To evaluate the destruction complex of beta-catenin by the expression of the proteins beta-catetenin, adenomatous polyposis coli, GSK3β, axin and ubiquitin in colorectal carcinoma and colonic adenoma. t, χ2, Mann-Whitney, and McNemar tests, and univariate logistic regression analysis were used. Tissue samples from 64 patients with colorectal carcinoma and 53 patients with colonic adenoma were analyzed. Tissue microarray blocks and slides were prepared and subjected to immunohistochemistry with polyclonal antibodies in carcinoma, adjacent non-neoplastic mucosa, and adenoma tissues. The immunoreactivity was evaluated by the percentage of positive stained cells and by the intensity assessed through of the stained grade of proteins in the cytoplasm and nucleus of cells. In the statistical analysis, the Spearman correlation coefficient, Student’s  In colorectal carcinoma, the expressions of beta-catenin and adenomatous polyposis coli proteins were significantly higher than in colonic adenomas . The immunoreactivity of GSK3β, axin 1 and ubiquitin proteins was significantly higher  in colorectal carcinoma than in the colonic adenoma and adjacent non-neoplastic mucosa. The immunohistochemistry staining of these proteins did not show significant differences with the clinical and pathological characteristics of colorectal cancer and colonic adenoma. These results suggest that, in adenomas, the lower expression of the beta-catenin, axin 1 and GSK3β proteins indicated that the destruction complex of beta-catenin was maintained, while in colorectal carcinoma, the increased expression of beta-catenin, GSK3β, axin 1, and ubiquitin proteins indicated that the destruction complex of beta-catenin was disrupted. Colorectal cancer (CRC) is considered the result of the cumulative effect of multiple mutations within the cell that allow it to escape growth control and regulatory mechanisms.,2 It has become apparent that the accumulation of gene mutations in a clonal cell results in the transition from the normal colon epithelial cell into colorectal carcinoma.All cells exist under strict regulation of signals for growth, apoptosis, differentiation, cell-cell interactions and cell-extracellular matrix interactions. Beta-catenin is a subunit of the cadherin protein complex and acts as an intracellular signal transducer in the Wnt signaling pathway.,4 The canonical Wnt signaling pathway, acting through beta-catenin, modulates a variety of cellular processes, including proliferation, survival, apoptosis, differentiation, cell adhesion and motility.Beta-catenin, a member of the catenin protein family, is a dual-function protein that regulates the coordination of cell-cell adhesion and gene transcription.,5 Mutations and the overexpression of beta-catenin are associated with many cancers, including hepatocellular carcinoma, colorectal carcinoma, lung cancer, malignant breast tumors, and ovarian and endometrial carcinomas.The Wnt effector beta-catenin is a transcriptional co-activator that can also mutate to a potent oncogene, while the canonical Wnt signaling pathway stabilizes beta-catenin transcription. In the absence of a Wnt signal, beta-catenin binds to the APC tumor suppressor protein and this protein is recruited to the destruction complex, which promotes its phosphorylation by axin, CK1 and GSK3β.,3,4,6 The phosphorylation of beta-catenin by GSK3β leads to its ubiquitination forming the complex polyubiquitin/beta-catenin and, subsequently, is degraded into the proteasome.,6The mutation of genes correspond to the formation of the complex destruction of the beta-catenin protein and triggers a cascade of events that lead to the disruption of the APC/axin 1/GSK3β complex and accumulation of unphosphorylated beta-catenin in the cytoplasm.,4 Then, the beta-catenin stabilized protein translocates into the nucleus, in which it activates the expression of a range of genes in association with the T-cell factor (TCF) and the lymphoid-enhancing factor (LEF), both transcription factors, that, in turn, activate the target genes that are related to carcinogenesis , casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β).ogenesis .-4 A large body of evidence has suggested that aberrant activation of Wnt signaling following APC loss is a major cause of colon adenoma formation.-6 Loss-of-function and gain-of-function studies of Wnt proteins and beta-catenin have suggested that aberrant Wnt signaling activation after APC loss is responsible for the initiation of intestinal adenoma.,7 Therefore, hyperactivation of Wnt signaling is generally viewed as the key event for the initiation of intestinal adenoma after APC loss.,8 An important step is to elucidate the molecular mechanisms underlying the cooperative functions of Wnt signaling, which may lead to the identification of novel therapeutic targets for the prevention and treatment of gastrointestinal cancer.,8The so-called adenoma-carcinoma sequence nowadays still represents the paradigm and the support of our understanding of the molecular and genetic basis of this disease. In human CRC, Wnt signaling activation is hypothesized to be the key event for adenoma initiation, whereas additional mutations are required for the progression from adenoma to carcinoma.,4,5 Still, little is known about the multiple interactions and mutual influences of this defective pathway, and about the mechanisms responsible for the genetic instability contributing to the establishment of the multiple genetic defects that are necessary to promote tumor progression and malignancy.,2,4The genetic mutations accumulated during tumor development are responsible for the deregulation of key signal transduction pathways, such as Wnt, which is responsible for uncontrolled cell growth, inhibition of apoptosis, and immortalization.,8By defining the molecular alterations involved in the development of the sporadic CRC, it is possible to expect the achievement of specific molecular targeting for the treatment of already-established tumors, as well as for chemoprophylaxis interventions.To evaluate the destruction complex of beta-catenin by the expression of beta-catetenin, adenomatous polyposis coli, glycogen synthase kinase 3β, axin, and ubiquitin proteins in colorectal carcinoma and colonic adenoma. By defining this protein alterations of the Wnt signaling pathway involved in the adenoma and colorectal cancer, we can to expect the achievement of specific molecular targeting for the treatment of already established tumors.This is an observational, longitudinal and retrospective study. A total of 64 patients with CRC and 53 patients with colonic adenoma were studied. Between 2006 and 2010, 64 patients with CRC were consecutively submitted to curative surgery with resection of their colorectal tumors. Patients with colonic adenoma underwent the removal of their adenomas by colonoscopy.The inclusion criteria were adult patients with CRC or colorectal adenoma confirmed by histological analysis. Exclusion criteria were the presence of colorectal hereditary nonpolyposis neoplasm (Lynch syndrome), CRC associated with inflammatory bowel disease, and intestinal polyposis syndromes.In patients submitted to surgery for CRC, 64 tissue samples were obtained from the tumor and from adjacent non-neoplastic colorectal mucosa, located 10cm from the upper margin of the neoplasm. In the 53 patients with colonic adenomas, there were 71 resected adenomas.Among the patients operated with CRC, 33 (51.6%) were females. The median age was 69.2±7.4 years (51 to 90 years). The location of the CRC was in the colon in 38 (59.4%) patients and in the rectum in 26 (40.6%).Regarding the adenomas, 27 (50.9%) patients were male. The median age was 60.7±3.4 years (29 to 88 years). The location of the adenomas was 46 (86.8%) in the left colon and 7 (13.2%) in the right colon. There were no rectal adenomas. presence of synchronous metastases, and immunostaining (staining intensity and percentage of stained cells scores) of the antibodies that were used in the colorectal tissue. In patients with colonic adenoma, statistics were recorded regarding the morphological characteristics of the neoplasm  and the immunostaining (staining intensity and percentage of stained cells scores) in the colonic tissue.Within patients with CRC, information was recorded regarding the location, size, level of invasion in the intestinal wall, inflammatory infiltrate degree, lymph node involvement, degree of tumor differentiation, lymphatic-vascular-neural invasion, Classification of Malignant Tumours (TNM),The tissues were fixed in formalin and routinely processed by using the paraffin-embedding method for histological analysis. Histological sections with 3µ thickness were obtained from each block. All slides were stained with hematoxylin-eosin (HE) and revised by the pathologist for confirmation of the diagnosis. The paraffin blocks were cut 3µm thick, and the slides were prepared for immunohistochemical study. The antibodies which were used included: rabbit polyclonal primary anti-beta-catenin; rabbit polyclonal anti-APC; rabbit polyclonal primary anti-GSK3β; rabbit polyclonal primary anti-axin; mouse polyclonal primary anti-ubiquitin . All antibodies were used at a dilution of 1:100. The positive controls used included normal colon tissue for APC and axin, HeLa whole-cell lysate for GSK3β and ubiquitin, and human colon cancer for beta-catenin. A similar slide was used as a negative control, subtracting the primary antibody from the reaction.In the stained slides, the areas of the tumor were identified for the preparation of tissue microarray (TMA). The TMA block was prepared by using Beecher™  equipment, in according to the previously described protocol that was used in our laboratory.The anti-beta-catenin, anti-APC and anti-GSK3β antibodies were used in CRC tissue, colonic adenoma and in adjacent non-neoplastic mucosa. The anti-axin and anti-ubiquitin antibodies were used in CRC tissue and adjacent non-neoplastic mucosa . Positivity was considered to be a score zero if less than 5% of epithelial cells were stained in the neoplasm; a score 1 if there was 5% to 25%; a score 2 if there was 26% to 50%; a score 3, if there was 51% to 75%; and a score 4, if more than 75% of epithelial cells were stained in the neoplasm. The intensity was considered to be score zero when there were no stained cells; a score 1 when the stain was weak; a score 2 when the stain was moderate; and a score 3 when the intensity of stained cells was strong. The final score of immunoexpression, which varied between zero and 12, was obtained by multiplying the scores of intensity and positivity. The immunoexpression was classified as reduced when the final score was among zero and 8, and strong if the final score was among 9 and 12.The immunoexpression score was prepared according to the method described by Hao et al.All slides were analyzed by two well-trained, independent researchers who had no access to any pathological information. The final score (zero to 12) was the result of a mean score by the researches. In most cases, there was agreement between the two evaluations of the final score. When there was not agreement, a consensus score was adopted.The loss by technical failure or inconclusive results has made it impossible to evaluate the immunohistochemical study of some samples, and these events were the reasons why they were excluded from the study.t, χ2, Mann-Whitney and McNemar tests were used to assess the significance of the differences in the clinical and pathological parameters, and associations with proteins’ immunoexpression. The univariate logistic regression analysis  and multivariate analysis were used to identify the dependent and independent variables. The statistical significance level was set at 5% (p<0.05), and the data were analyzed using the Statistical Package for Social Sciences® software , version 17.0.The quantitative results were described as the mean and standard deviation. Qualitative data were described as frequencies. The correlation between the scores of the immunohistochemical expression of the proteins with the clinicopathological parameters was calculated by Spearman’s correlation coefficient. The Student’s The present study was conducted according to the ethical principles of the Declaration of Helsinki from the World Medical Association and has been approved by the Research Ethics Committees of our institution, CAAE: 0042.0.338.000-09.In terms of CRC, the average tumor size was 5.2±1.3cm (0.5 to 12cm). The size of the tumor along its longest axis was >5cm in 43 (67.1%) patients and ≤5cm in 21 (32.8%). Lymph node metastasis was found in 31 (48.4%) patients. The invasion of blood vessels was observed in 21 (32.8%) patients, lymphatic vessel invasion in 24 (37.5%) and neural invasion in 12 (18.7%). Eleven (17.2%) carcinomas were well differentiated; 51 (79.7%), moderately differentiated; and 2 (3.1%), poorly differentiated. The CCR superficially infiltrated (T1 + T2) the intestinal wall in 18 (28.1%) patients and deeply (T3 + T4) in 46 (71.9%). The presence of synchronous metastases was observed in 14 (21.9%) patients. Relapse occurred in 13 (20.3%) patients and 12 (18.7%) of them died for this reason. The average follow-up time was 19.1±2.8 months (3 to 36 months).The average size of the polyps was 0.6±0.2cm (0.4 to 1.7cm). The polyps were <1.0cm in 34 (64.1%) patients and ≥1.0cm in 19 (35.8%). The histological type of adenoma was tubular in 49 (69%) and tubular-villous in 22 (31%). Moderate atypia were found in 39 (55%) adenomas and mild atypia was noted in 32 (45%).The expression of beta-catenin protein in CRC was significantly (p<0.001) higher than in the adenomas and significantly (p<0.001) higher than in the adjacent non-neoplastic mucosa. This increased beta-catenin expression occurred mainly in the cytoplasm of cells, but nuclear staining was also significant. In adenomas, the staining of beta-catenin was significantly (p<0.0001) lower than in adjacent non-neoplastic mucosa .The immunoreactivity of the APC protein in CRC was significantly (p<0.0001) higher than in the adenoma. The APC protein immunoreactivity in the adjacent non-neoplastic mucosa was significantly (p<0.0001) higher than in the adenoma. There was no significant difference (p=0.24) between the immunoreactivity of the APC protein in CRC and in its adjacent non-neoplastic mucosa.The immunoreactivity of the GSK3β protein in adenoma was significantly lower in the CRC and in the adjacent non-neoplastic mucosa . There was no significant difference (p=1.00) between the immunoreactivity of GSK3β protein in CRC and in the adjacent non-neoplastic mucosa.The immunoreactivity of axin 1 protein was significantly (p=0.039) lower in the adjacent non-neoplastic mucosa. The expression of ubiquitin showed a significant difference (p=0.03) between the CRC and the adjacent non-neoplastic mucosa.The immunohistochemical expression of beta-catenin protein APC, GSK3β, axin 1 and ubiquitin in colorectal adenomas and CRC did not show significant differences with the clinical and pathological characteristics of CRC and colonic adenoma. demonstrated an aberrant expression of beta-catenin mutations in the CCR, and this is an early event in human colorectal carcinogenesis. These authors verified that the adjacent normal epithelium to aberrant crypts showed a strong staining of beta-catenin, an event that was also observed in this study in CRC and in its adjacent non-neoplastic mucosa, but not in colonic adenomas.In humans, the beta-catenin protein is encoded by the gene CTNNB1, which maps at 3p22.1 . Hao et al.,12 The gene is mutated in 63% of sporadic adenomas and in over than 80% of sporadic CRC. The heterozygous mutation is inherent in all cases of familial adenomatous polyposis.-15 The APC protein forms molecular complexes that are capable of eliminating the intra-cytoplasmic beta-catenin, inducing its degradation.,4,12 In the epithelium of the large intestine, APC expression is restricted to regions in which cell replication has ceased, and terminal differentiation was established.The APC gene maps at position 21q in chromosome 5 and encodes a protein with multiple functional domains that interact with proliferation and apoptosis regulators.,3,12 Thus, since the physiological role of the APC protein is a growth advantage, the loss of its function will promote a cellular clonal expansion.,12 When the APC protein is mutated, it loses its binding site in the complex destruction of beta-catenin, which causes an increased expression of beta-catenin protein in the cytoplasm and nucleus.-17In the large intestine, isolated mutations in the APC gene are sufficient to provide a selective growth advantage, by reducing the function of the APC-specific degree sufficient to allow the accumulation of nuclear beta-catenin, promoting the cell proliferation without causing excessive apoptosis. examined the expression of APC and mutated in CRC proteins, the key regulators of beta-catenin, by immunohistochemistry in right-sided serrated polyps. This study implied that the molecular-specific form of beta-catenin may participate in the Wnt-signaling activation of right-sided serrated polyps. Moreover, the loss of mutated in colorectal protein but not APC expression may contribute to the early activation of Wnt signaling in right-sided serrated polyps. In the present study, a decreased immunoreactivity of the non-mutated APC protein was observed in most patients with colonic adenomas. On the other hand, the significant presence of non-mutated APC protein in CRC and in adjacent non-neoplastic mucosa may indicate that the mutated APC protein is not involved as an important component in the process of carcinogenesis in these specific tumors. Wong et al.studied 758 cases of colorectal adenomas and concluded that the malignant conversion of adenomas may not be related to the mutation of the APC gene alone. Thus, the development of colorectal carcinogenesis, even in the absence of the APC-mutated protein, is possible, as the results of the present study also suggested.Li et al. The GSK3β protein acts as an inhibitory key toward the canonical-Wnt signaling pathway. The expression of the GSK3β gene attenuates the proliferation of CRC cells or leads to early apoptosis. Therefore, under physiological conditions, the GSK3β phosphorylates and degrades protein transcription factors and oncoproteins, which suggests that this enzyme would be a suppressive factor in tumor development, which negatively interferes with oncogenic signaling.,23 It was also observed in the present study that the immunoreactivity of the GSK3β-mutated protein was shown to be increased in the CRC and in adjacent non-neoplastic mucosa regarding colonic adenoma.The GSK3β gene maps in chromosome 19 at position 13q.2. The GSK3β is a serine/threonine multifunctional protein, which is able to phosphorylate and inactivate glycogen synthase. The human homolog gene of axin 1 was mapped to chromosome 16p13.3. In sporadic CRC, an increased expression of axin protein suggests that mutation of the corresponding gene can participate in colorectal carcinogenesis.,27 In this way, the present study observed that the staining of the mutated protein GSK3β in CRC was significantly increased with respect to the non-neoplastic adjacent mucosa.Axin 1 has emerged as a major scaffold protein for regulating a variety of signaling pathways and biological functions. The human homolog gene was mapped to chromosome 17p16.38. In normal tissues, the phosphorylated beta-catenin is recognized by the E3 ubiquitin ligase complex forming the phosphorylated poly-ubiquitin/beta-catenin which is proteolyzed via the proteasome.,30The ubiquitin (E3 ubiquitin ligase) is a protein that recruits an E2 ubiquitin-conjugating enzyme, which recognizes the protein substrate and assists or directly catalyzes the transfer of ubiquitin from the E2 ubiquitin-conjugating to the protein substrate. found that the expression of ubiquitin in the CCR was significantly higher than in the adjacent non-neoplastic mucosa, and this result was also found in the present study. Moreover, they observed no significant relation between the expression of ubiquitin protein and the clinical and pathological features of CRC, a finding that was confirmed in this present study.Chen et al.The beta-catenin protein expression was increased in the cytoplasm and nucleus of the neoplastic cells, when compared to the colonic adenoma. The adenomatous polyposis coli protein was mainly altered in adenomas, and that, along with the increase in colorectal cancer of GSK3β, axin 1 and ubiquitin-mutated proteins, may prevent the phosphorylation of beta-catenin by the destroyer complex and its subsequent degradation in the proteasome. This event allows the stabilized beta-catenin protein to translocate into the cell nucleus, in which this protein can activate transcription factors, as well as the expression and activation of target genes related to colorectal carcinogenesis. Additional research is needed to determine whether the ongoing activity of signaling pathways is required for normal and neoplastic tissues, and whether or not these conditions differ sufficiently to allow for therapeutic intervention.


XAV939 and WXL-8 also attenuated rhWNT3A-induced TOPflash luciferase reporter activity in HCC cells, indicating reduced β-catenin transcriptional activity, consistent with decreased nuclear β-catenin levels. In vivo, intra-tumor injections of XAV939 or WXL-8 significantly inhibited the growth of subcutaneous HepG2 xenografts (P < 0.05). We suggest that tankyrase inhibition is a potential therapeutic approach for treating a subgroup HCC with aberrant WNT/β-catenin signaling pathway.Deregulated WNT/β-catenin signaling contributes to the development of a subgroup of hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide. Within this pathway, the tankyrase enzymes (TNKS1 and TNKS2) degrade AXIN and thereby enhance β-catenin activity. We evaluate TNKS enzymes as potential therapeutic targets in HCC, and the anti-tumor efficacy of tankyrase inhibitors  in HCC cells. Using semi-quantitative RT-PCR, we found significantly elevated levels of TNKS1/2 mRNA in tumor liver tissues compared to adjacent non-tumor livers, at protein levels only TNKS1 is increased. In HepG2, Huh7cells, siRNA-mediated knockdown suppression of endogenous TNKS1 and TNKS2 reduced cell proliferation, together with decreased nuclear β-catenin levels. XAV939 and WXL-8 inhibited cell proliferation and colony formation in HepG2, Huh7, and Hep40 cells ( Hepatocellular carcinoma (HCC) is the sixth most common cancer, and second leading cause of cancer deaths worldwide , accountThe WNT/β-catenin pathway is essential during the developmental stage, and in maintaining normal cellular homeostasis, cell proliferation, differentiation, and pluripotency . It is evia parsylation. Thus, these enzymes also promote mitosis and telomerase function. As such, tankyrases are promising candidate targets for anti-cancer molecular therapies ), fluorescence (mrfp or egfp), and positron emission tomography (ttk), at a multiplicity of infection of 5, as reported previously /2. The whole body weight of each animal was also monitored during the course of treatment.Animal studies were carried out in compliance with all federal and local institutional rules for the conduct of animal experiments. To generate subcutaneous xenografts, 1 × 10eviously . Opticalt-test was used to calculate statistical differences between paired groups. Differences between two or more experimental groups were analyzed by one-way ANOVA with Tukey's post-hoc test. P values of < 0.05 were considered significant. All statistical analysis was carried out using GraphPad Prism .All cell-based experiments were independently repeated at least three times, and representative experiments are shown. All quantitative data are reported as means ± SD. Paired


It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKβ induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFβ (transforming growth factor β)-activated kinase-1]-catalysed phosphorylation of Ser177 and, secondly, the IKKβ-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKβ to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO  component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKβ at Ser177 and IKKα at Ser176. These findings may be of general significance for the activation of other protein kinases.IKKβ {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β} is required to activate the transcription factor NF-κB, but how IKKβ itself is activated  We have discovered how a key enzyme that controls the immune system is switched on during infection by bacteria and viruses. Known by the acronym IKKβ, it triggers the production of many proteins that are needed to combat these pathogens. The canonical IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β} complex, consisting of the protein kinases IKKα and IKKβ  and a regulatory component called NEMO  –5. Never176 and Ser180 (IKKα) or Ser177 and Ser181 (IKKβ) ) was expressed as a GST fusion protein in HEK (human embryonic kidney)-293T suspension cells and, after cell lysis, was purified from the cell extracts by chromatography on glutathione–Sepharose. The GST-fusion protein was released from the glutathione–Sepharose by cleavage of the GST tag with PreScission protease. A catalytically active TAK1–TAB1 (TAK1-binding protein 1)-fusion protein  was expr177  or at both Ser177 and Ser181  were obtained from Cell Signaling Technology, whereas the antibody recognizing IKKβ phosphorylated at Ser181 was from Abcam . Antibodies that recognize p105/NF-κB1 phosphorylated at Ser933 , GAPDH  and all forms of p38α MAPK  and JNK  were from Cell Signaling Technology. Antibodies recognizing NEMO , all forms of IKKβ  and the HA (haemaglutinnin) tag  were from the sources indicated. An antibody raised in sheep against the full-length human IKKβ catalytic subunit  was produced and affinity purified by the Antibody Production Team of the MRC Protein Phosphorylation and Ubiquitylation Unit at Dundee (co-ordinated by Dr James Hastie). An antibody recognizing Met1-linked ubiquitin chains was generously provided by Vishva Dixit, Genentech, U.S.A. and the antibody recognizing Lys63-linked ubiquitin chains was purchased from Merck-Millipore .An antibody recognizing the HOIP {HOIL1 [haem-oxidized IRP2 (iron regulatory protein 2) ubiquitin ligase 1]-interacting protein} component of LUBAC (linear ubiquitin chain assembly complex) was raised in sheep and purified as described in . AntibodDNA encoding IKKβ (NCBI BAI45894.1) was amplified from total thymus RNA using the One Step RT PCR kit (Life Technologies). It was then cloned into pCR2.1 (Life Technologies), sequenced and sub-cloned into the Not1 site of pRetrox tight HA. Mutations were created following the QuikChange Site-Directed Mutagenesis method, but using KOD Hot Start DNA Polymerase (EMD Millipore).3Csk4 . Thereafter, cells were rinsed in ice-cold PBS and extracted in lysis buffer . Cell extracts were clarified by centrifugation (21000 g for 10 min at 4°C) and protein concentrations determined by the Bradford assay. Cell extract protein (20 μg) was separated by SDS/PAGE (8% gel), transferred on to PVDF membranes and proteins detected by immunoblotting and visualized by treating the blots with enhanced chemiluminescence (Amersham).MEFs and HEK-293 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 2 mM glutamine, 10% (v/v) FBS, and the antibiotics streptomycin (0.1 mg/ml) and penicillin (100 units/ml). DNA constructs were transfected into HEK-293 cells using polyethyleneimine (Polysciences). BMDMs (bone-marrow-derived macrophages) were obtained by culturing bone marrow from the tibia and femurs of mice in the presence of mouse M-CSF and replating for 24 h before stimulation. Kinase inhibitors (10 mM) dissolved in DMSO, or an equivalent volume of DMSO for the control incubations, were added to the culture medium of cells grown as monolayers. After 1 h at 37°C, MEFs were stimulated with IL-1α or TNF and BMDM with LPS or PamMice in which wild-type NEMO was replaced by the polyubiquitin-binding-defective mutant NEMO[D311N] were generated by Taconic-Artemis using conventional technology and their characterization will be reported elsewhere. Primary MEFs from NEMO[D311N] mice and wild-type littermates were generated at E11.5 (embryonic day 11.5), whereas MEFs from knockin mice expressing the inactive C879S mutant of HOIP were generated at E10.5 . ImmortaIKKα-deficient MEFs stably expressing HA-tagged empty vector (EV), wild-type HA–IKKβ (WT), HA–IKKβ[S177A], HA–IKKβ[S177E] or HA–IKKβ[D166A/S177E] were generated by retroviral transduction using an MMLV (Moloney murine leukaemia virus)-based system prepared with the VSVG (vesicular-stomatitis-virus glycoprotein) envelope protein. Retroviral particles were prepared according to the manufacturer’s instructions (Clontech). Viruses encoding the gene of interest and the Tet-On protein were harvested 48 h after transfection, diluted 4-fold with fresh medium and incubated for 24 h with IKKα-deficient MEFs in the presence of 2 μg/ml protamine sulfate (Sigma). Fresh medium containing 1 μg/ml G418 (Tet-On) and 3 μg/ml puromycin (gene of interest) was added to select the transduced cells. Cells were cultured for 16 h with doxycycline (0.1–1.0 μg/ml) to induce the expression of wild-type and mutant forms of IKKβ.2. Dephosphorylation was initiated by the addition of 100 μg of GST–PP1γ. After 60 min at 30°C the immunoprecipitates were collected, washed three times with 1.0 ml of lysis buffer containing 0.5 M NaCl, and three times with 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA and 0.1% 2-mercaptoethanol to remove the phosphatase.To immunoprecipitate transfected HA-tagged IKKβ, cell extract protein (40 μg) was incubated for 60 min at 4°C with 4 μg of anti-HA antibody, whereas for the endogenous IKKβ 0.2 mg of cell extract protein was incubated with 2.5 μg of anti-IKKβ antibody. Protein G–Sepharose was added  and, after mixing for 30 min at 4°C, immune complexes were collected by brief centrifugation, washed three times in cell lysis buffer plus 0.5 M NaCl, and three times with 50 mM Tris/HCl (pH 7.5), 0.05 M NaCl and 1.0 mM DTT, then resuspended in 0.03 ml of 50 mM Hepes, 10 mM NaCl, 2 mM DTT and 0.1% Brij35 (pH 7.5) containing 1 mM MnCl32P]ATP (5×105 c.p.m./nmol). After incubation for 10 min at 30°C on a shaking platform, the incorporation of 32P radioactivity into the peptide substrate was measured as described in  mutant . To investigate whether the phosphorylation of Ser177 could activate IKKβ in the absence of Ser181 phosphorylation, we incubated IKKα-deficient MEFs with BI605906 to supress the phosphorylation of Ser181 and assayed the endogenous IKKβ activity after its immunoprecipitation from the extracts of IL-1-stimulated cells. These experiments showed that IKKβ mainly phosphorylated at Ser177 had a much lower activity than IKKβ phosphorylated at both Ser177 and Ser181  and was not decreased significantly in Pam3CSK4- or LPS-stimulated BMDMs . This suggests that in wild-type cells, in which IKKα, IKKβ and NEMO form a single ternary complex, phosphorylation of IKKβ at Ser181 can be catalysed in trans by IKKα if IKKβ is inhibited by BI605906.The experiments presented above were carried out in IKKα-deficient MEFs or in BMDMs from knockin mice expressing the catalytically inactive IKKαS176A/S180A] mutant, because IKKα activity is unaffected by BI605906  mutant B, simila] mutant A.http://www.biochemj.org/bj/461/bj4610531add.htm), but was suppressed by the TAK1 inhibitors NG25 or 5Z-7-oxozeaenol . These control experiments indicated that activation of the TAK1 complex was unimpaired in MEFs from HOIP[C879S]- or NEMO[D311N]-knockin mice. The TAK1 inhibitor NG25 did not affect the IL-1-stimulated formation of Lys63-linked ubiquitin chains significantly and actually enhanced Met1-linked ubiquitin chain production in IKKα-deficient MEFs . Thus TAK1 activity is not required for the IL-1-stimulated formation of Lys63-linked or Met1-linked ubiquitin chains and NG25 does not suppress the phosphorylation of IKKβ by preventing formation of the ubiquitin chains. The enhanced formation of Met1-linked ubiquitin chains in the presence of NG25 implies the existence of a TAK1-dependent feedback control mechanism for restricting the formation of these ubiquitin chains.The IL-1-stimulated phosphorylation of JNK1/JNK2 and p38α MAPK in MEFs from HOIP[C879S] or NEMO[D311N] mice was similar to wild-type MEFs . It is therefore possible that the interaction of NEMO with IKKβ in the canonical IKK complex and/or the recruitment of the TAK1 complex to Lys63-linked ubiquitin chains are factors that prevent TAK1 from phosphorylating Ser181 in cells.Finally, it should be noted that although TAK1 phosphorylates the IKKβ–NEMO complex at Ser177, which is a priming event that permits IKKβ to phosphorylate itself at Ser181, which is needed before IKKβ can phosphorylate exogenous substrates, such as IκBα  permitted the IKKβ catalytic subunit to autophosphorylate at Ser181 and this induced activation even in cells that had not been stimulated with IL-1 or TNF. Interestingly, the other two members of the IKK subfamily of protein kinases, IKKε and TBK1 {TANK [TRAF (TNF receptor-associated factor)-associated NF-κB activator]-binding kinase 1}, both possess a glutamic acid at position 168 in their activation loops, which is the amino acid residue equivalent to Ser176/Ser177 of IKKα/β, and they are activated by the phosphorylation of Ser172, the site equivalent to Ser180/Ser181 of IKKα/IKKβ [in vivo and why they are instead activated by the canonical IKK complex and by autophosphorylation in response to IL-1 [The mutation of Ser to IL-1 .63-linked and Met1-linked ubiquitin oligomers [63-linked ubiquitin chains interact with the TAB2 and TAB3 components of TAK1 complexes, in-ducing the auto-activation of TAK1 [1-linked ubiquitin chains formed by the action of the E3 ubiquitin ligase LUBAC [1-linked ubiquitin chains formed in response to IL-1 are attached covalently to Lys63-linked ubiquitin chains, which may facilitate the TAK1-dependent activation of canonical IKK complex by recruiting both protein kinases to the same ubiquitin chains [176/Ser177, and hence the phosphorylation of Ser180/Ser181, was suppressed in MEFs that were unable to produce Met1-linked ubiquitin chains or that expressed a ubiquitin-binding-defective mutant of NEMO  sequences, enabling them to activate their cognate MAPKs. Similarly, most MAPKs are activated by the dual phosphorylation of a threonine and a tyrosine residue that are located in Thr-Xaa-Tyr within their activation loops. Although the activation of many MKKs and MAPKs is thought to be catalysed by a single protein kinase, the present study has shown that the requirement for one ‘upstream’ protein kinase does not exclude the possibility that a second protein kinase is also required. Indeed, we have shown that the activation of JNK requires the MKK7-catalysed phosphorylation of the threonine and the MKK4-catalysed phosphorylation of the tyrosine residue within the Thr-Xaa-Tyr motif . The act


To determine the clinical significance of C-X-C chemokine receptor type 4 (CXCR4) and β-catenin in osteosarcoma, their protein expression levels were assessed in 96 osteosarcoma and 20 osteochondroma cases using immunohistochemistry. Additionally, CXCR4 and β-catenin mRNA expression levels were measured in 16 fresh osteosarcoma and 16 adjacent healthy tissue samples using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the osteosarcoma samples, the positive CXCR4 protein expression rate was significantly higher than the rate in the osteochondroma samples . Furthermore, β-catenin protein expression was detected in 61.46% of osteosarcoma cases and 25.00% of osteochondroma cases. Similarly, the RT-qPCR data identified increased CXCR4 and β-catenin mRNA expression levels in the osteosarcoma compared with adjacent control tissues. It was determined that CXCR4 (P<0.01) and β-catenin (P<0.05) expression were significantly associated with the clinical Enneking stage, metastasis and survival of osteosarcoma. Furthermore, multivariate analysis identified CXCR4 and β-catenin protein expression levels, as well as clinical stage and metastasis, as significant risk factors for survival in patients with osteosarcoma (P<0.05). In conclusion, the present study determined that CXCR4 and β-catenin are abnormally expressed in osteosarcoma tissues, and, therefore, may be important during osteosarcoma progression. Osteosarcoma predominantly occurs in children and young adults , and is in vivo expression of CXCR4 and β-catenin in human osteosarcoma, and to analyze the association between the expression of these proteins and clinical prognosis.The Wnt/β-catenin signaling pathway is important in embryogenesis and organ development ,15, and 2 for 5 days/course), methotrexate (8 g/m2 for 1 day/course) and Adriamycin (50 mg/m2 for 1 day/course)]. Of the 96 patients, 44 were female and 52 were male, with a median age of 18 years . A total of 20 osteochondroma specimens were used as the normal controls, including 13 male and 7 female patients with a median age of 20 years . Follow-up care was provided for a minimum of three years. Following resection, formalin-fixed, paraffin-embedded blocks of the osteosarcoma and osteochondroma specimens were retrieved from the Department of Pathology of the Fourth Military Medical University. All samples were evaluated for diagnosis by two similarly experienced pathologists. In addition, 16 pairs of osteosarcoma and adjacent healthy tissue samples were obtained from 16 patients who underwent tumor resection at Tangdu Hospital of the Fourth Military Medical University between July 2013 and December 2013.All patients or their guardians provided written informed consent for participation in the present study. In addition, ethical approval was obtained from the Ethics Committee of the Fourth Military Medical University . Fresh osteosarcoma specimens were obtained from 96 patients who underwent tumor resection at Tangdu Hospital of the Fourth Military Medical University between March 2007 and November 2009. No patients received preoperative chemotherapy or radiotherapy, however, patients with Enneking stage I, II, III or IV disease  received−ΔΔCt). The PCR primers were as follows: Forward, 5′-AATAAAATCTTCCTGCCCACC-3′ and reverse, 5′-CTGTACTTGTCCGTCATGCTTC-3′ for CXCR4; forward, 5′-TGAGCACCTGTTTGCCTGAA-3′ and reverse, 5′-ATGAGCAGCACTCGGACCTT-3′ for β-catenin; and forward, 5′-TAGTTGCGTTACACCCTTTCTTG-3′ and reverse, 5′-TCACCTTCACCGTTCCAGTTT-3′ for β-actin. All experiments were independently performed in triplicate at least three times.Osteosarcoma and adjacent healthy tissue  were harvested, and TRIzol® reagent  was used to extract total RNA. First-strand complementary (c)DNA was synthesized using the Avian Myeloblastosis Virus First-Strand cDNA Synthesis kit and oligo(dT) primers , according to the manufacturer's instructions. RT-qPCR was performed using LightCycler®480 software  with the SYBR® Green PCR Master Mix . β-actin was used as the internal housekeeping gene and relative gene expression was calculated using the cycle threshold (Ct) method . Subsequently, 3% hydrogen peroxide was used to block endogenous peroxidase activity for 15 min. Slides were incubated overnight with primary antibodies [rabbit polyclonal anti-CXCR4  and rabbit polyclonal anti-β-catenin ] and then with secondary antibodies for 30 min at room temperature. Streptavidin-peroxidase was applied and EnVision™ and universal 3,3′-diaminobenzidine detection kits  was used with an extra washing step. The slides were counterstained with hematoxylin and mounted. Immunostaining was compared with osteochondroma samples as the normal controls. Negative controls were obtained by substituting the primary antibody with PBS for each protein.CXCR4 and β-catenin immunohistochemistry were examined as previously described . Cytopla2 test was used. Correlations between the target protein expression and clinicopathological features were assessed using the χ2 test. Furthermore, the Kaplan-Meier product limit method was used to evaluate survival after surgery and multivariate survival analysis was performed using the Cox proportional hazard model. P<0.05 was considered to indicate a statistically significant difference (two-tailed probability).A patient was defined as CXCR4- or β-catenin-positive if all respective evaluated samples exhibited strong positive CXCR4 or β-catenin protein expression. Thus, a tumor was negative if all samples from the patient were immunohistochemically negative. All statistical analyses were performed using SPSS software . To investigate the statistical association between CXCR4 and β-catenin protein expression in the same sample, Pearson's χ2 test demonstrated that the expression of these two markers was statistically different in osteosarcoma and osteochondroma  and the expression of β-catenin was significantly increased in osteosarcoma compared with osteochondroma samples. Furthermore, it was observed that cytoplasmic β-catenin expression was upregulated and membrane-associated β-catenin expression was downregulated in advanced stage tumors. Correlation analysis indicated that aberrant β-catenin expression was significantly associated with metastasis and decreased patient survival. Furthermore, an absence of β-catenin expression significantly correlated with increased patient survival. Aberrant β-catenin and CXCR4 expression were simultaneously observed in 53.1% of the samples. In addition, the current data revealed that CXCR4 and β-catenin mRNA expression were significantly higher in osteosarcoma compared with adjacent healthy tissue. To evaluate these proteins as biomarkers, Spearman correlation coefficient analysis was performed, revealing a significant association between CXCR4 and β-catenin expression.In conclusion, the present study demonstrated that strong CXCR4 and β-catenin expression were associated with advanced stage disease. Kaplan-Meier survival curves indicated significant differences in clinical prognosis between the β-catenin-positive and β-catenin-negative groups. Furthermore, statistical analysis revealed CXCR4 and β-catenin expression as a predictor of overall survival. Additionally, the present study identified high expression of at least one of the synergistically-regulated mRNAs (CXCR4 or β-catenin) in all osteosarcoma patients. Collectively, the current results indicate that CXCR4 and β-catenin expression may be used as biomarkers to predict prognosis in patients with osteosarcoma and allow for novel therapeutic strategies to be developed.


Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and  Elucidating the relevant mechanisms is important for understanding normal stem cell and tissue biology, and also because of the potential for developing therapies that can target stem-like cells in cancer.Stem cells of the adult mammary gland are predicted to have a capacity for self-renewal and to give rise to the two major epithelial cell lineages of mammary ducts: luminal and basal. Substantial progress has been made towards characterizing mouse mammary stem cell populations, both in vivo lineage tracing experiments have challenged some of these conclusions Evidence that adult mammary tissue contains multipotent self-renewing stem cells was first provided by classical transplantation studies in which a normal epithelial ductal tree, comprising both basal and luminal cell lineages, could be regenerated from small tissue fragments or individual cells Ex vivo assays of mammary epithelial cell sphere formation in suspension culture, mammospheres, offer a complementary approach to stem cell studies that is amenable to signaling pathway analysis. Originally developed for analysis of neuronal precursors, the ability of cells to form spheroids has been used as a stem cell assay for several other tissue types, including prostate and mesenchymal stem cells The canonical Wnt/β-catenin signaling pathway is one of the principal signaling mechanisms associated with regulation of stem cell behavior in numerous tissues Wnt1 mouse strain is a well characterized model for the studying the consequences of Wnt signaling in the mammary gland and its effects on stem cells Wnt1 transgene predominantly in luminal epithelium, and results in activation of canonical Wnt/β-catenin signaling in the basal layer Wnt1 mice display widespread mammary epithelial hyperplasia and are predisposed to carcinomas with nearly 100% penetrance Wnt1 was reported to contain larger numbers of CD24+ CD29HI cells, which are enriched for stem cell activity, in comparison to wild-type glands + CD29HI immunophenotype showed increased colony formation in response to purified Wnt3a The MMTV-Intracellular signaling elicited by members of the Wnt family of secreted ligands can be broadly classified into two modes: canonical and non-canonical. In the canonical pathway, Wnt ligand binding to receptor complexes containing Frizzled and Lrp5/6 proteins results in stabilization of cytoplasmic β-catenin and transcriptional activation mediated by β-catenin/TCF complexes While there is strong support for Wnt/β-catenin signaling promoting mammary stem cell properties, as described above, the roles of non-canonical signaling in the mammary gland are much less clear. Moreover, apparently conflicting data exists concerning functional interactions between canonical and non-canonical Wnt signaling in other experimental systems. Thus Wnt5a has been reported to act either act in opposition to, in concert with, or independently of Wnt/β-catenin signaling To elucidate the effects of canonical and non-canonical Wnt signaling on stem cell properties of mouse mammary epithelium, here we test the consequence of altered Wnt signaling activity on mammosphere cultures, specifically quantifying the number of secondary mammosphere-forming units (MFUs)et al. Mammospheres culture methods were adapted from Dontu Primary mammosphere cultures were disassociated with trypsin for 30 minutes in the presence of a vital cell labeling dye, . Cell suspensions were filtered through a 40 µM cell strainer and then diluted in mammosphere medium with 1% methylcellulose to limit cell aggregation, and 10 nM dexamethasone to maintain transcription from the MMTV promoter. Cells were plated in 96-well low adherence plates using 24 wells per treatment, and were scored for mammosphere formation after one week of growth. In all secondary mammosphere assays, cells were plated at 1000 cells per well except in lentiviral shRNA knockdown experiments, in which they were plated at 2000 cells per well. All exogenous treatments were given as a single dose to the dissociated cells at the time of secondary assay plating. Mammospheres were defined as colonies in methylcellulose suspension culture that contained 10 or more cells visible under phase contrast, of which fewer than 50% still retained the cell tracking dye, consistent with the dye being diluted out upon successive cell divisions.Secondary mammosphere cultures were harvested and washed with PBS. Mammospheres were fixed and permeabilized with a 1∶1 acetone:methanol fixation solution for 30 minutes at −20°C, and PBS was used to rehydrate the mammospheres for 10 minutes at room temperature. Non-specific antibody binding was blocked with a 3% BSA/PBS solution for 1 hour at room temperature. Primary antibodies to cytokeratin 8 , and cytokeratin 14 , both at a 1∶200 dilution in 3% BSA/PBS, were applied overnight at 4°C. Mammospheres were then washed twice in PBS. Cells were incubated in a 1∶1000 dilution of Alexafluor-conjugated secondary antibodies (Life technologies) in 3% BSA/PBS for 4 hours. Cells were washed twice with PBS and mounted in Vectashield mounting media (Vector laboratories) including 0.1 µg/ml DAPI.Numbers of mammospheres reported are the mean of 24 wells per treatment from representative experiments. All experiments were repeated 3 or more times, demonstrating consistent statistical relationship patterns. Comparisons between treatments were made using Student's t test with p<.05 required for significance.Mammospheres were prepared as for secondary mammosphere assays, except cells were plated in 6-well low adherence plates. After one week cells were harvested by centrifugation and total RNA was extracted using RNeasy mini kits (Qiagen). cDNA was produced using the iScript cDNA synthesis kit (Bio-Rad). Quantitech primers (Qiagen) were used with SYBR-Green mastermix (Quanta) for quantitative PCR using an MJ Opticon2 system (BioRad) according to manufacturer's instructions.Ror2 mRNA were obtained from Dr. T. Stappenbeck To infect mammosphere cultures with lentivirus, primary mammospheres were disassociated as for secondary mammosphere assays. Before plating, single cell suspensions in DMEM/F12 media were mixed with lentiviral particles in PBS at a multiplicity of infection of greater than 5∶1, plus a 1∶200 dilution of Transdux reagent (System Biosciences) for 30 minutes at 37°C. After infection, the cell suspension was plated as for secondary sphere assays. The 7TGC lentiviral reporter vector was obtained from Addgene (Plasmid#24304) In experiments using inhibitors and recombinant proteins in conjunction with secondary mammosphere assays, agents were added to single cell suspensions before plating. Recombinant Wnt3a (Peprotech), and Recombinant Wnt5a (R&D systems) were used at 200 ng/ml, except when noted. Recombinant Dkk1 (R&D systems) was used at 200 ng/ml. JNK inhibitor SP600125  was used at 10 µM, and iCRT3  This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committees of Weill Cornell Medical College (Protocol Number: 0052–11), and the New York Blood Center (Protocol Number: 267).ex vivo mammosphere cultures derived from primary mouse mammary epithelium. Dissociated epithelial cells were obtained from wild-type mice and from MMTV-Wnt1 transgenic animals. Such mammosphere cultures provide an assay system for stem cell-initiated sphere growth, independent of previously identified stem cell enrichment markers Wnt1 derived mammospheres Wnt1 and wild-type mammospheres have similar capacity to produce progeny cells expressing differentiation markers during mammosphere growth in vitro. In addition, wild-type and MMTV-Wnt1 mammospheres exhibited similar morphology in both shape and size  to suppress Ror2 expression, and validated its ability to knock down Ror2 mRNA in mammospheres by qRT-PCR , who observed that treatment of human breast epithelial cells with Wnt5a, in conjunction with activation of Wnt/β-catenin and TGFβ pathways, enhanced the efficiency of mammosphere formation as well inducing the expression of EMT markers in vitro, suggesting that its positive effect on mammospheres is not unique to breast tissues While elevated levels of canonical Wnt/β-catenin signaling are clearly associated with promoting hyperplasia and tumorigenesis in the mammary gland, both in mice and humans Ror2, a receptor tyrosine kinase that binds Wnt5a and transduces a non-canonical Wnt signal that includes activation of JNK Ror2 involvement, we found that Wnt5a-induced mammosphere formation was abolished by inhibition of JNK. We conclude that while Wnt3a promotes mammosphere formation via canonical Wnt signaling, Wnt5a does so by a non-canonical mechanism. Moreover, our data particularly implicate a Ror2-JNK pathway among the numerous other pathways that have been ascribed to non-canonical Wnt signaling Although Wnt5a typically signals via β-catenin-independent mechanisms, there are special circumstances in which it has been found to activate the canonical Wnt/β-catenin pathway in vivo have yielded data that may be inconsistent with those from classical mammary reconstitution assays in vivo, mammosphere assays provide a promising system for dissecting the responses of individual self-renewing cells to defined factors in vitro.It remains to be determined whether the independent and additive effects of canonical and non-canonical Wnt signaling on mammosphere formation are caused by both signals operating on the same population of mammosphere-forming cells, or whether they act on distinct target populations. Recent studies aimed at identifying mouse mammary stem cells by lineage tracing Figure S1Wild-type mammospheres can be serially passaged for multiple generations. Wild-type mammosphere cultures were serially passaged weekly. The number of mammospheres formed per 2000 cells plated was assayed at each passage. Passage one represents the number of secondary mammospheres resulting from passage from primary to secondary culture. Error bars show 95% confidence intervals.(TIF)Click here for additional data file.


Banana streak disease is caused by a variety of banana-infecting badnaviruses. A genome of the episomal form of a banana streak MY virus was recovered from an infected banana plant sampled on Vava’u Island, Tonga, and shares >98% pairwise identity with the six other genomes available in public databases. Musa spp. causing chlorotic streaks in leaves and yield reduction, with severe disease leading to the death of the plant. BSD is caused by multiple viral species of badnaviruses . Badnaviruses have double-stranded DNA (dsDNA) genomes (~7.5 kb) encapsidated in bacilliform particles 30 × 130 nm in size (Banana streak disease (BSD) is a disease of  in size . Banana- in size , 5.Dysmicoccus brevipes, Planococcus ficus, and Planococcus citri (––n = 2), BSMYV (n = 6), BSOLV (n = 6), BSVNV (n = 3), BSCAV (n = 2), BSIMV (n = 3), BSUAV (n = 1), BSULV (n = 1), BSUMV (n = 1), and BSUIV (n = 1).There are currently four recognized banana-infecting badnavirus (BIB) species: banana streak GF virus (BSGFV), banana streak MY virus (BSMYV), banana streak OL virus (BSOLV), and banana streak VN virus (BSVNV), as well as a further six yet to be assigned a species status: banana streak CA virus (BSCAV), banana streak IM virus (BSIMV), banana streak UA virus (BSUAV), banana streak UL virus (BSULV), banana streak UM virus (BSUMV), and banana streak UI virus (BSUIV). BIBs exist in two forms, either the infectious episomal form with a circular double-stranded DNA genome, or the endogenous form in which the viral genome is integrated into the host genome. BIBs are able to be spread via infected propagules, and the episomal form is vectored by a number of mealy bug species, including us citri –7. A nums citri ––12, whic citri ––. The genn = 134) collected from the islands of Vava’u, Tongatapu, and Ha’apai of Tonga in 2010 to 2011 using a GenCatch plant genomic DNA purification kit . Using the screening primers MysF1/R1 (n = 1) (n = 4) (n = 1)  , India ( (n = 4) , and an  (n = 1) .This report identifies the first episomal genome of BSMYV from Tonga.KR014107.The complete genome of BSMYV (isolate TO213) from Tonga has been deposited at GenBank under the accession no.


Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation. We found that the expression of Sgo1 mRNA was relatively low in normal tissues, but was upregulated in 82% of hepatocellular carcinoma (HCC), and correlated with elevated alpha-fetoprotein and early disease onset of HCC. The depletion of Sgo1 reduced cell viability of hepatoma cell lines including HuH7, HepG2, Hep3B, and HepaRG. Using time-lapse microscopy, we showed that hepatoma cells were delayed and ultimately die in mitosis in the absence of Sgo1. In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected. Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2. Thus, mitotic cell death induced upon Sgo1 depletion in hepatoma cells is mediated by persistent activation of the spindle assembly checkpoint. Together, these results highlight the essential role of Sgo1 in the maintenance of a proper mitotic progression in hepatoma cells and suggest that Sgo1 is a promising oncotarget for HCC. During mitosis, fidelity of chromosome segregation depends on precise control of sister chromatids separation in space and time. The maintenance of sister chromatid cohesion is one essential mechanism to ensure that premature chromosome separation does not occur prior to the onset of anaphase , 2. ShugHepatocellular carcinoma (HCC) develops with complex genetic backgrounds, and severe chromosome allelic losses and structural aberrations simultaneously contribute to genomic heterogeneity in advanced stages of HCC –19. The In the present study, we investigated the expression level of Sgo1 in various normal tissues and HCC specimens. In addition, we explored the essential roles of Sgo1 in the maintenance of cell survival on transformed HeLa cells and four different hepatoma cell lines, as well as two immortalized cell lines. Our results indicated that, in comparison to immortalized cells, transformed hepatoma cells were more sensitized to spindle assembly checkpoint (SAC)-dependent mitotic cell death induced upon Sgo1 depletion. These results highlight the essential role of Sgo1 in supporting proper mitotic progression and cell viability in hepatoma cells and therefore suggested that Sgo1 is a valuable therapeutic target for HCC.To investigate the status of Sgo1 expression in various human tissues, we measured the mRNA level of Sgo1 by quantitative real-time-PCR using primers covering the full-length A1/A2 isoforms. Sgo1 mRNA was mostly expressed in thymus, testis, and spleen. The mRNA level of Sgo1 is low in the majority of normal tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, colon, prostate, ovary, small intestine, and leukocytes Fig. . Next, tTo explore the clinicopathological significance of Sgo1, we examined Sgo1 mRNA in 60 HCC and adjacent non-HCC liver tissues by both conventional RT-PCR and quantitative real-time PCR. By conventional PCR, Sgo1 and actin mRNA was detected in 97% (58/60) of HCC and 73% (44/60) of adjacent livers Fig. . Next, rWe further examined protein expressions of Sgo1in lysates prepared from 21 paired-HCC and adjacent non-HCC tissues, using heat shock protein 70 (HSP70) and proliferating cell nuclear antigen (PCNA) as positive controls for HCC Fig. . WhereasSgo1 is essential for sister chromatid cohesion during mitosis and its depletion has been shown to induce cell death in cervical carcinoma and lung cancer cells , 21. WitIn HeLa cells, the depletion of Sgo1 resulted in precocious dissociation of cohesin complex from centromeres and thereby prevented the silencing of the SAC. As a result, cells were unable to exit mitosis and arrested in a prometaphase-like state for several hours , 8. To eThe present study identified Sgo1 as a promising and novel therapeutic target for HCC. Sgo1 mRNA was barely detected in the majority of normal tissues but was significantly expressed in various transformed hepatoma cell lines. Notably, upregulation of Sgo1 mRNA was detected in 82% of HCC tissues in comparison to adjacent non-HCC regions. In addition, nuclear staining of Sgo1 was detected in all HCC specimens, whereas adjacent livers displayed either weak or no Sgo1 staining. In addition, higher level of Sgo1 mRNA in HCC were linked to younger age and elevated serum alpha-fetoprotein, suggesting that upregulation of Sgo1 may contribute to an early disease onset of HCC. The most intriguing finding in the present study is the distinct sensitivity to Sgo1 depletion among hepatoma cell lines and immortalized cells. This suggests that hepatoma cells generally require a high level of Sgo1 to support proper mitotic progression and cell proliferation. Accordingly, Sgo1 may serve a selective therapeutic target for HCC without causing a significant defect on adjacent non-transformed hepatocytes.Besides HCC, as shown in this study, upregulation of Sgo1 was found in expression datasets of breast and pancreatic cancers , 11, 22.Several splice variants of the Sgo1 gene have been predicted but whether these mRNA variants are constantly spliced and translated in the cell remains unclear , 16, 24.Our findings indicate that Sgo1 is a common survival factor for hepatoma cells. During mitosis, Sgo1 is involved in the protection of centromeric cohesion by counteracting the actions of mitotic kinases and Wapl, through its association with protein phosphatase 2A –7, 26. IIt is not clear why hepatoma cells are more sensitive to Sgo1 depletion than immortalized cells. We suspect that chromosome instability in these cells may serve a genuine explanation. During mitosis, timely dissolution of sister chromatid cohesion ensures accurate chromosome segregation to guard against chromosome instability and tumorigenesis. Recent study suggests that Sgo1 binds to centromeric cohesin in a complex containing protein phosphatase 2A (PP2A), PDS5, and hypophosphorylated soronin, and thereby protects cohesin from the WAPL-mediated dissolution . It is pin vitro and in animal models, and some of them have reached clinical trials [The idea of targeting cancer cells through the activation of spindle assembly checkpoint is being actively exploited. Vinca alkaloids block microtubule polymerization and thereby prevent the formation of a proper spindle during mitosis, which subsequently lead to mitotic catastrophe . Inhibitl trials . So far,l trials . Thus, t2. Human multiple tissue panels of cDNA were obtained from Clontech . Paired HCC and adjacent non-HCC liver tissues were obtained from National Cheng Kung Hospital  with prior approval from the Institutional Review Board .HeLa, HuH-7, WRL-68, HepG2, Hep3B, 293T, HCT-116, and RPE-1 cells were grown in DMEM (Biowest) with 10% fetal bovine serum and 1% penicillin/streptomycin. NeHepLxHT cells were purchased from ATCC  and maintained under the suggested conditions (see details in ® Green PCR Master Mix (ABI) in ABI StepOnePlus™ Real-Time PCR Systems. The cycling steps are 95°C 10 min, 40 cycles of 95°C 15 s and 60°C 1 min.Total RNA was extracted with Trizol (Invitrogen) and reverse-transcribed to cDNA using SuperScript III (Invitrogen). The resulting cDNA was applied for conventional reverse-transcription (RT)-polymerase chain reaction (PCR) using the following primers: Sgo1, ACCAGGCCTCTAGCTAAAAGAGCAC and CGTCTCAAATCCTTTTTCTGCTTGA; actin, ATCATGTTTGAGACCTTCAA and CATCTCTTGCTCGAAGTCCA. For quantitation, cDNA prepared from cell lines or clinical tissues were amplified by the following primers: Sgo1, AACCTGCTCAGAACCAGGAA and CTGGAGCTGTCATCACTATTGG; gapdh, CAAGGCTGTGGGCAAGGT and GGAAGGCCATGCCAGTGA. Real-time PCR was used Power SYBRParaffin-embedded samples were pre-warmed at 65°C for 15 min, followed by treatment with xylene to remove the paraffin. The samples were rehydrated by sequential incubation in 100%, 95%, and 70% ethanol and then in water. Antigen retrieval was performed by boiling the samples in tissue pretreatment buffer solution (Invitrogen) for 30 min. The slides were then incubated with mouse anti-Sgo1 antibody (Abnova) for 1 hour and detected using the EnVision+ System-HRP kit (DAKO). The samples were counterstained with hematoxylin (DAKO) and imaged under a Zeiss LSM780 laser confocal microscope or a Leica DMI6000 microscope.RNA transfections were performed using Oligofectamine or RNAiMAX (Invitrogen) according to the manufacturer's instructions. A control GL2 siRNA (siCtrl) and a siRNA targeting Sgo1 were ordered from Dharmacon (Standard) using published sequences , 37. To We measured the cell proliferation activity with the water-soluble tetrazolium salt (WST-1) assay (Clontech). Cells were seeded in 96-well microtiter plates at a concentration of 2,000 cells/well. The WST-1 reagent was added to the cultures for 2.5 hr and the absorbance of samples was measured at 450 nm against the background using 650 nm as the reference wavelength.Cells were cultured in 3.5-cm dishes (BD) or 4-well chamber slides (ibidi). Multi-positional time-lapse imaging was performed using a Leica DMI6000 inverted microscope equipped with an HCX PL FL 20x/NA0.4 objective and an Andor Luca R EMCCD camera. DIC images were used to examine cell morphology and mitotic progression with a 10-min interval time for 24 hours. Image analysis and cell counts were performed using Metamorph software (Molecular Devices).405-nm excitation images were perform by a Leica DMI6000 inverted microscope equipped with an HCX PL FL 100x 1.4 NA oil-immersion.Cells were mitotic shaken-off and then treated with a hypotonic solution of 70 mM KCL for 15-min at room temperature. After cells swelling, the cell pellet was resuspended in 3:1 methanol:acetic acid and fixed for 30-min on ice. Centrifuged and washed cell pellet two times with 3:1 methanol:acetic acid, and then supernatant was replaced by fresh 3:1 methanol:acetic acid. Cells were dropped onto slides and dried at room temperature, followed by staining with DAPI for 5-min. p value less than 0.05 is considered significant .The quantitation results for the protein and mRNA levels and live cell imaging are presented as the mean ± standard error, except for the individual data sets. All statistical analyses were performed using Prism software (GraphPad). Any


Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC. Human hepatocellular carcinoma (HCC) is currently the fifth most common cancer and third leading cause of cancer death worldwide . HCC conet al. demonstrated that the CD90+CD44+ phenotype of liver CSCs may explain the aggressive growth pattern of HCC [Various stem cell markers are essential for identifying LCSCs. Previous studies have demonstrated that cluster of differentiation CD133 , CD90 77, CD13 , CD24 9 and EpitUnderstanding the pathways that regulate CSC self-renewal, differentiation and tumorigenicity may thus be critical to the development of effective anticancer therapies . DevelopIn this study, we found that simultaneous high expression of 4 different markers  correlates with poor prognosis in a total of 61 cases of HCC patients and serves as a promising predictor the prognosis of HCC patients. We also found that Notch and Wnt/β-catenin signaling pathways play a crucial role in maintaining the self-renewal of CD90, CD24, CD13, CD133 high expressed sphere-forming LCSCs. Notch1 may be downstream of Wnt/β-catenin signaling, and Notch1 negatively regulates Wnt/β-catenin signaling. There may also be a non-proteasome mediated feedback loop between those two signaling pathways.1To investigate whether cancer stem cell markers were over-expressed in HCC specimens, we retrospectively evaluated the expression levels of five cancer stem cell markers  using IHC in 61 matched human HCC specimens and adjacent liver specimens. The markers CD90, CD44, CD133, CD13, and CD24 were present diversely in all HCC samples. By contrast, their expression in non-tumor (NT) liver tissues was almost absent . The repp = 0.044 and p = 0.013, respectively, log-rank test, Figure p = 0.129 and p = 0.024, respectively, log-rank test). Patients whose tumors had significantly higher CD13 expression presented at more advanced TNM Stages  and the clinical outcomes of HCC patients. In a Kaplan-Meier survival analysis, patients with CD90CD24CD13CD133+/high primary tumors displayed worse overall and disease-free survival (estimated mean = 16 months) as compared to those patients with CD90CD24CD13CD133-/low primary tumors  in sphere-forming cells were up-regulated. The expression of CD90, CD24, CD133 and CD13 were increased to 12.2%, 15.9%, 15.2% and 80.6% respectively, comparing to the parental cells, in which the expression of all markers are less than 2.6% . We alsop = 0.0044 and p = 0.0071, respectively, t test, Figure in vivo, sphere-forming HCC cells and parental cells were inoculated subcutaneously into NOD/SCID mice. Higher tumor incidence was observed in the sphere-forming HCC cells group  are believed to possess the stem/progenitor properties of self-renewal , relativp Figure . Furtherp Figure , which sp Figure . Next, tp Figure .P < 0.01, To compare the relative quiescence of sphere-forming HCC cells and parental cells, cell cycle and apoptosis was measured by flow cytometry. We demonstrated that the proportion of sphere-forming PLC/PRF/5 cells in the G0/G1 phase was distinctly increased, while cells in the S and G2-M phase were significantly decreased, compared to the parental counterparts . To investigate the molecular mechanisms involved in self-renewal and metastasis activity of sphere-forming LCSCs, the mRNA and protein expression levels of Notch signaling pathway components  and Wnt/β-catenin signaling pathway components  were assessed. We found that the Notch and Wnt/β-catenin signaling pathway components are up-regulated in sphere-forming LCSCs HCC cells, compared to the parental counterparts Figure . To furt4in vivo using IHC . Furthermore, our data demonstrated that the cancer stem cells surface markers phenotype, CD90, CD44 and CD133 decreased when Wnt/β-catenin and Notch were blocked . Collectively, these results suggested that both Notch and Wnt/β-catenin were essential factors for maintaining the self-renewal and metastasis of LCSCs.To investigate further the roles of Notch and Wnt/β-catenin signaling pathways in maintaining the stem-ness characteristics and metastasis ability in sphere-forming LCSCs, we performed a sphere formation and colony formation assay. We demonstrated that DAPT, XAV939 and DAPT + XAV939 together could significantly inhibit sphere formation and colony formation in sphere-forming LCSCs derived from PLC/PRF/5 cells Figure . Furthersing IHC . Taken td Figure , indicatd Figure . Inhibit5p < 0.05, Figure Intriguingly, it has been demonstrated that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1 . To stud6To determine if Notch also regulates β-catenin protein levels in sphere-forming LCSCs, we blocked Notch endoproteolysis, which is mediated by the presenilin γ-secretase complex by DAPT, and investigated the regulation of β-catenin. Unexpectedly, we found that LCSCs treated with the γ-secretase inhibitor (GSI), DAPT, had a significant reduction of active β-catenin activity and protein levels in a dose-dependent fashion Figure . MoreoveThere is emerging evidence that HCC is driven and maintained by LCSCs that display stem cell properties. Therefore, tracing and “destemming” CSCs may be an effective strategy for treating HCC and improving patient outcomes.In this study, we found that HCC patients with different CSC markers  possessed distinct clinic-pathological features, suggesting that these cells with different cancer stem cell markers may be present in an identical HCC population. Furthermore, we have demonstrated that increased CD90CD24CD13CD133 expression in HCC not only correlates with advanced disease stage but also with larger tumor size and worse overall survival. This correlation suggests that CD90CD24CD13CD133+/high tumor cells have the potential to re-establish tumor growth in patients. Recently, several studies have also reported that successive passages of sphere-forming cells derived from mammary carcinoma cell line , pancreain vitro and in vivo. Furthermore, we found that CD90, CD24, CD44 or CD133 over-expression was also positively correlated with vascular infiltration, which is an important clinic-pathologic feature of HCC metastasis. The identification of sphere-forming HCC cells as a subpopulation involved in the CSC self-renewal of HCC might open up new perspectives for treatment.To determine whether sphere-forming (CD90CD24CD13CD133+/high) HCC cells were LCSCs, we detected the stem-ness associated characteristics. Our data demonstrated that the sphere-forming HCC cells possessed progressively increasing self-renewal and tumor-initiating ability Intriguingly, increasing evidence has shown that both Notch and Wnt signaling may play critical roles in the self-renewal of different CSCs , 41. In Chulan et al. recently demonstrated that Notch1 antagonizes Wnt/β-Catenin signaling by reducing levels of active β-Catenin in cardiac progenitor cells (CPCs) [Notch and Wnt/β-Catenin signaling often intersect in stem and progenitor cells and regulate one another transcriptionally . The effs (CPCs) . Converss (CPCs) . This iss (CPCs) . In the s (CPCs) . It is lIn this study, we identified sphere-forming LCSCs (CD90CD24CD13CD133+/high) within these small lesions, and they functioned to initiate tumor growth and self-renewal through Notch and Wnt/β-catenin up-regulation. In addition, Notch1 was downstream of Wnt/β-catenin. There may be a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs . Clinical data associated with those specimens was recorded without patient identification and all procedures were in accordance with the Huazhong University of Science and Technology Institutional Review Board protocols, and partial of human tissue samples. Informed consent was obtained from each subject. Patients were enrolled as described . SurvivaThe IHC stained Samples from patients with primary antibodies Rabbit monoclonal anti-CD90/Thy1 , Rabbit Monoclonal anti-CD44 , Rabbit Polyclonal anti-CD24 , Mouse monoclonal anti-CD13 , Rabbit Polyclonal anti-CD133  following the manufacturer. And the IHC stained tissue sections  were ana4 cells/10 ml.Human HCC cell lines  were obtained from American Type of Culture Collection (ATCC) and Cell Bank of Chinese Academy of Sciences . All of the cells were cultured as described . For sphFor suppression or inhibition of Notch in HCC cells, lentiviral particles  expressing Notch1-siRNA or NICD were used to regulate Notch signaling in sorted HCC cells. The siRNA sequence targeted Notch1 was listed as follows: 5â€²-GGAGCATGTGTAACATCAA-3â€². For optimization of transfection conditions with lentiviral vectors, HCC cells were infected with Lv-NICD or Lv-Notch1-si at different of multiplicity of infection (MOI) for 12 hours in the presence of 5 μg/ml of polybrene. Two days after infection, expression of green florescence protein was measured by FACS analysis. Infection of cells at MOI of 10 resulted in more than 90% of efficiency of infection without damaging cells (not shown in data).For the TCF activity assay, pSUPER8 × TOPFlash or pSUPER8xFOPFlash (Addgene) and Renilla plasmids, kindly provided by Dr. Timothy R. Billiar , were co-transfected into HCC cells. Twenty-four hours after transfection, the cells were serum-starved for 24 h and stimulated with 1% FBS in DMEM. Luciferase activity was measured at 24 h after stimulation unless indicated using the Luciferase Assay System . The assay was normalized with renilla as a transfection efficiency control.P < 0.05 was considered significant. Other data in cell experiment are presented as mean ± standard deviation. When two groups were com-pared, the Student's t test was used. P < 0.05 was considered significant statistically and is marked with an asterisk. P < 0.01 was considered highly significant statistically and is marked with a double asterisk.Statistical analysis and graphical presentation were performed using SPSS (v19.0) software for Windows . The logistic regression model was utilized to analyze the clinic-pathological parameters which were compared with log-rank test. The Cox regression model was used to perform univariate and multivariate analyses. The survival rate was calculated using the Kaplan–Meier method, and the resulting curves were compared by the log-rank test.