Moreover, NDRG1 silencing reduced β-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing β-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear β-catenin and suggests that NDRG1 is a significant therapeutic target.N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity  Colorectal cancer (CRC) is the third most common cancer and the fourth most prevalent cause of tumor-related death worldwide , 2. Patietc. It is commonly regarded that the gain of a mesenchymal phenotype is associated with some functional traits of cancer stem cell-like cells (CSCs) [The epithelial to mesenchymal transition (EMT) is an embryonic process during which polarized epithelial cells transform into a more motile and mesenchymal phenotype . This res (CSCs) , 10. In s (CSCs) –13. Howes (CSCs) , 15.N-myc downstream-regulated gene 1 (NDRG1) is a potent metastasis suppressor in multiple tumor-types , 17. Thein vitro and tumorigenesis in vivo through up-regulation of nuclear β-catenin and CD44 expression, while these phenotypes can be reversed by down-regulation of β-catenin. Importantly, NDRG1 positive CRC cases were found to have a better prognosis than NDRG1 negative cases. Finally, there was a close negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in the clinical CRC specimens. These findings demonstrate that the effect of NDRG1 on inhibiting nuclear β-catenin translocation and also CD44 expression plays an important role in preventing CRC progression.Herein, we demonstrate that low NDRG1 expression promotes CSC traits of CRC in vitro [etc. [Sphere formation is considered as one important characteristic of CSCs in vitro , 29. Thero [etc. , 31. To ro [etc. . These cp = 0.09) in HCT116 cells over-expressing NDRG1 when compared to its control group (NDRG1 Con). This effect of NDRG1 over-expression on inhibiting primary sphere formation was more pronounced in HT29 cells, where there was a marked and significant (p < 0.001) decrease relative to the NDRG1 Con  increased relative to the sh Con cells  result in lower rates of HCT116 and HT29 cell invasion when compared to the NDRG1 Con cells  greater rates of invasion compared to their relevant sh Con cells  more sensitive to this agent relative to the NDRG1 Con  decreased the sensitivity to 5-FU at concentrations of 1 μM or higher relative to the sh Con  between the cell lines examined when they were incubated with a low concentration of the cytotoxic agent 5-fluorouracil . However, increasing the concentration of 5-FU from 1 to 100 μM, revealed that both the HT29 and HCT116 cells over-expressing NDRG1 were significantly  decrease in colony number, there being approximately half as many colonies as when compared to NDRG1 Con cells  increase in colony formation (approximately 2-fold) relative to the sh Con cells  alteration in the proportion of CD44+ or CD133+ in both cell models (data not shown). However, for both HCT116 and HT29 cells, the mean fluorescent intensity (MFI) of CD44 in NDRG1 over-expressing cells was significantly (p < 0.001–0.01) lower than NDRG1 Con cells, while for sh NDRG1 cells, CD44 was significantly (p < 0.001–0.05) higher compared to sh Con cells  altered by NDRG1 over-expression or silencing in HCT116 cells  increased CD133 levels, while NDRG1 silencing had no significant (p > 0.05) effect on CD133 expression relative to the sh Con. These results indicate that NDRG1 consistently regulates CD44 expression in HCT116 and HT29 cells, while CD133 was not consistently affected in both cell-types.Our results demonstrated no significant  decreased OCT4 and NANOG mRNA levels, while NDRG1 silencing had no significant (p > 0.05) effect on the levels of these transcripts relative to the NDRG1 Con. Examining SOX2 mRNA expression, NDRG1 silencing significantly (p < 0.001) enhanced its expression relative to the sh Con, while NDRG1 over-expression had no significant (p > 0.05) effect  alterations were detected in ALDH1 mRNA levels upon modulation of NDRG1 expression. Overall, these results suggested that NDRG1 regulates CD44 expression, but other CSC markers were not consistently affected. Hence, in subsequent studies below, only CD44 was examined further.As shown in Figs. ect Fig. , 2C. No in vitro assays used to identify putative “stemness” of cancer cells must be verified by functional assays [in vivo, 3-week-old male nude mice were injected subcutaneously into the flank with 1 × 105, 1 × 106, or 1 × 107 HT29 cells/mouse  and the growth of the resultant tumor xenografts was monitored every 5 days for 30 days using Vernier calipers (n = 5–15 mice/group).Any l assays . In factl assays . Thus, iuse Fig. –3D. Nota7 cells/mouse yielded tumors in all mice after 5–20 days irrespective of the clone used  slower in the NDRG1 over-expressing group than those in the NDRG1 Con and all other groups  faster than their relevant sh Con cells  less than their relevant NDRG1 Con tumors. In contrast, the weight of tumors from sh NDRG1 cells was significantly (p < 0.01) greater than those derived from the sh Con cells  greater in NDRG1 cells relative to NDRG1 Con cells, and conversely, NDRG1 in sh NDRG1 cells was significantly (p < 0.001) less than that in sh Con cells , total β-catenin, p-GSK3β (Ser9), total GSK3β, cyclin D1 and c-myc in whole cell lysates Fig. . As demolls Fig. .p < 0.001) decrease in the ratio of β-catenin phosphorylation at Ser33/37,Thr41 to total β-catenin  increase in the ratio of β-catenin phosphorylation at Ser33/37, Thr41 to total β-catenin. Notably, total β-catenin increased slightly, but was not significantly (p > 0.05) altered in NDRG1 over-expressing cells relative to NDRG1 Con cells. However, total β-catenin expression did decrease significantly (p < 0.001) in sh NDRG1 clones for both the HCT116 and HT29 cell-types  increased the ratio of GSK3β phosphorylation at Ser9 to total GSK3β. In contrast, silencing NDRG1 significantly (p < 0.01) decreased this ratio. However, there was no significant (p > 0.05) difference in total GSK3β between the NDRG1 and NDRG1 Con clones, and also between the sh Con and sh NDRG1 clones. These observations suggest that NDRG1 can promote GSK3β phosphorylation at Ser9, leading to inhibition of β-catenin phosphorylation at Ser33/37,Thr4. Hence, these effects prevent the subsequent degradation of β-catenin, as shown previously by our laboratory [As the phosphorylation of GSK3β at Ser9 inactivates this protein leading to a decrease in phosphorylation of β-catenin at Ser33/37,Thr41 and the inhibition of β-catenin degradation , we nextboratory .p < 0.001–0.05) down-regulated in HCT116 and HT29 cells by the over-expression of NDRG1 relative to the NDRG1 Con. Conversely, NDRG1 silencing resulted in a significant (p < 0.001–0.05) increase of cyclin D1 and c-myc in both these cell-types relative to the sh Con  increase in membrane β-catenin expression and a significant (p < 0.01–0.05) decrease in nuclear β-catenin levels. In contrast, NDRG1 silencing had the opposite effect, leading to a significant (p < 0.01) decrease in the membrane β-catenin level and a significant (p < 0.001–0.05) increase in the nuclear β-catenin fraction relative to the sh Con  for the plasma membrane protein, Na+-K+-ATPase, nor the nuclear protein, lamin B1  decreased β-catenin transcriptional activity relative to the NDRG1 Con. On the other hand, silencing of NDRG1 in both HCT116 and HT29 cells resulted in significantly (p < 0.01–0.05) greater β-catenin transcriptional activity than the sh Con groups. Collectively, NDRG1 over-expression reduced nuclear β-catenin expression and inhibited its downstream signaling, while NDRG1 silencing had the opposite effect.Finally, we assessed the transcriptional activity of nuclear β-catenin in transfected HCT116 and HT29 cells using the TOP/FOP Flash assay . As showp < 0.001) decrease in total β-catenin expression relative to the control in both HCT116 and HT29 cells  decreased  inhibited in both HT29 and HCT116 cells in response to β-catenin silencing, as demonstrated by the TOP/FOP Flash assay  higher β-catenin transcriptional activity than the sh NDRG1 Con/sh β-catenin Con cells in both HCT116 and HT29 cell-types, the β-catenin shRNA  was still able to markedly and significantly (p < 0.001–0.01) inhibit this effect in both sh NDRG1 Con and sh NDRG1 cells  increased the sensitivity of both sh NDRG1 Con or sh NDRG1 cells to 5-FU by approximately 2-fold  or significantly (p < 0.05) reduced when β-catenin was silenced. This effect of β-catenin silencing on sphere formation was also observed for sh NDRG1/sh β-catenin Con cells, where a significant (p < 0.05) decrease was identified in the sh NDRG1/sh β-catenin cells  was significantly (p < 0.01–0.05) less than control cells treated with sh NDRG1 Con/sh β-catenin Con. Hence, decreasing β-catenin expression reduces invasion relative to the control. Studies examining HCT116 and HT29 cells treated with sh NDRG1/sh β-catenin Con led to a significant increase (p < 0.05) in invasion relative to the sh NDRG1 Con/sh β-catenin Con cells  significantly (p < 0.01–0.05) decreased invasion relative to these cells silenced with NDRG1 alone  less than their control groups in both cell lines.Finally, assays examining soft-agar colony formation Fig.  showed tp > 0.05) differences between the sh Con and sh NDRG1 cells once β-catenin was silenced. These results suggest that the low expression of nuclear β-catenin may inhibit the CSC phenotype in vitro.Interestingly, in all the assays examined above see Fig. –5F, therCollectively, nuclear β-catenin plays a vital role in maintaining stem-like traits in these cancer cell lines. The silencing of NDRG1, which increases nuclear β-catenin and its down-stream target CD44 , resultsMaterials and Methods: Clinical Colorectal Specimens and Immunohistochemistry), while 39 (33.6%) of the CRC cases expressed significantly (p = 0.001) positive NDRG1 (p = 0.031) better prognosis ; Fig. i.e., nuclear β-catenin(+); Fig. i.e., CD44(+); Fig. i.e., NDRG1(+); Fig. i.e., nuclear β-catenin(−); Fig. i.e., CD44(−); Fig. It is notable that in the CRC tissues examined, NDRG1 was mainly localized in the cytoplasm or on the membrane, with only rare nuclear expression, while β-catenin was mainly expressed in the cytoplasm and nucleus  had prominent nuclear β-catenin expression, while among 39 NDRG1 positive cases, 23 cases  were β-catenin negative, .Intriguingly, analysis of all 116 CRC patient cases demonstrated the regions with high nuclear β-catenin and total CD44 expression were well correlated to negative NDRG1 expression, while those samples with low β-catenin and total CD44 expression were correlated to high NDRG1 expression. In fact, in 77 NDRG1 negative cases, 70 cases  were CD44 positive  were CD44 negative .Assessing the relationship between CD44 expression and NDRG1, in the 77 NDRG1 negative cases, 61 cases  and lymphatic metastasis (p = 0.011). There was no significant (p > 0.05) relationship between NDRG1 expression and other parameters, including: gender, age, or tumor location  staging) [We further calculated the correlation coefficient between NDRG1 and β-catenin expression, and between NDRG1 and CD44 expression in CRC cases demonstrating different extents of invasion and lymphatic metastasis. The invasion and lymphatic metastasis was assessed in terms of staging criteria according to the 7staging) . The CRCp > 0.05) correlation detected between NDRG1, β-catenin and CD44 expression. However, in advanced invasive cases (T3+T4), NDRG1 had significant negative correlations with β-catenin and CD44 expression . In the lymph node metastasis assessment  in the N0 cases. In the N1 cases, more NDRG1 negative  cases were β-catenin (p < 0.001 sr = −0.572) and CD44 (p <0.001 sr = −0.408) positive .As demonstrated in Fig. ent Fig. , there wive Fig. . While iTaken together, these data in NDRG1 mRNA expression was found to be up-regulated in colorectal cancer data from the Cancer Genome Atlas (TCGA) [e.g., hepatocellular carcinoma), NDRG1 was associated with more aggressive phenotypes and a poorer prognosis [Increasing evidence demonstrates that NDRG1 functions as a metastasis suppressor and potentially could be a novel indicator for CRC prognosis , 25, 52.s (TCGA) . In addirognosis . These prognosis , 22.via its inhibitory effects on EMT transcription factors, such as Snail and Slug, which play a role in regulating cell invasion and metastasis [et al. also showed that NDRG1 interacts with the Wnt receptor, LRP6, which results in inhibition of the Wnt signaling pathway and its down-stream effects on promoting metastasis [In an earlier study, we demonstrated that NDRG1 can inhibit TGF-β-induced EMT tastasis . We alsotastasis . Studiestastasis .et al. showed that the features of the newly discovered EMT activator, Prrx1, surprisingly suppressed stem-like traits including mammosphere formation and self-renewal capacity [Many signaling pathways, including Wnt, Hedgehog and Notch, are commonly involved in maintaining the EMT and CSCs . Recentlcapacity . Hence, capacity . The specapacity , 58.e.g., cyclin D1 and c-myc), contributing to tumorigenesis [The Wnt/β-catenin pathway mediates a wide variety of processes, including cell proliferation, migration, differentiation, adhesion and apoptosis . Interesigenesis .in vitro and in vivo which could be linked to its ability to act as a negative EMT regulator [Our data herein indicate that NDRG1 expression can inhibit stem cell-like traits and tumorigenesis in CRC both egulator . Conversegulator . For insegulator . Moreoveegulator .p > 0.05) increase in total β-catenin expression  and stored in the Key Laboratory of Cell Differentiation and Apoptosis of the National Ministry of Education . NDRG1 over-expressing and silenced clones of the colorectal cancer cell lines, HT29 and HCT116, were constructed, as previously described . Both HT+-K+ATPase β2 ; rabbit anti-β-catenin, mouse anti-CD44 ; mouse anti-c-Myc, mouse anti-β-actin ; Phycoerythrin (PE)-labeled anti-human CD44  and PE-labeled anti-human CD133/1 (AC133) .The primary antibodies used were as follows: goat anti-NDRG1, mouse anti-cyclin D1, rabbit anti-lamin B1, mouse anti-Nai.e., 50 mL of DMEM/F12 containing 100 mg/mL EGF, 100 mg/mL bFGF, and 1 mL of B-27® Supplement; Gibco, USA). Then cells were filtered into a single-cell suspension and seeded  in an ultra-low attachment 24-well plate . The cells were cultivated for 12 to 14 days and the spheres were observed under a phase-contrast microscope . In order to assess the self-renewal ability of these cells, formed spheres were separated into a single cell suspension and then again seeded into serum-free culture and the sphere formation rate of “offspring” cells was calculated.The cells were detached from culture flasks with 0.25% trypsin and suspended in sphere formation medium  with Matrigel  according to the manufacturer's protocols. Then 8 × 103/well) were mixed in the top layer containing 0.7% agar in the same medium as the bottom. Cells were cultured for 20 days/37°C and colonies were counted and photographed.The soft agar assay was performed to determine transformation and anchorage- independent growth . In thisi.e., 0.1, 1, 5, 10, 50, 100 and 200 μM) in McCoy's 5A medium, then the cells were seeded in this medium at a density of 3000 cells/well in 96-well plates and cultured for 36 h/37°C. Then, 10 μL of CCK8 was added to each well and the absorption value (A) was detected at 450 nm with a UV-Vis spectrophotometer. The inhibition rate was calculated as: (Acontrol−Aexperiment)/Acontrol × 100%.As 5-FU (5-fluorouracil) is one of the widely used chemotherapeutic drugs for CRC treatment , we asseCultured cells were detached and washed twice with PBS (5 min at 1000 rpm/min at room temperature) and re-suspended in 100 μL PBS. PE-labeled anti-human CD44 and PE-labeled anti-human CD133/1(AC133) were added respectively, and incubated for 20 min at room temperature in the dark. Cells were washed twice with PBS and then the expression of CD44 and CD133 was detected by flow cytometry.Total RNA was extracted from cells using TriPure Isolation Reagent  according to the manufacturer's instructions. RT-PCR was performed by standard procedures  using thWestern analysis was performed using standard methods . Brieflyβ-catenin shRNAs  and the matching scrambled control sequences were purchased from GeneChemCo., Ltd  and used according to the manufacturer's protocol. β-catenin stably silenced cells were maintained in 5 μg/μL puromycin  and tested regularly by western analysis to ensure down-regulation.Immunofluorescence staining was performed as previously described , 21. TheRenilla luciferase. Transfection was performed with Lipofectamine 2000  and Opti-MEM reduced serum medium  according to the manufacturer's protocol. Cells were harvested 48 h post-transfection. Firefly and Renilla luciferase activities were measured by using the Dual Luciferase Reporter Assay  according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity and the transcript activity was shown as TOP fluorescent value/FOP fluorescent value.HCT116 and HT29 cells were seeded in 24-well plates. After an incubation of 24 h/37°C, the cells were transfected with 200 ng luciferase reporter plasmid, TOP Flash , containing two sets of three copies of the TCF-binding site (wild type), or its control FOP Flash , containing mutated TCF-binding sites in combination with 2 ng of pRL-TK vector  containing 5, 1 × 106 and 1 × 107 cells per mouse; n = 5–15 mice/group). Tumor growth was assessed by measuring the long and short diameters of the tumors every 5 days using calipers. Tumor volumes were calculated using the following formula: V = (length × width2)/2. Then 30 days post-inoculation, all the mice were sacrificed and the tumor xenografts were excised, weighed, fixed and embedded in paraffin.All animal experiments were approved by the Ethics Committee of Ruijin Hospital . Male nude mice  aged 3–4 weeks were randomly divided into cages and injected subcutaneously with different numbers of HT29 cells , and all patients were fully informed of the experimental procedures. A total of 116 cases of colorectal specimens were collected from 2006–2008 and used to generate a CRC tissue microarray. Continuous sections (5 μm thick) were cut from the paraffin-embedded tissue microarray. After de-paraffinization and hydration, sections were incubated with endogenous peroxidase blocking solution and normal non-immune serum. After blocking, the continuous sections were incubated overnight at 4°C with respectively goat primary anti-NDRG1 polyclonal antibody (diluted 1:75), anti-β-catenin rabbit polyclonal antibody (1:50) and anti-CD44 mouse polyclonal antibody (1:100). The sections were then incubated with horseradish peroxidase (HRP)-conjugated second antibody for 1 h at room temperature. Finally, the slides were visualized with DAB and counterstained with hematoxylin. The staining intensity (SI) was evaluated as follows: 0–1 (negative): no staining or slightly darker than the background; 2 (moderate positive): obviously darker than the background, and 3 (strong positive): deep-colored staining. The percentage of positive cells (PP) was classified from a score of 0 to 4. The final immunoreactive score (IRS) was calculated as: IRS = SI × PP and IRS > 4 was considered positive expression.t-test. The two-way ANOVA was adopted in the analysis of inhibition rate caused by different concentrations of 5-FU. The correlations between NDRG1, β-catenin and CD44 expression in CRC were estimated by rank correlation and Spearman coefficients (rs) were calculated. All tests were analyzed with SPSS Statistics version 16.0  and a p < 0.05 was considered statistically significant.Statistical differences between experiment groups and corresponding control groups in the number of cells, colonies, or spheres were analyzed using Student's


In this editorial, we focus on recent findings examining the involvement of β-catenin in cancer initiation and progression of epithelial tissues in thymus and prostate.β-catenin is a critical mediator of the Wnt canonical pathway for multiple cellular responses. Using transgenic mice, loss or activation of β-catenin has been studied in multiple tissues to elucidate its role β-catenin in the thymic epithelium and thymoma. In thymus, correct differentiation of cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) is required for thymocyte development and selection. Ablation of β-catenin in Keratin 5 (K5)-expressing mTECs results in thymic atrophy, mainly due to a defective IL-7 niche in β-catenin-deficient mTEC derivatives that are unable to support early thymocyte development [+ and MHCIIhigh mTECs from β-catenin-deficient mTECs is defective [T2) in mTECs, that could functionally rescue defects in thymopoiesis caused by β-catenin-deficient mTEC derivatives [elopment . Using aefective . AdditioT2 transgene in mTECs can activate Wnt/β-catenin canonical signaling and drive thymoma initiation and progression [T2 transgene are frequently identified at the cortical-medullary junction (CMJ) of the transgenic thymi. Interestingly, the CMJ of the thymus is hypothesized to be the anatomic location of bi-potential progenitors that can give rise to both cTECs and mTECs. Thus, this observation implies that the thymic epithelial progenitors at CMJ are susceptible to β-catenin oncogenic signaling during thymoma development. The thymoma lesions express K5, ∆Np63 (α and β isoforms), and β5t, which is a protease subunit used as a differential diagnostic marker of human type B3 thymomas. Consistent with expression patterns found in human thymomas, ∆N64Ctnnb1/ERT2-mediated thymomas showed loss of AIRE and downregulation of p21. Moreover, histologic characteristics of ∆N64Ctnnb1/ERT2 transgene-induced thymomas have a squamoid appearance and loose connective tissue in the perivascular space resembling that of human type B3 thymomas . Thus, our recent findings demonstrate that β-catenin oncogenic signaling drives K5-expressing thymoma initiation and progression  stem/progenitors in basal- and luminal-derived Pten-deficient prostate cancer, respectively. Our results show that aggressive cancer-associated EMT-TFs and LSCs are higher in basal-derived prostate cancer cells compared with luminal-derived cancer cells [al cells , 4. Thesal cells -5. To fuer cells . We furter cells . TherefoTaken together, our findings indicate that Wnt/β-catenin canonical signaling is essential for cellular differentiation of bi-potent epithelial progenitors in thymus and prostate. Aberrant β-catenin activation in the K5-expressing epithelial derivatives of the thymus and prostate gland promotes tumor progression.


However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/ KRAS [Colorectal cancer (CRC) is the third leading cause of cancer deaths in the United States, accounting for 49,700 estimated total deaths in the year 2015 alone [KRAS .Precursor lesions typically follow a polyp-adenoma- carcinoma sequence. The degree of nuclear β-catenin progressively increases during CRC progression, as a consequence of mutations in APC/β-catenin . Anotherwww.oncomine.org) shows that MUC4 is among the top 5% most significantly downregulated genes in colorectal cancers compared to normal tissue as per 3 studies . A recent study from our lab corroborated these findings via the immunohistochemistry of premalignant and malignant tissues, which showed MUC4 was significantly reduced in the adenoma-carcinoma sequence relative to normal tissue [Mucins are high molecular weight glycoproteins and usually line the epithelial surfaces of the digestive and reproductive tracts . MUC4 orl tissue . Thus, wMUC4 promoter analysis in the present study showed the presence of a putative Hath1 binding site at −3102/−3089. Thus, a number of factors collude to alter the expression of mucins in CRC.A number of studies have probed the effect of perturbations in the Wnt pathway on mucins in CRC. When a siRNA targeting β-catenin was used in the CRC cell line LS174, increased general mucin production (measured by Periodic-acid/Schiff (PAS) staining) was observed . The mosMUC4 promoter has been well characterized and is approximately 3.7 kilobases in size; a TATA box is present at −2672/−2668 upstream of ATG [MUC4 promoter analysis revealed the presence of three putative TCF/LEF sites; one in the proximal promoter at −2612 (Site #1), and two in the distal promoter at positions −3226 (Site #2) and −3408 (Site #3). In summation, studies thus far show that β-catenin KD increases mucin production in CRC. In addition, the MUC4 promoter was found to possess 3 putative TCF/LEF sites. We thus hypothesized that Wnt/β-catenin can repress MUC4 in CRC. In addition, the Wnt/β-catenin pathway has been shown to repress the transcription factor Hath1, which also governs expression of mucins such as MUC2 and MUC5AC. Notably, the MUC4 promoter also contains a putative Hath1 site. Thus, we further hypothesized that the β-catenin mediated repression of MUC4 may occur via the repression of Hath1, which ordinarily upregulates MUC4 in CRC.The m of ATG , dividinIn order to determine whether aberrant β-catenin expression/localization correlated with MUC4 expression in CRC, we first examined the expression of MUC4 and β-catenin in CRC cell lines. While all cell lines examined expressed β-catenin, only two of the 7 cell lines were found to express MUC4 abundantly; LS180 and HCT-8 Figure , while Min vivo, we examined Muc4 and β-catenin levels in colon tissue sections procured from three groups of mice: ApcMin mice that were treated with DSS (ApcMin/DSS), ApcMin mice overexpressing Claudin-1, which represents a more aggressive model of colorectal tumorigenesis, also treated with DSS (ApcMin/Cldn-1/ DSS) and lastly, ApcMin mice that were given water, representing a control group. Colon tumorigenesis can be induced in ApcMin mice by administration of the colitis inducing DSS [ApcMin/DSS sections alone. As expected, the section from the ApcMin/Cldn-1 mouse treated with DSS possessed 9 adenomatous polyps and displayed an increased cytosolic/nuclear β-catenin in the lesions in comparison to the adjacent normal regions. These lesions also displayed a virtual absence of Muc4, while adjacent normal regions showed intense Muc4 staining, particularly in goblet cells  β-catenin using lentiviral shRNA as well as siRNA in three CRC cell lines: HCT-8, HCT116 and LS180. Interestingly, we were unable to establish a stable β-catenin KD in all of CRC cell lines, likely due to the reliance of these cells upon β-catenin for survival. Upon the KD of β-catenin, there was an increase in MUC4 in all three cell lines Figure . Since tMUC4 transcript levels in all three cell lines and we confirmed that the MUC4 RNA was also increased upon β-catenin KD  for 6 hours. It was observed that there was a reduction in MUC4 , concurs Figure , thus inMUC4 transcript levels can be directly altered by β-catenin, we used the MUC4 promoter luciferase construct, p3778, which encompasses all three putative TCF/LEF sites. LS180 cells were transfected with a β-catenin si-RNA as well as the TOPflash plasmid and its negative control FOPflash, which are a measure of the β-catenin/TCF signaling. As expected, there was a decrease in the TOP/FOPflash luciferase activity in LS180 β-catenin siRNA transfected cells in comparison to the control (Si-ctrl) cells  in the p3778 construct. It was observed that while transfection with MUT1 caused a reduction in the luciferase activity  repressing MUC4 and low to moderate levels (as seen in pancreatic cancer) promoting MUC4 transcription via a differential recruitment of co-factors. One study has shown that the MUC4 promoter is methylated at certain key residues in the proximal promoter in the cell line Caco-2 and that treatment with the histone deacetylase inhibitor Trichostatin A and DNA methylation inhibitor 5-aza-2′-deoxycytidine caused increased MUC4 mRNA [The KD of β-catenin in the three cell lines that express MUC4 showed that there was a significant increase in MUC4 protein expression upon KD of β-catenin. This was consistent at the RNA level, where -catenin . However-catenin , thus poUC4 mRNA .MUC4 has been shown to be targeted by several miRNAs [MUC4 mRNA. From our Actinomycin D experiment, we concluded β-catenin KD does not increase MUC4 mRNA stability.The Wnt/β-catenin pathway has been shown to perturb the levels of numerous miRNAs in CRC , likely l miRNAs , 44. In MUC4 mRNA and protein levels, we decided to determine whether this β-catenin induced MUC4 repression occurs via a direct or an indirect mechanism. For this, we used a MUC4 promoter luciferase construct, p3778, which encompasses all three of the putative TCF/ LEF sites. It was observed that when LS180 cells were transfected with p3778 in the presence of β-catenin siRNA, there was an increase in the MUC4 promoter driven luciferase activity. However, this increase was not statistically significant. Mutational studies showed that site #2 and site #3 are repressive but site #1 may promote MUC4 transcription in the presence of 4ACAT. However, it is possible that binding of β-catenin does not occur at site #1 in CRC due to factors such as differential promoter accessibility. Despite the observed differences in MUC4 promoter luciferase activity due to mutations in TCF/LEF sites, the differences were statistically insignificant. Future studies could examine whether β-catenin/TCF forms a repressive complex on the MUC4 promoter via chromatin immunoprecipitation (ChIP).Having confirmed that the β-catenin KD induces increased MUC4. We focused on Hath1 because this well-established tumor suppressor gene in CRC has been shown to be repressed by β-catenin and regulate MUC2 in CRC [MUC4 RNA and protein in the HCT-8 cell line, suggesting that Hath1 may directly regulate MUC4 expression in CRC. Future studies could determine whether Hath1 binds the MUC4 promoter via ChIP.Owing to the fact that our promoter luciferase assays did not yield conclusive results, we went on to examine alternate pathways downstream of β-catenin that may regulate 2 in CRC . In conc2 in CRC , 18, it MUC4 and that during the course of CRC progression, both the Notch and Wnt pathways converge to repress Hath1 [MUC4 transcription. The conclusions from this study have been summarized in Figure MUC4 while the Wnt/β-catenin pathway is inactive. In CRC  and cultured in α-MEM containing 10% fetal bovine serum supplemented with 100 μg/ml penicillin and streptomycin at 37°C with 5% CO6 Lenti-X-293T cell line #632180  cells were seeded in 10 cm dishes, and transfection was performed using a calcium phosphate precipitation method  on the following day using the pLKO.1.sh. beta-catenin.1248 (Addgene plasmid #19761), pLKO.1 shSCR (Addgene plasmid #17920), packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG, which were a kind gift from Dr. Yuzuru Shiio . Supernatant was collected after 48 hours, and was concentrated using the Lenti-X™ Concentrators #631231 and #631232 from Clontech per manufacturer's instructions. After concentration, the lentiviral supernatant was used to infect 2×105 target cells seeded per well of a 12-well plate. Puromycin (5 μg/ml) was used to select for positive clones. The β-catenin siRNA was purchased from Thermo Fisher Scientific, Waltham, MA USA . For transfections, 4×105 cells were seeded per well in a six well plate. The following day, 125 pmoles of the β-catenin siRNA and 125 pmoles of Silencer® Negative Control siRNA  were transfected using Lipofectamine 2000 , per the manufacturer's directions, in serum free media. Fresh serum containing medium was added 4 hours following transfection. Cell lysates were made and RNA was extracted 72 hours after transfection.All transient transfections were performed with Lipofectamine 2000 , as per the manufacturer's directions. For lentiviral transfection, 2×10ApcMin mice treated with dextran sodium sulfate (DSS) tissues and ApcMin/Cld-1/DSS mice  were generated as described previously [loxP/+ mice, where CDX2P-NLS-Cre confers colon preferential expression of transgenes, were characterized and generated as described previously [tm2Rak/Nci (strain number: 01XAA) mice were obtained from the National Cancer Institute (NCI) mouse repository. These mice have the exon 14 of the Apc gene flanked by loxP sites. When crossed with mice expressing a tissue specific Cre-recombinase, the loxP sites are excised by Cre- recombinase resulting in truncated Apc protein, which is 605 amino acids long, of which only the first 580 are present in the normal protein. These mice also had a C57BL/6J genetic background. Animals were maintained in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committees (IACUC) of the University of Nebraska Medical Center. The animals were exposed to a 12 hour light/dark cycle and were allowed access to food and water ad libitum. The tails of mice were clipped at the age of 8 days and the DNA was isolated using the Maxwell 16 mouse tail DNA purification kit, Promega, Madison, WI, USA. Following DNA isolation, genotyping was performed using primers listed in et al [Tissues from eviously . The CDXeviously . The CDXeviously . We obtain et al  with thein et al . Images 5 cells were seeded in triplicate per well of 12 well plate and were transfected the next day. The pGL4.17 vector was gifted by Dr. Robert Bennett, UNMC. The M50 Super 8x TOPFlash contains seven TCF/LEF-binding sites upstream of firefly luciferase, while the negative control M51 Super 8x FOPFlash plasmid contains seven mutant TCF/LEF sites . The pRThe QIAGEN RNeasy mini kit  was used to isolate and purify RNA, as per the manufacturer's directions. The NanoDrop ND 1000 Spectrophotometer was used to measure RNA concentration. The purified and quantified RNA was used for cDNA preparation using the Oligo (dT) 12-18 Primer #18418-012 (Life Technologies) and Super Script II RNase reverse transcriptase . For Real-time PCR analysis, the Light Cycler 480 Real-Time PCR System  was used. A 2x SYBR® green mix (Life Technologies) along with the appropriate primers and nuclease free water was used in a master mix (9 μl), which was added to 20 ng of cDNA per reaction. Real time PCR primers are enumerated in 5 cells per well in a 6 well plate 24 hours prior to treatment. The γ-secretase inhibitor DBZ  was diluted in DMSO and treatment concentration was 500nM. Control cells were treated with an equal volume of DMSO. Following a freeze-thaw cycle, lysates were syringe-passed through a 215/8 gauge needle. The Bio-Rad protein assay kit  was used to quantify proteins in the lysates. For western blot analysis, a 10% SDS-PAGE gel was loaded with 20 – 40 μg of whole cell lysates for all proteins described, with the exception of MUC4, which was resolved on a 2% agarose gel due to its high molecular weight. The proteins were transferred onto a polyvinylidene difluoride membrane  and probed with primary antibodies overnight at 4°C. Details of the antibody suppliers and dilutions used are given in Western blot analysis was performed as previously described . LysatesMUC4 promoter construct has been described in a previous publication [The generation of the 4ACAT stabilized β-catenin construct, where the residues S33, S37, T41 and S45 were mutated to Alanine and the lication . For theMUC4 promoter was analyzed with the MatInspector software . Putative transcription factor binding sites with a matrix similarity score of > 0.85 were selected.The Data was analyzed using two-tailed T test with unequal variance using Microsoft® Office software. A p value of less than or equal to 0.05 was considered significant.


Abnormal activation of Wnt/β-catenin signaling promotes tissue fibrogenesis. In the present study, the role of β-catenin during liver fibrogenesis was analyzed and the functional effects of β-catenin gene silencing in hepatic stellate cells (HSCs) using small interfering (si)RNA were investigated. The expression of β-catenin in human hepatic fibrosis tissues of different grades and normal human hepatic tissues was examined using immunohistochemistry. To inhibit the Wnt/β-catenin signaling pathway, siRNA for β-catenin was developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. β-catenin expression was evaluated by quantitative polymerase chain reaction (qPCR) and western blot analysis. The expression of collagen types I and III was evaluated by qPCR and immunofluorescent staining. Cellular proliferation and the cell cycle were analyzed using a methyl thiazolyl tetrazolium assay. Apoptosis was assessed by Annexin V staining. A higher expression level of β-catenin was identified in the patients with high-grade hepatic fibrosis in comparison with that of the normal controls. Additionally, β-catenin siRNA molecules were successfully transfected into HSCs and induced inhibition of β-catenin expression in a time-dependent manner. β-catenin siRNA treatment also inhibited synthesis of collagen types I and I in transfected HSCs. Furthermore, compared with those of the control group, siRNA-mediated knockdown of β-catenin in HSC-T6 cells inhibited cell proliferation and resulted in cell apoptosis. This study suggests a significant functional role for β-catenin in the development of liver fibrosis and demonstrates that downregulation of the Wnt/β-catenin signaling pathway inhibits HSC activation. Thus, this study provides a novel strategy for the treatment of hepatic fibrosis. Hepatic fibrosis is an outcome of numerous chronic liver diseases, including hepatitis B and hepatitis C viral infections, alcoholic liver disease and non-alcoholic steatohepatitis . HepaticThe Wnt/β-catenin signaling pathway is critical in development and in adult tissue homeostasis. Wnt/β-catenin signaling initiates a signaling cascade critical in the normal development of a number of organs, including the brain, intestines, skin, liver and lung . The halRNA interference is a powerful tool for post-transcriptional gene silencing  and has All samples, from patients who underwent liver biopsies directed by ultrasonography within one week after inclusion in this study, were obtained from Shanghai Changzheng Hospital, Second Military Medical University  and Shanghai Xinhua Hospital, Shanghai Jiaotong University School of Medicine , between January 2006 and January 2007. The sample donation and application procedures were approved by the Health Human Research Ethics Committee of Shanghai Changzheng Hospital and the Health Human Research Ethics Committee of Xinhua Hospital. The study complied with the Declaration of Helsinki, 1995. The patients provided full written informed consent at the time of sample acquisition and patient anonymity has been preserved. All samples were treated by 10% formalin fixation and paraffin imbedding. Diagnosis and classification of the hepatic fibrosis were determined using Scheuer’s classification : F0, no 2O2 in methanol at 37°C for 10 min to suppress endogenous peroxidase activity. Subsequent to blocking at room temperature for 20 min with 10% bovine serum , the sections were incubated overnight at 4°C with antibodies against β-catenin , followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody  at 37°C for 20 min. The signal was amplified by avidin-biotin complex formation and developed using diaminobenzidine followed by counterstaining with hematoxylin. The samples were dehydrated in alcohol and xylene and mounted onto glass slides. Positive cells were counted in at least 10 fields at ×400 magnification. The incidence of immunoreactivity for β-catenin was evaluated based on the mean percentage of cells positive, as follows: High, >80% of cells positive; intermediate, 25–80% of cells positive; low, <25% of cells positive and negative, <5% cells positive.Part of all the paraffin-embedded liver tissues was stained with hematoxylin and eosin and another part underwent silver staining for reticular fibers. Immunohistochemical examination was conducted to detect the expression of β-catenin. Briefly, the paraffin sections of the left median hepatic lobes were incubated with 3% H2. The cells were divided into four groups: A normal group (NG), in which cells were maintained in RPMI-1640 medium without transfection; a control group (CG), in which cells were transfected with non-specific gene silencing effects; and two treatment groups: in which cells were transfected with β-catenin siRNA and analyzed after either 24 or 48 h, respectively.HSC-T6, an immortalized rat HSC line exhibiting a stable phenotype and biochemical characteristics of activated HSCs, was donated by Dr SL Friedman  . All celet al  according to the manufacturer’s instructions. The cells were transfected with a mixture of plasmid DNA and Lipofectamine 2000  in Opti-MEM I medium without serum (Invitrogen Life Technologies), as recommended by the manufacturer.β-catenin-specific siRNAs were designed as described by Elbashir et al . The senTotal RNA was extracted at different time points following siRNA transfection using a TRIzol kit (Gibco Life Technologies) according to the manufacturer’s instructions. The mixture of RNA and primers was loaded into the PCR amplifier . The following sense and antisense primers were used: Collagen I, 5′-GGTGGTTATGACTTCAGCTTCC-3′ and 5′-CATGTA GGCTACGCTGTTCTTG-3′; collagen III, 5′-GTCTTATCA GCCCTGATGGTTC-3′ and 5′-GCTCCATTCACCAGT GTGTTTA-3′; and β-actin, 5′-TGAAGGTCGGAGTCAACG GATTTGG-3′ and 5′-CATGTGGGCCATGAGGTCCAC CAC-3′. The PCR procedure was as follows: Predenaturate setting at 95°C for 5 min, denature at 94°C for 45 sec, annealing at 50 °C for 1 min and extension at 72°C for 1 min. The PCR was performed for 40 cycles followed by a final extension at 72°C for 10 min. The PCR product was then visualized by running it on a 1.5% agarose gel and was quantitatively analyzed with LabWorks 4.5 analysis software .Following transfection, the cells were harvested and immediately prepared for protein extraction. The protein content in the supernatant was detected using the bicinchoninic acid method . Equal quantities of protein were run on 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Following incubation with 10% non-fat milk for 1 h, the membranes were probed with polyclonal rabbit anti-rat β-catenin antibody  overnight at 4°C and then incubated with HRP-labeled goat anti-rabbit secondary antibodies . The protein levels were normalized using β-actin as a loading control. The relative optical density of the protein bands was measured using a Zeineh Laser Densitometer  after subtracting the film background.Expression of collagen types I and III in HSC-T6 cells infected with β-catenin siRNAs was examined by immunocytofluorescent staining using polyclonal antibodies against collagen types I and III . The fixed cells were treated with the primary antibodies (against collagen types I and III) overnight at 4°C, followed by incubation with secondary antibodies  at 4°C for 2 h. The cells were then stained for 30 min at room temperature with 4,6-diamidino-2-phenylindole. Following rinsing, the slides were viewed with a Zeiss LSM-510 Laser Scanning Confocal microscope . The fluorescence was quantified with semi-quantitative analysis by image scanning.5 cells were prepared to analyze the cell cycle phases by flow cytometry .The effect of siRNA-mediated downregulation of β-catenin on HSC-T6 cell proliferation was determined by 3--2,5-diphenyl tetrazolium bromide (MTT) assay. The cell suspension was placed into 96-well plates at 1,000 cells per well with eight repeat wells and incubated for 1, 2, 3, 4 or 5 days after transfection. The cells were then incubated with 20 μl methyl thiazolyl tetrazolium for 4 h. Following centrifugation, 150 μl dimethylsulfoxide was added to the precipitate and the absorbance of the enzyme was measured at 490 nm with a microplate reader . For the cell cycle analysis, HSC-T6 cells infected with β-catenin siRNA were harvested and fixed with 70% ethanol at 20°C for 24 h. Subsequently, 1×10To detect the cells in the early stages of apoptosis, the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit  was used according to the manufacturer’s instructions. Briefly, following transfection for 48 h, HSC-T6 cells were harvested and stained with propidium iodide and Annexin V-FITC in 100 μl staining solution at room temperature for 15 min in the dark. Samples were then diluted with binding buffer and were analyzed by the FACScalibur flow cytometer  within 1 h.Continuous data were expressed as the mean ± standard deviation. For statistical analysis, the group distributions were compared parametrically using Student’s t-test or one-way analysis of variance, and the group distributions were compared non-parametrically using the Mann-Whitney U test. The results were analyzed statistically using the Kruskal-Wallis test for the association between the incidence of immunoreactivity for β-catenin and the histological grade. P<0.05 was considered to indicate a statistically significant difference.Histologically, the hepatic fibrosis tissues exhibited connective tissue fibers extending from the central vein, and early septal formation was established in the livers of the F2 group. The expression levels of β-catenin were examined in the tissues by immunohistochemistry. The majority of β-catenin staining was observed in the high grade hepatic fibrosis tissues. Little specific β-catenin staining was observed in normal liver tissues . CompareForty-eight hours after transfection with β-catenin siRNA, the β-catenin transcript and protein levels were reduced in the transfected cells compared with those in the control group. This β-catenin gene-silencing effect was reproducible and specific as it failed to knock down the expression of an unrelated protein, β-actin. The β-catenin siRNA used in the present study was able to reduce β-catenin mRNA expression levels in HSC-T6 cells compared with the levels in the NG and CG . WesternTo investigate the effect of β-catenin siRNA on collagen degradation, the quantity of collagen types I and III in HSC-T6 cells transfected with β-catenin siRNA was examined using qPCR and immunofluorescent staining. The expression levels of collagen types I and III mRNAs were reduced in the HSC-T6 cells transfected with β-catenin siRNA, compared with those in the cells of the NG or CG . The fluTo investigate whether the reduction in β-catenin levels exhibits an effect on cell growth and proliferation, cell cycle distribution and MTT assays were performed. Knockdown of β-catenin by siRNA at 3, 4 and 5 days after transfection led to reduced cell proliferation in the treatment group than that in the control and mock groups . InvestiAnnexin V staining revealed that the proportion of apoptotic HSC-T6 cells transfected with β-catenin siRNA was significantly elevated when compared with the proportion of apoptotic cells in the control group . The ratin vitro cell model of hepatic fibrosis for over a decade (Hepatic fibrosis is a chronic pathological process that involves an inflammatory response characterized by an excessive release of inflammatory cytokines and infiltration of inflammatory cells ,15. All a decade . HSCs ar2+ pathway (The Wnt signaling pathway is an evolutionarily conserved, complex signaling pathway that is critical for development, differentiation and cellular homeostasis . Three s pathway . The can pathway . In the  pathway . As a re pathway ,21. Wnt  pathway .Wnt/β-catenin is an evolutionarily conserved cellular signaling system essential in the pathogenesis of numerous human diseases ,23. Wnt Inhibition of the Wnt pathway has also been analyzed in the regulation of fibrotic responses in tissues. When Dkk, the best characterized naturally secreted antagonist of the Wnt pathway, was administered locally in a naked plasmid vector for gene therapy, blockade of canonical Wnt pathway inhibited renal interstitial fibrosis . InhibitIn conclusion, β-catenin was upregulated during liver fibrogenesis. A reduction in the β-catenin expression levels in HSCs with siRNA suppressed the activity of HSCs and collagen synthesis, and enhanced collagen degradation. The results of the present study suggest a significant functional role for β-catenin in the development of liver fibrosis and that targeted knockdown of β-catenin mRNA may have a therapeutic effect on hepatic fibrosis.


The protein β-catenin exhibits a dual function in cells, by acting as a major structural component of cell-cell adherens junctions and as a central signaling molecule in the Wnt signaling pathway. However, how the regulation of β-catenin expression during tumor metastasis in non-small cell lung cancer (NSCLC) varies according to histological type remains unclear. To investigate the regulatory mechanism of β-catenin on tumor metastasis, the present study compared the expression of Wnt1, β-catenin and E-cadherin in 41 primary NSCLC tumors and their corresponding metastatic lesions by immunohistochemistry. Altered expression of β-catenin was more frequent in the metastatic tumors  than in the corresponding primary tumors . There were 12 cases [nine of adenocarcinoma (ADC) and three of squamous cell carcinoma (SqCC)] that revealed discordant β-catenin expression between the primary tumors and the corresponding metastatic lesions. Of these, 11 cases  demonstrated acquired β-catenin alterations in the metastatic lesions. Subgroup analysis of these nine ADCs revealed that six cases  were accompanied by E-cadherin loss but no Wnt1 overexpression. Subgroup analysis of the three SqCCs revealed discordant β-catenin expression. Two cases  demonstrated acquired β-catenin expression during metastatic progression with Wnt1 overexpression but no change in E-cadherin expression. One case of SqCC revealed normal β-catenin expression in the metastasis although the expression was aberrant in the primary tumor. The results of the present study revealed that the changes in β-catenin expression occurred during tumor metastasis by different mechanisms, depending on histological type. The alterations in β-catenin expression may be regulated by a cadherin-catenin system in ADCs with reduced membranous expression of E-cadherin, but mediated by Wnt1 overexpression in SqCCs with cytoplasmic or nuclear transition types. Lung cancer remains the leading cause of cancer-associated mortality in developed countries, although certain clinically-relevant advances have been achieved ,2. The mThe release of tumor cells from the primary tumor is the primary step of metastasis. Changes to cell adhesion molecules (CAMs) promote metastasis, which results in a deterioration of the disease prognosis. β-catenin, a multifunctional protein encoded in chromosome 3p21, is a crucial component of CAMs and is critical in cell-cell adhesion and tissue remodeling processes . In cellβ-catenin plays an important role in the Wnt/β-catenin signaling pathway by activating the transcription of target genes and leading to cell proliferation, invasion and metastasis . Wnt sigThus, the protein β-catenin exhibits a combined function in cells as a major structural component of cell-cell adherens junctions and as a central signaling molecule in the Wnt signaling pathway. However, few studies have analyzed the functions of β-catenin in patient-matched lung primary lesions and patient-matched distant metastases regarding either the cadherin-catenin system or the Wnt signaling pathway. In order to evaluate the dual role of β-catenin and to determine its importance as a predictor of metastasis and disease progression, the present study investigated the expression levels of Wnt1, β-catenin and E-cadherin in lung adenocarcinoma (ADC) and squamous cell carcinomas (SqCC) sections.The present study cohort comprised 460 patients with NSCLC who underwent surgical resection at Seoul National University Bundang Hospital  from May 2003 to April 2008. Of the 460 patients included, 112 patients had recurrence and from these, 41 specimens of the primary tumors and their corresponding metastatic lesions were available for study. The current study procedures were approved by the Institutional Review Board (IRB) at Seoul National University Bundang Hospital (B1008-109-301).® XT Slide Staining System Specifications . Antigen retrieval was performed by immersing the slides in citrate buffer (pH 6.0) for 15 min and endogenous peroxidases were blocked with 1% H2O2 for 10 min.Sections (4 μm thick) from formalin-fixed, paraffin-embedded tissue were deparaffinized in xylene and rehydrated in graded ethanol. The succeeding steps were performed automatically at 37°C using the BenchmarkThe sections were incubated with rabbit polyclonal antibody for Wnt1 , mouse monoclonal antibody for β-catenin  and mouse monoclonal antibody for E-cadherin  at 4°C overnight. The sections were subsequently washed with tris-buffered saline (pH 7.4).™ detection system  was used which included a peroxidase-conjugated polymer backbone and secondary antibody molecules directed against rabbit and mouse immunoglobulins.The biotinylated secondary antibody was incubated with the sections for 20 min. The slides were then stained using a diaminobenzidine detection kit and counterstained with hematoxylin. The EnVisionTwo pathologists (X.X and J.H.C.) independently performed blinded semiquantitative evaluations of the staining under a light microscope , without prior knowledge of the patient data. To calculate the expression levels of Wnt1, the scoring criteria used were based on a semiquantitative approach, in which the percentage of positive tumor cells (0–100%) was determined and multiplied by the staining intensity . A total score within the range of 0–300 was generated for each sample, where 0–100 was classified as negative and 101–300 was classified as positive for Wnt1 expression.For the expression of E-cadherin, a positive expression of <30% in the membrane was considered as reduced membranous expression. When evaluating the expression of β-catenin, a classification of staining patterns was used as follows: i) membranous pattern, the immunoreactivity was present solely in the cell membranes; ii) membranous-cytoplasmic pattern, the immunoreactivity was also present in the cytoplasm; iii) cytoplasmic pattern, the immunoreactivity was predominantly in the cytoplasm and in <20% of the nucleus; and iv) cytoplasmic-nuclear pattern, the immunoreactivity was present in the cytoplasm and concomitantly in >20% of the nucleus ,16. Stro2 test was used to evaluate the comparison of β-catenin, E-cadherin and Wnt1 expression between primary and metastatic tumors. P<0.05 was considered to indicate a medically statistically significant difference.The analyses were performed using the SPSS 17.0 software . The χOf the 460 patients, 112 patients  had recurrence and from these, 41 pairs of specimens of the primary tumors and the corresponding metastatic lesions were available for study. The origins of the 41 paired specimens used for analysis were the lungs (n=26), brain (n=6), bone (n=1), liver (n=1), pleura (n=3), spine (n=1), kidneys (n=2) and soft tissue n=1; . The patβ-catenin expression was normally localized to the cell membrane of the respiratory epithelium; however, reduced expression in the cell membrane, cytoplasm and/or nuclear translocation was considered an indication of its aberrant expression. Altered expression of β-catenin was more frequent in the metastatic tumors  than in their corresponding primary tumors . In the 12 discordant cases of β-catenin expression, where the β-catenin expression status differed between the primary tumor and the metastasis, 11 cases revealed acquired β-catenin alterations in the metastatic lesions; the exception was one case of SqCC . All nine discordant cases demonstrated acquired β-catenin alterations in the metastatic tumors .Subgroup analysis of the 10 cases of SqCC revealed three cases of discordance in β-catenin expression between the primary and matched metastases. Among them, two cases (case nos. 40 and 41) demonstrated acquired β-catenin alterations in the metastases but normal β-catenin expression in the primary tumors. The status of β-catenin expression in the primary and metastatic tumors is summarized in Immunohistochemical analysis revealed that E-cadherin expression was uniform in the cell membranes of the normal bronchial mucosa. E-cadherin expression was mainly observed on the membranes of the tumor cells and in certain cases, in the cytoplasm. Loss of E-cadherin expression had greater prevalence in metastatic tumors  than in their matching primary tumors . Among these, 15 (88.2%) cases acquired aberrant expression of E-cadherin in the metastases, although the expression was unaltered in the primary tumors. Altered β-catenin expression was associated with a decreased level of E-cadherin expression P<0.001; . Among tIn the subgroup analysis of the 10 cases of SqCC, there were five cases showing discordances in E-cadherin expression between the primary and matched metastases. Among these, four cases (case nos. 34–37) revealed decreased E-cadherin expression in the metastases but normal expression in the primary tumors . No signWnt1 expression appeared in the form of a cytoplasmic staining pattern . Wnt1 exIn the subgroup analysis of the 31 cases of ADC, discordance of Wnt1 expression between the primary tumors and metastatic lesions was observed in nine cases . Among the nine cases of ADC that revealed acquired β-catenin alterations in the metastatic lesion, only one case  had an accompanying change in Wnt1 expression. In a further seven cases  the expression levels of Wnt1 were preserved from the primary tumor to the metastatic lesions. The remaining case  overexpressed Wnt1 in the primary tumor but revealed no expression of Wnt1 in the metastatic tumor . No corrIn the subgroup analysis of the 10 cases of SqCCs, the expression of Wnt1 was preserved from the primary to metastatic tumors in 50% (5/10) of cases. There were three cases revealing discordance in β-catenin expression between the primary and matched metastases. Among them, two cases (case nos. 40 and 41) that demonstrated β-catenin alterations in the metastases and intact expression in their primary tumors exhibited acquired Wnt1 overexpression in the metastases. The remaining case (case no. 39) exhibited altered β-catenin expression in the primary tumors but intact β-catenin expression in the metastatic lesion. In these three cases, altered β-catenin expression was accompanied by altered Wnt1 expression, but no change in E-cadherin expression . HoweverThe present study aimed to investigate whether the distant metastases of lung cancer may have aberrant β-catenin expression compared with the primary tumor and how such β-catenin expression is regulated. Distant metastases are common and numerous studies that have investigated metastatic patterns in primary lung cancer have revealed large variations in the frequency of metastatic involvement between organ systems ,21,22. TIn the current study, aberrant expression of β-catenin in the primary tumors was preserved in the metastatic lesions for the majority of cases of ADC and SqCC , with the exception of one case of SqCC. The acquisition of changes in β-catenin expression during the metastatic progression of NSCLC were observed in 26.8% (11/41) of cases. These results suggest that the alteration of β-catenin expression is an important mechanism in tumor progression and the metastasis of NSCLC. Previous studies have also demonstrated that the aberrant expression of β-catenin is an independent prognostic factor of NSCLC, regardless of tumor, node and metastasis (TNM) stage ,25.The results of the present study also indicate that the loss of E-cadherin expression was much more prevalent in the metastatic tumors than in their matching primary tumors. Intact complexes of β-catenin/E-cadherin are important adhesion molecules and inhibitors of cancer invasion and metastasis. In the current study, the results indicate that changes in the stability and function of the β-catenin/E-cadherin complex resulted in the development of metastasis in tumors. A number of previous studies have also revealed a significant association between reduced E-cadherin expression and tumor differentiation grade, lymph node involvement, venous invasion and distant metastasis ,26. HoweThe results of the present study demonstrated that the alteration in β-catenin expression may be affected by signaling pathways that differ according to histological type. Alterations in β-catenin expression were significantly associated with the loss of E-cadherin in ADCs. Among the nine cases of ADC that acquired β-catenin alterations in the metastatic tumors, six cases (66.7%) were accompanied by a reduction in E-cadherin expression, and there was only one case (11.1%) in which Wnt1 overexpression was detected in the metastatic tumor. These results suggest that the aberration of β-catenin expression during tumor progression and metastasis in ADCs is more likely to be associated with the cadherin-catenin system than with the Wnt signaling pathway. Previous studies have also revealed that the aberration of β-catenin expression may induce epithelial-mesenchymal transition (EMT) during the development of cell lines and tumors –29. AnotAlterations in β-catenin expression between the primary and metastatic tumors in the cases of SqCC were accompanied by Wnt1 overexpression but not a loss of E-cadherin expression. This suggests that the aberration of β-catenin in tumor progression and metastasis in SqCCs may be associated with the Wnt signaling pathway rather than the cadherin-catenin system. However, it remains unclear as to whether the metastasis in SqCC is mediated by the Wnt signaling pathway due to the small number of cases analyzed. Therefore, further large-scaled studies are required to evaluate the role of the Wnt signaling pathway in the metastasis of SqCC.The present study had certain limitations. The sample size was relatively small, particularly that which dealt with the Wnt-1 regulated β-catenin expression. Of the original 460 patients, 112 had recurrences but only 41 of these (31 ADCs and 10 SCCs) had tissue samples that were available for further study. Since the majority of patients with extrathoracic metastasis did not undergo further biopsies or surgery, the primary and corresponding metastatic specimens were scarce and difficult to obtain. Thus, larger studies are required to confirm the observations made in the current study.In conclusion, three significant results were established in the present study. Firstly, primary lung carcinomas that develop into distant metastases may have aberrant β-catenin expression. Secondly, aberration of E-cadherin expression occurred frequently during the metastatic progression of NSCLC. Finally, changes in β-catenin expression may be regulated by the cadherin-catenin system in ADCs, but mediated by the Wnt pathway in SqCCs.


Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.Myoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca Summary: This novel approach seeks to overcome the limitations of studying the role of individual cadherins in skeletal muscle fusion in vitro by selecting for myoblasts that express dominant-negative E-cadherin, and provides evidence that cadherins play an essential role in myoblast fusion. The process by which muscle cells interact and fuse to produce syncytia has been studied for decades and yet remains poorly understood. Myoblasts of different species recognize one another, adhere and fuse to form heterokaryon myotubes, yet very rarely spontaneously fuse with cells from other tissues . A numbe2+-dependent cell–cell adhesion molecules. E-cadherin, a prototypical member of this family, is a transmembrane protein that forms adherens junctions between epithelial cells. The cytoplasmic domain of cadherins interacts directly with either β-catenin or plakoglobin. These two molecules interact with α-catenin, and α-catenin links the cadherin–catenin complex to the actin cytoskeleton through interactions with α-actinin, vinculin, formin, and actin filaments  exhibit reduced myogenesis but more slow myofibers  comprising a red fluorescent protein, DsRed, the E-cadherin cytoplasmic domain (ECT), and a C-terminal FLAG epitope tag  Fig. 1AAFig. 1A.yoblasts .Fig. 1.To further determine the localization of M-cadherin and N-cadherin at the outer and inner faces of the plasma membrane, we selectively biotinylated the cell surface of DsRed+ and DECT+ cells with a membrane-impermeable reagent, sulfoNHS-biotin. Biotinylated proteins were isolated with immobilized streptavidin before being subjected to immunoblot analysis for either M-cadherin or N-cadherin D. AlthouWe were unable to detect β-catenin on the surface membrane of DECT+ cells using immunofluorescence staining. Instead, we observed that β-catenin co-localized with DECT within intracellular compartments E. AlthouC2C12 cells can be induced to differentiate into muscle cells using low serum culture conditions (differentiation medium) . Under sThe measurement of the fusion index supports our hypothesis that DECT expression inhibits cell fusion and thus myotube formation C. AlmostMYOD, MYF5, and myogenin (MYOG)  , 1993. Tn (MYOG) . In estanduction . Consistnduction . Myogeninduction . As myognduction . TherefoThe Wnt/β-catenin signaling pathway has been reported to play key roles in myogenic fate determination and differentiation . CanonicThe cytoplasmic domain of E-cadherin sequesters β-catenin and prevents it from binding to LEF/TCF, thereby inhibiting β-catenin–dependent LEF/TCF transcription activity . The N-tThe β-catenin-engrailed chimera (β-EngMT)  was geneTo confirm that the failure of myoblast fusion was due to disruption of cell–cell junctions by depletion of the β-catenin/plakoglobin required for the correct localization of endogenous cadherins, we performed rescue experiments using chimeric E-cadherin–α-catenin molecules A. When tExpression vectors for these constructs, ELA, and ELAαC, were introduced into DECT+ C2C112 cells by transfection. Following selection with blasticidin, stable transfectants were isolated by immunostaining and immunoblotting with anti-HA B. AlthouThe cell surface localization of ELAαC in DECT+ cells suggests that cell–cell adhesions are established by expressing ELAαC, despite the presence of DECT, which sequestered β-catenin and prevented the cell surface localization of endogenous cadherins as well as exogenously introduced ELA protein. To determine the capacity for cell fusion, these cells were allowed to differentiate by culturing in differentiation medium. Although ELA expression did not increase the extent of myoblast fusion, ELAαC expression increased the fusion index by almost four times D and E. in vivo studies. The Cre/loxP system  fusion gene . Transfected cells were selected for puromycin. The selected cell clones were again tested for β-galactosidase and DECT expression. This time, these clones were found to be negative for β-galactosidase activity and positive for DsRed fluorescence  fused to make myotubes but cells expressing DECT (Δβ-geo) underwent decreased myoblast fusion  in C2C12 myoblasts inhibits the transport of endogenous cadherins, including N-cadherin and M-cadherin, as well as myoblast fusion. The former findings are consistent with our previous observations that when DECT is expressed in cells, it interacts with β-catenin and plakoglobin, reduces the levels of these proteins associated with endogenous cadherins, and inhibits the cell surface localization of endogenous cadherins. These results support our hypothesis that DECT can be used to analyze the role of cadherins in various cellular phenomena in which multiple cadherins are expressed. Under these circumstances, knocking out or knocking down a single cadherin species is not sufficient to abrogate cadherin function.Dominant negative cadherin not only inhibits the cell surface localization of endogenous cadherins but also inhibits β-catenin signaling . β-catenMYOD in P19 cells  (see β-geo) fused to make myotubes but cells expressing DECT (Δβ-geo) showed decreased myoblast fusion  see A and fout fusion D and E. The plasmid containing the E-cadherin cytoplasmic domain construct with an N-terminal DsRed tag and a C-terminal FLAG tag (pC-DECT), and the control plasmid containing DsRed with a C-terminal FLAG tag (pC-DsRed) have previously been described . pC-DECTlacZ reporter gene before Cre-mediated excision but expresses DECT following the removal of lacZ by Cre-mediated excision (pCALL-DECT)  as well as the puromycin resistance gene, was provided by Rolf Kemler, Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.An expression plasmid for DECT that expresses the ECT) see A was conL vector . pCAGGSnL vector . The CreC2C12 cells  were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (referred to as growth medium). Cells were transfected using the calcium phosphate precipitation method, and subjected to selection using either G418 (1 mg/ml), blasticidin (8 μg/ml), puromycin (5 μg/ml), or hygromycin (300 μg/ml). Stable transfectants were identified by fluorescence microscopy combined with immunoblotting, and were isolated as previously described . At leas6) with 10 μg ScaI–linearized pCALL-DECT was performed with the Amaxa nucleofector system  as previously described  were obtained from Abcam Japan  and R&D Systems , respectively. Mouse monoclonal anti-troponin T and anti-vinculin, along with FITC-labeled phalloidin, were purchased from Sigma-Aldrich Japan . Rat monoclonal anti-HA and anti-NCAM (MAB310) were purchased from Roche Diagnostics GmbH  and Merck Millipore , respectively. Other mAbs were purchased from Transduction Laboratories . All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories .Immunoblot analysis was carried out as previously described . Cells w5) were incubated twice with 0.5 mg/ml sulfo-NHS-biotin  at 4°C. Cells were washed with 50 mM NH4Cl in PBS at 4°C before they were stripped with 20 mM Tris-HCl (pH 8.0) containing 1% SDS, boiled for 3 min, passed 4–5 times through a 25-G needle, and finally added to nine volumes of 2% Triton X-100. Biotinylated proteins were collected using streptavidin-conjugated beads (Sigma-Aldrich Japan).Cells  and purified as previously described . cRNA wa


The regenerative ability of the heart is clinically very relevant, because adult teleost fish and amphibians can regenerate heart tissue, but we mammals cannot. Interestingly, heart regeneration is possible in neonatal mice, but this ability is lost within 7 days after birth. In zebrafish and neonatal mice, lost cardiomyocytes are regenerated via proliferation of spared, differentiated cardiomyocytes. While some cardiomyocyte turnover occurs in adult mammals, the cardiomyocyte production rate is too low in response to injury to regenerate the heart. Instead, mammalian hearts respond to injury by remodeling of spared tissue, which includes cardiomyocyte hypertrophy. Wnt/β-catenin signaling plays important roles during vertebrate heart development, and it is re-activated in response to cardiac injury. In this review, we discuss the known functions of this signaling pathway in injured hearts, its involvement in cardiac fibrosis and hypertrophy, and potential therapeutic approaches that might promote cardiac repair after injury by modifying Wnt/β-catenin signaling. Regulation of cardiac remodeling by this signaling pathway appears to vary depending on the injury model and the exact stages that have been studied. Thus, conflicting data have been published regarding a potential role of Wnt/β-catenin pathway in promotion of fibrosis and cardiomyocyte hypertrophy. In addition, the Wnt inhibitory secreted Frizzled-related proteins (sFrps) appear to have Wnt-dependent and Wnt-independent roles in the injured heart. Thus, while the exact functions of Wnt/β-catenin pathway activity in response to injury still need to be elucidated in the non-regenerating mammalian heart, but also in regenerating lower vertebrates, manipulation of the pathway is essential for creation of therapeutically useful cardiomyocytes from stem cells in culture. Hopefully, a detailed understanding of the  All organisms have evolved means of repairing tissue loss after injury or disease. In most species, healing of epidermal wounds and other epithelia is an efficient repair process, whereas the ability to recuperate the damage in other tissues varies widely. Mammals (including humans) can repair injury of skeletal muscle, regenerate large parts of the liver, and repair damage to the epithelia of the kidney and the lung but have limited regenerative capacity in other organs . In contOne of the pivotal signaling pathways regulating the regenerative process in many systems is the Wnt/β-catenin signaling pathway. In addition, Wnt/β-catenin signaling is activated in response to cardiac injury in adult mammals and plays important roles in hypertrophy and cardiac remodeling . Here, wi.e., hypertrophy [3H] thymidine, and this increased to only 3 in 36.000 nuclei (0.0083 %) in the injured heart [The adult mammalian heart has a very limited capacity to repair loss of cardiomyocytes (CMs) after infarction or cardiac overload disorders . In contertrophy , 19. Whiertrophy –22, otheertrophy –25. Indeed heart –25. The ed heart , 26.14C) was generated by nuclear bomb tests during the Cold War and rapidly declined after atmospheric tests were banned. Since 14C makes its way through the food chain into human cells, the 14C levels found in DNA of human CMs corresponds to the atmospheric levels at the time when these cells were born. Bergmann and colleagues have measured the 14C concentrations in DNA of human myocardial cells [14C concentrations were elevated compared to the levels before the tests, indicating that myocardial cells contained DNA synthesized years after birth. Thus, human CMs are capable of renewal during adulthood [Nevertheless, there is good evidence that some new CMs do form during adult mammalian life, including humans. A pulse of atmospheric carbon-14  that includes two serine/threonine kinases, namely Glycogen synthase kinase 3β (Gsk3β) and Casein kinase 1 (Ck1); the scaffolding protein Axin; and the tumor suppressor Adenomatous polyposis coli (Apc)  receptors and the coreceptor Low-density lipoprotein receptor-related proteins 5/6 (Lrp5/6) [Wnt/β-catenin signaling has vital functions during embryonic development, adult homeostasis, and tissue and organ regeneration , 48. The(Lrp5/6) , 55. Lrp(Lrp5/6) , 56–58. (Lrp5/6) , 59. Thi(Lrp5/6) . β-caten(Lrp5/6) , 61, 62.(Lrp5/6) , 64, som(Lrp5/6) .Fig. 1ThWnt/β-catenin signaling plays essential roles during vertebrate heart development . Severalin vivo comes from a genetic study in mouse. Inactivation of the Hippo pathway, which restrains cell proliferation and thus controls organ size in Drosophila, in the embryonic mouse heart results in development of enlarged hearts which display increased numbers of CMs and increased CM proliferation [nkx2.5+ cardiomyocyte precursor cells early during heart development in this study, it remains possible that these pathways act on precursor cell proliferation or differentiation and do not directly control CM proliferation.Less is known about potential roles of Wnt/β-catenin signaling in the regulation of CM proliferation. Treatment of neonatal or adult rat CMs in culture with the Gsk3 inhibitory small molecule BIO, which, among other effects, results in stabilization of β-catenin, can induce cell proliferation and mitosis . Evidencferation . IntriguHeart injuries caused by myocardial infarction, pressure/volume overload, inflammatory diseases, cardiomyopathy, chronic hypertension, or congenital heart disease mostly affect the ventricles and lead to complex responses including scarring, fibrosis, and CM hypertrophy in mammals, which are generally referred to as ventricular remodeling. These changes cause a decline in the ventricular performance and contractile function, which can result in heart failure.In addition to its role during cardiac development, Wnt/β-catenin signaling has also been implicated in regulation of cardiac remodeling and injury responses in mammals. While there is a body of work suggesting roles for Wnt/β-catenin signaling in protection of CMs from apoptosis and regulation of CM hypertrophy, there is little evidence supporting a direct role of the pathway in these processes. Furthermore, several of the Wnt pathway components shown to play a role in the cardiac injury response, in particular Gsk3 and sFrps, have additional functions unrelated to Wnt signaling, which perplex the situation.Several studies have shown that the expression of Wnt ligands and of the feedback regulators Dkk1 and Dkk2 is induced in response to injury of the mouse heart –78. AisaAlthough the studies by Aisagbonhi et al. and Duan et al. identified different primary sites of Wnt/β-catenin signaling induction in response to permanent LAD  and transient LAD (the epicardium), they surprisingly both define a functional role of Wnt/β-catenin signaling in promoting fibroblast formation from these cells in a process of endothelial or epithelial-to-mesenchymal transition (EndMT and EMT respectively) , 78. In Whether CMs activate Wnt/β-catenin signaling at later stages after infarction requires further investigation: while LacZ reactivity has been reported in heart regions containing CMs in the Axin2-LacZ mice, the identity of these cells was not confirmed by marker gene expression and the LacZ, which is targeted to the nucleus in these mice, was found in the cytoplasm in the presumptive CMs, raising questions about the validity of these results . Furtherin vivo has not been shown, functional evidence exists for an involvement of Wnt/β-catenin signaling in injury or stress-induced CM hypertrophy. However, conflicting data have been reported on whether β-catenin is required or actually inhibits CM hypertrophy. On one hand, several studies support a hypertrophy-promoting role for Wnt/β-catenin signaling. Conditional, CM-specific depletion of β-catenin in adult mice was found to impair CM hypertrophy in response to pressure overload induced by thoracic aortic constriction, while non-conditional transgenic overexpression of a dominant-negative Lef transcription factor in CMs throughout embryonic development resulted in CM hypotrophy [anf in response to phenylephrine is directly regulated by Lef1/β-catenin [Thus, while activation of the β-catenin-dependent pathway in CMs in response to myocardial infarction potrophy . Furtherpotrophy . β-catenpotrophy . β-caten-catenin . Contrad-catenin . On the -catenin . Further-catenin . Currentin vivo loss-of-function data will be required to clarify whether β-catenin function switches from CM proliferation-promoting in the embryo to hypertrophy-promoting in the adult.Interestingly, treatment of cultured neonatal and adult mammalian CMs with the small Gsk3 inhibitor BIO, which results in β-catenin stabilization, has been found to be sufficient to induce CM proliferation . Non-conExpression of several sFrps, which can act as inhibitors of Wnt signaling pathways, is elevated in mouse models of myocardial infarction (MI) generated by LAD and in overload-induced failing human hearts , 90, 91.Constitutive overexpression of sFrp-1 in mice has been shown to reduce infarct size and cardiac function after coronary artery ligation or cryoinjury . In contin vivo plus loss-of-function experiments will be required to clarify whether Wnt/β-catenin signaling is cardio-protective and whether sFrps have an endogenous role as modifiers of this function. A recent report on the function of the secreted Wnt inhibitor Dkk3 actually showed that Dkk3 knockout mice had greater infarcts and aggravated left ventricular function after infarct, while Dkk3 overexpression protected from infarction [Clearly, additional experiments using transcriptional readouts of β-catenin activity in culture and farction . This inIntriguingly, another Wnt signaling-independent function of sFrp has been uncovered in regulation of fibrosis in response to cardiac injury. Mammalian sFrp-2 has been found to enhance the function of the metalloproteinase Bmp1, which is a rate-limiting enzyme in the processing of pro-collagen during collagen fiber formation at physiological low concentrations (10–20 nM)  Fig. 3,,3.in vivo importance of cardiac progenitor cells for CM formation during adult homeostasis and in response to heart injury [in vivo relevance of this finding however has not been assessed using loss-of-function experiments.As outlined above, conflicting data exist on the t injury , 24. Somt injury . The Cret injury . Thus, βt injury . The in Human pluripotent stem cells (hPSCs), including hESCs and iPSCs, provide a potentially unlimited supply of progenitor cells that can differentiate into the vast majority of somatic cell types including CMs –102. TemIn contrast to the limited regenerative ability of the adult mammalian heart, urodele amphibia and teleost fish retain regenerative capacity of the heart throughout adult life , 112. MoAdult zebrafish hearts have been shown to display remarkable regenerative capacity in response to three types of injury: surgical removal of up to 20 % of the apex of the ventricle, cryoinjury-induced necrosis of up to 25 % of the apical ventricular region , 11, 116While assays for heart function, in particular assessment of cardiac hemodynamics using ultrasound, are difficult due to the small size of the zebrafish heart and thus not trivial to perform –119, thewt1) expression can also be re-activated by experimental treatment with the peptide thymosin 4 and wt1-positive cells have been reported to represent multipotent progenitors that can give rise to CMs after injury, albeit at a low frequency [tcf21 showed that epicardial cells give rise to perivascular cells but not CMs [gata4 promoter fragment appears to produce the bulk of the regenerated myocardial tissue [The cellular mechanisms activated after wounding and underlying heart regeneration appear to be highly similar between the injury models. A few days after injury, transcriptional responses occur in the entire epicardium, including upregulation of genes normally expressed during development (including Wilms’ tumor 1b), which is followed by proliferation and expansion of the epicardium into a multilayered structure , 124. Inrequency . In cont not CMs . Thus, n not CMs , genetic not CMs , 15. In l tissue , 129. Thsimplet (smp), which we have identified to be necessary for β-catenin-dependent signal transduction [Although a large body of data shows that Wnt/β-catenin signaling plays important roles in mammalian cardiac remodeling and regeneration processes occurring in response to heart injury, its potential role in regulation of zebrafish heart regeneration remains to be tested. We have previously found that a transgenic reporter of Wnt/β-catenin signaling is upregulated in the injured ventricle after ventricular resection . Besidessduction , is alsosduction . Thus, wsduction , 132, arsduction , 134.Heart regeneration is a complex process that only occurs in lower vertebrates, but not in mammals. Several processes such as dedifferentiation, proliferation, redifferentiation, and patterning should take place in concert in a highly regulated manner. Wnt/β-catenin signaling is crucial for orchestration of the regeneration process in other systems , 135. Th


Objects. To investigate the effect of CXCL13 (C-X-C motif chemokine 13) on hepatocellular carcinoma and clarify the potential mechanisms. Methods. 32 patients with hepatocellular carcinoma and 12 healthy controls were recruited for analyzing the expression of CXCL13 by RT-PCR (reverse transcription-polymerase chain reaction). ELISA (enzyme-linked immune-sorbent assay) was used to test the concentration of serum CXCL13. The interaction between CXCL13 and Wnt signaling was analyzed by western blot. In vitro PBMCs cultured with HepG2 supernatant, the levels of IL-12, IL4, IL-6, and IL-17, and four IgG subclasses were detected by ELISA. Results. The rate of high expression CXCL13 was 63.4% in advanced HCC patients, and the serum CXCL13 was also at a high level in stage IV HCC patients. Meanwhile CXCL13 level was positively correlated with serum ALT (Alanine Transaminase) and AST (Aspartate Aminotransferase). CXCL13 and Wnt/β-catenin signaling shared a positive feedback loop. Furthermore, CXCL13 could obviously promote the expressions of IL-12 and IL-17, and induce IgG4 secreted by B cells. Conclusions. The effect of CXCL13 on promoting liver cancer is related to the activation of Wnt/β-catenin pathway and the facilitation of IL-12, IL-17 and IgG4. CXCL13 plays an important role in the progression of HCC, and it may act as a potential target for the diagnosis and treatment of HCC. Hepatocellular carcinoma (HCC) is the most primary malignant tumor of the liver cells  and the third most deadly tumor globally, following lung and stomach cancers . Unlike The molecular pathogenesis of HCC is relative to multiple influences such as tumor microenvironment , 10 and CXCL13 (B-lymphocyte chemoattractant (BLC)), which is the only chemokine binding to its receptor CXCR5, is mainly secreted by follicular dendritic cells (FDCs), monocyte-like mature macrophages, and stromal cells in the B-cell area of the secondary lymphoid tissues , 27, wheβ-catenin signaling could increase the expressions of CXCL13 and its receptor CXCR5, while CXCL13 could stimulate the Wnt/β-catenin pathway. By culturing PBMCs with HepG2 culture supernatant, CXCL13 was identified to significantly induce the production of IL-12 and IL-17 secreted by T cells, and moreover, it could upregulate the concentration of IgG4 secreted by B cells. These results indicated that the effect of CXCL13 on promoting liver cancer development might be related to the activation of Wnt/β-catenin pathway and the facilitation of IL-12, IL-17 and IgG4. CXCL13 played an important role in the progression of HCC, and it might serve as a potential target for the diagnosis and treatment of HCC.In this study, we confirmed the high level CXCL13 was existed in both tissue and serum of advanced liver cancer patients. We also found the activation of Wnt/A total of 32 patients with new onset HCC and 12 gender- and age-matched health controls (HC) were recruited from the Hepatopancreatobiliary Department of the First Hospital of Jilin University . To avoid the interference of other diseases, the participators with any autoimmune diseases, hepatitis virus infection, or those who had received drug therapies within the past 6 months were excluded from the study. Written informed consent was obtained from individual subjects and the experimental protocol was approved by the Ethical Committee of the First Hospital of Jilin University. Their demographic and clinical characteristics of these subjects are show in Fresh liver tumor samples were collected immediately after surgical resection and stored in liquid nitrogen until further use. The corresponding serum samples were stored in −80°C freezer.Peripheral blood mononuclear cells (PBMCs) from venous blood samples (10 mL) of health controls were sorted by Ficoll-Paque  density-gradient centrifugation. PBMCs were washed three times in Hanks balanced salt solution , counted, and suspended in Dulbecco's Modified Eagle Medium , containing 10% FBS  and penicillin streptomycin solution . The liver cancer cell line HepG2 was obtained from the Cell Bank of the Chinese Academy of Sciences  and cultured as mentioned above.5 cells/well) were seeded in a 24-well plate , culturing using the supernatant of HepG2 cells. After 72 h, the supernatant was taken and levels of cytokines and IgG subclasses were determined by ELISA. For the proliferation of T lymphocytes, PBMCs were initially cultured in a 24-well plate precoated with CD3/CD28 mAbs . To activate B cells, 3 μg/mL CpGB and 10 ng/mL IL-2  were used to stimulate B cells for 72 h.To analyze the effect of CXCL13 on the immune status of HCC, PBMCs cells  acted as a stimulator of this pathway. Recombinant human CXCL13 (100 ng/mL) [100 ng/mL DKK-1  was obtained from PeproTech, using to generally block Wnt/0 ng/mL) , 32 was 0 ng/mL) .According to the manufacturer's instructions, enzyme-linked immune-sorbent assay (ELISA) of serum and cell culture supernatant was performed on 96-well plates. The serum or supernatant levels of human CXCL13 and IL-12/4/6/17 were determined by ELISA kit from R&D, USA. The concentrations of IgG1, IgG2, IgG3, IgG4, and total IgG were determined by ELISA  as described previously , 35. Theμg total RNA, 0.5 μg oligo (dT), and Superscript II enzyme . The gene-specific primers for RT-PCR were listed as follows: CXCL13 forward 5′-CTGGTCAGCAGCCTCTCTC-3′, and reverse 5′-TTCTCAATACTTCCATCATTCTTT-3′; GAPDH forward 5′-CACCAACTGGGACGACAT-3′, and reverse 5′-ACAGCCTGGATAGCAACG-3′. The mRNA expression of interested gene in each sample was normalized against housekeeping gene.To test the mRNA expression in liver cancer tissues, total RNA from HCC tissues were extracted using TRIzol  according to the manufacturer's instructions. RNA pellets were stored in sterile ribonuclease-free water. Reverse transcription was carried out using 1–3 6 cells/well). After 8 h treatment, cells were harvested and lysed on ice for 30 min in RIPA  buffer and protein extracts were quantitated. 20 μg of total protein was then subjected to 10–15% SDS-PAGE, electrophoresed and transferred to a nitrocellulose membrane. After using 5% nonfat milk in Tris-buffered saline for blocking, the membrane was washed and incubated with the indicated antibodies. The primary antibodies for β-catenin, phospho-GSK-3β (Ser9), GSK3β, CXCL13, CXCR5, and GAPDH were all from Santa Cruz, USA. The animal-matched horseradish peroxidase-conjugated secondary antibody was purchased from Santa Cruz as well.To illustrate the interaction between CXCL13 and Wnt pathway, HepG2 cells were plated in 6-well plates  or AST . No other obvious difference was observed in any of the clinical parameters  than that in health control as shown in  Figures . Thus thβ-catenin signaling in HCC and the effect of Wnt/β-catenin pathway on CXCL13 was still obscure. In HepG2 cells, by treated with Wnt/β-catenin inhibitor DKK-1, we found decreased expressions of CXCL13 and CXCR5 compared with that in control, whereas upregulated levels of CXCL13 and CXCR5 were observed in stimulant LiCl treated group as shown in β-catenin and an increased p-GSK-3β expression were observed in the dexamethasone treated group, while inverse expressions of these two factors were founded in the CXCL13 stimulated group  and lipoprotein receptor-related protein 5/6 (LRP5/6), Axin/adenomatous polyposis coli (APC)/glycogen synthase kinase (GSK)3β complex is inactivated, which leads to the accumulation of β-catenin. Then β-catenin translocates to the nucleus and interacts with transcription factors of the T-cell factor (TCF) and lymphoid-enhancing factor (LEF) families, promoting the transcription of many oncogenic factors, such as c-MYC, cyclin D1, and VEGF [β-catenin transduction pathway was found to be stimulated by CXCL13 and blocked by dexamethasone as well. These data showed a positive relationship between CXCL13 and Wnt/β-catenin pathway in HCC and indicated that the mechanism of CXCL13 in promoting proinflammatory reaction might be mediated by the mutual feedback with Wnt/β-catenin signaling.The progression of HCC is a result of a complex cellular system with reciprocal signaling. Three regulatory networks, PI3 K/Akt, ERK/MAPK, and JNK/c-Jun pathways, are confirmed to be dramatically induced by CXCL13 in prostate cancer . Wnt/β-cand VEGF , 37. By γ (IFN-γ) production [P < 0.05), while minimal changes of IL-4 and IL-6 level were found in our experiment. The abnormally high production of IL-12 and IL-17, which presented Th1 and Th17 cell, respectively, indicated an immunological imbalance in CXCL13 stimulated group and that might be related to the HCC progression.Considering CXCL13 plays a central role in the positioning, cooperation and activation of T and B cells within lymphoid and extralymphoid sites, especially in the differentiation of B cells into antibody-producing plasma cells , we alsooduction , 40. IL-oduction . In PBMCCompared with that in T lymphocytes, CXCL13 may have a crucial function in B lymphocytes as well. To clarify the effect of CXCL13 on B lymphocytes, we analyzed the proportions of IgG subclasses, which were reported to be associated with many cancers' progression , 43. Aftβ-catenin signaling shared a positive feedback loop. Subsequently, we also found CXCL13 might function through activating Th1 and Th17 reaction and inducing IgG4 production. Thus CXCL13 may act as a promising prognostic marker and a potential therapeutic target in HCC.In conclusion, this study underlines the prognostic relevance of CXCL13 to HCC. We observed a considerable overexpression of CXCL13 in both liver cancer tissues and serum, and its level was positively correlated with serum ALT and AST. Furthermore, CXCL13 and Wnt/