Since β-catenin phosphorylation by glycogen synthase kinase 3β (GSK3β) and casein kinase 1ε (CK1ε) results in its degradation, mutations affecting these phosphorylation sites cause β-catenin stabilization. However, the relevance of missense mutations in non-phosphorylation sites in exon 3 remains unclear. The current study explores significance of such mutations in addition to addressing the clinical and biological implications of β-catenin activation in human HCC.CTNNB1, gene expression of β-catenin targets such as glutamate synthetase (GS), axin2, lect2 and regucalcin (RGN), and protein expression of β-catenin were examined in 125 human HCC tissues.Gene alteration in exon3 of p = 0.0461). Of these patients with nuclear β-catenin localization, loss of described β-catenin target gene upregulation showed significant poorer overall survival than others (p = 0.0001).Sixteen patients (12.8%) showed conventional missense mutations affecting codons 33, 37, 41, and 45. Fifteen additional patients (12.0%) had other missense mutations in codon 32, 34, and 35. Induction of exon3 mutation caused described β-catenin target gene upregulation in HCC cell line. Interestingly, conventional and non-phosphorylation site mutations were equally associated with upregulation of β-catenin target genes. Nuclear localization of β-catenin was associated with poor overall survival (CTNNB1 lead to β-catenin activation in human HCC. Additionally, the mechanism of nuclear β-catenin localization without upregulation of described β-catenin target genes might be of clinical importance depending on distinct mechanism.This study suggests that both conventional and other missense mutations in exon 3 of  CTNNB1) mutation and mutation in AXIN1 are major oncogenic gene alterations in HCC, seen in 20–40% and 10%, respectively [CTNNB1 mutations cause stabilization of β-catenin protein through loss of codon 33, 37, 41, and 45 phosphorylation sites for glycogen synthase kinase 3β (GSK3β) and casein kinase 1ε (CK1ε) resulting in upregulation of β-catenin target genes such as glutamine synthetase (GS), axin2 and regucalcin [Primary liver cancer which is predominantly hepatocellular carcinoma (HCC), is the sixth most common cancer worldwide and the third most frequent cause of cancer-related mortality . β-Catengucalcin .CTNNB1 exon 3 are theoretically considered to affect β-catenin signaling because of the structural change of protein caused by alteration of amino acid close to the GSK3β binding. Austinat et al. reported that transfection of P44A or H36P, mutants of CTNNB1 that are not direct phosphorylation sites of CK1ε or GSK3β enhanced the promotor activity of TCF4/β-catenin [In addition to mutations directly affecting phosphorylation sites, there are a substantial number of patients who have other missense mutation sites in exon 3  . These m-catenin . HoweverIn the current study we validate that missense mutations not directly affecting GSK3β or CKε binding sites in β-catenin gene indeed show active β-catenin signaling in human HCC. We also show that those HCC that exhibit nuclear β-catenin localization have worse prognosis. Immunohistochemical and transcriptomic analysis revealed that some patients whose tumor showed nuclear β-catenin localization but didn’t have determined target gene upregulation turned out to show the poorest survival. We eventually discuss the implication of targeting β-catenin signaling in patients with aberrant β-catenin activation in HCC caused by genetic alterations , 10.One hundred twenty five patients with HCC were registered and underwent curative first-line surgery at the Department of Gastroenterological Surgery, Kumamoto University Hospital, between 2005 and 2010. Specimens of primary HCC and adjacent normal liver tissues were obtained from the patients after their written informed consent was obtained. This study was approved by the Human Ethics Review Committee of the Graduate School of Life Sciences, Kumamoto University .Axin2 forward 5′- GCTGACGGATGATTCCATGT -3′, Axin2 reverse 5′- ACTGCCCACACGATAAGGAG -3′, and universal probe #56; Lect2 forward 5′- GCTATCCATGGAATATTAGAACTTGA -3′, Lect2 reverse 5′- TGCATCTCTCATTTCTTTAGTGTGA -3′, and universal probe #58; Regucalcin forward 5′- TCAATGATGGGAAGGTGGAT -3′, Regucalcin reverse 5′- TGGTGCCGCTCAAGAACT -3′, and universal probe #30; GS forward 5′- AAGAGCGGAGCGTGTGAG -3′, GS reverse 5′- TCATGGTGGAAGGTGTTCTG -3′, and universal probe #55; HPRT forward 5′-TGACCTTGATTTATTTTGCATACC-3′, HPRT reverse 5′- CGAGCAAGACGTTCAGTCCT-3′, and universal probe #73. HPRT was used as internal control gene. For amplification, an initial denaturation at 95°C for 10 min was followed by 15 s at 95°C, 15 s at 60°C, and 13 s at 72°C.RNA extraction, reverse transcription, and qRT-PCR was performed as previously described . PrimersCTNNB1, extraction of PCR products, and sequencing are performed as previously described [Genomic DNA was extracted using QIAamp DNA Micro Kit (Qiagen). Amplification exon 3 of escribed .Cultured cells were washed, and fixed in 4% paraformaldehyde for 15 min. Subsequently, the samples were washed and nonspecific protein binding sites were blocked by 30 min incubation in 3% bovine serum albumin. Sections were incubated for 1 h with rabbit anti-β-catenin antibody (SantaCruz). After 3 washes in PBS, the sections were incubated for 30 min at room temperature in the dark, with the appropriate secondary antibody. After 2 washes in PBS, Hoechst staining was performed and the mounting reagent was applied.Hep3B-S33Y and Hep3B-pCI cells were plated in 24 well plate. TCF4 reporter plasmid was transfected with lipofectamin 2000 (Invitrogen) into them. Cells were lysed and prepared by using the luciferase reporter assay system . Activity was read on a luminometer .Statistical analyses were performed using JMP ver. 8.0 . Values are expressed as the mean ± SD. Differences between groups were calculated by the Wilcoxon test. P < 0.05 was defined as significant.CTNNB1 revealed 16 patients with conventional missense mutations in the GSK3β and CK1ε phosphorylation sites affecting codons 33, 37, 41, and 45. Additional 15 patients showed mutations affecting neighboring codons that were not direct phosphorylation sites. Thus overall, 31 (26.9%) patients had CTNNB1 mutations exon 3, which included transitions, transversions, and two very short deletions affecting codon 33  using Hep3B cell line. Hep3B transfected with S33Y (Hep3B-S33Y) shows nuclear translocation of β-catenin , Lect2 (p = 0.0009), and regucarcin (p<0.0001) expressions (CTNNB1 mutations in phosphorylation sites in exon 3 (group C); (2) patients with other non-phosphorylation site CTNNB1 mutations in exon 3 (group O); (3) patients with wild-type CTNNB1 but β-catenin target gene upregulation (group NU); and (4) patients with wild-type CTNNB1 and no activation of β-catenin signaling (group N) (CTNNB1 mutations (n = 31) is significantly higher than patients without them (n = 94)  . Intrigu(n = 94) . We also(n = 94) . GS exprCTNNB1 mutations but also others without such mutations showed GS overexpression. We aimed to assess β-catenin activation in the two-sided ways by immunohistochemical analysis forcusing on nuclear translocation of β-catenin and by qRT-PCR analysis forcusing on downstream target gene upregulation. Nuclear translocation of β-catenin was seen in 28 patients (22.4%)  . The per (22.4%) . Target  (22.4%) . Collect (22.4%) .CTNNB1 other mutation group (p = 0.0164). Since there were many patients in our prospective database who received repeated treatments, we only analyzed patients who underwent curative surgery as an initial treatment for HCC in order to more meaningfully determine the prognostic impact of β-catenin signaling. This group of patients showed five year survival rate of 69.1%. There was no difference in prognosis between patients with HCC harboring wild-type or mutated β-catenin gene (p = 0.2855). Given the previous reports suggesting that GS expression is a good prognostic indicator [p = 0.0461) in the current dataset (p = 0.0001), suggesting that β-catenin of which function is still undetermined in the current study might have a distinct oncogenic role in this subgroup.Next, we questioned if there were any correlations between aberrant β-catenin signaling and clinical parameters of HCC patients. We compared clinicopathological background of patients in the four groups . There wndicator , we firs dataset . Since wCTNNB1 exon3 missense mutations not directly affecting phosphorylation sites but adjacent non-phosphorylation codons, have an equally robust effect on β-catenin activation in HCC patients. Gene expression analyses revealed that β-catenin target gene expressions were remarkably similar in these two groups of HCC. Additionally, we reaffirm the existence of a subset where β-catenin signaling is active in absence of CTNNB1 mutations.The current study was focused on β-catenin signaling in HCC in clinical settings. We demonstrated that in vitro studies have previously addressed the function of some of these mutations [We aimed to determine the significance of mutations in exon 3 of β-catenin gene that did not directly affect a phosphorylation site. Indeed utations . Howeverutations .CTNNB1 were compared to the rest. Instead, when patients were divided into group with or without nuclear β-catenin translocation, the patients with nuclear β-catenin translocation showed a worse overall survival. Among these patients, we unexpectedly found that some patients completely lost described β-catenin target gene upregulation and showed the poorest overall survival. Hoshida et al. recommended that Wnt-TGF-β subclass could be discriminated from CTNNB1 subclass which contained CTNNB1-mutation-signature. In Wnt-TGF-β subclass, most patients showed membraneous β-catenin expression with GS overexpression, suggesting that molecular background of patients with the poorest prognosis in the current study might be distinct from Wnt-TGF-β subclass [According to previous observations, the phenotype of β-catenin mutated tumor is mutually exclusive to p53 mutation and characterized by low rate of loss of heterozygosity, less fibrosis in tumor, lacking steatosis, well-differentiated tumor type, and cholestasis –17. Moresubclass , which iin vivo [CTNNB1 mutation but also those who don’t have mutation are presumably candidates for β-Catenin targeted therapy [CTNNB1 mutation is AXIN1 mutation which is reported to be seen in more than 10% of HCC patients [CTNNB1 mutation are issues of great importance to be addressed.Aberrant β-catenin signaling in solid cancers including HCC is well known, and has been discussed as an attractive and therapeutic target to date , 19–21. in vivo . Howeverin vivo . β-caten therapy , 18. In patients , 25. TheCTNNB1 exon3 mutation would be desirable candidates for β-Catenin targeted therapy irrespective of mutation cites, and the mechanism of β-Catenin activation in the remaining patients is an urgent issue to be argued. This study would be of great help to understand the β-Catenin activation in patients with HCC.In conclusion, patients with S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Notum encodes a secreted Wnt deacylase that inhibits Wnt activity and thereby restricts the zone of activation of Wnt/β-catenin signaling. An important role of NOTUM has been described in development in drosophila, planaria and zebrafish, but its role in the mammalian liver is unknown. Notum is required for spatial control of the Wnt/β-catenin signaling in several animal models and the Wnt/β-catenin pathway contributes to liver patterning involved in metabolic zonation. Therefore, Notum may be involved in the liver patterning induced by the Wnt/β-catenin signaling during the adult stage.Fine tuning of the Wnt/β-catenin signaling pathway is essential for the proper development and function of the liver. Aberrant activation of this pathway is observed in 20%-40% of hepatocellular carcinomas (HCC). Notum knockout mouse mutant to study the effect of the deletion of Notum in the liver. We show that Notum is a direct target of the Wnt/β-catenin signaling in the liver. Liver-specific deletion of Notum did not modify liver zonation, but Notum deletion had a long-term effect on mouse physiology. In particular, male mutant mice developed metabolic disorders.We generated a conditional Notum is not a key actor of Wnt/β-catenin-dependent liver patterning of adult mice, but has role in liver glucose homeostasis. Male mice deficient in Notum specifically in the liver develop metabolic dysfunctions implicating Notum in the development of Type 2 diabetes.We show that  The morphogenic Wnt/β-catenin signaling pathway is essential in numerous physiological processes during embryonic development and in the maintenance of self-renewing tissues in adulthood . It is aCTNNB1, the gene encoding β-catenin, are present in 20–40% of hepatocellular carcinomas (HCC), the most frequent primary liver cancer . Ho. Ho20]. T2D risk  22]..NOTUM isNotumKOLiv mice are gender-specific, the metabolic phenotype was not present in NotumKOLiv old female. Sexual dimorphism in obesity is well described in humans [NOTUM might be a new factor responsible for sex differences in metabolic disorders.Importantly, we observed that the obesity and glucose intolerance developed in n humans . While tNotum is strongly expressed by hepatocytes and is a direct target of the Wnt/β-catenin signaling in liver. Disruption of its expression in the liver does not have significant consequences on liver development and metabolic zonation. We also show that obesity and deregulation of glucose homeostasis appeared progressively with age specifically in male Notum-deficient mice, implicating NOTUM in the physiopathology of obesity and T2D in a gender-specific manner.In conclusion, we report that


H be a Hilbert space and let C be a closed convex nonempty subset of H and C is a strictly convex set and T is an inward mapping. Meanwhile, they proposed a open question for a countable family of non-self nonexpansive mappings. In this article, authors will give an answer and will prove the further generalized results with the examples to support them.Let  C be a closed, convex and nonempty subset of a Hilbert space H and let T is a self-mapping, for T under the following assumptions: Let T to be non-self at the price of strengthening the requirements on the sequence C.Many authors are interested in lowering condition f is a contraction and P can be a resource consumption task and it may require an approximating algorithm by itself, even in the case when P is the nearest point projection.Historically, the inward condition and its generalizations were widely used to prove convergence results for both implicit . Then the following properties hold: for anyandif and only iffor anyand anyifTis an inward mapping, thenfor anywhenever and such thatThenweakly converges to a pointMoreover, ifthen the convergence is strong. satisfies S-condition.Let H into itself with nonempty common fixed point set F.Let LetXbe a uniformly convex Banach space, be two sequences, if there exists a constantsuch thatthewhereanda, bare two constants.Reich  LetXbe aThe following theorem is main result which is also a answer to the open question of Colao and Marino.LetCbe a convex, closed and nonempty subset of a Hilbert spaceHand letbe a uniformly weakly closed countable family of non-self nonexpansive mappings. Then the algorithm (4) is well defined. Assume thatCis strictly convex and eachsatisfies the inward condition and such thatThen the following conclusions hold:if there existsuch thatfor alltheweakly converges to a common fixed pointifand  satisfiesS-condition, theconverges strongly to a common fixed pointd such thatH satisfies Opial’s condition, we have(1) for any N such that VG, we obtain that S-condition implies (2) SinceThe proved theorem is a partial answer to the open question that it is not completely satisfactory. In fact the assumption that can not approach to 1, imposes a restriction a priori on F(T) is nonempty andA mapping By using the same method of proof as in Theorem 7, we can prove Theorem 7 is still right for quasi-nonexpansive mappings. Therefore, we can get the further generalized result as follows.LetCbe a convex, closed and nonempty subset of a Hilbert spaceHand letbe a uniformly weakly closed countable family of non-self quasi-nonexpansive mappings. Then the algorithm (4) is well defined. Assume thatCis strictly convex and eachsatisfies the inward condition and such thatThen the following conclusions hold:if there existsuch thatfor alltheweakly converges to a common fixed pointifand  satisfiesS-condition, theconverges strongly to a common fixed pointA be a multi-valued operator from H into it-self with domain A is said to be monotone ifA is said to be maximal if it’s graph A is a maximal monotone operator, then Let LetHbe a Hilbert space and letAbe a monotone operator fromHinto it-self. ThenAis maximal if and only iffor allwhereIis the identity operator. satisfies S-condition.Let D, C) satisfies S-condition.Let C be a convex, closed and nonempty subset of a Hilbert space H and let C is strictly convex and each if there exist F, C) satisfies S-condition, the if Let


In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF  and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases. In multicellular organisms, cell–cell communication is critical for development, homoeostasis, and response to injury. Long and short-range signalling molecules (ligands) bind to cellular receptors and convey messages by initiating a cascade of cytoplasmic signalling events. These signals can lead to dramatic changes in cellular protein abundance, localization and activity, as well as the changes in the transcriptome, and ultimately, in the epigenome. All steps in these pathway can potentially be regulated and the intricate interactions among the ligands, receptors, and intracellular signalling molecules add both complexity and robustness into the system.The Wnt family of signalling ligands and associated downstream mediators play key roles in short range cell–cell signalling within specific tissues that is essential for both developmental and homoeostatic processes. Adding to their importance, dysregulation of Wnt pathways can result in diverse pathologic disorders (recently reviewed by Clevers and Nusse ). The clFree cytoplasmic β-catenin is a key player in a major subset of Wnt signalling cascades. When specific Wnt ligands are absent, cytoplasmic β-catenin levels are kept at low via constant targeting by a multiprotein destruction complex, recently comprehensively reviewed by Niehrs . The desConversely, when Wnt ligands bind to Frizzled (Fzd) and its co-receptor, the low-density LRP (lipoprotein receptor-related protein)5/6, the receptors multimerize and form larger multiprotein complexes, which are visible as distinct punctate structures in the cytoplasm . These aAlternative models for how Wnt/GSK3 signalling regulates cell fate have recently been proposed. The Wnt/LRP6-mediated down-regulation of GSK3 activity can occur through internalization of GSK3 into multivesicular bodies –16. ThisDetailed kinetic studies have been carried out in cell culture to investigate how β-catenin levels are regulated after Wnt stimulation . By careThe exact mechanisms underlying how the signals perceived by the receptors are transduced to the destruction complex remain controversial. Upon Wnt stimulation, the PPPSP motif at the cytoplasmic tail of LRP6 is phosphorylated, leading to the recruitment of Axin to LRP6 . MultiplThe interaction of Wnts with Frizzled and LRP5/6 at the membrane is regulated by a dense network of additional signalling agonists and antagonists. Diverse non-Wnt proteins also bind to the Wnts and to the Frizzled receptors to either activate or inhibit Wnt/β–catenin pathway. For example, Norrie disease protein Norrin binds to Fzd4 receptor with high affinity to activate the Wnt pathway and mutations of either Norrin or Fzd4 lead to incomplete retinal vascularization . Norrin The Wnt-binding CRD of the frizzled family of Wnt receptors is also found in the single pass Ror2 (receptor tyrosine kinase-like orphan receptor 2). Wnt5a may interact with Ror2 through this domain . FurtherA complex new mechanism that controls the abundance of Wnt receptors on the cell surface has recently emerged. Ubiquitination of Frizzled receptors induces their endocytosis, thus reducing their surface receptor abundance and decreasing sensitivity to Wnts ,51. RNF4ex vivo survival and proliferation of intestinal stem cells [The RSPO/LGR5/RNF43/Frizzled pathway is critical in both normal stem cell maintenance and in cancer. Inactivating mutations in RNF43 and ZNRF3 occur in ~5% of pancreatic, ovarian, gastric and biliary cancers –58. Pancem cells ,61. The em cells . Notablyem cells ; this trDrosophila, it has been suggested that UBPY could be the deubiquitinating enzyme for Frizzled [The crystal structure of R-Spondin binding to the ectodomains of LGR5, RNF43 and ZNRF3 has been solved recently ,65. TogeFrizzled  and mighThe downstream Wnt signalling cascades have been extensively studied in the past three decades. More recently, the study of the production and secretion of Wnt proteins has garnered increased attention as well.55 in XWnt8) was lipid modified, subsequent investigation supports the conclusion that Wnts contains only O-linked palmitoleate, on a conserved serine corresponding to Ser209 of WNT3A [Protein maturation and proper folding require several cellular processes after its translation. Wnt proteins contain 22 or 24 conserved cysteine residues that are important for proper folding or oligomerization of the proteins through the formation of 11 or 12 intra–molecular disulphide bonds ,71. Postof WNT3A ,72,75. DXenopus WNT8 (XWNT8) was co-expressed, co-purified and co-crystallized with the Wnt-binding CRD of Fzd8 [The hydrophobic nature of Wnt made its purification difficult, and its crystallization near impossible, until  of Fzd8 . The str of Fzd8 .Drosophila Wnt family member that does not undergo lipid modification [Notably, the palmitoleated serine residue is conserved in all Wnt members across different species except in WntD, a fication . Comparification ,79.Drosophila, PORCN belongs to the superfamily of MBOATs (membrane-bound O-acyltransferases) [Porcn in mice is embryonic lethal, as the embryo fails to complete gastrulation [Sox2-Cre driver [Porcn mutant (PORCNΔ/+) mice showed a range of abnormalities in limbs and dermis that resemble the human FDH disease [The ER resident PORCN is both necessary and sufficient to catalyse the lipid modification of Wnts –84. Initferases) . Since Wferases) . Inheritferases) ,87. Globrulation . This exe driver . Female  disease . These fPORCN allele in the X-chromosome was inactivated in male HT1080 fibrosarcoma cells [PORCN appears to be the only acyl-transferase capable of modifying Wnts. Using specific zinc-finger nuclease technology, the single ma cells ,84. In tma cells .Studies with PORCN knockouts are consistent with prior studies of Wnt, β-catenin and LRP5/6 germline mouse knockouts that show that cell-autonomous Wnt signalling is not essential for the proliferation of embryonic stem cells. While several Wnt mRNAs are detectable in blastocysts, knockout of PORCN, WLS and the double knockout of LRP5 and LRP6 all demonstrate that the embryonic requirement for Wnt protein function only appears at gastrulation, and then later in a subset of tissues in the adult mouse. ,89,91–95Drosophila system. The multipass transmembrane protein Wntless (WLS) is a Wnt-sorting receptor that carries Wnt out to the cell surface for release [A second essential Wnt secretion protein was identified through screening in the  release –99. It i release .Xenopus, WLS is required for XWNT4 secretion and morpholino-mediated knockdown of WLS resulted in eye development defects [Wls knockout is embryonic lethal similar to the Porcn knockout with failure of mesoderm induction and gastrulation [Drosophila larval neuromuscular junction, Wingless  is secreted in the WLS-containing exosome-like vesicles from the presynaptic neurons [In  defects . In micerulation . It has  neurons . Jin et  neurons . These dWls knockout mouse models have been developed that highlight the essential role of WLS in development. When the floxed Wls allele is excised by Wnt1-Cre, the resulting mice exhibit a phenotype similar to the Wnt1 null mice, with developmental abnormalities in the midbrain and hindbrain [Wls was specifically deleted in mature osteoblast and osteocytes, the mice developed severe low bone mass due to reduced bone formation and increased matrix resorption [Wls with Keratin 14-Cre driver results in the loss of hair follicles and infiltration of immune cells into the skin together with up-regulation of inflammation-related genes [Several conditional indbrain . Recentlindbrain . Consistsorption . These med genes ,108.PORCN, WLS-coding region mutations are not reported to date in any human diseases. WLS expression levels vary widely, however, and are elevated in some cancers, including astrocytic gliomas and glioblastoma cell lines [WLS expression is reduced in melanoma tumours as compared to the normal skin and benign lesions [WLS in melanoma cell lines increased cell proliferation in vitro and promoted lung metastasis in a xenograft model [Unlike ll lines . On the  lesions . Knockdoft model , consistft model . These ftrans-Golgi network [Caenorhabditis elegans, as the vps-35 mutant produced an EGL-20/Wnt-defect phenotype [Saccharomyces cerevisiae, the retromer complex is composed of two subcomplexes, cargo-selection subunits consisting of Vps35p, Vps29p and Vps26p; and structure subunits, consisting of Vps5p and Vps17p dimer [At the molecular level, WLS is a cargo receptor that transports Wnt from the Golgi apparatus to the plasma membrane. The interaction of Wnt and WLS is dependent on the palmitoleation of Wnt at the serine residue mediated by PORCN ,84,112.  network –117. Thehenotype . In the 7p dimer . WLS int7p dimer . In the 7p dimer . Therefo7p dimer . To date2) commonly found on the early endosomes [C. elegans [2, which could potentially affect endosomal trafficking. However, in contrast to the conventional SNX1-/SNX-2 and SNX-5/SNX-6 (homologous to yeast Vps17p) dependent retromer pathway, an unrelated SNX-3 appears to participate in the retrograde transport of WLS [The retromer complex is associated with the endosomal membrane, probably through interaction with lipid-enriched elements found in the endocytic network . SNX-1  to the Golgi complex relies on a sequence of protein-sorting events in the early secretory pathway. Specific COP (coat protein) complexes are involved in this bidirectional trafficking of proteins. Generally, COPII mediates the anterograde transport from the ER to the Golgi, whereas COPI generates the retrograde trafficking from the Golgi back to the ER. However, there is also evidence showing that anterograde-directed cargos are present in a distinct population of COPI vesicles .Drosophila revealed the a role for the p24 family of proteins in Wnt secretion [Drosophila. However, there is only limited evidence supporting the role of p24 in mammalian Wnt secretion and the redundancy of p24 family members complicates their study.Genome-wide RNAi screening in ecretion ,128. p24ecretion . In mammecretion ,131. SimWLS cycles from the Golgi to the plasma membrane and then back to the Golgi via the retromer complex after endocytosis, but it is not known how the palmitoleated Wnts in the ER meet up with WLS in the Golgi. A high-affinity monoclonal antibody against human WLS was developed that specifically recognizes endogenous WLS . Using aAs noted above, Wnt secretion is a complex process involving specialized cellular machinery that controls the export of Wnt. In addition, the presence of a fatty acid chain on Wnts also increases their hydrophobicity, posing challenges to the spreading of Wnt in the extracellular matrix that is required to exert signalling as a morphogen. Although this process is still poorly understood, a few models have been proposed to better account for Wnt extracellular trafficking. Some examples of these models are described below.Drosophila wing imaginal disc, filopodia-like cellular extensions called cytonemes have been observed in outlying  cells oriented towards the disc centre  cells [In g) cells . This tyHSPGs (heparan sulphate proteoglycans) are another group of molecules that mediate the lateral diffusion of Wnt proteins. Glypican Dally and Dlp  are involved in gradient formation by transferring Wg to neighbouring cells, thus inhibiting the binding of Wg to its local receptor for signalling .Drosophila to facilitate their movement [Drosophila Wg with high affinity [in vitro [Alternatively, a complex Wnt-containing structure might be formed, which shields the lipids attached to the protein from the extracellular environment. Self-aggregation into a micelle might reduce the hydrophobicity of the Wnt molecule. However, monounsaturated palmitoleate, unlike palmitate, might not favour micelle formation. This mechanism has been reported for the Hedgehog protein, another palmitoylated signalling protein that requires long-range signalling during development . It has movement ,136. Secaffinity . More imin vitro .Drosophila [Drosophila S2 cells, Wg is also secreted on exosome-like vesicles, which depends on the Rab11 function as well [Recent studies from the Budnik laboratory provide strong evidence that Wg is transported across synapses in WLS-containing exosome-like vesicles at the larval NMJ (neuromuscular junction) in osophila . The relosophila . In cult as well . However as well . In mamm as well . Exosome as well –143.Since Wnt signalling controls cell growth, differentiation and migration, the aberrant activation of Wnt signalling can lead to uncontrolled cell growth and contribute to tumourigenesis. The molecular evidence that Wnt is involved in both embryogenesis and cancer development demonstrates the convergence for these two fields .A well-known example that links the Wnt pathway with cancer development is a hereditary disease condition known as FAP . FAP patients inherit a mutated copy of the APC gene, and the second APC allele is often lost later in life –147. TheGiven the therapeutic potential in the treatment of Wnt-related cancers, there are diverse attempts to target the Wnt pathway with small molecule inhibitors. The identification of valid drug targets is challenging, although an increasing number of proof-of-concepts studies in animal models have been reported and recently reviewed . The effDownstream inhibitors include tankyrase inhibitors. This class of compounds stabilizes Axin protein and thus promotes the function of the destruction complex. The earliest examples of these include IWR-1  and XAV9ICG-001 inhibits the interaction of β-catenin with the histone acetyltransferase and transcriptional co-activator CBP, hence blocking transcriptional activation of a subset of β-catenin regulated genes. A related compound, PRI-724, is now in clinical trials for acute myeloid leukaemia  and advanced solid tumours  .Inhibition of upstream Wnt activity can be achieved by small molecule inhibition of PORCN activity . NovartiDrosophila wing imaginal disc has been challenged [Wg was expressed in Drosophila in the absence of the endogenous alleles, the resulting flies were viable with almost normal wing size [Drosophila. The authors reasoned that the Wg transcription is active during the initial stage of wing patterning, and only restricted to the dorsoventral boundary later on. Nevertheless, the expression of target genes such as vestigial and Distal-less persists at a lower level. This partially explains the slow growth phenotype of flies with membrane-bound Wg [As we learn more about Wnt biology, new questions continue to arise. There continues to be controversy about how Wnts are transported outside the cell. Lipoproteins and argosomes were identified first, while the exosome model is gaining traction. More work is needed to define the molecular origin of the exosomes and how Wnts are incorporated into these vesicles. Even the concept of Wg as a secreted morphogen in the allenged . When a ing size . This imbound Wg . This stLimited studies have been done to investigate the Wnt secretory pathway in a polarized cell system, which is more relevant to normal physiology. At least for WNT3A and WNT11, glycosylation seems to regulate whether Wnt is secreted from the apical or basolateral surface of polarized epithelial cells . HoweverLGR5-positive CBC (crypt base columnar) cells [ex vivo data. However, several recent in vivo studies call this model into question. In the absence of Paneth cells, or if epithelial Wnt secretion is blocked by knockout of PORCN or WLS, intestinal stem cells are still functional in vivo [In the context of the tissue or organ, it will be important to identify the source of the Wnt. For example, Paneth cells at the bottom of the small intestine crypts were reported to be the critical Wnt-producing cells that support the r) cells , based o in vivo ,163,164.Because of their central role in both development and disease, only a few signalling pathways have received as much scrutiny over the years as Wnt signalling. This intensive study continues to yield surprising insights and unexpected mechanisms. Our ability to target the pathway therapeutically is gaining in sophistication, and the number of clinical trials ongoing suggests we will learn in the coming years if therapeutic intervention in the Wnt pathway is safe and effective.