Oridonin (ORI) can inhibit proliferation and migration in various types of cancer cell lines. However, the exact mechanism remains unclear. We investigated the migration inhibitory effect of ORI on human pancreatic cancer SW1990 cells and dissected the possible molecular mechanism(s).CCK-8 assay was used to observe the cell viability. Wound healing assay, transwell assay and spontaneous metastasis model were used to observe the migration activities. Real-time PCR, immunofluorescence, western blot analysis and immunohistochemistry methods were used to observe the expression of genes or proteins.6 SW1990 cells into nude mice intraperitoneally to test whether ORI affects tumour metastasis. Metastatic formation was inhibited by ORI (5 and 10 mg/kg) compared with the control group. Tumour sections stained with anti-E-cadherin, anti-vimentin and anti-β-catenin antibodies revealed that ORI inhibited EMT, as well as the Wnt/β-catenin pathway in vivo.ORI inhibited the migration of SW1990 cells. Real-time PCR and immuno-fluorescence analyses of epithelial-to-mesenchymal transition (EMT) markers were compared between control group and ORI group. The expression of mesenchymal molecular markers, such as vimentin, snail and slug decreased. The expression of epithelial-related marker E-cadherin increased. Wnt/β-catenin signalling was inhibited by ORI using luciferase reporter assay. ORI can decrease the β-catenin protein level not only in the nucleus, but also in the cytoplasm and the whole cell after the treatment with ORI and glycogen synthase kinase 3β (GSK3β) was increased in the ORI-treated group. CHIR could attenuate the effects of ORI in SW1990 cells. We established a mice model by injecting 1 × 10ORI can inhibit pancreatic cancer cell SW1990 migration and EMT by down-regulating Wnt/β-catenin signal transduction in vitro and in vivo. Therefore, it can be potentially and effectively used in the clinical management of pancreatic cancer. Pancreatic cancer, which is one of the most common cancers of the digestive system, is a leading cause of morbidity and mortality in both developed and developing countries. Pancreatic cancer is characterised by a covert anatomical structure, aggressive biology, resistance to conventional therapeutic agents, and no early detectable biomarkers were observed . ConsequThe epithelial-to-mesenchymal transition (EMT) is among the most critical processes that occur during the progression of tumour metastasis. EMT-induced adenocarcinoma cells can acquire malignant features, such as invasion, metastatic capabilities and chemo-resistance , 4. In hRabdosia rubescens, has anti-inflammatory, antibacterial and antitumour effects ) method. Each plate was run in triplicate.Total cellular RNA was extracted using TRIzol (Invitrogen) reagent. The reverse transcription was performed with 2 μg of total RNA, using an oligo (dT) primer and other reagents, and cDNA was synthesized using the Revert Aid First-Strand cDNA Synthesis kit  according to the manufacturer’s protocol. Real-time PCR was carried out using the miScript SYBR Green PCR kit . The cycle conditions for the PCR reaction were as follows: 95 °C for 15 min followed by 40 cycles of 60 °C for 20 s, and 72 °C for 40 s. The expression of each RNA was normalized to that of the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The specific primer pairs used in this PCR reaction were: E-cadherin (NM_004360.3), forward: 5′-ACAGCCCCGCCTTATGATTCTC-3′, reverse: 5′-AAGCGATTGCCCCATTCG-TT-3′; vimentin (NM_003380.3), forward: 5′-AGATGGCCCTTGACATTGAG-3′, reverse: 5′-CCAGAGGGAGTGAATCCAGA-3′; snail (NM_005985.3), forward: 5′-AATCGGAAGCCTAACTACAGCG-3′, reverse: 5′-GTCCCAGATGAGCATT-GGCA-3′; slug (NM_003068.4), forward: 5′-GTCCGCTCTGCCGCACCTGA-3′, reverse:5′-GCTTGAGGGTCTGAATCTTGCT-3′; GAPDH (BC026907.1), forward: 5′-CTGCACCACCAACTGCTTAG-3′, reverse: 5′-GTCTTCTGGGTGGCAGTGAT-3′. The changes of expression were calculated using the  paraformaldehyde and permeabilized with 0.1 % Triton-X 100 in PBS. E-cadherin , vimentin  and slug  were visualized by sequential incubation with indicated antibody overnight at 4 °C. Pictures were acquired by using a fluorescence microscope (Olympus).40 μg proteins were separated by 10 % sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies against Histone H1 , Non-phospho (Active) β-catenin , β-catenin , GSK-3β, GAPDH  at 4 °C overnight separately, and further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody  for 1 h. Bands were visualized by Western Blotting Reagents .Sub-confluent SW1990 cells were seeded in 24-well plate and transfected with 0.5 μg β-catenin/Tcf4 luciferase reporter (pTop-luc) per well with Lipofectamine (Invitrogen). After incubating for 12 h, cells were treated with 0.1 % DMSO or 15 μM ORI. 24 h later, cells were lysed and subjected to luciferase assays using luciferase assay kit (Promega). Each assay was done in triplicate.6) in 50 μl PBS were injected into the nude mice intraperitoneally. Based on the different treatments, mice were segregated into 3 groups (each group has 5 mice). ORI (5 mg/kg and 10 mg/kg) was injected intraperitoneally every day for 10 days starting from the second day of SW1990 injection. Lungs were dissected and fixed in 10 % formaldehyde after 1 month. Paraffin-embedded lungs were cut for H & E staining.SW1990 cells  and β-catenin (1:200).t test (between two groups) or one-way analysis of variance (ANOVA) (among three or more groups) under the computer software SAS 9.2 . A two-tailed P value <0.05 was considered to be statistically significant.Quantitative results were expressed as mean ± standard deviation (SD). The statistical analysis was performed by using two-tailed unpaired After treatment with different concentrations  of ORI for 12, 24, 36 and 48 h, the proliferation rates of four human pancreatic cancer cell lines  were assessed via CCK-8 kit. As shown in Fig. IC50 of ORI in SW1990 of 24 h was 63.47 μM (Table EMT is an early event in tumour migration. It is related to the structural changes in the intercellular junction, which is essential for migration . We hypoGiven that stabilization and nucleus translocation of β-catenin are key events in transduction of the canonical Wnt/β-catenin signalling, which is highly involved in cancer metastasis, we conducted western blot analysis to explore whether oridonin can suppress β-catenin activity. The results indicated that ORI can decrease the β-catenin protein level not only in the nucleus, but also in the cytoplasm and the whole cell after the treatment with ORI for 24 h Fig. b. LucifeTo further validate the role of β-catenin and GSK3β in the migration inhibitory effect of ORI on SW1990 cells, SW1990 was tested with CHIR, a GSK-3-specific inhibitor. CHIR attenuated the inhibitory function of ORI. WB results indicated that CHIR can reverse the decrease of the β-catenin in the cytoplasm, nucleus and the whole cell Fig. d. As sho6 SW1990 cells into nude mice to test whether ORI affects tumour metastasis. As shown in Fig. We injected 1 × 10Rabdosia rubescens. It exerts immune protective effects in severe inflammatory diseases. ORI has shown remarkable anti-proliferative and pro-apoptotic effects against leukemia and some types of solid tumours [ORI is a tetracycline diterpenoid compound extracted from the traditional Chinese medicinal plant  tumours –23. It a tumours –26. ORI EMT is defined by epithelial cells taking on a mesenchymal phenotype, characterised by loss of apical-basal polarity, increased cellular motility and reorganization of cytoskeleton . There aWnt/β-catenin signalling pathway plays a critical role in various cancers , 38. In Our data suggest that ORI can be effective for human pancreatic cancer. The anticancer effect of ORI in SW1990 cells may result from the suppression of EMT by increasing GSK-3β activity and inactivating Wnt/β-catenin signal transduction. Future studies should be directed to the identification of more ORI target proteins, which are essential to elucidate the molecular mechanisms underlying the anti-cancer capacity of ORI.


Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in some tumors, but its role in hepatocellular carcinoma (HCC) is still unknown.PRMT5 level in HCC specimens was determined by immunohistochemical staining and the association with clinicopathologic features was evaluated. PRMT5 was inhibited by AMI-1  or small interference RNA (siRNA). The proliferation of HCC cells was tested by Cell Counting Kit-8, cell migration was evaluated by Transwell assay and cell cycle and apoptosis were analyzed by flow cytometry. The effect of AMI-1 on HCC in vivo was examined by mouse xenograft model.PRMT5 expression was markedly upregulated in HCC tissues, and correlated inversely with overall patient survival. Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells. Furthermore, β-catenin was identified as a target of PRMT5. Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells. AMI-1 strongly inhibited HCC growth in vivo, increased the ratio of Bax/Bcl-2, and led to apoptosis and loss of migratory activity in several HCC cells. Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5.PRMT5 plays an important role in HCC. PRMT5 may be a promising target for HCC therapy.The online version of this article (doi:10.1186/s12967-015-0721-8) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is the fifth leading cancer and the third most common cause for cancer death worldwide –3. The oProtein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, is localized in both cytoplasm and the nucleus of mammalian cells , 8. PRMTIn this study we aimed to investigate the role of PRMT5 in hepatocarcinogenesis by using in vitro and in vivo models. We showed that PRMT5 protein is overexpressed in HCC tumor tissues, and its elevated level is associated with worse prognosis in patients with HCC. In addition, we found that specific deletion of PRMT5 by small interference RNA (siRNA) or the inhibition of its activity by arginine methyltransferase inhibitor 1 (AMI-1) significantly decreases HCC development. Furthermore, we demonstrate that the deletion of PRMT5 significantly down-regulates the expression of β-catenin and its downstream effector Cyclin D1 in HCC cells.Fifty-four pairs of HCC tissues and corresponding normal adjacent tissues (NATs) were purchased from the National Engineering Center for Biochips . All human materials were obtained with informed consent, and this study was approved by Ethics Committee of Lanzhou University. Four human HCC cell lines , one rat hepatoma cell line CBRH-7919 and one normal liver cell line HL-7702 were purchased from American Type Culture Collection , and cultured under conditions recommended.Tissue arrays were constructed using 54 pairs of HCC tissues and corresponding NATs. Immunohistochemical staining was performed on 4 μm sections of paraffin-embedded human HCC tissues and matched NATs to determine the expression level of PRMT5 protein. Briefly, the slides were incubated with PRMT5 antibody at 4 °C overnight. After extensive washing, the sections were incubated with secondary antibody, followed by staining with DAB . The slides were then washed and counterstained with hematoxylin. A semiquantitative scoring method according to the overall staining of the cells was applied for immunohistochemistry (IHC) , 17.Three different siRNA each were synthesized against human PRMT5 by Ribobio , and the most effective one (target sequence: CCGCTATTGCACCTTGGAA) was selected for the subsequent experiments with test siRNA. Cells were transfected with PRMT5 siRNA  or scramble siRNA  using Lipofectamine 2000  according to manufacturer’s protocol.3 cells per well) in triplicate. After 24 h, cells were treated with si-NC, si-PRMT5 or AMI-1. Then cell proliferation was assessed using the Cell Counting Kit-8  according to the manufacturer’s protocols. The efficacy of the treatment was expressed as best tumor growth inhibition [T/C value was calculated as follows: %T/C = (median tumor weight of treated tumors/median tumor weight of control tumors) × 100. For the colony formation assay, HepG2 and HL-7702 cells were placed in 60 mm dishes at 200 cells per dish and maintained in the absence or presence of si-PRMT5 for 16 days. Cells were fixed with fixative  for 15 min and then stained with crystal violet (0.2 g/L) for 30 min.Cells were seeded in flat-bottom 96-well plates .Cell cycle and apoptosis were analyzed by flow cytometry as described previously , 21. BriCell lysates were separated in 12 % SDS–polyacrylamide gel electrophoresis, and then transferred to PVDF membrane (Bio-Rad). After being blocked with 5 % non-fat milk for 1 h, the membranes were incubated with the following primary antibodies at 4 °C overnight: PRMT5 , PRMT7 , Cyclin D1 , CTNNB1 , H4R3me2s , H3R8me2s , Bax , Bcl-2 , PRMT7  or β-actin , and then incubated with secondary antibodies conjugated to horseradish peroxidase . Immuno-reactivity was visualized using ECL Western blotting detection reagents and then analyzed by densitometry.4 cells per well) were placed in upper chamber of insert in triplicate and medium with 600 µL 10 % FBS was used as chemo-attractant in lower chamber. AMI-1 or vehicle control was added to inner chamber. After 24 h, the cells remaining on the top surface of the membrane were removed with a cotton swab. The cells on the bottom surface of the membrane were fixed in methanol and stained with 0.2 % crystal violet.Cell migration assay was performed in 24-well transwell chambers, 8 µm pore size Transwell plate  , 23. In 6 HepG2 cells were inoculated subcutaneously (s.c) in the right flank of 5–6-week old healthy BALB/c nude mice. When tumors reached an average volume of about 70 mm3, the mice bearing too large or too small tumors were eliminated and the left were randomly divided into two treatment groups : AMI-1 (0.5 mg in 100 µL of 0.9 % NaCl) or 100 µL of 0.9 % NaCl were administered intratumorly (i.t) twice a week. Control animals and animals treated with AMI-1 were sacrificed after 36 days and tumor were excised and weighted. Tumor volumes were calculated according to the following formula: (length × width2) × 0.52.Female athymic BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd  and maintained under specific pathogen-free room with a 12-h on/off light cycle, and fed autoclaved chow and water. Mice were manipulated and housed according to protocols approved by the Institutional Animal Care and Treatment Committee of Lanzhou University. 5 × 10t test. Differences were considered statistically significant as *P < 0.05, **P < 0.01 or ***P < 0.001.Unless otherwise stated, data were presented as the mean ± SD and subjected to student’s P > 0.05, Table To evaluate the clinical significance of PRMT5 in HCC development, we analyzed PRMT5 level and its distribution in HCC tissues by immunohistochemical (IHC) staining. The array included the cancer tissue samples and corresponding normal adjacent tissues (NATs) from 54 HCC patients, which consisted of 48 males and 6 females, with a medium age of 53 years (range 38–72 years). Among the patients 31 (57.4 %) died of tumor related to causes, and 23 (42.6 %) were still alive as of the last follow-up  and one normal liver cell (HL-7702), and then cell proliferation was measured by CCK-8 assay. As shown in Fig. To explore the mechanism by which PRMT5 knockdown inhibits HCC cell proliferation, we performed cell cycle analysis. As shown in Fig. To understand underlying mechanism of cell cycle arrest, the levels of several cell proliferation/cycle-related proteins in si-NC and PRMT5-knockdown HCC cells were analyzed by Western blot analysis. As shown in Fig. Because PRMT5-driven methylation of arginine residues leads to H4R3me2s and H3R8me2s, we then measured H4R3me2s and H3R8me2s in HCC cells treated with si-PRMT5. We found that the levels of H4R3me2s and H3R8me2s were significantly decreased compared with si-NC  activity in vitro . InteresTo further explore the mechanism of PRMT5 action in HCC, Western blot analysis was performed to determine protein levels of Bax and Bcl-2 in HCC cells. The results showed that AMI-1 increased Bax/Bcl-2 ratio associated with apoptosis relative to control cells Fig. c. As shoInhibition of PRMT5 overexpression can induce apoptosis in different types of cancer , 31, 32.PRMT5 emerges as an important regulator of multiple cellular processes and is expressed aberrantly in different types of cancer –15. HoweWnt/β-catenin signaling is frequently activated in HCC, but the causes of its activation are not well understood . CytoplaThere are several strategies to inhibit PRMT5 overexpression in cancer. In this study, we demonstrated that knockdown of PRMT5 by siRNA effectively inhibited HCC cell proliferation and colony formation in vitro, but treatment with siRNA is still in the experimental stage , 40. MorPRMTs are classified as either type I or type II. Both types can catalyze the formation of omega-N monomethylarginine (MMA) as an intermediate, and type I PRMTs  lead to the production of asymmetrical dimethylarginine (aDMA), whereas type II PRMTs (PRMT5 and PRMT7) catalyze the formation of symmetrical dimethylarginine (sDMA) . In thisThese results strongly indicate that PRMT5 is frequently up-regulated and inversely correlated with poor prognosis in HCC patients. PRMT5 might function as an oncogene partly by mediating the repression of β-catenin and its downstream target Cyclin D1 in HCC. Therefore, PRMT5 might be a potential biomarker and a promising therapeutic target for HCC.


Neosartorya pseufofischeri, acted as inhibitors of the Wnt signaling pathway. In addition, we found that GTX downregulated the β-catenin levels in colorectal cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or activating mutations of β-catenin. Furthermore, we demonstrated that GTX induced growth inhibition and apoptosis in multiple colorectal cancer cell lines with mutations of the Wnt signaling pathway. Together, we illustrated a practical approach to identify small-molecule inhibitors of the Wnt signaling pathway and our study indicated that GTX has therapeutic potential for the prevention or treatment of Wnt dependent cancers and other Wnt related diseases.The discovery of new bioactive compounds from marine natural sources is very important in pharmacological research. Here we developed a Wnt responsive luciferase reporter assay to screen small molecule inhibitors of cancer associated constitutive Wnt signaling pathway. We identified that gliotoxin (GTX) and some of its analogues, the secondary metabolites from marine fungus  Inappropriate activation of Wnt signaling has been observed in numerous human cancers ,2. The hAspergillus sp. Plinabulin is shown to be a vascular disrupting agent that elicits tumor vascular endothelial architectural destabilization leading to selective collapse of established tumor vasculature and is currently under clinical trials [Neosartorya pseudofischeri, have previously been shown to exert potential cytotoxic effects on CRC cell lines [Marine-derived fungi has been proven to be a prolific source of secondary metabolites with interesting structural properties and biological activities ,12,13. Fl trials ,15. In rl trials ,17,18,19In this study, we developed a Wnt responsive luciferase reporter assay to screen small molecule inhibitors of cancer associated constitutive Wnt/β-catenin signaling pathway and identified gliotoxin (GTX) and some of its analogues as small molecule Wnt inhibitors. GTX blocked the Wnt/β-catenin signaling pathway and repressed the expression of β-catenin dependent genes. In addition, GTX decreased the β-catenin levels in CRC cells with inactivating mutations of APC or activating mutations of β-catenin. Furthermore, we demonstrated that GTX induced growth inhibition and apoptosis in several CRC cell lines. Together, we illustrated a practical approach to identify small molecule inhibitors of Wnt signaling pathway in the field of marine anti-cancer drug development. Our study demonstrated that GTX has therapeutic potential for the prevention or treatment of Wnt dependent cancers and other Wnt related diseases.We developed a Wnt responsive luciferase reporter assay in HCT 116 cells to identify the small molecule inhibitors of cancer associated constitutive Wnt signaling. HCT 116 cells, which harbor activating mutations of β-catenin, were stably transfected with a luciferase reporter plasmid which contained eight copies of a TCF/LEF response element that drives transcription of the luciferase reporter gene. Luciferase activity was measured to evaluate the modulation of the Wnt/β-catenin transcriptional activity. To increase luciferase assay sensitivity, a single clone with high luciferase expression was chosen to conduct further experiments. Since this system could be used for compound screens, it would be of paramount importance to demonstrate that small molecules were capable of modulating Wnt signaling pathway. Previously, XAV-939, which selectively inhibits β-catenin-mediated transcription, has been identified as an inhibitor of Wnt signaling . TherefoNext we used these HCT 116-Wnt-luc cells to screen small molecule derived from secondary metabolites of marine fungi , which wSince β-catenin is a key mediator of the Wnt pathway, we then tested whether GTX inhibited the Wnt pathway by regulating β-catenin. First, we examined its effect on the expression of β-catenin in mRNA levels by real-time PCR assay. As shown in Since GTX inhibited Wnt signaling in CRC cells, we examined whether this compound could inhibit the proliferation of six CRC cell lines and one non CRC cell line using the colony formation assay. β-catenin expression levels of a number of cell lines were detected by western blot. As shown in To examine the ability of GTX to induce apoptosis in Wnt related cancer cells, HCT 116 (harboring activating mutations of β-catenin), HT-29, and SW620 (harboring inactivating mutations of APC mutations) were chosen for further study. Initially, the capacity of GTX to induce cell apoptosis was evaluated by flow cytometry with Annexin V-FITC/PI dual staining. The proportion of apoptotic cells, in concentration dependent manners, increased in CRC cells a. To furNeosartorya pseudofischeri, exerted potential cytotoxic effect on CRC cell lines [Marine-derived fungi has been proven to be a promising source of bioactive metabolites. In recent years, we used an alternative cultivation parameters strategy to isolate and identify a series of novel and/or bioactive metabolites from marine fungi ,17,18,19ll lines ,23,24,25GTX, an epipolythiodioxopiperazine metabolite with immunosuppressive properties ,27,28 ha2 at 37 °C.Human epithelial colorectal adenocarcinoma HCT 116, SW620, HT-29, HCT-8, Caco-2 and LS174T cells were purchased from Culture Collection of Chinese Academy of Science . Human normal hepatic L-O2 cells were kindly provided by Professor Fang . Cells were cultured in RPIM 1640 medium  supplemented with 10% inactivated fetal bovine serum , 100 units/mL penicillin and 10 µg/mL streptomycin  in a humidified atmosphere of 5% CONeosartorya pseudofischeri by following the procedure described previously [GTX was isolated from the secondary metabolites of marine fungus, eviously . It was Reporter plasmids pRL-TK and pGL4.49 were purchased from Promega . The pGL4.49 vector contains eight copies of a TCF-LEF response element (TCF-LEF RE) that drives transcription of the luciferase reporter gene. Cell transfection used lipofectamine3000  according to the manufacturer’s instructions. Single transfected colonies were picked in the presence of hygromycin B .Luciferase activity assay were performed following the manufacturer’s instructions developed by Promega. In brief, HCT 116 cells stably transfected with pGL4.49 plasmid were seeded and cultured in 24-well plates for 24 h. Cells were incubated with compounds. After 24 h, cell lysate was prepared by employing Luciferase Assay Kit  and luciferase activity was measured using a Thermo Scientific Varioskan Flash Multimode Reader .2 at 37 °C for the indicated times. Medium was changed every three days. The cells were washed with PBS and fixed in ice-cold methanol for 5 min, and stained with crystal violet. Images of the colonies were photographed. Each treatment was evaluated in triplicates, and representative images were shown.Cells were seeded in 6-well plate, with 500 cells per well. The cells were treated with either different concentration of compounds or 0.1% DMSO as vehicle control and cultured in an atmosphere of 5% COTotal RNA was isolated with Trizol reagent  following the manufacturer’s instructions. The first-strand of cDNA was synthesized from 2 μg of total RNA using the PrimeScript RT reagent Kit  and random primers. Real-time PCR was carried out using SYBR Green Premix Ex Taq II Kit  in the ABI 7500 system . Gene expression was normalized to GAPDH and relative quantitation was calculated by using the ΔΔCt method. The specific primers were used as follows: MYC-forward: GGACCCGCTTCTCTGAAAGG, MYC-reverse: TAACGTTGAGGGGCATCGTC, GAPDH-forward: GCACCGTCAAGGCTGAGAAC, GAPDH-reverse: TGGTGAAGACGCCAGTGGA.Western blot was performed as previously described . BrieflyFor cell apoptosis, cells treated with GTX for the indicated times, were collected, washed with binding buffer, then incubated in working solution (100 μL binding buffer with 0.3 μL Annexin V) for 15 min in the dark. Cells were then washed and resuspended with binding buffer. PI  was added just before flow cytometric analysis, apoptotic cells were determined by BD FACScanto II flow cytometry  and the resulting data were analyzed by BD FACSDiva software version 6.1.3 .t test between two groups and by one-way ANOVA followed by Fisher’s LSD test for multiple comparison involved. The Statistical analyses were performed using GraphPad Prism software . A p value of <0.05 was considered to be statistically significant.All experiments were done at least three times. Data were presented as “mean ± SD”. All the data were analyzed by two-tailed unpaired Student’s This is the first study to identify GTX and some of its analogues as potential Wnt pathway inhibitors. Our results show that GTX inhibits Wnt pathway activity by promoting the degradation of β-catenin. In addition, GTX inhibits the proliferation and induces apoptosis in CRC cells which display high Wnt signaling activity. Collectively, these results revealed that the potential of GTX and it analogues as inhibitors for Wnt pathway and provide evidence for their anticancer activity in CRC cells.


Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative, nonmotile, facultative intracellular pathogen. Although glanders has been eradicated from many parts of the world, the threat of B. mallei being used as a weapon is very real. Here we present draft genome assemblies of 8 Burkholderia mallei strains that were isolated in Turkey. Burkholderia mallei is a host-adapted bacterium that is not known to persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East, and Central and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent . Turkey 10 strain had Illumina short- (ca. 300-bp) and long-insert  paired data; all the other strains had Illumina short-insert paired data and Roche 454 long-insert paired data. Short- and long-insert paired data were assembled together in both Newbler and Velvet, and computationally shredded into 1.5 kbp overlapping shreds. All data were additionally assembled together in Allpaths , 7. EachGenome assemblies ranged from 12 to 302 contigs and 2 to 15 scaffolds . As expeAccession numbers for all 8 genomes are listed in


The aetiology of PD is poorly understood but work performed over the last two decades has identified a growing number of genetic defects that underlie this condition. Here we review a growing body of data connecting genes implicated in PD—most notably the PARK genes—with Wnt signaling. These observations provide clues to the normal function of these proteins in healthy neurones and suggest that deregulated Wnt signaling might be a frequent pathomechanism leading to PD. These observations have implications for the pathogenesis and treatment of neurodegenerative diseases in general.Wingless/Int (Wnt) signaling pathways are signal transduction mechanisms that have been widely studied in the field of embryogenesis. Recent work has established a critical role for these pathways in brain development, especially of midbrain dopaminergic neurones. However, the fundamental importance of Wnt signaling for the normal function of mature neurones in the adult central nervous system has also lately been demonstrated by an increasing number of studies. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide and is currently incurable. This debilitating disease is characterized by the progressive loss of a subset of midbrain dopaminergic neurones in the  Individcompacta . Howevercompacta . Since tIn this article we summarize recent data from the study of genes linked to PD that suggest a central importance of Wingless/Int (Wnt) signaling pathways for the normal function of dopaminergic neurones. These hereditary clues indicate that perturbation of these cell signaling cascades might represent a key event in the early pathogenesis of PD. Deregulation of Wnt signaling has the potential to explain many of the commonly observed features of neurodegeneration, while the sheer number of genes implicated in PD that are also involved in Wnt signaling is compelling. In light of the vital requirement for improved PD treatments and the therapeutic potential of targeting signal transduction cascades, these connections between PD and Wnt signaling make a persuasive and timely case for research into the role of Wnt pathways in the adult brain, both in health and disease.2+) cascades. This article will focus on the canonical pathway since the majority of research reviewed focuses on this signaling cascade. Nonetheless, it should be stressed that roles for the PCP and Wnt/Ca2+ pathways in the pathogenesis of PD cannot be excluded.Wnt signaling pathways are a group of highly conserved cell signaling cascades with a well-established importance to animal development and cancer biology . There ahttp://www.stanford.edu/group/nusselab/cgi-bin/wnt/). Further signaling complexity is conferred by membrane co-receptors. In the case of the canonical Wnt pathway, these receptors are low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). The binding of Wnt ligands to the combination of FZD and LRP5/6 proteins allows the extracellular signal to be relayed across the plasma membrane, leading to the activation of intracellular signaling.The term Wnt describes the secreted ligand proteins that bind to the extracellular domains of frizzled (FZD) receptors on target cells. Wnt ligands are usually glycosylated and are relatively large for secreted morphogens, suggesting that the sites of secretion and action tend to be relatively close . In humans there are 19 genes encoding Wnt ligands and 10 genes encoding FZD receptors . The canonical pathway is unusual as several events take place in the absence of a stimulus. In the basal state, β-catenin is sequestered into an inhibitory cytosolic complex known as the β-catenin destruction complex. Within this complex β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β), resulting in the targeting of β-catenin for proteosomal degradation. Thus, in the absence of stimulation by Wnt ligand, β-catenin is continually degraded. The activation of FZD receptors causes the recruitment of dishevelled (DVL) proteins to the plasma membrane, where DVLs are thought to associate with the intracellular surface of FZD receptors. Through additional interaction with components of the β-catenin destruction complex, DVL proteins mediate the relocalization of the β-catenin destruction complex to juxtamembrane sites. The resultant formation of large membrane-spanning complexes, containing extracellular ligand, transmembrane receptors, and a multitude of intracellular proteins, has been termed a Wnt ‘signalosome’. Wnt signalosomes have one basic purpose: the inhibition of β-catenin phosphorylation, allowing β-catenin to become resistant to proteosomal degradation. As a result, β-catenin can accumulate and spread throughout the cell. Most importantly, β-catenin binds to a number of transcription factors, and via transcriptional co-factor activity, modulates the expression of downstream genes. The best described targets of β-catenin are T-cell transcription factor/lymphoid enhancer binding factor family transcription factors, although other unrelated transcription factors can also be regulated by β-catenin. The mechanism by which β-catenin phosphorylation is repressed in signalosomes is not clear, but appears to involve two processes. Firstly, the intracellular C-termini of LRP5/6 co-receptors are able to compete with β-catenin as phosphorylation substrates for GSK3β. Secondly, signalosome formation triggers internalization of the entire complex from the plasma membrane into the endosomal system. This complex continues to signal from the cytosolic face of intracellular membranes until it is eventually sequestered into multi-vesicular bodies. It is believed that this removes the canonical Wnt signaling pool of GSK3β from the cytosol, which in turn protects newly synthesized β-catenin from proteosomal targeting  Figure .Figure A key role for Wnt cascades during embryogenesis is well established. It is thus unsurprising that Wnt signaling has been implicated as a master regulator of neurogenesis including adult neurogenesis . Nonethe2+ channel Cav3.1 in the thalamus, leading to enhanced T-type Ca2+ currents  (LRRK2 mutations to display aggregates of tau protein (Mutations in  (LRRK2) . Intrigu (LRRK2) . LRRK2 m (LRRK2) . It has  (LRRK2) . In addi protein . Tau pat protein . These o protein .LRRK2 is a complex 2527 amino acid protein, containing both kinase and GTPase activities as well as a number of protein–protein interaction domains. LRRK2 GTPase activity is conferred by the presence of adjacent Roc (Ras of complex proteins) and COR  domains, which together can be considered a single RocCOR tandem domain since Roc and COR domains are always expressed together in nature . CuriousVia interactions with a number of established Wnt signaling components, LRRK2 has been strongly implicated as a key scaffold protein in the canonical pathway. Initial work using yeast two-hybrid, co-immunoprecipitation, and confocal microscopy reported a direct interaction between LRRK2 and all three mammalian DVL proteins . In lighThe capacity for LRRK2 to enhance DVL1-driven Wnt signaling increased further by targeting LRRK2 to membranes. In agreement with this, LRRK2 was discovered to bind directly to the intracellular domain of the LRP6 membrane receptor, while treatment of HEK293 cells with recombinant Wnt3a increased the amount of endogenous LRRK2 protein present in membrane fractions. These observations are consistent with the Wnt signalosome hypothesis and suggest that LRRK2 affects canonical pathway activity at cellular membranes . SimilarLRRK2 knockout mice also display enhanced canonical Wnt activity compared with wild-type controls (unpublished data). Importantly, the effect of knocking down LRRK2 expression was observed under Wnt3a- and DVL1-stimulated conditions but also basally, suggesting an inhibitory role for LRRK2 in the β-catenin destruction complex. Consistent with this idea, co-immunoprecipitation of endogenous LRRK2 from mouse brain revealed LRRK2 to exist in complex with multiple components of the β-catenin destruction complex, including Axin1, GSK3β, and β-catenin , and also interact genetically in animal models  is the product of the  in 2011 . The ove in 2011 . Howeverer LRRK2 . Secondler LRRK2 , while mer LRRK2 . In thiser LRRK2  and animutations . Thus ittrans-Golgi network , encoded by smission . Even thse of PD , one mutse of PD , and decse of PD . In addise of PD . Nurr1 hynuclein . Therefoynuclein . In partynuclein . The posGSK3B) has been implicated in PD risk in two genetic studies  have also been reported to bind the LRP6 intracellular domain  and the Michael J Fox Foundation.We are grateful for the support of our work on PD and Wnt signaling by the Conflict of interest: none declared.


Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of  Aspergillus and Penicillium catalyzes the oxidation of β-D-glucose using molecular oxygen as electron acceptor, to yield D-gluconolactone and hydrogen peroxide. The enzyme belongs to the glucose-methanol-choline (GMC) oxidoreductase family. It is also classified within Auxiliary Activities family 3 (AA3) in the carbohydrate active enzymes (CAZy) database [Glucose oxidase (GOX) characteristically produced by species of database . GOX actdatabase .GOX has a wide range of industrial applications in diverse sectors such as food, pharmaceutical, energy and textile . As a foAspergillus niger GOX is the most commonly used enzyme for industrial applications. Early studies with this enzyme showed that it was a glycosylated dimeric protein, which had a mass of about 160 kDa and used FAD as cofactor [Among glucose oxidases, cofactor , 5. Its cofactor . The proWhile being used for many applications, intrinsic properties of GOX, particularly thermal stability and optimum pH for activity, are limiting factors. Therefore, directed evolution approaches have been used to improve enzyme performance –11. AlteAspergillus niger. The DNA was purified by a standard procedure  from strain CECT 2775 obtained from Colección Española de Cultivos Tipo (www.cect.org). The GOX coding sequence, excluding its signal peptide, was amplified by PCR with oligonucleotides JM823 (TACGCTAGCAGCAATGGCATTGAAGCCAGC) and JM824 (CACAAGCTTTCACTGCATGGAAGCATAATCTTC), containing NheI and HindIII restriction sites, respectively (underlined). PCR was carried out with a proofreading polymerase , according to the specifications of the manufacturer. Plasmid pSTRD-Bgl1 [S. cerevisiae. In pSSP-GOX, the GOX coding sequence is fused to the STA1 signal peptide from Saccharomyces cerevisiae var. diastaticus and is under the control of the galactose-inducible GAL10/CYC1 promoter (see Saccharomyces cerevisiae BY4741 (MATa his3 leu2 met15 ura3) was used as the host strain.The GOX-encoding gene, which lacks introns , was cloTRD-Bgl1  was usedSite directed mutagenesis was carried out by PCR as previously described  using plNheI / HindIII and re-cloned into pSSP-GOX. E. coli was transformed with the resulting ligation product. The population of transformant cells was enriched by growing the transformed culture overnight in liquid LB medium supplemented with ampicillin (25 μg/ml). Restriction analysis of the pool of plasmid DNA molecules isolated from this culture (named pSSP-GOX*) indicated that at least 90% of the plasmids contained a GOX insert.Random mutagenesis was carried by PCR with a polymerase without proofreading activity  with oligonucleotides JM823 and JM824. The PCR product was digested with S. cerevisiae. Transformant colonies were selected in SD  medium with all auxotrophic requirements except uracil.Plasmids encoding the wild-type GOX or different mutant versions of the enzyme, were used to transform O-dianisidine. Colonies transformed with the same plasmid lacking the GOX encoding gene were used as a negative control. The plates were incubated at 80°C for 30 minutes, a condition that causes inactivation of the wild type enzyme, and then cooled back to room temperature. To assay for GOX activity, 10 ml of reaction mix P, containing 10 μg/mL horseradish peroxidase  and 90 mM glucose (Sigma) in 50 mM phosphate buffer pH 6, were added per plate. After 30 minutes of incubation at 37°C, the plates were washed with deionized water and kept at 4°C for 20 minutes. Active transformants were detected by the formation of an orange halo.A plate assay was carried out to determine activity in yeast colonies grown on solid medium. Transformant colonies were grown on petri dishes of YPD (complete) medium supplemented with 0.5% galactose and 2.8 mM leu2d gene present in the plasmid [1 and t2) before chilling on ice. For each set of experiments, wild-type and mutant enzymes were assayed in parallel under the same conditions. Incubation times (t1 and t2) were selected to render a residual wild-type activity of ca. 40% and 25%. Subsequently, the samples were incubated at 37°C in a reaction mix L, containing 85 mM glucose, 12 μg/mL HRPO, 0.17 mM O-dianisidine in 50 mM pH 6. The reaction was stopped by addition of 0.2 M HCl and cooling on ice. Absorbance at 400 nm was measured and the amount of oxidized glucose was determined by interpolation in a standard curve. The glucose standard curve was prepared as follows: known amounts of glucose were incubated with 12 μg/mL HRPO, 0.16 mM O-dianisidine and 25 μg/ml of commercial GOX from A. niger , for 30 minutes at 37°C. The reaction was stopped with 0.2 M HCl and the absorbance at 400 nm was measured. A400 was plotted versus glucose concentration. GOX activity was determined as the amount of oxidized glucose per unit of time.phosphate bufferActivity in liquid medium (culture supernatant) was assayed as follows. Colonies were firstly grown on SD plates supplemented with requirements lacking both uracil and leucine, to force the maintenance of the plasmid in high number of copies as an effect of the defective  plasmid . Individ600 = 0.014. The flasks were incubated for 2 days at 30°C with orbital shaking at 250 rpm. After this time, the culture medium was separated from the cells by centrifugation and concentrated 30–60 fold by ultrafiltration through a membrane with a 20 KDa cutoff (Thermo Sci). An aliquot from the concentrated sample was incubated with 3 U/μl EndoH (New England Biolabs) in 50 mM phosphate buffer pH 6, 90 mM β-mercaptoethanol and protease inhibitor cocktail  for 16 hours at 37°C. A control sample was treated in the same conditions but without EndoH. Both deglycosylated and control samples were analyzed by SDS-PAGE, as previously described [Yeast transformants were grown for 2 days in 5 mL of liquid SD medium supplemented with auxotrophic requirements except uracil and leucine. These cultures were used to inoculate 100 ml flasks containing 50 ml of YPD with 0.5% galactose at a cell density equivalent to ODhttp://www.expasy.org/spdbv/).Multiple sequence alignment was carried out with Clustal W . StructuA. niger wild type GOX gene sequence that corresponding to GenBank KJ774107.1. The encoded amino acid sequence (AID16306.1) shows two changes (V167T and K282E) with respect to the GOX sequence reported in crystallographic structures . These residues are located on the protein surface, are fully exposed to solvent and do not show significant interactions with other residues in the protein.Oligonucleotides used as primers for PCR amplification of the GOX coding region were designed on the basis of the GOX sequence with code X16061 in GenBank. However, the sequence of the DNA fragment resulting from the amplification showed higher similarity to another GOX sequence, GenBank KJ774107.1. The amplified GOX differed from KJ774107.1 only in two nucleotides and encoded the same amino acid sequence. Therefore, in this communication, we consider S. cerevisiae clones expressing randomly mutated GOX coding sequences were subjected to the semi-quantitative plate assay, using as a control a S. cerevisiae transformant with the wild-type version of the gene. 274 clones showed GOX activity after incubation of at 80°C. Thermal stability of GOX produced by these clones was further analyzed. They were individually cultivated in liquid medium where the expression of the enzyme was induced by the addition of galactose. The crude enzyme preparation from each clone was heated at 60°C for 45 or 80 minutes and the residual activities (R1 and R2 respectively) were determined. Clones expressing wild type GOX gave R1 ca. 40% and R2 ca. 25%.  of the rest of mutants except for Q142R/L569E which showed an almost 2-fold increase, compared to the wild-type enzyme  displayed an unexpected effect resulting from Thr to Met substitution at position 554. The analysis of the protein structure shows that mutation T554M disrupts a hydrogen bond between T554 and W122 Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized.50-values were calculated.Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally ECT.CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity. Small molecules are attractive chemical compounds to control pluripotency in murine embryonic stem cells -6. They The Wnt/beta-catenin pathway controls miscellaneous biological processes during tissue development by autocrine and paracrine activities . When WnIn this study the effect of four commonly used GSK3 inhibitors, namely BIO, SB-216763, CHIR-99021, and CHIR-98014 was analyzed in a comparative fashion in two different mouse embryonic stem cell lines (ES-D3 and ES-CCE). Specifically, the cytotoxicity, the ability to activate the Wnt/beta-catenin-pathway, and the changes in gene and protein expression were analyzed in a defined serum-reduced medium devoid of LIF.The results show that one of the four tested compounds, CHIR-99021, is optimally suited for strong activation of the Wnt/beta-catenin-pathway without significant concomitant toxicity.RPMI1640 advanced, KO-DMEM, NEAA, and glutamax were obtained from Life Technologies  and fetal calf serum was from . The GSK3 inhibitors BIO, SB-216763, and CHIR-99021, were from Tocris Bioscience  and CHIR-98014 was purchased from Axon Medchem . Wnt3a was obtained from Peprotech  and Matrigel was from Corning . All primers were synthesized by Life Technologies. The RevertAid™ H-Minus M-MuLV reverse transcriptase was purchased from Thermo Fisher Scientific . The GoTaq® Taq polymerase was from Promega  and dNTPs from Genecraft . Unless mentioned otherwise, chemicals were obtained from Sigma-Aldrich .To maintain the pluripotency of the mouse ES cell lines ES-D3  and ES-CThe viability of the mouse ES cells was determined after exposure to different concentrations of GSK3 inhibitors for three days using the MTT assay . The decThe Wnt/beta-catenin reporter assay was performed with the M50 Super 8× TOPFlash and M51 Super 8× FOPFlash vector containing the firefly luciferase gene under the control of TCF/LEF binding sites (M50 Super 8x TOPFlash) or mutated bindings sites (M51 Super 8× FOPFlash) . 12,500 T fw: catcggaacagctctccaacctat, rev: gtgggctggcgttatgactca, Nanog fw: ccctgaggaggaggagaacaaggtc, rev: ccactggtttttctgccaccgc, and Pou5f1 fw: aggcccggaagagaaagcgaacta, rev: tgggggcagaggaaaggatacagc. Each qPCR amplification was performed in triplicates and the gathered data were normalized with qBasePlus  against the housekeeping genes G6pdx, Tbp, and Tuba1a[Total RNA was isolated from the cells using the RNeasy Kit . Briefly, the cells were lysed in Qiazol (Qiagen), the hydrophilic phase was loaded onto RNA spin columns, and RNA was then prepared as instructed. cDNA synthesis was performed with random hexamer primers and 2 μg of the isolated total RNA following the manufacturer’s instructions. 10–20 ng of cDNA was then loaded in each well of a 384-well plate and specific primers were mixed with the GoTaq® PCR master mix according to the manufacturers’ instructions. Primer sequences were (5’-3’): 4. Primary and secondary antibodies were diluted in PBS with 0.1% Triton X-100 and 0.1% BSA. Primary antibodies were incubated on the slides for 2.5 h at room temperature (RT) or overnight at 4°C. Secondary antibodies were incubated on the slides for 1 h at RT. The following primary antibodies were used: anti-Oct3/4  and anti-Brachyury . Secondary antibodies were obtained from Dianova . Finally, the slides were mounted with immunoselect antifading mounting medium containing DAPI (Dianova) to counterstain the nuclei. The stained cells were examined using an Olympus IX81 inverted microscope .Immunofluorescence was performed according to standard procedures. ES cells were treated on 6-well plates with different GSK3 inhibitors and after 48 h 100,000 cells were re-seeded in each cavity of Matrigel-coated glass slides . After 24 h in medium the cells were fixed in 4% (w/v) paraformaldehyde for 45 min at 4°C. Subsequently, the cells were blocked for 20 min in PBS plus 0.2% Triton X-100, 6% BSA, and 1 mg/ml NaBHStudent’s t-test or ANOVA followed by Bonferroni’s or Dunnett’s post hoc test for multiple comparisons.Data were expressed as mean values ± SEM unless stated otherwise. Statistical analyses were performed using the GraphPad Prism software  applying Two mouse stem cell lines, ES-D3 and ES-CCE, were exposed to different concentrations of the GSK3 inhibitors BIO, SB-216763, CHIR-99021 and CHIR-98014 in defined medium devoid of LIF and with low FCS. Their toxicity was tested using the MTT assay; Bio at concentrations of 0.1 to 1 μM and all other compounds at 1 – 10 μM. As controls the cells were treated without compounds in the medium with the solvent DMSO as vehicle control. Differentiation in the presence of DMSO did not significantly decrease cell viability in both mouse ES cell lines  and Nanog upon differentiation [It has been previously reported that double-knockout of both GSK3 isoforms in mouse ES cells yielded an induction of ntiation . In the ntiation , gene ex5F1 Oct3/ and Nanontiation . Severalntiation ,33. Thesntiation .T was observed upon GSK3i. The strongest effect was detected for CHIR-99021 and CHIR-98014, which confirms the reporter assay results. The increased gene expression yielded Brachyury-positive cells predominantly together with Oct3/4 in case of both CHIRs but not in the presence of BIO and SB-216763. Interestingly, only a small subpopulation accounted for the 2,500-fold increase in T expression as measured by qPCR. This observed heterogeneity raises the question why some cells did not acquire Brachyury-positivity whereas another subpopulation showed a significantly increased Brachyury expression.Additionally, a strong increase in gene expression of Small molecule inhibitors of GSK3 have become valuable reagents for studies on pluripotent stem cells. In murine ES cells they may be used to maintain pluripotency whereas in human ES cells GSKi was shown to be useful in differentiation experiments ,25. The The authors declare that they have no competing interests.ON designed the study, analyzed and interpreted the data and wrote the manuscript. JL collected and interpreted the cytotoxicity and reporter assay data. UD analyzed and interpreted the data and wrote the manuscript. CD performed immunofluorescence and real-time PCR and wrote the manuscript. SL designed the study, wrote and finally approved the manuscript. All authors read and approved the final manuscript.


In patient CRC samples, high S100A4 levels predict metastasis and reduced patient survival. Our results link pathways important for tumor progression and metastasis: the Wnt signaling pathway and S100A4, which regulates motility and invasiveness. S100A4 suppression by interdicting Wnt signaling has potential for therapeutic intervention. As proof of principle, we applied S100A4 shRNA systemically and prevented metastasis in mice. Furthermore, we identified small molecule inhibitors from high-throughput screens of pharmacologically active compounds employing an S100A4 promoter-driven reporter. Best hits act, as least in part, via intervening in the Wnt pathway and restricted metastasis in mouse models. We currently translate our findings on restricting S100A4-driven metastasis into clinical practice. The repositioned FDA-approved drug niclosamide, targeting Wnt signaling, is being tested in a prospective phase II clinical trial for treatment of CRC patients. Our assay for circulating S100A4 transcripts in patient blood is used to monitor treatment success.The aberrant activity of Wnt signaling is an early step in the transformation of normal intestinal cells to malignant tissue, leading to more aggressive tumors, and eventually metastases. In colorectal cancer (CRC), metastasis accounts for about 90% of patient deaths, representing the most lethal event during the course of the disease and is directly linked to patient survival, critically limiting successful therapy. This review focuses on our studies of the metastasis-inducing gene S100A4, which we identified as transcriptional target of β-catenin. S100A4 increased migration and invasion  In colorectal cancer (CRC), metastasis accounts for about 90% of patient deaths, representing the most lethal event during the course of the disease. Metastasis is directly linked to patient survival, and critically limits successful therapy ,2. The di.e., β-catenin dependent) or non-canonical  signaling, subgrouped in non-canonical planar cell polarity (PCP) and non-canonical Wnt/calcium pathway. However, cross-talk downstream of both pathway classes is reported, based on the availability of extracellular Wnts, cellular context, and the types of Wnt receptor [Wnt-mediated signaling is one of the crucial signaling pathways in CRC. The molecular composition of the Wnt pathway and its role in signal modulation has been reviewed extensively in the last few years ,8,9,10, receptor .Without active signaling, β-catenin has a rather short half-life in the cytoplasm, since it gets phosphorylated by the so called “destruction complex”, consisting of glycogen synthase kinase (GSK) 3β, casein kinase (CK) Iα, Axin, and APC, subsequent ubiquitinylation and eventual degradation by the proteasome .The canonical Wnt pathway is triggered by the interaction of Wnt with the Frizzled receptor and the co-receptors low-density lipoprotein receptor-related proteins (LRP) 5/6. This inactivates the β-catenin destruction complex, when Axin is recruited by the segment polarity protein dishevelled homolog (Dvl) to the activated Wnt/Frizzled/LRP complex. β-catenin starts to accumulate in the cytoplasm and eventually translocates into the nucleus, where it activates the transcription of target genes under the control of a T-cell factor (TCF) binding motif along with other factors .The activation of the Wnt signaling pathway is tightly controlled at the cell surface. Secreted Wnt-antagonists of the Dickkopf (DKK) family form a ternary complex by binding to the DKK receptor family Kremen (Krm) and the Wnt co-receptor LRP5/6, thereby inhibiting the formation of active Wnt/Frizzled/LRP signaling complexes . The tigThe S100 calcium binding protein A4 was first discovered by Ebradlize and colleagues in 1989 and was initially named metastasin (mts1) . Already2+ binding proteins with a molecular mass of 10–12 kDa, which share 50% of their amino acid sequence. This high degree of homology suggests a common ancestor, that might have evolved 500 million years ago [2+ binding helix-loop-helix domains [2+ with higher affinity [2+ to the EF hand motif results in a movement of the two helices giving access to hydrophobic protein-protein interaction sites, which were previously hidden in the protein structure. The Ca2+ bound S100 proteins are regarded as ”open” and constitute the active form [S100A4 belongs to a family of S100 proteins, named due to their solubility in saturated ammonium sulfate. The first member of the group of S100 proteins was described back in 1965 by Moore . Currentears ago . All S10 domains . The N-taffinity . Bindingive form ,28. S100ive form ). All S1ive form .Most of the S100 family members are involved in or initiating biological functions contributing to malignant disease such as proliferation, metastasis, angiogenesis and immune evasion. These proteins represent promising candidates for cancer diagnosis and prognosis as well as therapeutic targets with first inhibitors already identified and tested in clinical trials (reviewed in ).wt cells, a HCT116 derived cell line expressing monoallelic wt β-catenin [wt cells. This result was confirmed in HCT116-derived cell lines HAB-68mut, expressing monoallelic Δ45-mutant β-catenin, and NCI-H28null, nullosomic for β-catenin, verifying, that functionally active β-catenin is necessary for S100A4 induced expression in CRC. Forced overexpression or knock-down of S100A4 and/or mutated β-catenin further proved, that transcriptionally active β-catenin enhances the S100A4-induced migration and invasion of HCT116, HAB-92wt and NCI-H28null cells. In a CRC xenograft mouse model, intrasplenic or intracardiac transplantation of HAB-92wt cells, with or without stable mutant β-catenin expression, demonstrated that due to GOF in the mutant β-catenin, this group showed more metastatic lesions in liver and lungs, compared to vector control cells. With this evidence we have designed a comprehensive study to determine whether S100A4 is a direct transcriptional target of β-catenin. Sequence analysis of the human S100A4 promoter revealed a putative TCF binding motif at −679 to −673. Luciferase experiments with or without TCF binding site mutations showed that S100A4 is a direct transcriptional target of β-catenin. Further, the binding of β-catenin to the S100A4 promoter was confirmed by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analysis [Since Wnt signaling activity is altered in almost all CRC tumors, we addressed the question, which genes are modified in their expression by a gain-of-function (GOF) mutation in β-catenin . We perf-catenin . In thisanalysis . This dain vitro [S100A4 is associated with both non-malignant and malignant human diseases. Several groups have reported a role of S100A4 in inflammation. Human articular chondrocytes upregulate S100A4 expression levels during rheumatoid- and osteoarthritis. Elevated S100A4 levels lead to increased phosphorylation of protein tyrosine kinase (Pyk)-2, mitogen-activated protein (MAP) kinases, and activated nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), that in turn results in elevated matrix metalloproteinase (MMP)-13 secretion. These effects are mediated at least in part via the receptor for advanced glycation end products (RAGE), as the inhibition of RAGE resulted in decreased S100A4 dependent signaling . When anin vitro .in vitro and in mice [The cellular functions of S100A4 were mainly characterized in cancer, promoting tumor progression and metastasis formation, reviewed by Boye and Mælandsmo, and recently by Bresnick and colleagues ,38. Enha in mice . The exp in mice , breast  in mice , lung sq in mice , pancrea in mice , gastric in mice , and col in mice . S100A4  in mice . Interes in mice . S100A4  in mice ,47.EMT is an important step in cancer development, characterized by aberrant signaling activities, including the Wnt pathway (reviewed in ). Before2+, which in turn triggers the fusion of non-secretory vesicles to seal the wound and the shedding of the injured part [2+-binding annexin A2, filamentous (F-) actin and myosin II, which were described to establish the wound closure in cooperation with S100A11, were also reported to interact with S100A4 [When focusing on the molecular mechanisms of S100A4 and its role in cancer, a number of cancer related protein-protein interaction partners have been described, including cytoskeletal proteins such as actin, myosin, and tropomyosin (reviewed in ). An impred part . Interesh S100A4 ,58. IndeS100A4 is also secreted into the intercellular fluid—by the tumor cell itself or by cells in the local tumor environment—and can exert multiple functions by interaction with receptors like RAGE ,60,61. RTaken together, it is well established that S100A4 has profound impact in many types of solid cancers, where its upregulation causes tumor progression and metastasis formation. S100A4 expression levels in tumors are considered as a biomarker for the prognosis of both metachronous metastasis and survival of cancer patients.When looking at CRC prognosis in general, there is a decrease of the 5-year survival rate of CRC patients after resection of the primary tumor from approximately 85%, when tumors were diagnosed at stages I and II, to less than 50%, when lymph node metastases occurred (stage III) . In caseToday, S100A4 expression in tumor tissue is a valid and valuable biomarker for determining the risk for metastasis formation of CRC patients, and much effort is made to evaluate S100A4 expression in tumors as a predictor for therapy response.The link of reported prognostic value of high S100A4 levels in the primary tumor for metachronous metastasis and reduced patient survival, documented in a large body of studies, is mainly based on snapshot analyses due to tissue availability. Thus, we established for the first time a non-invasive, plasma-based assay for the quantification of circulating S100A4 transcripts in blood of colon, rectal, and gastric cancer patients, that allows clinical application routinely for diagnosis, prognosis and for monitoring treatment success . We deteCombinatorial detection of relevant transcripts might even enhance diagnosis, prognosis, and/or prediction for cancer patients. Thus we combined detection of circulating transcripts of S100A4 with those of the MACC1 gene for CRC and gastric cancer patients ,75. We dElevated S100A4 expression is altering the genetic expression pattern of cells, leading to a more malignant phenotype . For CRCin vitro as well as in vivo [Early attempts to reduce the mRNA and protein level of S100A4 in the late 1990s reported successful reduction of the S100A4-induced metastatic phenotype for osteosarcoma,  in vivo . Ribozym in vivo . Our gro in vivo . This tr in vivo . Additio in vivo . Other r in vivo ,88. But  in vivo . For CRCin vivo, as well as in less metastasis formation in vivo. Applying RNAi-based therapeutics to decrease S100A4 expression in the clinic might be an approach to reduce the metastatic burden of CRC patients and may prolong their disease-free survival.Taken together, the reduction of S100A4 expression, either in the tumor itself or in its environment, has been proven to reduce the metastatic potential of CRC, shown by decreased cell motility Sulindac is long known as a nonsteroidal anti-inflammatory drug (NSAID), which inhibits the cyclooxygenase (COX) activity of the prostaglandin endoperoxide synthase (PTGS1 and PTGS2) enzyme that is involved in inflammation processes by converting arachidonic acid to prostaglandin H2. However, in the 1970s it came into focus that such drugs, including sulindac, also exert anti-tumor effects, which was repeatedly confirmed by clinical studies ,92. The More detailed studies on the molecular mechanism of action of sulindac revealed that the anti-tumor and the chemopreventive activity of this compound is rather COX-independent, as shown in different cancer cell lines with varying levels of COX-expression . More inin vitro and in vivo experiments demonstrate that S100A4-mediated tumor progression and metastasis formation, driven by β-catenin signaling, can be mitigated with administration of sulindac. This further exemplifies the effective interference of a NSAID compound in Wnt signaling as anti-tumor and anti-metastatic mode of action. The attractiveness of such an approach is supported by the fact, that meanwhile new sulindac derivatives such as sulindac-benzylamine were developed, which also inhibit colon cancer cell growth by suppression of transcriptional activity of β-catenin [In our studies we also used the pharmacological inhibitor sulindac known to intervene in the Wnt signaling pathway. We demonstrated the reduction of β-catenin-mediated S100A4 activation and expression in GOF as well as in loss of function (LOF) β-catenin variant carrying human CRC cell lines treated with sulindac . The pro-catenin . Another-catenin .The knowledge on the importance of S100A4 for metastasis formation and its involvement in the Wnt signaling pathway is the driving force for continued search for novel inhibitors. This search is accelerated by the use of high throughput assays, which provide the technological platform to identify promising new drug candidates from drug libraries . Such li50) values. By this HTS-based workflow two drug candidates were identified, which possessed high inhibitory efficacy of S100A4 expression: the antibiotic calcimycin and the anti-helminthic niclosamide.Regarding the Wnt target S100A4, the key prerequisite for high throughput screening (HTS) was the availability and molecular characterization of the human S100A4 promoter to create the appropriate luciferase-reporter system for the drug screens. The analysis of the S100A4 promoter revealed its link to the Wnt signaling pathway at transcriptional level by the identification of TCF-4 binding sites as functional units of TCF-4/β-catenin-mediated regulation of this gene. For identification of S100A4 transcription inhibitors we performed HTS, using the Library of Pharmacologically Active Compounds (LOPAC) of FDA-approved compounds ,105. We in vivo studies in mice after intrasplenical application of calcimycin-treated CRC cells to analyze its anti-metastatic activity. These studies showed that calcimycin reduced metastasis formation to 30% compared with control mice. Via bioluminescence imaging and immunohistochemistry, smaller and less frequent liver metastases were seen in the calcimycin treated animals. S100A4 levels in the metastases were also reduced by calcimycin. These in vivo studies provide strong indication, that the calcimycin-mediated reduction of S100A4 expression is able to restrict metastasis formation in vivo via transcriptional inhibition [Using the HTS approach with the S100A4-promoter driven reporter assay the polyether antibiotic drug calcimycin was identified with effective inhibitory activity ,107. Furhibition .Out of the 1280 well-characterized small molecules of the LOPAC library we identified niclosamide as the most efficient inhibitor of S100A4 promoter activity. Niclosamide is an anti-helminthic drug and is approved for human use since the middle 1960s. It restricts glucose uptake, oxidative phosphorylation and anaerobic metabolism in its target cells . NiclosaSelecting a candidate drug from the HTS is dependent on the ratio of luciferase activity inhibition and cell viability. Niclosamide inhibited luciferase activity at 0.78 μM and higher concentrations, but reduced cell viability at 3.1 μM and higher concentrations. For target gene validation, we analyzed the concentration- and time-dependency of niclosamide to modify endogenous S100A4 mRNA and protein levels in cell culture. To achieve the maximum S100A4 expression inhibition accompanied by minimum toxicity, we went further with a daily application of 1 μM niclosamide resulting in constantly reduced S100A4 expression. Since S100A4 is a major inducer of cell motility, we tested for the effects of niclosamide on S100A4-induced migration and invasion in a panel of human CRC cells. Niclosamide reduced significantly migration, invasion and also impaired wound closure in a wound healing assay in the different CRC cell lines, compared to solvent-treated controls. In rescue experiments using ectopically CMV-promoter driven S100A4 overexpressing CRC cell lines, neither reduction of S100A4 expression nor of cell motility inhibition was seen after niclosamide treatment. Niclosamide also inhibited anchorage-dependent and -independent proliferation of the CRC cells, however, this effect was—in contrast to the inhibition of cell motility—not S100A4-specific. Thus, niclosamide inhibited S100A4-dependent cell motility and invasiveness in CRC cells.in vivo luminescence imaging and at the experimental endpoint at day 22 by ex vivo imaging of the isolated organs spleen and liver. Whereas solvent-treated control mice formed metastases in the livers, no metastasis development was seen in niclosamide-treated animals. In the spleen tumors of the niclosamide-treated mice, expression of S100A4 was reduced indicating that niclosamide reduced S100A4 levels in vivo as well. We also investigated metastasis formation in vivo under continuous and discontinuous  niclosamide treatment. Both niclosamide-treated mouse groups showed enhanced overall survival compared with the solvent-treated mice linked to long-term inhibition of tumor growth and liver metastasis formation, and to reduction of S100A4 expression. No statistically significant differences were found between the continuously and discontinuously treated animals. We conclude that niclosamide has the potential for the clinical treatment or prevention of CRC metastasis in humans.Further, we demonstrated that niclosamide restricted liver metastasis formation by using an intrasplenically xenografted mouse model. Niclosamide was administered intraperitoneally on a daily basis. Tumor growth and metastasis development was monitored by non-invasive A large variety of studies demonstrated actions of niclosamide as anti-cancer agent in different tumor entities, including CRC, breast cancer, prostate cancer, ovarian cancer, non-small cell lung cancer (NSCLC), glioblastoma, osteosarcoma, multiple myeloma, and leukemia. There is growing evidence that anti-cancer actions of niclosamide are predominantly mediated via the Wnt/β-catenin signaling pathway, which is known to represent a major regulatory pathway for cancer initiation, growth, cell differentiation and metastasis . With moWe also analyzed the effect of niclosamide on the Wnt signaling pathway since we identified the metastasis inducing gene S100A4 as a transcriptional target gene of Wnt/β-catenin signaling . We used50) values less than 1 μM. They concluded that niclosamide has potential as chemopreventive and therapeutic agent for human cancer. Osada and colleagues demonstrated anti-proliferative actions of niclosamide in CRC cells regardless of mutations in APC [in vitro cytotoxicity was enhanced, and growth of orthotopic tumor xenografts was suppressed. The authors conclude that niclosamide or its congeners might be beneficial for the treatment of basal-like breast cancers. The effect of niclosamide as a potential therapeutic agent interfering with the Wnt signaling pathway was also investigated for ovarian cancer [Also by employing libraries of FDA-approved drugs by HTS, Chen and colleagues identified niclosamide as a drug able to interfere with the Wnt signaling pathway. They demonstrated that niclosamide is able to inhibit Wnt/Frizzled-1 signaling in osteosarcoma cells ,114. Thes in APC . This efs in APC . The auts in APC . Niclosas in APC . Again, n cancer . The autn cancer . Niclosan cancer . The aut50 values below 1 μM [Although all of these studies demonstrate the interference of niclosamide in the Wnt signaling pathway, leading to reduced target gene expression and resulting in reduced cell proliferation and motility in several cancer types, it has to be mentioned that niclosamide is able to block multiple signaling pathways, which play a major role in cancer initiation, progression, and metastasis in a panel of different cancer types. This broad anti-cancer activity is also reflected by a screen with niclosamide on the NCI-60 human tumor cell line panel . Niclosalow 1 μM . Severallow 1 μM . Despitelow 1 μM ,126): nilow 1 μM ; inhibitlow 1 μM ,129,130;low 1 μM ,132, andlow 1 μM ; inactivlow 1 μM , and simlow 1 μM .Based on the promising anti-cancer effects of niclosamide, niclosamide chemotypes have been identified or synthesized, and tested for their anti-cancer effects and for their interference with the Wnt signaling pathway. When we discovered the anti-migratory and anti-invasiveness effects of niclosamide via inhibiting the Wnt signaling pathway and the metastasis gene S100A4, we extended our analyses to structural derivatives of this drug . In contBased on the niclosamide-related findings reviewed here, we now aim at the repositioning of the anti-helminthic drug niclosamide as anti-cancer agent for the clinical treatment or prevention of colon cancer metastasis. Niclosamide is well tolerated in humans, approved for human use, and 2 g are administered daily in adults to treat tapeworm infections. We are translating our findings on restricting S100A4-driven metastasis into clinical practice. Together with the Charité Comprehensive Cancer Center, we are evaluating the repositioned FDA-approved drug niclosamide targeting Wnt signaling for treatment of CRC patients in a prospective phase II clinical trial: Drug Trial to Investigate the Safety and Efficacy of Niclosamide Tablets in Patients with Metastases of a Colorectal Cancer Progressing after Therapy (Nikolo). This clinical trial is registered with ClinicalTrials.gov (NCT02519582) and the European Clinical Trials Database (EudraCT 2014-005151-20). We initiated this first phase II clinical trial in August 2015 evaluating the safety and efficacy of orally applied niclosamide in patients who are progressive with metachronous or synchronous metastases of CRC after the previous therapy. Two grams niclosamide per day are given to the CRC patients of this monocentric open-label clinical trial, received until progression  or unacceptable toxicity. The primary endpoint is the progression-free survival (PFS) at 4 months. Secondary outcome measures are overall survival , time to progression , disease control rate , the number of adverse events > grade 2 toxicities according to NCI Common Toxicity Criteria for Adverse Effects v4.03 , and the number of serious adverse events . The first patients are already enrolled; a total of 37 patients will be enrolled in this interventional trial. The primary outcome measure, PFS at 4 months, will be analyzed in a first evaluation after treatment of the first 17 patients. Paralleling this clinical trial, translational research is performed, e.g., determining S100A4 expression in tumor tissues and metastases, and monitoring treatment success by quantifying the circulating S100A4 transcripts in patient blood. The trial is planned to be completed in February 2018.Very recently, in February 2016, another clinical trial testing niclosamide in CRC patients was initiated by the Duke University Medical Center: A Study of Niclosamide in Patients with Resectable Colon Cancer. This clinical trial is registered with ClinicalTrials.gov (NCT02687009). The authors are performing this phase I clinical trial to obtain safety data along with pharmacokinetic data and with the determination of the maximum tolerated dose (three dosage levels of niclosamide) in patients with colon cancer who undergo primary resection of their tumor. Further, they wish to obtain information on the changes in the Wnt pathway signaling following niclosamide administration in humans. They aim at future studies in patients with more advanced CRC or other cancers with dysregulation of the Wnt pathway.Taken together, these recently initiated clinical trials will provide first insights in the niclosamide-induced inhibition of Wnt/β-catenin signaling and the clinical consequences thereof. The outcome of these clinical trials on niclosamide for treatment of CRC patients will contribute to the evaluation of the repositioning of this anti-helminthic drug niclosamide for the clinical treatment or prevention of CRC metastasis.Metastasis formation is the major hurdle in CRC therapy. To identify patients at high risk for metastasis formation, early diagnosis and molecular characterization of the primary tumor is crucial to define prognostic and therapeutic targets. S100A4 has been shown to contribute to both demands in CRC: serving as biomarker, which allows the prognosis of patient survival and metachronous metastasis already in early stages being determined in tumor tissue or non-invasively in liquid biopsies; and serving as therapeutic target, since its down-regulation by e.g., small molecules restricts metastasis formation in mice and is currently tested in clinical trials. Other strategies of therapeutic interventions tested in preclinical studies, like S100A4-specific antibodies or drug/peptide-based interference of S100A4-protein interactions, further point to the relevance of treating CRC by targeting S100A4.in vitro and metastasis formation in vivo and are currently evaluated in clinical trials to reduce the risk of metastasis formation in CRC thereby improving patient survival and quality of life. In recent years, further approaches have been demonstrated to interfere with the Wnt signaling pathway upstream of β-catenin [As S100A4 is a direct target of β-catenin/TCF-mediated transcription in CRC, interfering in the Wnt signaling pathway thereby reducing expression and/or nuclear accumulation of β-catenin has emerged as new option to restrict S100A4-induced cell motility and metastasis. Promising, already FDA-approved drugs were identified to reduce β-catenin-mediated S100A4 gene transcription resulting in diminished cellular motility -catenin ,140. The


While β-catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and β-catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti-β-catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. β-Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear β-catenin. β-Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic β-catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited β-catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of β-catenin and that β-catenin and Rad6 play independent roles in melanoma development.We have previously demonstrated that Rad6 and  Consequently, β-catenin accumulates in the cytoplasm and translocates to the nucleus. Nuclear β-catenin stimulates transcription of a large number of TCF/β-catenin responsive genes that include cyclin D1, c-myc [β-catenin as observed in several cancer types is considered a marker of canonical Wnt/β-catenin pathway deregulation and unfavorable prognosis [The Wnt/r cancer –3. Underradation . Increas1, c-myc , 6, and 1, c-myc . Thus, arognosis .β-catenin accumulation and melanoma progression and suggested nuclear β-catenin to be a marker of poor prognosis [β-catenin expression [β-catenin may be used as biomarkers of good prognosis in melanoma [Previous studies have reported an association between nuclear rognosis , 7. Howepression , 10. Mormelanoma –14.β-catenin signaling [β-catenin by polyubiquitin modifications that render β-catenin resistant to 26S proteasomal degradation [β-catenin [β-catenin-mediated activation of Rad6 gene expression and Rad6-induced β-catenin stabilization.Recently, HHR6, a human homologue of the yeast Rad6 gene and a principal component of the postreplication DNA repair pathway, has been identified as an important regulator of canonical Wnt/ignaling , 16. HHR-catenin , thus re in situ (DCIS) and invasive breast carcinomas [β-catenin pathway [β-catenin [β-catenin work in concert to promote melanoma pathogenesis. Furthermore, Rad6 expression in the skin has not been investigated, and there are no data on the role of Rad6 in the pathogenesis of benign (nevi) and malignant (melanoma) melanocytic lesions. It is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because Rad6 and β-catenin have been identified as therapeutic targets [Rad6 expression is low in normal breast tissues; however, increases in Rad6 protein expression are detected in hyperplastic, ductal carcinomarcinomas . We have pathway . Since t-catenin , 10, rat targets , 20.β-catenin expressions in serial sections of nevi, primary, and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression. Our data suggest that membranous relocation of β-catenin and upregulation of Rad6 are independent markers of melanoma development and progression. We also offer a hypothesis that explains the role of membranous β-catenin relocation and decreasing cytoplasmic β-catenin in melanoma development, a phenomenon that has been linked to unfavorable prognosis [In this study, we examined Rad6 and rognosis , 21, 22.Cases were retrieved from the files of the Pinkus Dermatopathology Laboratory (PDL), a private dermatopathology laboratory located in Monroe, MI. Preserved paraffin-embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information. Nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator (Stata/MP 13.1). The study groups consisted of 30 cases of melanocytic nevi, 29 cases of primary cutaneous melanoma, and 29 cases of metastatic cutaneous melanoma. The melanoma and melanocytic nevus subtypes are listed in β-catenin (IS702) was purchased from Dako  and used in an undiluted form; and (ii) anti-Rad6 (ab31917) was purchased from Abcam  and used at a 1 : 500 dilution. In humans, the yeast homologous Rad6 gene is duplicated and the proteins encoded by the two genes HHR6A (or Rad6A) and HHR6B (Rad6B) from chromosomes Xq24-q25 and 5q23-q31, respectively, share 95% identical amino acid residues [Primary antibodies used in the study are as follows: (i) anti-residues , nor anyImmunohistochemistry was performed as previously described . Brieflyβ-catenin antibody, and breast cancer tissues were included as positive controls for staining with anti-Rad6 antibody. Tissue sections incubated without primary antibody served as negative controls.Lung and colon cancer tissues were included as positive controls for immunostaining with anti-β-catenin. A tumor was considered to be stained with high intensity if >50% of the cells in a specimen expressed Rad6 or β-catenin, similar to the criteria used by Mineta et al. [Stained sections were independently enumerated by two coauthors (D. R. Mehregan and M. Campbell), who were blinded to patient medical records for each case. Blinded enumeration was performed under light microscopy at 400x magnification, and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section. Per section, the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields. The mean value of the three fields was used to estimate the relative density of cells in a specimen. To increase assessment accuracy, all positively and negatively stained melanomas or nevus cells in a visual field were individually counted as opposed to being graded as ranges of percentages. Enumeration data were reviewed independently by each evaluator. When independent readings for positively stained cells differed by 20% for a given section, both evaluators reviewed the section together to establish a consensus reading. A specimen was considered negative if less than 4% of the cells were immunostained for Rad6 or a et al. .β-catenin localization. Spearman's rank correlation was used to assess the pairwise association between age and percent of Rad6 positive and β-catenin positive cells. Multinomial logistic regression was used to assess the simultaneous association of Rad6 and age with diagnostic group. Adjustments were made for multiple comparisons using Wilcoxon rank sum tests with Bonferroni correction for pairwise comparisons.Kruskal-Wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells. Chi-square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and P = 0.08) and significantly in age (P = 0.0001). Significant age differences were observed between individuals with nevi and those with either primary melanomas (P = 0.02) or metastatic melanomas (P = 0.0001). In contrast, no age difference is observed between individuals with primary and metastatic melanomas (P = 0.27). Significant differences in age were also observed between groups defined by β-catenin localization (P = 0.007). Individuals with β-catenin localized in the cytoplasm were significantly younger than individuals with β-catenin localization at the cell membrane (P = 0.02) and marginally younger than individuals with β-catenin localization at both the cytoplasm and the cell membrane (P = 0.05). When age was categorized as <50, 50–60, and >60 years, there were statistically significant differences in Rad6 expression between the groups (P = 0.0008), although there is substantial variability (P = 0.04) and <50 years old group (P = 0.001). A 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases, and these cases were evaluated together to establish a consensus reading.Our analysis included 30 individuals diagnosed with nevi, 29 with primary melanoma, and 29 with metastatic melanoma . These giability . Median β-catenin. β-Catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas. Also, the percentages of nevi (93%), primary melanoma (97%), and metastatic melanoma (93%) that expressed β-catenin in more than 50% of the cells did not differ significantly  or metastatic melanoma (26%) (data not shown).Melanoma development and progression were not associated with significant changes in percentage of specimens expressing  Figures , and 3. β-catenin in the nucleus  to primary and metastatic melanomas  to primary and metastatic melanomas  or the cytoplasm  did not differ in β-catenin localization as β-catenin was localized at the plasma membrane, in the cytoplasm, or in both  did not express Rad6. Conversely, all primary melanomas 100%) and the majority of metastatic melanomas (96%) exhibited greater than 50% Rad6 expression. The increase in tumor populations expressing Rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant (00% and t% did notP < 0.001) even when age group (P = 0.65) and gender (P = 0.24) are included in the model. The model predicts that every 1% increase in Rad6 expression results in a 9% increase in the probability that a lesion is melanoma. If we assume that a predicted probability of >0.5 indicates melanoma, the model with only Rad6 has sensitivity of 93% and specificity of 80%. These results are very encouraging; however, they need to be validated in a larger study.Interestingly, only one of the 30 nevi  expressed Rad6 in >80% of the cells, and none of the primary and metastatic melanomas expressed Rad6 in <40% of the cells . These rβ-catenin and Rad6 differed considerably in nevi. Approximately 93% of nevi expressed β-catenin in more than 50% of the cells, whereas only 27% of the same population of nevi expressed Rad6 . There is a 2.7-fold difference in the percentage of primary melanomas (100%) expressing Rad6 compared to nevi (37%) and virtually no difference in β-catenin expression between primary and metastatic melanomas (100%). Accordingly, Rad6 and β-catenin expressions in primary melanoma were not correlated . A significant correlation between Rad6 and β-catenin positive cells was observed in metastatic melanoma . However, this association diminished  following the exclusion of two observations which are disproportionally influential (one with <50% positive Rad6 and one with <50% positive β-catenin).The expression profiles of sed Rad6 . β-Catenβ-catenin expressions in benign and malignant melanocytic tumors to determine whether Rad6 works in concert with β-catenin to influence melanoma development and progression. Rad6 and β-catenin positively regulate each other in breast cancer [β-catenin has been implicated in the pathogenesis of melanoma and other cancer types, data about the role of Rad6 in cancer pathogenesis are mostly limited to breast cancer. Approximately 30% of melanomas develop in preexisting benign melanocytic neoplasms (nevi) [β-catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [This is the first study to characterize Rad6 expression in cutaneous benign and malignant melanocytic tumors. In this study, we examined the association between Rad6 and t cancer , 18. Hows (nevi) . Therefot cancer , 28.β-catenin has been implicated in driving the development and progression of several cancer types  [β-catenin do not contribute to melanoma initiation and progression since no difference in β-catenin levels was found between nevi, primary melanoma, and metastatic melanoma . The high expression levels of β-catenin are in line with the crucial role of β-catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells [β-catenin staining in nevi (100%) and primary melanoma  but are higher than reported in metastatic melanoma  [β-catenin levels in metastatic melanomas between the studies can be attributed to differences in the type of metastatic tissues. While we studied only melanoma metastases to the skin, other studies either obtained 58% of their specimens from lymph nodes, tonsil, and liver or did not identify the anatomical site of their metastases [Accumulation of nuclear and cytoplasmic cancers) –31. Howema cells . Also, o5%, 68%) . The vartastases . Furthertastases , 34.β-catenin in nevi than in melanoma  [β-catenin suggest poor prognosis and decreased overall survival of melanoma patients [β-catenin in all the nevi and melanomas analyzed in our study was surprising. Usage of different anti-β-catenin antibodies may explain in part the discrepancy in nuclear β-catenin expression observed between the studies. However, our results are consistent with the lack of nuclear β-catenin reported in four studies which comprised 57 nevi, more than 55 primary melanomas, and 20 metastatic melanomas [β-catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas [β-catenin was reported in only 6.4% of the melanomas [β-catenin was reported in only 13 cases (5.6%) and therefore those cases were excluded from analysis [β-catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for β-catenin in nevi and melanoma. This notion is supported by a role for cytoplasmic β-catenin to execute functions that do not require nuclear translocation  [Previous studies have shown higher percentages of nuclear sus 15%) , 22. Thopatients , 22. In elanomas , 35–37. elanomas . In anotelanomas . Finallyanalysis . Taken td NF-kB) .β-catenin. The percentage of cases that expressed β-catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas, respectively. Concurrently, the percentage of cases that expressed cytoplasmic β-catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas, respectively  as in our study, Bachmann et al. also reported an association between nevus to melanoma development and relocation of β-catenin to the cell membrane [β-catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in β-catenin distribution corresponded with the malignant transition of nevi [β-catenin relocation from cytoplasm to cell membrane [β-catenin relocation from cytoplasm to cell membrane has been reported to block β-catenin signaling in a human embryonic kidney (HEK293) cell line [β-catenin expression in melanoma [β-catenin is selectively decreased, a phenomenon that has been associated with unfavorable melanoma prognosis [β-catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated β-catenin at the membranous site.A major finding of this study is the association between melanoma development and intracellular redistribution of ectively . We hypomembrane . Neverthmembrane ; and (ivell line . Of notemelanoma , cytoplarognosis , 22. Our in situ to invasive primary carcinoma and metastatic cancer [Rad6 has been implicated in early breast cancer development since an increase in Rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . In contc cancer , 28. In c cancer , 17, we β-catenin, and, in turn, β-catenin positively upregulates Rad6 transcription [β-catenin and Rad6 expression does not appear to be conserved in melanoma as the expression profiles of β-catenin and Rad6 differed considerably in nevi. Approximately 93% of nevi expressed β-catenin compared to only 27% of nevi that expressed Rad6 in more than 50% of their cells (β-catenin expression in nevi is likely driven by regulators other than Rad6. At first glance, it would appear that β-catenin and Rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas. Also, these findings correspond to the 80% correlation between Rad6 and β-catenin expressions in primary breast cancer [β-catenin expression, because Rad6 expression is low in nevi despite the presence of high cytoplasmic β-catenin expression that is comparable to primary melanoma. This notion is confirmed by lack of statistical correlation between Rad6 and β-catenin expressions in primary melanoma. β-Catenin is not the only activator of Rad6; for instance, Rad6 is activated by nerve growth factor in nervous tissue [β-catenin and Rad6 expressions. Taken together, our study does not support a direct positive interaction between β-catenin and Rad6 in either benign or malignant melanocytic tumors.In benign and malignant breast tumors, Rad6 stabilizes cription –17. Howeir cells . These os tissue . Therefoβ-catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors. Taken together, in contrast to the Rad6 β-catenin positive relationship in breast cancer [β-catenin and Rad6 in benign or malignant melanocytic tumors. Finally, our findings suggest a role for the cytoplasmic to membrane translocation of β-catenin in the development of primary melanoma. Future studies will determine whether newly generated β-catenin at the membranous site coincide with β-catenin translocation from the cytoplasm.We characterized for the first time Rad6 expression in cutaneous benign and malignant melanocytic tumors. We are showing a striking upregulation of Rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas. These findings strongly suggest a role for Rad6 in the development of primary melanoma and metastatic disease. We show that in contrast to Rad6, t cancer –17, our