Dysregulation of Wnt/β-catenin signaling has been associated with the development and progression of many cancers. The stability and subcellular localization of β-catenin, a dual functional protein that plays a role in intracellular adhesion and in regulating gene expression, is tightly regulated. However, little is known about the transcriptionally active form of β-catenin, Active Beta Catenin (ABC), that is unphosphorylated at serine 37 (Ser37) and threonine 41 (Thr41). Elucidating the mechanism by which β-catenin is activated to generate ABC is vital to the development of therapeutic strategies to block β-catenin signaling for cancer treatment. Using melanoma, breast and prostate cancer cell lines, we show that while cellular β-catenin levels are regulated by the Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway and are dependent on the phosphatase activity of the protein phosphatase PP2A. Furthermore, we demonstrate that although the PI3K/PTEN pathway does not regulate total β-catenin protein levels within the cell, it plays a role in regulating the subcellular localization of β-catenin. Our results support a novel functional interaction/cross-talk between the PTEN/PI3K and Wnt pathways in the regulation of the subcellular/nuclear levels of ABC, which is crucially important for the protein's activity as a transcription factor and its biological effects in health and disease. Activation of the Wnt pathway results in stabilization and accumulation of β-catenin in the cytosol, and its subsequent localization in the nucleus. Under normal conditions, cytosolic levels of β-catenin are regulated by the ubiquitin-proteasome system. Targeted phosphorylation of highly conserved serine (Ser) and threonine (Thr) residues  at the NH2-terminal domain of β-catenin, by glycogen synthase kinase-3 beta (GSK-3β) and Casein Kinase (CK)-1 [Wnt signaling pathway results in increased β-catenin levels in the nucleus and consequently, the constitutive transcription of Wnt target genes [Beta (β)-catenin is a multifunctional protein that serves a structural role at the adherens junctions  and a reet genes .Xenopus embryo explants [In 2002, Staal and colleagues demonstrated that β-catenin molecules that were dephosphorylated at residues Ser37 and Thr41 at its N-terminal had enhanced transcriptional activity , 9. Thisexplants , 11.While cellular stability of β-catenin is known to be regulated by the canonical Wnt pathway, several mechanisms have been implicated in the regulation of the transcriptional activity of β-catenin. EGF stimulation of cells results in translocation of β-catenin to the nucleus and increases its transactivation function without altering its stability and phosphorylation levels by GSK3β . In addiAn increasing body of evidence suggests that deregulation of the PI3K pathway is strategically used by tumors to attain growth advantage and survival . InteresThe Wnt/β-catenin and PI3K/PTEN signaling pathways include GSK3β as a common point of intersection . HoweverIn this study we used several melanoma, breast and prostate cancer cell lines to understand the mechanism by which ABC is regulated. In light of the frequent deletion or inactivation of the PTEN gene in these cancers as well as the evidence for crosstalk between these pathways, we investigated the regulation of ABC by the Wnt and PI3K pathways. Currently, the involvement of the Wnt/β-catenin and/or PI3K pathways in the generation of ABC is unknown. Our study demonstrates that ABC levels are elevated with PTEN loss/inactivity. Furthermore, our results suggest that while cellular levels of β-catenin are regulated by the canonical Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway, and involve the phosphatase activity of PP2A.We used a panel of four cell lines representing melanoma progression, including the melanocytic line (HeMa-Lp), radial growth phase (WM35), vertical growth phase (WM793B) and metastatic melanoma (A2058), to assess the cellular levels of total β-catenin and ABC. Total β-catenin was elevated in WM35, WM793B and A2058 cell lines compared to HeMa-Lp cell line Figure . SimilarInterestingly, there was a corresponding loss of PTEN expression in the WM793B and A2058 cell lines with high P-PKB Ser473 levels supporting a constitutively active PI3K pathway Figure . AlthougGiven the inverse relationship between PTEN and β-catenin/ABC expression in the melanoma cell lines, we compared the levels of β-catenin/ABC and PI3K/PKB in PTEN-positive or PTEN-deficient prostate and breast cancer cell lines. β-catenin/ABC levels were substantially higher in the PTEN-null cell lines relative to cell lines with PTEN Figure .To further investigate the relationship between the PI3K/PTEN pathway and β-catenin/ABC, A2058 metastatic melanoma cells were transiently transfected with pEGFP-PTEN-wild-type (pEGFP-PTEN). Total cellular β-catenin and ABC were evaluated by Western Blot and immunofluorescence (IF) analysis and levels of β-catenin phosphorylated at Ser33, Ser37, Thr41 or Ser45 were evaluated by Western Blot.ABC levels decreased upon reintroduction of pEGFP-PTEN into A2058 cells Figure . HoweverAlterations in cellular levels and subcellular localization of β-catenin and ABC were further investigated with IF analysis. As shown in Figure Collectively, these results show that the PI3K/PTEN pathway plays a role in regulating cellular/nuclear levels of ABC in cells. The results also indicate that although the PI3K/PTEN pathway does not regulate cellular β-catenin levels, it does regulate the subcellular localization of β-catenin.To further assess involvement of the PI3K pathway in regulating ABC levels, we treated A2058 cells with PI3K inhibitor, Wortmannin. As shown in Figure To investigate the contribution of the Wnt pathway in modulating ABC levels, we treated A2058 cells with purified recombinant Wnt-3a protein (100ng/ml). Activation of the Wnt pathway resulted in an increase in total β-catenin levels within 1hour of treatment Figure . InteresCollectively, the results from Figures Since our results indicate that ABC levels are regulated by the PI3K pathway, we studied the effect of inhibiting the PI3K pathway on β-catenin/TCF transcriptional activity using the TOPFlash-Luciferase reporter Figure . We usedWe next studied the effect of inhibiting the PI3K/PTEN pathway on the mRNA expression of selected β-catenin/TCF regulated target genes, including cyclin D1 Figure , VEGF-A Since the generation of ABC requires partial dephosphorylation of β-catenin, our next aim was to elucidate the identity of the phosphatase involved in this pathway. PP2A has been previously suggested as a regulator in the generation of ABC , 9. We mSince our data suggest that both ABC protein level and PP2A activity are regulated by the PTEN/PI3K pathway, we explored the putative role of PP2A in regulating ABC levels in the cell. A2058 cells were treated with or without okadaic Acid (OA) to inhibit PP2A activity and ABC levels were evaluated by Western Blot. Treatment of A2058 cells with OA (5nM and 10nM) resulted in a significant decrease in ABC levels Figure . Importaβ-catenin, the central player of the canonical Wnt signaling pathway, functions as a potent oncoprotein that promotes malignant transformation, increases migratory capacity and invasiveness. The PI3K pathway has been identified as a metabolic signaling pathway that is integral to driving the malignant progression of many tumors. Here, we characterize a link between these two pathways in the regulation of the transcriptionally active form of β-catenin, ABC. We show a dramatic reciprocal relationship between PTEN and ABC in melanoma cells where loss/inactivation of PTEN is accompanied by a marked elevation in levels of ABC Figure . We furtWe investigated the putative regulation of β-catenin by the PI3K pathway by inhibiting PI3K signaling in PTEN-null melanoma cell lines via two methods: (i) re-introduction of PTEN, and (ii) inhibition of PI3K signaling using an established inhibitor of PI3K. Taken together, we noted decreased cellular/nuclear ABC levels independent of changes in total cellular β-catenin levels Figures , 3A, 3B.To investigate whether the Wnt pathway plays a role ABC regulation, we treated cells with Wnt3A, a canonical Wnt ligand that binds Frizzled (Fz) and consequently disables the destruction complex. Classically, activation of the canonical Wnt pathway has been associated with accumulation of β-catenin and a corresponding increase in nuclear β-catenin transcriptional activity. We observed that while treatment with Wnt 3A resulted in increasing levels of total cellular β-catenin, there was no identifiable change in levels of ABC Figure . Collectet al. were Chinese-hamster ovary derived (CHO) cells, which express very low levels of PTEN (with high PI3K pathway activity) [At first glance, our findings are partly discordant with data published by Staal et al. , whose gctivity)  and a rectivity)  present ctivity) . Converset al. used a β-catenin/AR co-transcriptional model of nuclear β-catenin activity, which may differ from the canonical β-catenin/TCF-4 mediated activity that we used as our model.Several studies have previously explored connections/cross talk between the Wnt and PI3K pathways. A suggested intermediate in this cross talk is GSK-3β, a common component to both pathways. Sharma et al.  charactePrevious studies have identified PP2A as a Ser/Thr phosphatase that may regulate the dephosphorylation of β-catenin to generate ABC . PP2A anThere are a number of mechanisms by which PI3K signaling could influence PP2A signaling, both in terms of activation and in role specification by subunit composition. PP2A and PKB have been shown to interact both directly and indirectly: PP2A can dephosphorylate PKB  or may aCollectively, our results support a heterogeneous role for PP2A in cancer and also a rather complex regulatory system underlying its activation. We predict that the PR55α B subunit of PP2A plays a dominant role in modulating PP2A activity in malignant melanoma and potentially in other cancers with dysfunctional or null PTEN, promoting an oncogenic role of PP2A as opposed to its classical tumor suppressor function. This model is supported both by the previous identification of PR55α as tumorigenic in other cancers , as wellIn summary, we have shown that the PI3K/PTEN pathway modulates β-catenin activation to form the transcriptionally active ABC in the setting of PTEN-null/inactive cell lines, while the canonical Wnt pathway regulates β-catenin stability. We also show, in agreement with work published by Bos et al. , a role Human Epidermal Melanocytes (HeMa-Lp) (Cascade Biologics™) were grown in Medium 254, supplemented with 10% Human Melanocyte Growth Supplement, 10 μg/ml gentamicin and 0.25 μg/ml amphotericin B (Cascade Biologics™). Cell lines A375 A2058, DU145 and PC3 (ATCC) were grown in Dulbecco's Modified Eagle Medium (DMEM)/10% Fetal Bovine Serum (FBS). WM35, WM793B , MCF7 and MDA-MB-468 were grown in Hyclone's RPMI-1640 medium/5% FBS. HEK 293 cells that were stably transfected with pTA-TCF/LEF-luciferase reporter vector containing 6 repeats of TCF/LEF binding sites, a minimal promoter upstream of the firefly luciferase coding region (Signosis). HEK 293T were grown in Dulbecco's Modified Eagle Medium (DMEM)/10% FBS. All cells were incubated at 37°C and 5% CO2. Where indicated, cells were treated with 2nM Wortmannin (Sigma) or 100 ng/ml purified recombinant human Wnt protein derived from CHO cells (R & D Systems). For okadaic acid treatment, cells were serum starved overnight followed by treatment with 5nM or 10nM okadaic acid in serum-free media for 2 hours.Cell lines were grown to 50% confluence and transfected with pEGFP-C3-hPTEN or pEGFP-C3 vector control (4 μg). All transfections utilized Lipofectamine2000 (Invitrogen) according to the manufacturer's protocols. Transfected cells were analyzed by Western blot, IF or quantitative RT-PCR.Whole cell lysates were obtained by incubating with 100μl of lysis buffer , 80 ng/ml aprotinin, 40 ng/ml chymostatin, 40 ng/ml antipain, 40 ng/ml leupeptin, 40 ng/ml pepstatin) on ice for 10 minutes. Cellular debris was removed by centrifugation for 5 minutes at 16,000 x g. Nuclear and cytosolic extracts were separated using the NE-PER Nuclear Cytoplasmic Extraction Kit (ThermoScientific) according to the manufacturer's protocols. Protein concentrations were determined using Bicinchoninic (BCA) Protein Determination Assay (ThermoScientific).We determined β-catenin/TCF-induced transcriptional activity using HEK 293T cells (Signosis). HEK 293T cells were treated with 10mM LiCl  in the presence or absence of Wortmannin (2nM). Reporter activity was measured using a luminometer (Fluor Star OMEGA: BMG Labtech).Total RNA was isolated using RNeasy Kit (QIAGEN) as per the manufacturer's protocol. 1μg of total RNA was used for reverse transcription of Oligo (dT) (Invitrogen) and Superscript III reverse transcription (Invitrogen). Real time quantification of Cyclin D1, VEGF-A and MMP-2 were assessed using power SYBR Green PCR Master Mix (Applied Biosystems). GAPDH was used as the endogenous control. Samples were amplified with a precycling hold at 95°C for 15 seconds, 30 cycles of annealing and extension at 60°C for 1 minute. The following primers were used: Cyclin D1: sense (5`-CAT CTA CAC CGA CAA CTC CAT C-3`); Cyclin D1: anti-sense (5`-TCT GGC ATT TTG GAG AGG AAG-3`); VEGF-A: sense (5`-AGT CCA ACA TCA CCA TGC AG-3`); VEGF-A: anti-sense (5`-TTC CCT TTC CTC GAA CTG ATT T-3`); MMP-2: sense (5`-GGC CCT GTC ACT CCT GAG AT-3`); MMP-2: anti-sense (5`-GGC ATC CAG GTT ATG GGG GA-3`); GAPDH: sense (5`-TCAACGACCACTTTGTC AAGC TCA-3`); GAPDH: anti-sense (5`-GCTGGTGGTCCAGGG GTCTTACT-3`). Each measurement was performed in triplicate with Rotor-Gene-3000 instrument  and analyzed using Rotor-Gene-6 Software. Gene expression was determined using the relative standard curve method normalized to GAPDH-binding protein expression. Histograms are reported as fold change of control which was set at 1.® 555-conjugated secondary antibody (Invitrogen) for visualization. Cells were visualized with IF imaging using a Carl Zeiss Laser Scanning Microscope and analyzed using LSM510 software.Cells were cultured on coverslips, washed with phosphate buffered saline (PBS) and fixed with 4% formaldehyde. Cells were permeabilized with 100% ice cold methanol at −20°C for 10 minutes and stained with primary antibody followed by treatment with Alexa Fluor® Plus-ECL . Densitometric analysis normalized to the respective actin was performed by Image J software. Histograms are representative of four or more independent experiments and are reported as a percentage of control set at 100%. Statistical analysis was performed by Student's t-test (p<0.005) and Mann-Whitney Rank Sum test (p<0.05). Antibodies used: Antibodies to β-catenin, PKB, P-PKB-Ser-473, PTEN, S33/37/T41-phospho-β-catenin, S45-phospho-β-catenin, PP2A and PR55α , ABC (Millipore), GFP and Actin (Santa Cruz Biotech Inc.).Cells were lysed with 50mM Tris buffer (pH 8.0) . Protein concentration was quantified by BCA Assay. Proteins were separated by SDS polyacrylamide Gel (SDS PAGE) or Tricine Gel electrophoresis and transferred onto PVDF membranes (Millipore). PVDF membranes were blocked with 5% milk, probed with specific primary antibodies followed by peroxidase-conjugated secondary antibodies  and visualized using Western LightningCellular PP2A activity was assayed using a PP2A immunoprecipitation phosphatase assay kit (Upstate). Cells were washed in TBS, lysed with phosphatase assay buffer . PP2A-C subunit was immunoprecipitated from total cell lysates (500 μg) using 4 μg of anti-PP2A-C antibody  and Protein A agarose for 2 h at 4°C. PP2A activity was assayed by incubating the immunoprecipitated protein with the synthetic phosphopeptide K-R-pT-I-R-R at 30°C for 10 min prior to detection with malachite green phosphate detection solution, according to the manufacturer's instructions. Phosphatase activity was normalized to the relative amount of immunoprecipitated C subunit using Western blot quantitation with Image J software.


C-terminal-truncated hepatitis B virus (HBV) X (HBx) (ctHBX) is frequently detected in hepatocellular carcinoma (HCC) through HBV integration into the host genome. However, the molecular mechanisms underlying ctHBx-associated oncogenic signaling have not yet been clarified. To elucidate the biological role of ctHBx in hepato-oncogenesis, we functionally analyzed ctHBx-mediated regulation of the activin membrane-bound inhibitor bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) through transforming growth factor-β (TGF-β) or β-catenin (CTNNB1) in HCC cells and in an animal model, and we compared its role to that of the full-length HBx protein. Ectopic ctHBx expression generated more colonies in anchorage-dependent and -independent growth assays than did HBx expression alone. ctHBx downregulated BAMBI to a greater degree than did HBx in HCC cells. HBx activated the Wnt/β-catenin pathway, which positively regulated the BAMBI expression through T-cell factor 1 signaling, whereas ctHBx negatively regulated the Wnt/β-catenin pathway. BAMBI downregulated the β-catenin and TGF-β1 signaling pathways. TGF-β1 positively regulated BAMBI expression thorough Smad3 signaling. Furthermore, knockdown of BAMBI was more tumorigenic in HCC cells. Therefore, downregulation of both β-catenin and TGF-β1 signaling by BAMBI might contribute to tumor suppression in mice xenotransplanted with HepG2 or SH-J1 cells. Taken together, ctHBx may have a more oncogenic role than HBx through its inhibition of tumor-suppressive β-catenin/BAMBI signaling. Among the four proteins translated by HBV, the hepatitis B virus X (HBx) oncoprotein has been implicated in HBV-mediated hepato-carcinogenesis.4 During hepato-carcinogenesis, cancer cells gain an advantage through the selective reduction of the tumor-suppressive activity of transforming growth factor-β (TGF-β), together with augmentation of its oncogenic activity.5 Furthermore, HBx has been reported to shift human TGF-β signaling from tumor suppression to oncogenesis in early chronic hepatitis B.6 Therefore, alterations in the TGF-β signal transduction pathway may be involved in HCC development. HBx mutants, particularly C-terminal-truncated mutants (ctHBx), are frequently detected in the tumor tissues of HCC patients but are rarely observed in surrounding non-tumor tissues.8 Furthermore, these mutant forms of HBx can also enhance the transformation abilities of ras and myc.9 However, little is known regarding whether or how ctHBx might be involved in carcinogenesis and tumor progression, especially in association with bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI)/TGF-β signaling in hepato-carcinogenesis. The assumption that ctHBx is oncogenic through altered BAMBI/TGF-β signaling was the basis for this study.Hepatocellular carcinoma (HCC) is a highly malignant tumor type and is the third most frequent cause of cancer-related death in the United States and Europe.Xenopus to Homo sapiens, is highly homologous to TGF-β/BMP type-1 receptors (TGFR1/BMPR1), except that it lacks an intracellular serine/threonine kinase domain. BAMBI is incorporated into complexes with TGF-β/BMP/activin type-2 receptors (TGFR2/BMPR2) and, as a pseudoreceptor, antagonizes all TGF-β/BMP and activin signaling.11 BAMBI is tightly co-expressed with BMP4 during embryonic development in zebrafish, Xenopus, birds and mice14 through TGF-β15 and Wnt signaling.16BAMBI, a transmembrane glycoprotein evolutionally conserved in species ranging from 17 Its elevated expression has been suggested to attenuate TGF-β-mediated growth arrest in colorectal and HCC cells16 as well as induce the growth and invasion of human gastric carcinoma cells.18 BAMBI expression is also associated with Toll-like receptor 4- and lipopolysaccharide-mediated hepatic fibrosis.19 Therefore, we first assessed the relationship between ctHBx or HBx and BAMBI/β-catenin signaling. Finally, we investigated the tumor-suppressive role of BAMBI in hepato-oncogenesis.Human BAMBI can also promote Wnt signaling by enhancing its interaction with the receptor Frizzled-5.All patients provided informed consent for a protocol that was reviewed and approved by the institutional review board of Chonbuk National University Hospital. Written informed consent was obtained from all patients. The study using experimental animals was approved by the Institutional Animal Care and Use Committee at Chonbuk National University (CUH-IACUC-140121-12).E. coli XL2-Blue cells . Two thousand colonies were randomly selected. The plasmids were purified on Qiaprep Spin Columns , and the insert sequences were determined by restriction endonuclease digestion with EcoRI. The final sequence homolog searches were performed using the GenBank (nr) and EST (dbEST) databases employing the BLASTN algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST).SSH was performed between paired HCC and non-HCC tissue with the Clontech PCR-Select cDNA Subtraction Kit  according to the manufacturer's protocol for HBV-associated HCC patients using two-way subtraction. In the forward subtraction, the codes from HCC were used as the tester, and the codes from paired non-HCC tissues were used as the driver; in the reverse subtraction, the codes from the paired non-HCC tissues were used as the tester, and the codes from HCC were used as the driver. Ten nanograms of the PCR product was cloned into the pGEM-T Easy Vector plasmid  and transformed into competent Genomic DNA was isolated from 50 pairs of HCCs from patients with positive serum hepatitis B surface (HBs) antigen. For PCR amplification of HBx, sets of PCR primers  were used for full-length and COOH-truncated HBx, respectively.Enh1/Xp-tHBx was completed through two cycles of RT-PCR and overlap primer extension after cDNA amplification of the mRNA. First, ctHBx was extended by searching the human HBV database; the extended sequence was confirmed using RT-PCR (primers: 5′-CACCTCCTTCCCATGGCT-3′ and 5′-CACTTCCCCTGTAATCCC-3′), and it contains the initiation codon. Next, Enh1/Xp was PCR-amplified using the specific primers (5′-GRAAAYTGCCTGTAAAT-3′ and 5′-GGGTCGTCCGCGGGATTCAG-3′). Finally, full-length Enh1/Xp-ctHBx was obtained by overlapping primer extension using primers containing KpnI and XhoI restriction enzyme sites (5′-cggggtaccGRAAAYTGCCTGTAAAT-3′ and 5′-ccgctcgagCACTTCCCCTGTAATCCC-3′), respectively, and was then ligated into pGEMT-Easy Vector (Promega). The full-length ctHBx gene was synthesized using PCR and specific primers for HBx: 5′-ccggaattcCCTTCCCATGGCTG-3′ (forward) and 5′-ccgctcgagCACTTCCCCTGTAATCCC-3′ (reverse). The reaction conditions were 94 °C for 5 min; 40 cycles of 94 °C for 1 min, 45–55 °C for 1 min, 72 °C for 1 min; and 72 °C for 7 min.Initially, we obtained an EST clone that contained a partial HBx cDNA fused to a cDNA for the 5′ side of dual specificity phosphatase 26 in the chromosome 8 genomic contig. The HBx cDNA was truncated at the C-terminus by the stop codon at the junction of two genes, suggesting that a ctHBx was produced through HBV integration into the DNA and was functionally active in HCC. The cloning of full-length cDNAs of BAMBI promoter (−255 to +82 bp) and its mutants  CAG(A/C)C sequences)11 and for the TGF-β1 promoter (−1362 to +11) and its mutant (mutation of the 3′ motif of TGF-β1-DR1)20 were conducted using luciferase reporters driven by the respective promoters. T-cell factor (TCF)-1/4-dependent transcriptional activation was also examined through a luciferase reporter gene assay using pTOP-Flash and its mutant pFOP-Flash. The dual luciferase assay was performed according to the manufacturer's instructions (Promega).Transcriptional activation assays for the A total of 30 μg of each cell or tissue lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to Hybond membranes . The membranes were blocked and then incubated with the designated primary antibodies, and signals were detected using an ECL Western Blotting Kit (Amersham Pharmacia Biotech). Goat anti-BAMBI polyclonal antibody , anti-TGF-β1  anti-β-catenin , anti-HBx  and β-actin  were used as the primary antibodies.−1). Cells were examined using laser scanning microscopy  as previously described.21HepG2 and Hep3B cells were grown on glass coverslips, fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. Coverslips were incubated for 1 h in phosphate-buffered saline containing 0.1% bovine serum albumin and a mixture of antibodies. After two washes in phosphate-buffered saline, the cells were incubated for 1 h with TRITC-conjugated anti-rabbit antibody (1:100 dilutions) . DNA was labeled using the Hoechst 33258 dye , and the GFP-tagged BAMBI insert was transferred to the pAd/CMV/V5-DEST vector (Invitrogen) by the Gateway system using LR Clonase (Invitrogen). Plasmids were linearized with PacI (Promega) and transfected into 293 A cells using Lipofectamine 2000 transfection reagent. The resulting 293A cells were cultured for 1–2 weeks in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and the medium was replaced every 2 days. As a control, the pAd/CMV/V5-GW/lacZ vector (Invitrogen) was used to produce lacZ-bearing adenovirus.To prepare the BAMBI-expressing adenovirus, a GFP-tagged BAMBI cDNA fragment was cloned into the 6 cells in 0.08 ml of culture medium and 0.04 ml of 10% Matrigel were injected into the subcutaneous tissues of both shoulders of Balb/c nude mice . Tumor growth was monitored once a week for 4 weeks using the Xenogen IVIS 100 Imaging System . Mice were anesthetized with 3% isofluorane after administration of 150 mg kg−1 firefly D-luciferin  via intraperitoneal injection for imaging.We established an orthotopic nude mouse subcutaneous tumor model with SH-J1-Luc cells overexpressing BAMBI by adenovirus delivery. Briefly, 3 × 10Lentivirus vectors encoding a shRNA targeting BAMBI, β-catenin or TGFβ1  as well as the shRNA non-specific control (SHC002V) were used for transduction of HepG2 cells according to the manufacturer's instructions . In brief, 5 × 10t-tests. Values of P<0.05 were considered significant.The data are expressed as the mean±s.d. Differences were analyzed using dependent or independent 23 To select genes associated with HCC and functional HBx, a total of approximately 2,000 subtracted cDNA fragments were obtained from a two-way (forward and reverse) subtraction library. We identified candidate genes based on more than four appearances (HBx and the middle of HBs mRNA were expressed in HCC (tumor) tissue but were only minimally observed in the non-tumor tissue in Case 1 (data not shown), in which an integrated stage of chronic HBV infection was observed (low HBV-DNA and positive anti-HBe Ab). In contrast, neither HBx nor the middle of HBs was expressed in the HCC or non-tumor tissues in Case 2. Among the candidate genes, we were interested in the BAMBI expressed at a low frequency in HCC because it is a negative regulator of TGF-β1.14Clinical characteristics of tumor and non-tumor tissues were summarized in two patients with HCC . All immearances . The digHBx gene was fused to the 5′ end of dual specific phosphatase 26 from the SSH EST library  and seven missense mutations , using PCR primers that covered 44–465 nt . The remaining 20 (40%) HCC samples without full-length HBx showed ctHBx . We iden library . Based of Case 1 . Enh1/Xputations . Notablyutations . ConsistG2 cells , in whiccolonies , left. Tpression . In contwith HBx . TherefoThe effects of HBx, ctHBx and BAMBI on Wnt/β-catenin signaling were examined using a reporter plasmid containing multimerized TCF-4-binding sites linked to the luciferase gene. BAMBI efficiently inhibited TCF-4-dependent transcriptional activation, while HBx increased this activity, and ctHBx had no effect . To deteBAMBI transcription. Mutant β-catenin increased the promoter activity of BAMBI by more than twofold in ALX  cells with wild-type β-catenin, whereas wild-type β-catenin decreased this promoter activity in HepG2 cells containing mutant β-catenin , which contained a 3′ motif consisting of a direct repeat of the hexameric nucleotide sequence AGGTCA that is a peroxisome proliferator-activated receptor response element.20 Co-transfection of BAMBI resulted in a significant decrease in either wild-type or mutant TGF-β1 promoter activity, although the basal promoter activity of wild-type TGF-β1 was low  was transduced with either Ad-BAMBI/GFP or Ad-LacZ (3 in volume within 4 weeks. However, the growth of SH-J1 cells transduced with Ad-BAMBI was markedly suppressed (in vivo. Next knockdown in HepG2 cells was established by lentiviral delivery of BAMBI, β-catenin or TGF-β1 shRNAs (Finally, we determined the functional role of BAMBI in HepG2 cells. The stable transfectants that expressed BAMBI showed morphological changes, including polygonal and epitheloid monolayer types . In the  Ad-LacZ , left, a Ad-LacZ . SH-J1 cppressed , suggest1 shRNAs .16 ctHBx mutants found in HCCs have been shown to confer a selective clonal advantage in preneoplastic or neoplastic hepatocytes through the abrogation of p53-mediated apoptosis, which contributes to hepatocellular carcinogenesis.25 We speculated that functional ctHBx/HBx modulates the expression of BAMBI and TGF-β1, thereby influencing the development of HCC.BAMBI, a negative regulator of TGF-β activation, is thought to have a role in hepato-oncogenesis because the expression of BAMBI is aberrantly elevated in some HCCs relative to its expression in corresponding non-cancerous tissues.HBx was analyzed using a reporter construct of CRE-Luc and a luciferase assay, and the C-terminal region of HBx is crucial for transcriptional function.9 The specific transcriptional activity of ctHBx remains unclear; however, our data revealed that both HBx and ctHBx are abundantly localized in the nucleus and less in the cytoplasm of HepG2 and ALX cells. An immunoblot analysis showed that β-catenin accumulated in the cytoplasmic fraction in HBx transfectants but less in ctHBx transfectants. Cytoplasmic HBx appears to modulate Wnt/β-catenin signaling. In HepG2 cells, the basal level of BAMBI activation was relatively high, while that of TGF-β1 was low, as previously observed in other HCC cases.16 BAMBI is a transmembrane protein that lacks an intracellular kinase domain but has high sequence similarity to the extracellular domain of TβRI and thus inhibits TGF-β signaling by forming a heterodimer with TβRII.10 However, our data revealed that BAMBI also transcriptionally regulates TGF-β1 expression.Our data consistently revealed that ctHBx overexpression enhanced the colony-forming ability and proliferative capacity in immortalized hepatocytes more than HBx overexpression. The transactivation activity of the C-terminal-deleted mutant of 11 However, our cell culture system showed that Smad3 stimulated BAMBI promoter activity, whereas Smad4 depressed it. This Smad3-mediated BAMBI expression can contribute to hepato-carcinogenesis through TGF-β signaling, as previously reported.6 Furthermore, a mutation in the SBE abrogated the Smad3-stimulatory effect but did not change the inhibitory activity of Smad4. Therefore, the SBE in BAMBI seems to be the binding site for Smad3, but not for Smad4.BAMBI is also directly induced by TGF-β signaling through three tandem repeats of a 13-bp sequence containing SBEs. In other words, BAMBI transcription is regulated through the direct binding of Smad3 and Smad4 to its promoter.28 In addition to mutations of β-catenin, ectopic expression of HBx along with Wnt-1 has also been reported to activate Wnt/β-catenin signaling in Huh7 cells by stabilizing cytoplasmic β-catenin, which was achieved by suppressing glycogen synthesis kinase 3 activity via the activation of Src kinase.29 Our study showed that HBx activated Wnt/β-catenin signaling, which in turn positively regulated BAMBI through TCF-1. BAMBI negatively modulated Wnt/β-catenin signaling in HepG2 HCC cells, which differs from previous data found in HEK293T cells.17 Although, β-catenin activation increased the promoter activity of BAMBI, the deletion mutants of ctHBx and HBx decreased the promoter activity of BAMBI in HepG2 and ALX cells because they have transactivation domains and regulate the transcriptional activity of other target genes in addition to β-catenin target genes.31 ctHBx profoundly suppressed BAMBI promoter activity owing to the lack of activated Wnt/β-catenin signaling, whereas HBx less profoundly suppressed BAMBI promoter activity through activated Wnt/β-catenin signaling. Thus our data suggest that HBx stimulates Wnt/β-catenin signaling, which is followed by tumor-suppressive β-catenin/BAMBI signaling. Furthermore, ectopic overexpression of BAMBI negatively downregulated both TGF-β1 and β-catenin signaling in HepG2 cells, which consequently appeared to be associated with the suppression of oncogenicity. Thus the induction of BAMBI may function in part as a tumor-suppressor mechanism in HCC. In contrast, ctHBx seems to be more oncogenic than HBx through the inhibition of tumor-suppressive β-catenin/BAMBI signaling.β-Catenin mutation leads to the functional activation of Wnt/β-catenin signaling in hepatoma cells, and the amount of literature on the stabilizing mutation of β-catenin in HCC is increasing.In conclusion, the data presented here provide a novel mechanism by which ctHBx might contribute to strong hepato-oncogenesis via the loss of tumor-suppressive β-catenin/BAMBI signaling.


Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a ‘whole animal’ developmental framework to approach this question. Here we identify a C-terminally truncated  β-catenin transcriptional activity requires a fine-tuned regulation to enable the multiple context-specific responses that it performs. These nuclear mechanisms that regulate the Wnt/β-catenin signaling remain poorly understood. Here we report the existence of C-terminal truncated forms of β-catenin in planarians (β-cat3 and 4), which, in vitro, do not show transactivation activity and compete with the canonical planarian β-catenin (β-cat1), thus acting as competitor inhibitors. Functional analyses in planarians indicate that β-cat4 acts as a negative regulator of β-cat1 during planarian eye photoreceptor specification. We provide evidence to suggest that this novel mechanism for the regulation of nuclear β-catenin activity could be conserved across animal evolution.The Wnt signaling pathway is essential for proper intercellular communication in every developmental process since it controls basic cellular events as cell fate or proliferation. The key element of the Wnt signaling is β-catenin, which controls the transcription of multiple genes in the Wnt receiving cell. A main level of regulation of the Wnt/β-catenin signaling occurs in the cytoplasm, where β-catenin protein levels depend on the activity of the β-catenin destruction complex. However, once it reaches the nucleus,  The Wnt/β-catenin pathway is an evolutionarily conserved intercellular signaling pathway with essential roles in virtually every developmental process –3 and liC. elegans. However, their specific role in nuclear signaling appears extremely complex and, apparently, divergent [Planarians, flatworms with an almost unlimited ability to regenerate and remodel their tissues during their whole life span –30, haveivergent , 42. Thuivergent , planariβ-cat1 and the main elements of the Wnt/β-catenin signaling pathway demonstrate an essential role for this pathway in the specification of the antero-posterior (A-P) axis during planarian regeneration and homeostatic cell turnover [β-cat1 silencing generates a range of anteriorized phenotypes, from “tailless” to “radial-like hypercephalized” planarians [β-cat1 have been reported in planarian brain and eye regeneration, and in gonad development [Functional analysis of turnover –34, 44. anarians , 45. Recelopment , 46. Impβ-catenin3 (β-cat3) and β-catenin4 (β-cat4), which have a truncated C-terminal transactivation domain and are expressed primarily in the nervous system. β-cat3 and β-cat4 can bind to TCF but do not activate the Wnt signal in vitro. Functional analysis in planarians indicates that β-cat4 acts as a negative regulator of nuclear β-cat1 in planarian eye photoreceptors probably by competing for binding to TCF-2, a new TCF found in planarian photoreceptor cells. We provide evidence to suggest that this novel mechanism for the regulation of nuclear β-catenin activity could be conserved across animal evolution.Here we report the existence of two new planarian β-catenins, β-catenin family members in the Schmidtea mediterranea transcriptomes revealed two new genes with protein sequences indicating that they were β-catenin homologs (Smed-β-catenin3 (β-cat3) and Smed-β-catenin4 (β-cat4), since two β-catenin paralogs had been already reported in this species [A search for homologs . We name species –33, 40.  species , 40.Since β-cat3 and 4 contain several conserved domains or residues involved in Wnt signaling and cell adhesion, we further tested the interaction of β-cat3 and 4 with the main components involved in these two processes in mammalian cell lines. Co-immunoprecipitation experiments indicated that β-cat4 strongly interacts with the cell-cell adhesion elements E-cadherin and α-catenin , whereasβ-catenins in Xenopus embryos  in intact and regenerating animals showed that β-cat3 and 4 are expressed in the parenchyma and in the central nervous system of intact animals, as well as in the new regenerating brain  analysis demonstrated that it is exclusively expressed in opsin+ cells (β-cat3 (RNAi) planarians (β-cat4 (RNAi) animals regenerated smaller eyes, with smaller pigmented spots and missing the periglobular unpigmented epidermis, which corresponds to the photoreceptor area (β-cat1 (RNAi) animals [β-cat4 (RNAi)  . The effd 3 mRNA . Thus, wβ-cat4 (RNAi) planarians by analyzing the expression of opsin and tph, specific markers of photoreceptor and pigment cells, respectively [β-cat4 resulted in reduced photoreceptor cells early in regeneration (R4d), whereas pigment cells did not show significant differences at this stage (β-cat4 (RNAi) animals showed a significantly reduced number of pigment cells compared to control (β-cat4 (RNAi) animals did not show the proper negative phototaxis behavior (β-cat4 (RNAi) organisms seemed to move normally, most of them remained in the clearest zone and did not reach the darkest zone in the same time period  animals always have fewer photoreceptor cells (β-cat4 (RNAi) animals showed a defective negative phototaxis response that got worse as the experiment progressed  and correspond to the specialized eye stem-cells [β-cat4 influences eye regeneration, β-cat4 expression was further studied with FISH. Besides its expression in eye photoreceptor cells, β-cat4 was found to be expressed in the trail of eye precursors posterior to the eye both in intact animals and during regeneration  animals using the specific markers otxA and sp6-9, which label differentiating photoreceptor and pigment cells, respectively [β-cat4 (RNAi) animals had a reduced number of otxA+ cells in the eye from early regeneration stage and a later decrease in sp6-9+ cells  resulted in failure of photoreceptor progenitor-cell specification from the early regeneration stage (R3d), whereas pigmented cells appeared reduced at a later stage (R7d)  . Thus, βtem cell .β-cat1 and β-cat4 expression in the planarian eye field. FISH for β-cat1 followed by immunostaining of VC-1, which labels the rhabdomeres of photoreceptor cells [β-cat1 was expressed in regenerating photoreceptor cells  [β-cat1 (RNAi) led to a disordered eye structure, in which photoreceptor and pigment cells formed larger eyes and in which ectopic eye cells appeared  planarians, which had a decrease in the number of photoreceptor cells, thus supporting the opposing role of these β-catenins.Next, we performed RNAi experiments to analyze the functional relationship between the two  by qPCR . Newly f by qPCR . The obs1 (RNAi) . FISH wiappeared  Movies.  animals . This reAPC-1, the APC homolog in planarians [APC-1 (RNAi) leads to β-cat1 up-regulation and nuclear accumulation [APC-1-knockdown planarians, we performed RNAi experiments in intact animals, since it is known that APC-1 silencing during two weeks does not lead to tail-head transformation [APC-1, β-cat4, and APC-1;β-cat4 for 2 weeks. The efficiency of the inhibition was analyzed by qPCR (β-cat4 (RNAi) caused smaller eyes with a decrease in both photoreceptor and pigment cell numbers compared to controls (APC-1 (RNAi) generated the same phenotype   . Co-sileor cells . This laβ-cat1 gain of function through APC-1 (RNAi) results in the same phenotype of diminished eye cell numbers as β-cat4 (RNAi), whereas β-cat1 loss of function causes the opposite effect. Thus, β-cat1, as a key downstream transcriptional co-activator in Wnt signaling, plays a negative role in planarian photoreceptor development.β-cat1 and β-cat4 causes extremely disorganized eyes, with abundant delocalized eye cells, which show a reduction in the number of photoreceptor and pigment cells  in the Planmine   phenotype in the eyes phenocopies the β-cat1 (RNAi) phenotype with respect to the number of photoreceptor cells and is consistent with the hypothesis that Wnt/β-cat1 signal inhibition is required for correct planarian photoreceptor specification. To note, TCF-2 RNAi animals showed bigger eyes but not the appearance of ectopic eye cells, suggesting that this defect is caused in a TCF-2 independent manner.The eyes of  by qPCR . Analysicontrols . The brad normal , which sor cells . The numor cells , probablβ-cat4 function in photoreceptor specification also depends on TCF-2, we performed a double RNAi assay to inhibit β-cat4 and TCF-2 simultaneously during planarian regeneration. The efficiency of the inhibition was tested by qPCR (β-cat4 (RNAi) planarians had smaller eyes with a reduced number of photoreceptor cells, whereas TCF-2 (RNAi) resulted in larger eyes with an increased number of photoreceptor cells. Remarkably, the size of the eyes in double β-cat4 and TCF-2 RNAi animals, and the number of photoreceptor and pigment cells, resembled the phenotype observed with TCF-2 (RNAi) alone  alone . This obi) alone .β-catenin paralogs is not exclusive to planarians. The β-catenin family has undergone a vertebrate-specific subphylum duplication (β-catenin and plakoglobin) [β-catenins in C. elegans) [β-catenin family members of different Lophotrocozoa species shows the existence of a unique bi-functional β-catenin in all of them except for Platyhelmintes  , two nemelegans) , and mulelegans)  , a C-terminally truncated Arm named Neural Armadillo (NArm) has been reported to occur through alternative splicing [NArm is expressed in the brain from larval stage [Drosophila NArm could have an inhibitory function, we designed in vitro Super-TOPflash experiments. Results showed that while β-catenin (S37A), a stabilized β-catenin that cannot be captured by the cytoplasmic destruction complex, could highly activate Wnt signaling, Plakoglobin activity was very low , thus representing an example of convergent evolution.β-cat4, a C-terminally truncated β-catenin generated by gene duplication within the planarian group, and we demonstrate that it modulates β-cat1 activity during eye photoreceptor cells specification but not during axial patterning. Wnt/β-catenin activity is modulated through a novel mechanism in which β-cat4, a truncated form of β-catenin that lacks the transactivation domain, exerts a negative regulatory effect on a β-cat1/TCF-2 dependent process. The existence of this new Wnt/β-catenin activity regulatory mechanism would ensure the appropriate fine-tuning of nuclear β-catenin/TCF activity in specific cell types. Genome searches and in vitro assays indicate that use of an inhibitory form of β-catenin could be a conserved mechanism to regulate β-catenin activity in specific contexts.Here we show a new role for the Wnt/β-catenin pathway in planarian eyes and demonstrate the existence of a novel mechanism to regulate its activity in a context-specific manner. We have identified The multiple roles and complexity of the Wnt/β-catenin signaling necessitate regulation at different levels. Extracellularly, secreted proteins interact with Wnts or their receptors  to inhibit the ligand-receptor interaction , 66, andβ-cat4 is required for normal specification of photoreceptors from the common eye stem cell and that its function relies on the inhibition of β-cat1 activity. Importantly, β-cat4 has no role in A-P axial polarity, in contrast to β-cat1.Here we found two new β-catenins (β-cat3 and 4) in planarians, after the two β-catenins (β-cat1 and 2) already described , which 1β-cat4 or APC, which in planarians is demonstrated to increase β-cat1 activity [β-cat1 produces the opposite phenotype, indicating that β-cat4 exerts a negative regulatory effect on a β-cat1-dependent transcriptional activity. The finding that inhibition of TCF-2, the TCF factor identified in the present study, leads to an increase in the number of photoreceptor cells, as observed after β-cat1 RNAi, and that RNAi inhibition of β-cat4 together with TCF-2 leads to the same phenotype as TCF-2 RNAi alone, indicates that TCF-2 is the downstream effector of the β-cat1 and β-cat4 action. Although we cannot rule out the possibility that β-cat1 and β-cat4 could interact in the cytoplasm, since β-cat4 conserves the essential domains to interact with cytosolic β-catenin destruction elements, our results support the regulative interaction between β-cat1 and β-cat4 at nuclear level. Thus, both β-cat1 and β-cat4 are found in the nucleus of photoreceptor cells  , 22–27, β-cat3 and 4 are mainly expressed, the complexity of which requires a more sophisticated regulation. Phylogenetic analysis of the β-catenin family in platyhelminthes supports the hypothesis that a β-cat1, β-cat2 and β-cat3/4 ortholog was present in their common ancestor. Although the expression pattern and functional analysis of the β-cat3/4 ortholog in other platyhelminth species should be done, our data predicts that the β-cat3/4 ortholog would modulate β-cat1 activity in Platyhelminthes.Here we have found a new planarian β-catenin (β-cat4) which represents a novel mechanism to fine tune the activity of nuclear β-catenin in a context-specific manner. Not surprisingly, this mechanism acts in neural tissues, where β-catenin with dual adhesion and signaling functions has been proposed to be present in the last common animal ancestor [β-catenin family are more common than previously thought. In vertebrates, plakoglobin is a genomic duplication of β-catenin that has suffered a functional specialization, since it is predominantly involved in desmosomes [Importantly, although the presence of a unique ancestor , genomicsmosomes , 77. Howsmosomes , 78 and,smosomes , 80. Takβ-catenin family has undergone several species- and phylum-specific duplications [C. elegans has four β-catenins with different roles in cell adhesion and nuclear signaling [Drosophila a unique β-catenin is found (Arm), an alternative splicing which occurs mainly in neural cells has been reported, which is known as Neural Armadillo (NArm) [β-cat4 towards β-cat1. Although the role of NArm as modulator of Arm should be demonstrated ‘in vivo’, it should be stressed that the presence of the alternative splicing isoform deleting exon 6 is conserved across all insect homologs [β-catenin of different species did not arise from any homolog of a common ancestor but originated in different animal groups from different mechanisms of gene diversification, as gene duplication or alternative splicing. Thus, the presence of a β-catenin with limited transactivation activity, which could compete with the canonical β-catenin, in different species represents an example of convergent evolution and supports its importance as a regulatory mechanism.In invertebrates, the ications . The nemignaling , and sevignaling . Importao (NArm) . Intereshomologs , supportThe existence of inhibitory β-catenins and its expression in specific cell types provides a new answer to the important question of how the same transcription factor elicits so distinct responses. It is assumed that the different transcriptomic/proteomic context of each cell could result in differential activation of β-catenin target genes. However, few tissue-specific Wnt/β-catenin regulators have been found, leading to the idea that not only Wnt/β-catenin regulators but the interplay with the other conserved signaling pathways as BMP would be essential for their complex regulation . Our resβ-cat4 is a new essential element for photoreceptor cell specification in planarians. Planarian eyes are true cerebral eyes and their simplicity makes them an excellent system to study eye development. They are composed by two cell types: rhabdomeric photoreceptor neurons, which express evolutionarily conserved photoreceptor genes such as otxA or opsin [Tryptophan hydroxylase (tph), an enzyme involved in the production of melanin, the pigment found in planarian eyes. Several studies demonstrate that both cell types arise from a common eye stem cell that express stem cell markers (h2b) and eye determinants as the transcription factors ovo, eya and six1/2 [ovo is the only factor exclusively found in the eye cell and specifically essential for eye regeneration and maintenance [sp6-9 and dlx in the common eye progenitor determines the pigment fate, while otxA specifies photoreceptors [β-cat4 is a new factor that specifies the photoreceptor fate and that it is expressed in the stem cell precursors (h2b+/ovo+), a fact that has not been yet demonstrated for otxA. Inhibition of β-cat4 results in a decrease in number of photoreceptor progenitor and differentiated cells from the earliest time points analyzed (3–4 days of regeneration), while the number of progenitors and differentiated cells of the pigment lineage only decrease several days after. This observation agrees with the already described mutual dependence of both cell populations in planarians [β-cat4 is specifically required for photoreceptor determination, according to its expression in the photoreceptor lineage, the reduced number of photoreceptor cells would lead to the subsequent decrease of the pigment lineage. Whether the origin of this mutual dependence is at the level of the common stem cell progenitors, or at the level of the differentiated cells in the optic cup, remains to be studied. Although not much is known about the mutual dependence between photoreceptor and pigment cells in vertebrates, some studies suggest that it also exists [Our results demonstrate that or opsin , 55, andd six1/2 , 55, 83.ntenance . The expeceptors , 55. We anarians . Thus, ao exists .β-cat1 inhibition results in larger eyes, while APC-1 silencing causes a reduction of photoreceptor cell number. The simplicity and approachability of planarian eyes has allowed the identification of a new mechanism to fine regulate Wnt/β-catenin pathway activity in the eyes. Thus, the truncated β-cat4 that competes with the canonical β-cat1 for binding to TCF-2 allows the required fine-tuning of the Wnt/β-catenin pathway in planarian photoreceptor cells. Since the in vitro experiments showed that β-cat4 could bind to E-cadherin and α-catenin, a role of β-cat4 in cell adhesion should be considered. However, β-cat4 protein was only found in the nucleus of photoreceptors  and vertebrates (Plakoglobin) can compete for Arm or β-catenin binding, respectively, to the TCF co-factor ‘in vivo’ and assess whether this mechanism provides a tissue-specific manner to modulate β-catenin/Arm once reaches the nucleus; 2) to study the existence and function of possible β-catenin isoforms present in other animal species, as a product of gene duplication or alternative splicing, and understand their real contribution to the regulation of the Wnt/β-catenin signal; and 3) to test whether new drugs mimicking the inhibitory action of β-cat4 could be used to fine tune the activity of nuclear β-catenin in human diseases.Our finding that planarian β-cat4 functions as a modulator of β-cat1 activity in the nucleus by regulating TCF-2 transcriptional activity has two deep implications: 1) considering its specific role in modulating β-cat1 activity during planarian photoreceptor specification, and not in other Wnt/β-catenin dependent processes such as axial patterning, it appears as a tissue-specific manner to fine-tune nuclear β-catenin activity; and 2) the finding of C-terminal truncated/non-conserved β-catenin forms in other species (NArm or Plakoglobin), which inhibit β-catenin activity in TOPflash experiments, suggests that an inhibitory β-catenin could be an evolutionarily conserved mechanism to regulate the Wnt/β-catenin signaling. The identification of a novel mechanism of Wnt/β-catenin signaling regulation has important implications in view of the complex and essential roles of this pathway in development and diseases. Thus, the present research represents a starting point to design further studies: 1) to demonstrate whether the C-terminal truncated/non-conserved β-catenins found in S. mediterranea BCN-10 biotype. The animals were maintained at 20°C in PAM (1X) water [S. polychroa collected from Sant Celoni  was used in The planarians used in this study belong to an asexual clonal strain of X) water . Animalsβ-Catenin sequences from Planarian species were identified in the Planmine database [β-Catenin sequences from Echinococcus multilocularis and Kronborgia cf. amphipodicola were found in the available databases [http://mafft.cbrc.jp/alignment/server/). Neighbor joining distance-based analyses were conducted using MEGA version 6 [database . β-Catenatabases , 96. Theersion 6 , and theHEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. To obtain pCS2+-6Myc-Smed-β-catenin3 and 4, the full length Smed-β-catenin3 and 4 was amplified by PCR and inserted into pCS2+-6Myc vector at StuI/XbaI sites. HEK293T cells were seeded in 96-well plates and transfected in triplicates with pCS2+-6Myc-Smed-β-catenin1/2/3/4 plasmids, together with Super-TOPflash and pRL-TK as the internal control. Firefly and Renilla luciferase activities were measured 36h after transfection using the Dual-Luciferase assay kit (Promega). TOPflash luciferase activity was normalized to that of Renilla. 15 ng of Super-TOPflash and 0.5 ng of pRL-TK reporter plasmids were added per well. pCS2+ empty vector was used to adjust the total DNA amount to 150 ng/well. All experiments were repeated at least three times.HeLa cells were cultured in DMEM with 10% fetal calf serum and grown on glass coverslips. Thirty-six hours post transfection of pCS2+-6Myc-Smed-β-catenin3/4 and pCS2+-HA-mAxin, cells were fixed with4% paraformaldehyde/PBS for 20 min, permeabilized with 0.2% Triton X-100/PBS for 10 min and then blocked with 3% BSA/PBS for 30 min before primary antibodies were applied. A PBS rinse for 5 min between each step was performed. The primary antibodies anti-Myc  and anti-HA  were incubated for 1h at RT diluted in 1%BSA, 0.1%Tween 20/PBS at 1:100. After washing 5min X 3 times in 3%BSA, 1%TritonX-100/PBS, the fluorophore-conjugated secondary antibodies diluted at 1:400 in 3%BSA/PBS were incubated for 1h. Donkey anti mouse–Alexa Fluor 568 (Molecular Probes) was used to visualize β-catenin3/4, and goat anti rabbit–Alexa Fluor 488 (Molecular Probes) to visualize Axin. Cell nuclei were visualized with DAPI staining. Cells were mounted in Prolong Gold antifade reagent (Thermo Fisher Scientific) and stored at 4°C before imaging. Images were recorded using a Zeiss LSM710 confocal microscope.HEK293T cells were seeded into 6-well plates and the following plasmids transfected the following day: pCS2+-6Myc-Smed-β-catenin1/2/3/4, pCS2+-flag-xTCF1, pcDNA3.1-flag-β-Trcp, pcDNA3.1-flag-E-cadherin and pcDNA3.1-flag-α-catenin. Forty-eight hours after transfection, cells were lysed and sonicated in 400 μl of lysis buffer/well  containing protease inhibitor mixture (Roche Applied Science) at 4°C. After centrifugation at 14000 rpm, 4°C for 15min, 40 μl of supernatant was mixed with 10 μl 5x SDS-loading buffer and treated at 95°C for 5min. The remaining supernatant for each well was incubated with 10 μl of FLAG-M2 beads (Sigma) at 4°C for 6h. The beads were then washed three times with lysis buffer at 4°C for 10min each, and bound proteins were eluted with 40 μl of 2x SDS loading buffer at 95°C for 5min. Immunoprecipitates and total lysates were separated by SDS-PAGE and analyzed by immunoblot with anti-Myc or anti-Flag specific antibodies.Xenopus embryos were cultured under standard conditions. mRNA was synthesized using a mMESSAGE mMACHINE SP6 kit  according to the manufacturer’s instructions. Synthetic mRNAs were microinjected into embryos cultured in 2% Ficoll 400 in 0.3× MMR  at 8-cell stage, and fixed at stage 20.in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) were performed as elsewhere described [in vitro transcription kit (Roche): Smed-β-catenin1/3/4, Smed-TCF1/2/3; Smed-opsin, Smed-otxA, Smed-sp6/9 [Smed-tph [Smed-ovo [Smed-h2b [Smed-th (tyrosine hydroxylase), Smed-tbh (tryptophan hydroxylase) [Smed-pc2 (prohormone convertase 2) [sp6/9 (1:200). Samples were observed through Leica MZ16F , Zeiss Stemi SV6 stereomicroscopes and a Zeiss Axiophot microscope ; images were captured with a ProgRes C3 camera from Jenoptik , sCMEX 3.0 camera  and Leica DFC300FX camera . Confocal laser scanning microscopy was performed with a Leica TCS-SP2  adapted for an inverted microscope.Colorimetric whole-mount escribed , 99. Theed-sp6/9 ; Smed-tpSmed-tph ; Smed-ovSmed-ovo ; Smed-h2Smed-h2b ; Smed-thoxylase) , Smed-pcrtase 2) . Primersβ-cat4  or anti-VC-1  antibody incubation. PBSTx (PBS with 3% TritonX-100) wash for 15min x3. Goat-anti-mouse-488 conjugated antibody  and Goat-anti-rabbit-HRP conjugated antibody  were used as secondary antibody. HRP signal was developed with a tyramide signal amplification kit following manufacturer’s recommendations (Perkin Elmer). anti-β-cat1 immunohistochemistry was performed as previously described [After FISH, samples were rinsed with TNTx 5min, 50%PBSTx, 50%TNTx  10min, then PBSTx 10min. 1% BSA or 10% Goat serum were used as blocking reagent for 2 hours, followed by anti-escribed . Nuclei Smed-β-catenin1/3/4, Smed-TCF2 and Smed-APC-1 were synthesized and delivered as described elsewhere [Smed-β-catenin3/4 the same procedure was performed during 2 weeks to improve the penetrance of the phenotype. For experiments in intact planarians, animals were injected 3 consecutive days per week for 5 weeks.Double-stranded RNAs (dsRNA) for lsewhere . Primerslsewhere . dsRNA wlsewhere  followinTotal RNA was extracted from a pool of 4 planarians each for every RNAi condition. Quantitative real-time PCR was performed as previously described , and datPhototactic assay was carried out as described previously . The behS1 FigABW79875.1; Smed-bcat2, ABW79874.1. The accession numbers of the new planarian β-catenins are: β-cat3, KY196224; and β-cat4, KY196225. Abbreviations: Hv, Hidra vulgaris; Pd, P. dumerilii; Hs, H. spiens; Sp, Strongilocentrotus purpuratus; Dm, Drosophila melanogaster; Smed, S. mediterranea.Protein regions and amino acids required for Wnt signaling are indicated in red. β-cat3 and 4 conserve the GSK3 phosphorylation sites in the N terminus, the hydrophobic pocket formed by Phe 253 and some of the surrounding residues, essential for Axin and TCF binding . The red(TIF)Click here for additional data file.S2 Fig(A-B) Co-immunoprecipitation assays in 293T cells showed the ability of β-cat4 to interact with E-cadherin and α-catenin, while β-cat3 could only interact with E-cadherin. β-cat1 was used as a control since it is reported its interaction with E-cadherin [C) Axis duplication assays in Xenopus embryos. β-cat1 mRNA injection induces a secondary axis in Xenopus embryos, as reported [β-cat3 or 4 mRNA injection. However co-injection of β-cat3/4 together with β-cat1 rescued the “double axis” phenotype. The percentages of embryos with the indicated phenotypes are shown (n = 50).cadherin . (C) Axireported , 40, whe(TIF)Click here for additional data file.S3 Fig(A) Both β-cat3 and β-cat4 were mainly expressed in the CNS after WISH and FISH (red). β-cat4 was also expressed in photoreceptors (see magnification in the orange box). Analyzed planarians correspond to intact animals. (B)β-cat3 and β-cat4 expression pattern during regeneration of trunk fragments, which must regenerate the head and the tail. β-cat3 and β-cat4 were expressed in the newly formed brain (arrows in R3d) and β-cat4 is also expressed in the regenerating eyes (see magnification in the orange box). (C) Phenotype of control and β-cat3 and 4 RNAi animals after dsRNA injection and induced regeneration. No polarity defects were observed in β-cat3 or β-cat4 RNAi animals. No obvious affection in the brain was neither observed through analysis of brain markers, 3C11 (green), Smed-th (tyrosine hydroxylase) (red), and Smed-tbh (tryptophan hydroxylase) (green) [(D)β-cat1 RNAi caused anteriorization of regenerating head fragments, as expected [β-cat4 RNAi planarians show a normal regenerated posterior region, indicating that β-cat4 RNAi does not cause antero-posterior defects. (E) Relative expression levels of β-cat1/2/3/4 after β-cat4 RNAi by qRT-PCR. Values represent the means of three biological replicates. Error bars represent standard deviation. Data were analyzed by Student′s t-test. *p<0.05; **p<0.01; ***p<0.001. Scale bars = 250 μm  and 50 μm (magnification in A and B). (green) . Planariexpected –33, whil(TIF)Click here for additional data file.S4 Figopsin (red) and tph (green) in control and β-cat4 (RNAi) animals, at 4, 7, and 14 days of regeneration. Scale bar = 50 μm.Double FISH of (TIF)Click here for additional data file.S5 Fig(A)β-cat4 (RNAi) planarians after 5 weeks injection show smaller eyes with small photoreceptor area. FISH of opsin (red) and tph (green) in control and β-cat4 (RNAi) intact animals along the 5 weeks of β-cat4 dsRNA injection and its quantification, showing a decrease mainly in photoreceptor cells. opsin+ cells in control h2w, 51.80±4.71 ; β-cat4 (RNAi) h2w, 30.75±5.20 ; control h3w, 45.40±6.26 ; β-cat4 (RNAi) h3w, 25.50±2.62 ; control h4w, 39.40±4.25 ; β-cat4 (RNAi) h4w, 18.70±1.64 ; control h5w, 37.08±4.14 ; β-cat4 (RNAi) h5w, 12.50±1.35 . tph+ cells in control h2w, 18.60±1.90 ; β-cat4 (RNAi) h2w, 16.38±1.41 ; control h3w, 18.80±2.00 ; β-cat4 (RNAi) h3w, 13.25±0.89 ; control h4w, 17.40±1.43 ; β-cat4 (RNAi) h4w, 10.80±1.32 ; control h5w, 16.00±3.10 ; β-cat4 (RNAi) h5w, 10.00±1.33 . *p<0.05, ***p<0.001 (t test). (B) Phototaxis assay of β-cat4 (RNAi) intact animals. Graphical representation of the percentage of control and β-cat4 (RNAi) planarians found in the different regions in 2 minutes. The scheme of the container with the different zones is shown. β-cat4 (RNAi) animals became more insensitive to photophoby along the experiment. (C) FISH of β-cat4 (red) in intact animals. Yellow arrows indicate isolated β-cat4+ cells in the trail posterior to the eyes, corresponding to eye precursor cells. (D) Quantification of β-cat4+, sp6-9+ and ovo+ cells in the eye of 7 days regenerating animals (n = 6 eyes). Scale bars = 50 μm.(TIF)Click here for additional data file.S6 FigotxA (red) and sp6-9 (green) in control and β-cat4 (RNAi) animals at indicated regeneration time points and its quantification in the eye structure. otxA+ cells in control R7d, 52.9±2.96 ; β-cat4 (RNAi) R7d, 21.00±1.58 ; control R14d, 48.25±4.95 ; β-cat4 (RNAi) R14d, 15.80±0.92 . The sp6-9+ cells number for control R4d, 17.00±3.74 ; β-cat4 (RNAi) R4d, 18.75±3.01 ; control R7d, 21.38±1.85 ; β-cat4 (RNAi) R7d, 17.00±2.78 ; control R14d, 20.00±2.16 ; β-cat4 (RNAi) R14d, 9.20±2.20 . **p<0.01, ***p<0.001 (t test). Scale bar = 50 μm.FISH of (TIF)Click here for additional data file.S7 Fig(A) Left, scheme of a planarian eye showing the eye photoreceptors (in blue) and the pigment cells (in brown). middle, FISH of β-cat1 (green) combined with immunostaining with anti-VC-1 (red), which labels rhabdomeres of photoreceptor cells (red), demonstrates the localization of β-cat1 mRNA (green) in the photoreceptor area. Double FISH of β-cat4 (red) and β-cat1 (green) demonstrates their localization in the photoreceptor area (dashed line) (B) Immunostaining of control and β-cat4 RNAi planarians with the anti- β-cat4 antibody (green) to demonstrate its specificity. Quantification of β-cat4 signal per cell shows that it decreases significantly in β-cat4 RNAi animals compared to controls. ***p<0.001 (t test). n = 140 cells of 5 different animals per condition. β-cat4 signal was measured to obtain the raw integrated density (RID) for each individual nuclei. (C) Relative expression level of β-cat1 after β-cat1 RNAi by qRT-PCR. Values represent the means of three biological replicates. (D) FISH of opsin (red) and tph (green) in control and β-cat1 (RNAi) at 9 days of regeneration. β-cat1 (RNAi) planarians show larger eyes and the appearance of ectopic photoreceptor (yellow arrows) in 50% of the eyes analyzed. Nuclei are stained with DAPI. The respective quantification of opsin+ and tph+ cells per eye is shown. Ectopic cells were not included in the analysis. opsin+ cells in control R9d, 29±3 ; β-cat1 (RNAi) R9d, 38±8 . tph+ cells in control R9d, 13.5±3.5 ; β-cat1 (RNAi) R9d, 18,2±1.6 . *p<0.05, ***p<0.001 (t test). (E) Relative expression level of β-cat4 and APC after their RNAi by qRT-PCR. (F) Relative expression level of β-cat4 and β-cat1 after their RNAi by qRT-PCR. In (D), (E) and (F) values represent the means of three biological replicates. Error bars represent standard deviation. Data were analyzed by Student′s t-test. *p<0.05; **p<0.01. Scale bars = 20 μm , 50 μm (D).(TIF)Click here for additional data file.S8 Fig(A) Phylogenetic analysis of TCF homologs from different species. Confidence values are shown at the main nodes. Accession number of the analyzed sequences: Aq-TCF ADO16566.1; NBf-TCF AAZ77711.1; Cs-TCF BAB68354.1; Dm-Pangolin P91943.1; Dr-LEF-1 NP_571501.1; Dr-TCF-1 NP_001012389.1; Dr-TCF-3 Q9YHE8.1; Dr-TCF-4 NP_571334.1; Eg-TCF-1b EUB55217.1; Eg-TCF-2 EUB60264.1; Eg-TCF3 CDS20600.1; Hs-LEF-1 NP_057353.1; Hs-TCF-1 AAH48769.1; Hs-TCF-3 NP_112573.1; Hs-TCF-4 NP_110383.2; Pd-TCF ANS60442.1; Sd-TCF CAH04889.1; Sp-Tcf/Lef AAD45010.1; Tc-Pangolin XP_008191151.1; Ts-TCF-1b OCK35857.1; Ts-TCF-2 OCK32932.1; Ts-TCF-3 OCK34187.1. TCF sequences from planarian species were found in Planmine [Smed-TCF1, Smed-TCF2 and Smed-TCF3 are: KY196226, KY196227, KY196228. Abbreviations: Aq, Amphimedon queenslandica; Bf, Branchiostoma floridae; Cs, Ciona savignyi; Dlac, Dendrocelum lacteum; Dm, Drosophila melanogaster; Dr, Danio rerio; Eg, Echinococcus granulosus; Hs, Homo sapiens; Pd, Platynereis dumerilii; Pnig, Polycelis nigra; Pten, Polycelis tenuis; Ptor, Polycelis torva; Sd, Suberites domuncula; Smed, Schmidtea mediterranea; Sp, Strongylocentrotus purpuratus; Spol, Schmidtea polychroa; Tc, Tribolium castaneum; Ts, Taenia saginata. (B) Expression pattern of TCF-1 and TCF-3. Both of them are predominantly expressed in the CNS. (C) Relative expression level of TCF-2 after RNAi by qRT-PCR. (D) Double FISH of opsin (red) and tph (green) in control and TCF-2 (RNAi) animals at 5 days of regeneration with the corresponding quantification opsin+ and tph+ cells. opsin+ cells in control R5d, 25.38±2.07 ; TCF-2 (RNAi) R5d, 41.88±2.59 . tph+ cells number for control R5d, 14.38±1.77 ; TCF-2 (RNAi) R5d, 20.63±1.51 . ***p<0.001 (t test). (E) FISH of pc2 (prohormone convertase 2) (red) [TCF-2 (RNAi) at 7 days of regeneration. No difference is observed between control and TCF-2 (RNAi) brains. (F) Relative expression levels of TCF-2 and β-cat4 after their RNAi by qRT-PCR. In (C) and (F) values represent the means of three biological replicates. Error bars represent standard deviation. Scale bar = 250 μm (B), 50 μm (C) and 200 μm (D).(TIF)Click here for additional data file.S9 FigS. mediterranea TCF proteins (Smed-TCF-1/3). The β-catenin binding domain [Alignment of the three g domain , underlig domain , underlig domain , in red,(TIF)Click here for additional data file.S10 FigABW79874.1; Sp-bcat NP_001027543.1; Tc-Arm1 EFA10737.1; Tc-Arm2 NP_001164124.1. β-Catenin sequences from Echinococcus multilocularis and Kronborgia cf. amphipodicola were found in the available databases [Aplysia californica; Aq, Amphimedon queenslandica; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Cl, Cerebratulus lacteus; Crfor, Crepidula fornicata; Cv, Chaetopterus variopedatus; Dm, Drosophila melanogaster; Dr, Danio rerio; Em, Echinococcus multilocularis; Hs, Homo sapiens; Hv, Hydra vulgaris; Kram, Kronborgia amphipodicola; Mm, Mus musculus; Osc, Oscarella carmela; Pd, Platynereis dumerilii; Ph, Parhyale hawaiensis; Pnig, Polycelis nigra; Pten, Polycelis tenuis; Sci, Sycon ciliatum; Sk, Saccoglossus kowalevskii; Sm, Schistosoma mansoni; Smed, Schmidtea mediterranea; Sp, Strongylocentrotus purpuratus; Spol, Schmidtea polychroa; Tc, Tribolium castaneum.β-catenin sequences from planarian species were identified in the Planmine database . Platyheatabases , 96. β-catabases . Accessi(TIF)Click here for additional data file.S11 FigThe functional domains are indicated. Arm splicing isoform  and Plakoglobin, highlighted in yellow, conserve all functional domains but show a shorter C-terminal transactivation domain. Accession numbers and abbreviations are indicated in (PDF)Click here for additional data file.S1 MovieControl animals moved away from the light and remained in the darkest zone (zone 3 in (AVI)Click here for additional data file.S2 Movieβ-cat4 (RNAi) organisms seemed to move normally, most of them remained in the clearest zone and did not reach the darkest zone in the same time period than controls (Although controls .(AVI)Click here for additional data file.S3 Movieopsin (red) and pigment cells are labeled with an RNA probe of tph (green). Scale bars = 10 μm.Animated Z-Stack of the eye corresponding to a control animal. Nuclei are labeled with DAPI (blue), photoreceptors are labeled with an RNA probe of (AVI)Click here for additional data file.S4 Movieβ-cat1 (RNAi) animal. Nuclei are labeled with DAPI (blue), photoreceptors are labeled with an RNA probe of opsin (red) and pigment cells are labeled with an RNA probe of tph (green). Scale bars = 10 μm.Animated Z-Stack of the eye corresponding to a (AVI)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Developmental Cell, The scaffold protein APC has a well-known function in ensuring β-catenin destruction. In this issue of  Developmental Cell, Saito-Diaz et al. (2018) uncover another role for APC in Wnt signaling: to prevent clathrin-dependent signalosome formation in the absence of ligand.The scaffold protein APC has a well-known function in ensuring β-catenin destruction. In this issue of  Developmental Cell, activation of β-catenin signaling by loss of APC still responds to manipulation of upstream components. In a series of elegant experiments, Saito-Diaz et al. demonstrate that loss of APC leads to spontaneous signalosome formation without Wnt ligand in a manner that requires clathrin, DVL, and the receptor complex , and two scaffold proteins, Axin and APC—ensures that β-catenin is continuously targeted for proteasomal degradation through phosphorylation by GSK3 and subsequent ubiquitylation . In the  complex .Figure 1The authors first show that inhibition of LRP6 in colorectal-cancer cell lines (CRCs) carrying mutations in APC, or in cells lacking APC by CRISPR-mediated knockout, reverses β-catenin signaling. They further validate this requirement for LRP6 with a blocking antibody that they developed against a central region of the extracellular region of this receptor. Perturbation of other signalosome components, Fz or DVL, also prevented pathway activation in APC mutant cells, indicating that they, too, are required for β-catenin signaling in APC mutant cells. Moreover, LRP6 was found in a dense fraction indicative of signalosome in lysates prepared from APC mutant cells. Therefore, it appears that, in APC-deficient cells, the signalosome forms and depletion of signalosome components allows the destruction complex to recover its function, most likely through the activity of APC2, a paralog of APC . These eex vivo organoid cultures derived from intestinal adenomas of APCmin mice and to the Drosophila adult midgut. As expected, LRP6 suppression inhibited the growth of APCmin organoids. In the Drosophila adult midgut, as in the adult mammalian intestine, loss of APC aberrantly activates the Wnt pathway, leading to increased expression of Wnt target genes, overproliferation of intestinal stem cells, and disruption of epithelial polarity. All three phenotypes were reversed by genetic disruption of signalosome components, but not by mutations that target the ligand (Wingless) or its secretion, mirroring the findings from cell culture. How does signalosome assembly induced by loss of APC occur in the absence of ligand? As the authors show, this involves an APC-modulated, clathrin-dependent mechanism.To confirm their findings in a physiological context, the authors turned to 2), which in turn recruits clathrin and its adaptor AP2. AP2 and clathrin are then further enriched at these domains through the AP2-binding motifs present in LRP6 and DVL  , leading and DVL . One mod and DVL . An alte and DVL . These p and DVL .Clathrin is known to be required for ligand-induced Wnt signaling but it will come as a surprise to many that, as shown by The results of


APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs.The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the   •APC CID protects β-catenin from USP7-mediated deubiquitination•APC lacking CID exposes β-catenin to USP7 for deubiquitination•APC mutant CRC by restoring β-catenin ubiquitinationUSP7 depletion inhibits Wnt in •USP7 inactivation suppresses xenograft tumor growth and is tumor specific APC-truncating mutation activates Wnt signaling by promoting USP7-mediated β-catenin deubiquitination. USP7 inhibition significantly suppresses tumor growth of APC-mutated colon cancer cells but does not affect cells with wild-type APC, suggesting that it can be used as a tumor-specific drug target.Novellasdemunt et al. find that an  The canonical Wnt/β-catenin signaling pathway is evolutionarily conserved and plays crucial roles in many biological processes, such as stem cell maintenance and cell fate decision . The keyAPC truncating mutation is the hallmark of the vast majority of human CRCs. Its loss initiates adenoma formation through constitutive Wnt activation (APC somatic mutations occur in the “mutation cluster region” (MCR) between codons 1,286 and 1,513  . Mutatiotivation . Most APnd 1,513 . Region-tibility . Differetibility . Howevertibility . The CIDtibility . A more tibility .APC mutation in β-catenin ubiquitination and Wnt activation. We find that the DUB enzyme USP7 is crucial in sustaining pathological, but not physiological, Wnt activation in APC-truncated CRCs by mediating β-catenin deubiquitination.Protein ubiquitination-mediated degradation is a reversible process that is tightly regulated by E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). The role of DUBs in regulating β-catenin ubiquitination is not well defined, particularly in the context of CRC. Here, we sought to investigate the mechanistic role of the APC truncating mutations endogenously in wild-type (WT) HEK293T cells with intact Wnt signaling cascade using the CRISPR/Cas9 genome editing technique. Different APC truncating deletions (APC1–6) were generated by targeting the central region of the APC protein sequentially, as shown in AXIN2  treatment in WT cells, while β-catenin was stabilized in all CID-deleted mutants APC3–6, again with a gradual difference between the various mutants C. ImmunoAPC mutations in CRC cells abrogate Axin-bound β-catenin ubiquitination  and CK1-mediated (S45) β-catenin phosphorylation statuses in WT versus APC2-5 cells in the absence of MG132. In contrast to the previous finding (APC mutations at the MCR (APC3 and APC4). Our APC CRISPR mutant isogenic cells provide unique tools for direct quantitation of endogenous β-catenin phosphorylation for the first time in a single APC mutation event.A previous study proposed an alternative mechanism where rylation . To dete finding , our dat finding G. The reAPC mutant SW480 CRC cells (amino acid [aa] 1338) and measured the Wnt transcriptional activity. TopFlash luciferase assay showed that expression of APC ΔCID failed to suppress Wnt activation in SW480 cells as compared to APC WT  expression constructs. To test their Wnt inhibitory roles, we expressed the constructs in the o APC WT A. IP-couo APC WT B and S2ATo examine the compositional differences of the β-catenin destruction complex upon the loss of CID, WT and ΔCID APCs were individually expressed in human HEK293T and mouse L cells for tandem affinity purification (TAP) followed by mass spectrometry (MS) analysis C. IntensConsistent with our MS data, we observed a significant increased binding of USP7 to the destruction complex in the CID-deleted APC mutants (APC3–5) as compared to the WT and the CID-containing APC2 cells E and 2F.APC mutation promotes β-catenin deubiquitination by (1) reduced binding of the E3 ligase β-TrCP from the destruction complex and (2) exposing β-catenin to the DUB enzyme USP7. This led us to hypothesize the direct interaction between USP7 and β-catenin proteins.Based on our current data, we speculate that APC CID may, indeed, function to protect β-catenin from binding to the DUB enzyme USP7 in the destruction complex. Deletion of CID will then expose β-catenin to USP7 for deubiquitination, leading to subsequent β-catenin accumulation and aberrant Wnt activation. To test this hypothesis, we co-expressed FLAG-tagged β-catenin and MYC-tagged USP7 in APC4 cells with or without additional CID expression. β-catenin was pulled down using FLAG antibody, and the binding of USP7 in the presence or absence of CID was examined. Consistent with our hypothesis, expression of CID, indeed, suppressed the binding of USP7 to β-catenin E. TogethAPC mutated (APC4) cells using Axin1-specific antibody. In contrast to the reported data, we were unable to detect RNF220 in the endogenous Axin destruction complex  using CRISPR/Cas9 gene targeting. Wnt activation of APC4 cells was significantly suppressed upon USP7 mutation . Unlike the parental cell line, USP7 depletion in HCT116 APCCRISPR cells showed significant Wnt suppression   B and 5C. Caco-2) D.Figure APC mutation (DLD1) and WT HEK293T treated with HBX19818 did not suppress Wnt activation  was administered at 30 mg/kg daily by intraperitoneal injections . TreatmeTo validate the tumor-suppressive effect of USP7 inhibitors, we repeated the xenograft experiment by comparing tumor growth between the parental and USP7 CRISPR-targeted SW480 cells D. ConsisAPC mutation (HCT116 APCCRISPR) in both p53 WT and null backgrounds. Strikingly, the HCT116 APCCRISPR USP7CRISPR cells demonstrated remarkable suppression in tumor growth when compared to the HCT116 APCCRISPR cells  screening in HEK293T cells . Anotherp53 mutation (SW480 and HCT116 p53−/−). Our results clearly demonstrated that USP7-mediated β-catenin deubiquitination is p53 independent. We confirmed that targeting USP7 can suppress tumor growth in vivo by inhibiting Wnt activation. To our knowledge, this is the first intervention against USP7 in CRC in vivo in the context of the Wnt signaling pathway.Previous studies have identified other proteins as potential substrates of the DUB enzyme USP7 . The tumAPC-mutated CRC cells indicates the essential role of USP7 in cancer cell survival. On the other hand, incomplete KO of USP7 was sufficient to inhibit Wnt signaling and suppress tumor growth. Our data echo a recent study showing that minimal APC restoration is sufficient for tumor suppression  according to the manufacturer’s instructions. For the TOPFlash luciferase assay, cells in a 48-well plate were transfected with 100 ng of the reporter plasmid TOP or FOP and 10 ng of TK/Renilla for each well. 100 ng of additional plasmid was cotransfected in each well when indicated. After 16 hr of transfection, cells were treated with Wnt3a-conditioned medium (when indicated) or control medium or with USP7 inhibitor treatment or DMSO for an additional 18 hr. The luciferase activity was measured using a luminometer. For transfections for IP, cells were also seeded 24 hr before transfection in a 10- or 15-cm plate, and 7–15 μg plasmid was transfected in each plate dependent on each plasmid. 48 hr after transfection, cells were lysed.HEK293T, SW480, DLD1, HCT116, and Caco-2 cells were maintained in DMEM GlutaMAX (GIBCO) supplemented with 5% fetal bovine serum (FBS) (GIBCO) and with 100 U/mL penicillin (GIBCO) and 100 μg/mL streptomycin (GIBCO). All cells were maintained at 37°C in an incubator with 5% COCells were pre-treated with 10 μM MG132 proteasome inhibitor for 4 hr prior to lysate collection for all IP experiments. Cells were washed and collected with cold PBS and lysed in cold lysis buffer as previously described .All animal-regulated procedures were carried out according to Project License constraints and Home Office guidelines and regulations.See the L.N. and V.S.W.L. designed the experiments and analyzed the data. L.N., V.F., L.C., P.A., A.K., V.E., and A.P.S. conducted the experiments. L.N. and V.S.W.L. wrote the paper.


Hepatitis C virus (HCV) infection is one of the leading causes of hepatocellular carcinoma (HCC) worldwide but the mechanistic basis as to how chronic HCV infection furthers the HCC process remains only poorly understood. Accumulating evidence indicates that HCV core and nonstructural proteins provoke activation of the Wnt/β-catenin signaling pathway, and the evidence supporting a role of Wnt/β-catenin signaling in the onset and progression of HCC is compelling. Convincing molecular explanations as to how expression of viral effectors translates into increased activity of the Wnt/β-catenin signaling machinery are still largely lacking, hampering the design of rational strategies aimed at preventing HCC. Furthermore, how such increased signaling is especially associated with HCC oncogenesis in the context of HCV infection remains obscure as well. Here we review the body of contemporary biomedical knowledge on the role of the Wnt/β-catenin pathway in the progression from chronic hepatitis C to cirrhosis and HCC and explore potential hypotheses as to the mechanisms involved. Hepatitis C virus (HCV) is estimated to infect up to 2% of the global population (around 180 million people worldwide) . FollowiDistinct from hepatitis B virus (HBV), a DNA virus that can integrate into the human genome and thus directly provoke genomic alterations potentially leading to cancer , HCV is The Wnt/β-catenin pathway is an attractive candidate to mediate the HCV-specific effects leading to hepatocyte oncogenic transformation. Activation of this pathway clearly contributes to hepatocarcinogenesis as indicated by the detection of recurrent genetic mutations of Wnt/β-catenin signaling pathway components in HCC that appear especially frequent in HCV-related tumors. HCV-derived viral proteins appear to be capable of autonomous activation of Wnt/β-catenin signaling, although the underlying molecular mechanisms remain poorly understood. Here we explore potential hypotheses explaining these effects and summarize documented interactions of Wnt/β-catenin signaling components in HCC patients with HCV infection. We propose that the Wnt/β-catenin signaling pathway constitutes a rational target for the prevention and treatment of HCV-associated HCC.Homo sapiens DNA is now quite complete, it is unlikely further Wnt paralogues will be discovered. Wnts can provoke different modes of cellular signaling, either mediated by β-catenin or independent of this protein. According to the dependence on β-catenin for provoking cellular effects, Wnts are classified into canonical (β-catenin-dependent) and noncanonical (β-catenin-independent) subgroups [Wnt/β-catenin signaling is a pivotal morphogenetic pathway and accordingly is associated with a host of physiological and pathophysiological processes, including embryonic patterning, cell proliferation, cell differentiation, angiogenesis, and especially cancer –12. Wnt ubgroups , 16. In Except for several stem cell niches, canonical Wnt/β-catenin signaling is typically not active in tissues of adult individuals , despiteCYCLIND1, AXIN2, MYC, RNF43 , and ZNRF3  [On binding of Wnt ligands to a complex consisting of the FZD receptor and coreceptors, which include low-density lipoprotein receptor related protein 5/6, the scaffolding protein disheveled is recruited to the membrane, an event that in turn causes the disassembly of the multiprotein β-catenin destruction complex. This results in rescue of β-catenin from proteasomal degradation and thus the accumulation of β-catenin in the cytoplasm, eventually causing β-catenin translocation to the nucleus . In the , ZNRF3) , 22. RNF, ZNRF3) . This E3, ZNRF3) . Hence rCDH1 gene encoding E-cadherin [Further complexity is added by the role of β-catenin in cell–cell adhesion, where it acts, independent of its transcriptional activity, by forming a complex with cadherins and facilitating the formation of cellular junctions between adjacent hepatocytes. The β-catenin captured in these cell-adhesion complexes represents a dynamic pool of β-catenin capable of nuclear signaling following several stimuli. One of these stimuli is β-catenin tyrosine phosphorylation by receptor tyrosine kinases activated by growth factors produced by epithelial and stromal cells. In particular, phosphorylation of the β-catenin residue Tyr654 results in its release from cadherins and an increase in T-cell-factor-mediated transcriptional activity –28. Furtcadherin , 31, thicadherin , 33. HenCTNNB1 (which encodes β-catenin), followed by loss-of-function mutations in AXIN1, AXIN2, and APC. The relative mutation frequencies of these various Wnt/β-catenin signaling elements are different in HCC as compared with other cancers . The reason that these differences emerge may result from different causes of HCC and thus the type of mutations induced in liver genomes as compared with other sites in the body, but may also derive from the fact that in different organs, optimal cancer-driving Wnt/β-catenin signaling mutations may be substantially different, resulting in selection pressure for different types of mutations [CTNNB1. Missense, insertion, or partial deletions within CTNNB1 exon 3 lead to the generation of a mutant β-catenin preventing the proper phosphorylation of amino acids Ser33, Ser37, Thr41, and Ser45, resulting in compromised degradation and thus stabilization of β-catenin in the cytoplasm. Less frequently, loss-of-function mutation of AXIN1, AXIN2, or APC is found in 3.2%, 0.4%, and 0.2% of HCCs respectively, evidently contrasting with the situation in colorectal cancer, where up to 80% of cancers display mutated APC [Important in the context of potential modulation by HCV infection in relation to HCC is that aberrant signal transduction in general and β-catenin signaling in particular is one of the key characteristics of hepatocarcinogenesis . Functioutations , 36. As ated APC . Thus atCTNNB1  in HCV-related HCC is especially striking, in the view of the relative absence of such mutations in HBV-related liver cancers but also in the view of their paucity in non-virally-associated HCC  or that observed in total non-virally-associated HCC (21.2%). Furthermore, we noticed that, differently from colorectal cancers, which mainly show Thr41 and Ser45 mutations [CTNNB1 mutations from Asp32 to Ser37 residues [CTNNB1 mutations, suggesting that activating mutations occurring at the Asp32 to Ser37 residues lead to higher signaling levels than mutations at Thr41 and Ser45 [HCV infection presents a substantial clinical challenge, for which only direct antiviral medication appears to be a suitable solution . If leftCC Table . Around utations , HCV-relresidues , 70, 71 nd Ser45 . This maCTNNB1 mutation appears to be a late-stage event in the progression to HCC [CTNNB1 mutations observed may be directly and causally related to the HCV infectious process as in vitro studies show that both acute and chronic HCV infections provoke specifically CTNNB1 mutations in hematological model systems and HCCs [CTNNB1 mutations may prove exceedingly useful for the design of strategies aimed at preventing HCV-associated HCC.Although n to HCC , the higand HCCs . EvidentThe HCV genome is a single-stranded positive-sense 9.6-kb RNA molecule, which includes a single open reading frame encoding a polyprotein of approximately 3000 amino acids that following translation is cleaved into ten mature proteins by both host and viral proteases. These proteins are the structural proteins , viroporin p7, and the nonstructural proteins . The pro-oncogenic pathogenesis of HCV appears mainly mediated by the core protein and two of the nonstructural proteins, NS3 and NS5A . These pCDH1 (E-cadherin) gene promoter [The 21-kDa core protein is the major component of HCV. Despite lacking obvious organelle localization signals in the primary sequence, it is detected not only in the cytosol but also in the Golgi apparatus, in lipid droplets, and in the nucleus , 81. Rempromoter . ReducedThe notion that HCV is under selection pressure to counteract apoptosis is further reinforced by observations that NS5A not only functions as a component of the HCV RNA replication complex  but alsoSOCS1) [CTNNB1 mutations. Support for this idea can be found in the observation that in experimental animals the hepatocarcinogenic nitrosamine diethylnitrosamine provokes cancer by inducing CTNNB1 mutations [The hypotheses that successful HCV infection critically depends on its potential to stimulate Wnt/β-catenin signaling is further supported by observations that, in addition to direct activation, HCV infection leads to elevation of the levels of miR-155  and miR-SOCS1) , which dSOCS1) . MoreoveSOCS1)  by supprutations , 97, andApc−/−Lect2−/−) mouse model [The HCV battles with the immune system. Thus negative modulation of inflammatory responses through enhanced Wnt/β-catenin signaling could conceivably provide further selection pressure for HCV to acquire Wnt/β-catenin signaling activating properties. However, the effect of Wnt/β-catenin signaling on hepatocyte immune responses remains controversial. On one hand, Wnt/β-catenin signaling could suppress the immune response by blunting T-cell activation , 99, redse model . In potese model . Thus thChronic inflammation evoked by HCV infection may culminate in liver fibrosis. Such fibrosis progresses gradually and disrupts liver physical structure and function over the course of several decades, finally resulting in fatal diseases such as cirrhosis and HCC . Given H1–S phase transition for hepatocyte proliferation [The EGFR pathway controls a variety of signals ranging from cell proliferation, cell motility, and apoptosis decrease to epithelial–mesenchymal transition, upregulation of matrix metalloproteinases (MMPs), and even stem cell maintenance . EGFR isferation . EGFR paferation , and is feration , 110. EGferation , 112. Foferation . The virferation . Bindingferation . Activatferation . Conversferation , 117. Thferation , 118–120feration –125 FGF18 andCDC73), acting as a tumor suppressor inhibiting CYCLIND1 and MYC, together with suppressor of variegation 3-9 homolog 1. As a result, parafibromin acquires the ability to bind β-catenin stably, overriding the repression effect and inducing the expression of Wnt target genes [It has been reported that the Src homology region 2 domain containing phosphatase 2 (SHP-2) can be activated by HCV structural protein E2 . Thus coet genes  Fig. 3)CDC73), aAs one of the important cascades involved in HCV-related HCC initiation and development, Wnt/β-catenin signaling is aberrantly activated by HCV viral core and NS5A proteins. In turn, stimulated Wnt/β-catenin signaling promotes progression of hepatitis C during inflammation and fibrosis, eventually promoting cirrhosis and HCC. This interaction is further aggravated by a vicious circle involving the EGFR and FGF pathways.