogenous promoters. Therefore, β-catenin and Slit2 are overexpressed several fold more . This could lead to nonphysiological effects.Agree. However, to achieve muscle-specific expression prior to NMJ formation, HSA remains to be an ideal promoter. The other well-characterized muscle-specific promoter, MCK, is not active until neonatal stage and is thus unsuitable for studying NMJ formation. We would like to point out that our conclusion is not simply based on transgenic expression, rather a comprehensive set of in vivo and in vitro experiments. A parsimonious explanation of the results is that Slit2 serves a retrograde factor downstream of muscle β-catenin for presynaptic differentiation.


The wingless (Wnt) signaling is suggested as a fundamental hierarchical pathway in regulation of proliferation and differentiation of cells. The Wnt ligands are small proteins of about 40 kDa essentially for regulation and initiation of the Wnt activity. They are secreted proteins requiring acylation for activity in the Wnt signaling cascade and for functional interactivity with transmembrane proteins. Dual lipidation is important for posttranslational activation of the overwhelming number of Wnt proteins and is probably involved in their spatial distribution. The intestinal mucosa, where Wnt signaling is essential in configuration and maintenance, is an established model to study Wnt proteins and their role in carcinogenesis and cancer. The intestinal crypt-villus/crypt-plateau axis, a cellular system with self-renewal, proliferation, and differentiation, is tightly coordinated by a Wnt gradient. In the review, some attention is given to Wnt3, Wnt3A, and Wnt2B as important members of the Wnt family to address the role of lipidation and modifiers of Wnt proteins in intestinal carcinogenesis. Wnt3 is an important player in establishing the Wnt gradient in intestinal crypts and is mainly produced by Paneth cells. Wnt2B is characterized as a mitochondrial protein and shuttles between mitochondria and the nucleus. Porcupine and ACSL5, a long-chain fatty acid activating enzyme, are introduced as modifiers of Wnts and as interesting strategy to targeting Wnt-driven carcinogenesis. Among the Wnt-related pathways, the canonical Wnt pathway is the best characterized signaling cascade and consists of hierarchical organized highly specialized proteins signaling extracellular events to the nucleus. By this mechanism, Wnt signaling is able to initiate transcriptional changes [Wingless (Wnt) proteins are important mediators of cell proliferation and differentiation by activation of cell surface receptors. In Wnt responder cells, the Wnt signaling includes the canonical β-catenin pathway and several types of non-canonical pathway activities. This signaling is described as a non-canonical Ca changes ,2,3.The central protein in canonical Wnt signaling is β-catenin which is able to translocate into the nucleus in its dephosphorylated state. Glycogen synthase kinase-3β (GSK-3β) acts in a complex with casein kinase 1 (CK1), axin, and adenomatous polyposis coli (APC) and is responsible for β-catenin phosphorylation and inactivation of the canonical Wnt-pathway . In the Secreted Wnt proteins, the most important inducers of Wnt signaling activities, control growth and patterning during development of multicellular structures, tissues, and organisms. It has been suggested that the important developmental function of Wnt proteins depends on the establishment of Wnt gradients, acting either as a short- or long-distance paracrine modifier of gene transcription . The endSince Wnt signaling is essential in several biological phenomena including proliferation and development of cells, organs as well as complex biological systems, increasing evidence is given that this important signaling pathway is involved in the pathogenesis of many types of diseases ,11. InteIn this review, the current understanding of the emerging roles of Wnt proteins in intestinal carcinogenesis and cancer is given with a focus on Wnt lipidation. Wnt2B, Wnt3, and Wnt3A are discussed as models for Wnt family members to address lipidation and modifiers.2+ pathway. In addition, there are cross activities with other signaling cascades including JNK, RYK, CaMKII, and ROR [In the evolutionary conserved Wnt pathway, induction of signaling is given by a small number of Wnt proteins. In humans, 19 Wnt genes that encode respective Wnt proteins have been identified so far ,13. Sign and ROR ,11,14.The canonical Wnt-pathway is characterized by Wnt binding to its co-receptor complex that is substantiated with LRP-5 or -6. The signaling activity is further modified by intracellular proteins which influence extracellular antagonists such as secreted frizzled-related proteins (SFRP), the inhibitory factor WIF-1, and members of the dickkopf (DKK) family of secreted proteins. DKK1 and DKK3 are able to bind to LRP-5 and LRP-6 inhibiting the Wnt signaling by degrading the co-receptor . In FiguAs stated above the Wnt ligand type in addition to the expression domain and further determinants contributes essentially to the diversity and tissue specificity of Wnt functions. For example, Wnt16 has been found strongly associated with skeletal development and bone homeostasis . The WntImmature Wnts undergo strong posttranslational modifications in the ER including attachment of oligosaccharide chains in N-linked addition on the protein backbone. Some evidence is given that glycosylation makes protein secretion more efficiently and glycosylated Wnts are able to leave the ER. In the ER, the chaperone immunoglobulin heavy-chain-binding protein BiP, a member of the Hsp70 family, is able to associate with Wnt species and facilitate further sorting and final secretion of mature proteins, but can sequester defective Wnts in the ER . The WntIn addition to glycosylation, lipidation is a common feature of Wnt proteins and especially found in the N-terminal protein part. With the exception of WntD, Wnt proteins are posttranslationally modified by lipid adducts . SpecifiIn addition to C77, which is conserved in most other Wnt proteins, another hotspot of lipidation, the serine residue at position 209 (S209), was revealed in Wnt3A. In contrast to C77, S209 is highly conserved among Wnts  and modified with palmitoleic acid, a monounsaturated fatty acid . The S20As stated above hydrophobic activity of Wnts is due to their posttranslational addition of palmitate/palmitoleic acid to one or two residues. These lipid modifications of Wnt proteins are assumed as essential for the trafficking of secreted Wnts as well as Wnt recognition by Fzd receptors. It has been speculated that the Wnt acylation may directly engage the N-terminal Fz cysteine-rich domain and could also mediate binding to WIF-1 ,27. RegaIn the ER, the protein porcupine is found, which is a member of the membrane-bound O-acyltransferase family (MBOAT) and involved in N-glycosylation ,31,32. PIn addition to permit N-glycosylation, experimental evidence is given that porcupine catalyzes the lipidation of Wnts especially at S209 and equivalents resulting in hydrophobicity and membrane association ,34. The + T-cells, however, porcupine is not required for the release of Wnt proteins 1, 3, 5b, and other [Porcupine is essential in Wnt lipidation and no mechanism has so far been identified to fully compensate the enzymatic function . Deletiond other . A speciWnt proteins are mostly modified with palmitoate and plamitoleate. Thus, the relative cytoplasmic abundance of palmitoyl—and palmitoleoyl-CoA with other acyl-CoA species in addition to active site determinants and specialized domains of porcupine protein—Wnt protein interaction are assumed to translate and dictate enzyme selectivity . This poACSL5 is a nuclear-coded and mitochondrial expressed enzyme involved in the regulation of cellular apoptosis  and jeju+ mitochondria), the content of palmitoyl-CoA is significantly increased and strong plamitoylation of Wnt2B with intramitochondrial protein accumulation is found. The palmitoylated Wnt2B is not able to leave mitochondrial membranes and does not translocate into the nucleus for promoting the intranuclear Wnt signaling activities [The Wnt2B protein  is a positive regulator of Wnt signaling . The protivities . PorcupiThe surface lining epithelial cells of the intestinal mucosa display a very high turnover rate with cellular replacement within a few days. Lineage-tracing studies and cell labeling experiments revealed that the intestinal crypt is the niche for proliferative, multipotent intestinal precursor cells. Interestingly, several key intestinal stem cell marker genes like Lgr5, Ascl2, and Sox9 are Wnt targets . This ishigh cells and moderate Wntlow cells [Similar to intestinal stem cells, cancer stem cells are suggested to give rise to progenitor cells populating the majority of an intestinal tumor. The Wnt is essentially involved in the establishment and proliferating activities of these stem cells. Two important concepts have been established describing intestinal carcinogenesis: the “top-down” and the “bottom-up” model. In both scenarios, Wnt signaling is considered as an important driver and regulator in the development of autonomy in tumor cell proliferation. In the “bottom-up” concept the stem cells at the crypt base acquire genetic alterations and migrate upwards constituting the tumor. The model has been substantiated by mice with conditional loss of APC in crypt base stem cells . The losow cells ,49. Wnt In intestinal adenocarcinomas, heterogeneity of Wnt activity exists with increased proliferative activities in tumor cells with nuclear β-catenin expression. These cells are preferentially found at the invasive front of the tumor and apparently perform an epithelial- to -mesenchymal transition. The transition is suggested as important molecular and cellular mechanism to promote the malignant phenotype with establishment of tumor budding, drug resistance, and cancer stem cells . ActivatIt is suggested that ACSL5 is involved in the early genesis of colorectal cancer, most likely by changing transport and pool formation of long-chain acyl-CoA thioesters . In suchModifiers of Wnt proteins and Wnt signaling are suggested as interesting molecular targets for anti-cancer strategies, because activation/dysregulation of Wnt signaling pathways are strongly involved in initiation and progression of neoplasias ,59. In pFor therapeutical intervention it is of importance that the modification of Wnt proteins at the two lipidation sites  is apparently regulated differentially . WhereasTargeting Wnt protein lipidation is a promising strategy in the development of anti-cancer agents. Increasing evidence is given that lipidation is found in a variety of disease-related proteins which are not involved in carcinogenesis and cancer . For exaAmong the different types of Wnt signaling, the canonical pathway is the best known and plays important roles in morphogenesis, cell development, and carcinogenesis. Wnt proteins, which are essential in initiating and promoting Wnt activities, are molecules with posttranslational modifications including intensive lipidation by modifiers like porcupine and ACSL5. Lipidation of Wnt proteins is essential for their maturation/segregation, secretion, and protein function. Within the canonical Wnt pathway, the activated Wnt proteins are essentially involved in carcinogenesis and cancer. Therefore, Wnt lipidation is suggested as an interesting strategy to target Wnt-driven carcinogenesis.


In a recent issue of Cell Death and Discovery, we reported Isopropyl-ethyl-1--9H-pyrido-indole-3-carboxylate (Z86) as a novel Wnt/β-catenin signaling inhibitor with selective proliferation inhibitory activity on cancer cells in vitro and in vivo.3The evolutionarily conserved Wnt/β-catenin signaling is frequently activated by mutated APC or β-catenin, it seems that the ideal antagonist of the pathway would be the transcriptional complex of TCF and β-catenin in the nucleus. Accordingly, small molecules first identified as Wnt/β-catenin inhibitors targeted exactly the level of transcriptional complexes, such as NC043,4 Henryin5 and ICG001.6 However, there are recently some experimental results showing that, at least in some cases, targeting the upstream components of the Wnt signaling pathway can also have a role. The IWP compounds interfered the Wnt/β-catenin signaling through attenuating the production of Wnt ligands by targeting Porcupine, a member of the membrane-bound O-acyltransferase (MBOAT) family, which is essential for Wnt secretion and signaling ability.7 Recently, several small molecule inhibitors targeting the destruction complex composed of APC, Axin, GSK3β and other proteins of Wnt/β-catenin signaling were discovered. IWR,8 XAV939,9 JW55,10 J67 and J7411 promoted the phosphorylation and subsequent degradation of β-catenin by stabilizing of Axin, whereas the compound Pyrvinium12 enhances casein kinase to promote the phosphorylation of β-catenin and disturbs the stabilization of β-catenin.In colorectal cancers where Wnt/β-catenin signaling inhibitor that belongs to β-carboline structure-type compound by using a cell-based luciferase reporter assay system.3 The inhibitory activities of the derivatives on Wnt/β-catenin signaling were investigated and the structure–activity relationship was characterized. The Wnt/β-catenin signaling inhibitory activity of Z86 was further confirmed in HEK293T cells transiently transfected with Wnt1, and cell lines with abberant activation of Wnt/β-catenin signaling, HCT116 and SW480 cells. Furthermore, Z86 inhibited the expression of endogenous Wnt/β-catenin signaling target genes and antagonized the second axis formation of Xenopus embryos induced by Wnt8. Further mechanism studies showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its over-activation and promoted the phosphorylation and degradation of β-catenin  phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel chemotype of antagonists of the canonical Wnt signaling and highlight a promising candidate for further colorectal cancer therapeutics development.Constitutive Wnt/


S33Y)-β-catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1) mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28±22.32 ng/mL  that were insignificantly different from patients with non-neoplastic chronic liver disease  or healthy volunteers . Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels . While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients.We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC). By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO) livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3B CTNNB1) mutations are one of the major oncogenic gene alterations in HCC seen in 10–40%, while mutations affecting Axin1 are seen in around 10% of all HCCs CTNNB1 mutations are observed in exon-3 that contain phosphorylation sites essential for β-catenin degradation leading to its stabilization and enhanced expression of target genes such as glutamine synthetase (GS), axin2 and regucalcinPrimary liver cancer, which is predominantly hepatocellular carcinoma (HCC), is the sixth most common cancer worldwide and the third most frequent cause of cancer mortality LECT2 is a 16 kDa chemotactic protein purified from human T-cell line Lect2 expression to be decreased in hepatocyte-specific β-catenin knockout livers in vitro analysis in human HCC cells, we demonstrate that indeed LECT2 expression and its protein levels reflect β-catenin activity and hence hypothesize that it may be a good biomarker for HCC with β-catenin activation. The utility of LECT2 as a biomarker was validated first in a mouse liver tumor model where exon-3 mutation in β-catenin gene and ensuing β-catenin activation is implicated in HCC pathogenesis CTNNB1 or without CTNNB1 mutations when compared to patients with chronic liver disease or healthy volunteers. Furthermore, despite β-catenin activation observed in an additional subset of non-CTNNB1 mutated HCC, which showed high LECT2 expression, serum LECT2 levels were not predictive for active β-catenin signaling in the tumor. Interestingly though, irrespective of molecular aberrations, LECT2 levels were significantly higher in all HCC patients versus patients with cirrhosis or healthy controls. In fact, serum LECT2 ≥ 50 ng/ml indicated HCC with high specificity and positive predictive value.Here, screening a previously published Affymetrix gene array, we identify CTNNB1) or scrambled Negative Control siRNAs  as previously described WT and Hep3BS33Y, respectively, as described previously Human HCC cell lines, Hep3B, HepG2, SNU449, SNU398, and HuH7, were obtained from the American Type Culture Collection . Cells were cultured in Eagle's minimal essential medium (EMEM) or RPMI supplemented with 10% vol/vol FBS at 37°C in a humidified 5% carbon dioxide atmosphere. For siRNA knockdown experiment, the cells were transfected using Lipofectamine 2000  with β-catenin  at a dose of 90 µg/gram body weight at 6 weeks of age and 3 weeks later putting them on a diet containing 0.05% phenobarbital (PB) until sacrifice at 6–8 months. Serum was collected at time of euthanasia and simultaneously, liver tissues were collected for histology and protein analysis.CTNNB1) using polymerase chain reaction (PCR), gel extraction, purification of PCR products, and direct sequencing were performed as described previously Genomic DNA was extracted using DNA Micro Kit  from both the frozen liver sections after hematoxilin and eosin (HE) staining  to identify the tumor and from patient's frozen liver tumor tissues. Amplification of exon 3 of β-catenin gene  forward, 5′- CTCGCTCTCCTGACCTGTTC -3′ and reverse, 5′- TTCAAGTGGGAACTTGCTGA -3′; LECT2 forward, 5′- CCCACAACAATCCTCATTTCA -3′ and reverse, 5′- GTTAGCCCATGGTCCTGCTA -3′; GAPDH was used as an internal control.In mice analyses, total RNA was extracted from frozen sections after H&E staining using a mirVana microRNA isolation kit (Ambion) in accordance with the manufacturer's instructions. After reverse transcription and DNase treatment were performed, qRT-PCR was performed on a StepOne Plus using 2× SYBR Green PCR Master Mix . The primers' sequences are as follows: In human analyses, total RNA was extracted from frozen tissues and qRT-PCR analysis performed as described previously Serum LECT2 levels were measured by either human or mouse LECT2 ELISA kit  Co, Ltd, Niigata, Japan) according to the manufacture's protocol.All tissues and materials used in this study were obtained under an approved Institutional Review Board protocol at the University of Pittsburgh and Kumamoto University. Specifically, frozen tissues and serum samples were obtained from HCC patients in the Department of Surgery, University of Pittsburgh  with a written informed consent approved by the University of Pittsburgh Institutional Review Board. Frozen tissues and serum samples from HCC patients were also collected by the Department of Gastroenterological Surgery, Kumamoto University , with a written informed consent approved by the Institutional Review Board at the Kumamoto University. Additional serum samples were obtained from patients with chronic liver disease (n = 15) in the Department of Medicine that did not have any evidence of HCC as determined by normal serum α-fetoprotein and negative abdominal ultrasound or CT scan within 6 months of serum collection. These patients also signed informed consent prior to providing serum samples and the University of Pittsburgh Institutional review board approved the study.WT and Hep3BS33Y cells . 3×106 cells were fixed with 1% formaldehyde for 10 min, incubated with glycine (0.125 M) for 5 min, and then washed twice with PBS. After a short spin, the pellets were resuspended in cell lysis buffer  by pipetting. After centrifugation, the pellets were resuspended in nuclear lysis buffer  containing protease inhibitors by pipetting, and sonicated to break chromatin into fragments of around 0.3–1.0 kb length. Subsequent IP experiment was performed with ChIP-IT High sensitivity kit . The diluted DNA-protein complex (25 µg of protein) was incubated overnight at 4°C with different antibodies  in the presence of herring sperm DNA and protein A/G agarose beads. PCR was performed using primers for LECT2 promoter region: 5′- CAGCCCAGAAGACTGTCGAT -3′ (forward) and 5′- GATTAGAGTTGCCCCCACAC -3′ (reverse); albumin promoter region, 5′- TGGAGAAAACAGTTCCAGATGGT -3′ (forward) and 5′- CGTGTGGGGTTGACAGAAGA -3′ (reverse).ChIP Assay was performed using Hep3BSNU449 cells were plated in 24 well plate and treated with either 50 nM control si-RNA or 50 nM β-Catenin si-RNA with lipofectamine iMAX (Invitrogen). TOPFLASH reporter plasmid was transfected using the lipofectamine 2000 (Invitrogen) a day after the siRNA treatment. Subsequently, cells were treated with TGF-β1 (5 ng/mL) (R&D system) for 24 hours. Cells were lysed and prepared using the luciferase reporter assay system . Reporter activity was read on a luminometer .Statistical analysis was performed using the JMP 8.0 software . Values were presented as the mean ± standard deviation (SD). Differences between two variables were calculated using the Wilcoxon test. Multiple samples were compared by ANOVA followed by Tukey-Kramer post hoc test. P<0.05 was considered to indicate a statistically significant difference.lect2 showed a 117-fold lower expression in KO as compared to WT  and Hep3B cells expressing S33Y-mutated β-catenin (Hep3BS33Y), the latter showing highest β-catenin levels . Whole cell lysates also showed increased LECT2 protein levels in Hep3BS33Y as compared to Hep3BWT cells  with that of wild type (WT) ed to WT . To valin levels  [17]. WeWT cells . To see n levels .WT and Hep3BS33Y cells and subjected to ELISA assay. Significantly higher LECT2 protein was detected in conditioned media from Hep3BS33Y (0.29±0.13 ng/mL) as compared to Hep3BWT cells (0.023±0.046 ng/mL)  . Basal mLECT2 expression, chromatin immunoprecipitation (ChIP) analysis was performed on extracts from Hep3BS33Y and Hep3BWT cells. As shown in LECT2 was pulled down with TCF4 and not control IgG in Hep3BS33Y cells. This indicates promoter activity driven by TCF4-β-catenin in Hep3BS33Y cells. Albumin, whose expression is β-catenin-TCF independent, was pulled down by HNF1α in both Hep3BS33Y and Hep3BWT cells. These results show occupancy of Lect2 promoter by TCF4, thus demonstrating its regulation by the β-catenin-TCF signaling.Lastly, to specifically determine if β-catenin-T cell factor (TCF) signaling was indeed regulating p<0.01) higher serum Lect2 levels (55.9±19.9 ng/mL) as compared to the 4 non-tumor bearing mice (24.9±5.5 ng/mL)  expression in these tumor nodules. All 3-tumor nodules had high Lect2 and Glul expressions as compared to the background liver  . To conf nodules . Direct  nodules . We nextnd liver . Thus, iCTNNB1) alterations in the form of missense mutations in exon-3  or healthy volunteers . On the other hand, patients who did not harbor CTNNB1 mutations showed significantly higher LECT2 level  than those with cirrhosis and from healthy volunteers   .LECT2 expression in the tumors, we selected a group of 28 patients for which had serum and corresponding tumor tissue , 2) patients with WT β-catenin gene but active Wnt signaling (+), and 3) those with WT β-catenin gene but lacking Wnt signaling (−). Patients in MT and (+) groups were insignificantly different from each other in expression values of AXIN2, REGUCALCIN, LECT2 and GLUL, although the expression was always highest in the MT group (LECT2 expression than MT and (+)  patients with MT group . HoweverMT group . Intrigu and (+) .LECT2 expression in tumors, they unexpectedly still showed high serum LECT2 level with the values ranging from 50–80 ng/mL.Taken together, these data indicate that despite some HCC patients showing low Next, due to heterogeneity in HCC, we investigated if serum LECT2 levels may be a biomarker of HCC irrespective of a precise molecular basis. Intriguingly, Tukey-Kramer analysis revealed a significant difference in serum LECT2 levels in HCC patients when compared to chronic liver disease patients with cirrhosis (p = 0.0017) and healthy volunteers (p = 0.0078) . Area UnLECT2 expression was indeed β-catenin-dependent. The major goal of the study was to investigate if serum LECT2 levels could reflect β-catenin activity in HCC and hence could be a biomarker to detect HCC-driven by Wnt signaling for prognostic and therapeutic implications. β-Catenin therapeutic targeting is indeed actively discussed in HCC CTNNB1 mutations is often not possible. Thus there is a significant need for biomarker discovery for personalized medicine to identify a subset of HCC patients with β-catenin activation. We hypothesized that serum LECT2 may be a non-invasive way to identify β-catenin gene mutation-harboring HCC. Indeed previous investigations have shown LECT2 to be observed systemically, suggesting that it can be a serum predictor LECT2 is a secreted protein from hepatocytes functioning like a chemokine, and is known to be directly regulated by β-catenin Lect2 expression in the tumor that occurred secondary to β-catenin gene mutations in a murine HCC model Using mouse model, we confirmed that serum LECT2 levels indeed correlated with upregulation of CTNNB1-mutated versus non-mutated group. In fact, we did not find any correlation between expression of Lect2 in the tumors and its corresponding serum levels in a patient. Since, β-catenin activation in HCC can be due to multiple independent mechanisms such as overexpression of Wnt inhibitors, increased expression of Wnt3 and Frizzled-7, TGF-β activation or other reasons CTNNB1 mutations in exon-3. While, we did observe β-catenin activation in around 50% more HCC samples, LECT2 serum levels remained ambiguous in this subset as well.In patient analysis however, serum LECT2 levels were not statistically different in Based on the observation of relatively high levels of LECT2 in serum of a major subset of HCC patients, irrespective of molecular aberration, we were able to address its utility as a general biomarker for HCC. A recent study also identified an increase in serum LECT2 levels in obese individuals and patients with fatty liver Lect2 upregulation in HCC, both downstream of CTNNB1 mutations and due to mutation-independent β-catenin activation. While LECT2 serum levels coincide with Ctnnb1 mutations in mice, this relationship was ambiguous in patients and Lect2 expression and serum levels did not correlate with β-catenin activation due to either mutations or otherwise. However, serum LECT2 by itself turned out to be an important biomarker of HCC (AUC  = 0.82). Further, serum LECT2 levels of greater than 50 ng/ml successfully diagnosed HCC in patients irrespective with specificity of 96.1% and positive predictive value of 97.0% and thus should be investigated prospectively for its diagnostic value.Thus, in summary we have identified


The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of β-Catenin was shown to occur towards the source of a localized extracellular cue. A key constituent of the central nervous system of all jawed vertebrates is the cerebellum . The steDrosophila melanogaster, neuroectodermal cells that divide parallel to the axis of apical-basal polarity results in both daughter cells retaining their neuroectodermal identity. In contrast, when the plane of division is rotated 90 degrees and is perpendicular to apical-basal polarity, the basal daughter cell becomes the neuroblast and delaminates, whereas the apical cell remains in the neuroectodermal layer  regulates GNP proliferation . Therefosupplementary material Fig. S4). Two representative z-stack images are shown, with the L-R ratio for PH3 given on the left side and the L-R ratio for β-Catenin on the right side for each z-plane image . The frequency histograms show that the distribution of β-Catenin in GNPs changes across developmental ages and the number of cells showing highly asymmetric values increased and was present at a great frequency than that for PH3. For this analysis, all data from analyzed cells were included, even those that had a large ratio for PH3. It is possible that large asymmetric values for PH3 are due to the fact that some of the cell pairs may undergo cell death.The distribution of β-Catenin during GNP division was investigated A,B. The ne image C. Furthene image D. The rene image A-C. In cne image D. To visA recent study  demonstrIn this study, the orientation of dividing GNPs at different stages of postnatal cerebellar development has been characterized. Previous studies have shown that laminin that is expressed in the pia may provide a signal for outer EGL cells that strongly express the integrin alpha-6 receptor for laminin . In addiin vivo was perturbed. Blocking Shh with a Smoothened inhibitor, Cyclopamine resulted in a reduction in cerebellar size in mice. Treatment with a Smoothened agonist, SAG resulted in an increase in surface area. These results are in agreement with previous studies that have disrupted Shh signalling . We did not address whether cells in which β-Catenin dividing symmetrically were confined to particular layers of the EGL since the characteristic of the EGL is continually changing during the first two postnatal weeks, for example the thickness of the PCNA positive or the NeuroD1 positive layers . However, a study by Espinosa and Luo . Further, since which of the two anaphase cells received β-Catenin was random the role of extrinsic cues in this process is not clear. This study is of particular importance because while cell fate determination in the VZ has been widely studied, the precise mechanisms that maintain the balance between GNP proliferation and differentiation are yet to be elucidated. This study has characterized the plane of division in a secondary zone of proliferation. While previous studies have linked intrinsic factors to spindle orientation, this study points to the role of extrinsic factors such as Shh that may directly or indirectly influence plane of cell division by increasing proliferation. Further analysis of the mechanisms that control mitotic spindle orientation and asymmetric distribution of molecules within the EGL and establishing a causal role of these molecules in cell fate will help in better understanding of how brain size is regulated. This in turn could help us decipher the factors contributing to disorders involving hypoplasia and overgrowth.A recent study showed that localization of Wnt signalling by using beads to deliver the ligand in a spatially controlled manner resulted in an asymmetric distribution of β-Catenin . SimilarAll animal experimentation in this study was done in accordance with the guidelines laid down by the Institutional Animal Ethics Committee, of the National Brain Research Centre, and the Indian Institute of Science, India. Cerebellar tissue from C57BL/6J mice of ages P0–P14 was dissected out following hypothermia, and subsequent perfusion. These were fixed in 4% paraformaldeyde (PFA) for 24 hours and then placed in 30% sucrose. Midsagittal cryo-sections of 20–60 μm were taken. For alteration of Shh pathway in the developing mouse cerebellum, P0 animals were given a daily dose of Cyclopamine  or SmootThe following primary antibodies were used in the study; β-Catenin , Phosphohistone H3 (PH3) , PCNA , N-Cadherin , NeuroD1 , β-III Tubulin , Shh , Wnt3a  and Cell Mask Deep Red Plasma membrane stain . Alexa Fluor anti-rabbit and anti-mouse 488 and 594 secondary antibodies were used at a dilution of 1:1000 (Molecular Probes). Sections from control, cyclopamine and SAG treated mice were stained with Nissl stain. The protocols for IHC and Nissl staining have been described previously .L and R denote proximal and distal positions respectively in a parallel division and as left and right cell respectively in perpendicular division, to get the normalized ratio for PH3 and β-Catenin. The normalized ratios of PH3 and β-Catenin immunofluorescence were compared. The normalized ratios for β-Catenin and PH3 are either positive or negative. For parallel divisions, positive values denote that the cell proximal to the pial surface has more β-Catenin compared to the cell distal to the pial surface.Mitotic cells were identified using anti-PH3 antibody. Midsagittal sections of 20–60 μm each were used in the study. The number of animals and cells analyzed per age is given in For classifying β-Catenin expression in outer and inner EGL, based on PCNA/NeuroD1 staining, the first three layers in a P6–P8 cerebellum were taken as outer EGL and the three innermost layers as the inner EGL since these layers were almost exclusively either PCNA or NeuroD1 positive respectively.P6 pups were sacrificed by hypothermia; their cerebellar cortex dissected aseptically in CMF-Tyrode solution. Meninges were removed; the tissue was chopped into smaller pieces and collected in CMF-Tyrode. These were treated with trypsin-DNAse and then dissociated in the same solution by triturating to make a single cell suspension, pelleted and resuspended in serum containing media . After 12 hours, the media was replaced with serum free media [Dulbecco's modified Eagle's medium (DMEM)], B27 supplement, N2 supplement and penicillin-streptomycin (Invitrogen). Cells were seeded on Poly-D Lysine coated coverslips that were printed with Shh and Wnt3a. After 24 hours of incubation at 37°C in 5% CO2, cells were washed with 1× PBS and then fixed in 4% PFA.Glass coverslips  were cleaned and coated with Poly-D-Lysine (PDL) (0.5 mg/ml), for a minimum of 2 hours. Silicon masters were fabricated lithographically according to standard clean room protocols using SU-88 as a photoresist . Shh 25.All images were captured at room temperature. Apart from minor adjustment of contrast and brightness, no additional image alteration was done. All fluorescent images were captured using the Zeiss Apotome Imager M2 microscope and Zeiss Confocal LSM 510 Meta. All bright field images were captured on a Leica DFC 320.t-test. To calculate whether there is a statistical difference between the ratio of L versus R obtained for PH3 as compared to the ratio of L versus R obtained for β-Catenin, Wilcoxon matched pair one tailed test was performed for all values obtained for each age. Mann-Whitney U test was done to test whether the L versus R ratios for β-Catenin changes with age. Statistical analyses by student t-test were used wherever appropriate.The percentage of parallel divisions was calculated for different animals and weighted mean and variance for the percentages for each age was determined. Statistical difference was evaluated by unpaired