The Wnt/β-catenin signaling pathway is crucial in animal development from sponges to humans. Its activity in the adulthood is less general, with exceptions having huge medical importance. Namely, improper activation of this pathway is carcinogenic in many tissues, most notably in the colon, liver and the breast. On the other hand, the Wnt/β-catenin signaling must be re-activated in cases of tissue damage, and insufficient activation results in regeneration failure and degeneration. These both medically important implications are unified by the emerging importance of this signaling pathway in the control of proliferation of various types of stem cells, crucial for tissue regeneration and, in case of cancer stem cells – cancer progression and relapse. This article aims at briefly reviewing the current state of knowledge in the field of Wnt signaling, followed by a detailed discussion of current medical developments targeting distinct branches of the Wnt pathway for anti-cancer and pro-regeneration therapies. The Wnt signaling plays instrumental roles in animal development. This tyThe β-catenin-dependent Wnt signaling pathway described above is often referred to as the "canonical" Wnt signaling. In addition to it, a group of "non-canonical", β-catenin-independent pathways can be initiated by Wnt/FZD complexes. They caThe Wnt/β-catenin signaling is repeatedly used during animal development. Given this important developmental function, the role of this pathway in regulation of stem cell proliferation and differentiation, which emerges as a unifying function of this pathway in the adult, both in the physiological and pathological contexts, is not surprising. Initially Wnt/β-catenin signaling has been shown to be crucial for the maintenance and self-renewal of hematopoietic cells. This diThe Wnt/β-catenin signaling pathway as a target of anti-cancer drugs has attracted attention of biotech companies relatively recently. This field received a special attention when it became clear that this signaling plays a major role in CSC proliferation. CSCs have been implicated in tumor maintenance and relapse after surgical resection. The CSC pool is self-renewing and this process is largely driven by re-activation of embryonic programs mediated by Wnt, Hedgehog and Notch signaling pathways and the mTOR signaling hub, 28.A large number of preclinical experiments demonstrated that inhibition of Wnt/β-catenin signaling can affect cancer cell growth and survival (for review see). While Most current anti-cancer drugs, small molecule inhibitors and monoclonal antibodies (mAbs), are designed to target rapidly proliferating cells which represent committed cancer cells but not CSCs. Sorafenib, a small molecule inhibitor of multiple tyrosine kinases involved in tumor proliferation, is used in the treatment of acute myeloid leukemia (AML). There it is supposed to inhibit FMS-like kinase overexpressed in almost all cases of AML. As has been recently demonstrated, sorafenib effectively reduces the number of mature AML progenitors but fails to eradicate AML stem cells and primitive progenitors. TrastuzSignificant efforts are made world-wide to develop potent inhibitors of the Wnt/β-catenin signaling, but only few of these have made it to reach clinical trials. Small-molecule inhibitors identified in a number of high-throughput screens can be classified into four groups: β-catenin/TCF-antagonists, modulators of transcription co-activator, Dvl binders, and other mechanism-based inhibitors Figure .Table 1http://www.clinicaltrials.gov).Among small molecules the leading class is the Porcupine inhibitors, as exemplified by LGK974 (Novartis). PorcupiAnother class of compounds inhibits tankyrases (TNK1 and 2) – enzymes among other functions found to destabilize Axin. TNK inhibition results in prevention of β-catenin accumulation. Examples are XAV939 from Novartis and G007in vitro and substantially inhibiting tumor growth in animal studies .During activation of β-catenin-dependent gene transcription, the complex of β-catenin and LEF/TCF recruits additional factors for chromatin remodeling, like CBP and p300, which possess the histone acetyltransferase activity. Association of β-catenin with histone acetylases can be antagonized by several compounds. One of them, PRI-724 (Prism Pharma) has reached clinical trials in AML and advanced solid tumors. It is a small molecule that selectively inhibits the histone deacetylase CBP/β-catenin complex, blocking expression of the Wnt/β-catenin pathway-dependent pro-growth and pro-survival genes. PRI-724 exhibits a selective antiproliferative effect, inhibiting various cancer cell lines 1[In vitro, niclosamide treatment reduced the levels of LRP6 and β-catenin, and in vivo it had a suppressive effect on basal breast cancer xenografts[Already approved drugs with well-established safety profile find a far easier way to clinical trials for a different indication. Niclosamide is an anti-helminthic drug used in humans for nearly 50 years. In 2009, it emerged as a compound that inhibits Wnt3a-stimulated β-catenin stabilization and TCF/LEF reporter activity in osteosarcoma cell line. This was a result of screening of an FDA-approved drug library for compounds that would promote endocytosis of FZD1. In vitrnografts, 43. Desnografts.http://www.oncomed.com/Pipeline.html) (but see section "Safety of Wnt pathway targeting" below).Although there has been a number of reports where Wnt proteins were targeted directly by antibodies , noThe FZD family of GPCRs provides a large and practically untapped source of potential targets for therapeutic interventions. A numbein vitro experiments indicate that these peptides can compete with full-length Wnts and attenuate canonical signaling. However one can doubt their value even in animal model preclinical studies, since the rapid clearance of non-modified peptides would prevent any lasting effect on cancer cells. Such antagonist mimetics of Wnts would need to be modified, for example by PEGylation or formylation, to effectively increase their half-life, before considering them as therapeutic candidates. A hexapeptide Box5, derived from Wnt5a and stabilized by the N-butyloxycarbonyl group, has been developed to antagonize Wnt5a-stimulated metastasis in melanoma[Peptide fragments of Wnt ligands, binding to the CRD domain of FZD receptors, have been proposed as potential therapeutic agents. Indeed, melanoma. In contmelanoma, 53; thi10 has a very restricted expression pattern; it is undetectable in normal human tissues except placenta, but up-regulated in synovial sarcomas. Taking this opportunity, OncoTherapy Science has developed a chimeric humanized mAb against FZD10, named OTSA101. Non-radiolabeled OTSA101 antibody has only weak antagonistic activity on synovial sarcoma cell growth. However, Yttrium 90-radiolabeled OTSA101 (OTSA101-DTPA-90Y) showed significant antitumor activity following a single intravenous injection in mouse xenograft model[FZDft model and is n7 is the Wnt receptor most commonly up-regulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma (HCC) and triple negative breast cancer[7 displayed anti-cancer activity in vitro and in vivo due to the inhibition of the canonical Wnt signaling pathway[7’ CRD domain and ability to inhibit Wnt3a-induced signaling in a cell-based assay. In addition to FZD7, vantictumab also binds to four other FZD receptors (out of ten encoded by the human genome) – restricting its selectivity (see below). In mouse xenograft models of several types of solid tumors , vantictumab as a monotherapy demonstrated just a decrease in the tumor growth rate. On the other hand, combined treatment with cytotoxic chemotherapy produced in some tumor types not only a dramatic tumor shrinkage but also tumor cell differentiation and reduction of CSC numbers[FZDt cancer. It has  pathway. While s numbers. The intin vitro and inhibit activation of β-catenin and nuclear factor of activated T-cells c3 (NFATc3) in endothelial and tumor cells. sFRP2 mAb treatment of angiosarcoma allografts or MDA-MB-231 breast carcinoma xenografts in nude mice significantly reduced tumors volume. Given that this mAb preferentially accumulates in sFRP2-positive tumors and is long circulating in the blood, it may be a good candidate for clinical studies.Secreted Frizzled-related proteins (sFRPs) have also been in focus as targets for cancer therapy. There are conflicting reports in the literature as to whether sFRPs are antagonists or agonists of Wnt/β-catenin signaling–60. For Even more perspective would be an approach combining two or more targeted therapies. In this respect a recent study by Birchmeier and colleagues is of a The Wnt/β-catenin pathway is known to be essential for stem cell proliferation. While life-threatening in the context of CSCs, this fact underlies the importance of the pathway in regenerative processes in many tissues, such as the liver, brain, muscle, skin and bone. This makes Wnt pathway agonists desirable candidates for regeneration-enhancing therapies, and some have already shown their regenerative effects. We will review the role of different branches of the Wnt pathway (but mostly Wnt/β-catenin) separately for different organs and tissues, paying special attention to the therapeutic potential and successful practical applications of activators of the Wnt signaling Figure .Figure 2The Wnt/β-catenin signaling is known to be involved in liver organogenesis during embryonic development and is aIn the normal adult liver the Wnt/β-catenin pathway is generally inactive, and β-catenin undergoes phosphorylation and subsequent degradation. NeverthIn the liver, the role of stem cells is known to be played by adult hepatic progenitor cells also known as oval cells. The ovaThe importance of Wnt/β-catenin signaling for the liver regeneration has been shown in a series of other observations focusing on different levels of regulation of the Wnt cascade. For example, it has been demonstrated that Wnt-dependent regeneration in the liver can be controlled by an endogenous long non-coding RNA, which activates Wnt/β-catenin signaling by inhibiting expression of Axin. This action of the non-coding RNA leads to enhanced hepatocyte proliferation during liver regeneration, showingSeveral examples demonstrating the interplay of the Wnt/β-catenin pathway with other signaling cascades during liver regeneration have recently been described. The TGFβ family-related protein SMAD6 is able to suppress the Wnt/β-catenin signaling in the liver acting at the β-catenin-TCF-promoter interaction; down-regulation of SMAD6 leads to increased Wnt-dependent proliferation and self-renewal of the hepatic progenitor cells. The Wntin vitro from single cells and form transplantable "organoids", retaining many characteristics of the original epithelial architecture, under the influence of a Wnt synergist R-spondin1 which acts as a Dkk1 competitor[de novo growth of transplantable organs.Given the importance of Wnt/β-catenin signaling for hepatocyte proliferation and differentiation, therapeutic intervention in the Wnt signaling pathway appears as a promising tool for liver regeneration. The first successful attempts have been already made. Liver cells expressing Lgr5, a marker of actively dividing stem cells in Wnt-driven self-renewing tissues, are able to clonally expand mpetitor. Such apA therapeutic effect of a small molecule Wnt/β-catenin pathway agonist, 2-amino-4-benzylamino)-6-(3-methoxyphenyl)pyrimidine (2-AMBMP), has been demonstrated in the hepatic ischemia model in rats. The drug blunted the ischemia-induced elevation of aspartate aminotransferase and alanine aminotransferase levels, increasing cell proliferation rate and decreasing negative effects such as inflammation and apoptosis, and reduced the death rate in general. As smal1, 2, 4 and 5, the co-receptors LRP5 and LRP6, β-catenin, and the Wnt target genes, such as the osteoblast differentiation-associated transcription factor Runx2, are up-regulated in the fracture calyx during bone regeneration[9 is significantly up-regulated during the early stages of osteoblast differentiation[Fzd9-/- mice[Wnt5a+/- mice demonstrate decreased trabecular bone mass in the femurs, and mice homozygous for the loss-of-function mutation in Wnt5a show truncation of the proximal skeleton and lacking of distal digits[Wnt signaling is known to be a key regulator of bone tissue growth in embryos and in the adult. It is eneration–94. It nntiation and is r-/- mice. The β-c-/- mice. Wnt5a+/l digits. The β-cl digits.Taken together, these data suggest that Wnt signaling modulators could be used as therapeutic agents to stimulate bone formation after injury or disease. Currently two strategies are exploited to find an effective Wnt-based bone regenerative therapy. The first involves reduction of the action of native Wnt signaling inhibitors endogenously present in the organism, which in turn would enhance the intrinsic Wnt activity. In this regard, activation of Wnt signaling by neutralizing antibodies against the Wnt inhibitors Dkk1 and sclehttp://clinicaltrials.gov/show/NCT01323894).Another way to promote Wnt signaling in order to boost the bone tissue growth is a direct use of exogenous Wnts or other pathway agonists. Although native Wnt proteins are hard to use due to their low solubility, a successful attempt has been recently made with liposome-loaded Wnt3a, which upon application to the injury site has caused a ca. 3.5-fold increase in the bone regeneration rate in mice. A clini2CO3[+ is a well-known inhibitor of GSK3β – a key enzyme targeting β-catenin for proteosomal degradation – and thus activator of the Wnt pathway; however, it is less specific than Wnt proteins or other pathway agonists and therefore may cause more side effects. In general it can be stated that Wnt signaling in the bone tissue is well studied, leading to a big variety of possible artificial modulations of the pathway to improve bone healing and to treat bone diseases.Stimulation of murine bone regeneration has been also obtained by application of LiCl, and sim2CO3. Li+ is 7, is capable of activation of distinct pathways at different stages of myogenesis. First, the Wnt7a-FZD7 interaction leads to activation of the Wnt-PCP (planar cell polarity) pathway which is responsible for symmetric expansion of satellite cells in the muscle tissue[7 by Wnt7a in differentiated myofibers directly activates the Akt/mTOR growth pathway through Gαs and PI3K, thereby inducing myofiber hypertrophy[7 interactions have been shown to increase the polarity and directional migration of murine and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1, resulting in improved muscle strength[The role of Wnt/β-catenin signaling in the skeletal muscle remains controversial. In the adult, the β-catenin-dependent Wnt pathway has been suggested to control myogenic lineage progression by limiting Notch signaling and thus promoting differentiation, 109, ine tissue – a smale tissue. Over-exertrophy. Thus Wnstrength.Taken together, these findings identify Wnt7a as an important drug candidate against muscle wasting diseases like sarcopenia, cachexia or muscular dystrophies, and for improvement of stem cell-based muscle regenerative therapy. Therapeutic potential of Wnt7a has indeed been tested in several systems, like the mouse model of Duchenne muscular dystrophy, where the Wnt7a treatment stimulated satellite cell expansion and myofiber hypertrophy and even led to a significant increase in muscle strength. Furthermore, Wnt7a decreased the level of contractile damage, most likely by inducing a shift in the fiber type toward the slow-twitch. In othe7 and stimulation of symmetric expansion of satellite stem cells through the PCP pathway, as well as induction of myofiber hypertrophy by signaling through the AKT/mTOR pathway[Interestingly, a truncated Wnt7a variant consisting of only 137 C-terminal amino acids and devoid of the conserved palmitoylation sites has recently been shown to preserve the full biological activity of the native protein in skeletal muscles. It reta pathway. This fiIn the skin, β-catenin-dependent Wnt signaling is one of the dominant pathways regulating the patterning and determining the fate of embryonic and adult stem cells during their differentiation, as well as subsequently controlling the function of differentiated skin cells. An impoThere is evidence pointing out an involvement of Wnt signaling in the skin repair and hair follicle regeneration. For example, a Wnt synergist R-spondin2 promotes cell proliferation in the adult epidermis which isArtificial up-regulation of Wnt/β-catenin signaling in regenerating hair follicles through adenoviral infection of mice with Wnt10b has led to an increase in the size of regenerating follicles and to excessive proliferation of follicle cells. The observed effects were reduced when the mice were co-infected with Dkk1 and Wnt10b. These fAnother example of effective use of external Wnt treatment to improve the skin wound healing rate is the injection of Wnt3a-carrying liposomes into the skin of injured mouse ears. The liposomal Wnt3a has demonstrated the ability to enhance Wnt signaling in the damaged tissue and to dramatically improve the wound healing rate. Hair grFinally, more robust ways to up-regulate Wnt/β-catenin signaling have been successfully applied to skin and hair regeneration. Valproic acid, an inhibitior of GSK3β, has shown its efficacy in boosting the cutaneous wound repair in mice as well 1[2+-dependent signaling could modulate mitochondrial dynamics and prevent the changes induced by the Aβ-peptide oligomers in mitochondrial fission and fusion usually present in neurodegenerative diseases[Wnt signaling is actively involved in neurogenesis and modulation of the synaptic function in the adult. Further1. Recent 1. Moreovediseases.in vivo and in vitro, enhancing it through depletion of isoprenoid synthesis  and improving the rate of adult hippocampal neurogenesis, making it a potential neuroprotective drug[Other small molecule Wnt/β-catenin signaling agonists have also demonstrated an ability to increase adult neurogenesis or inhibit the neurodegenerative effects of the Aβ-peptides. Simvastatin has been shown to synergize with Wnt/β-catenin signaling ive drug. In a moive drug.In silico molecular docking studies suggest the possible mechanisms of curcumin action through interaction with Wnt antagonists Wif-1, Dkk, and the β-catenin inhibitor GSK3β[Another interesting example of small molecule agonists of Wnt/β-catenin signaling able to act in the brain is curcumin. Cucrumin-coated nanoparticles could activate the Wnt/β-catenin pathway in rat brains and reverse learning and memory dysfunctions in the Aβ-induced rat model of the Alzheimer’s disease by stimulation of neurogenesis. or GSK3β. These oHowever, in case of the brain tissue, over-activation of Wnt pathways can also be counterproductive in therapeutic applications, as Wnts are known to stimulate pro-inflammatory processes in microglia, thus complicating the Alzheimer’s disease treatment. An increase in Wnt/β-catenin signaling has been observed in microglia of mice with Alzheimer’s-like pathology; in cultured microglia, treatment with Wnt3a activated the β-catenin-dependent pathway and led to increased expression of pro-inflammatory genes. ExperimGenerally, the regeneration processes in most tissues are regulated by re-activating the Wnt/β-catenin signaling, which leads to stem cell proliferation and/or differentiation, although in some cases β-catenin-independent Wnts take on the leading role, as it is the case with the skeletal muscle. In spite of a complex interplay of Wnt signaling with other signaling cascades, straightforward approaches like ectopic treatment of the injured organs or tissues with Wnt agonists have shown their effectiveness in different tissues, improving the regeneration rate. Combining these findings with possibilities to further enhance therapeutic potential of Wnts through viral or liposomal delivery or by engineering of small Wnt-related peptides allows us to look optimistically towards the direct use of Wnt cascade agonists in regenerative medicine. In addition to application of the truncated Wnt7a in the muscle, short peptide analogs of Wnt5a or their more stable formylated derivatives have shown to mimic the biological activity of the native Wnt5a protein by blocking cell migration in breast epithelium, 53. StrLiver, brain, skin and bones are the well-studied organs for Wnt/β-catenin regenerative applications, but they do not limit the potential of possible targets for the Wnt-based regeneration therapy. It is known that Wnt/β-catenin signaling is also involved in angiogenesis, indicatApproaches for the future regenerative medicine do not stop just at the increasing of the natural regeneration rate. A highly desired goal is to develop technologies of tissue engineering and of growth and transplantation of artificial organs. Although no complete functional organs ready for transplantation have been grown so far, first efforts have already been made in this direction. Such complex tasks would involve multiple steps and demand compound cocktails of different growth factors, where Wnt signaling activators are essential components in most cases. A successful attempt to grow an optic cup  structure from a three-dimensional culture of mouse embryonic stem cell aggregates has been performed, which involved a treatment with Wnt/β-catenin pathway agonists at certain steps. As a coAs exemplified above, the Wnt pathways are involved not only in many developmental processes but also in the maintenance of adult tissue homeostasis. Thus a careful safety assessment of drugs directly or non-directly targeting the Wnt signaling is required. It is clear that a general inhibition of the Wnt/β-catenin pathway is potentially unsafe, mainly because of: (i) broad Wnt/FZD expression pattern; (ii) the role of the pathway in the maintenance of the differentiated epithelium and its interaction with mesenchymal cells; (iii) its involvement in stem cell pluripotent state maintenance; (iv) its role in bone homeostasis. A recent review providesin vivo administration of an LRP6 antagonist Mesd markedly suppressed growth of MMTV-Wnt1 tumors without causing undesirable side effects[The first example is about the FZD co-receptor antagonists. Blockade of Wnt/β-catenin signaling by adenovirus-mediated expression of Dkk1  in mice has been shown to suppress epithelium proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Further effects.Another example concerns the potential of using Wnt proteins as drugs. Indeed, some Wnts have tumor suppressing activity. Wnt5a, for example, is often viewed as a "non-canonical" Wnt capable of antagonizing the Wnt/β-catenin signaling and has a role in limiting B-cell proliferation and functions as a tumor suppressor in hematopoietic tissue. Mice hehttp://www.reuters.com/article/2014/06/13/us-oncomed-study-idUSKBN0EO1B920140613). These were observed in 8 out of 63 (13%) patients treated with vantictumab and in 2 out of 41 (5%) with OMP-54F28. Since the increase in bone turnover has been stated as one of the vantictumab pharmacodynamics effects, it is logical to assume that the mentioned percentage of patients developed osteoporosis. Lowering the dosage and frequency of the drugs might neutralize such side effects – but this could also have a negative impact on the drug efficacy.In many cases, the side effects whose presence or seriousness has been ruled out during pre-clinical studies appear during clinical trials, despite all pre-cautionary measures. For example, the enthusiasm initially shown for both of Oncomed Pharmaceuticals’ Wnt pathway biologics, vantictumab and OMP-54F28, diminished when clinical trials for both were put on hold. The quoted reasons for halting patient enrollment dosing are bone-related side-effects ranging "from mild to moderate"  while not affecting the FZDs indispensable for osteogenesis. These considerations highlight perhaps the most important issue in targeting the Wnt pathways – safety is likely to result from higher specificity in targeting a particular sub-pathway implicated in a certain pathogenic condition, rather than bluntly suppressing all or most of the sub-pathways[The root of the current problem with vantictumab in particular may lie in the initial design of the drug molecule. It binds to 5 out of 10 FZD family members pathways, 159.As discussed above, Wnt signaling plays important functions in cancer progression and tissue regeneration. Ironically, what is therapeutically good for one (activation of the pathway in regeneration) is bad in for the other (anti-cancer treatment). Thus, extreme care must be taken when developing Wnt-stimulating pro-regenerative drugs, to exclude the risks of cancer complications. This demand appears not un-achievable through design of highly specific and/or local activators of the Wnt pathways. On the other hand, the expected side effects in targeting the Wnt/β-catenin pathway in anti-cancer treatments are myelo- and gastrointestinal suppression, exactly due to the adverse effects of anti-Wnt treatments on proliferation of hematopoetic and intestinal stem cells, as well as progenitor cells in other organs. Here again, a hope is for designing specific agents targeting the Wnt/FZD sub-pathways activated in a given cancer, rather than blocking the Wnt/β-catenin signaling altogether. In this


The β-catenin is an important effector in WNT/β-catenin signaling pathway, which exerts a crucial role in the development and progression of hepatocellular carcinoma (HCC). Some researchers have suggested that the overexpression of β-catenin in cytoplasm and/or nucleus was closely correlated to metastasis, poor differentiation and malignant phenotype of HCC while some other researchers hold opposite point. So far, no consensus was obtained on the prognostic and clinicopathological significance of cytoplasmic/nuclear β-catenin overexpression for HCCs.Systematic strategies were applied to search eligible studies in all available databases. Subgroup analyses, sensitivity analyses and multivariate analysis were performed. In this meta-analysis, we utilized either fixed- or random-effects model to calculate the pooled odds ratios (OR) and its 95% confidence intervals (CI).A total of 22 studies containing 2334 cases were enrolled in this meta-analysis. Pooled data suggested that accumulation of β-catenin in cytoplasm and/or nucleus significantly correlated with poor 1-, 3- and 5-year OS and RFS. Moreover, nuclear accumulation combined with cytoplasmic accumulation of β-catenin tended to be associated with dismal metastasis and vascular invasion while cytoplasmic or nuclear expression alone showed no significant effect. Besides, no significant association was observed between cytoplasmic and/or nuclear β-catenin expression and poor differentiation grade, advanced TNM stage, liver cirrhosis, tumor size, tumor encapsulation, AFP and etiologies. Additional subgroup analysis by origin suggested that the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression was more validated in Asian population. Multivariate analyses of factors showed that cytoplasmic and/or nuclear β-catenin expression, as well as TNM stage, metastasis and tumor size, was an independent risk factors for OS and RFS.Cytoplasmic and/or nuclear accumulation of β-catenin, as an independent prognostic factor, significantly associated with poor prognosis and deeper invasion of HCC, and could serve as a valuable prognostic predictor for HCC. Hepatocellular cancer (HCC) is a global health problem and its incidence has been increasing dramatically since 20 years especially in developed countries CSF1R, CTNNB1, KRAS, BRAF, NRAS, ERBB2, MET, PIK3CA, JAK1, and SMO genes CTNNB1 gene account for the majority of somatic mutations and appear to be the most common cause for activation of WNT signaling pathway. CTNNB1 is the coding gene for β-catenin, a multifunctional protein that integrates the intercellular E-cadherin–catenin adhesion system with intracellular WNT signaling pathway.Several molecular pathways are implicated in the hepatic oncogenesis such as β-catenin, p53, EGF, HGF, TGF β and others CTNNB1 mutations occurred, the functional residues of β-catenin may be affected so that the targets usually become invalid of priming phosphorylation by GSK-3β and subsequent catalyzation by proteasome system c-myc, cyclin-D1, and survivinCTNNB1 mutations, though the aberrant activation may also occurred in the absence of CTNNB1 mutations.In normal hepatocytes, the great majority of β-catenin is located in cytomembrane where it directly connects the E-catherin to a-catenin, which is in turn bound to the actin-based cytoskeleton, forming an adhesion complex The normal hepatocytes are transformed to the malignant ones when the WNT pathway is initiated. Aberrant activation of WNT/β-catenin signaling prevents the formation of the β-catenin destruction complex and further leads to the accumulation of β-catenin in the cytoplasm and/or nucleus Abnormal cytoplasmic and/or nuclear accumulation of β-catenin has been demonstrated in 17–40% of HCCs In this paper, a meta-analysis was performed based on retrospective studies to evaluate the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression in patients with HCC. In addition, the association between cytoplasmic and/or nuclear β-catenin expression and etiology (HBV and HCV) was also analyzed.The Pubmed, Elsevier, Embase, Cochrane Library and Web of Science databases were searched systematically for identified articles published until June 7th, 2014. The terms used in search was as follows:“β-catenin, Beta-catenin, or CTNNB1”, “WNT/β-catenin signal pathway”, "prognostic, prognosis, or survival", “HCC”, “hepatocellular carcinoma”, “hepatic tumor”, “hepatic cancer”, “liver cancer”, “liver tumor” and “liver neoplasms” with all possible combinations. The searched articles were filtered out and the reference lists of eligible ones were taken under scrutiny for additional available studies. The systematic literature search was carried out independently by two reviewers (JC and JL).To make this meta-analysis meet the high standards, studies had to fulfill the following criteria: (1) patients with distinctive hepatocellular carcinoma diagnosis by pathology but without restriction on age or ethnicity; (2) β-catenin expression was measured by immunohistological chemistry (IHC) or other methods in primary HCC tissue; (3) clinical trials or reports were published in English; (4) sufficient information or valid data about β-catenin expression and OS or RFS and the other clinicopathological features were provided directly or could be calculated indirectly; (5) the study with the highest quality assessment was enrolled when several studies reported by one individual author were conducted on the same patients population; (6) the patients were followed-up for at least 3 years.Abstracts, editorials, letters and expert opinions, conference records, reviews without original data, case reports and studies lack of control groups were excluded. Studies and data were also excluded if: (1) articles about animals or cell lines; (2) the outcomes or parameters of patients were not clearly reported; (3) no related data required for necessary analysis; (4) overlapping articles.In this process, the title and abstract of studies were first screened to see whether they met the including criteria. Second, the full text was subjected to further examination following the initial screening. Finally, the eligibility of studies was verified by 2 reviewers (JC and JL) after resolving the disagreements by the third reviewer (RJ).Independently, valid data were retrieved from the set of eligible studies by two reviewers (JC and JL) and relevant characteristics were listed as follows: (1) first author and publication year; (2) number of cases; (3) characteristics of subject population, such as age, gender, origin and clinicopthological features; (4) disease stage; (5) methods of evaluating ORs and 95%CI between β-catenin levels and OS or RFS or other clinicopathological parameters; (6) accumulated percentage of β-catenin expression; (7) the number of cases with accumulated β-catenin expression in specific location . Any divThe quality of each included study was assessed by two reviewers (JC and JL) using Newcastle-Ottawa scale (NOS). NOS evaluated various aspects of methodology, which were grouped into the three dimensions of selection, comparability, and outcome. Final scores ranged from 0 (lowest) to 9 (highest); the higher value, the better eligibility of methodology. The study would not be enrolled in the meta-analysis if its value of quality assessment was too low.I2 statistic, ranging from 0% to 100%, was used to estimate the proportion of total variation caused by inter-study heterogeneity . By heterogeneity test, according to the result of Q statistical test, a fixed-effects model was selected if P>0.05 and a random-effects model was selected if P<0.05. P<0.05 was considered statistically significant. The funnel plots were made by utilizing Egger's test and Begg's test to examine the risk of potential publication bias. Then, trim and fill analyses were used to evaluate the stability of our meta-analysis results if the plots were asymmetrical.This meta-analysis was performed using the Review Manager (RevMan) software  and STATA  As shown in Methodological quality of the 22 studies was assessed according to NOS. The results of quality assessment were shown in the ‘level of evidence’ column of I2 = 0% for 1-, 3- and 5-year RFS). Taken together, the above results suggested that β-catenin accumulation in cytoplasm and/or nucleus was significantly correlated with a worse prognosis of HCC. That is to say, patients with HCC showing no β-catenin overexpression in cytoplasm and nuclear were found to have a better prognosis than those patients with HCC showing cytoplasmic and/or nuclear β-catenin overexpression.Some of the enrolled studies provided the ORs and 95%CI directly or indirectly when they investigated the correlation between cytoplasmic and/or nuclear β-catenin expression and overall survival (OS) or recurrence-free survival (RFS) . By pool2I = 0%). This result suggested that there was significant correlation between cytoplasmic and/or nuclear accumulation of β-catenin and metastasis of HCC. As depicted in We performed the meta-analysis to evaluate the correlation between β-catenin expression in cytoplasm and/or nucleus with clinicopathological parameters, namely, metastasis, vascular invasion, differentiation grade, TNM stages, liver cirrhosis, tumor size, tumor encapsulation and alpha fetal protein (AFP). Eight studies I2 = 0%, 4%, respectively). The results suggested that no significant correlation was observed between cytoplasmic and/or nuclear β-catenin expression and etiology (HBV and HCV).In this meta-analysis, the correlation between cytoplasmic and/or nuclear β-catenin expression and etiology (HBV and HCV) was evaluated basing on the retrospective studies which provided the relevant data. Just as depicted in To critically investigate prognostic and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression, subgroup analysis by positive β-catenin expression rate was performed and the results showed that in the group of studies with higher aberrant β-catenin expression rate, a more significant correlation was observed between cytoplasmic and/or nuclear β-catenin expression and poor prognosis . The detIn Begg's test indicated that there was no evidence of significant publication bias after assessing the funnel plot  is the third most fatal cancer worldwide and a major health threat Despite new therapies of HCC arising continually, researchers still felt gloomy for its poor prognosis mainly caused by metastasis and recurrence. That's why many researchers have been dedicated to finding out more reliable and exact predicators of prognosis for patients with HCC. The β-catenin protein has been reported to be located at the cell membrane, in the cytoplasm, or in the nucleus Although we obtained substantial results in this meta-analysis, no more significant conclusions could be reached due to the weakness of the enrolled studies. It was clearly shown from taxonomic studies that different clusters of HCCs with WNT activation can be split depending on CTNNB1 mutation or not. However, the enrolled studies in this meta-analysis failed to provide adequate information about relationship between cytoplasmic and/or nuclear β-catenin expression and CTNNB1 mutation. Thus, we couldn't investigate their further relationship by grouping depending on CTNNB1 mutation or not. What's more, during the process of our study selection, we learned that tremendous studies have been done about CTNNB1 mutation and WNT pathway but only a very limited number of clinical studies reported the qualitative relationship between CTNNB1 mutation and its prognostic value. Of the total 22 studies, only 2 ones In addition, in this meta-analysis, there also remained some limitations worthy of further concern. First, heterogeneity was inevitable among the groups due to differences of patient characteristics such as age, gender and chemotherapies in each study. On the one hand, we used a random-effects model in order to eliminate variations across studies. On the other hand, sensitivity analysis was also used to observe whether omission of any single study would have significant impact on the combined OR estimates. Although the effort could not necessarily rule out the effect of heterogeneity among studies, the adverse influence will be weakened to some degree. Second, bias was unavoidable for clinical evidence because some relevant data were retrieved indirectly from several studies or by reading the survival curve. The inaccurate reading might produce errors and bring about bias. Third, studies performed with positive results or significant outcomes will be apt to be published, suggesting a potential publication bias. Fourth, reports in other languages than English were excluded, so potential language bias may be present in our meta-analysis. Fifth, a significant heterogeneity might also be brought about in this meta-analysis by the difference of the antibodies used for test of ectopic cytoplasmic/nuclear β-catenin expression. Besides, the difference of sample size in each study may induce potential risks of weakening the results of large sample with better quality and strengthening the effect of the small sample with worse quality. All these bias or risks should not be neglected and further investigation should be given to determine whether these factors influence the results of the meta-analysis.The prognostic and clinicopathological significance of β-catenin expression in cytoplasm or nucleus for patients with HCC was investigated in this meta-analysis. The result suggested that patients with HCC showing cytoplasmic and/or nuclear expression of β-catenin appeared to have a poorer OS and RFS, in comparison with those patients with HCC showing normal membranous expression of β-catenin. Additionally, it showed that nuclear combined with cytoplasmic accumulation of β-catenin significantly correlated with metastasis and vascular invasion, while no significant relationship was observed between β-catenin overexpression in cytoplasm and/or nucleus and advanced differentiation grade, TNM stage, liver cirrhosis, tumor size, tumor encapsulation and AFP level. Besides, no significant correlation was observed between cytoplasmic and/or nuclear β-catenin expression and etiology (HBV and HCV). Moreover, cytoplasmic and/or nuclear β-catenin expression was an independent prognostic factor. In summary, β-catenin accumulation in cytoplasm and/or nucleus, as an independent prognostic factor, closely associated with poor prognosis and deep invasion for patients with HCC. Thus, cytoplasmic and/or nuclear expression of β-catenin could serve as a potential predictor for progression and prognosis of patients with HCC and act as a novel target of the developed therapies.Checklist S1PRISMA checklist(DOC)Click here for additional data file.File S1Supporting figures. Figure S1, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 1-year overall survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S2, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 3-year overall survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S3, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 5-year overall survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S4, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 1-year recurrence-free survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S5, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 3-year recurrence-free survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S6, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with 5-year recurrence-free survival by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S7, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with metastasis by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S8, Forest plot of odds ratio for the association of β-catenin expression in cytoplasm and/or nucleus with vascular invasion by subgroup analysis. Sub1: ≥50% expression rate; Sub 2: <50% expression rate. Figure S9, Funnel plot to assess publication bias. A, Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 1-year overall survival (OS) in the meta-analysis. B, Begg's publication bias plot showed the presence of publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 3-year OS in the meta-analysis. C, Begg's publication bias plot showed the presence of publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 5-year OS in the meta-analysis. Figure S10, Funnel plot to assess publication bias. A, Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 1-year reccurrence-free survival (RFS) in the meta-analysis. B, Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 3-year RFS in the meta-analysis. C, Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and 5-year RFS in the meta-analysis. Figure S11, Funnel plot to assess publication bias. Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and metastasis in the meta-analysis. Figure S12, Funnel plot to assess publication bias. Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and vascular invasion in the meta-analysis. Figure S13, Funnel plot to assess publication bias. Begg's publication bias plot showed the presence of publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and differentiation grade in the meta-analysis. Figure S14, Funnel plot to assess publication bias. Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and TNM stage ((III/IV versus I/II)) in the meta-analysis. Figure S15, Funnel plot to assess publication bias. Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and liver cirrhosis in the meta-analysis. Figure S16, Funnel plot to assess publication bias. Begg's publication bias plot showed the presence of publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and tumor size in the meta-analysis. Figure S17, Funnel plot to assess publication bias. Begg's publication bias plot showed the presence of publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and tumor encapsulation in the meta-analysis. Figure S18, Funnel plot to assess publication bias. Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and AFP in the meta-analysis. Figure S19, Funnel plot to assess publication bias. A, publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and HBV in the meta-analysis. B, Begg's publication bias plot showed no publication bias for studies regarding cytoplasmic and/or nuclear expression of β-catenin and HCV in the meta-analysis.(RAR)Click here for additional data file.


Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the β-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding β-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected β-catenin levels and some β-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected β-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis. Ctnnb1 and Apc gene variations, rather than somatic gene targeting, and the work focused on small intestine tumors and no effects on colon tumor phenotypes were described. Furthermore, definitive insights were lacking into how reduced Ctnnb1 gene dosage affected Apc mutation-dependent tumorigenesis. Here, we show somatic inactivation of one Ctnnb1 allele dramatically inhibits mouse colon adenomatous polyposis induced by somatic bi-allelic Apc inactivation. In contrast, Ctnnb1 hemizygous inactivation does not affect mouse ovarian endometrioid adenocarcinoma development arising from Apc- and Pten-inactivation. Ctnnb1 hemizygous gene dose dramatically reduces the active pool of β-catenin, leading to the significant inhibition of β-catenin/TCF-regulated target gene expression, including those encoding key stem cell regulatory and crypt compartmentalization factors in colon epithelium. Tissue-specific differences for expression of selected β-catenin/TCF-regulated genes, such as Myc, may contribute to the context-dependent effects of Ctnnb1 gene dosage in Apc mutation-driven colon and ovarian tumors.Enhanced Wnt signaling contributes to colorectal and other cancers. β-catenin functions in Wnt signaling as a T cell factor (TCF) transcriptional co-activator. Previous studies showed specific β-catenin dosage favors Wnt signaling-dependent tumorigenesis for some tumor types. However, earlier studies emphasized the role of constitutional  APC (adenomatous polyposis coli) and TP53 tumor suppressor genes are found in roughly 80% and 60% of CRCs, respectively [APC allele underlie the familial adenomatous polyposis (FAP) syndrome, where affected individuals often develop hundreds to thousands of colon adenomas during their second or third decades of life. The wild type APC allele is somatically inactivated in adenomas arising in those with FAP [Apc, such as the ApcMin mutation, may develop 50–100 small intestinal tumors and occasional colon tumors by 140 days of age and nearly all of the tumors are adenomas. Similar to the situation in FAP tumors, intestinal tumors in ApcMin mice show somatic inactivation of the wild type Apc allele [Colorectal cancers (CRCs) harbor accumulated mutations in tumor suppressor genes and oncogenes along with epigenetic alterations. Many CRCs arise from precursor lesions, such as adenomatous polyps or serrated epithelial lesions with dysplasia. Inactivating mutations in the ectively . Oncogenectively . Constitwith FAP , 2. Mice3β factors and other proteins—promotes phosphorylation of conserved serine/threonine residues in the β-catenin amino (N)-terminal region. The N-terminally phosphorylated β-catenin can then be β ubiquitinated and degraded by the proteasome. Activating Wnt ligands inhibit degradation of the “free” or Wnt signaling pool of β-catenin via binding at the cell surface to the frizzled and LRP5/6 (low density lipoprotein-related proteins 5 and 6) cognate receptor complex, resulting in inhibition of β-catenin phosphorylation and/or ubiquitination by the destruction complex [APC alíeles are defective, destruction of the free pool of β-catenin is impaired and active β-catenin accumulates in the cytoplasm and nucleus, where it can complex with DNA binding proteins of the TCF (T-cell factor family)/Lef (lymphoid enhancer family) family. β-catenin functions as a transcriptional co-activator for TCFs [MYC gene has been highlighted as a potentially key target gene regulated by β-catenin/TCF in CRCs. Genes encoding negative-feedback inhibitor proteins functioning in the Wnt/β-catenin/TCF pathway, such as AXIN2, DKK1, and NKD1, are also activated by β-catenin/TCF (see http://www.stanford.edu/~rnusse/pathways/targets.html for a list of candidates). In APC-mutant neoplastic cells, the ability of these induced regulator proteins to inhibit the Wnt signaling pathway is abrogated because the factors function upstream of or at the level of the APC protein in the pathway [The best understood function of the roughly 300 kD APC protein is regulation of the pool of β-catenin protein that functions in the canonical (β-catenin-dependent) Wnt signaling pathway –6. In th complex , 6. In cfor TCFs . Normallfor TCFs . Constitfor TCFs , 10. Actfor TCFs , 12. The pathway .APC inactivation may promote cancer development through β-catenin dysregulation. For instance, while most CRCs harbor APC mutations, a subset of CRCs and other cancers lacking APC mutations have CTNNB1 gene mutations resulting in production of oncogenic β-catenin proteins that are resistant to regulation by the destruction complex and that activate β-catenin/TCF transcription [Ctnnb1 gene dosage on liver, small intestine, and mammary gland tumor phenotypes in mouse models as well as effects of Ctnnb1 hemizygous inactivation state (Ctnnb1+/-) in Apc-mutation induced mouse embryonic development phenotypes [Ctnnb1+/- constitutional state can inhibit intestinal and liver tumorigenesis in mice carrying mutations in the Apc gene  [Apc1638NCtnnb1+/- mice, perhaps because Ctnnb1 functions as a tumor suppressor gene in the mammary gland tumors via β-catenin’s role in E-cadherin-dependent tumor suppression [Apc mutation-induced tumorigenesis in some tissues, the prior work did not assess the role of Ctnnb1 dosage in Apc mutation-induced colon tumorigenesis, the chief site of APC mutation-dependent tumorigenesis in humans. Moreover, the work used mice constitutionally deficient in β-catenin, not just in Apc-mutant epithelial cells, and the findings did not highlight specific factors and mechanisms that might account for effects of Ctnnb1 dosage in Apc mutation-instigated tumorigenesis in different contexts. We report here on studies of the effects of Ctnnb1 gene dosage on β-catenin protein expression and β-catenin/TCF transcription in Apc mutation-induced colon and ovarian mouse tumors and cell culture models. We provide evidence that Apc mutation-induced tumorigenesis in the colon is inhibited by Ctnnb1 hemizygous gene status through marked effects on the free pool of β-catenin in the cytoplasm and nucleus and its ability to activate key β-catenin/TCF-regulated target genes, including those encoding key stem factors, such as Lgr5, and regulators of crypt compartmentalization, such as the EphB2/B3 receptors. We also uncovered a novel feed-forward mechanism where β-catenin protein stabilization and β-catenin/TCF transcription appear critical in regulating Ctnnb1/CTNNB1 transcription in the setting of Apc inactivation in mouse colon and human colon cancer cells. Moreover, we found that differences in the ability to activate Myc expression may underlie colon versus ovary tissue-specific differences in Apc mutation-instigated tumorigenesis in the setting of Ctnnb1 hemizygous gene dosage.Besides these findings, other evidence indicates that cription , 13. Alsenotypes , 15. The Apcfl) , 15. In pression . NonetheCDX2P-G22Cre transgenic mice, in which human CDX2 regulatory sequences and an out-of-frame Cre transgene allele, carrying a 22-basepair guanine nucleotide repeat tract affecting the Cre open reading frame, manifest mosaic Cre recombinase expression in caudal embryonic tissues and in epithelium of the distal ileum, cecum, colon, and rectum during adult life [CDX2P-CreERT2 transgenic mice that express a tamoxifen (TAM)-regulated Cre protein (CreERT2) under control of human CDX2 regulatory sequences, allowing for TAM-inducible targeting of loxP-containing alleles in adult terminal ileum, cecum, colon, and rectal epithelium [CDX2P-G22Cre or CDX2P-CreERT2 transgenic mice, we have described the phenotypic consequences in colon epithelium of somatic, bi-allelic, inactivating mutations in Apc [CDX2P-G22Cre Apcfl/fl mice lived only for 8–20 days after birth  and CDX2P-CreERT2Apcfl/fl mice (only 20 days after TAM induction) were dramatically thickened and many polypoid lesions were seen  axis  is thought to generate two daughter cells with equivalent luminal  and basement (extracellular matrix) surfaces. If the spindle axis is not oriented parallel to the crypt axis, cytokinesis generates daughter cells with differences in luminal and basement membrane surfaces and the potential for resultant differences in the fates adopted by the two daughter cells. We previously reported significant increases in the percentage of epithelial cells where the mitotic spindle axis was oriented orthogonal to the planar axis in e crypts . Consistpt) axis . The finApc inactivation acutely induces hyperproliferation and dysplastic alterations in mouse proximal colon epithelium, with the altered epithelium arising in part from expansion of the crypt progenitor compartment at the expense of the differentiated compartment, along with frequent crypt fission/branching [Apc inactivation in colon epithelium not only increased EphB2 and EphB3 expression, but also perturbed the gradient of EphB2 and B3 receptor expression along the crypt axis, with EphB2/B3 expression seen even at the crypt surface in Apc-mutant crypts  expressed from the Lgr5 locus (Lgr5-EGFP)  in colon epithelium of TAM-treated CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice compared to the control mice, paralleling the subtle increase in crypt fission/budding seen, the expanded population of EYFP-positive cells remained restricted to the crypt base region   in Apc-dpression . In Apc- protein , anothere region , consistng cells .CDX2P-CreERT2Apcfl/fl mice  observed in Apc-deficient colon epithelium was significantly suppressed in colon epithelium from CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice  or stem cell fate  is likely to underlie the dramatic abrogation of adenoma formation in CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice.Consistent with our studies of Lgr5 and Msi expression patterns in colon epithelium, the levels of transcripts encoding presumptive stem cell markers, including Lgr5, CD44, Msi1, and Hopx, were also found to increase dramatically in the colon tissues of /fl mice . The indl/+ mice . Taken tMyc is a well-known β-catenin/TCF-regulated target gene [Myc gene expression in mouse colon epithelium seen following Apc bi-allelic inactivation was abrogated when Apc bi-allelic inactivation occurred concurrently with somatic inactivation of one Ctnnb1 allele  and E-cadherin (Cdh1) were similar in the mouse colon tissues independent of genotype  studies from the ENCODE project indicate that the TCF4 protein, encoded by the TCF7L2 gene, is bound in the proximal promoter and exon 1 region of the CTNNB1 gene in selected cell lines. The mouse Ctnnb1 and human CTNNB1 promoter regions lack known TCF family protein consensus binding elements. Nonetheless, to further explore the role of CTNNB1 transcript and β-catenin levels in regulating CTNNB1 transcription, we generated a reporter gene construct in which a 555 bp fragment of human CTNNB1 upstream and exon 1 sequences (-336 to +219 relative to the transcription start site) were cloned upstream of a firefly luciferase sequence  line, through use of a doxycycline (DOX)-regulated shRNA against APC, we reduced endogenous APC gene and protein expression in the cells to less than 10% of control levels  Wnt signaling is dependent on increases in the levels and localization of a hypo- or un-phosphorylated pool of β-catenin, which is often termed the “free” or “active” pool of β-catenin in the cytoplasm and nucleus of cells with Wnt pathway activation , 28. Actl levels . Followiwas seen . Concurr protein . The maroducible . Followiin HCECs . These A-catenin . We alsoof HCECs . Concurrytoplasm , consistCTNNB1 transcript levels were reduced in HCECs was further studied in three human colon cancer cell lines stably transduced with the two DOX-inducible CTNNB1 shRNAs. These included a colon cancer cell line with a gain-of-function mutation in CTNNB1 (HCT116) and two colon cancer cell lines with APC loss-of-function mutations (DLD1 and SW480). At 7 days after DOX-induction of the CTNNB1 shRNAs, in the three colon cancer cell lines, we found moderate (HCT116) to dramatic (DLD1 and SW480) decreases in the active pool of β-catenin protein with only modest changes in total β-catenin protein levels  arising from bi-allelic inactivation of both the Apc and Pten genes [PTEN mutations are often seen in the OEAs with Wnt pathway mutations [Apc and Pten genes following injection of AdCre into the right ovarian bursa of Apcfl/flPtenfl/fl mice [Apcfl/flPtenfl/fl mice and Apcfl/flPtenfl/flCtnnb1fl/+ mice, adenocarcinomas morphologically similar to human OEAs formed following AdCre injection, with 100% penetrance and no difference in tumor latency between mice with two wild type Ctnnb1 alleles or one wild type and one floxed Ctnnb1 allele -surface (low) gradient of EphB2/B3 expression appears to restrict high levels of EphB2/3 expression to the crypt base. Activated β-catenin/TCF transcription has been implicated in repression of ephrinB expression [Apc-mutant crypts when one Ctnnb1 allele was inactivated. In contrast, Apc-mutant crypts with intact Ctnnb1 dosage markedly down-regulated ephrinB1/B2 expression and dramatically upregulated and expanded EphB2/B3 expression throughout the colon crypts. Because the EphB and ephrin molecules mediate critical repulsive interactions in intestinal crypts, the maintenance of the normal inverse EphB/ephrinB gradient from crypt base to cell surface in Apc-mutant crypts where one Ctnnb1 allele is inactive restricts the expansion of the Lgr5-positive crypt stem cell pool and the crypt fission/branching that would result from unrestrained crypt stem cell expansion and altered migration [Apc mutant crypts with reduced Ctnnb1 dosage, stem cell expansion and the dysplastic and adenomatous changes induced by Apc inactivation in colon epithelium are potently inhibited, even though Lgr5 and some other stem cell marker genes are modestly increased in expression in the targeted crypts of CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice relative to crypts in normal mice.Besides highlighting tissue-specific differences for pression , 23. We igration , 23. As Ctnnb1 transcripts are up-regulated in Apc-mutant mouse colon epithelium as well as in Apc-mutant mouse OEAs, together with our findings that Ctnnb1 hemizygous gene dosage inhibited Apc mutation-dependent Ctnnb1 transcript induction in colon and ovarian tumor models imply that the Ctnnb1 gene is subject to feed-forward activation by β-catenin levels and β-catenin/TCF-regulated transcription. Of interest with regard to a role for β-catenin and β-catenin/TCF transcription in regulating Ctnnb1 transcription are chromatin immunoprecipitation (ChIP) studies from the ENCODE project reporting that the TCF4 protein, encoded by the TCF7L2 gene, is bound in the promoter region of the CTNNB1 gene in selected cell lines. Based on our mouse colon tissue studies and the ENCODE project findings, we generated a CTNNB1 reporter gene construct containing 555 bp of human CTNNB1 upstream and exon 1 sequences and found that shRNA-mediated inhibition of CTNNB1 endogenous gene expression in the APC-mutant DLD1 human colon cancer cell line led to inhibition of the activity of the CTNNB1 reporter gene. These data demonstrate that CTNNB1 transcript levels affected CTNNB1 transcription in colon cancer cells. The lack of known TCF consensus element binding sites in the mouse Ctnnb1 and human CTNNB1 promoter regions currently limits support for the argument that β-catenin/TCF transcription directly regulates Ctnnb1 transcription, though further studies to address the point will need to be pursued.Our findings showing that Myc induction in Apc-mutant colon epithelium with one Ctnnb1 allele, Myc induction was retained in the Apc-mutant mouse OEAs with one functional Ctnnb1 allele. Of note, in prior studies, it has been shown that hemizyous inactivation of Myc dramatically inhibited Apc mutation-induced small intestine tumor phenotypes, but not Apc mutation-induced effects on liver cell proliferation and size [Myc as perhaps one of the key β-catenin/TCF-regulated genes with tissue-specific differences in its regulation by β-catenin/TCF that may account for why Ctnnb1 hemizygous state abrogates Apc mutation-induced effects in some tissues  but not in other tissues . The identification of a possible feed-forward mechanism for β-catenin and β-catenin/TCF transcription in regulating Ctnnb1 transcript levels following Apc inactivation are also potentially interesting with regard to Myc, because the ENCODE project work also indicates that the Myc protein is bound in the promoter and intron one regions of the Ctnnb1 gene in selected cell lines. As such, β-catenin/TCF transcription may cooperate in some fashion with Myc, itself encoded by a β-catenin/TCF target gene, in a more complex feed-forward loop to activate Ctnnb1 transcription in certain cell types when Apc is inactivated.In contrast to the near complete abrogation of and size , 38. Henand size –39 highlCtnnb1/CTNNB1 transcription in the setting of Apc/APC inactivation. Besides Myc, other β-catenin/TCF target genes may also be differentially regulated in a tissue- and context-dependent fashion, perhaps contributing in some fashion to the tissue-specific differences of Ctnnb1 hemizygous gene dosage on Apc mutation-instigated tumorigenesis observed. In addition, the basis for the dramatic changes in the free or active pool of β-catenin protein relative to the more modest effects on total β-catenin protein levels when Ctnnb1/CTNNB1 transcript levels are reduced in colon epithelial cells with Wnt pathway dysregulation remains to be elucidated. Nonetheless, our findings highlight the possibility that novel approaches and/or agents that can reduce CTNNB1 transcript levels and/or the free pool of β-catenin protein might have quite dramatic effects on the development and perhaps persistence of neoplastic cells with Wnt pathway defects.Further studies are needed to better understand the details of the apparent feed-forward mechanisms through which β-catenin and β-catenin/TCF transcription may regulate Apc and/or Ctnnb1 alleles in colon tissues, CDX2P-G22Cre transgenic mice [CDX2P-CreERT2 transgenic mice [Lgr5-EGFP-IRES-CreERT2 (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J) transgenic mice [Apc targeted alleles  [Ctnnb1-targeted alleles  [Apcfl/+Ctnnb1fl/+ mice were then crossed to Apcfl/fl mice in order to target two alleles of Apc and one allele of Ctnnb1 (Apcfl/flCtnnb1fl/+) or only alleles of Apc (Apcfl/fl), respectively. The Cre positive Apcfl/flCtnnb1fl/+ and Apcfl/fl littermates were compared and the Cre negative littermates served as normal control. The CDX2P-CreERT2Lgr5-EGFP-IRES-CreERT2Apcfl/fl compound mice or CDX2P-CreERT2Lgr5-EGFP-IRES-CreERT2Apcfl/flCtnnb1fl/+ compound mice were constructed by crossing Lgr5-EGFP-IRES-CreERT2Apcfl/fl mice to CDX2P-CreERT2Apcfl/fl mice and CDX2P-CreERT2Apcfl/flCtnnbfl/fl littermates, respectively. To assess Cre-mediated recombination or Wnt signaling in colon epithelium, mice carrying the Gt(ROSA)tm1(EYFP)Cos/J26Sor  reporter allele (EYFP) [B6.129P2-Axin2tm1Wbm/J allele (Axin2-LacZ) [CDX2P-CreERT2Apcfl/fl mice or CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice. To assess the role of β-catenin function in another Apc mutation-dependent mouse tumor model, we used the previously described mouse model of ovarian endometrioid adenocarcinoma (OEA), arising from bi-allelic inactivation of both the Apc and Pten genes (Apcfl/flPtenfl/fl) [Ctnnb1 allele, Ctnnbfl/fl mice were first crossed to Apcfl/flPtenfl/fl mice to generate Apcfl/+Ptenfl/+Ctnnbfl/+ mice, and then Apcfl/+Ptenfl/+Ctnnbfl/+ mice were bred to Apcfl/flPtenfl/fl mice to generate Apcfl/flPtenfl/fl, and Apcfl/flPtenfl/flCtnnbfl/+ mice. All mice were on a mixed C57BL/6 and 129 background, which were backcrossed to C57BL/6 mice for at least 10 generations, except the EYFP reporter mice and the CDX2P-CreERT2 transgenic mice, which were backcrossed for 7 and 3 generations, respectively. All experimental compound mice were on a mixed C57BL/6 and 129 background, and littermates with similar genetic background and different genotypes were used for comparison (see breeding scheme above). Animal husbandry and experimental procedures were carried out under approval from the University Committee on Use and Care of Animals, University of Michigan and according to Michigan state and US federal regulations. All the mice were housed in specific-pathogen free (SPF) conditions. After weaning, rodent 5001 chow and automatically supplied water were provided ad libitum to mice. Animals were euthanized and analyzed at the specified time points, based on particular study design parameters or defined humane treatment and euthanasia guidelines.To target nic mice , or CDX2nic mice , or Lgr5nic mice    and Ctnnem/KnwJ) . The rese (EYFP)  or the Bn2-LacZ)  (The JacAPC (targeting sequence: 5’-CAAATCATATGGATGATAA-3’) or a non-silencing scramble shRNA (Scrmbl). Cells were selected with 1μg/mL of puromycin (Sigma-Aldrich) for 5 days. The resulting stable cell lines (HCEC/APC shRNA or HCEC/Scrmble) were further transduced with TRIPZ lentiviruses driving expression of two different shRNAs targeting CTNNB1  or a non-silencing scramble shRNA (Scrmbl). Human colon cancer cell lines, HCT116, SW480, and DLD1, and human OEA-derived cell line, TOV-112D, were grown in 5% CO2 with DMEM containing 10% fetal bovine serum and penicillin/streptomycin. HCT116, SW480, DLD1 and TOV-112D cells stably expressing the shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl) were made in the same way as HCEC cells. Expression of shRNAs was induced by incubation of cells with doxycycline  at 2 μg/ml or a solvent control for 3 days (for HCEC cells) or 7 days . The degree of inhibition of the shRNAs on APC transcripts and protein and CTNNB1 transcripts and the respective β-catenin protein was assessed by qRT-PCR and Western blotting assays.Human colonic epithelial cells (HCEC)  were kinCTNNB1 sequences from −336 to +219 relative to the transcription start site was obtained by PCR amplification of genomic DNA, and was subcloned upstream from the luciferase reporter gene in the pGL3Basic reporter vector , using the MluI and XhoI sites. The forward primer for generating the CTNNB1 reporter construct was 5′-ACGCGTGCTGCTCTCCCGGTTCG -3′; the reverse primer for generating the CTNNB1 reporter construct was 5′- CTCGAGCAGGGGAACAGGCTCCTC-3′.DNA fragment containing human CDX2P-CreERT2 transgene or Lgr5-EGFP-IRES-CreERT2 were injected intraperitoneally with TAM (Sigma-Aldrich) dissolved in corn oil (Sigma-Aldrich). For two TAM daily dosing, we used 150mg/kg weight per dose; for three consecutive daily doses, we administered TAM at 100mg/kg weight per dose. Mice were injected with TAM at 2- to 3-months of age. For OEA induction, 5 x 107 plaque-forming units of replication-incompetent recombinant adenovirus expressing Cre recombinase  were injected into the right ovarian bursal cavities of 6–10 week old female mice as previously described [Mice with the escribed .Mouse tissues were prepared for paraffin-embedding or cryosectioning as described previously . For assTo assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K  at 37°C for 15 min. The nicked DNA was labeled by using terminal transferase (TdT)  and Biotin-16-UTP (Roche Applied Sciences) according to the manufacturer’s recommendation. The signal was detected by using the Vectastain ABC kit  according to the manufacturer’s suggestion.The spindle angles were defined by the orientation of mitotic spindles, based on α-tubulin staining, relative to the most adjacent apical membrane, as indicated by Crb3 staining. The mitotic spindle axis angle relative to the planar axis of the cells  was measured by ImageJ (NIH).Western blot analyses on lysates from HCEC, HCT116, SW480, DLD1 and TOV-112D cells were performed as described . The folcDNA was synthesized using a high capacity cDNA reverse transcription kit . qRT-PCR was performed with an ABI Prism 7300 Sequence Analyzer using a SYBR green fluorescence protocol (Applied Biosystems). See CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl), were treated for 4 days with DOX at 2 μg/ml or a solvent control. At second day during DOX treatment, cells were plated in 35-mm six-well plates. After 12—24h cells were then transfected with 0.5 μg of CTNNB1 reporter or TOPflash, 1 μg of pCDNA3 (Invitrogen) and 0.05 μg of PRL-CMV Renilla luciferase reporter vector (Promega) using Mirus TransIT-LT1 transfection reagent  according to the manufacturer's protocol. Cells were harvested 45h later and luciferase activities were measured using a Dual-luciferase kit and GloMax-Multi Detection System from Promega.DLD1 cells, stably expressing two different doxycycline-inducible shRNAs targeting P < 0.05. Error bars denote standard deviations (S.D.). Kaplan-Meier survival curves were compared by log-rank (Mantel-Cox) test. Chi-Square test was used to determine significance when mitotic spindle angles were compared among mice with different genotypes. P < 0.05 is considered statistically significant.All data for qRT-PCR were evaluated by Student's t test and asterisks denote significance with Animal husbandry and experimental procedures were carried out under approval from the University Committee on Use and Care of Animals, University of Michigan (PRO00005075) and according to Michigan state and US federal regulations. All the mice were housed in specific-pathogen free (SPF) conditions. After weaning, rodent 5001 chow and automatically supplied water were provided ad libitum to mice. Animals were euthanized and analyzed at the specified time points, based on particular study design parameters or defined humane treatment and euthanasia guidelines.S1 FigApcfl/fl mouse (lacking Cre). (B and C) Shown are extensive regions of thickened mucosa and polyposis (indicated by the bracket), extending from the terminal ileum  through much of the proximal colon in a 20-day old CDX2P-G22Cre Apcfl/fl mice (B left panel) and in CDX2P-CreERT2Apcfl/fl mice at 20 days after the third of three daily doses of TAM (C left panel). In contrast, in a 6-month old CDX2P-G22Cre Apcfl/flCtnnb1fl/+ mice (B right panel) or in CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice at 2 or 6 months following TAM induction (C middle and right panels), only occasional isolated adenomas in the cecum were observed (indicated by arrows). Scale bars, 1 cm. (D) Left panels, diagram of the conditionally targeted alleles of Apc and Ctnnb1  and the deletion-mutant alleles of Apc and Ctnnb1 that result from the Cre-mediated recombination of loxp sites . The involved exons are shown as white boxes and the introns are shown as solid lines. Black triangles indicate loxP sites. Positions of PCR primers used for the detection of each allele are indicated . Right panels, Cre-mediated recombination of Apc and Ctnnb1 genes was assessed by PCR using genomic DNA isolated from grossly normal-appearing proximal colon or cecum tissues and rare cecal tumors from three different CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice  at 6 months following TAM treatment of the mice. Proximal colon or cecal tissue from a CDX2P-CreERT2Apcfl/fl mouse (labeled as CA), obtained 20 days after TAM induction, or from an Apcfl/flCtnnb1fl/+ mouse (lacking the CDX2P-CreERT2 transgene) (labeled as AB) served as controls. All of the tissues were genotyped for Apc and Ctnnb1. Flox alleles represent alleles that did not undergo Cre-mediated targeting, whereas the floxdel alleles have undergone Cre-dependent targeting. Due to the PCR primers used to detect the Cre-targeted Ctnnb1 allele , the PCR product for the floxdel allele is larger than that for the flox allele. Tissues were manually dissected. Cecum tumor DNA preparations likely include DNA from some adjacent colon epithelial cells with Ctnnb1 gene targeting by Cre to generate floxdel alleles. Both epithelial and non-epithelial elements were present in grossly normal-appearing proximal colon and cecum tissue samples from CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice. The Cre transgene is only expressed in epithelial elements. Both flox and floxdel alleles for Apc and Ctnnb1 are therefore seen in grossly normal-appearing tissue samples from CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice; and rare Apc flox alleles are seen in proximal colon and cecal tumors due to non-neoplastic elements in the tumors.(A) The intestinal and colon tissues, extending from the terminal ileum through much of the proximal colon, from a control (TIF)Click here for additional data file.S2 FigCDX2P-CreERT2Apcfl/fl (middle), or CDX2P-CreERT2Apcfl/flCtnnb1fl/+ (right) mice, obtained at 27 days after TAM dosing, were co-stained for α-tublin (green), lysozyme (red), and Crb3 (red), and counter-stained with Hoechst 33342 (Blue). Representative low magnification (upper row panels) or high magnification (lower row panels) images are shown. Scale bars, 20 μm for low magnification images, and 10 μm for high magnification images. Double-headed arrows indicate the orientation of mitotic spindles; solid lines indicate the most adjacent apical membrane of the mitotic cell, which was used as a reference to assess the spindle angle. (B) The spindle angles were defined by the orientation of mitotic spindles relative to the most adjacent apical membrane, as indicated by Crb3 staining. Quantification for the tissues is shown and for CDX2P-CreERT2Apcfl/fl and CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice, only the lysozyme-positive crypts, indicating Apc gene targeting had occurred, were scored. *P < .01 compared CDX2P-CreERT2Apcfl/fl mice to CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice; n.s., not significant, compared CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice to the normal tissues using Chi-Square test (n = 4 and >70 crypts were counted).(A) Proximal colon tissues from wild type , (TIF)Click here for additional data file.S3 FigCDX2P-CreERT2Apcfl/flCtnnb1fl/+ mouse (middle) and a CDX2P-CreERT2Apcfl/fl mouse (bottom) were analyzed 20 days after the third of three daily doses of TAM. The normal colon mucosa from a Cre-negative mouse was used as a control . Scale bars, 20 μm. (B) Detection of Axin2 locus-regulated LacZ reporter (Axin2-LacZ) gene expression in mouse proximal colon tissues, as assessed by X-gal staining with H&E counter-staining. Tissues from the CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mouse (middle) and the CDX2P-CreERT2Apcfl/fl mouse (bottom) with the reporter gene Axin2-LacZ were analyzed 20 days following two daily doses of TAM. The normal colon mucosa from a wild-type mouse (Cntrl) and a Cre negative reporter mouse (Axin2-LacZ) were used as controls (top). Scale bars, 50 μm.(A) Proximal colon tissues were co-stained for EphB3  and Sox9 , and counter-stained with Hoechst 33342 (Blue). The tissues from a (TIF)Click here for additional data file.S4 FigCtnnb1 inactivation on Apc mutation-induced expression of selected Wnt target genes, Axin2, Nkd1, Ccnd1, and Irs1. Gene expression was assessed by qRT-PCR in mouse proximal colon tissues obtained from Cre negative control mice (cntrl), CDX2P-CreERT2Apcfl/flCtnnb1fl/+ mice (Apcfl/flCtnnb1fl/+) and CDX2P-CreERT2Apcfl/fl mice (Apcfl/fl) at 20 days after TAM dosing to activate Cre to target alleles. Gene expression was normalized to β-actin expression. (B) Effects of Ctnnb1 inactivation on Apc mutation-induced expression of selected candidate stem cell markers, Lgr5, CD44, Msi1 and Hopx . RNA preparations used for the work in panel A were studied by qRT-PCR and normalized to β-actin expression. Two asterisks denote P < 0.01 and one asterisk denotes P < 0.05 in Student's t test, and error bars denote S.D. (n = 3 for each group).(A) Effects of (TIF)Click here for additional data file.S5 FigAPC shRNA or a non-silencing scramble shRNA (Scrmbl) were further transduced with lentiviral vectors (ShRNA V2) driving expression of two different shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl). RNA and protein were collected from the HCECs at 3 days in the presence of doxycycline  at 2 μg/ml or simply in a solvent control lacking DOX (“-“). (A) Inhibition of APC gene expression by the DOX-inducible APC shRNA construct was demonstrated by gene expression analysis by qRT-PCR, with normalization to U6 expression . (B) Inhibition of APC protein expression by the DOX-inducible APC shRNA construct was demonstrated by Western blot analysis with two different antibodies against APC (Ab-5 and C-20). β-actin was used as a loading and transfer control.HCECs stably transduced with a lentiviral vector (ShRNA V1) expressing an (TIF)Click here for additional data file.S6 FigAPC shRNA or a non-silencing scramble shRNA (Scrmbl) were further transduced with lentiviral vectors (ShRNA V2) driving expression of two different shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl). Proteins were collected from the HCECs at 3 days in the presence of doxycycline  at 2 μg/ml or simply in a solvent control lacking DOX (“-“). CTNNB1 shRNA-mediated changes in the expression of the total (right) and active  pools of β-catenin in the HCECs were assessed by Western blot analysis ((A) HCECs stably transduced with a lentiviral vector (ShRNA V1) expressing an analysis  and the (TIF)Click here for additional data file.S7 FigAPC shRNA or a non-silencing scramble shRNA (Scrmbl) were further transduced with lentiviral vectors (ShRNA V2) driving expression of two different shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl). These cells were grown on chamber slides and were induced for 3 days for shRNA expression by addition of DOX at 2 μg/ml. Cells were then subjected to Immunohistochemical staining for β-catenin. Scale bar, 20 μm.HCECs stably transduced with a lentiviral vector (ShRNA V1) expressing an (TIF)Click here for additional data file.S8 FigCTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl). β-catenin protein levels were determined after 3-day exposure of the cells to DOX (“+”) at 2 μg/ml or a solvent control (“-“). β-actin protein levels served as a loading and transfer control. The density of Western blotting bands for each cell lines was quantified using AlphaImager HP system (from ProteinSimple). The protein levels of active and total β-catenin were normalized to β-actin level, and the expression from cells expressing scramble shRNA in the presence of DOX was set as 1.Western blot analysis of protein levels of non-phosphorylated, active β-catenin and total β-catenin in human colon cancer cell lines, DLD1 (A), HCT116 (B) and SW480 (C) , stably expressing two different doxycycline-inducible shRNAs targeting (TIF)Click here for additional data file.S9 FigCTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl) were treated for 7 days with DOX (“+ DOX”) at 2 μg/ml or a solvent control (“- DOX”). Analysis of the β-catenin dosage effects on gene expression are shown: CTNNB1 (A); and Wnt target genes—AXIN2 (B), BMP4 (C), NKD1 (D), LGR5 (E) and CD44 (F). Gene expression was assessed by qRT-PCR and normalized to HPRT expression. Error bars denote S.D. **P < 0.01 and *P < 0.05.DLD1 and HCT116 cells stably transduced with two different doxycycline-inducible shRNAs targeting (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Chem. Mater. 2010, 22, 2804–2813], we carried out a wide set of reactions using Mn(II) carboxylates and Mn2(CO)10 as precursors with varying amounts of sulfur and carboxylic acid. MnS NCs were obtained provided that the S/Mn ratio was larger than the L/Mn ratio, otherwise MnO NCs were produced. Since MnS can crystallize in three distinct phases , we also investigated whether the surfactant affected the NC polymorphism. We found that MnS polymorphism can be controlled by appropriate selection of the surfactant. γ-MnS nanocrystals formed when a 1:2 mixture of long chain carboxylic acid and amine was used, irrespective of the presence of carboxylic acid as a free surfactant or ligand in the metal precursor. When we used a single surfactant , α-MnS nanocrystals were obtained. The peculiar role of the amine seems to be related to its basicity. The nanocrystals were characterized by TEM and electron diffraction; ATR-FTIR spectroscopy provided information about the surfactants adsorbed on the NCs.We investigated how the outcome of the solvothermal synthesis of manganese(II) sulfide (MnS) nanocrystals (NCs) is affected by the type and amount of long chain surfactant present in the reaction mixture. Prompted by a previous observation that a larger than stoichiometric amount of sulfur is required [Puglisi, A.; Mondini, S.; Cenedese, S.; Ferretti, A. M.; Santo, N.; Ponti A.  Eg ≈ 3 eV) –360 °C) , it can 2(CO)10 (10–20 nm) to MnSt2 (up to 30 nm). α-MnS NCs are larger (10–65 nm), more polydisperse (15–35%) and have irregular  shape.A complete collection of the NC morphological data can be found in 2(CO)10 or MnSt2, respectively. This outcome does not conform to the above view of a diagonal boundary separating α-MnS and MnO regions. We therefore decided to further investigate the S/L/Mn = 2:4:1 reaction in more detail by selecting L from a wide set of long chain surfactants of different chemical nature.Recalling 2) carried out with the S/L/Mn molar ratio fixed at 2:4:1. The long chain surfactant, L, was selected from a set including carboxylic acid, amine, alcohol, and thiol surfactants. This set allowed us to understand how basicity, nucleophilicity and ability to undergo condensation reaction with stearic acid affect the structure directing ability of the surfactant. The crystal structure of the resulting NCs was determined by analyzing the electron diffraction patterns. Selected ED patterns and TEM images are displayed in In this subsection, we report on the synthetic outcome of the thermal decomposition of manganese(II) distearate 10 as a precursor and restricting ourselves to amines as a free surfactant. The main NC features are collected in 2 vanished when Mn2(CO)10 was used, pointing at an important role of the stearate ligand in the polymorphism control. The NCs were smaller and had a lower aspect ratio than the corresponding ones prepared starting from MnSt2.To understand the role that the stearate ligand presents in the precursor in the above reactions leading to γ-MnS NCs, we carried out synthetic experiments similar to those described in the previous subsection, employing Mn2(CO)10 was decomposed using a mixture of free surfactants including, as above, an amine  and a carboxylic acid in Lacid/Mn = 2:1 molar ratio . The S/Mn ratio was 2, as before. These reactions mimicked those carried out using MnSt2 except that StAc is here present as a free surfactant and not as a ligand within the Mn precursor. The main features of the resulting NCs are collected in amine = HdAm case can be found in 2 and had a similar aspect ratio (1.35–1.45).To gain more insight into the role of stearate, we carried out reactions where MnOlAm represented an exception in that α-MnS NCs were obtained. This could depend on the presence of a double bond in the OlAm chain, which is the main structural difference between OlAm and the other amines. To investigate this issue, we performed a similar reaction using a mixture of OlAc and HdAm, which yielded γ-MnS NCs. Thus, the presence of a double bond in the free surfactant chain does not favor the formation of α- vs γ-MnS NCs. Considering that OlAm also showed a peculiar behavior in the reaction where StAc was absent see , one migThe observation that both OlAc/HdAm and StAc/(saturated amines) surfactant mixtures direct the synthetic outcome towards γ-MnS NCs supports the view that the formation of γ-MnS NCs is insensitive to the structural details of both surfactants.In conclusion, the data presented herein are evidence that to prepare γ-MnS NCs from a solution of elemental sulfur and a manganese precursor in octadecene, it necessary that the reaction mixture comprises both amine and carboxylic acid surfactants, otherwise α-MnS NCs are formed. Whether the carboxylic acid is present as free surfactant or as carboxylate ligand within the Mn precursor is irrelevant to the crystal structure of the resulting NCs.2  and Mn2(CO)10  as a precursor  spectra of the NCs was recorded for several reactions (Tables 1–3) to investigate the organic species present on the NC surface at the end of the reaction. In particular, we analyzed NCs from reactions using both MnStrsor see . For the2 as a precursor, both StAc and DdTh gave α-MnS NCs. The spectra of these NCs are very similar (as(C=O) and νs(C=O) of carboxylate moiety are located at 1564, 1552 and 1471, 1462 cm−1, respectively, and a doublet of ν(C=O) of the COOH group is located at 1705 and 1699 cm−1. The bands located at 1462 and 1564 cm−1 closely resemble those of M2+ stearates. The occurrence of a splitting of ν(C=O) bands of StAc and stearates is reminiscent of the Davydov splitting occurring in highly ordered structures such as Langmuir–Blodgett films [2 with free DdTh can be ruled out by the absence of absorption bands ascribable to DdTh and by the close similarity of this spectrum to the spectrum of NCs prepared in the presence of free StAc.Using MnSt similar ,b. The ptt films . This sutt films  reported2 in the presence of an amine (yielding γ-MnS NCs) gave similar spectra, where bands corresponding to both stearate and amine can be discerned. For example, when OlAm or HdAm were used (−1 (ascribable to the γw(NH2) wagging vibration [−1 (due to νs(C=O) and νas(C=O) of the carboxylate group). Hence, the NCs seem to be coated with both surfactants. The presence of oleylammonium or hexadecyammonium stearate salts cannot be excluded; however, there is no positive evidence, since the presence of carboxylate is not sufficient proof. Similar conclusions also apply to the spectrum of γ-MnS NCs prepared using a DdTh/OlAm mixture , while  mixture .2(CO)10 as a precursor. We first note that the strong CO stretching absorption bands of the precursor  [−1, originated by νs,as(C–H) vibrations of aliphatic CH2 and CH3 groups, and two complex system of bands located between 800–1200 and 1300–1700 cm−1, respectively. The latter system, even if significantly broadened, closely resembles the 1600–1300 cm–1 spectral region of pure OlAm and HdAm . Conversely, the 800–1200 region is not easily attributable to any vibration of pure amines. We can conclude that these NCs are coated with the corresponding amine, as expected.We now turn to NCs prepared using Mn45 cm–1)  were not2(CO)10 using the mixture StAc/HdAm and StAc/OlAm  can be predicted on the basis of the S/Mn and L/Mn molar ratios: MnS is obtained when S/Mn is larger than L/Mn. More interestingly, the polymorphism of the MnS NCs can be controlled by appropriate choice of the surfactant. α-MnS NCs are obtained when no or a single surfactant is present, irrespective of the chemical nature of the latter. When a 1:2 mixture of carboxylic acid and amine is used, γ-MnS NCs are instead obtained.The α- vs γ-MnS control does not depend on the details of the chemical structure of amine and carboxylic acid and is also independent of whether the carboxylic acid is present as a free surfactant or is contained in the manganese precursor. The peculiar role of the amine (no other surfactant in combination with carboxylic acid is able to direct the synthesis to γ-MnS NCs) seems to be related to its basicity. The ATR-IR results provided some information about the surfactants stabilizing the final NCs: those prepared in absence of amines show the presence of both stearic acid and stearate, whereas in presence of stearic acid and an amine, both stearate and amine  could be detected although carboxylic acids are usually stronger ligands than amines.2S but it was also shown that long chain alkylamines react with sulfur following several two-step pathways, all of which produce H2S at 130 °C [2S. It is clearly difficult to understand the peculiar chemistry leading to γ-MnS NCs as many reacting species are simultaneously present. However, two brief considerations can be made about the specificity of the amine/carboxylic acid mixture. First, we found that the carboxylic acid is equally effective both as a carboxylate ligand in the precursor and free acid. This could be explained by the formation of a manganese stearate intermediate when Mn2(CO)10 is heated in the presence of StAc, similar to the case of Fe(CO)5 and oleic acid that give an iron oleate intermediate in the synthesis of iron oxide NCs [1–NH3+ R2–COO−), which were reported to form in an apolar solvent such as cyclohexane [The chemistry occurring in the reported reactions is quite complex, especially in the formation of γ-MnS NCs, where both stearic acid and amine are present. As we have seen, 1-octadecene can react with sulfur at 180 °C to produce Ht 130 °C . To add xide NCs . Second,lohexane , may plaManganese(II) chloride , tetramethylammonium hydroxide pentahydrate, sulfur powder (99.98%), dimanganese decacarbonyl (98%), oleic acid (90%), stearic acid (99%), oleylamine , dodecylamine (98%), hexadecylamine (90%), octadecylamine (≥99%), dodecanthiol (98%), oleylalcohol (≥85%), tryoctylphosphine (97%), and octadecene  were purchased from Sigma-Aldrich. Stearic acid (99%) was purchased from Calbiochem. All chemicals were used as received without further purification.2) was synthesized by a modification of the procedure reported in [2  in methanol (100 mL) was slowly added. A white precipitate immediately formed, which was collected by filtration using a Buchner funnel previously cooled to −20 °C and washed with cold methanol . Drying under vacuum (p ≈ 10−2 torr) for 6 h gave MnSt2 as a white powder. The average yield was ≈50%. IR (KBr), ν (cm−1): 2955, 2917, 2849, 1552, 1468, 1440, 1320, 1113, 949, 721.Manganese distearate (MnStorted in . In a thManganese monooleate hydroxide (Mn(Ol)(OH)) was synthesized following the procedure reported in .The procedure to prepare MnX NCs is described with reference to the S/L/Mn = 2:4:1 case. In a two-necked flask, a solution of manganese(II) distearate , sulfur , and stearic acid  in 1-octadecene was prepared. The solution was heated to 320 °C (heating rate = 10 °C/min) under nitrogen and magnetic stirring. After 1 h, the reaction mixture was cooled to room temperature. The NCs were precipitated by adding the reaction crude with a five-fold excess of ethanol and then collected by centrifugation . Next, the NCs were washed with ethanol and collected by centrifugation (three times) and then washed with acetone and collected by centrifugation (three times). The precipitate was finally dispersed in hexane (5–10 mL) by sonication . The obtained NC dispersions are stable for several months.Several variants of this procedure were carried out by changing:2) or Mn2(CO)10);the manganese precursor (manganese distearate (MnStthe S/Mn molar ratio (from 0:1 to 4:1);the free surfactant (L = stearic acid (StAc), hexadecylamine (HdAm), dodecylamine (DdAm), octadecylamine (OdAm), oleylamine (OlAm), oleylalcohol (OlAl), or dodecanethiol (DdTh)); andthe L/Mn molar ratio (from 0:1 to 4:1).−1 resolution, 50 scans) were collected using an ATR platform  mounted in a spectrophotometer  equipped with a KBr beam splitter and a MCT detector operating between 400 and 4000 cm−1. For ATR-FTIR spectroscopy, the purified NCs were further washed (2×) with a large excess of acetone and then collected by centrifugation . NCs with L = hexadecylamine were washed with CHCl3 to effectively remove the residual surfactant.Conventional transmission electron microscopy (TEM) images (medium resolution), high resolution TEM (HRTEM) images and electron diffraction (ED) patterns were recorded by a Zeiss LIBRA 200FE-HR TEM. The samples for microscopy were prepared by evaporating a drop of the nanocrystal dispersion in hexane on a carbon-coated TEM grid. ATR-FTIR spectra (4 cmFile 1Additional TEM images and ED patterns of MnO and MnS nanocrystals.