Wnt/β-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/β-catenin pathway modification of the cellular apoptosis.To identify potential genes regulated by Wnt/β-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of β-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR.Stably integrated inducible RNAi vector could effectively suppress β-catenin expression and the transcriptional activity of β-catenin/TCF. Meanwhile, depletion of β-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of β-catenin.Coupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/β-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified β-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth. Defecpoptosis . Howeverpoptosis . Even leet al. developed a doxycycline (DOX)-inducible form of the RNA polymerase III H1 promoter to drive siRNA expression, which could inducibly knockdown the expression of target genes [vs. after induction. A series of differential expression genes due to reduced expression of β-catenin was identified and confirmed by RT-PCR. These genes have been implicated in apoptosis but never linked to Wnt/β-catenin pathway before. Since apoptosis is critical for normal embryonic development and for homeostasis in adult tissues, cancellation of this process with increased resistance to cell death is a common feature of malignant cells and represents a significant obstacle to therapy of human cancers [The advent of RNA interference (RNAi)-directed knockdown has sparked a revolution in somatic cell genetics, allowing the inexpensive, rapid analysis of gene function in mammals -10. RNAiet genes . With th cancers ,13, some2, fed every 3 days with complete medium supplemented with 5 μg/ml blasticidin, and subcultured when confluence was reached.The parental T-REx™-HeLa cells , stably expressing the tetracycline repressor protein were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco BRL), 50 units/ml penicillin, 50 units/ml streptomycin and 5 μg/ml blasticidin. These cells were maintained in a humidified 37°C incubator with 5% COThe plasmid pTER-β-catenin, which encodes a short hairpin RNA (shRNA) against human β-catenin driven by a DOX-inducible form of the RNA polymerase III H1 promoter, was a kind gift from Dr. M. van de Wetering and Prof. H. Clevers  . TOPFLAS5 cells were seeded into each well of a six-well tissue culture plate (Costar).The next day when the cells were 70–80% confluent the culture medium was aspirated, and the cell monolayer was washed with prewarmed sterile phosphate buffered saline (PBS). Cells were transfected with the appropriate plasmids using LipofectAMINE™ 2000 Reagent (Invitrogen) according to the manufacturer's protocol. The cells were harvested at different time points. Western blot analysis or other experiments were performed.Transfections were performed in 6-well or 24-well plates. A total of 2 × 10For the establishment of stable transfectants of pTER-β-catenin, after transfection, HeLaT cells were selected with 200 μg/ml Zeocin, and resistant clones were mixed and cultured. Zeocin-resistant clones were tested for their ability to downregulate β-catenin by Western blot and RT-PCR. The resulting resistant clones populations were designated HeLaT-β-catenin-RNAi.Total RNA was extracted from cells by using TRIZOL Reagent (Invitrogen) according to the standard protocol. The integrity of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under UV light. RNA was stored at -80°C in RNase-free water until reverse transcription or fluorescence labeling.HeLaT-β-catenin-RNAi cells were grown in the presence or absence of DOX (2 μg/ml). For Western blot analysis, cells were harvested at different time points and lysed in lysis buffer, then Western blot analysis was performed with the use of conventional protocols as described previously . In brieFor RT-PCR, total RNA (5 μg) was used for cDNA synthesis by reverse transcription using mouse-mammary tumor virus (M-MLV) Reverse Transcriptase (Promega), according to the manufacturer's protocol in a total volume of 25 μl. All PCR reactions were performed using standard PCR conditions: 95°C 5 min, 95°C 1 min, annealing at different temperatures for each gene respectively 1 min  and the internal control plasmid pRL-TK (5 ng) using LipofectAMINE™ 2000 Reagent (Invitrogen). The TOPFLASH and FOPFLASH reporters contain two sets of three copies of wild-type or mutant β-catenin/TCF binding sites respectively as well as the Thymidine Kinase (TK) minimal promoter upstream of the Firefly Luciferase open reading frame. After transfection, cells were treated with or without DOX (2 μg/ml) for 3 days. Then, luciferase activity was determined using the Dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. All results are expressed as means ± SD for independent triplicate cultures.in situ cell death detection kit, Alkaline Phosphatase (Roche Applied Science), in accordance with the manufacturer's instructions. In brief, HeLaT-β-catenin-RNAi cells were grown on coverslips. The next day, cells were treated with or without DOX (2 μg/ml) for 3 days. Coverslips with adherent cells were fixed in 4% paraformaldehyde for 1 h at room temperature and permeabilized with 0.1% Triton X-100 for 2 min on ice. DNA fragments were labeled with the TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. The coverslips were then incubated with Converter alkaline phosphatase for 30 min at 37°C in a humidified chamber, rinsed in PBS, and incubated with nitro blue tetrazolium/5-bromo-4-chloroindol-3-yl phosphate (Roche Applied Science) for 10 min. Cells were mounted cell side downward on a microscope slide, and the apoptotic cells (dark blue staining) were counted under a microscope. Three fields were randomly counted for each sample.Apoptotic cells were confirmed with the A total of 1384 apoptosis-related genes were selected. Probes against these genes were designed with OligoArray2_1 (University of Michigan). The oligonucleotide probes were synthesized and spotted as described previously . The olit test was used to compare the statistical significance of the differences in data from two groups. Values of P < 0.05 were considered statistically significant  cells that stably expressed the tetracycline (Tet) repressor were transfected with pTER-β-catenin and selected with Zeocin. Stable transfectants were analyzed for β-catenin expression by Western blot and RT-PCR before and after DOX induction. The protein level of β-catenin was reduced within 1 day after treatment and completely suppressed on the third day Figure  and micrWe then investigated the effects of β-catenin knockdown on the transcriptional activity of β-catenin/TCF . The lucWe found that the spontaneous activity of the TCF reporter, TOPFLASH, was reduced to background (FOPFLASH) levels on reduction of β-catenin levels by the induced expression of pTER-β-catenin by DOX Figure . There wVascular endothelial growth factor (VEGF) [Cox2) [Inhibitor of differentiation protein 2 (Id2) [Gastrin [Matrix metalloproteinase-7 (MMP7) [CD44 [β-actin, were all reduced after treatment with DOX  , cycloox) [Cox2) , Inhibit 2 (Id2) , Gastrin[Gastrin , Matrix 7 (MMP7) ,22 and C7) [CD44 , but notX Figure ; Table 1These results indicate that inducible knockdown of β-catenin by the stably integrated RNAi vector pTER-β-catenin in HeLaT cells results in the downregulation the β-catenin/TCF-dependent gene expression.P < 0.01). These data suggested that depletion of β-catenin by RNAi in HeLaT-β-catenin-RNAi cells made the cells more sensitive to apoptosis.Previous studies demonstrated that inhibiting the activity of Wnt/β-catenin pathway induced apoptosis . To deteet al. found that Wnt-mediated β-catenin/TCF transcription was responsible for providing protection against apoptosis [vs. control HeLaT-β-catenin-RNAi cells. Duplicate experiments were carried out on a single total RNA preparation from the cells.The data we show above and previous studies suggest that Wnt/β-catenin pathway may be associated with cellular apoptosis. However, the molecular mechanisms by which Wnt/β-catenin pathway regulate apoptosis are unclear until now. Chen poptosis . To systvs. induced cells. We found that the mRNA level of β-catenin (CTNNB1) was markedly reduced after induction with DOX, demonstrating the effectiveness of the RNAi system. Meanwhile, several apoptosis-related genes, such as MYBL2, BAG3, PTEN, PDCD6IP, HIF1A, BAG2 and DAP3, were significantly upregulated (P < 0.05) . The significance of the oligonucleotide microarray expression data is further supported by RT-PCR. Confirmation of 8 regulated genes by RT-PCR provides experimental support for the reliability of the microarray data.It is well known that Wnt/β-catenin pathway regulates the transcription of a suite of genes controlling numerous aspects of development and human diseases, ranging from cellular proliferation, differentiation and apoptosis ,4. HowevPTEN, which negatively regulates the PI3K-AKT survival pathway, was upregulated after DOX treatment. Elevated expression of PTEN may inhibit the activity of PI3K-AKT pathway and induce these cells to apoptosis. Consistent with our results, it has been demonstrated that Wnt pathway regulates cellular apoptosis by PI3K-AKT pathway [NFKBIA, the gene coding for IκBα, which retains NF-κB dimers in the cytoplasm to prevent the activation of NF-κB pathway [et al. They demonstrated that expression of Wnt-1 increases survival of PC12 cells in the absence of serum by activating the anti-apoptotic factor NF-κB [TP53BP1 and a p53-induced gene TP53I11 were elevated after the inhibited activity of Wnt/β-catenin by treatment. The 53BP1 encoded by TP53BP1 gene was found to be able to bind p53 protein and enhanced p53-mediated transcriptional activation [TP53I11, a direct p53 target gene, was proved to induce cell apoptosis and enhance the apoptotic effects of arsenic trioxide [Programmed cell death 5 (PDCD5) [Death-associated protein 3 (DAP3) [Fas-associated via death domain (FADD) [The overall pattern of gene expression observed in response to the inhibition of Wnt/β-catenin pathway by inducible RNAi vector against β-catenin has important implications for elucidating the role of this pathway in apoptosis. Of particular interest are those genes that have been implicated in several cell-survival pathways, such as Phosphatase and tensin homolog (PTEN)-Phosphatidylinositol 3-kinase (PI3K)-AKT pathway -47, NF-κ pathway ,40. Simi pathway , was als pathway ,49. Thisor NF-κB . We alsotivation ,53. Meantrioxide . These dtrioxide . Other p (PDCD5) , Death-a3 (DAP3)  and Fas-n (FADD) , were aln (FADD) ,30,31. HAs evident from the examples above, assessment of differentially expressed genes with known functions is useful to monitor pathways or biologic processes that are triggered in expression profiling experiments. In addition, microarray expression profiling of β-catenin-decreasing cells identified at least 13 regulated genes that are, so far, not annotated with an experimentally verified function, predicted biologic process or molecular function. This provides a basis for further experimental studies to provide more direct information on gene function.In summary, we successfully downregulate the expression of β-catenin and also the transcriptional activity of Wnt/β-catenin pathway in HeLa cells in a stably inducible manner. Microarray data suggest that a series of pro-apoptotic genes, which may be involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway may contribute to the enhanced apoptosis. Further studies are needed to identify the precise mechanism underlying the Wnt/β-catenin pathway in apoptosis.APC = adenomatous polyposis coli; DAP3 = death-associated protein 3; DOX = doxycycline; FADD = Fas-associated via death domain; GSK3β = glycogen synthesis kinase 3β; PDCD5 = programmed cell death 5; PI3K = phosphatidylinositol 3-kinase; PTEN = phosphatase and tensin homolog; RNAi = RNA interference; siRNA = small interfering RNA; TCF = T cell factor; TUNEL = TdT-mediated dUTP nick end labeling.The author(s) declare that they have no competing interests.WYH established the stable transfectants of pTER-β-catenin, performed western blot and reporter assays, and wrote the manuscript. HML designed, annotated the microarray, performed the statistical analysis and constructed website. SDC, YYB and WSQ were responsible for construction of microarray. ZHX performed the RNA extraction. ZW and YSB performed the RT-PCR assay. QLP and BJF conducted the cell culturing. CQ provided some ideas and suggestions on dealing the parental T-REx™-HeLa cells. XNZ and LSG designed the study and were corresponding authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:all microarray data and annotation. The data provided all probes annotation in the microarray and expression valuesClick here for file130 differential expressed genes from microarray. The data provided differential expressed genes with ID, name, location and p-values.Click here for fileSAM result of microarray data. The data was producted by SAMClick here for file


The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines , and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease. Both hyper- and hypoactivation of this pathway have been linked to disease High-throughput RNA interference (RNAi) screening in cultured human cells represents a powerful, unbiased, and comprehensive genetic approach for dissecting the contribution of individual genes to signaling pathways or cellular functions. By use of libraries of chemically synthesized small interfering RNAs (siRNAs), or small hairpin RNAs (shRNAs) expressed from vectors and packaged as pseudotyped viral particles, we and others have shown the utility of RNAi screens to define modulators of clinically relevant and investigative compounds We screened RKO colon carcinoma cells that express a β-catenin responsive firefly luciferase reporter (BAR) P value less than 0.01  added third shRNAs targeting GSK3β and DHFR to this list of BIO enhancers , and packaged them into lentiviral particles. As expected, in concert with a sub-optimal dose of BIO, the DHFR shRNAs induced robust activation of BAR to levels similar to that induced by shRNAs targeting GSK3β . The thrTo investigate how DHFR inhibition enhanced the effect of GSK3 inhibition, we tested whether inhibition by shRNA silencing or methotrexate led to an increase in β-catenin mRNA and protein levels. To examine these possibilities, we transduced RKO-BAR cells with DHFR shRNA and control viruses, exposed cells to BIO or vehicle control, and then measured the accumulation of β-catenin protein by Western blot and β-catenin mRNA by Q-PCR. Consistent with an impact on the β-catenin destruction complex, silencing of DHFR increased β-catenin protein levels in concert with BIO, but no increase in the transcript was observed . This reDHFR regulates the regeneration of tetrahydrofolate, a carrier of one-carbon units used in a variety of biosynthetic processes including synthesis of pyrimidines, S-adenosylmethionine (SAM), and purines Given that addition of SAM blocked methotrexate enhancement of BIO-induced β-catenin protein accumulation, we questioned whether aberrant methylation may impact expression of GSK3β or other members of the destruction complex. Measurement of mRNA levels of Axin1, Axin2, β-TrPC, CK1α, CUL4b, and GSK3β by Q-PCR revealed no substantial changes following shRNA- or methotrexate-induced inhibition of DHFR alone or in combination with BIO compared to controls . AlthougWe next examined whether inhibition of DHFR could interfere with the ability of GSK3 to phosphorylate substrates such as β-catenin. Because GSK3 regulates β-catenin stability by phosphorylating β-catenin serine and threonine residues (Ser33/37 and Thr41) we examined whether DHFR inhibition affected this phosphorylation event. To enable a comparison of the phosphorylation status of β-catenin between cells with active GSK3 versus potentially inhibited GSK3, β-catenin degradation was blocked in all samples by treatment with the proteasome inhibitor MG132. In line with our previous experiments, exposure to methotrexate had no affect on β-catenin protein levels or phosphorylation status compared with control cells treated with MG132 alone. In contrast, treatment of cells with a fully active concentration of BIO (2.5 µM) completely inhibited GSK3-mediated phosphorylation of β-catenin (Ser33/37/Thr41) as well as laddering of total β-catenin on the protein gel, which is suggestive of decreased β-catenin ubiquitination. Importantly, cells treated with the dose of BIO used in our screen (312 nM) exhibited only modest decreases in β-catenin phosphorylation and laddering, and these effects were significantly enhanced by co-treatment with methotrexate . SimilarPhosphorylation and inactivation of GSK3β occur in response to growth factor stimulation of receptor tyrosine kinases and subsequent activation of PI3K signaling. Since enhancement of GSK3 inhibitor-mediated BAR activation by methotrexate correlated with increased inhibitory phosphorylation of GSK3β , we examGSK3 promotes pro-inflammatory signaling downstream of Toll-like receptor (TLR) activation, and GSK3-specific inhibitors have been shown to significantly decrease TLR-mediated inflammatory responses. Pre-treatment of primary immune cells with GSK3 inhibitors substantially increases production of the anti-inflammatory cytokine IL-10 while suppressing production of pro-inflammatory cytokines including TNFα, IL-6, and IL-12 High-throughput RNAi screening provides an unbiased and powerful means to interrogate attractive drug targets with complex signaling functions. Such screens can reveal unexpected interactions that impact mechanisms of target regulation and can ultimately promote advancement strategies for target-specific inhibitors in the clinic. A growing body of literature indicates that positive modulation of β-catenin signaling has potential in the treatment of a wide range of diseases, and small molecule inhibitors of GSK3 represent one means to potentiate this pathway To our knowledge, this is the first report of modulation of GSK3 activity by inhibition of DHFR. However, links between folic acid intake and β-catenin have been observed previously. Patients with rectal adenomas treated with dietary folic acid supplementation for one year exhibited reduced nuclear translocation of β-catenin and reduced cellular expression of inactive phospho-Ser9-GSK3β compared with placebo treated controls The mechanism of the interaction between DHFR inhibition and β-catenin/GSK3 is likely to be complex and due to cross-talk between these signaling pathways rather than direct activation of GSK3 by DHFR. Although DHFR inhibition increased the potency of BIO, in the absence of BIO, methotrexate and RNAi-induced silencing of DHFR had no effect BAR activity, β-catenin accumulation, or β-catenin phosphorylation. Evidence shown here suggests that alterations in methylation caused by DHFR inhibition may underlie the synergy with BIO. However, to what extent the observed synergy is due to changes in DNA methylation versus changes in protein methylation, or both, remains to be determined. The observation that exposure to adenosine blocked the synergistic effect of combined DHFR and GSK3 inhibition opens the possibility that signaling events outside of those perturbed by changes in methylation may play a role in regulating pathway cross-talk. To date, four key mechanisms have been identified that contribute to regulation of GSK3. These include regulation by phosphorylation of GSK3 itself, the subcellular localization of GSK3, protein-protein interactions with GSK3, and the phosphorylation status of GSK3 substrates Given the recent demonstration of anti-inflammatory properties of GSK3 inhibitors, and the use of low dose methotrexate as the gold standard treatment for rheumatoid arthritis, our data raise several interesting possibilities for the use of these inhibitors in the clinic. The observation that dual inhibition of GSK3 and DHFR led to a synergistic anti-inflammatory response suggests that the combination of DHFR and GSK3 inhibitors may have merit for use in the clinic. However, it is unclear whether the potent anti-inflammatory effect of combined inhibition requires activation of β-catenin signaling. The ability to separate the anti-inflammatory and pro-oncogenic effects of dual DHFR and GSK3 inhibition will ultimately determine the utility of the proposed combination therapy for treatment of chronic inflammatory conditions. Interestingly, rheumatoid arthritis patients treated with methotrexate were found to have a 50% increased risk of developing certain cancers 5′- GTAGACATGGTCTGGATAG-3′), DHFR2 (5′-GACTTTGAAAGTGACACGT-3′), and DHFR3 (5′- GGTAAACAGAATCTGGTGA-3′).293FT producer cells  were maintained as per the manufacturer's specifications in DMEM supplemented with 10% FBS, and 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, 1% Pen/strep, and 500 µg/ml Geneticin (Invitrogen). HT29  and RKO-BAR cells engineered to express a β-catenin responsive firefly luciferase reporter (BAR)Automated high-throughput lentiviral vector production was performed as described previously Infection conditions were optimized for screening in 384-well format. Robot-assisted lentiviral infection of RKO-BAR cells was carried out as follows. One set of 180 96-well infection-ready plates (IRPs) comprising the screening library was thawed on ice and kept at 4°C prior to infection. 384-well screening plates were primed for infection by addition of 15 µl of plating medium  to each well with a WellMate® bulk dispensing apparatus . Next, four IRPs were then transferred (10 µl of virus/well) into each screening plate in triplicate by use of a Biomek® FX Laboratory Automation Workstation . RKO-BAR cells were harvested by trypsinization and reverse transduced by seeding of the virus-containing screening plates at a density of 1200 cells/well in the presence of 6 µg/ml polybrene in a final volume of 35 µl of growth medium using the bulk dispenser. Forty-eight hours after infection, BIO was added to a final concentration of 312 nM and firefly luciferase was measured by use of a BriteLite luciferase assay  24 hours later.P value of testing no mean difference P values were then used to calculate the false discovery rate (FDR) by use of methods described by Strimmer Smoothing methods were used to adjust for positional effects including systematic edge effects mRNA silencing was quantified by real-time PCR with an ABI PRISM 7900HT Sequence Detection System and Assays-on-Demand™ gene expression products as described previously Human IL-6, IL-10, and TNFα in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA) kits from Invitrogen. IL-12 was measured with an ELISA kit from Pierce, Rockford, IL.Figure S1DHFR inhibition does not decrease mRNA expression of known components of the β-catenin destruction complex. RKO-BAR cells were transduced with DHFR shRNAs or were exposed to methotrexate with or without BIO and RNA was harvested for Q-PCR analysis with probes specific for Axin1, Axin2, β-TRPC, CK1α, CUL4b, and GSK3β. Of the genes tested, a modest, albeit consistent, reduction in Axin2 expression alone was observed upon silencing and methotrexate inhibition of DHFR. However, as expected with activation of β-catenin, exposure to the combination of BIO and methotrexate led to an increase in Axin2 expression, and Axin2 shRNAs did not lead to GSK3 inhibitor enhancement in our screen. It is therefore unlikely that a reduction in Axin2 levels alone underlies the synergistic interaction between DHFR and GSK3 inhibitors.(0.04 MB DOC)Click here for additional data file.Figure S2Establishment of minimal titer values in GFP units required to detect enhancement of BIO-mediated activation of BAR by GSK3β shRNA. Because it is difficult to normalize viral titers across a large collection of packaged vectors we established a minimal titer threshold that would be expected to result in BAR activation if gene silencing enhanced BIO potency. To establish a minimum effective titer, we exposed HT29 cells to serially diluted control virus  or virus targeting GSK3β. GFP expression was measured 96 h post transduction to determine titer values in terms of fluorescent light units on an Acumen Explorer laser scanning microplate reader . In parallel with the HT29 infections, RKO-BAR cells were infected with same serially diluted control virus or lentiviral shRNA targeting GSK3β. Forty eight hours after infection, cells were exposed to BIO at a concentration (312 nM) that induces luciferase-based luminescence only upon infection with an shRNA capable of negatively modulating GSK3 function. Significance of enhanced activation of luciferase by the GSK3β shRNA infected cells over that observed in control vector infected cells is plotted on the Y-axis. GFP values as Log total intensity fluorescent units as a measurement of viral titer is plotted on the X-axis. Each data point represents the comparison of 4 replicate wells for each dilution of U6-TERM to GSK3β shRNA vector. As expected, we observed a strong correlation between titer of GSK3β shRNA virus and enhancement of luciferase activation by BIO. The arrow denotes the titer value in GFP units (1.7×107 FLU) required for 5-fold enhancement of luciferase activation with a P value of 1×10-4. We set this as our minimal titer value for effective lentiviral shRNA production for this screen.(0.03 MB DOC)Click here for additional data file.Figure S3Analysis of titer values for the lentiviral shRNA screening library. Titer values were established based on GFP expression as described in (0.07 MB DOC)Click here for additional data file.Table S1Statistical analysis of top shRNA screen hits. Gene Symbol and ID numbers for the intended targets of each shRNA screen hit are provided. Hits are ranked by strictly standardized mean difference (SSMD) using a cut-off of SSMD >1.6. All statistical metrics were calculated as described in procedures. Each hit listed represents a distinct shRNA sequence and the three DHFR shRNA vectors are highlighted in bold.(0.03 MB DOC)Click here for additional data file.


In the rectal carcinomas, there was a significant inverse association between the expression of p120-catenin in cell membranes of the primary tumors and the occurrence of local recurrence, while membranous protein expression of β-catenin was inversely related to distant metastases.The E-cadherin-catenin complex provides cell-cell adhesion. In order for a carcinoma to metastasize, cancer cells must let go of their hold of neighboring cells in the primary tumor. The presence of components of the E-cadherin-catenin complex in 246 rectal adenocarcinomas was examined by immunohistochemistry and compared to their presence in 219 colon carcinomas. The expression data were correlated to clinical information from the patients' records. There were statistically significant differences in protein expression between the rectal and the colon carcinomas regarding membranous  It is located at the cell membrane of epithelial cells and consists of a chain of proteins. The outermost element is E-cadherin, also called uvomorulin, which is a transmembrane glycoprotein . E-cadheto actin . α-caten-catenin , 11 or γkoglobin , which i-catenin  and γ-ca-catenin  also areβ-catenin, that is not bonded, translocates from the cytosol to the nucleus. Here, in a complex with T cell factor (TCF) and Lymphoid enhancer-binding factor (LEF), β-catenin controls expression of several genes, among them Snail1 [β-catenin that would otherwise be combined with E-cadherin in the E-cadherin-catenin complex by the cell membrane [β-catenin can enter the Wingless pathway, so causing increased expression of Snail1.In the Wingless (=Wnt) pathway, m Snail1 . Snail1 m Snail1 . In EMT,m Snail1 , 17. Themembrane . The libβ-catenin signaling, whereas higher levels of Kaiso expression inhibit β-catenin signaling [p120-catenin stabilizes E-cadherin by controlling E-cadherin turnover at the cell membrane. p120 absence dramatically accelerates E-cadherin degradation , 20. p12ignaling .Adherens junctions are cellular structures found near the apical surface of polarized epithelial cells. They provide cell-cell adhesion. The E-cadherin-catenin complex is typically found in adherens junctions , 26, butIn order for a carcinoma to metastasize, cancer cells must detach from neighboring cells in the primary tumor. This process requires a malfunction of the E-cadherin-catenin complex. Several studies have demonstrated reduced expression of E-cadherin –35 and cA simultaneous investigation of the protein expression of both E-cadherin and all the catenins in the E-cadherin-catenin complex in only rectal carcinomas has so far never been performed. Most of the previous studies have included both rectal and colon carcinomas. Since there are clinical differences in prognosis and outcome between patients operated for rectal and colon carcinomas, as well as reports about tumor biological differences between these two tumor types , we aimeAvailable paraffin-embedded tumor samples from a consecutive series of 274 rectal adenocarcinomas removed surgically at Akershus University Hospital in the years 1992–2000 were scrutinized for inclusion into the survey. These surgical treatments were all primary operations. Tumors at a level of 15 centimeters (5.9 inches) or less from the anal verge  were included (246 patients), in accordance with the somewhat arbitrary range of 15 to 18 centimeters commonly used to define the border between rectum and colon. The most restrictive border was chosen in order to avoid unintentional inclusion of sigmoid tumors. The clinico-histopathological characteristics of the patients are shown in Out of 246 patients, 25 experienced a local recurrence of their rectal cancer. Among those included in the statistical analyses, mean time from primary operation to local recurrence was 2.4 years. Minimum time was six months, maximum 6.0 years. For patients with no local recurrence, mean observation time was 6.7 years (range 4 days–14.5 years). Out of 246 patients, 49 developed distant metastases from their rectal cancer. Among those included in the statistical analyses, mean time from primary operation to distant metastases was 2.3 years. Minimum time was two months, maximum 12.9 years. For patients without distant metastases, mean observation time was 7.2 years. Minimum observation time was four days, maximum 14.4 years.This material of rectal carcinomas was compared to a material examined by Bondi et al. of 219 colon carcinomas operated on at Akershus University Hospital during the years 1988, 1990, and 1997–2000 , 50.Serial sections (3-4 micrometers) from formalin fixed, paraffin wax embedded archive tumor tissue were applied to coated slides before immunohistochemical staining, where different methods were used.β-catenin, sections were dried at 50–60°C overnight, deparaffinized and rehydrated before antigen retrieval was performed using microwave technique (20 minutes at 100°C). Staining was done in Dako Autostainer .For α-catenin, p120-catenin, and E-cadherin were dried overnight at 50–60°C before pretreatment with Dako PT link (20 minutes in 98°C), and staining in Dako Autostainer , using Dako's EnVision Flex-system, with mouse-linker in p120-catenin and E-cadherin.Sections for γ-catenin, sections were fixed in incubator 30–40 minutes at 56°C, and overnight at 37°C. The immunostaining was performed in Ventana ES Benchmark automated slide stainer , antibody diluent (251-018). The details of each antibody used are shown in For The antibodies were visualized for light microscopy with Envision Plus-System and diaminobenzidine (DAB), and with Detection Kit Ventana iViewTM DAB, respectively. Counter-staining was done with Hagen's haematoxylin for visualization of tissue structures. Positive control was a test block with normal colon mucosa and multiple colonic adenocarcinomas with diverse differentiation.The percentage of positive cell membranes was counted semiquantitatively by applying four defined grades of immunopositivity. When 60% or more of the cancer cells were stained, we scored the tumor as grade three. Staining of 30%–59% of the cells was classified as grade two. When 5%–29% of the cells were stained, the score was grade one. Positivity in less than five percent of cells qualified for grade zero. The protein expression was regarded positive regardless of whether the expression was mainly at the apical portion of the cell , 52 or iβ-catenin was done in the same way as for the membranes, except for that the border between grade zero and grade one was set to zero percent of the nuclei being stained. The reason for this is that normally, there should be no staining of nuclear β-catenin at all. Only clearly nuclear staining was recorded as positive.Scoring of nuclear staining of We intended to compare our results from rectal cancers to results from a similar examination of colon cancers previously performed. Therefore, our method for quantifying proteins in question had to be the same, with the same cut-offs between the scored grades. Originally, the choice of four levels of staining were applied as a compromise between the wish of detailed measurements and the wish of not giving the impression of being more accurate than manual human judgment is in real life. One intention with the system was that alterations in protein staining in a cancer should be larger than alterations naturally occurring in normal mucosal tissue before they were recorded as pathological. For most slides, we were able to check that this intention was followed, because almost all slides contained normal adjacent mucosa in addition to the cancer. The normal mucosa served as an internal control. In other, earlier, similar investigations, varying cut-offs have been used, and no unified, generally applied standard seems to exist for this. So, in addition to the above mentioned, the original cut-offs were somewhat arbitrarily chosen.At least 100, usually more than 1000 cells, were examined in each slide. All slides were primarily judged by RAA. In addition, judgment of slides was done by AB, IRKB, JB, or SNA, depending on the type of slide in question. When necessary, consensus was achieved by simultaneous examination by two examiners in a double microscope.P < .05 was chosen.Statistical analyses were performed by SPSS version 16.0 running on Windows XP. Binary logistic regression analysis and Cox regression analysis were performed. We made test plots for proportional hazards for the Cox analyses and found them satisfactory. An alpha level of statistical significance of α-catenin, β-catenin, γ-catenin, p120-catenin, and E-cadherin, and also nuclear β-catenin, into the same multivariate analysis.In all statistical analyses, we included patient gender, tumor differentiation grade, Dukes' tumor stage, and patient age at surgery as well as the five membrane proteins Figures β-catenin in the rectal and the colon adenocarcinomas are shown in β-catenin, γ-catenin, p120-catenin and E-cadherin, and regarding nuclear staining of β-catenin . There was also a significant inverse relationship between the expression of β-catenin in cell membranes of the primary tumors of the rectal cancers and the occurrence of metastases . In the rectal carcinomas, there was a significant inverse relationship between the expression of p120-catenin in cell membranes of the primary tumors and the occurrence of local recurrence , a process crucial for the metastatic process. The present study indicates that the EMT process may be different in rectal and colon adenocarcinomas within the same tumor stage.Results from the present study strongly indicate that rectal and colon adenocarcinomas should be separated before analyzing the prognostic value of proteins involved in the adhesion complexes of the cells.The present research is one of the first studies demonstrating tumor biological differences with significance on protein level between rectal and colon adenocarcinomas, regarding the metastatic process. These differences in tumor biology may explain some of the differences in clinical behavior of these two tumor types, but such differences are so far not taken into account when planning the treatment options. The observation that p120-catenin may be related to decreased risk of experiencing local relapse may help stratifying patients, together with other modalities, in risk groups for relapse.


Three related proteins of the plakophilin family (PKP1_3) have been identified as junctional proteins that are essential for the formation and stabilization of desmosomal cell contacts. Failure of PKP expression can have fatal effects on desmosomal adhesion, leading to abnormal tissue and organ development. Thus, loss of functional PKP 1 in humans leads to ectodermal dysplasia/skin fragility (EDSF) syndrome, a genodermatosis with severe blistering of the epidermis as well as abnormal keratinocytes differentiation. Mutations in the human PKP 2 gene have been linked to severe heart abnormalities that lead to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the past few years it has been shown that junctional adhesion is not the only function of PKPs. These proteins have been implicated in cell signaling, organization of the cytoskeleton, and control of protein biosynthesis under specific cellular circumstances. Clearly, PKPs are more than just cell adhesion proteins. In this paper we will give an overview of our current knowledge on the very distinct roles of plakophilins in the cell. Cellular adhesion is mediated by distinct protein complexes at the cytoplasmic membrane, termed junctions, that have been characterized by their morphology on the ultrastructural level . Desmosoctn-group, which are associated with classical cadherins, such as E-cadherin, in adherens junctions. The PKPs are more distantly related to the classical catenins, β-catenin and plakoglobin [Plakophilins are probably the most basic proteins identified in cellular adhesion complexes so far with an isoelectric point (pI) of about pH 9.3. Based on their primary sequences, PKPs have been classified as a distinct subfamily of the armadillo repeat proteins (for review see ). The cakoglobin , 13. PKPstratum corneum of stratified squamous epithelia though  is less abundant in stratified epithelia. The additional amino acid sequence contained in PKP1b is encoded by exon 7 which is spliced out of the PKP1a mRNA . The PKPβ-catenin, plakoglobin, and p120ctn, have been postulated for PKP 1, but proof is still lacking [Both the nuclear and desmosomal PKP 1 pool are degraded by caspases rapidly during apoptosis of keratinocytes suggesting that this protein is involved in the remodeling of the cytoskeleton under these conditions . Signali lacking , 22. A t lacking . The mecVarious in vitro approaches revealed that the binding of desmosomal PKP 1 to other desmosomal proteins such as DSP, DSG 1, DSC 1, and different keratins is mediated by its head domain sequence –28. The Important clues for the understanding of PKP 1 function came from a report of an autosomal-recessive genodermatosis that is caused by mutations in the PKP 1 gene . The ectThese findings in conjunction with cell biological data obtained in transfection studies convincingly illustrate that PKP 1 is essential for the recruitment of desmoplakin to the desmosomal plaque and probably is involved into lateral enlargement of the plaque structure in skin, explaining the structural and functional defects in epidermal desmosomes lacking PKP 1. Evidently, integration of PKP 1 in the desmosomes provides the epidermal keratinocytes with stability against mechanical stress. A sequence stretch in the HR2 domain of PKP 1 is thought to be essential for the recruitment of DSP and represents a conserved motif of all the PKPs, suggesting that DSP recruitment is a common function of all PKPs . pemphigus group in which autoantibodies target desmosomal cadherins. Binding of autoantibodies to the desmosomal cadherins seems to trigger intracellular signaling pathways that lead to the reorganization of the cytoskeleton involving the disconnection of desmosomal cadherins of adjacent cells , for anchorage of keratins  suggestiling see ). The saAnalysis of keratinocytes derived from patients suffering from EDSF syndrome exhibits some interesting properties. Quantitative analyses of the desmosome size in cultured cells revealed that reintroduction of PKP 1 increases the lateral extent of desmosomes. As proposed by others , 40, desPKP 2 is, with a predicted mass of 92.756 Da and an apparent molecular weight of 100 kDa , the largest of the three plakophilins and it is also the prevailing isoform since it is expressed in all cell types with desmosomal junctions . PKP 2 iLike PKP 1, PKP 2 has been detected in the nucleus of many cell types . Its preβ-catenin or p120ctn, which are translocated into the nucleus upon specific signals and have been shown to be involved in gene regulation [What does PKP 2 do in the nucleus? Recent experiments by Mertens and colleagues provided some insights . Immunopgulation , 22. β-catenin [β-catenin. However, when bound to PKP 2, β-catenin cannot associate to E-cadherin, which may reduce the pool of β-catenin available to function in cell adhesion. Overexpression of PKP 2 in colon carcinoma cells leads to an increase in β-catenin/TCF signaling suggesting a regulatory role of PKP 2 in Wnt signaling and providing a potential functional link between desmosomal adhesion and signaling [Besides these nuclear functions, PKP 2 may be involved in cytoplasmic signaling, which is based on the observation that it can bind -catenin , a key d-catenin . Using tignaling . α  phosphorylation site. Different isoforms of PKC have been implicated in the regulation of cellular processes such as migration, cellular adhesion, or cytoskeletal reorganization . Bass-Zview see . The autn by PKC . This in areae compositae  that co animals . This isβ-catenin-signaling [β-catenin-signaling pathway [The essential function of PKP 2 in the heart was also demonstrated by the identification of a haplo-insufficiency of PKP 2 in a hereditary human disease, autosomal-dominant arrhythmogenic right ventricular cardiomyopathy . In ARVignaling , 54. Thi pathway , suggest areae compositae in heart muscle cells.The loss of PKP 2 may also contribute to the abnormal electrical conductance of the heart . Gap junPKP 3 has a calculated mass of 87,081 Da and is detected with an apparent molecular weight of approximately 87 kDa on Western blot analysis , 58. StrA better understanding of the functions of PKP 3 came from the analyses of PKP 3 knockout mice . In cont in silico the most extensive interaction rate of desmosomal proteins, as predicted for keratinocytes by Cirillo and Prime [Surprisingly, among the three plakophilins, PKP 3 exhibits the most extensive binding repertoire to other desmosomal components  and it dnd Prime . It is cnd Prime . This imnd Prime .Further PKP 3 interaction partners are emerging, that are not linked to cell adhesion, suggesting a broader biological role of PKP 3. PKP 3 has been shown, for example, to interact with RNA-binding proteins such as poly-A binding protein C1 (PABPC1), FXR1 , and G3BP (GAP SH3 domain-binding protein) in stress granules . Stress Another PKP 3-binding protein identified is dynamin-like protein DNM-1L . DNM-1L β-catenin, have been shown to be important in the development, progression, and metastasis of tumors [Cellular adhesion molecules, especially components of the adherens junctions such as E-cadherin and f tumors . Likewisf tumors ). Reliabf tumors . Tumor cf tumors . In solif tumors , 70. Thif tumors , who fouInterestingly, in a part of oral and pharyngeal SqCCs analyzed by Schwarz et al. , nuclearAn analysis of adenocarcinomas from different organs such as colon and pancreas revealed that PKP 1 is not detected whereas PKP 2 and PKP 3 are frequently expressed , 72, somIn summary, in the past few years PKPs have been recognized to be essential for desmosomal adhesion and tissue integrity. Nevertheless, recent data suggest that PKPs exert cellular functions unrelated to cell adhesion. Further questions like the ability of individual PKPs to compensate for the loss of one isoform and the role of PKPs in cell signaling and in tumor development need to be further investigated.


Gamma motor neurons (γ-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from α-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate γ-MNs. Recently, it was reported that early embryonic γ-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing γ-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which γ-MNs are selectively lost.Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfrα1, we demonstrated that small motor neurons with high Gfrα1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of γ-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfrα1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of α-MNs with β-skeletofusimotor collaterals. These molecular markers can be used to identify γ-MNs from birth to the adult and to distinguish γ- from β-motor axons in the periphery. We also found that postnatal γ-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of γ-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of γ-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development.In mice heterozygous for both the Neonatal γ-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfrα1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of γ-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of γ-fusimotor activity in motor behaviors. Muscle spindles provide proprioceptive information required for motor control. Unlike other mechanoreceptors, the sensitivity of muscle spindles is actively regulated by a specialized fusimotor system. This allows for continuous control of the mechanical sensitivity of spindles over the wide range of lengths and velocities that occur during normal motor behaviors . FusimotMost motor pools contain both α- and γ-MNs, which derive from common progenitors and then differentiate to form specific cell types that differ in morphology, physiology and connectivity . InveRecent work has shown that survival of γ-MN precursors during embryonic development is selectively dependent on glial cell line-derived neurotrophic factor (GDNF) ,12. AbseGDNF gene (GDNFFLOX) demonstrates that the selective dependence of γ-MN survival on GDNF continues after birth and that muscle spindles are a critical source of this factor in the postnatal period. The conditional elimination of GDNF expression from muscle spindles in the mouse results in selective γ-MN loss with no obvious effect on other spindle components or α-MNs, and so provides a model to investigate the specific role of γ-fusimotor activity in motor behaviors.To address these issues, we investigated the expression pattern of the GDNF receptor Gfrα1 and that of several other markers in postnatal MNs and defined molecular and genetic criteria that can be used to identify postnatal γ-MN somata independent of size and to distinguish γ- from β-fusimotor axons in the periphery. Furthermore, genetic elimination of spindle-derived GDNF using a novel conditional allele of the GDNFFLOX allele was constructed from an approximately 8 kb SphI/NcoI fragment containing the GDNF coding sequence of exon 3. loxP sites were placed in the intronic sequence just upstream of exon 3 and in the 3' untranslated region of the GDNF gene. A neomycin-resistance expression cassette flanked by FRT sites was inserted upstream of the 5' loxP site. The linearized targeting construct was electroporated into W9.5 embryonic stem cells, selected with G418 and screened for homologous recombinants by Southern analysis (EcoRV digest) using as probe a 2-kb fragment downstream of the 3' end of the targeting construct. The frequency of recombination with this construct was low (<1%). Recombinant clones were injected into C57BL/6J blastocysts to generate chimeric founders. After germline transfer of the GDNFFLOX+NEO allele was confirmed, the neomycin cassette was excised by crossing F1 animals to ACTB-FLPe mice [GDNFFLOX allele. The GDNFFLOX allele was maintained on a predominantly C57BL/6J strain background.Mouse GDNF genomic clones from a 129sv/J genomic library were kindly provided by Jose Pichel . The tarLPe mice  to generHlxb9-GFP1Tmj (Hb9::GFP) [Egr3NULL [ErbB2FLOX [ErbB2NULL [myf5-CRE [GDNFLacZ [Other mouse lines used in this study were previously characterized and generously shared, and include Gfrα1-TLZ , Hlxb9-Gb9::GFP) , Egr3NULEgr3NULL , ErbB2FLrbB2FLOX , ErbB2NUrbB2NULL , myf5-CRmyf5-CRE , and GDNGDNFLacZ . All aniAt postnatal day 18, animals were deeply anesthetized by halothane induction, and individual muscles were surgically exposed and pressure injected using a glass micropipette with 1 to 2 μl of a 2.5% solution of Fast Blue diluted in 0.01 M phosphate buffered saline . After recovery from surgery, animals were held for 48 to 72 hours, and then transcardially perfused with 4% paraformaldehyde diluted in 0.01 M PBS. Isolated spinal cords were immersion-fixed overnight in the same fixative solution and washed in PBS before processing for immunocytochemical analysis.Spinal cords were dissected from postnatal day (P)5, P20 or P60 animals that were transcardially perfused with PBS followed by 4% paraformaldehyde and postfixed overnight, as above. For sectioning, the spinal cords were embedded in warm 5% agar and serial transverse sections (75 μm thick) cut in a vibratome and processed free-floating. The sections were blocked with 10% normal donkey serum diluted in PBS with 0.1% Triton X-100 (pH 7.4) and incubated overnight at room temperature in primary antisera diluted in PBS with 0.1% Triton X-100. The following day, immunoreactive sites were revealed with different species-specific goat or donkey secondary antibodies, depending on the experiment, and coupled to Alexa 488, 555, 647 , Cy3 or Cy5 . All fluorescent sections were mounted in anti-fading solution Glycerol:PBS (3:7) or Vectashield .For analysis of neonatal spinal cords and muscle spindles, tissues were fixed overnight in 4% paraformaldehyde, washed in PBS, cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB) and then frozen in OCT . Cryostat sections were cut at a thickness of 14 to 20 μm and placed on glass slides for immunostaining using the same conditions as above.Primary antibodies used in this study were specific for vesicular glutamate transporter 1 , choline acetyl transferase , vesicular acetylcholine transporter ; β-galactosidase . Hb9::GFP fluorescence in MNs was detected without the use of immunocytochemical amplification, but in the periphery, green fluorescent protein (GFP) in motor axons was visualized with a sheep anti-GFP polyclonal antibody . Muscle spindles and motor axons in muscle sections were labeled with antibodies against the peripheral axon marker protein gene product 9.5  and acetylcholine receptor clusters at intrafusal and extrafusal neuromuscular junctions were labeled with Cy5-bungarotoxin .2 bins. In each animal distribution, histograms represent pooled data from six ventral horns. Depending on age, genotype and section thickness, approximately 40 to 80 MNs were counted per ventral horn. A minimum of three distribution histograms from three different animals of similar age/genotype were averaged in each experiment (exact numbers provided in the Results section). Average histograms were fit to either single or dual Gaussian distributions using Clampfit . From the fitted distributions we estimated the average cross-sectional area and standard deviation (SD) of the small and large size MN populations. From the raw histogram data we obtained relative percentages for each population according to cell size or phenotype. In the histograms, error bars always represent ± standard error of the mean (SEM). Depletions in certain genotypes were calculated against all ChAT+ MNs or the number of cells identified by a particular set of markers or cells below a certain threshold cutoff size. Cutoff sizes for the small population were estimated as the average (μ) + 2 SD (σ) of the fitted small population distribution in control animals of similar age.At each postnatal age, counts of MNs were performed on the lateral motor column of lumbar spinal segments L4 through L5 from z-series of confocal optical sections obtained at a magnification of 20× . Motor neurons labeled with a combination of ChAT, β-galactosidase and GFP were counted and measured using Neurolucida . All ChAT+ MNs imaged in each stack were outlined in the confocal plane where each exhibited the maximum cell body cross-section and classified according to their differential expression of Gfrα1-TLZ and Hb9::GFP. Distribution histograms were constructed for each animal by grouping cell body cross-sectional areas in 50 μmt-tests.Quantitative analysis of VGluT1 and VAChT immunoreactive  synaptic densities on MNs at postnatal day 20 were performed on z-series of optical confocal sections obtained at high magnification  throughout the entire cell body and proximal dendrites of randomly selected large diameter, Hb9::GFP+/Gfrα1-TLZ-/ChAT+ and small Hb9::GFP-/Gfrα1-TLZ+/ChAT+ MNs. VGluT1+ and VAChT+ contacts were counted over the surface of each MN. Contact densities on cell somata were estimated by measuring the diameter of each MN cell body in all three planes and calculating the surface area of each cell - modeled as an ellipsoid - as previously described . ContactGfrα1-TLZ/Hb9::GFP compound heterozygous animals were used to reconstruct individual Hb9::GFP+/Gfrα1-TLZ-/ChAT+ and Hb9::GFP-/Gfrα1-TLZ+/ChAT+ MNs using Neurolucida software . The MN cell body was traced in a middle cross-sectional optical section. Dendrite origins were located at the points at which the membrane changed from convex out to concave out. Each dendrite was manually traced by making discrete measurements along their paths and manually entering each branching point. Dendrite thickness was first entered at dendrite origins (by adjusting cursor size) and then readjusted at each measurement point. Reconstructed neurons were analyzed with Neuroexplorer software  to obtain information on the number of primary or higher order dendrites, their average thickness, branching patterns, total dendritic tree length, total dendrite surface and dendritic lengths and surfaces of individual dendrites or dendritic segments at different Sholl distances. Sholl analysis calculated the amount of surface membrane in dendritic segments at different distances from the cell body. For this purpose a set of nested concentric spheres was centered at the cell body with the spheres separated by 50 μm, creating a series of shells of increasing distance from the cell body. The total surface area of all dendrite segments contained within each shell was added to obtain an estimate of the distribution of dendritic surface membrane at increasing distances from the cell body. Fine caliber distal dendrites were better resolved with the Gfrα1-TLZ marker compared to Hb9::GFP-labeled dendrites. As a result, longer dendritic segments were analyzed in Gfrα1-TLZ+ MNs. Therefore, the most meaningful comparisons focused on the more proximal dendritic tree that was equally sampled in both MN cell types.To analyze MN morphology and dendritic structure, confocal images obtained from P20 In situ hybridization analysis was performed with digoxigenin-labeled cRNA probes [in vitro transcription of the GDNF cDNA.A probes  specificA probes  and GDNFGDNFFLOX/FLOX Egr3CRE/CRE double homozygote and one P18 GDNFFLOX/+ Egr3+/+ control animal were transcardially perfused with 4% paraformaldehyde and 4% glutaraldehyde diluted in 0.1 M PB. The tibialis anterior muscle (studied here) and several others were dissected, washed, postfixed with 2% OsO4 in 0.1 M PB and embedded in Spurr resin. Serial semithin sections were obtained transversally through the blocks, contrasted with 1% toluidine blue/borax and the equatorial and polar regions of muscle spindles imaged with bright field light microscopy. Images were digitally recorded with a 60× or 100× objective and using a RT-Spot Camera .One P18 Hb9-GFP1Tmj transgene (Hb9::GFP). In Hb9::GFP animals, the murine HB9 promoter drives expression of enhanced GFP in MNs and a subpopulation of ventral interneurons [Previous studies have demonstrated that Gfrα1 is expressed in a subset of MNs -28 and trneurons ,29,30.2 ( ± SD) while the large size population showed a wider distribution around a mean of 755 ± 220 μm2. The distribution of all Gfrα1-TLZ+ neurons was also best fit by a bimodal distribution (correlation = 0.9) with the same estimated parameters as the entire population of ChAT+ neurons . The percentage of ChAT+ neurons that were Gfrα1-TLZ positive was significantly different in the small versus large size groups. We used 485 μm2 in area  as the cutoff point to distinguish the small and large populations. Using this criterion, small MNs represent 34 ± 1.4%  of all ChAT+ MNs, of which 91 ± 2% are strongly Gfrα1-TLZ positive  and represent approximately 66 ± 1% of all large MNs at this age  ChAT+ neurons and 24 ± 3% of large (>485 μm2) ChAT+ neurons at P20.We also observed that small ChAT+ MNs that displayed strong Gfrα1-TLZ immunoreactivity lacked Hb9::GFP expression, while MNs that were larger and weakly immunolabeled with Gfrα1-TLZ were frequently Hb9::GFP positive Figure . At P20,e Figure . Double-e Figure . Thus, se Figure , but theThe expression of Gfrα1-TLZ and the expression of Hb9::GFP in MNs are independently regulated in postnatal development and become increasingly restricted with age to distinct but overlapping subpopulations Figure . At P1 -In the course of our MN cell counts, we also made the unexpected observation that small Gfrα1-TLZ+/Hb9::GFP- MNs generally lack immunoreactivity for the neuronal nuclear protein NeuN  Figure . The larIn conclusion, small MNs are generally characterized by strong Gfrα1-TLZ expression, low NeuN immunoreactivity and lack of Hb9::GFP. We hypothesize that these markers define new molecular criteria by which to identify γ-MNs at birth, well before other distinguishing features are expressed in mature α- and γ-MNs.In addition to small size, mature γ-MNs are characterized by a distinct dendritic morphology -35, lackTo confirm the γ identity of Gfrα1-TLZ+/Hb9::GFP- MNs, we first compared their dendritic arborization with that of Hb9::GFP+ MNs Figure . At P20,P < 0.001, t-tests). The somatic density of VAChT-IR contacts on Gfrα1-TLZ+/Hb9::GFP- MNs . These data describe a cholinergic input to Gfrα1-TLZ+/Hb9::GFP- MNs that is distinct from the large C-type cholinergic inputs previously identified by electron microscopy only on α-MNs. The dendritic morphology and synaptic input of the Gfrα1-TLZ+/Hb9::GFP- neurons are therefore consistent with their identification as γ-MNs.Second, using VGluT1 as a marker of primary afferent contacts -46 and Vs Figure  was onlyIn the rat, the incidence of γ and β efferents differ from muscle to muscle, but the large majority of motor inputs on intrafusal fibers in hindlimb muscles are γ motor axons . Our finUsing these markers, we analyzed extrafusal motor endings (n = 105) in the tibialis anterior muscle of a P30 double heterozygous Gfrα1-TLZ/Hb9::GFP mouse and found that all were GFP+. In contrast, 91% (30 of 33) of intrafusal neuromuscular junctions identified in 12 individual tibialis anterior muscle spindles were innervated by motor axons that were Hb9::GFP-. Since all motor axons that innervate extrafusal tibialis anterior muscle are Hb9::GFP+, so too must be any β-skeletofusimotor collateral. Hb9::GFP- fusimotor axons must therefore originate from γ-MNs, providing further evidence that postnatal γ-MNs do not express Hb9::GFP.KO) mice, early muscle spindle development is abnormal, and muscle spindles degenerate in the postnatal period [KO mice that were heterozygous for Gfrα1-TLZ+/-  or double heterozygous for Gfrα1-TLZ+/- and Hb9::GFP+/- (n = 3) and analyzed the size distributions of L4-5 lumbar MNs expressing the different genetic markers. These were compared to Egr3 wild-type (Egr3WT) controls (n = 3) . This loss could be accounted for by 89% loss of small diameter, Gfrα1-TLZ+ MNs . There was no significant decrease in the number of large diameter MNs in the Egr3KO animals. Therefore, the proportion of small MNs (<485 μm2) that were Gfrα1-TLZ+ decreased from 31% ± 1 ( ± SEM) to 5 ± 1% of all ChAT+ MNs , identical to the large MN population observed in wild-type controls.In Egr3 mutant  Figure . In Egr3 Figures . In equis Figure . Moreove% Figure , and the% Figure 1. There s Figure . As a coKO mutants double-labeled with Gfrα1-TLZ and HB9::GFP . In contrast, the number of large MNs that were Gfrα1-TLZ+ and HB9::GFP+ declined by 78%. Since the total number of large ChAT+ and Hb9::GFP+ MNs is unchanged in Egr3KO mutants, the decrease in the number of dual-labeled Gfrα1-TLZ+/and HB9::GFP+ MNs is best explained by downregulation of Gfrα1 expression in surviving MNs. This spindle dependence of Gfrα1 expression in large MNs together with our finding that all motor axons innervating extrafusal tibialis anterior muscle fibers are Hb9::GFP+ suggest that large Gfrα1-TLZ/Hb9::GFP MNs may be the source of β-skeletofusimotor axons.In Egr3P Figure , there wErbB2NULL/FLOX/myf5CRE spindle mutants (ErbB2Δ in future text and figures), we also observed a marked 34% loss of ChAT+ MNs , and a decrease of small (<485 μm2) Gfrα1-TLZ+ MNs to 15 ± 2% of the total. When expressed as a percentage of all ChAT+ MNs, there appears to be an intermediate loss of γ-MNs in the ErbB2Δ spindle mutant that is significantly different from that found in control and Egr3KO animals . This difference can be explained by a significant decrease in the total number of large diameter MNs and the survival of some small MNs in the ErbB2Δ mutant  and this cell loss is distributed over all size bins. At P5, when a bimodal distribution of MN cell bodies is first evident, MN loss increases to 16%  mainly because of the selective loss of 65%  of small diameter γ-MNs  , roughly equivalent to the loss observed at P20 , P5 and P10 spinal cords from Egr3t Figure , there i) Figure . The los0 Figure . These dKO and ErbB2Δ animals using a targeted allele of GDNF (GDNFLacZ) in which β-galactosidase (lacZ) expression replaces GDNF [GDNFLacZ was crossed into the Egr3KO background, and hindlimb muscles analyzed at P5 for GDNF (lacZ) expression. Histochemical staining for β-galactosidase activity revealed an absence of GDNF in postnatal Egr3KO mutant spindles  line [GDNFFLOX/FLOX/Egr3CRE/CRE mutant animals had no apparent phenotype at birth and are viable and mature into adulthood.To address the question of whether spindle-derived GDNF is required for γ-MN survival, we generated a conditional allele of RE) line  to selecCRE could effectively eliminate GDNF expression in the GDNFFLOX/FLOX/Egr3CRE/CRE mice, we used in situ hybridization analysis to examine the expression of GDNF and the transcription factor Egr3, expressed in nascent intrafusal fibers upon muscle spindle induction at E15.5 [GDNFFLOX/FLOX (no CRE) controls, in situ hybridization analysis performed on contiguous sections of P5 hindlimb muscle revealed the co-expression of Egr3 and GDNF in muscle spindles  revealed that mutant muscle spindles each contain four intrafusal fibers Figure , two chaGDNFFLOX/FLOX controls (n = 3) and no significant difference in the calculated size average or SD of the small and large populations in these animals compared to the fitted distributions of age-matched wild-type animals analyzed previously  showed a significant  decrease in the number of small (<485 μm2) ChAT+ MNs  with a mean average cross-sectional area of 708 μm2 ± 200 ( ± SD), which is similar to that estimated for the large MN population in GDNFFLOX/FLOX controls (758 ± 201 μm2). Similarly, selective loss of γ-MNs was also observed in mutants carrying a conditional (GDNFFLOX) and null (GDNFLacZ) allele of GDNF and a single copy of Egr3CRE  MNs  GDNF allele and both muscle- and spindle-specific Cre drivers . Even wet al.  it was as Figure . The resIn the absence of GDNF signaling, γ-MNs degenerate selectively and no loss of α-MNs is observed . Yet, GfKO mutant, which supports a role for GDNF in the specification of β-skeletofusimotor neurons. In Egr3KO animals, all large MNs survive, but downregulate Gfrα1, perhaps because of the loss of spindle-derived GDNF. This spindle-dependence of Gfrα1 expression argues that large Gfrα1+ MNs interact directly with muscle spindles and therefore must represent those MNs that send a β-skeletofusimotor collateral to intrafusal muscle fibers. Our analysis also shows that β efferents are Hb9::GFP+, and together these findings argue that the subpopulation of MNs that co-express Gfrα1-TLZ and Hb9::GFP are β-skeletofusimotor neurons that innervate both intra- and extrafusal muscle. The variable numbers of mature Gfrα1-TLZ+/Hb9::GFP+ MNs we observed in different pools may then reflect differences in the amount of β-innervation in different muscles. Spindle-derived factors may maintain β-skeletofusimotor collaterals and regulate aspects of γ-MN differentiation - for example, strong Gfrα1 expression, Err3 expression or downregulation of NeuN - but the degeneration of muscle spindles and the selective loss of γ-MNs in the Egr3KO mutant precludes this analysis.An additional role for GDNF signaling in MNs is suggested by our analysis of large Gfrα1+ MNs in the Egr3Hb9::GFP transgene in large diameter MNs is regulated independently of Gfrα1 expression and is not influenced by spindle-derived factors. Moreover, Hb9::GFP transgene expression does not faithfully reflect the expression of the endogenous Hb9 gene, which analysis of the Hb9-NLS-LacZ knock-in mice demonstrates is expressed in both α- and γ-MNs [Hb9::GFP transgene has been reported in non-Hb9 interneurons in lumbar segments [Hb9 gene are missing in the transgene that could also account for its consistent, selective downregulation in postnatal γ-MNs.The differential expression of the nd γ-MNs . Extensisegments ,30, suggOur data provide further evidence that reciprocal interactions between the muscle spindle and the sensory and MNs that innervate it are critical to establish and maintain the circuits that provide proprioceptive sensory feedback during motor behaviors. Primary afferents induce muscle spindles through a mechanism dependent on neuronal Neuregulin 1 (Nrg1) . In respSome GDNF actions on MN development are mediated by induction of the ETS transcription factor Pea3 ,55,57. IThe transcriptional profile of γ-MNs may also be reflected in the selective downregulation of NeuN, a predominantly nuclear protein that is able to bind DNA and is expressed exclusively in postmitotic neurons . The recKO animals [FLOX/FLOX/Egr3CRE/CRE mice. The absence of an obvious motor phenotype in animals in which γ-MNs are significantly depleted could reflect residual γ-fusimotor activity or functional compensation by β-skeletofusimotor inputs to muscle spindles. However, the lack of an overt phenotype during normal locomotion on level ground, as observed, for example, during slow speed treadmill locomotion, may reflect low level recruitment of γ-control during this type of locomotion. Further study of these mutants using locomotor and other behavioral tasks that require dynamic regulation of muscle spindle sensitivity [In contrast to Egr3 animals  and muta animals , mice wisitivity  are needHb9::GFP transgene. Together, these define a unique molecular criterion for γ-MN identity. The strong expression of Gfrα1 in postnatal γ-MNs correlates with our finding that γ-fusimotor neurons depend selectively on muscle spindle-derived GDNF for their survival. In demonstrating this trophic dependence in mice, we created a novel animal model in which γ-MNs are selectively lost. Unlike other animals with muscle spindle or proprioceptor defects, this mutant preserves muscle spindle structure, sensory afferent innervation, and functional sensorimotor connectivity with no α-MN loss and so provides a genetic model to study the specific role of γ-MNs in motor control.At birth, γ-MNs express high levels of Gfrα1 and low levels of NeuN and the ChAT: choline acetyl transferase; E: embryonic day; Gfrα1: GDNF receptor; GDNF: glial cell line-derived neurotrophic factor; GFP: green fluorescent protein; IR: immunoreactive; MN: motor neuron; NeuN: neuronal nuclear protein; P: postnatal day; PBS: phosphate buffered saline; PGP9.5: protein gene product 9.5; SD: standard deviation; SEM: standard error of the mean; VAChT: vesicular acetylcholine transporter; VGluT1: vesicular glutamate transporter 1.The authors declare that they have no competing interests.GDNFFLOX mouse. All authors read and approved the final manuscript.NAS and FJA conceived the study, designed the experiments, analyzed the data and wrote the manuscript. NAS, MNB and CAS performed the experiments. CAS performed all neurolucida analyses of cell size and morphology. JP and NAS generated the


Aberrant β-catenin signaling has been linked to various disease pathologies, including an important role in tumorigenesis. Here, we review the regulation of the Wnt signaling pathway as it relates to β-catenin signaling in tumorigenesis, with particular focus on the role of microRNAs. Finally, we discuss the potential of β-catenin targeted therapeutics for cancer treatment. Altered function of components of the canonical Wnt/β-catenin signaling pathway is associated with cancer, as multiple Wnt/β-catenin target genes are regulators of cell proliferation, metastatic potential and tumorigenesis ,2. In thUp to now, there have been more than ten thousands of β-catenin related publications in MEDLINE (Pubmed with: beta catenin). Over the last 10 years, the number of new entries about β-catenin in MEDLINE has grown at a 9.3% compounded annual growth rate, and the number of new entries in MEDLINE each year has grown at a compounded annual growth rate of 3.1% . FurtherActivation of EGFR induces phosphorylation and activation of CK2α via ERK. CK2α activation signals phosphorylation of α-catenin at S641, and triggers loss of α-catenin binding to β-catenin and subsequent activates β-catenin/Lef/Tcf transcriptional activity . AdditioAKT1, a serine/threonin kinase also known as PKB, functions as a key mediator of the PI3K/AKT pathway. Activated AKT1 is phosphorylated at Thr308 and Ser473, thereby regulating β-catenin by inducing phosphorylation of effector molecules GSK3β, mTOR and BAD -27. SpecThe c-Jun N-terminal kinase (JNK) is a stress-activated protein kinase that is a member of an evolutionarily conserved sub-family of the mitogen-activated protein kinase (MAPK) family of serine/threonine protein kinases. In early Xenopus embryos, high level of nuclear JNK negatively regulates the canonical Wnt/β-catenin signaling pathway by expelling β-catenin from the nucleus . A recenLoss of JNK 1/2 results in increased expression of β-catenin, and β-catenin/Tcf complex target genes, including c-myc ,32. PhosMicroRNAs are single-stranded noncoding RNAs of 21 to 23 nucleotides in length that repress translation or induce cleavage of target mRNAs that they are partially complementary to at the 3'or 5' untranslated region (UTR). MiR-200a was recently reported to downregulate β-catenin-mediated transcription via two different mechanisms. MiR-200a targets the mRNA of the E-cadherin repressor proteins ZEB1 and ZEB2 , subsequently increases the total E-cadherin available for binding to β-catenin and induces formation of the cell-cell adhesion complex . β-catenProtein and mRNA analyses identified that WNT1 is translationally repressed by miR-21. Antagonism of the effects of miR-21, either by transfection with miRNA inhibitors or by exogenous addition of Wnt-1, inhibits human monocyte-derived dendritic cell (MDDC) differentiation, suggesting that miR-21 has a key regulatory role in MDDC differentiation . InteresMiR-203 directly targets the Wnt signaling transcription factor Lef1 in zebrafish . The 3'UWhile it is known that mammalian miR-8 family members promote adipogenesis, possibly by inhibiting Wg signaling, the mechanism of this event remains elusive. Three potential mechanisms have been reported. Expression of miR-8 may potently antagonize Wg signaling by directly binding the 3'UTR of wntless (wls) and inhibiting Wg signaling in part by preventing Wg secretion . MiR-8 aMiR-135a and miR-135b target the 3'UTR of APC and suppress its expression, subsequently increasing Wnt signaling by stabilizing β-catenin . SimilarThe Tcf family consists of four members in vertebrates, including Tcf1, Tcf3, Tcf4 and Lef1. Each member contains a DNA-binding high mobility group (HMG) box, and a highly conserved β-catenin interacting region. In the absence of β-catenin, Tcf4 recruits co-repressors HDAC1, CtBP, and Groucho/transducin-like enhancer of Split (TLE) to silence expression of target genes -54. GrouHistone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from ε-N-acetyl lysine amino acids on a histone. HDACs attenuate β-catenin transcriptional activity by binding to Lef, forming a HDAC-Lef complex that hypoacetylates the promoter region of β-catenin/Tcf/Lef complex target genes ,61. UponRecent evidence identifies that HDAC3 expression may enhance transcription activity of the Wnt/β-catenin pathway . Stable c-Jun is well known as a downstream effector of the β-catenin/Tcf complex . c-Jun hMultiple novel upstream regulators of β-catenin/Tcf/Lef complex transcriptional activity have been identified. Positive modulators of β-catenin activity include Nck-interacting kinase (TNIK) and Snail. TNIK expression is enriched in the nuclei of Wnt-activated intestinal crypts, but not cells of the villus, where it is specifically recruited to the promoters of Wnt target genes Axin2 and c-Myc . In vitrhttp://www.stanford.edu/~rnusse/pathways/targets.html. Here, we summarize the novel downstream targets of β-catenin and the positive feedback loops between β-catenin and the downstream targets  suppress β-catenin expression in human cancer cell lines, including aspirin, indomethacin, sulindac and etodolac -79. NitrChIP and transgenic analysis identified that the Gbx2 regulatory elements that drive expression in the neural crest (NC) respond directly to Wnt/β-catenin signaling. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC protein expression and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. Previous studies identified a region of 500 bp upstream of Gbx2 that contains three (1-3) Lef/Tcf consensus sequences, termed the Gbx2 enhancer . SimilarPreviously, WNT/β-catenin was suggested to regulate STAT3 at the mRNA and protein level, suggesting that STAT3 maybe a direct target of β-catenin . FurtherWnt/β-catenin signaling regulates miR-15/16 maturation rather than its transcription, as overexpression of β-catenin inhibits the expression of mature miR-15 and miR-16 isoforms. The mechanism of Wnt control of miR-15 and miR-16 maturation is unknown, but perhaps works through a protein complex controlled by or containing β-catenin. β-catenin control of the intensity and spatial pattern of Nodal responsiveness is thought to be regulated by miR-15 and miR-16 expression . UpregulThe β-catenin complex plays a critical role in tumorgenesis, angiogenesis, and progression of metastasis, making it an attractive therapeutic target for chemotherapy. Current drugs that target β-catenin include Quercetin, which inhibits the transcriptional activity of β-catenin by disrupting the binding of β-catenin and Tcf4 and suppressing their translocation to the nucleus ,97. SimiAlthough Wnt/β-catenin transcriptional activity has been studied over the past several years, molecular regulation of Wnt/β-catenin pathway is complex and summarized in Fig. The authors declare that they have no competing interests.KH, JXZ, LH and YPY drafted and wrote the manuscript. TJ and PYP revised the manuscript critically for important and intellectual content. CSK contributed to the writing of the manuscript and supervised the project. All authors read and approved the final manuscript.


Protein interactions were examined by sequential immunoprecipitation and immunoblotting with antibodies to E-cadherin, α, β and γ-catenin, p120ctn and APC. Abnormalities of E-cadherin, α- and β-catenin expression, were associated with disturbance of E-cadherin–catenin complex composition, loss of membranous localization and loss of calcium-dependent aggregation in six gastric carcinoma cell lines. APC protein expression and interaction with β-catenin was preserved in five cell lines. We demonstrate frequent abnormalities of expression and function of E-cadherin and catenins, and associated disturbance of E-cadherin-mediated intercellular adhesion in gastric carcinoma cell lines. These findings support the tumour suppressor role of the E-cadherin and its contribution to the development and progression of the neoplastic phenotype in gastric carcinoma. © 1999 Cancer Research CampaignDysfunction of the cadherin–catenin complex, a key component of adherens junctions, is thought to confer invasive potential to cells. The aim of this study is to examine the expression and function of the E-cadherin/catenin complex in gastric carcinoma cell lines. Expression of E-cadherin, α, β and γ-catenin and p120


Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and their loss has been correlated with poor prognosis in human cancer. Mutational activation of protein kinases and loss of cell adhesion occur together in human lung adenocarcinoma but how these two pathways interconnect is only poorly understood. Mouse models of human lung adenocarcinoma with oncogene expression targeted to subtypes of lung epithelial cells led to formation of adenomas or adenocarcinomas that lacked metastatic potential. Conditional genetic abrogation of epithelial tumour cell adhesion in mice with benign lung tumours induced by oncogenic RAF kinase has been demonstrated to induce intratumourous vascularization (angiogenic switch), progression to invasive adenocarcinoma and micrometastasis. Importantly, breaking cell adhesion in benign oncogene-driven lung tumour cells activated β-catenin signalling and induced the expression of several genes that are normally expressed in intestine rather than the lung. I will discuss potential routes to nuclear β-catenin signalling in cancer and how nuclear β-catenin may epigenetically alter the plasticity of tumour cells during malignant progression. Lung cancer has become the most prevalent neoplasm throughout the world with 1,2 million deaths per year . Cigaret2, whereas the conducting airways cover less than 1% of this surface. Early studies [The major function of the lung is gas exchange. The epithelial cells which line the tree of tubules and alveolar units arise from progenitor cells present in the lung bud of the ventral foregut endoderm. During embryonic development, they form the epithelial lining of the conducting airways and the distal vascularized sac-like structures. At birth, fluid is resorbed from these sacs to expose a functional gas exchange surface. The subsequent postnatal maturation of the lung involves the formation of septa in the terminal sacs  leading to an expansion of the gas exchange surface . Thus, i studies  on the n studies ,15. Alve studies . Type II studies . The dou studies ). BASCs  studies  and in b studies ,20. Duri studies .I will focus here on genes specifying components of the classical mitogen activated protein kinase (MAPK) signalling cascade which are among those most frequently overexpressed, amplified or mutated in NSCLC Figure . StartinRAS . The send Mec1; .NKX2.1 has been found to be amplified in a significant number of lung adenocarcinoma samples [Nkx2.1 deficient mice have only two main bronchi and instead of an epithelial tree, cystic structures with columnar cells form and surfactant protein genes fail to be expressed [In another NSCLC model for oncogenic RAF, the SP-C promoter-directed the expression of an oncogenic variant of C-RAF, called C-RAF BXB . The hom samples ,50. Convxpressed . The SP-xpressed ,53. In txpressed . The tumxpressed ,56. Progxpressed , similarxpressed . No furtxpressed , not evexpressed . Thus, txpressed  and moreK-RasG12D oncogene activation with a PI3K unable to interact with RAS only formed premalignant lesions but no macroscopic lung tumours [Increased growth factor receptor or RAS signalling not only activates RAF but in addition phosphoinositide-3-kinase  cascade . In the YC gene, ; see belYC gene, ,84. AnotCdh1 gene in mice with a "floxed" Cdh1 gene [We addressed the functional consequences of weakening or destroying cell-to-cell adhesion in the adenomas of SP-C C-RAF BXB mice using two different genetic approaches, conditional ablation of the dh1 gene  or switcdh1 gene . In addidh1 gene .Cdh1 gene, massive formation of blood and lymph vessels, termed the "angiogenic switch" [Increased tumour cell proliferation was observed in vivo, in line with the up-regulation of cyclin D1 and c-Myc in tumour cells 36 hours after induction of dominant negative E-cadherin . After i switch"  was obse switch" . Vascula switch" . The fin switch" . Vascula switch" . Progres switch"  but also switch" . Combine switch" .CDH1) have been found in gastric and colon cancer but have not yet been reported in lung cancer. The most frequent colorectal cancer-associated genetic lesions are loss-of-function mutations in the APC gene and they result in sustained β-catenin signalling [CTNNB1 gene, which encodes β-catenin, in colon cancer cells [CTNNB1 have been detected in colorectal carcinomas and transcripts lacking exon 3 are translated into a stabilized β-catenin with dominant nuclear β-catenin signalling [APC and are rare in CTNNB1 (3%) [Frequent mutational activation of Wnt/β-catenin signalling has been observed in various types of human cancers . With regnalling . Activater cells  as well er cells . In addignalling ,95. Colognalling . In humaNB1 (3%) . ActivatNB1 (3%) . WhetherNB1 (3%) .CDH1 expression by epigenetic mechanisms might lead to a loss of E-cadherin function in the absence of CDH1 loss-of-function mutations. Aberrant methylation of normally unmethylated CpG islands in the promoter regions of genes has been correlated with transcriptional inactivation of several genes in human cancer. Silencing of E-cadherin expression by promoter CpG methylation was found in a variety of human epithelial carcinoma cell lines [I will now discuss alternative mechanisms that may result in nuclear β-catenin signalling in cancer cells in the absence of overexpressed Wnt ligands or mutational activation in the canonical Wnt/β-catenin signalling cascade. Repression of ll lines . In primll lines . Secondlll lines . The conll lines . miR-10bll lines .As a further possibility we have to consider that β-catenin may be released by phosphorylation from complexes at the membrane and subsequently enter the nuclear signalling pool. The three-dimensional structure of β-catenin and the structure of the cadherin/β-catenin complex have been elucidated ,104. β-cK-RasG12V allele in conjunction with a loss-of-function allele of APC was shown to increase nuclear β-catenin signalling [Cell surface transmembrane tyrosine kinase receptors modulate the adhesion complex by phosphorylation, as demonstrated for the EGFR . This wognalling . This cognalling . Activatgnalling . Src hasgnalling . Furthergnalling . Using agnalling . In addignalling . In fibrgnalling .MET [Tyrosine-142 is present in the first armadillo repeat which overlaps with the α-catenin binding region of β-catenin. Phosphorylation of this residue induced the dissociation of α-catenin from the E-cadherin/β-catenin complex with concomitant loss of cell aggregation . TyrosinMET . They leMET . AnotherMET . In sum Cdx1, Atoh1) that are normally expressed in the intestine indicating that an epigenetically-driven change in cell fate had occurred [Why did the disseminated tumour cells forming a micrometastasis in compound mice with oncogenic RAF and disrupted adherens junctions fail to colonize and grow into a macrometastasis? One obvious explanation may be the failure of the micrometastasis to acquire a "metastasis virulence gene" that would allow for colonization in the new tissue . A seconoccurred . As it ioccurred . During occurred . Modificoccurred ,129. TheTherefore, it appeared that there may be no way back for a differentiated committed cell to an earlier fate after it has lost its pluripotent capacity. However, there are examples for transdifferentiation, the transformation of one differentiated cell into another . FurtherMYC proto-oncogenes are essential activators of cell proliferation and have emerged as some of the genes most commonly deregulated in cancer [MYC genes in human cancer occurs by amplification or loss of transcriptional control, which results in MYC protein overexpression. In NSCLC, MYC gene copy number is found to be amplified in a fraction of specimen [The n cancer . Activatspecimen . The up-specimen . The halspecimen . Active specimen .In transgenic mice expressing Nmyc from the SP-C promoter progenitor cells in the developing distal lung expanded in numbers and their differentiation was blocked leading to disturbed lung function and perinatal death . SP-C prAccording to a classical model, c-Myc is a basic-helix-loop-helix zipper transcription factor that dimerizes with Max leading to the activation of genes that contain binding sites for Myc-Max complexes. The identification of thousands of putative c-Myc target genes , the finCdx1, Atoh1 and other genes normally expressed in intestinal epithelium. Another example of reprogramming has been observed upon introduction of activated β-catenin into either the common lymphoid or common myeloid progenitors [A form of epigenetic plasticity similar to that noted in adult mice with oncogenic RAF-driven lung tumours with disruption of adherens junctions and nuclear β-catenin signalling  had beengenitors . Multipogenitors . Therefogenitors ,148. In genitors . Howevergenitors ,151 thatTwo recent studies have evaluated the consequences of the loss of E-cadherin in conjunction with either oncogenic RAF kinase expression or loss of p53 for malignant progression of epithelial tumours. The investigation of the breast cancer model revealed that combined loss of E-cadherin and p53 in mammary epithelial cells induced metastastic carcinoma that resembles invasive lobular carcinoma. In the lung NSCLC model, disruption of E-cadherin led to vascularized tumours that grew rapidly and gave rise to micrometastasis. Nuclear β-catenin signalling was instrumental for up-regulation of vascular endothelial growth factor expression and epigenetic plasticity. Published data are being reviewed on how receptor and non-receptor kinases impinge on the stability of the adherens junction complex. Evidence suggests that kinases may modulate nuclear β-catenin signalling. In a cooperative manner, kinases and nuclear β-catenin may be instrumental for the expression of gene targets in the lung which in turn is involved in malignant progression. I finally discuss what mechanisms may block the step from micrometastasis to macrometastasis and how E-cadherin function, together with oncogenic and nuclear β-catenin signalling might control this important step of malignancy.APC: adenomatous polyposis coli; BADJ: bronchio-alveolar duct junction; BASC: bronchio-alveolar stem cell; CC: Clara cell; c-Myc: cellular Myelocytomatosis; EGFR: epidermal growth factor receptor; ERK: extracellular signal-regulated kinase; HGF: hepatocyte growth factor; LEF: lymphoid enhancer factor; MAPK: mitogen-activated protein kinase; MEK: MAP kinase extracellular signal-regulated kinase; NSCLC: non-small-cell lung cancer; PCR: polymerase chain reaction; PI3K: phosphoinositide-3-kinase; RT-PCR: reverse transcription- polymerase chain reaction; rtTA: reverse transcriptional transactivator; SCID: severe combined immune deficiency; SP-C: surfactant protein-C; Sca1: stem cell antigen 1; TCF: T-cell factor; VEGF: vascular endothelial growth factor; Wnt: wingless/int.The author declares that they have no competing interests.