However, the mechanisms responsible for β-catenin overexpression have remained elusive.Here we show that the deubiquitinase USP9X stabilizes β-catenin and thereby promotes high grade glioma cell growth. USP9X binds β-catenin and removes the Lys 48-linked polyubiquitin chains that normally mark β-catenin for proteasomal degradation. Increased USP9X expression correlates with increased β-catenin protein in high grade glioma tissues. Moreover, patients with high grade glioma overexpressing USP9X have a poor prognosis. Knockdown of USP9X suppresses cell proliferation, inhibits G1/S phase conversion, and induces apoptosis in U251 and A172 cells. Interestingly, c-Myc and cyclinD1, which are important downstream target genes in the Wnt/β-catenin signal pathway, also show decreased expression in cells with siRNA-mediated down-regulation of USP9X. Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo.In summary, these results indicate that USP9X stabilizes β-catenin and activates Wnt/β-catenin signal pathway to promote glioma cell proliferation and survival. USP9X could also potentially be a novel therapeutic target for high grade gliomas. Gliomas account for the majority of all primary brain and central nervous system tumors in adults , 2. HighThe Wnt/β-catenin signal pathway plays a key role in development, tissue homeostasis and cancer –8. β-catHere, we hypothesize that USP9X interacts with and stabilizes β-catenin in glioma cells. As a result, the Wnt/β-catenin signal pathway is activated and promotes glioma cell proliferation.P= 0.001) . Importantly, there were no significant differences between the two groups in terms of patient age, gender, KPS, histologic grade, surgery and chemotherapy (P = 0.002) with a relative risk of 0.365  in a Cox multivariate analysis, which was performed with the following variables for each case: USP9X expression, radiotherapy and tumor size  Figure . Meanwhiotherapy . USP9X emor size . These rTo further explore the molecular mechanisms of USP9X in high grade gliomas, we studied the key molecular pathways involved in action. The co-localization of USP9X and β-catenin was detected by using laser confocal microscopy. As shown in Figure To obtain direct evidence that USP9X affected β-catenin stability, we transfected short interfering RNAs (siRNAs) targeting USP9X into U251 and A172 cells. The results of the western blot suggested that knockdown of USP9X decreased β-catenin protein Figure , but theAs shown in Figure P <0.05)  (P <0.05) (P <0.05)  . In contP <0.05) c,d. We aP <0.05) . The resP <0.05) . HoweverP <0.001) in high grade glioma tissues. The positive rates of c-Myc and cyclinD1 were 48.1% and 40.7 %, respectively. We found that c-Myc and cyclinD1 positive reactions primarily occurred in the cell nuclei and produced brownish yellow granules . Moreover, the rate of apoptosis in USP9X positive cases was 5.2 ± 0.8%, and the rate of apoptosis in USP9X negative cases was 28.7 ± 1.2%, as detected by TUNEL method. As shown in Figure Among 54 glioma cases, there were 26 USP9X positive cases, representing a positive rate of 48.1% . Using is Figure . The expTo determine the role of USP9X in modulating glioma tumorigenesis in vivo, we implanted primary glioma cells and lentivirus infected primary glioma cells into the dorsal side of the left hind legs of mice. The efficiency of USP9X shRNA was also examined using anti-USP9X antibody Figure . As showet al. found that low USP9X protein and mRNA expression in pancreatic ductal adenocarcinoma (PDA) correlated with poor survival after surgery and USP9X levels were inversely associated with metastatic burden in advanced disease [USP9X is a member of the peptidase C19 family. A few studies have explored the relationship between USP9X and tumors, but the results have been controversial. Immunohistochemical staining of follicular lymphomas and normal lymphoid tissue samples have shown that USP9X levels are increased in the lymphomas . Increas disease . These rNext, we demonstrated that USP9X inhibition retarded the Wnt/β-catenin signal pathway through β-catenin. As transcriptional regulation of c-myc and cyclin D1 may be manifested indirectly by reduced cell cycle progression induced by USP9X knockdown, we need to clarify whether USP9X can regulate c-myc and cyclin D1 levels through the Wnt/β- catenin pathway. By evaluating signaling in U251 cells treated with a Wnt ligand, we found that USP9X knockdown partly blocked the activation of Wnt/β- catenin pathway by regulating the stability of β-catenin Figure . c-Myc pHowever, β-catenin plays an important role in the Wnt/β-catenin signal pathway. Knockdown of β-catenin in glioma cells inhibited cell proliferation and resulted in cell apoptosis , 32. PreIn summary, our study indicated that USP9X expression appeared as a significantly independent prognostic factor of high grade glioma patients, and USP9X interacted with and stabilized β-catenin and activated the WNT/β-catenin signal pathway to promote glioma cells proliferation and survival in vitro and in vivo. Our findings may provide new evidence that regulation of USP9X could potentially be a plausible method for improvement of prognosis in high grade glioma patients. USP9X could also potentially be a therapeutic target in high grade gliomas.A total of 54 paraffin-embedded, high-grade, WHO grading system III to IV glioma tumor samples were used in this study. They were obtained from the Department of Pathology, Wuhan General Hospital of Guangzhou Command. The samples were collected from patients undergoing surgery for high grade gliomas from 2004 to 2009. After resection of tumor, some patients received radiotherapy and chemotherapy, and the other patients who rejected postoperative treatment were treated with supportive care. This study was approved by the Ethics Committee of Wuhan General Hospital of Guangzhou Command.The paraffin embedded stage III to IV glioma tumor tissues were stained with a commercially available anti-USP9X antibody  and anti-β-catenin antibody . Immunohistochemistry stained slides were independently reviewed by two pathologists who were blinded to all clinical data. Staining was graded  and the percentage of positively stained cells was counted . The final score was determined by the combined staining score (intensity + extent). A score <3 was defined as negative expression, and ≥3as a positive staining pattern.U251and A172 human malignant glioma cell lines were obtained from the Cell Bank of Chinese Academy of Science, Shanghai, China. USP9X was knocked down by using 3 validated, commercially available USP9X-specific siRNAs  and a nontarget, scrambled siRNA (USP9X si-NC) sequence as a negative control (Shanghai GenePharma Co.Ltd). The sequences were as follows: USP9X si-1, USP9X si-2, USP9X si-3, USP9X si-NC . Transfection was carried out using siRNA duplexes at a final concentration of 100nmol/L and Lipofectamine RNAiMAX reagent (Life Technologies).U251 and A172 glioma cells were seeded in 96-well plates (2000 cells/well). After serum starvation, the cells were further transfected with USP9X-specific siRNAs. The proliferative potential of cells was analyzed according to the protocol of CCK-8 . After 24h, 48h, 72h and 96h, the absorbance at 450 nm was measured using a microplate reader with the wavelength correction set to 630 nm.The HEK293T cell line was obtained from the Cell Bank of Chinese Academy of Science, Shanghai, China. The full-length β-catenin gene was delivered into the HEK293T cells via the pCMV-β-catenin plasmid. Forty-eight hours after transduction, cells were harvested. The lysates were then resolved by SDS-PAGE and immunoblotted with indicated antibodies.U251 and A172 cells were cultured in 40 mm dishes and further transfected with USP9X-specific siRNAs. After a 48 h culture, the total cell RNA in each dish was extracted and quantitative realtime RT-PCR was performed according to the protocol . Primer sequences are shown in data Primary cell cultures  were established from glioblastoma multiforme tissues obtained during surgery and diagnosed according to the WHO classification. The protocol was approved by the Ethics Committee of Wuhan General Hospital, Guangzhou Command. Selected shRNA lentivirus vector (pGMLV-SC5) against USP9X (targeting sequence: 5′-CAATCAAGTTCAATGATTA-3′) and the negative control shRNA with GFP were obtained as a kit from Genomeditech Co., Ltd. . After lentivirus infection of GBM1and GBM2, GFP+ cells were sorted by flow cytometry and single cell colonies were grown.6 in 100 μL media) were injected subcutaneously into the left dorsal leg of the mice. To follow tumor growth, tumor size was measured with a caliper each week, and tumor volume was calculated using the formula: volume = length × width2 × π/6. The endpoint of observation was at the end of the eighth week.Animal experiments were performed in accordance with animal care ethics approval and guidelines. BALB/C nude mice aged 4 to 6 weeks were purchased from Shanghai Experimental Animal Center . GBM1 and GBM2 cells  was considered significant.SPSS software  was used to conduct all statistical tests. All in vitro experiments were repeated independently in triplicate. Data were pooled with mean and SE of the mean values calculated for all end points where appropriate. The 2-tailed Student t test was used to test the significance of differences in sample means for data with normally distributed means. Alternatively, the Mann–Whitney U test was used for the analysis of nonparametric data. Kaplan–Meier survival curves were generated and compared using a 2-sided log-rank statistic. The Cox proportional hazards model was used for the multivariate analysis of patient survival. The Pearson correlation coefficient was used to test linear associations. A


Jasus edwardsii) industry in Australia favours red lobsters, which are usually caught in shallow waters, over paler (brindle) lobsters. This preference is driven partly by the Chinese market, where red is associated with luck and prosperity, and additionally, by the widely held perception within the industry that brindles have greater mortality rates during out of water transport than reds. Limited scientific evidence supports these industry observations; however, these studies did not evaluate the initial condition of lobsters. This study aimed first, to determine which variables better describe condition in J. edwardsii and second, to compare condition among lobsters in several sites around Tasmania at the typical time of high transport mortality. Male lobsters were collected from the South West, South East, East and North coast of Tasmania in late November/December 2014, which correspond to the start of the Tasmanian fishing season. A comprehensive condition assessment was applied by measuring tissue proximal composition, Brix index, Total Haemocyte Count, pH, haemocyanin and another 16 haemolymph parameters of interest. A useful framework to compare condition in J. edwardsii was established by first, using Brix index as a measure of nutritional condition, second, using pH, magnesium, and bicarbonate to evaluate differences in physiological condition and finally, using THC counts as a proxy for lobster health condition. Lobsters from different sites had different nutritional, physiological and health condition, consistent with industry observations, however our results indicate that some red shallow water lobsters exhibited poorer nutritional and health condition, while some deep water brindle lobsters were in good condition. Differences in condition could not be directly associated to catch depth of lobsters and was related to other spatially discrete factors which sometimes vary over distances <3 km.The southern rock lobster ( Jasus edwardsii) from Tasmania, Victoria and South Australia to markets in Asia is currently 3,500–4,000 tonnes with a gross revenue of approximately AUD200 million [The annual export of southern rock lobsters  for measuring “nutritional condition” in lobsters –8. ProceJ. edwardsii and second, to assess differences in condition among lobsters from several sites around Tasmania at the typical time of high transport mortality. A comprehensive condition assessment was applied by measuring tissue proximal composition, Brix index, THC, pH, haemocyanin and another 16 haemolymph parameters of interest.Blood biochemistry panels developed for veterinary use provide the opportunity of analysing a wide range of haemolymph constituents from the same animal , 15 and Male lobsters were collected from the South West, South East, East and North coasts of Tasmania in late November/December 2014 with two to three sites sampled from each area . The comn = 156) from all sites in each area were sampled non-destructively for Brix index. A total of 100 μl of whole haemolymph was added directly to an automatic temperature compensated digital refractometer to measure the Brix index (%) . The refractometer was zeroed using deionised water. Biochemical composition analyses of tissues were used to calculate various biochemical indices of nutritional condition and to analyse the relationship between these and haemolymph biochemistry parameters as well as the Brix index for this species. A subsample of 54 male lobsters from all areas were destructively sampled immediately after taking the haemolymph sample by bringing them to a chill-coma on ice and were kept frozen until dissection at −40°C. Lobsters were weighed and the hepatopancreas (HP) and abdominal muscle  dissected out and weighed (± 0.01 g).In order to determine if differences in nutritional condition of male lobsters differed amongst areas around Tasmania, lobsters  was determined by the method of Bligh and Dyer (1959) using chloroform: methanol: water (1: 1: 0.9) and expressed in mg g-1 of hepatopancreas wet tissue (HPWW) for 40 lobsters.Total lipid (TL) in the HP (HP2 and three pictures of different grids were recorded using 100x magnification. THC was estimated using the software Image J (v1.48c) and calculated with the following equation:Haemolymph (2.5 ml) was collected from the base of the 5th leg of 78 lobsters collected at sites F2, O1, O2, O3, SE1, SE2, SW2, and SW3 using a 3 ml syringe and 26 G Terumo needle pre-chilled on crushed ice. Whole haemolymph pH was measured using a Testo 205 temperature compensated pH probe. A total of 200 μl of whole haemolymph was extracted from each lobster and added to 200 μl Baker’s Formol Calcium anticoagulant  to estimate total haemocyte count (THC). Samples were placed on a slide haemocytometer with grids of 0.0625 mmWhere ŷ = average count of cells per grid, dilution factor of sample , and the volume factor of a grid was defined by the haemocytometer as 80000.-1) sodium (Na), chloride (Cl), potassium (K), magnesium (Mg) and bicarbonate (bicarb); minerals (mmol l-1) calcium (Ca) and phosphorus (P); metabolites (mmol l-1) glucose (Gluc), lactate (Lact), cholesterol (Chol), triglyceride (Trig), total protein in the haemolymph , urea, and uric acid . The second aliquot was used as a backup and to determine haemocyanin. Twenty μl of plasma were diluted 30 times in deionised water for analysis of oxygenated haemocyanin (OxyHc) at 334 nm [Two aliquots of 1000 μl were extracted from a subsample of 52 lobsters originating from sites F2, O2, O3, SE2, SW2, and SW3, centrifuged at 3,000 × g for 4 min, the haemocyte-free plasma removed into clean cryovials, and snap-frozen in liquid nitrogen. Samples were then stored at − 80°C. The first aliquot was shipped frozen to Diagnostic Services at the Atlantic Veterinary College, University of Prince Edward Island, Canada, and analysed using a Cobas c501 automated biochemistry analyzer  for a full blood profile consisting of the electrolytes , respectively. Spatial differences in the Brix index , were assessed using a Nested ANOVA due to the hierarchical nature of the sampling design (sites inside areas) [To assess whether Brix index was a good indicator of nutritional condition of e areas) . The maiJ. edwardsii, based on replicability of sampling or existing literature available for discussion. These three variables were: pH, bicarb, and Mg. This step was essential as collinearity between variables is a problem when using MANOVA (see subsequent section) and not including highly correlated (redundant) variables will lessen this negative impact [Haemolymph biochemistry data were explored using factor analysis, which is a statistical method that groups variables that are highly correlated to each other and is used to simplify the interpretation of trends down to only a few factors  . Haemolye impact .Differences in physiological condition among sites were assessed using a MANOVA test. A sequential Bonferroni (Holm’s method) was used to adjust the p-values from the pairwise contrasts among the sites . The ShaTHC means were compared using an ANOVA, followed by a Tukey’s post-hoc test to determine if there were any differences amongst sites or lobster colour. Normality of residuals was assessed visually by plotting the residuals. Homogeneity of variances was assessed using the Bartlett’s test . Tukey’sLobsters from Flinders were significantly smaller and weighed less on average than lobsters collected from the other three areas . MicroscTL , AMDM,  and TP  (2 = 0.88), followed by abdominal muscle dry matter content (r2 = 0.69), and hepatopancreas lipid reserves (r2 = 0.49).The Brix index was significantly related to HPp<0.001)  and was There was significant variation in the Brix index between the replicate sites within each sampling area but there was no significant difference in the Brix index among areas . SignifiFactor analysis of the haemolymph biochemistry data resulted in four main factors explaining 75% of the cumulative variation : Factor Physiological condition was different among sites . We then used this framework to demonstrate that male lobsters originating from different sites in Tasmania differed in nutritional, physiological and health condition. Nevertheless, these differences were not always evident between lobsters of different catch depths or colouration. For example, brindle lobsters from one site at Orford  were in poorer nutritional condition than red lobsters from the other two sites at Orford , however, no such differences were detected between these two sites and brindle lobsters from South East and South West Tasmania . Similarly, while overall differences in THC between shallow water red and deep water brindle lobsters were detected, these differences were not always consistent at the level of site . Furthermore, THC for both brindle and red lobsters had overall high THC consistent with good health. THC below 4 million cells/ml have previously been reported as an indication of poor condition or health in P. cygnus lobsters [-1. Nevertheless, comparisons between studies need to be made with caution, as THC varies between species and due to a range of environmental factors  [J. edwardsii which warrants future research. Our observations strongly suggest that site-specific characteristics , are driving the differences in lobster condition in Tasmania. The presence of sites with lobsters in poor nutritional and health condition (e.g. site O1) is consistent with observations by the industry in Tasmania that lobsters from some areas have greater mortality rates during out of water transport. However our results indicate that the effect of site is complicated, and is not sufficiently explained by site depth alone.By evaluating a wide array of haemolymph constituents from the same animal, we were able to establish a useful framework to compare nutritional, physiological and health condition of the southern rock lobster, lobsters . Only onerature) . There iJ. edwardsii showed no significant change after 28 days of starvation [J. edwardsii.The Brix index correlated strongly to haemolymph total protein as well as to both hepatopancreas and abdominal muscle tissue composition in our study. Previous studies have also demonstrated refractometry  to be an easy and affordable field method to estimate nutritional condition of crustaceans including lobsters , 15, 25.arvation  suggestiHomarus americanus [Variation in nutritional condition based on the non-invasive Brix index assessment was larger between sites within broad areas of the coast than between broad areas or within pots. For example, lobsters collected from the deeper Orford site (O1), which were brindle-coloured, were in lower nutritional condition as measured by the Brix index than in other shallower sites of Orford and deeper sites in SE and SW. This suggests that if measurements were taken from lobsters to guide harvesting, data would need to be collected across small (fine) spatial scales. The close relationship between the Brix index and nutritional condition, combined with the ease of sampling suggests that further sampling of this index through the course of routine observer catch sampling for stock assessment, which is already conducted at fine spatial scales in Tasmania and samples lobsters caught in about 12000 pots per year , could be worthwhile to guide harvesting patterns. These data could conceivably also help in fisheries assessment, for example to identify years with unusually high or low growth, which would affect the productivity of the stock independently of recruitment. Linking the Brix index to condition of lobsters is complex however as the relationship may vary from year to year depending on changes in factors such as water temperature, productivity and timing of the moult cycle. For example, in our sampling, male lobsters across all areas on the whole were in better nutritional condition  in late November-December 2014 (average Brix index of 12.2) than previously noted from similar areas in November 2007 and 2008 (RI of 1.34–1.346 = Brix index of 6.5–10.0) . Additioericanus . MeasuriLobsters collected from deeper areas (South East and South West) were associated with greater values of magnesium, compared to lobsters from shallower areas (Orford or Flinders). Similarly, Chandrapavan et al  found grJ. edwardsii showed that the blood pH initial drop from 7.8 to 7.3 is associated with limited lactate build up (<5mmol l-1) but decreases in pH < 7.2 are characterised by much higher lactate levels >10 mmol l-1 linked to much greater reliance on anaerobic metabolism and are therefore of greater biological significance Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC. Wnt/β-catenin signaling is an important developmental pathway that is often hijacked by cancer cells to sustain their unrestricted proliferation, promote survival and facilitate metastatic spread. Wnt family of secreted proteins is comprised of 19 members that are ubiquitously expressed and bind to their cognate receptors Frizzled/Lipoprotein Receptor-related Proteins (LRPs) to initiate a series of events leading to stabilization and nuclear entry of intracellular signal transducer β-catenin. Once in the nucleus, β-catenin associates with TCF/LEF family of transcription factors resulting in the expression of a multitude of downstream target genes, including well known oncogenes c-myc, cyclin D1, MET as well as promoters of tumor growth including VEGF, survivin, matrix metalloproteinases (MMPs) and many others .CTNNB1, gene that encodes β-catenin, or components of its regulatory network, such as APC and AXIN, have been detected in human cancers. For example, APC is functionally inactivated in the overwhelming majority of colorectal tumors, as well as in a significant fraction of gastric and uterine cancers [CTNNB1 have been detected in a number of tumors, but the highest frequency is observed in hepatocellular carcinoma (HCC) [Under normal physiological conditions, and in the absence of the Wnt signal, β-catenin protein is targeted for ubiquitin-mediated proteasomal degradation by the components of the “destruction box” complex AXIN, adenomatous polyposis coli (APC), glycogen synthase kinase 3b (GSK3b) and casein kinase 1 alpha (CK1a). However, many human malignancies display constitutive activation of Wnt/β-catenin signaling pathway through genetic and epigenetic mechanisms leading to stabilization of β-catenin protein. Frequent somatic alterations in  cancers . Somaticma (HCC) . These mma (HCC) . HCC is ma (HCC) . HCC is ma (HCC) . Importama (HCC) , 11. Mulma (HCC) .CTNNB1 [CTNNB1 occur early in HCC pathogenesis and are considered to be a prerequisite for benign hepatic adenoma transformation into frank HCC [CTNNB1/ β-catenin mutations [To further enable the development of effective targeted therapies for HCC, a number of groups have conducted detailed molecular characterization of liver cancer based on both gene sequencing and gene expression analysis. These studies revealed a major role for the Wnt/β-catenin pathway in HCC pathogenesis. For example, study by Guichard et al. revealed that more than 30% of HCC harbor somatic activating mutations in CTNNB1 . Importarank HCC . Gene exutations . These fStudies in animal models of liver cancer have confirmed the central role of Wnt/β-catenin signaling in the onset and maintenance of HCC. Forced overexpression of activated β-catenin in mouse liver progenitor cells results in the development of liver tumors and lung metastases . Similarin vitro and to HCC tumor maintenance in vivo in diverse cellular models. Our data provide evidence of wide-spread β-catenin activation in HCC and suggest a correlation between activation status and oncogenic dependency on β-catenin in HCC.In this study, we aimed to systematically examine activation status of β-catenin at both gene and protein level in a large cohort of human HCC patient samples, as well as in patient-derived xenografts and in established HCC cell lines. Using highly validated genetic tools, we defined the contribution of β-catenin to HCC cell viability CTNNB1/β-catenin itself, APC, AXIN and FGF19. The latter was included since it is part of the 11q13.3 amplicon in HCC and is thought to mediate its oncogenic effects through stabilization of β-catenin [APC, AXIN1 and FGF19, were also identified, albeit with lower frequency. Taken together, these results suggest that genetic alterations in CTNNB1/β-catenin itself or its regulatory network constituents occur in > 30% of HCC.Dysregulation of Wnt/β-catenin signaling has been suggested to play a key role in the pathogenesis of HCC. To evaluate the extent of genomic alterations that might influence β-catenin stability and activity in HCC, we interrogated two large HCC data sets, The Cancer Genome Atlas  and HCC -catenin . SomaticCTNNB1/β-catenin or components of its regulatory network, it is now well accepted that β-catenin protein stabilization can occur through multiple other mechanisms, including epigenetic changes. As previously mentioned, β-catenin protein is kept at low basal levels in the cell under normal physiological conditions. An increase in cytoplasmic and nuclear β-catenin typically signals increased Wnt/β-catenin pathway activity. To study β-catenin protein abundance in HCC, we acquired 102 paraffin-embedded HCC and adjacent normal liver tissue samples from commercial sources and analyzed them using β-catenin immunohistochemistry. Our data show that in the majority of HCC samples (91 out of 102) β-catenin was elevated in both cytoplasm and nucleus, while in the normal liver, β-catenin staining was localized to cell membrane due to a well-known association with E-cadherin   . CumulatCTNNB1/β-catenin is often mutated and its protein levels are significantly upregulated in substantial fraction of HCC patient samples. To select appropriate cellular models for functional analysis of β-catenin, we assembled a panel of 25 HCC and cholangicarcinoma (CC) cell lines and probed their cell lysates using antibodies recognizing total as well as non-phosphorylated form of β-catenin. Lack of GSK-3b-mediated phosphorylation on Ser33, Ser37 and Thr41 of β-catenin typically signals resistance to ubiquitin-mediated proteolysis and is thought to mark an “active” β-catenin fraction capable of entering the nucleus and activating downstream gene expression program [Our data have demonstrated that  program . As showCTNNB1/β-catenin, while HepG2 carries a deletion in exons 3–4 of CTNNB1/β-catenin gene resulting in the deletion of amino acids 25–140. These somatic alterations interfere with β-catenin phosphorylation and protein turnover and lead to its constitutive activation. As a consequence, the antibody specific for non-phosphorylated, active β-catenin fails to recognize these mutant proteins. In contrast, Hep3B, JHH5, JHH6 and SNU449 are known to harbor mutations in AXIN1, a critical component of β-catenin degradation machinery, which leads to stabilization and accumulation of β-catenin protein. Finally, Huh7, JHH7 and SNU387, contain 11q13.3 amplification, which includes FGF19, a known positive mediator of β-catenin activity [Next we selected a subset of cell lines for further functional studies. Three CC cell lines  were excluded since cholangiocarcinoma was not in the scope of this investigation. Certain HCC cell lines  that proved difficult to grow and manipulate in culture (data not shown) were excluded as well. We decided to focus on 18 HCC cell lines with known genetic alterations in β-catenin as well as on those with “high”, “intermediate” and “low” levels of active, non-phosphorylated β-catenin . For 18 activity .AXIN1 and amplification in FGF19 were also detected in HCC PDX , suggesting translational relevance of these disease models. Our results also highlight the fact that genetic alterations are not the only cause of β-catenin activation in HCC as accumulation of β-catenin protein was also observed in the absence of detectable somatic mutations in CTNNB1 gene.HCC is a complex human malignancy characterized by deregulation of multiple signaling pathways, among which Wnt/β-catenin occupies the place of central importance. This is due to high frequency of somatic activating mutations in Nuclear β-catenin has been previously identified as a powerful predictor of overall survival and clinical outcomes in a number of human cancers, including colorectal –28, non-Cognizant of multiple genetic and epigenetic events that may lead to β-catenin activation, we decided to focus on non-phosphorylated (active) form of β-catenin and use it as a marker of pathway activation. In our studies, we utilized an antibody that specifically recognizes non-phosphorylated β-catenin to screen a large panel of HCC cell lines and PDX models. Since established cell lines are more amenable to manipulation in culture, we deployed siRNAs to establish oncogenic dependency on β-catenin in these models. Importantly, in each cell line screened we documented both β-catenin target knock-down and phenotypic effects of siRNA in two different assays . Since siRNA activity is sometimes associated with off-target effects that confound cell growth data interpretation , we embaCTNNB1/β-catenin was significantly affected by β-catenin siRNA in our study. Our results also demonstrate that dependency on β-catenin is not restricted to mutant β-catenin HCC cells, but extends to many contexts where “active” β-catenin protein is elevated. While not all “high” cell lines active β-catenin were sensitive to β-catenin knock-down, the majority displayed reduced survival and proliferation when treated with β-catenin siRNA. The rest of the HCC cell lines with elevated active β-catenin levels likely depend on other oncogenic drivers. For example, SNU449 cells, in addition to AXIN1, harbor genetic alterations in tumor suppressor PTEN leading to reduced PTEN protein expression, increased levels of phospho-AKT (Ser473) and significant sensitivity to inhibitors of PI3K/mTOR signaling [AXIN1-mutant HCC cell line, JHH6, has been reported to contain deletions in exon 2 of NFE2LD (Nrf2) and display strong oncogenic dependence on Nrf2 [Our data suggest a link between the presence of activating mutations in CTNNB1 or increased “active” β-catenin levels and sensitivity to β-catenin ablation. To our knowledge, this is the first study that systematically examined both activation status and oncogenic dependency on β-catenin in such an extensive panel of human HCC cell lines. Survival of all HCC cell lines harboring somatic alterations in ignaling . Similar on Nrf2 .in vitro findings to HCC tumor xenografts in vivo, once again utilizing genetically validated inducible shRNA to knock-down β-catenin. The use of inducible shRNA system in these studies provided crucial evidence for the role of β-catenin in HCC tumor maintenance rather than in xenograft implantation. This implies that targeting of β-catenin in human HCC may have therapeutic benefit. Taken together with previously published data, our results establish an important role for β-catenin in human HCC. They also suggest that, in addition to somatic activating mutations in CTNNB1, non-phosphorylated β-catenin can be used as a patient selection marker. This approach may significantly broaden the reach of β-catenin-directed therapies in HCC.We extended our in vivo has not been demonstrated. Therapeutic siRNA represents an appealing approach to directly inhibit β-catenin [The development of potent and specific β-catenin therapeutics continues to represent a significant challenge. Activated β-catenin is a transcriptional modulator that exerts its effects in the nucleus by altering the expression of genes associated with cell proliferation and survival. Attempts to directly target β-catenin with low molecular weight inhibitors have not succeeded thus far. Stapled peptides for β-catenin have been reported , but the-catenin , 36; howCTNNB1, APC, AXIN1), as well as FGF19, were determined based on whole-exome next-generation sequencing data and comparative genomic hybridization (CGH) data from two HCC patient cohorts: TCGA [CTNNB1 gene, only activating mutations or in-frame deletions in exon 2/3 region were counted. For FGF19, only copy number amplification (defined as copy number > = 5) was considered.Frequencies of genetic alterations, including non-synonymous mutations, small indels and copy number amplification, for genes in the Wnt pathway  on 4 μm sections of human Hep3B and HepG2 liver xenografts and human HCC TMAs. HCC TMAs  XPFor IHC of Ki-67, unstained sections of formalin-fixed paraffin-embedded sections of Hep3B tumor samples were subjected to bright field IHC using BondMax automated immunostainer (Leica) as described previously . A rabbiIHC was performed to detect β-catenin protein in the nucleus, cytoplasm and cellular membrane in 102 HCC patients as described above. Any patient with detectable nuclear β-catenin staining was considered as having “Bcat activation” and otherwise as “No Bcat activation”. Based on IHC results, 9 patients were assigned to “Bcat activation” group and 93 were assigned to “No Bcat activation” group. Log-rank test was performed to compare the survival distribution of the two groups and Kaplan-Meier curves were plotted.2.HCC cell lines were obtained from the DSMZ, ATCC or JCRB. Short Tandem Repeat (STR) DNA profiling was performed to verify cell identity. Cells were grown in DMEM (Invitrogen), supplemented with 10% FBS (Invitrogen), and cultured at 37°C in a humidified incubator with 5% COLentiviral constructs (shRNA or cDNA) were packaged in HEK293T cells using the Trans-Lentiviral packaging kit (Open Biosystems). Virus-containing media were collected and filtered. Target cells were transduced at a multiplicity of infection of 1 in the presence of 8 μg/ml polybrene. After a 24 h infection, the culture media was supplemented with puromycin at a final concentration of 1 μg/ml. The cells were selected for 5 days.® (Thermo Fisher Scientific). This mixture was combined with 300 μl of siRNA mixture prepared in OptiMEM®. The RNAiMAX/siRNA mixture was incubated for 10 min, and the entire 600 μl was added to a single well of a 6-well tissue culture plate. Cells (6 × 105 in 2400 μl of growth media) were added to the transfection mixture. At 24 h post-transfection, the media were replaced with fresh culture medium. After an additional 24 h, the cells were trypsinized and suspended at a dilution of 5 × 104 cells/ml and 100 μl of cell suspension was dispensed into duplicate 96-well plates.siRNAs used in this study are listed in shRNAs used in this study are listed in ® reagent (Promega) to cell cultures for 10 minutes. Endpoint readings were collected using SpectraMax Pro V5 plate reader (Molecular Devices). The intensity of the luminescence signal was directly proportional to the number of live cells.Cell viability/proliferative expansion was assessed by the addition CellTiter-GloApproximately 4000 cells that were previously transfected with siRNAs were seeded into each well of a 6-well plate. After incubation at 37°C for 12∼14 days, the cells were washed with PBS twice, fixed with methanol and then stained with 0.1% crystal violet.The plates were scanned for quantification. Full-well brightfield images were acquired on a Nexcelom (formerly Cyntellect) Celigo automated microscope. Images were analyzed in Definiens  using a custom-developed ruleset: Images were scaled down to 25%, initial colony objects were identified with the auto threshold algorithm. Artifacts and non-colony objects were subsequently filtered out and the number and area of colony objects was exported.For rescue experiments, silent mutations were introduced into cDNA encoding β-catenin (siRNA#1 resistant) in plenti9_IRES-BlastR_DEST . JHH5 and Hep3B cells stably expressing siRNA#1-resistant β-catenin cDNA or empty vector control were established using the pLEN.T.I9-IRES_BLASTR lentiviral expression system. Lentiviral constructs were packaged in HEK293T cells using the Trans-Lentiviral packaging kit (Open Biosystems). Virus-containing media were collected and filtered. JHH5 and Hep3B cells were infected with the virus at multiplicity of infection of 1, followed by blasticidin selection (6 g/ml). Stable cell lines were then transfected with N.T. siRNA and β-catenin siRNAs, and the cells were monitored for viability as described above./spin protocol without the optional DNase treatment. RNA was eluted into nuclease-free water and quantified by ultraviolet absorbance using a NanoDrop 8000 (Thermo Scientific). cDNA synthesis was performed using the High Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer’s recommendations. The cDNA was diluted 1:4 with nuclease-free water and 2.5 μl was added to a 9 μl Taqman reaction mixture prepared using the TaqMan Gene Expression Master Mix (Applied Biosystems). Taqman assays were performed on a ViiATM7 (Applied Biosystems) or a QuantStudioTM 12K Flex Real Time PCR System (Applied Biosystems) using the following conditions: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene expression was normalized to the endogenous control GAPDH for mRNA expression. Taqman primers and probes are listed in RNA was isolated using the 96RNeasy kit (Qiagen), following the vacuumg for 20 min and the supernatant was collected. Protein lysates were quantified using a Bicinchoninic Acid Assay (Pierce). Protein lysates were mixed with NuPAGE LDS Sample Buffer (Life Technologies), loaded onto a 4–12% Bis-Tris gradient gel (Life Technologies), and subjected to electrophoresis in MES buffer (Life Technologies). The proteins were transferred to a nitrocellulose membrane using an iBlot gel transfer device (Life Technologies). The nitrocellulose membrane was blocked with a 5% non-fat milk solution prepared in TBS-Tween . Primary antibodies to total β-catenin , active non-phoshpo-β-catenin , β-Actin  were incubated overnight at 4°C with rocking in 2% non-fat milk/TBS-Tween solution. Membranes were extensively washed and incubated with secondary antibody . Membranes were developed with Super SignalWest Pico Chemiluminescent Substrate (Pierce) and exposed to film. Quantification of the Western blot analysis was performed using ImageJ computer program.Total cell lysates were prepared in RIPA Buffer (Pierce) supplemented with HALT Protease and Phosphatase Inhibitor (Pierce). Lysates were pelleted at 12 000 × For immunoblot analysis of Hep3B and HepG2 tumor lysates, tissue extracts were separated by an automated capillary-based electrophoresis system . All procedures were performed according to the manufacturer’s recommendations using the supplied reagents. Briefly, after determination of protein concentration of each lysate, 1 μg of total protein (4 μL) was mixed with 1 μL of 5X fluorescent master mix and heated at 95°C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies, secondary antibodies, and chemiluminescent substrate were dispensed into designated wells in the manufacturer provided microplate. Primary antibodies used are as follows: total β-catenin , pan-actin . Following plate loading, the separation and immuno-detection was performed automatically using default settings. The resulting data were analyzed using Compass software (ProteinSimple). β-catenin signal was normalized by pan-actin signal.in vivo experiments were performed according to the institutional guidelines and approved by the Institutional Animal Care and Use Committee. 5 × 106 Hep3B or HepG2 cells stably expressing doxycycline-inducible β-catenin shRNA#1 or Control (non-targeting shRNA) were suspended in 50% matrigel and implanted subcutaneously (s.c.) into a flank of 9∼11 week old female Severe Combined Immuno Deficiency (SCID) mice (Charles River Laboratories). The tumors were allowed to establish until average tumor volume reached about 150 mm3. Subsequently mice were randomized into two groups (n = 8/group), and the +DOX group mice were switched to 400 ppm doxycycline diet (Harlan), while animals in the -DOX group were maintained on standard mouse diet. Tumor growth was monitored over time using caliper measurements. Subcutaneous tumor volume was calculated using the formula: length × width2/2.All


Activation of Wnt/β-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/β-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/β-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active β-catenin was decreased and expression of β-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/β-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs. KRAS, p53, and SMAD4, activation of the canonical Wnt/β-catenin signaling pathway is a critical event in colorectal tumorigenesis. The Wnt/β-catenin signaling activation is mainly caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene  supplemented with 1× protease inhibitor cocktail  and PhosSTOP phosphatase inhibitor cocktail (Roche). Western blot analysis was performed as described previously . AntibodCells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) and were permeabilized with 0.5% NP-40 in PBS. The fixed cells were blocked in PBS containing 1% bovine serum albumin (BSA) and reacted with the rabbit anti-non-phospho (active) β-catenin (S33/S37/T41) antibody . This primary antibody was detected using the Alexa 488-conjugated anti-rabbit immunoglobulin (IgG). DNA was stained with 0.2 μg/mL 4′,6-diamidino-2-phenylindole (DAPI).Total RNA was extracted using the RNeasy Mini kit (Qiagen). cDNA was synthesized using SuperScript III First-Strand Synthesis SuperMix for RT-qPCR (Thermo Fisher Scientific). RT-qPCR was performed using LightCycler 480 Probes Master (Roche) and detected using a LightCycler 96 (Roche). A Universal ProbeLibrary Human ACTB Gene Assay was used to detect a reference gene to normalize for differences in the amount of RNA in each sample. All probes were purchased from Roche. Primers and probes for RT-qPCR are shown in the http://software.broadinstitute.org/gsea/index.jsp).Total RNA was extracted as described above. cDNA microarray analysis was performed with the RNAs as described previously , using tThe gene expression data have been deposited in Gene Expression Omnibus (GEO) and are accessible through the accession number GSE86061. The data were released on November 1, 2016.


PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of  PrKD1 through hypermethylation of the gene promoter has been reported as the mechanism of PrKD1 down regulation in breast and gastric cancer [Protein Kinase D1 (PrKD1) is a novel serine threonine kinase and a founding member of the protein kinase D family. There is increasing evidence that loss of PrKD1 expression contributes to progression of several human cancers including prostate cancer. Several regulatory mechanisms modulate PrKD1 activity such as autoinhibition, phosphorylation, proteolytic degradation, subcellular localization, methylation and various cell-context–dependent PrKD1 activation mechanisms . Epigenec cancer , 5. HowePrKD1 expression through MYC/MAX transcription factors and thereby perpetuating down regulation of PrKD1 leading to increased nuclear beta-catenin. The study for the first time identifies a novel auto-repressive loop for PrKD1 expression that perpetuates PrKD1 down regulation contributing to disease progression in prostate cancer.Transcriptional regulation of genes is under the control of transcription factors, co-activators and co-repressors. Beta-catenin, a known transcriptional co-activator , is invoPrKD1 gene regulation, we performed CHIP assays using high PrKD1 expressing LNCaP prostate cancer cell lysate and beta-catenin antibody. The assay demonstrated recruitment of beta-catenin to the promoter site of PrKD1 gene as well as other downstream regions in the gene  by adding LNCaP nuclear protein extract (pulled down with beta-catenin) with or without PrKD1 promoter sequence to 48-TF array plate  mice (n=3) and littermate controls (n=3) [PrKD1 KO mice compared to litter-mate controls  . Qualitas Figure .PrKD1 promoter activity was measured in LNCaP prostate cancer cells co-transfected with either T120 mutated or wild type beta-catenin (WT) and PrKD1-promoter reporter . The results showed a significant decrease in PrKD1 promoter activity in T120 mutant transfected cells compared to WT beta-catenin transfected cells suggesting that nuclear beta-catenin has a negative regulatory effect on PrKD1 promoter  expression, down regulation of PrKD1 led to AR mediated increased PSA expression . As the In order to study the role of PrKD1 on AR expression and transcriptional activity through changes in beta-catenin phosphorylation and subcellular localization, we used AR reporter assays and cell line transfection experiments. The AR expression did not show significant changes in T120 beta-catenin mutant transfected LNCaP cells compared to control transfected cells Figure . HoweverMYC/MAX heterodimerization leads to activation of MYC mediated transcription, heterodimerization of MXD1/MAX leads to inhibition of MYC activity [PrKD1 gene expression through activation or inhibition of MYC activity respectively. Beta-catenin was not included because we have shown that only subcellular nuclear translocation of beta-catenin, and not changes in the beta-catenin gene expression, are involved in PrKD1 regulation.We interrogated publicly available DNA microarray expression data sets derived from human prostate benign and cancerous tissues using National Center for Biotechnology Information’s Gene Expression Omnibuses . We focuactivity . Our anaPrKD1 gene expression seems varied among the cancer types. There is paucity of data regarding regulation of PrKD1 gene expression in the published literature other than DNA methylation being involved in PrKD1 down regulation in gastric cancers and by nuclear beta-catenin in colon cancer [While PrKD1 is up regulated in pancreatic and skin cancers, it is down regulated in breast, gastrointestinal and advanced prostate cancers . Whereasn cancer , 15. In PrKD1 is a target gene under MYC transcriptional control. In fact, the PrKD1 gene contains the consensual sequence for MYC/MAX binding in several regions including the promoter region and further investigation in the area can elucidate the effects of MYC binding to other PrKD1 binding sites. In addition, the function of MYC is targeted by several upstream proteins including receptor tyrosine kinase stimulated Ras/Raf signaling and PI3K (phosphoinositide 3-kinase) mediated AKT signaling pathways. Interestingly, the MYC gene has several Wnt responsive elements and one of the upstream regulators of MYC is nuclear beta-catenin, which is increased by down regulation of PrKD1. The complex network of mutually dependent protein expression and activity suggests built in auto-regulatory mechanisms to regulate critical cellular functions, and nuclear beta-catenin is a key player in the study network.MYC is a well-studied transcription factor known to be involved in cell proliferation and apoptosis. Whereas the transcriptional activity of MYC is induced by obligate heterodimerization with MYC-associated protein X (MAX), the heterodimerization of MAX with MAX dimerization protein 1 axis (MXD1 or MAD1) inhibits MYC transcriptional activity . The MYCPrKD1 gene has several Wnt responsive DNA elements, the beta-catenin did not bind to the putative TCF/LEF factor in the transcription array  family of sequence-specific transcription factors . Althoug-catenin . While t-catenin , 21. On  as well  and that as well , 23, 24 PrKD1 promoter site. Interestingly, the recruitment of beta-catenin is independent of TCF4 and AR, which have overlapping interaction domain on beta-catenin and demonstrate competitive binding [AR is a key driver of prostate cancer progression and has been an effective therapeutic target in managing patients with advanced prostate cancer . The act binding .PrKD1 expression and thereby perpetuates the down regulation of PrKD1 in advanced prostate cancer  used in the Migration experiment  and its more metastatic and less androgen dependent derivative, C4-2 cells (Urocor), were cultured in RPMI 1640 medium plus 10% Fetal Bovine serum . For androgen treatment and control, charcoal stripped serum  was used instead of FBS, with or without R1881 , using 1 nM R1881 for androgen treatment. Enzalutamide , a second generation AR antagonist, at 10 μM final concentration, was used for AR inhibition. LNCaP cells were stably co-transfected with either wild type beta-catenin (WT), T120 mutated beta-catenin (T120 mutant) or empty vector (DsRed2-C1) constructs as previously described [t Figure  were preThe non-transfected and stably transfected cells were seeded in 96 well plates. The cells were co-transfected with ARE-dual luciferase formatted reporters gene using Cignal reporter assay kit . The co-transfected cells were recovered in growth medium (RPMI1640+ CSS 10%) with or without androgen for 24 hours. The luciferase assay was developed using Dual-Luciferase Reporter Assay system . The AR activity was calculated as the ratio of Luciferase/Renilla Bioluminescence. For beta-catenin transfected cells, the results were normalized against empty vector-transfected cells. All transfections were performed in triplicates. Positive and negative controls (provided in the kit) were included in each experiment.PrKD1 promoter reporter construct  was co- transfected with beta-catenin constructs (WT and T120 mutant) in LNCaP cells using Fugene reagent. After an overnight co-transfection and 24 hour recovery, a luciferase assay was performed on co-transfected LNCaP cells using LightSwitch Luciferase Assay kit . The measured luminescence was representative of PrKD1 promoter activity in each cell line. Luminescence in transfected cells were normalized against the empty vector.LightSwitch/The RNA was extracted from the cell lines using RNeasy Plus Mini Kit . Four μg of RNA was used for cDNA synthesis using Transcriptase III . Primer sets for androgen response genes and controls were designed using NCBI primer pick tool for Sybr green assays  or selecThe CHIP assay was performed using Simple CHIP kit . Briefly, cultured cells were incubated with 37% fomaldehyde to crosslink proteins to DNA for 10 minutes, collected and centrifuged after adding glycine. After nuclear extract preparation, the chromatin was digested using Micrococcal Nuclease, immunoprecipitated overnight with beta-catenin , MYC  or MAX  antibodies followed by elution through CHIP-Grade protein G Magnetic Beads  for 2 hours. Antibody against histone H3  was used as positive control, mouse and rabbit IgGs as negative controls. Chromatin was eluted from the antibody/protein G complex using SDS containing elution buffer. DNA was isolated and quantified by qPCR using promoter-specific primers .Part of the DNA prepared (pulled down with either beta-catenin or IgG) for CHIP assay was used in CHIP sequencing. DNA sequencing was performed on an Illumina NextSeq 500 sequencer in the Wake Forest Cancer Genomic Core Laboratory. Each DNA library was barcoded to allow optimized pooling in order to reduce sequencing cost. We carried out single-end DNA sequencing at up to 75 base pair (bp) single end, which was determined based on estimated fragment capture sizes. At the 75 bp scale and indexing of 12 libraries per flow cell, we had the capacity to generate up to 30 million sequencing tags/sample with an output of >∼ 26 gigabase per flow cell.The data set (GSE8511) with significant down regulation of PrKD1 in metastatic samples, was downloaded and analyzed using the statistical language R , 32. AllPrKD1 Knock down  mice was described previously [PrKD1 knock-out efficiency was confirmed by mRNA expression and at protein level [lox/Lox genotype were used as PrKD1 KO and animals without PB-Cre4 used as control because they lack PrKD1 down regulation [2 inhalation followed by cervical dislocation (secondary method). The studies were done following approval by the Institutional Animal Care and Use Committees (IACUC).Generation of prostate specific eviously . PrKD1 kin level . Male migulation . Mouse ePrKD1-promoter oligonucleotide were incubated with a biotin tagged probe mix to generate TF/probe complex. After spin column purification to separate un-bounded probes from complexes, the captured probes are then detected with Streptavidin-HRP and chemiluminescent substrate. PrKD1 promoter region regulated by beta-catenin was amplified by real-time PCR and the amplified region was used as PrKD1-promoter-specific-oligonucleutide. If the DNA fragment contains a TF binding sequence, it will compete for the biotin-labeled probe to bind to the TF in the sample. When the changes in reading between two samples were ≥1.5 fold, the results were considered significant for activated TFs.The potential activity of 48 transcription factors (TFs) were measured using TF activation plate array 1  for promoter binding TF profiling. Briefly, nuclear extract of LNCaP cells (pulled down with beta-catenin) with or without 8. Mouse prostate tissue was fixed in 4% paraformaldehyde. The paraffin-embedded paraformaldehyde-fixed mice tissue was cut at 5 micron thickness. The first slice of staining sections was used for histological confirmation by Hematoxylin and Eosin (H&E) staining. The tissue slides and fixed cells were treated with 0.1% Triton-X-100 for 3 min, followed by blocking nonspecific binding using protein block solution , overnight incubation with primary antibody at 4°C and one hour incubation with fluorescent secondary antibodies AF594 (Life technologies #SA5-10088) or AF488  and contra-stained with DAPI for 5 minutes. Goat isotype IgG (Santa Cruz) was used as negative controls. Tissue slides were examined using Olympus Fluoview (FV10i) confocal microscope and cells were analyzed by IN Cell analyzer 2000 cell imaging system .Immunohistochemical detection of beta-catenin, and PrKD1 was performed on mouse prostate tissue and transfected and non-transfected cell lines using anti-beta-catenin  and anti PKC mu The nuclear proteins were extracted from different cell lines using nuclear extraction kit ; whole cell lysate was extracted using Pierce RIPA buffer  and other subcellular proteins were extracted using subcellular protein fractionation kit for cultured cells . Lysates containing equal amounts of protein were separated on 10% SDS-PAGE gels and transferred onto a PVDF membrane . All primary antibodies were obtained from Santa Cruz Biotechnologies. Total protein loading was determined by probing the membranes for beta-actin or PCNA. The bands were visualized using horse-radish peroxidase (HRP) conjugated secondary anti-mouse or anti-rabbit antibody  in conjunction with chemiluminescence substrate  via LAS 3000 imaging system (Fuji Photo Film). Image J software was used to analyze the density of electrophoretic western blot bands. The values were normalized against beta-actin or PCNA expression.3 cells per well were seeded into 96-well plates. After overnight incubation, medium was changed to treatment (MYC inhibitor 100 μM) in half of the wells. Cell proliferation was evaluated after 72 hours by incubating cells in MTS/PMS mix  for 1 hour at 37° C. Absorbance was detected at 490 nm with a microplate reader . The culture medium was used as a blank. Experiments were done in triplicate. Cell viability in different treatment groups compared using two-way analysis of variance (ANOVA) with Bonferroni post-test correction.Approximately 3 ×104 cells per insert. Growth medium with 10% FBS was used as chemoattractant. After 24 hours, the cells on the lower surface of the membrane were stained with Diff-Quick stain following the manufacturer’s protocol. The invading cells were counted using 10X magnification on a Zeiss imager M1 microscope. The percentage of invading cells for each of the conditions was calculated as the mean number of invading cells through Matrigel coated inserts divided by mean number of cells migrated through uncoated insert membrane. The experiments were done in triplicates.Cells were seeded in Matrigel coated cell culture inserts  and non-coated inserts (as control) in serum free growth medium with or without MYC inhibitor treatment at 2.5 x 104 per well. A scratch wound was produced using IncuCyte wound maker . After two washes, cells were cultured in standard growth medium in the presence or absence of MYC inhibitor treatment for 96 hours and migration is captured for 48 hours by two hour scans intervals in IncuCyte ZOOM system and analyzed briefly using ZOOM software.The cells were seeded on a black/clear Falcon 96 well plate at 2 x 10


Wnt/β-catenin signaling is highly conserved throughout metazoans, is required for numerous essential events in development, and serves as a stem cell niche signal in many contexts. Misregulation of the pathway is linked to several human pathologies, most notably cancer. Wnt stimulation results in stabilization and nuclear import of β-catenin, which then acts as a transcriptional co-activator. Transcription factors of the T-cell family (TCF) are the best-characterized nuclear binding partners of β-catenin and mediators of Wnt gene regulation. This review provides an update on what is known about the transcriptional activation of Wnt target genes, highlighting recent work that modifies the conventional model. Wnt/β-catenin signaling regulates genes in a highly context-dependent manner, and the role of other signaling pathways and TCF co-factors in this process will be discussed. Understanding Wnt gene regulation has served to elucidate many biological roles of the pathway, and we will use examples from stem cell biology, metabolism, and evolution to illustrate some of the rich Wnt biology that has been uncovered. Intense investigation into the mechanisms of this pathway has uncovered some of the basics of how Wnts influence gene expression. A better understanding of how this signaling cascade operates has also provided genetic tools to explore various aspects of Wnt biology (reviewed inThe Wnt/β-catenin (Wnt/β-cat) pathway is conserved throughout metazoans and is essential for development and tissue homeostasis in adult organisms  are secreted proteins that potentiate Wnt/β-cat signaling  occupancy at promoters of Wnt targets, but it does increase phosphorylation of the C-terminal domain of Pol II, indicating that Wnt/β-cat signaling stimulates transcriptional elongation22. This study provides the clearest description to date of some of the chromatin events that tie the binding of TCF and β-cat to enhancers with the initiation of transcription at Wnt target loci, and it will be interesting to see if they are typical for Wnt gene activation beyond the human embryonic stem cells used in this report.In the nucleus, TCF/β-cat can act through enhancers that can be hundreds of kilobases away from the proximal promoters of Wnt targets e.g–26. EnhXenopus gastrulating embryos had no detectable effect on gene expression30. There was a strong overlap of the >10,000 β-cat ChiP-seq peaks identified in this report with TLE and p300 peaks from prior studies32, suggesting that many of these regions are functioning according to the standard model of Wnt-regulated enhancers. The authors propose a model of β-cat recruitment to regulatory DNA acting as a primer, with inputs from other signaling pathways required for activating transcription33. Interestingly, a priming role for β-cat has previously been proposed to occur at Wnt targets at an earlier developmental stage inXenopus, prior to the onset of zygotic transcription at midblastula transition34.The traditional assumption is that the recruitment of β-cat to chromatin results in transcriptional activation of nearby promoters  is present in the destruction complex and methylates YAP, which is required for its ability to promote the nuclear accumulation of β-cat55. Hippo and Wnt/β-cat signaling are connected through multiple mechanisms, and understanding the cell-specific cues that favor one interaction over another will be an important goal for future studies.Subsequent reports on the intersections between Hippo and Wnt/β-cat signaling support a complex and context-dependent relationship between the pathways. For example, LATS2 has been shown to directly inhibit β-cat’s interaction with other co-activatorsDrosophila andCaenorhabditis elegans have oneTCF gene, which plays a dual role on Wnt targets, inhibiting expression in the absence of signaling and mediating transcriptional activation when bound by β-cat 4a expression to inhibit spinal cord neurogenesis in a Wnt/β-cat signaling-independent manner60. Recently, Merrill and co-workers reported a dramatic genetic interaction betweenTCF3 alleles in mice that also supports a major role for β-cat-independent repression61.TCF3 null mutants die during gastrulation59, whileTCF3 mutants lacking the β-cat binding domain (∆NTCF3) die during late embryogenesis62. Surprisingly,nullTCF3/∆NTCF3 heterozygotes survive into adulthood with no obvious defects61. This result demonstrates that TCF3 has an essential role in development that is independent of binding to β-cat.Invertebrates such as64. In mouse embryonic stem cells, several papers have reported a downregulation of TCF3 in backgrounds where Wnt/β-cat is elevated66. Interestingly, the three reports differ on whether this effect acts at the level of transcription and/or post-transcriptionally. Under conditions in which mouse embryonic stem cells differentiate into endoderm, TCF3 downregulation coincided with elevated expression of the endodermal marker Forkhead Box A2 (FoxA2) and a loss of TCF3 on FoxA2 regulatory chromatin66. Indeed, mouse embryonic stem cells lacking TCF3 have elevated FoxA2 expression and can differentiate into endoderm  in the absence of Wnt stimulation66. While this indicates that derepression of Wnt targets is a major driver for endoderm differentiation, another endoderm marker, Sox17, is directly activated by TCF4/TCF7L2 and β-cat67. In sum, it appears as if derepression as well as β-cat activation of Wnt targets contribute to endoderm differentiation in vertebrates.How do some Wnt target genes undergo a transcriptional switch from repression by TCF3 to β-cat-dependent transcriptional activation by other TCFs? One model is that Wnt/β-cat signaling activates homeodomain-interacting protein kinase 2 (HIPK2), a kinase which phosphorylates TCF3, removing it from chromatin68, to the degree that HMG-DNA recognition cannot be sufficient to drive TCF distribution on chromatin -expressing cells located in the nail matrix were identified as nail stem cells (NSCs) through lineage tracing94. A conditional knockout of β-cat in+K14 cells impaired nail growth, with the entire nail epithelium showing elevated levels of NSC markers. Surprisingly, overexpressing a stabilized β-cat in+K14 cells did not impact nail growth94. In addition to its being a continuously growing tissue in adults, the nail epithelium has been studied for its role in digit tip regeneration. A population ofWntless (Wls)-expressing cells—Wls is an acyltransferase required for the secretion of Wnt proteins95—flanks the NSCs and is essential for digit tip regeneration. Digit tip regeneration does not happen after amputations that remove this population, but this defect can be rescued by the expression of β-cat in+K14 cells. In this context, the Wnt/β-cat pathway appears to be a permissive signal that is essential for differentiation but has no influence on NSCs. Consistent with this, NSCs do not express high levels of Axin294.In contrast to the stem cells of the intestine and liver, stem cells of the nail epithelium are seemingly agnostic to Wnt/β-cat signaling but require the pathway for differentiation.96. A colon cancer cell line with elevated Wnt/β-cat signaling expressing a ∆NTCF4 isoform containing a C-clamp showed reduced proliferation and a metabolic shift towards oxidative respiration. Consistent with this, genes controlling the cell cycle and metabolism were downregulated by this dominant negative TCF497. Interestingly, ∆NTCFs lacking a C-clamp did not affect proliferation but still caused the metabolic shift97. Pyruvate dehydrogenase kinase 1 (PDK1), which blocks oxidative respiration, was found to be the key Wnt target promoting aerobic glycolysis in these cancer cells97. PDK1 and the lactate transporter monocarboxylate transport protein (MCT)-1 are direct targets of the Wnt pathway in this context98 and lactate dehydrogenase, another enzyme driving the Warburg effect, was indirectly activated by the Wnt targetc-Myc99. Interestingly, fluorescence lifetime microscopy (FLIM), which can provide an indicator of the relative rates of glycolysis and oxidative phosphorylation in live tissue, found that aerobic glycolysis also occurs in Wnt-dependent Lgr5+ intestinal stem cells100.The Warburg effect or aerobic glycolysis is seen in cancer cells, which preferentially metabolize glucose through lactic acid fermentation instead of the TCA cycle, even in the presence of oxygen103. Characterization of marine/freshwater hybrids identified theEctodysplasin (Eda) locus as a major gene responsible for the loss of lateral armor plates in the freshwater species104. Further refinement identified a single point mutation in an enhancer just downstream ofEda105. This enhancer is a target of Wnt/β-cat signaling, and the freshwater allele has reduced activation105. While this 3.2 kb enhancer contains several putative TCF binding sites, they are not close to the polymorphism , and it is not clear whetherEda is a direct target of the pathway. A similar story exists for the evolution of wing spots inDrosophila guttifera, where theyellow gene is activated by Wnt/β-cat signaling in the pupal wing, though the activation appears to be indirect106.In contrast to transcriptional profiling, Wnt targets that are important in animal evolution have been identified through linkage studies. The three-spined stickleback has become a premier system for studying the evolution of physical traits, since the marine species has repeatedly lost its body armor and ventral spines after colonizing freshwater lakesKITLG locus that is responsible for blond hair in humans107.KITLG encodes a ligand for the KIT receptor, known to control pigmentation in mammals108. Interestingly, the polymorphism resides in a predicted TCF binding site, with the blond allele showing reduced activation by Wnt/β-cat signaling109. This study directly links the regulation of a direct Wnt target to an important physical trait. This is reminiscent of a polymorphism 335 kb upstream of thec-myc locus, also in a functional TCF site, where the higher-affinity allele is linked to increased risk in colorectal and other cancers110. Both examples illustrate how a detailed understanding of Wnt gene regulation can facilitate the molecular understanding of polymorphisms in the human population.Another link between Wnt/β-cat and evolution comes from a genome-wide association study (GWAS) which identified a polymorphism near theAn increasing number of molecular approaches can now be employed to identify Wnt transcriptional targets in cells or tissues. Continued definition of Wnt transcriptional programs will further the understanding of how the Wnt/β-cat signaling pathway achieves its varied roles in development, stem cell maintenance, and metabolic regulation as well as in disease states and molecular evolution. It is clear that the activation of Wnt targets is highly context dependent, and the emerging picture is that a combination of TCFs and a diverse assortment of other TFs work together in different cells at different times. Unraveling the molecular mechanisms behind context specificity in Wnt responses will not only address a central question of gene regulation but also enhance our knowledge of the diversity of Wnt biology.Eda,Ectodysplasin; FoxA2, Forkhead Box A2; Fzd, Frizzled;K14,keratin-14; LATS, large tumor suppressor kinase; Lgr, leucine-rich repeat-containing G-protein-coupled receptor; NSC, nail stem cell; PDK1, pyruvate dehydrogenase kinase 1; Rnf43, ring finger protein 43; Rspo, R-spondin; Sox, Sry-related HMG box; TAZ, Tafazzin; TCF, T-cell factor; TF, transcription factor; TLE, transducin-like enhancer of split; β-TrCP, β-transducin repeat-containing E3 ubiquitin protein ligase;Wls,Wntless; Wnt/β-cat, Wnt/β-catenin; YAP, yes-associated protein; Znrf3, zinc and ring finger 3.ChIP-seq, chromatin immunoprecipitation sequencing;


Apc and β-catenin at different stages of mouse liver development. In normal development, the ammonia metabolising enzymes carbamoylphosphate synthetase I (CPSI) and Glutamine synthetase (GS) begin to be expressed in separate hepatoblasts from E13.5 and E15.5 respectively and gradually increase in number thereafter. Restriction of GS expression occurs at E18 and becomes increasingly limited to the terminal perivenous hepatocytes postnatally. Expression of nuclear β-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expression of nuclear β-catenin throughout the developing liver and increased number of cells expressing GS. Conversely, conditional loss of β-catenin resulted in loss of GS expression. These data suggest that the Wnt pathway is critical to the development of zonation as well as maintaining the zonation in the adult liver.While the Wnt/β-catenin pathway plays a critical role in the maintenance of the zonation of ammonia metabolizing enzymes in the adult liver, the mechanisms responsible for inducing zonation in the embryo are not well understood. Herein we address the spatiotemporal role of the Wnt/β-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of  This refers to the heterogeneous distribution of key rate-limiting enzymes and metabolic pathways across the liver lobule or acinus3. The most striking example of zonation is observed in the enzymes and pathways of ammonia detoxification4. The urea cycle and the enzyme glutamine synthetase (GS) are two systems responsible for the removal of ammonia in the liver. Carbamoylphosphate synthetase I (CPSI) is the rate-limiting enzyme in the urea cycle and is expressed in the periportal, intermediate and first few layers of the perivenous zone. GS is expressed in a complementary fashion to CPSI and catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. In the developing liver the onset and pattern of expression of CPSI and GS has been documented7. Expression of CPSI and GS is first detected in a small number of cells at embryonic day 13 (E13) and embryonic day 15 (E15) respectively, thereafter expression gradually increases throughout development with a decline in CPSI accumulation in the hepatocytes around the central vein occurring at E17.55. In contrast, Shiojiri et al. documented that CPSI is first detected at E15 while GS is first expressed in the perivenous regions of the postnatal liver three days after birth7. This discrepancy has been attributed to the fixation conditions used during tissue preparation5. Both studies showed that the strict compartmentalization of these two enzymes observed in adult liver occurs postnatally.The adult liver exhibits a remarkable phenomenon known as metabolic zonation9. Deletion of the Wnt effector Apc, which leads to ligand independent activation of β-catenin, or inhibition of Wnt signalling by virally-mediated hepatic overexpression of the inhibitor DKK1 disrupts the normal zonation of ammonia metabolizing enzymes8. Loss of Apc results in a reduction of CPSI and an expansion of GS expression. Deletion of c-Myc does not rescue the liver phenotype when Apc is deleted suggesting that β-catenin regulates zonation through a c-Myc-independent mechanism9. The sensitivity of hepatocytes to a threshold of Wnt/β-catenin signaling in the regulation of zonation has also been demonstrated in Apcmin mice in which expression of full length APC is partially attenuated10.The Wnt/β-catenin pathway has been proposed to play a key role in the maintenance of metabolic zonation in the adult liverApc, through expression of Cre-recombinase under the control of a chimeric alpha-fetoprotein-albumin enhancer/promoter, results in the accumulation of β-catenin in the liver from E11.5 and subsequent embryonic lethality between E16.5 and E18.511. Apc loss also resulted in liver hypoplasia and failure of hepatoblasts to differentiate towards the hepatocyte lineage. Apc-deficient liver also exhibited an increase in bile duct morphogenesis indicating a preference towards biliary differentiation in the presence of activated β-catenin11. Deletion of β-catenin in hepatoblasts resulted in significant underdevelopment of the liver from E12 with lethality occurring at around E1712. β-catenin-deficient embryonic liver lack hepatocyte and bile duct compartments. Collectively, these observations suggest a critical role for β-catenin in the survival, maturation and differentiation of hepatoblasts.The Wnt/β-catenin pathway is also important in liver development. Hepatoblast-specific inactivation of 12. A role for the Wnt/β-catenin pathway in hepatic zonation of ammonia metabolizing enzymes has previously been established in adult liver but the question remains as to whether this pathway is also involved in establishing hepatic zonation during embryogenesis13. Here, we use inducible conditional deletion of Apc and β-catenin to dissect the role of Wnt/β-catenin signaling in the development of metabolic zonation of GS and CPSI.The early embryonic lethality observed in these studies has precluded a detailed analysis of the effects of disrupting the Wnt/β-catenin pathway on the expression of ammonia metabolizing enzymes in the developing mouse liveret al. described the onset of CPSI and GS expression at E13 and E15 with a gradual increase as development proceeds, followed by a decline in CPSI protein in hepatocytes surrounding the central vein prior to birth5. However, Shiojiri et al. observed that the onset of CPSI expression occurred at E15 while GS expression appeared postnatally6. The discrepancy in GS staining has been attributed to differences in tissue preparation while the observation that CPSI accumulation exhibits intralobular heterogeneity may account for differences observed in CPSI staining5. We first determined the expression pattern of CPSI and GS in relation to that of β-catenin at different stages of liver development (from E12.5 up to E18.5) in wild-type mice using immunostaining and western blotting . The inducible Ahcre model provides an efficient approach for controlling Cre-mediated gene deletions in the hepatoblasts of the mouse liver in a time-specific manner. Ahcre mice were intercrossed with mice carrying Apc Apc580S (Apcfl/fl) or β-catenin (β-Catfl/fl) loxP flanked alleles16. Pregnant females were identified and injected intraperitoneally with a single dose of β-NF at E13.5, E14.5 or E16.5 and embryonic tissue isolated either 2 or 4 days following recombination. We did not attempt inducing recombination at E13.5 and analysing at E18.5. Adult Ahcre + Apcfl/fl mice rapidly deteriorate within 4 days following APC deletion thus for consistency the latest we analysed genotypes was 4 days post-recombination.Previous studies of early βApc and β-catenin, immunohistochemistry for β-catenin was performed on different recombinants: AhCre+Apc+/+β-Catl++, AhCre+Apcfl+/β-Catl++, AhCre+Apcfl/flβ-Catl++, AhCre+Apc+/+β-Catlfl+ and AhCre+Apc+/+β-Catfllfl , and the liver enriched transcription factors Hnf4α and Hnf6  and Hnf4α (1.4-fold) was detected  decrease in gene expression. Immunohistochemical analysis of AhCre+Apc+/+β-Catfllfl liver following recombination at E16.5 revealed the loss of GS zonation. The pattern of CPSI staining remained unchanged but the intensity of staining was increased throughout the liver. However, qPCR showed a reduction in mRNA levels of 1.7-fold and 2.3-fold for GS and CPSI respectively , following recombination at E14.5 and E16.5, resulted in a similar pattern of staining for GS and CPSI to that observed in the wild type liver and nuclear staining for β-catenin was marginally more widespread. Conversely fewer cells exhibited nuclear β-catenin staining in AhCre+Apc+/+β-Catlfl+ liver sections accompanied by less robust staining for GS following recombination at both gestational time points. No significant differences were observed by qRT-PCR in GS and CPSI levels. Thus overall these results show that dose-dependent disruption of Wnt signalling either by ablation or activation of β-catenin affects both the pattern and intensity of CPSI expression and results in disruption of GS.Deletion of yos Fig.  and a 4.yos Fig. . In contyos Fig. . Interesely Fig. . HeterozAhCre+Apcfl/flβ-Catl++ embryos following recombination at E13.5 we saw robust nuclear accumulation of β-catenin  and Hnf4α expression was reduced (1.4-fold)  Fig. . This da13. However, the mechanism involved in regulating the zonal expression of these enzymes in the developing liver remain unclear. Previously, Notenboom et al. established that the expression of CPSI begins at around E13.5 and becomes expressed throughout the liver by E18.5. Our data is consistent with these observations. Restriction of expression of CPSI from the terminal perivenous hepatocytes, as seen in adult liver, is reported to occur postnatally at weaning17. In contrast, the onset of expression of GS is less certain with its first appearance being reported from E15.5 up to 3 days postnatally6. We observed the robust and ordered induction of GS expression at E17.5-E18.5. Expression of GS and nuclear β-catenin were observed within the same cells in discrete areas of the liver (associated with vascular structures). For the first time, our data provides evidence that the zonation of GS during development is regulated through the Wnt/β-catenin pathway. Previously, Apc deletion in mouse embryos at E11.5 caused severe liver hypoplasia, a reduction in expression of hepatoblast/hepatocyte markers  and embryonic lethality between E16.5–E18.511. The authors also showed a partial commitment toward the biliary lineage, based on the presence of duct-like structures overexpressing the biliary marker integrin β411.The zonal regulation of ammonia metabolising enzymes in the adult liver is controlled by the Wnt/β-catenin pathwayGS expression on β-catenin activation, we used the previously reported AhCre Apc model. Here, Cre transgene expression is driven by the inducible Cyp1A1 promoter9. All of the embryos (littermates) came from mothers that were treated with β-napthoflavone. We did not use animals that were either untreated or AhCre−. The observed changes in zonation therefore correlate with genotype (reduced gene expression) and not with the treatment. We cannot exclude the possibility that the changes are due to hypomorphic effects of the floxed alleles. Disruption of Apc at each of the time-points examined, resulted in nuclear β-catenin in the majority of cells throughout the embryonic liver demonstrating efficient recombination and activation of the Wnt signalling pathway. A concomitant increase in GS staining and mRNA levels was revealed by immunohistochemical and qPCR analysis respectively for all time points examined. Following recombination at E14.5, we observed a complementary loss of CPSI staining and gene expression in E18.5 livers suggesting a disposition for hepatoblasts to become perivenous-like at the expense of the periportal phenotype. We also examined the effect of forced β-catenin activation at E13.5 and found that GS expression was induced in the E15.5 liver  while CPSI mRNA levels were reduced. Interestingly, recombination at E16.5 did not alter CPSI expression (either at the protein or mRNA level) indicating differential sensitivity of the periportal phenotype to β-catenin activation in a temporal manner. This difference may be due, in part, to the expression of Hnf4α. It has been proposed that Hnf4α plays a crucial role in regulating zonation in adult mouse liver19. In one of these studies, Hnf4α directly binds to the upstream enhancer of GS suggesting that suppression of GS expression in periportal hepatocytes is mediated through Hnf4α-dependent recruitment of repressor complexes18. In a complementary study, expression of GS was repressed by the binding of Hnf4α to its own consensus sequence within the promoter19. Hnf4α-mediated repression is inhibited upon β-catenin activation through the displacement of Hnf4α by the Wnt signalling mediator LEF1. However, activation of the Wnt pathway and binding of LEF1 results in the displacement of Hnf4α and a consequent decline in expression19. These studies reveal a dependence of zonation on the convergence of Hnf4α on Wnt signalling pathway. In the context of our study, the relatively unaltered levels of Hnf4α mRNA following activation of β-catenin at E16.5, may be responsible for the observed lack of change in CPSI expression. In addition, the persistent staining for CPSI in the Apc null liver following recombination at E16.5 may be due to stability of the enzyme.To determine in more detail the dependence of 12. Foxa3 Cre β-cateninfl/fl embryos exhibit a reduction in liver size and embryonic lethality occurring at E17.5. Crucially, we have extended these studies to determine the effect of deletion of β-catenin on expression and zonation of GS in a conditional and temporally controlled manner. β-catenin deletion at E14.5 and E16.5 caused almost complete loss of GS staining and a reduction in gene expression in the E18.5 liver, demonstrating a requirement for β-catenin in induction and zonation of GS during development. While CPSI staining in the E15.5 liver remains unchanged following recombination at E13.5, mRNA levels were elevated. Conversely, inactivation of Wnt signalling at E14.5 and E16.5 caused decreased CPSI gene expression at E18.5. These observations imply that maturation of hepatoblasts toward a periportal phenotype is influenced by the status of β-catenin activation between E14.5-E18.5. This was unexpected given that previous work supported the idea that the maintenance of the periportal phenotype in the adult requires inhibition of β-catenin9. During normal development, bi-potential hepatoblasts at E13.5 begin to differentiate to hepatocytes or biliary cells. The disruption of Wnt signalling either through stabilisation or inhibition of β-catenin prior to this stage has a profound effect on liver development by impairing normal hepatoblast fate decisions12. Stabilisation of β-catenin appears to drive hepatoblasts toward a biliary fate. In the present study, we noted the presence of duct-like structures in AhCre Apcfl/fl embryos following recombination at E14.5. Ectopic staining and increased gene expression for the biliary progenitor marker Sox9 was observed following activation of β-catenin  (80 mg/kg) at the gestational time points indicated . Tissue samples were harvested either 2 or 4 days after recombination. Mice were humanely killed by a schedule 1 procedure. Ethical approval was granted from Cardiff University’s Animal Welfare and Ethical Review Body (previously known as the ERP), and all animal procedures were conducted in accordance with UK Home Office regulations.The loxP flanked alleles used were as follows 9.Embryonic liver tissue was fixed and processed as described previously9 with the following changes;Immunostaining for GS, CPSI, and β-catenin was carried out as described previouslyHigh temperature antigen retrieval was performed in EDTA buffer for CPSI, GS, and β-catenin. Following antigen retrieval sections were immuno-histochemically labelled for β-catenin or immunofluorescently labelled for CPSI and GS.For immunohistochemistry sections were blocked in 10% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in PBS. Antibodies were diluted in 1% NGS/1%BSA in PBS: anti-mouse β-catenin . Antibody detection was carried out using HRP conjugated secondary antibodies  and DAB (Vector Labs). All immunohistochemical staining was imaged using an Olympus microscope and CellP Olympus software.For immunofluorescent staining with CPSI and GS sections were blocked in 5% BSA/PBS. Antibodies were diluted in 1% BSA/PBS CPSI , GS , anti-mouse Alexa 488  and anti-rabbit Alexa 594 . Fluorescent staining was imaged using a Zeiss LSM Meta confocal microscope and LSM software.st strand cDNA synthesis and reverse transcriptase polymerase chain reaction (RT-PCR) was performed as described previously23. Primer sequences used are listed in Table RNA extraction, 1G for 5 min at 4 °C. The supernatant (containing the cytosolic fraction) was stored at −80 °C. The nuclear pellet was washed twice in 500 μl of cytosolic extraction buffer, dissolved in 50 μl nuclear extraction buffer  and rotated at 4 °C for 60 min. The lysate was centrifuged at 16300 G for 15 min at 4 °C and the supernatant (containing the nuclear fraction) was stored at −80 °C. Western blotting was carried out as previously described23. Antibodies used were obtained and diluted as follows: anti-mouse β-catenin , anti-mouse GS , anti-rabbit CPSI . Anti-mouse glyceraldehyde-3-phosphate dehydrogenase , anti-goat-Lamin B1  and peroxidase labelled anti-rabbit or anti-mouse both at 1:2000 . The signal was detected with the ECLTM Western blotting analysis system (Amersham) and developed on HyperfilmTM (Amersham). Autoradiographs were scanned on a Sharp MX-3570 printer/scanner and images manipulated using Photoshop Elements 10. Original uncropped autoradiographs are provided in Supplementary Figures Fractionation of liver proteins was performed as follows; liver tissue was lysed and homogenized in 500 μl of cytosolic extraction buffer . The lysate was rotated at 4 °C for 30 min and further homogenized by 10 passes through a 27 G needle and syringe. The nuclear fraction was isolated by centrifugation at 3000 All data generated or analysed during this study are included in this published article.Supplementary information