Hematopoietic stem cell transplantation is an intensive therapy used to improve survivorship and cure various oncologic diseases. However, this therapy is associated with high mortality rates and numerous negative side-effects. The recovery of the immune system is a special concern and plays a key role in the success of this treatment. In healthy populations it is known that exercise plays an important role in immune system regulation, but little is known about the role of exercise in the hematological and immunological recovery of children undergoing hematopoietic stem cell transplant. The primary objective of this randomized-controlled trial (RCT) is to study the effect of an exercise program (in- and outpatient) on immune cell recovery in patients undergoing an autologous stem cell transplantation. The secondary objective is to determine if an exercise intervention diminishes the usual deterioration in quality of life, physical fitness, and the acquisition of a sedentary lifestyle.This RCT has received approval from The Conjoint Health Research Ethics Board (CHREB) of the University of Calgary (Ethics ID # E-24476). Twenty-four participants treated for a malignancy with autologous stem cell transplant (5 to 18 years) in the Alberta Children’s Hospital will be randomly assigned to an exercise or control group. The exercise group will participate in a two-phase exercise intervention (in- and outpatient) from hospitalization until 10 weeks after discharge. The exercise program includes strength, flexibility and aerobic exercise. During the inpatient phase this program will be performed 5 times/week and will be supervised. The outpatient phase will combine a supervised session with two home-based exercise sessions with the use of the Wii device. The control group will follow the standard protocol without any specific exercise program. A range of outcomes, including quantitative and functional recovery of immune system, cytokine levels in serum, natural killer (NK) cells and their subset recovery and function, and gene expression of activating and inhibitory NK cell receptors, body composition, nutrition, quality of life, fatigue, health-related fitness assessment and physical activity levels will be examined, providing the most comprehensive assessment to date.We expect to find improvements in immunological recovery and quality of life, and decreased acquisition of sedentary behavior and fitness deconditioning. The comprehensive outcomes generated in this RCT will provide preliminary data to conduct a multisite study that will generate stronger outcomes.Gov identification # NCT01666015 Hematopoietic stem cell transplantation (SCT) is an intensive therapy used in the treatment of various oncologic diseases . This trThere is increasing evidence that exercise (EX) is safe, beneficial and feasible in different stages of cancer treatment -6, incluHuang et al.  in a recDespite robust scientific evidence describing the positive effects of EX in the adult cancer population, limited research efforts have focused on exploring the translation of EX therapy to children. Specifically, the role of EX in the immune system recovery of pediatric patients undergoing SCT has not been examined sufficiently to date, however the results from the adult population -18 are pThe purposes of the present study are:Study the effect of an EX program (in- and outpatient) on immune cell recovery in patients undergoing an autologous SCT.Determine if an EX program performed in patients undergoing autologous SCT diminishes the deterioration of physical fitness, physical activity levels and improves QOL.Establish the program’s feasibility (uptake and adherence) to provide the impetus to run multi-centre research with larger populations in the future.SCORE “Stem Cell patients Ongoing Recovery through Exercise” is a RCT of pediatric cancer patients undergoing autologous SCT. Assessors will be blind to participant’s condition (EX or control) and all the statistical analyses will be performed maintaining blindness to condition. Blinding of assessors and statistical analyses ensures limited bias on outcomes, especially with subjective outcomes of interest. Flow through the study is depicted in Figure This study will examine the effect of two phases of an EX program on several health related outcomes in pediatric patients undergoing autologous SCT Figure . The firThe second phase will be a 10-week outpatient intervention, beginning once the child is discharged. We will utilize a mixed EX program, including supervised  and home-based training incorporating the use of the Nintendo ® Wii device™ .Assessments include measures of QOL, fatigue, health-related fitness (HRF), physical activity levels and hematological and immunological reconstitution. Assessments will be completed at baseline, 30, 85 and 180 days post infusion day (day 0+). Hematological and immunological reconstitution will be assessed at the time points described above, plus at 7, 15 and 56 days post reinfusion. The baseline assessment will be completed within the two-month period prior to the conditioning regimen. The 30 day assessment is intended to capture the effect of Phase 1, the EX program during the hospitalization period (from conditioning regimen up to discharge). The 85 day assessment is intended to capture the early survivorship recovery period after an autologous SCT and the effect of Phase 2, EX program during the outpatient phase. The 180 day assessment will provide a mid-term follow-up of physical activity, QOL, fatigue and hematological and immunological reconstitution. These outcomes will allow us to examine the impact of an EX program implemented early during the SCT process 3 months after finishing the EX intervention on (i) physical activity levels (ii) QOL and fatigue iii) hematological and immunological recoveryhttp://www.socialpsychology.org). Allocation concealment will be ensured. After baseline assessments each participant will choose a sealed, non transparent envelop, which contains a number that will allocate the subjects to the control or EX group. The research coordinator will be responsible for the random allocation.A total of 24 children will be recruited for this RCT; 12 patients will be assigned to an EX training program and 12 subjects will be assigned to a control group (without any specific training). A sequence to allocate participants randomly will be generated by the software Research Randomizer, a research tool provided by the Social Psychology Network (Eligibility criteria includes: (1) autologous SCT at Alberta Children’s Hospital (ACH) for malignancy, (2) age 5 to 18 years of age, (3) will be receiving myeloablative conditioning regimen, (4) no evidence of cardiac or pulmonary failure associated with treatment (shortening fraction (SF) ≥28%, ejection fraction (EF) ≥ 50%), (5) no functional nor cognitive limitation that would prohibit performance of the home-based training, (6) approval by treating oncologist for participants, (7) a parent or legal guardian must sign the consent form, and (8) children should express verbal assent to participate. The entire team of assessors and the persons performing the statistical analyses will be blinded to group assignment.Children undergoing autologous SCT will be recruited through the Section of Pediatric Oncology and Blood and Marrow Transplantation at ACH. ACH is the SCT centre for Alberta. The referring oncologist will identify potential subjects and the research nurse will contact them to present the study at least 4 weeks before the hospitalization to start autologous SCT treatment. Those subjects who are interested in the study will be asked to sign the consent form by the research nurse and will be contacted by the research assistant to set up the baseline assessment.This on-site patient recruitment method will be more likely to enhance recruitment efforts than a mail out or other off-site method. Also, prospective subjects might feel more supported if the physician supports the research protocol. In order to obtain a homogenous sample, is known that the recruitment phase will take between 2-3 years to reach the sample described.Children in the intervention group will participate in an inpatient and outpatient mixed EX program including both resistance and aerobic training.These subjects will not participate in any scheduled EX and will perform the same battery of tests as the intervention group. They will be offered an EX program upon completion of phase 2, to be held in the Thrive Centre, University of Calgary.High dose chemotherapy and autologous SCT will be performed for a given child as per the recommended therapy for a given disease and stage risk group. Only children receiving myeloablative conditioning will be eligible. Most children will have a history of Sarcoma, Lymphoma, Neuroblastoma or Germ cell tumor. Supportive care will be as per institutional practice.Power calculations were derived using GPower 3.1  The sampThe raw data analysis will be performed using SPSS (v. 20.0) and will include descriptive, correlations, and repeated measures analysis of variance (ANOVA). Specifically, for the primary and second objectives, correlations will be run to examine the degree of relationship between the variables, and planned doubly multivariate repeated measures ANOVA’s will be performed to examine group differences between the intervention and control group. Descriptive statistics will be conducted for demographics, medical variables, adherence to the program, and physical activity behavior using means, standard deviations, and ranges as appropriate. As was previously mentioned, the statistician will be blinded to participant condition.This RCT will be a biphasic EX intervention.Phase 1 – The inpatient phase will be performed at the ACH during conditioning and the isolated phase of autologous SCT.Phase 2 – The outpatient phase will take place after discharge. The participants will participate in a 10 week mixed supervised and home-based EX program utilizing the Wii device.In both phases participants will wear a portable heart rate monitor (RS300X Polar) when at the EX sessions to ensure safety and target the aerobic EX intensity between 50% and 70% of heart rate reserve (HRR), depending on the physical status of each child.The training session will be cancelled if the child has platelet levels below 10.000/ μl, hemoglobin levels below 8 g/dl, fever greater than 38°C, pain, diarrhea, hemorrhage or any other complication(s) the oncologist thinks may worsen with EX. Additionally, a session may be cancelled if the child doesn’t want to participate.This program will consist of combined aerobic (20-30 minutes) and resistance training, designed on the basis of our previous experience with pediatric patients with cancer, and following the rules of the institutions for global benchmarks for strength training and aerobic EX in children and adolescents ,14,22. DThe strength EXs, will engage the major muscle groups , performing one set of 12–15 repetitions per EX.Following the in-patient EX program, the participants of the intervention group will engage in a 10-week EX program consisting of 3 weekly sessions. The participants from Calgary will perform a combined supervised and unsupervised/home-based exercise program (2 unsupervised and 1 supervised sessions/week) with an average duration of 60 minutes/session. The participants from outside of Calgary will perform only the home-based EX program 3 days/week. The supervised and unsupervised EX plans will be individualized based on fitness assessment outcomes and current health status.The supervised training session  will be performed at the Thrive Centre at the University of Calgary 1 time/week and will be similar to the inpatient intervention.Designed by exercise professionals and supervised by the parents, it will include 20-30 minutes of aerobic EXs and 30 minutes of strength and stretching EXs using Nintendo’s Wii console and Wii Fit/Wii Sports/Wii Dance games .Parents and children are going to be instructed on how to control the intensity of the EXs. Training adherence will be reported in a logbook, including reported training days and reasons for missed EX sessions. Training sessions will be declared valid if 80% of the planned session were performed. Participants will be reminded and encouraged to engage in their home-based workouts when they come to the Thrive Centre, and all participants  will receive a weekly phone call (from the research assistant) to encourage the participants to continue performing the EX program and answer any questions they or their parents may have.Once consent has been obtained, the research nurse will be responsible for instructing participants (parents and pediatric patient) (1) on the use of the accelerometers and log book, (2) on how to perform the food record, and (3) on the QOL assessment. The research coordinator will be responsible for contacting participants to perform the baseline HRF assessment. All baseline assessments will be performed before any conditioning regimen is administered. Additionally, the research coordinator will ensure all assessments are administered according to the protocol.We will perform: (1) Hematological and Immunological Recovery, (2) Health and Lifestyle Assessment and (3) Health-Related Fitness Assessments (HRF).At baseline 2 Buccal Swabs will be collected to use for deoxyribonucleic acid (DNA) extraction. DNA specimens will be used to perform genotyping of activating and inhibitory KIR genes. The patient should not eat or drink anything for one hour prior to the specimens being taken. We will take one swab and rub the inside of right and left patient’s cheek for approximately 10 seconds. The swab will air dry for 1-2 minutes prior to replacing it into its container.The baseline blood draw will be performed at the Unit 1 in the ACH (within 7 days prior to conditioning and transplant). The maximum blood volume to be drawn will be determined based on patient weight and other ordered blood work. A minimum of 7 mL will be required. The blood sample collection will be performed at baseline, day 7, 14, 30, 56, 85 and 180.To avoid any acute EX effects, we will discourage any specific EX training 24 h before blood draws in the samples that will be collected at baseline and after transplant on day 14, 30, 56, 85 and 180. Research samples may be collected via central line or peripheral draw. For this study, we will store 1 aliquot each for peripheral blood mononuclear cells (PBMNCs) for every blood specimen serum. The aliquoted serum samples will be stored in our -86°C freezers and PBMNCs will be cryo-preserved at -150°C using liquid nitrogen based cell storage facility. All immune functional assays will be performed on cry preserved PBMNCs.Assessment of the impact of the EX intervention on the immune recovery of pediatric autologous SCT patients will be based on four parameters (a) Recovery of different leukocyte cell subset  in peripheral blood, (b) Expression of activating and inhibitory KIRs (c) Function of NK cells and their subsets and (d) Levels of different pro-inflammatory and regulatory cytokines in serum,a) Recovery of NK cells and other immune cells subsets will be performed by flow cytometry enumeration of different blood leukocyte subsets including NK cells and its subsets (cytolysis and regulatory NK cells), T cells and its subsets , B cells, monocytes, neutrophils and dendritic cells.b) Gene/allele carriage analysis for 8 inhibitory KIR genes ; 6 activating KIR genes  and two pseudo genes (2DP1 and 3DP1) will be performed for all 24 patients. Gene expression analysis of 14 KIR genes will be done by RNA based real-time PCR analysis. Total RNA will be extracted from the PBMNCs for each time point and cDNA will be prepared using random primers and M-MLV reverse transcriptase enzyme. A quantitative real time PCR analysis will be performed to assess the expression of genes encoding for 8 inhibitory and 6 activating KIR genes. Quantitative PCR will be carried out with SYBR Green PCR method. The mRNA expression of different KIR genes will be normalized against GAPDH, and fold change in expression will be determined using ddCt method. Additionally, a flow-cytometry based protein expression analysis for 7 KIRs (4 activating and 3 inhibitory KIRs) on NK cells will also be performed. PBMNCs from each subject for each time point will be stained with fluorochrome labeled antibodies against different KIR molecules and markers specific for NK cells. Cells will be analyzed by multi-color flow cytometry. To quantify the amount of KIR expression on NK cells, we will determine the mean fluorescence intensity (MFI) for each KIR on NK cells.2. To measure degranulation, fluorochrome-conjugated antibody against human CD107a (a surrogate marker of degranulation) will be added to the cells at the start of culture as CD107a is only transiently expressed on T cell surface following activation-induced degranulation and it becomes internalized over time. Monessen will also added to the culture from beginning to ensure blockade of cytokine export from cells to the extra-cellular space and to provide neutral pH that maintains the fluorescence of FITC-conjugated CD107a antibody bound to CD107a, when internalized into the cell. At the end of the culture, cells will be stained with fluorochrome labeled antibodies against CD, CD3, CD56, CD16 and IFNγ and will be analyzed by multi-color flow cytometry. A cytolysis NK cell will be defined as CD3neg CD56dim CD16pos NK cell while a regulatory NK cell will be defined as CD3neg CD56dim CD16pos IFNγpos NK cell. Positivity for IFNγ and CD107a will correspond to the cytokine producing and degranulation functions respectively.c) NK function will be assessed by measuring in vitro cytokine secretion and degranulation by different NK cell subsets (cytolysis and regulatory NK cells) on incubation with K-562 cell lines. PMNCs will be cultured with and without K562 cell in RPMI supplemented with 5% fetal bovine serum, glutamine (2 mM), Penicillin (100 U/ml) and Streptomycin (0.1 mg/ml) at 37°C in a humidified atmosphere containing 5% COd) Assessment of cytokine levels in serum: A total of 30 cytokines including TNF-α, IFN-γ, IL-10, IL-6, IL-1α, IL-1β, IL-1R, IL-2, IL-12, TGF-β IL-15, and IL-17 etc will measured in the serum specimens for each time point using a bead-based array on Luminex.Baseline demographic and health characteristics of the study participants will include demographic characteristics, health record information  and information related to SCT . These questionnaires will be filled out by the physician at baseline, 15, 30, 60, 85 and 180 days after stem cell infusion.Usual dietary intake will be assessed at four time points  using a 3-day dietary record . DietaryQuality of life will be assessed using the Pediatric quality of life inventory (PedsQL) general and cancer module as a self-report, and PedsQL and Behavior Assessment System for Children (BASC-2) as parent proxy report. The PedsQL Measurement Model is a modular approach to measuring QOL in healthy children and adolescents and those with acute and chronic health conditions. The PedsQL Measurement Model integrates seamlessly both generic core scales and disease-specific modules into one measurement system. The 23-item PedsQL Generic Core Scales were designed to measure the core dimensions of health as delineated by the World Health Organization, including physical, emotional, social and school functioning. In addition, the cancer module will be assessed to provide greater measurement sensitivity. The reliability and validity of the PedsQL general and cancer module has been shown in pediatric cancer populations . ParticiThe BASC-2  is a staFatigue will be assessed using the multidimensional PedsQL fatigue scale. The recently developed 18-item PedsQL Multidimensional Fatigue Scale was designed to measure fatigue in pediatric patients and comprises the General Fatigue Scale (6 items), Sleep/Rest Fatigue Scale (6 items), and Cognitive Fatigue Scale (6 items). Reliability and validity of the pediatric multidimensional fatigue scale has been shown in cancer population . ParticiA triaxial accelerometer  will be used as an objective assessment of physical activity levels. Actual measures and records time-stamped acceleration in all directions, providing an index of physical activity intensity. The digitized values are summed over a user-specified interval of 1 min, resulting in a count value per minute (counts per minute). Accelerometer signals are also recorded as steps per minute. This gold standard measure provides a reliable and objective measure of physical activity levels, previously validated and tested for reliability ,27.The activity monitor (AM) will be put on when the participant gets up from bed each day and worn until the participant is ready to fall asleep. Participants can go about their normal, daily activities, including rigorous EX, swimming or bathing. Participants will be instructed to wear the activity monitor over their right hip on an elasticized belt for seven consecutive days. The AM can be worn over or under clothing. Additionally, participants will be asked to record, in a daily log, the time they put on and took off the monitor each day. This protocol and device was recently used in a study to asses physical activity among healthy Canadian children between 6-19 yr . The resThe HRF assessments will be performed by a certified exercise physiologist. Participants will complete the assessments in the following order: resting blood pressure (BP), heart rate (HR) and electrocardiogram; body composition; musculoskeletal fitness ; functional mobility test and cardiorespiratory fitness. Adequate recovery and hydration will be provided, with the total fitness assessment time approximately 2.0 hours.Resting HR will be measured using a heart rate monitor  and resting BP (mmHg) will be measured following the Fourth Report published by the U.S. Department of Health and Human Services . All par2), respiratory exchange ratio (RER), and Respiratory Quotient (RQ) will be recorded.All the children will be verbally encouraged during the test. Gas-exchange data will be measured in a mixed metabolic cart  and specific pediatric face-masks . The duration of the test, the maximal load reached (velocity and slope), and the maximal distance walked will be recorded. In addition, parameters including ventilation (VE), ventilatory equivalent of carbon dioxide (VCO2-1) and end-tidal pressure of oxygen (PetO2) with no increase in the VCO2 while RER remained below 1.0 . Estimated children’s fat-free mass (FFM) will be calculated from the following equation (1): FFM = body mass – fat mass (FM), where FM = 4.95 x Body density (BD) x body mass. Children’s BD will be estimated from skin fold thickness at the triceps, biceps, sub scapular, and suprailiac area. In all subjects, each skin fold will be obtained in duplicate by the same researcher using standard equipment  and the mean value per skin fold utilized for statistical analysis [Standing height and body mass will be measured. We will measure standing height to the nearest 0.1 cm with a clinical stadiometer , while children stand barefoot. Body mass will be measured to the nearest 0.01 kg using a balance scale  with the subjects in their underwear and will calculate body mass index (BMI) as weight height [mass (kg)/height  . The cli2. Exercise, health-related fitness, quality of life and fatigue questionnaires. The specific objectives of this study are to examine the effect of an EX program on (a) QOL and fatigue outcomes across the SCT process and immediately after discharge, (b) to examine the associations between HRF indices, physical activity and QOL and fatigue outcomes.Pediatric cancer patients undergoing SCT have an elevated risk for poor QOL and fatigue both during treatment and into survivorship, however this issue has to be studied in depth . Some evSystematic reviews support the promising role of EX as a safe and effective intervention to improve HRF and quality of life and fatigue outcomes in children with cancer ,4. Howev3. The effects of an EX intervention on body compositionThe aim of this study is to determine if an early EX intervention during SCT will (a) avoid loss of body weight. Survivors of pediatric patients who went undergoing a SCT are at an increased risk to have less lean mass and an excess of fat mass compared with healthy peers .Given the scarcity of literature on the effect of an EX program on the immune system recovery, QOL, physical activity levels and HRF measures for pediatric cancer patients undergoing SCT, the SCORE study will generate new knowledge and be instrumental in developing future clinical and community-based programming for this population. This will be the largest known study in children undergoing autologous SCT and will provide us with valuable information for a future multisite study. This research could add biologic plausibility to the association between EX and immune system recovery after autologous SCT that may identify new targets for interventions, and inform clinical recommendations for improving survival. Ultimately, this project will enable us to achieve more effective targeted interventions that help pediatric cancer patients achieve healthy levels of PA and HRF and with the hope of optimizing disease and treatment related outcomes.SCORE, (Stem Cell patients Ongoing Recovery through Exercise); SCT, Hematopoietic stem cell transplantation; QOL, Quality of life; EX, Exercise; RCT, Randomized controlled trial; NK, Natural Killer; KIR, Killer immunoglobulin like receptor; HRF, Health-related fitness; PBMNC, Peripheral blood mononuclear cell; DNA, Deoxyribonucleic acid; MFI, Mean fluorescence intensity; PedsQL, Pediatric quality of life inventory; BASC-2, Behavior assessment system for children; AM, Activity monitor; BP, Blood pressure; HR, Heart rate; VE, Ventilation; VCO2, Ventilatory equivalent of carbon dioxide; VT, Ventilatory threshold; RER, Respiratory exchange ratio; RQ, Respiratory quotient; PetO2, End tidal pressure of oxygen.The entire authors listed in this manuscript declare that they have no competing interests.CC-V conceived the study and participated in its design and coordination as well as helped draft the manuscript. GG participated in the design of the study, facilitated the in-hospital setting and helped to draft the manuscript. FK was responsible for choosing the methods to carry out the immune system assessment. Additionally he will carry out the immunoassays, data analysis of immune system assessment and he helped to draft the manuscript. KM as a research nurse who will help us in the recruitment of the participants and ensure the entire participant will have done the blood draw properly. She helped to draft the manuscript. FS will participate in the analysis of the quality of life and fatigue questionnaires. AW will participate in the development of the exercise intervention and will help coordinate testing times. She also helped to draft the manuscript. TW will be responsible to perform and interpret the health-related fitness assessments. RR will be responsible for analyzing the 3 day dietary intake records. NCR as a PI will be responsible to oversee the study and data analysis and all subsequent manuscripts and dissemination of the work. She also conceived and participated in the design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/401/prepub


The elevated MYC levels in colon cancer cells are attributed in part to ß-catenin/TCF4 transcription complexes that are assembled at proximal Wnt/ß-catenin responsive enhancers (WREs). Recent studies suggest that additional WREs that control MYC expression reside far upstream of the MYC transcription start site. Here, I report the characterization of five novel WREs that localize to a region over 400 kb upstream from MYC. These WREs harbor nucleosomes with post-translational histone modifications that demarcate enhancer and gene promoter regions. Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops. The chromatin loops are not restricted to colon cancer cells, but are also found in kidney epithelial and lung fibroblast cell lines that lack de-regulated Wnt signaling and nuclear ß-catenin/TCF4 complexes. While each chromatin loop is detected in quiescent cells, the positioning of three of the five distal enhancers with the MYC promoter is induced by serum mitogens. These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that ß-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells.Inappropriate activation of  MYC gene is frequently over-expressed in numerous cancers including up to 80% of colon carcinomas The c-Myc transcription factor (MYC) is a critical regulator of cellular growth, proliferation and apoptosis APC) gene, are found in most cases of colon cancer MYCMYC expression in colon cancer cells through two proximal Wnt/ß-catenin responsive enhancers (WREs) MYC transcription start site (TSS) and was identified using a screen for genes whose expression was negatively regulated by APCMYC transcription stop site and was identified using a functional genomics screen for ß-catenin binding sites MYC expression in response to serum mitogens MYC gene expression in colon cancer cells.The Wnt/ß-catenin signaling pathway controls cell fate decisions and tissue architecture in the gastrointestinal (GI) tract MYC expression. An approximately 500 kb span of chromosome 8q24 is a hot spot for single nucleotide polymorphisms (SNPs) that confer risk to colon, prostate and breast cancers MYC promoter MYC can influence MYC expression levels.A careful analysis of data generated by several genome-wide association studies led to the identification of another WRE that regulates MYC gene expression however, was readily detected MYCMYC. These findings suggest that the gene desert region of chromosome 8 is prone to forming chromatin loops and that additional enhancer elements within this region might align with the MYC promoter through long-range interactions.Two recent studies have expanded upon the idea that the gene desert on chromosome 8q24 harbors additional enhancer elements and that these enhancers may loop to distal target genes. Jia et. al. profiled a 5 Mb segment of chromosome 8 for RNA expression, histone modifications and RNA polymerase two (RNAP) binding MYC transcription start site. I show that nucleosomes that co-localize with the ß-catenin/TCF4 binding sites contain histones that are decorated with modifications on their amino-terminal tails that are known to demarcate both enhancer and promoter elements. Using chromatin conformation capture (3C), I find that these novel WREs are positioned next to the MYC promoter through large chromatin loops. Surprisingly, these loops are not restricted to colon cancer cells, but are also found in cells that lack ß-catenin/TCF4 chromatin associated complexes. The interactions between three of the five distal enhancers with the MYC promoter were induced by serum mitogens. Together these data suggest that long-range chromatin loops position distal enhancer elements to the MYC promoter and that this conformation is poised to recruit ß-catenin/TCF4 complexes to regulate MYC expression in colon cancer cells.In the present study, I characterize five novel ß-catenin/TCF4 binding sites that were identified using a ChIP-Seq screen for ß-catenin binding regions in the human HCT116 colon cancer cell line 2.HCT116 human colon cancer cells (ATCC number CCL-247) and HEK293 cells were cultured as previously described 7 cells and were conducted as reported with some modifications MYCAntibodies used in ChIP assays included: 3 µg of anti-ß-catenin , 3 µg of anti-TCF4 , 5 µl of anti-RNAP , 3 µg of anti-TBP , 3 µg of anti-RNAPS5 , 5 µl of anti-H3K4me3 , 5 µl of anti-H3K4me 1 , 3 µg of anti-H3K9,K14 Ac  and 2.5 µl of anti-PanCT H3 . ChIP assays contained 1×10The sequential ChIP assays were conducted as described in Shang et. al. with some modifications The ß-catenin ChIP-Seq screen in HCT116 cells is described in Bottomly et al. RNA extraction, cDNA synthesis and real-time PCR were conducted as described http://rsbweb.nih.gov/ij/).HEK293 or HCT116 cells were suspended in 0.5 ml of Radio-Immunoprecipitation Assay (RIPA) buffer  containing protease inhibitors . The cells were lysed by brief sonication using a Misonix XL-2000 liquid processor . The lysates were clarified by centrifugation for 10 min at 14,000 rpm and the protein content was quantified using protein assay reagent . For each sample, 20 µg of protein was subjected to polyacrylamide gel electrophoresis and the proteins were transferred to a nitrocellulose membrane. Standard western blot methodology was used to visualize proteins. Antibodies used in the analysis included anti-c-Myc 9E10 , 1∶250 dilution; and anti-ß tubulin , 1∶1000 dilution. Band intensities were quantified using ImageJ software (6 HCT116 cells using an Amaxa nucleofector II device.The dominant negative TCF4 (pcDNAdnTCF4) was obtained from Addgene (kindly deposited by Dr. B. Vogelstein). 10 µg of pcDNAdnTCF4 or a GFP expressing plasmid were electroporated into 5×10in vitro were then mixed at equal molar concentrations. This mixture was serially diluted and used as a control in the quantitative real-time PCR reactions. The 2 ΔCt method was used to represent amplified products where 10−8 ng of TOPO plasmids harboring control PCR products served as the reference. Control experiments were conducted as described and it was confirmed that all 3C DNA products analyzed in this report required the addition of Hind III and ligase to the reaction Chromatin conformation capture was conducted as described with minor modifications c-Myc proto-oncogene (MYC). In addition to the well-characterized 5′ and 3′ proximal WREs, at least seven putative binding sites localized to regions far upstream of the MYC transcription start site  with ß-catenin specific antibodies. These assays were conducted in HCT116 colorectal cancer cells that contain abundant levels of nuclear ß-catenin and, as a control, in HEK293 cells that contain much reduced levels of nuclear ß-catenin ectively . Only maMYC promoter. To determine whether TCF4 was recruiting ß-catenin to these sites, a dominant negative TCF4 plasmid (dnTCF4) was introduced into HCT116 cells and ß-catenin ChIP assays were conducted 24 hours later. TCF4 protein produced from this plasmid lacks the ß-catenin interaction domain and its expression should therefore compete with the formation of functional ß-catenin/TCF4 complexes on elements A through E ß-Catenin recruitment to enhancer elements in colon cancer cells occurs predominantly through interactions with the T-cell factor family member TCF4 MYC promoter in both cells as MYC is expressed in each cell type  and tri-methylated at lysine 4 (H3K4me3) were analyzed by ChIP. H3Ac is commonly found at both enhancer and promoters of expressed genes whereas H3K4me3 preferentially localizes to gene promoters promoter . IncreasMYC allele, or two distinct populations of alleles (or cells) may have been interrogated in the ChIP assays. Sequential ChIP assays were conducted to distinguish between these possibilities. First standard ChIP assays were performed to isolate H3K4me3-associated chromatin. The captured complexes were eluted from the H3K4me3 antibody, diluted in ChIP buffer and then re-precipitated with antibodies against H3K4me1 or antibodies against H3Ac. Irrelevant IgG was used as control in the second precipitation to monitor non-specific interactions. Results from sequential ChIP assays clearly indicated that H3K4me3 and H3K4me1 or H3Ac modifications reside on the same allele for each of the distal enhancers A through E  although interactions between regions on distinct chromosomes (in trans) have been described MYC enhancers and the MYC promoter are depicted in MYC promoter is used as “bait” to test for specific interactions with Hind III fragments adjacent to enhancers A through E (the “preys”). For each PCR reaction, the forward primer anneals upstream of the distal enhancer and the reverse primer, which is common to each reaction, anneals downstream from the Hind III site in the MYC promoter. Interactions between the MYC promoter and enhancers A, C and D were readily detected in HCT116 cells  is a powerful technique used to identify potential long-range interactions between distal elements 16 cells . InteracC assays , Ctrl. SMYC promoter were dependent on chromatin-associated ß-catenin/TCF4 complexes, 3C assays were next conducted in HEK293 cells. HEK293 cells lack ß-catenin and TCF4 binding to enhancers A through E  RNAs were isolated from HCT116 and HEK293 cells and cDNAs were synthesized using reverse transcriptase. Real-time PCR with primers specific to the MYC gene was used to detect MYC expression. Error bars represent SEM. (B) Proteins isolated from each cell type were separated by SDS-PAGE and analyzed by western blot using antibodies specific for MYC or ß-tubulin. (C) Relative MYC protein levels in HCT116 and HEK293 cells. MYC and ß-tubulin bands in B were quantified using ImageJ software.(EPS)Click here for additional data file.Table S1PCR primer sequences, chromosomal positions of ß-catenin ChIP-Seq peaks and the positions of consensus TCF4 motifs within the ß-catenin bound peak regions.(XLSX)Click here for additional data file.


In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.We have placed six copies of a TCF/Lef responsive element and an TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The  Wnt signaling is a key, evolutionarily conserved, cellular signal transduction pathway required and reiteratively used for diverse biological functions. Precise regulation of pathway activity is required for proper embryonic development, and in adulthood, for tissue homeostasis. By contrast, impaired Wnt signaling activity can lead to embryonic defects and disease progression. Wnt proteins encompass a large family of secreted glycoproteins that trigger their outcome through different downstream cascades, among them the canonical Wnt/ß-catenin pathway, which activates transcription of target genes by the stabilization and nuclear localization of ß-catenin, a transcriptional co-activator protein.T cell-specific transcription factor/lymphoid enhancer-binding factor 1 (TCF/Lef) family of transcription factors induces the transcription of downstream genes  and LRP5/6, Dishevelled (Dvl) is recruited by Fz leading to LRP5/6 phosphorylation and Axin recruitment. Loss of Axin from the degradation complex dismantles the complex and releases ß-catenin. Once stabilized, ß-catenin translocates to the nucleus. As a transcriptional coactivator, ß-catenin together with the TOPFLASH design [n), which together with a minimal promoter (min)promoter, drive expression of a reporter gene. Thus, in principle, such n-promotermin:reporter TCF/Lefconstructs label cells that are actively transducing a Wnt signal.Over the past fifteen years several transgenic mouse strains have been established to monitor Wnt/ß-catenin pathway activity during development, homeostasis and disease progression (reviewed in ). These H design . They con-promotermin:reporter TCF/Lefconstructs, or derivative mouse strains, facilitate single-cell resolution imaging of Wnt/ß-catenin pathway activity that can be quantified in live as well as in fixed tissues.As a validation of their utility, several variant Wnt/ß-catenin reporter mouse strains have been characterized, are readily available and have been used to determine Wnt signaling status in a broad spectrum of applications. First generation constructs usually comprised a LacZ reporter (e.g. ), whereaTCF/Lef response elements and the hsp68 minimal promoter in a configuration identical to previously reported TCF/Lef-LacZ reporter mice [TCF/Lef:H2B-GFP mouse strain.We therefore sought to generate an improved, third generation, Wnt/ß-catenin reporter, that would incorporate a bright fluorescent reporter which could be live imaged at single-cell resolution and also quantified, but which would withstand fixation and therefore could also be visualized in tissue sections. To do so, we designed a reporter construct that combined the Wnt/ß-catenin signaling read-out efficiency of multimerized TCF/Lef DNA binding sites with the single-cell resolution and quantifiable reporter expression afforded by fluorescent histone fusions. Fluorescent proteins fused to histones, for example human histone H2B, are localized to the nucleus because they remain bound to chromatin, and as such allow the visualization and tracking of individual cells. H2B fusions also provide details of cell divisions, including the plane of division and identification of daughter cells. They also reveal nuclear fragmentation which is often associated with cell death ,10. We pter mice . We thenTCF/Lef:H2B-GFP reporter expression was independent of integration site. Characterization of the TCF/Lef:H2B-GFP strain of mice revealed bright single-cell resolution reporter expression that spatio-temporally recapitulated TCF/Lef-LacZ reporter expression during mouse embryonic development. Moreover, given its improved sensitivity, the TCF/Lef:H2B-GFP strain revealed additional sites of reporter expression, in the visceral endoderm and epiblast of the pre-gastrula stage mouse embryo, tissues suggested through genetic and expression analyses to possess active Wnt/ß-catenin signaling, that is not reflected by existing Wnt/ß-catenin signaling reporters.We recovered several founder transgenic lines which exhibited equivalent expression, demonstrating that TCF/Lef:H2B-GFP reporter currently represents an improved tool for imaging the in vivo processes triggered by canonical Wnt signaling pathway activation.In summary, we have generated a transgenic mouse strain that serves as a quantitative, non-invasive single-cell resolution read-out of Wnt/ß-catenin signaling in the mouse. The TCF/Lef-LacZ reporter lines, since both have been extensively used in recent years as reliable read-outs of canonical Wnt activity. In the BAT-gal transgene construct, the promoter elements driving reporter expression consist of 7 TCF/Lef binding sites together with a 130 bp fragment containing the minimal promoter-TATA box of the gene siamois [TCF/Lef-LacZ construct was generated by fusing six TCF/Lef response elements upstream of the hsp68 minimal promoter  founder animals produced identical patterns of reporter expression in F1 progeny. We then compared the pattern of expression of the TCF/Lef:H2B-GFP reporter with that observed in stage-matched embryos of the related TCF/Lef-LacZ strain  the embryo implants into the maternal uterus. This event is followed by lineage expansion, which results in formation of a cup-shaped egg cylinder. Close apposition of epiblast, extraembryonic endoderm and visceral endoderm (VE) facilitates signaling cross-talk between these layers, leading to the formation of the distal visceral endoderm (DVE), migration of the anterior visceral endoderm (AVE) and establishment of the anterior-posterior axis of the embryo . CanonicTCF/Lef:H2B-GFP embryos GFP reporter expression was detected specifically in the VE tissue layer around the time of DVE specification (E5.5), and throughout the period encompassing AVE migration (E5.75). VE-specific localization was confirmed by colocalization with the pan-VE marker HNF4α [Hex:GFP transgenic embryos  stage embryos Figure  confirmeIn the data depicted in Figure We selected 4 cells that did not divide during the time-lapse as a reference set  from the pluripotent epiblast, and the elaboration of the axes . In the mouse, the onset of gastrulation is marked by the appearance of the primitive streak (the source of mesoderm and definitive endoderm) which represents a morphologically-distinct structure which breaks the bilateral symmetry of the epiblast, and in doing so, defines the posterior of the embryo at E6.5. The site of primitive streak formation has been proposed to be regulated by Nodal and Wnt3 signaling activities likely emanating from the overlying visceral endoderm -20. ThisTCF/Lef:H2B-GFP reporter expression reproduced Wnt3 early expression in the proximal epiblast before the onset of gastrulation (pre-streak (PS) stage, ~E6.0) and later on, in early streak  stage embryos, became restricted to the posterior side, where single cells within the epiblast became positive for the reporter and could be visualized as they underwent gastrulation traversing the primitive streak , the level of streak-specific expression of the H2B-GFP reporter increased, and by the time the streak had fully elongated and reached the distal tip of the embryo, H2B-GFP fluorescent cells could be seen along the posterior body axis  stages, a second population of H2B-GFP-positive cells located in the proximal part of the conceptus within the extra-embryonic region emerged. These small patches of H2B-GFP-positive cells resembled the pools of primitive erythroid cells, or "blood islands", a site at which Wnt signaling has been proposed to play a role in specifying hematopoietic cell populations  robust levels of transgene expression were evident along the presomitic mesoderm and newly formed somites Figure . This stogenesis -26. By togenesis .GFP expression was maintained in the neural tube, somites, heart, nasal and pharyngeal region  were established both exhibiting similar patterns and levels of reporter expression. However, only one line (TCF/Lef:H2B-GFP #61) was further characterized in detail. Subsequent generations exhibited Mendelian transgene inheritance, stable transgene activity and comparable levels of reporter expression. Animals were maintained in accordance with National Institute of Health guidelines for the care and use of laboratory animals and under the approval of the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.To generate the onstruct  were insTCF/Lef-LacZ [TOPGAL [Embryos were dissected in DMEM/F12 (1:1) containing 5% calf serum and fixed in 4% paraformaldehyde. Presomitic embryos were staged by morphological landmarks according to Downs and Davies ; somite Lef-LacZ  and TOPG [TOPGAL  reporterex utero culture embryos were dissected in DMEM/F12 (1:1) containing 5% calf serum and cultured in medium comprising 50% DMEM/F12 (1:1), 50% rat serum and 1% Penicillin/Streptomycin [For ptomycin .For vibratome sections, embryos were embedded 0.5% gelatin/15% BSA/20% sucrose  and 4.5% glutaraldehyde in PBS and cut on a Leica vibrating microtome (VT1000) at 35-175 μm. For cryosections, samples were equilibrated in PBS/10% sucrose followed by PBS/30% sucrose overnight and snap-frozen in OCT (Tissue-Tek). Sections were cut in a Leica cryostat at 12 μm and counterstained with Hoechst for nuclei  and Phalloidin Alexa546 for F-actin (Invitrogen).2, 100 mM sodium phosphate pH 7.3) for 20 min and incubated in 1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(Cn)6, 0.02% NP-40, 0.01% Sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate pH7.3 overnight. They were finally rinsed with PBS and fixed for 30 min in 4% paraformaldehyde.Mouse embryos were dissected in PBS, fixed for 20 min in 0.35% glutaraldehyde in PBS and processed as whole mounts or embedded in the gelatin/albumin mix and sectioned. Samples were washed 3 times with X-Gal rinse , anti-Eomes (abcam), anti-GFP (Invitrogen), anti-HNF4a (Santa Cruz), and anti-Oct4 (Santa Cruz). Secondary Alexa Fluor (Invitrogen)-conjugated antibodies were used at a dilution of 1/500. DNA was visualized using Hoechst 33342 .http://www.improvision.com/ and a Perkin-Elmer RS3 Nipkow-type scan head mounted on a Zeiss Axiovert 200 M with Hamamatsu C4742-80-12AG camera. GFP was excited using a 488 nm Argon laser. Images were acquired using a Zeiss plan-Neofluar 25×/0.8 DIC korr objective. 10-20 xy planes were acquired, separated by 3-4 μm. Time intervals between z-stacks were 15 minutes. For live imaging experiments, embryos were maintained in a humidified, temperature-controlled chamber with 5% CO2 atmosphere. Sections were mounted in Fluoromount-G (Southern Biotech) and imaged through glass coverslips. Confocal images were acquired as z stacks of xy images taken at 1 μm z -intervals.Widefield and epifluorescence images were acquired using a Zeiss Axiocam MRc camera coupled to a Leica M165FC dissecting scope or a Zeiss A1 Axioscope. Laser scanning confocal data were acquired using a Zeiss LSM510 META using a PlanApo 20×/NA0.75 objective. Fluorophores were excited using a 405 nm diode laser (Hoechst), 488 nm Argon laser  or 543 nm HeNe laser (Alexa 546). Embryos were imaged whole mount in MatTek dishes (Ashland). Spinning disc confocal 3D timelapse data was acquired using Volocity acquisition software http://www.jax.org/imr/index.html will be distributing the TCF/Lef:H2B-GFP mouse line. The provisional reference name for the strain is: STOCK Tg(TCF/Lef1-HIST1H2BB/EGFP)61Hadj/J.The Jackson Laboratory Induced Mutant Resource TCF/Lef:H2B-GFP reporter strain, and wrote the manuscript. AP - carried out experiments to characterize the mouse TCF/Lef:H2B-GFP reporter strain. GT - carried out experiments to generate TCF/Lef:H2B-GFP constructs. RJA - performed image data analysis. DD - provided reagents making this study possible. AKH - conceived, designed, funded and supervised the project and wrote the manuscript. All authors critically read and revised the manuscript, and approved the final version.AFV - carried out experiments to characterize the mouse 3D time-lapse movie of E5.5 TCF/Lef:H2B-GFP embryos. Rendered images of 3D time-lapse data acquired on a spinning disc confocal (A-D) of a litter of embryos obtained from Tg/+ TCF/Lef:H2B-GFPx ICR (wild type) cross. Half the embryos were hemizygous for the transgene. Embryos were dissected at E5.5 and exhibited variability in stage, which resulted in variability of the level of the fluorescent reporter and its localization. The time-lapse experiment was carried out for over 9 hours.Click here for fileTCF/Lef:H2B-GFP embryosTracking H2B-GFP reporter expressing cells in the visceral endoderm of . Registered rendered images focusing on a Tg/+ TCF/Lef:H2B-GFP embryo which remained in the field of view for the duration of the 3D time-lapse experiment depicted in the previous movie (A-D). Duration of time-lapse experiment was 9 hours 46 minutes and 21 seconds (t = 9:46:21). Individual cells identified by H2B-GFP nuclear-labeling were color-coded  and tracked using the spots function in Imaris . The movie consists of 37 frames each depicting one time-point. The first frame (t = 0) depicts the initial state, with 4 non-dividing cells used as a reference for both position and distance. At frame 15 (t = 3:48:02), the first tracked cell division occurs below the bottom reference cell. Thereafter, tracked cell divisions occur in frames 21, 23, 25, 27, 28, 31, 33 and 37. Throughout the observation period, nearest-neighbor relationships are preserved, despite the substantial growth of the embryo. The change in the angle between the reference cells, as well as the distance between them, suggests that circumferential  expansion of the embryo is greater than the proximal-distal  growth. Scale bar: 30 μm depicted in lower left.Click here for file


RNF43 is regulated by the Wnt-signaling pathway. However the regulatory region(s) for its transcriptional activation has not been clarified. In this study, we have shown for the first time that RNF43 is a direct target of TCF4/β-catenin complex, and that its expression is regulated by a regulatory region containing two Wnt-responsive elements (WREs) in intron2. A reporter gene assay revealed that nucleotide substitutions in the WREs decreased the reporter activity in colon cancer cells, suggesting that both WREs are involved in the transcriptional activation. Knockdown of β-catenin by siRNA suppressed the reporter activity. In addition, ChIP assay showed that both elements associate with TCF4/β-catenin complex in colon cancer cells. These data indicate that expression of RNF43 is regulated by the canonical Wnt/β-catenin pathway through binding of the WREs with TCF4/β-catenin complex. These findings should be useful for the understanding of the regulatory mechanism of RNF43 and may contribute to the clarification of signaling pathways regulated by RNF43.RING finger protein 43 (RNF43), an E3-type ubiquitin ligase, is frequently up-regulated in human colorectal cancer. It has been shown that expression of  In the US, it is estimated that 142,820 of new cases will be diagnosed and that 50,830 patients will die of this disease in 2013 CTNNB1 and AXIN1c-myccyclinD1MMP-7 (matrilysin) urokinase-type plasminogen activator receptor (uPAR) connexin 43CD44PPAR-deltaAF17ENC1Laminin-5 γ2Claudin-1MT1-MMPIn colorectal cancer cells, frequent mutations are observed in RNF43 mutations were identified in a subset of pancreatic cancer RNF43 was frequently enhanced in colorectal cancer as well as hepatocellular carcinomas RNF43 expression was also elevated in adenomas of the colon RNF43 was induced by Wnt3a conditioned media RNF43 is a downstream gene regulated by the Wnt-signaling pathway, but none has clarified the regulatory regions of its expression. In this study, we identified two Wnt-responsive elements (WREs) in intron2 of RNF43 and found that these WREs are crucial for its transcriptional regulation through interaction with Tcf4/β-catenin complex. This is the first report of RNF43 as a direct target of Tcf4/β-catenin complex and our data may be useful to understand the precise mechanism of RNF43 regulation.Earlier assigned as a hypothetical protein FLJ20315, RNF43 was shown to be an ubiquitin E3 ligase that associates with a nuclear protein, HAP95 Human colorectal cancer cell lines, HCT116 and SW480, were obtained from the American Type Culture Collection . HCT116 cells were cultured in McCoy’s 5A medium containing 10% fetal bovine serum  and antibiotic/antimycotic solution . SW480 cells were cultured in Leibovitz’s L-15 medium containing 10% FBS and antibiotic/antimycotic solution.CTNNB1-specific siRNA were purchased from Dharmacon . ON-TARGETplus Non-targeting Pool (D-001810-10) was used as a control. HCT116 or SW480 were seeded a day before treatment of siRNA. Cells were transfected with 15 nM of CTNNB1-specific or control siRNA using Lipofectamine RNAiMAX (Life Technologies). After 48 hours incubation, total RNAs were isolated with miRNeasy Mini Kit  according to the manufacture’s instruction. The silencing effect was evaluated by quantitative RT-PCR and western blotting. Complementally DNA was synthesized from 1 µg of total RNA with Transcriptor First Strand cDNA Synthesis Kit . Real-time PCR was performed using SYBR Green technology with sets of primers  for RNF43 on StepOnePlus (Life Technologies). Amounts of transcripts were determined by relative standard curve method, and GAPDH was used as internal control.Human ACGCGTCTACAGGGGAAACAATGTTGAAGGTCAATAGGCT-3′5′-AAA, and reverse primer, CTCGAGTGGCCAGGTTTCTAGGCCCACTGC-3′5′-AAA or CTCGAGTGGCAAAGAGAATGCCAACTGGTGCTGT-3′5′-AAA, containing a recognition site of MluI or XhoI restriction enzyme (underlined nucleotides). PCR products were digested with the restriction enzymes and cloned into the appropriate enzyme sites of pGL3-Basic vector . In addition, putative intronic enhancer region was amplified by PCR with sets of primers . PCR products were digested with the restriction enzymes, and cloned into the appropriate enzyme sites of pGL3-Promoter vector (Promega). Site-directed mutagenesis was carried out for both putative TCF4 binding sites, replacing CTTTGWW by CTTTGGC with the QuickChange II XL Site-Directed Mutagenesis Kit  according to the manufacture’s instruction.Putative promoter regions in the 5′-flanking region of RNF43 were amplified by PCR with two sets of primers  at the concentration of 15 nM and incubated for an additional 36 hours. The cells were harvested and luciferase activities were measured using dual luciferase assay system .HCT116 or SW480 cells seeded on six-well plates were transfected with 1 µg of reporter plasmid and 0.1 µg of pRL-TK plasmid (Promega) by FuGENE 6 reagent (Roche) and incubated for 12 hours. Then the cells were further transfected with 5′-TCAACTCTCTGGATAAGGTGGAATAGC-3′, and reverse primer, 5′-GACTTTTGGGGTGGGTGGGAAATA-3′; RNF43-int2-3′, forward primer, 5′- TCGGGCACCTGGCCAAGATACA-3′, and reverse primer, 5′- TGGACGCCCTGGCTTCTGAG-3′. Specificity of the assay was determined by the amplification of a 5′-flanking region located from −4861 to −4768 of RNF43 transcriptional start site using the following primers; forward primer, (−4861) 5′- CAAGGCTAGTCTGCCTCCAG-3′, reverse primer, (−4768) 5′- AGCGCTTTCCAAAGGAGGAA-3′. In addition, the amplifications of c-Myc (MYC) enhancer was used as a positive control .A ChIP assay was performed according to the Agilent Mammalian ChIP protocol with slight modifications. HCT116 or SW480 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature and quenched with 0.4 M glycine. Chromatin extracts were sheared by Micrococcal nuclease digestion, and subsequently protein-DNA complexes were immunoprecipitated with 3 µg of anti-TCF4 monoclonal antibody  or anti-β-catenin monoclonal antibody  bound to anti-mouse IgG-coated Dynabeads (Life Technologies). Non-immune mouse IgG  was used as a negative control. The precipitated protein-DNA complexes were purified with the conventional DNA extraction method, and the DNAs were subjected to quantitative PCR analysis with the following primer sets; RNF43-int2-5′, forward primer, RNF43 is regulated by the Wnt/β-catenin pathway, we measured expression levels of RNF43 with or without β-catenin siRNA in HCT116 and SW480 cells .To confirm that 80 cells . HCT116 se cells . Consistse cells . RNF43 pRNF43, we constructed three forms of reporter plasmids  containing the 5′-flanking region and intronic regions of RNF43. RNF43-5′-1 contained approximately 1.6 kb , RNF43-5′-2 approximately 2.5 kb  and RNF43-5′-3 approximately 5.1 kb  of RNF43, respectively  between –274 and −268, and between –54 and −48 of TSS, respectively. The reporter plasmids were transiently transfected with or without β-catenin siRNA in HCT116 cells, and luciferase activities were measured. As a result, we found that the luciferase activity of the cells transfected with RNF43-5′-1 or RNF43-5′-2 was significantly higher  than that with empty vector, and that the activity of RNF43-5′-3 was similar to the empty vector (To identify the regulatory element(s) of Wnt-signaling in ectively . These ry vector . Howevery vector . These dRNF43 in public databases. A search in the ChIP-seq data of the ENCODE project , identified four TCF4-enriched regions in the RNF43 gene; one between −517 and +100 of TSS, two in intron2, and one in intron3. The two regions in intron2, but not the one in intron3, overlapped with RNA pol II-enriched regions. It is of note that these three regions were enriched with histone H3K4 mono-methylation. On the other hand, the region between –517 and −100 of TSS was enriched with pol II, histone H3K4 tri-methylation, and histone H3K27 acetylation, but not with histone H3K4 mono-methylation. Therefore we focused on the two regions in intron2 and tested whether they encompass TCF4-mediated transcriptional enhancer(s).We next searched for putative regulatory regions in We then carried out a reporter assay using plasmids (RNF43-int2) containing a genomic region of 4.2 kb  encompassing the two TCF4-enriched regions in intron2. As we expected, the plasmids showed approximately 5-fold higher activity than empty promoter vector in HCT116 cells . Importa5′-CTTTGAA-3′ and 5′-CTTTGAT-3′, which were termed putative Wnt-responsive element 1 and 2 (WRE1 and WRE2), respectively . Although the luciferase activities of RNF43-int2-A and RNF43-int2-B were decreased at 57% and 65% of RNF43-int2, respectively, their activities were significantly higher than the control plasmids. In addition, co-transfection of β-catenin siRNA significantly suppressed both reporter activities  in the regions; ectively . To clartivities . We furttivities . Comparetivities . Similartivities . Since cRNF43.To confirm whether TCF4 and β-catenin associates with WRE1 and WRE2, we conducted ChIP assays with TCF4-specific or β-catenin-specific antibody in HCT116 cells. Immunoprecipitation and subsequent quantitative PCR analysis revealed that the regions containing WRE1 and WRE2 were enriched by 7.3-fold and 28.1-fold with TCF4-specific antibody, respectively . Of noteRNF43 is a direct target gene of Tcf4/β-catenin complex. Our initial challenge to identify regulatory region(s) using reporter assay containing the 5′ flanking region successfully showed that the region is involved in its transcriptional activation, but failed to find region(s) associated with Wnt/β-catenin signaling. Subsequent search of the ENCODE database helped us to explore candidate regions that may interact with Tcf4. Regarding the decrease of reporter activity with RNF43-5′-3 plasmids containing the 5′ flanking region, intron 1, and a part of intron 2, the 3′ region of intron 1 and/or the 5′ region of intron 2 may have a repressive element(s) for the transcription.In this study, we have identified regulatory regions of RNF43 transcription and showed that et al. found a total of 6,868 enriched regions using tiling array RNF43 , this peak was close to WRE2  suggesting a consistency of transcription factor-binding regions detected by ChIP-on chip in spite of different cell lines they used. They also reported that the Tcf4-binding sites are distributed along the genome, and 18% of them were located in intragenic regions further than 10 kb from the TSS. Consistently, the two WREs of RNF43 were located at approximately 22 kb downstream of TSS. In addition, their data unveiled that 31% of peaks were located in the 5′-flanking region at a great distance from TSSs. In the case of c-Myc, WREs localized to a region over 400 kb upstream from the gene are involved in chromatin looping in response to the activation with serum mitogens Genome-wide approaches including ChIP-on-Chip and ChIP-seq analyses have helped to illustrate a global association map of transcription factors, chromatin occupancy, and histone modifications. As for the Tcf4-interacting regions, Hatzis In addition to Tcf4, genome-wide approaches including serial analysis of chromatin occupancy (SACO) and ChIP-seq have been applied to identify the regions interacting with β-catenin In this study, we incorporated the data of histone modifications and occupancy of RNA pol-II. Consistent with the view that histone H3K4 mono-methylation and an interaction with RNA polymerase II are the hallmarks of transcriptional enhancers, the two WREs in intron2 played a vital role as a transcriptional enhancer in our luciferase assay. Meanwhile, the 5′-flanking region of 2.4 kb was associated with RNA polymerase II and histone H3K4 tri-methylation suggesting that this region served for the constitutive transcriptional activation through the remodeling of heterochromatin complex to euchromatin state. Although the ENCODE data depicted a peak of Tcf4-binding in the 5′-flanking region, enrichment of multiple transcription factors was observed in the same region. Therefore the peak of Tcf4 may be a false positive. Alternatively Tcf4 may interact with that region without the recruitment of β-catenin. Since the data of histone modifications and RNA pol-II occupancy are useful information to predict the chromatin structure of interacting regions and their transcriptional activity, future studies on a global association map of β-catenin and Tcf4 with histone modifications will gain an insight into the dynamic transcriptional regulation played by Tcf4/β-catenin and chromatin modification enzymes such as Brg1, TRRAP, TIP60, CBP/p300, and MLL AXIN2, and DKK1, which are also downstream targets of the canonical Wnt signaling pathway and negatively regulate the signals in different manners RNF43 are supposed to abrogate Wnt signaling including canonical and non-canonical pathways. However, the effect of negative feedback by the enhanced expression of RNF43 has not been clarified in colorectal or liver cancer. Although the augmented transcriptional activity of Tcf4/β-catenin complex by inactivation mutations in APC or AXIN2, or activating mutations in CTNNB1 may not be affected by the suppression of frizzled receptors, complex network of signaling pathways may render undetermined characteristics to colorectal and liver cancer cells, as restoring expression of SFRP4 and DKK1 in colorectal cancer cells attenuates Wnt signaling Recently, it was reported that RNF43 and ZNRF3, transmembrane E3 ubiquitin ligases, selectively ubiquitinate frizzled receptors and targets them for degradation. RNF43 and ZNRF3, a homologue of RNF43, are highly conserved in vertebrates, and associate in the membrane with frizzled receptors and low density lipoprotein receptor-related proteins, LRP5/6. RNF43 promotes endocytosis of frizzled receptors including FZD1 and FZD3, and suppresses Wnt/β-catenin responses


The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling. TherePathways induced by Wnt ligands are highly evolutionarily conserved, with recognizable Wnt signaling pathways seen in animals as primitive as Hydra . Given tThis review will summarize our current knowledge of the mechanisms underlying Wnt signal transduction see  and discAbbreviations: Wls/Evi (Wntless/Evenness interrupted); LRP5/6 (low density lipoprotein-related proteins 5/6); Dvl (Dishevelled); GSK-3 (glycogen synthase kinase 3); Apc (adenomatous polyposis coli); Ck1α (casein kinase 1α); JNK2 ; TCF (T cell factor - highly related to LEF1 or lymphoid enhancer factor); SOST (sclerostin).Wnt ligands are produced in the endoplasmic reticulum, lipid modified by the action of Porcupine, and then trafficked to the surface in a Wntless/Evi-dependent manner. Once secreted, they activated signaling in target cells via engaging a member of the Frizzled family and either LRP5 or LRP6. In the absence of a Wnt ligand, β-catenin is phosphorylated on serine and threonine residues near its N-terminus, targeting it for ubiquitin (Ub)—dependent proteosome-mediated degradation. In the presence of an upstream Wnt signal, the multi-protein complex required to target β-catenin for degradation is localized to the cell membrane via an interaction between Axin and the phosphorylated (P) C-terminus of Lrp5 or Lrp6. This inhibits the GSK3-dependent phosphorylation of β-catenin, stabilizing its levels in the cytoplasm. A concurrent signal is transmitted that results in the phosphorylation of other serine residues near the C-terminus of β-catenin, which facilitates its nuclear entry. At least in some contexts, this event is mediated by JNK2. Once in the nucleus, β-catenin interacts with a member of the LEF/TCF family of DNA binding proteins, leading to release of transcriptional repressors like Groucho proteins from target genes and the recruitment of various co-activators to the site. The net result is activation and/or repression of target gene transcription. The pathway is regulated by several other inhibitors including extracellular proteins such as Dickkopfs, secreted Frizzled-related proteins (SFRPs), sclerostin (SOST), and Wise/SOSTDC1. In addition to the pathway controlling the regulation of β-catenin stability and nuclear localization, Wnt ligands can also activate so-called “non-canonical” pathways. One example of this is the ability of Wnt5a to bind to the transmembrane receptor ROR to transduce downstream signals. 2.2.1.Wnt1, was originally isolated during analysis of chromosomal insertion sites enriched in murine mammary tumors induced by mouse mammary tumor virus (MMTV) [Drosophila melanogaster. The foundation for this work was provided by the pioneering work of Nusslein-Volhard and Wieschaus, which identified and categorized a number of genes required for embryonic development in Drosophila [Drosophila Wingless gene, which had been identified as being required for segment polarity in Drosophila, was a close homolog of the int-1 protein [Wingless gene had been identified as a component required for segment polarity in Drosophila allowed for epistasis analyses to order many components within the pathway. After subsequent work in Drosophila [Xenopus axis induction [Int1 gene as Wnt1 (a combination of Wingless and Int1), with all subsequently identified homologous genes carrying the Wnt name [There are 19 Wnt genes found in humans. Wnts are cysteine-rich glycoproteins that share a high degree of sequence homology. The prototypic Wnt gene, s (MMTV) . The gens (MMTV) . The proosophila . Subsequosophila  and othenduction ), a consWnt name .2.2.Wnt1 gene was first identified in 1982, it took until 1996 to identify a putative receptor [Drosophila had shown that Wnt secretion requires the activity of the acyltransferase Porcupine [Drosophila embryonic development and causes accumulation of Wingless protein within the endoplasmic reticulum [Several characteristics of Wnt proteins made their biochemical purification challenging. Thus, while the receptor -15 and ureceptor . Some ofreceptor  and palmreceptor . Earlierorcupine -20. Losseticulum . In addieticulum -24. Wntleticulum . More reeticulum .Once secreted, Wnt ligands remain tightly associated with the extracellular matrix, with a particularly strong affinity for heparin sulfate proteoglycans -29. In a2.3.+-ATPase [Wnt ligands initiate signaling pathways via engagement of several types of cognate receptors. These signaling pathways are often referred to as “canonical” and “non-canonical” pathways, although classifying Wnt ligands neatly into these categories may not be advisable . The so-+-ATPase -45. Fina+-ATPase -53.2.4.The phosphorylation of the cytoplasmic tail of Lrp6 leads to the recruitment of the scaffolding protein Axin to the receptor complex ,54. Thiset al. [Once β-catenin enters the nucleus, it interacts with members of the LEF/TCF family of DNA binding proteins to bind to specific promoter targets . β-catenet al. .2.5.In addition to regulating the cytoplasmic stability of β-catenin, Wnt-induced inhibition of GSK3 can also directly regulate the activity of the mTOR pathway . In the Recent work has also greatly increased our knowledge regarding pathways downstream of Wnt ligands that are independent of GSK3 regulation. As stated previously, these diverse pathways have historically been referred to as “non-canonical” Wnt signaling. These include pathways that control the activation of protein kinase C (PKC), protein kinase A, the small GTPase RhoA, and several others . In some3.3H-TdR DNA labels throughout long periods of time, which means that +4 cells are either in a prolonged quiescent state or are in a slow-cycling state. In addition, the +4 cells are extremely sensitive to radiation [The intestinal epithelium is regenerated throughout adult life with robust proliferation. The mammalian intestine is lined by a monolayer of epithelial cells (or mucosa), which contains a crypt compartment at the base maintained by multipotent stem cells and a villus compartment facing the lumen. In the colon, there is a flat surface epithelium instead of a villus compartment. The crypt contains a group of undifferentiated and actively dividing cells with stem cell properties that will eventually invaginate into the mucosa to form crypts and villi ,71. Epitadiation ,73. Ther3.1.in vitro [It is well accepted that Wnt/β-catenin signaling plays a key role in the homeostasis and maintenance of intestinal stem cells ,71. Therin vitro . Lgr5 main vitro . A recenin vitro . Therefo3.2.APC) gene is a well-known tumor suppressor that plays a central role in the Wnt signaling pathway by targeting β-catenin for degradation. Germline loss-of-function mutations in the APC gene were originally identified to be associated with familial adenomatous polyposis (FAP), about 1% of which progress to CRC [The majority of colorectal cancer (CRC) is caused by mutations in key components of the Wnt signaling pathway -85. The s to CRC -88. Furts to CRC . Althougs to CRC .Min/+ mice are so-called because the heterozygous APC mutation induces multiple intestinal neoplasias, while homozygous APCMin/Min mice die at gastrulation. The APCMin/+ mouse was originally identified in chemical mutagenesis screens and proved to carry a premature stop/truncation mutation in the Apc gene [Min/+ mice raised on a high fat diet developed adenomas throughout the intestinal tract and mostly died by 120 days of age. The presence of multiple lineages in the adenomas of APCMin/+ mice suggests that tumorigenesis may be initiated in a multipotent stem cell [Several mouse models of FAP and intestinal carcinoma have been generated by inducing mutations at different amino acid positions in APC -93. The Apc gene ,93. The tem cell .et al. showed that Apc inactivation in Lgr5-positive stem cells at the crypt bottom could lead to transformation within days [Min/+ mice attenuates intestinal tumorigenesis [Until recently, it was not clear which cell type sustained the cancer-initiating mutation, but due to the well-determined stem cell markers of the intestine, significant progress has been made in identifying the origin of intestinal adenomas induced by activated Wnt signaling. Critical work by Barker hin days . In conthin days . These shin days . Furtherigenesis ,98. OverSFRP) is hypermethylated often in CRC, and this epigenetic silencing contributes to overactive Wnt signaling, as evidenced when reintroduction of SFRP to colon cancer cells reversed Wnt signaling [SFRP by silencing the gene that induces its promoter methylation. Another example is the inhibition of DACT3 (a member of the Dpr/Frodo family) methylation. DACT3 is a negative regulator of Wnt signaling that has its expression suppressed in CRC via histone methylation and deacetylation [Many studies have shown that inhibition of Wnt signaling can reverse tumorigenic properties of CRC cells. The inhibition of the β-catenin interaction with its transcription factor TCF in colon cancer cells caused an arrest of the cell cycle in the G1 phase ,100. Theignaling . This sttylation . When DA4.hiCD29hiCD24+Sca1- subpopulation [hiEpCAM- subpopulation [The mammary gland is a complex organ that undergoes the majority of development postnatally and extensive tissue remodeling during reproductive estrous cycles. These unique features, in addition to the ability to perform epithelial reconstitution assays, have allowed a more thorough analysis of stem cell differentiation within the mammary gland. Recent studies have identified several mammary stem cell (MaSC) surface markers that have laid the groundwork for understanding the hierarchical organization of the mammary epithelium. In mice MaSCs are highly enriched in the CD49fpulation -105, whepulation ,107. In 4.1.i.e., Wnt1, Wnt2, Wnt3, Wnt3a, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt10a, Wnt10b, Wnt11), receptors , and downstream DNA-binding proteins  [Several studies in mice have revealed the integral role of Wnt signaling in the development of the normal mammary gland. Numerous components of the Wnt signaling cascade are expressed during embryonic mammary morphogenesis, including Wnt ligands (nd Lef1) . Mammarynd Lef1) . Embryosnd Lef1) -112. Theet al. have shown that Wnt2, Wnt5a and Wnt7b expression is enriched in the terminal end bud microenvironment [At the time of birth, the rudimentary gland consists of ducts containing two differentiated cell types, an inner layer of luminal epithelial cells, which secrete milk, and myoepithelial cells, which are located on the basal surface of the luminal cells adjacent to the fat pad. Most of the development of the mammary gland takes place postnatally and depends on cues from the hormonal and local microenvironment. In juvenile animals, the tips of growing ducts have a club-shaped structure known as the terminal end bud, which is enriched in stem cells. The hormonal environment stimulates active proliferation of the terminal end bud and induces invasion of the surrounding adipose tissue to form complex branching structures. Werb ironment . Knockouironment ,114. Conironment . Given tironment . These sDuring estrous cycles, pregnancy, and lactation, there is an expansion of the mammary epithelium, and during involution, extensive remodeling reduces the gland to a pre-pregnancy state. The hormonal and local factors that orchestrate these changes during development are still marginally understood. In the luminal epithelium of the mammary duct, cells are either positive or negative for both the estrogen receptor α (ERα) and progesterone receptor (PR), while the basal, myoepithelial cells express neither receptor . Despite4.2.Int-1 (Wnt1) locus, and overexpression of Wnt1 induced mammary tumorigenesis [et al. have demonstrated that distinct Wnt signaling components affect distinctive mammary cell populations [et al. demonstrated that aberrant Wnt signaling alters MaSC populations during tumorigenesis [Studies in both mouse models and human breast cancers have revealed that Wnt signaling is critical to mammary tumorigenesis. Wnt signaling was first implicated in mammary tumors when the mouse mammary tumor virus (MMTV) was found to integrate into the igenesis ,121. Mouigenesis . In addiulations . MMTV-ΔNigenesis . An expaigenesis . Using tet al. have demonstrated that overexpression of Lrp5 also correlates with basal breast cancers [CTNNB1 (the gene encoding β-catenin) [In spite of the strong evidence for Wnt signaling in mouse mammary tumor models, there are conflicting reports on the importance of Wnt activation in human breast cancer. Numerous reports have identified dysregulation of the Wnt pathway in breast cancer -130; how cancers . Down-re cancers -137. In catenin) ,139. TheStudies in breast cancer cell lines have shown that stem cell populations are more resistant to radiation treatment and that Wnt/β-catenin signaling mediates resistance -142. Rec5.The function of the adult human prostate gland is to secrete proteolytic enzymes into the semen to facilitate conception. The prostate (which surrounds the urethra at the base of the urinary bladder) develops out of the embryonic urogenital sinus (UGS), a derivative of the endoderm layer. Testosterone production begins in the human fetus about 8 weeks into gestation, and at 10-11 weeks, solid epithelial buds begin to extend and branch into a complex series of ducts that form the adult prostate and open into the urethra . The anaPSA), transmembrane protease serine 2 (TMPRSS2), and others [Full development of the prostate depends on the presence of androgens, which are secreted by the testes during puberty. Androgens bind to and induce the homodimerization and nuclear translocation of the androgen receptor (AR), thereby causing expression of target genes such as prostate-specific antigen  [in vitro and in vivo [Other Wnt pathway members are dysregulated in prostate cancer. Frizzled-4  is co-expressed in human prostate tumor samples with the ETS-related gene (ERG) . FurtherERG) , and ind in vivo . Also, w in vivo . Further in vivo .PB) has been widely used to control Cre recombinase expression and subsequently activate or inactivate genes flanked by loxP regions [APC) is mutated and hypermethylated to a silent form, and β-catenin is also frequently mutated to an active form [Apc gene was inactivated (referred to as Cre/PB+flox/floxApc) resulted in prostatic hyperplasia and adenocarcinoma [Cre/PB+lox(ex3)Catnb), and adenocarcinoma also developed in these mice [Conditional mouse models have shown that aberrant Wnt signaling in the prostate is tumorigenic. The prostate epithelial-specific gene for Probasin , which are prostate cancer cells with stem-like properties ,183-186.6.As discussed thus far, Wnt signaling is active in intestinal, mammary, and prostate cancers, which consist primarily of epithelial tissue. Wnt has also been implicated in skin cancer, lung cancer, bladder cancer, and leukemia, and it may be oncogenic in other cancers ,191-193.6.1.NSAIDs reportedly have some anti-cancer effects, particularly in colon cancer, and it is widely believed that the mechanism is related to the inhibition of cyclooxygenase (COX) activity. However, this may only be part of the answer, as COX and Wnt signaling appear to be linked . It was 6.2.Vitamin D  may protect against some cancers. Colon cancer xenograft studies, in which nude mice were treated with EB1089, have shown that tumors decreased in volume due to an increase of apoptosis -201. The6.3.et al., one that inhibited Wnt1-Lrp6 interactions and one that inhibited Wnt3a-Lrp6 interactions [et al. also discovered two novel Lrp6 antibodies that inhibited Wnt3- and Wnt3a-Lrp6 interactions [Endogenous Wnt inhibitors such as sclerostin, DKK1, Wise, and Mesd bind to and inactivate the Wnt co-receptors Lrp5 and Lrp6 ,205,206.ractions . These aractions . When Wnet al. has done a great deal of work with Wnt1 and Wnt2 antibodies in various cancers [in vitro  and in vivo  [in vitro in lung cancer, mesothelioma, and malignant melanoma [Jablons  cancers -213. Wntibodies) ,213. Thimelanoma -212. Alsmelanoma . Other Wmelanoma .6.4.c-myc and p21/CIP1), and this interaction is one such target [Several Wnt pathway members have been targeted for inhibition via small molecule inhibitors. β-catenin interacts with the TCF transcription factor to induce expression of target genes . Giv


The Wnt/β-catenin signaling pathway is crucial for human organ development and is involved in tumor progression of many cancers. Accumulating evidence suggests that the expression of β-catenin is, in part, regulated by specific microRNAs (miRNAs). The purpose of this study was to determine the expression of a recently identified epithelial to mesenchymal transition (EMT)-associated tumor suppressor microRNA (miR)-200a, in cancer cells. We also aimed to identify specific miR-200a target genes and to investigate the antitumor effects of miR-200a on the Wnt/β-catenin signaling pathway. We employed TOP/FOP flash luciferase assays to identify the effect of miR-200a on the Wnt/β-catenin pathway and we confirmed our observations using fluorescence microscopy. To determine target genes of miR-200a, a 3′ untranslated region (3′ UTR) luciferase assay was performed. Cell viability, invasion and wound healing assays were carried out for functional analysis after miRNA transfection. We further investigated the role of miR-200a in EMT by Western blot analysis. We found fluctuation in the expression of miR-200a that was accompanied by changes in the expression of members of the Wnt/β-catenin signaling pathway. We also determined that miR-200a can directly interact with the 3′ UTR of CTNNB1 (the gene that encodes β-catenin) to suppress Wnt/β-catenin signaling. MiR-200a could also influence the biological activities of SGC790 and U251 cells. Our results demonstrate that miR-200a is a new tumor suppressor that can regulate the activity of the Wnt/β-catenin signaling pathway via two mechanisms. MiR-200a is a candidate target for tumor treatment via its regulation of the Wnt/β-catenin signaling pathway. Constitutive activation of the Wnt/β-catenin signal pathway promotes uncontrolled cell growth and survival, and can consequently drive cancer formation ,2. DysreOur previous review on the Wnt/β-catenin pathway described multiple genes involved in its regulation, with special focus on the function of miRNAs. Several miRNAs have been found to be regulators, either as oncogenes or tumor suppressor genes that regulate the activity of the Wnt/β-catenin pathway . MiR-2002. The miRNA mimic, miRNA inhibitor and negative control were synthesized by GenePharma . For transfection, trypsinized cells were plated in 6-well plates at 2-3×105 cells per well. MiRNA transfections were performed using Lipofectamine 2000 . For each well, miRNA (100 pmol) in 250 μl of serum and antibiotic-free medium was mixed with 5 μl of Lipofectamine 2000 in 250 μl of the same medium and allowed to stand at room temperature for 20 min. The mixture was then added to cells and after 4 h the medium was changed to complete medium.Human stomach adenocarcinoma cell lines, SGC7901 and U251, were obtained from the Laboratory of Neuro-Oncology, Tianjin Neurological Institute. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). All cultures were maintained at 37°C in a humidified atmosphere containing 5% COThe mimic and inhibitor sequences were: miR-200a mimic sense: 5′-UAA CAC UGU CUG GUA ACG AUG U-3′; anti-sense: 5′-AUC GUU ACC AGA CAG UGU UAU U-3′; negative control sense: 5′-UUC UCC GAA CGU GUC ACG UTT-3′; anti-sense: 5′-ACG UGA CAC GUU CGG AGA ATT-3′; miR-200a inhibitor: 5′-ACA UCG UUA CCA GAC AGU GUU A-3′; inhibitor negative control: 5′-CAG UAC UUU UGU GUA GUA CAA-3′.Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the standard protocol. A nanodrop spectrophotometer (Gene) was used to detect the concentration of total RNA. Total RNA (1 μg) was used to synthesize cDNA by reverse transcription using MMLV reverse transcriptase , according to the manufacturer’s instructions. Real-time PCR analysis was performed to determine the abundance of miR-200a in SGC7901 and U251 cells 48 h after transfection with miR-200a mimic or inhibitor or scrambled negative control. The expression of u6 was used as an internal control. We performed qRT-PCR for 40 cycles, comprising 95°C for 10 min, 95°C for 15 sec, 65°C for 30 sec, 72°C for 30 sec and an extension at 72°C for 10 min.Real-time PCR analysis was also performed to determine β-catenin mRNA levels and data were normalized to GAPDH, which was used as an internal control. Both reverse transcription and qRT-PCR primers were purchased from GenePharma.XbaI and FseI sites of the pGL3 control vector  and were named pGL3-CTNNB1 and pGL3-CTNNB1-mt, respectively.TOPflash and FOPflash reporters contain wild-type (WT) and mutated TCF-4 consensus binding sites, respectively, and are widely used to evaluate β-catenin-dependent signaling events that drive the expression of TCF. These reporters have been described previously . The wil5 cells/well) were plated in 24-well plates 1 day prior to transfection. The miR-200a mimic/inhibitor transfection was performed according to the Lipofectamine 2000 instructions (Invitrogen), and 48 h after reporter plasmid transfection, luciferase activity was measured using a luciferase reporter assay system (Promega).Cells  three times and were lysed for 30 min on ice in RIPA buffer in the presence of a proteinase inhibitor cocktail, then centrifuged at 12,000 rpm for 15 min at 4°C. Proteins were harvested and 40 μg from each sample was subjected to SDS-PAGE separation, and then transferred to a PVDF membrane . The membrane was incubated with primary antibodies against β-catenin, ZEB1, ZEB2 , E-cadherin, N-cadherin, Tcf-4, Fra-1, MMP-9 and Cyclin D1 , followed by incubation with an HRP-conjugated secondary antibody . Specific proteins were detected using a SuperSignal protein detection kit . The membrane was stripped and re-probed with a primary antibody against GAPDH .Twenty-four hours after transfection, cells were plated on glass cover slips and 48 h post transfection the cover slips were washed extensively in phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS. After additional washing, the cells were permeabilized with 1% Triton X-100 in PBS for 10 min. The cover slips were then washed and blocked with 1% BSA for 30 min. Cells were incubated in the appropriate primary antibodies (β-catenin or TCF-4) overnight at 4°C. Samples were then washed and incubated with species-specific secondary rhodamine-labeled antibodies (TRITC) in PBS (1:100 dilution) for 60 min. Nuclei were stained with DAPI at RT for 10 min and cover slips mounted with Antifade solution prior to imaging on a confocal microscope .5) or U251 cells (1×105) were seeded in 6-well plates. When cell confluence reached about 90%, approximately 24 h post-transfection, an artificial homogenous wound was made onto the monolayer using a sterile plastic 200 μl micropipette tip. After wounding, debris was removed by washing cells with PBS. At different time points, cells that migrated into the wounded area or cells with extended protrusions from the wound border were photographed at ×200 magnification under a light microscope.Cell culture and transfection conditions were optimized to ensure a homogeneous and viable cell monolayer prior to wounding. One day before transfection, equal numbers of SGC7901 cells .The top chamber of a transwell chamber was incubated with 60 μl Matrigel diluted with DMEM  at 37°C for 30 min. The Matrigel solidified and acted as the extracellular membrane (ECM) for tumor cell invasion analysis. Transfected cells were trypsinized, adjusted to 5×10Forty-eight hours after transfection, cells were trypsinized and collected by centrifugation, washed in PBS and fixed with 75% ethanol overnight at 4°C. Cells were then washed twice with PBS, and incubated with 200 μl RNase A (1 mg/ml) at 37°C for 30 min. Cells were then stained in the dark with 800 μl propidium iodide staining solution for 30 min at 4°C. Analysis was performed on a FACSCalibur flow cyto-meter .Cells were seeded in a 96-well plate at a density of 3000 cells per well, and 24 h before transfection, cells were incubated with 20 μl MTT solution (5 mg/ml). At 24, 48, 72, 96, 120, 144 and 168 h following transfection at 37°C for 4 h, the solution was aspirated, and 200 μl DMSO was added to each well. The Optical density (OD) was measured at a wavelength of 570 nm. The data are presented as the mean ± SD, which are derived from triplicate samples of at least three independent experiments.2 test was used to analyze the significance between groups. The LSD method of multiple comparisons with parental and control vector groups was used when ANOVA showed statistical significance. Statistical significance was determined at the level of p<0.05.A commercially available software package, SPSS16.0, was used for statistical analysis. One-way analysis of variance (ANOVA) and the χTo monitor the expression of miR-200a in target cells, the miR-200a mimic, inhibitor and scrambled control were delivered into SGC7901 and U251 cells. The level of miR-200a expression was then examined 48 h after transfection by qRT-PCR. Expression of miR-200a was up-regulated by approximately 40-fold in cells transfected with the miR-200a mimic. Meanwhile, in cells transfected with the miR-200a inhibitor, miR-200a expression was reduced by about 80% compared with control . These rRecent studies have reported that miR-200a targets the mRNA of the E-cadherin repressor proteins, ZEB1 and ZEB2. This results in an increase in the level of E-cadherin available for binding to β-catenin and induces formation of the cell-cell adhesion complex . We consWe also used the miR-200a inhibitor to down-regulate the expression of miR-200a in SGC7901 and U251 cells. Using luciferase assays, Western blot analysis and fluorescence microscopy we showed that Wnt/β-catenin signaling activity was negatively correlated with the level of miR-200a .It is well known that the Wnt/β-catenin pathway has essential functions in the regulation of cell growth and differentiation. Here, we used Western blot assays to investigate the expression of some downstream targets of Wnt/β-catenin signaling, such as Fra-1, Cyclin D1 and MMPs –14 Fig.. These rWe next investigated a functional outcome for the miR-200a-mediated suppression of β-catenin/Wnt signaling. The expression level of miR-200a clearly influenced the biological activity of SGC7901 and U251 cells. Therefore, we investigated two major biological activities of tumor cells, namely migration and invasion potential and growth ability. Migration and invasion potential are important biological characteristics of malignant tumor cells. Representative micrographs of wound healing assay and of transwell filters are shown in The proliferation rate of variously transfected SGC7901 and U251 cells was measured using the MTT assay. The miR-200a mimic group proliferated at a significantly lower rate than the other groups. Cell cycle analysis confirmed these results. The miR-200a mimic led to G0/G1 entry, while the miR-200a inhibitor blocked G0/G1 entry .MicroRNA-200a, a member of the miR-200 family, stands out as an inhibitor of EMT. Direct evidence for the EMT-inhibitory actions of miR-200a has been revealed in several cancer cell lines, including nasopharyngeal carcinoma, endometrial serous adenocarcinomas, bladder cancer and meningiomas –18. The CTNNB1 (the gene which encodes β-catenin) has been suggested to be a target of miR-200a ,20. AccoRecently, ZEB1 and ZEB2 have both been suggested to be targets of miR-200a, and miR-200a was demonstrated to cause changes in E-cadherin expression ,21,22. TThis study highlights two different mechanisms by which miR-200a regulates the activity of β-catenin. First we showed that miR-200a can inhibit the activity of the Wnt/β-catenin signaling pathway. Then we found that miR-200a can regulate the expression of β-catenin through not only direct interaction with the 3′ UTR of CTNNB1, but also via interaction with the cadherin/catenin complex. In an effort to further investigate the effect of miR-200a on the phenotype of tumor cells, we used epithelial tumor cell lines, SGC7901 and U251, and found that over-expression of miR-200a significantly inhibited SGC7901 and U251 cell growth, invasion and induced G0/G1 phase arrest, while reduced expression of miR-200a promoted tumor cell growth and invasion. The induction of apoptosis by inhibiting the activity of the Wnt/β-catenin signaling pathway in tumor cells was not detected in our studies and further investigations are required to explain the role of miR-200a in tumor cells.et al (As the most common primary brain tumor, gliomas account for more than 70% of all primary central nervous system neoplasms. Accumulating evidence suggests that aberrant activation of Wnt/β-catenin signaling is involved in glioma development and progression . We demoet al  showed tet al ,26, so oWe identified for the first time in gastric adenocarcinoma SGC7901 cells that over-expression of miR-200a reduced levels of ZEB1, ZEB2 and N-cadherin and increase E-cadherin levels. ZEB1 and ZEB2 are transcription factors that activate EMT by binding to E-box elements present in the E-cadherin promoter, suppressing transcription. While E-cadherin has a widely acknowledged role in cell-cell adhesion, it also functions as an invasion suppressor protein. During EMT E-cadherin is down-regulated, while N-cadherin is induced and β-catenin is released from junctional complexes and is translocated to the nucleus –29. The In summary, β-catenin is an important functional target for miR-200a in gastric adenocarcinoma SGC7901 cells and glioblastoma U251 cells. MiR-200a regulates the activity of β-catenin through two kinds of mechanism , and up-