A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT.Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay.ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration.ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.     

An experimental model of the epithelial to mesenchymal transition and pro-fibrogenesis in urothelial cells related to bladder pain syndrome/interstitial cystitis

BackgroundSuitable in vitro models are needed to investigate urothelial epithelial to mesenchymal transition (EMT) and pro-fibrogenesis phenotype in bladder pain syndrome/interstitial cystitis (BPS/IC). This study is to establish a novel experimental BPS/IC cell model and explore how different concentrations of tumor necrosis factor (TNF)-α influence the EMT and pro-fibrogenesis phenotype of urothelial cells.MethodsSV-HUC-1 urothelial cells were cultured with 2, 10, or 50 ng/mL TNF-α to mimic chronic inflammatory stimulation. The EMT and pro-fibrogenesis phenotype, including production of collagen I and pro-fibrosis cytokines, were estimated after 72 h of culture.ResultsThe bladder urothelial cells of BPS/IC exhibited upregulated vimentin, TNF-α and TNF receptor, downregulated E-cadherin, and increased collagen I. Higher concentrations of TNF-α (10 and 50 ng/mL) produced an obvious mesenchymal morphology, enhanced invasion and migratory capacity, increased expression of vimentin, and decreased expression of E-cadherin. Collagen I was increased in cells treated with 2 and 10 ng/mL TNF-α after 72 h. Secretion of interleukin (IL)-6 and IL-8 was promoted with 10 and 50 ng/mL TNF-α, while that of IL-1β or transforming growth factor-β was unaffected. Slug and Smad2 were upregulated by TNF-α after 72 h. The Smad pathway was activated most strongly with 10 ng/mL TNF-α and Slug pathway activation was positively correlated with the concentration of TNF-α.ConclusionsSustained 10 ng/mL TNF-α stimulation induced the EMT and pro-fibrogenesis phenotype resembling BPS/IC in SV-HUC-1 cells. Minor inflammatory stimulation induced the pro-fibrogenesis phenotype while severe inflammatory stimulation was more likely to produce significant EMT changes. Different degrees of activation of the Slug and Smad pathways may underlie this phenomenon.     

E-cadherin和Slug在胃癌组织中的表达及意义

目的：探讨胃癌患者肿瘤组织中E-cadherin及Slug的表达与肿瘤病理特征及预后的关系。方法：免疫组化检测82例胃癌患者瘤组织E-cadherin及Slug的表达,并分析它们与患者的病理特征及生存时间的关系。结果：E-cadherin保留表达率为47.6%,Slug阳性表达率为39.0%。E-cadherin的表达率差异与患者的N分期有关,Slug的阳性表达率差异与患者的性别、T分期及N分期有关（均P<0.05）;在E-cadherin表达保留组,Slug的表达与N分期、M分期、静脉侵袭、血行性转移和腹膜复发有关,而在E-cadherin表达降低组,Slug的表达与T分期有关（均P<0.05）;E-cadherin表达保留患者5年生存率高于E-cadherin表达降低患者（79.5% vs. 60.5%）,Slug阳性患者的5年生存率低于Slug阴性患者（59.4% vs. 74.0%）（均P<0.05）;E-cadherin表达保留组中,Slug阳性患者5年生存率低于阴性患者分别为（48.4% vs. 87.5%）（P<0.05）,而E-cadherin表达降低组中,Slug阳性及阴性患者生存率无统计学差异（58.3% vs. 68.4%）（P>0.05）。结论：E-cadherin及Slug的表达主要与胃癌TNM分期有关;E-cadherin表达保留的患者中Slug的阳性表达对不良预后有预测意义。

     

Evaluation of thymosin β4 in the regulation of epithelial-mesenchymal transformation in urothelial carcinoma

ObjectivesTo study the underlying alteration in the expression of epithelial markers involved in epithelial-mesenchymal transition (EMT), and elucidate the potential mechanism(s) for Tβ4-induced EMT-like phenotypic changes in bladder cancer cells.Materials and methodsAll tissue samples in this study were obtained from clinical patients of the Union Hospital of Tongji Medical College, and were confirmed by surgery and pathology. Of these, normal bladder tissues (control), primary urothelial carcinoma of different grades (Stage pTa, Stage pT3), bladder paracancerous tissues, accompanied with 2 bladder cancer cell lines (BIU-87 and T24), were divided into 6 groups. Quantitative RT-PCR, Western blotting, and immunohistochemical study of adhesion molecules Tβ4, ILK, E-cadherin, and β-catenin involved in EMT were carried out. A lentiviral gene transferring vector containing the RNA polymerase III-dependent U6 promoter to express short hairpin RNA (shRNA) directed against Tβ4 was also applied. In the present study, all agents were evaluated using commercial kits.ResultsA strong correlation between the expression levels of Tβ4, ILK, E-cadherin, and β-catenin was found in the bladder transitional cell carcinoma (TCC) patients. In the BIU-87 and T24 bladder cancer cells overexpressing Tβ4, which were accompanied by a loss of E-cadherin as well as a cytosolic accumulation of β-catenin, up-regulation of ILK was also revealed. The inhibition of the Tβ4 expression with lentiviral shRNA vector could raise EMT-like phenotypic changes, significantly depressed motility, and subsequent invasiveness of bladder cancer cells.ConclusionsOur results imply that the Tβ4 is likely to play a crucial role in EMT progression, and that inhibition of the Tβ4 expression or interactions with other genes should be novel therapeutic targets for bladder cancers with high invasive and metastatic potential.     

Expression profile of epithelial-mesenchymal transition markers in non–muscle-invasive urothelial carcinoma of the bladder: Correlation with intravesical recurrence following transurethral resection

ObjectivesTo evaluate the expression of molecular markers involved in epithelial-mesenchymal transition (EMT), a key process mediating the progression of malignant tumors, in non–muscle-invasive urothelial carcinoma of the bladder (NMIUCB) to clarify the significance of these markers as predictors of intravesical recurrence in patients treated with transurethral resection (TUR).Materials and methodsExpression levels of 13 EMT markers, including E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP)-2, MMP-9, Slug, Snail, TWIST, vimentin, ZEB1, and ZEB2, in TUR specimens obtained from 161 consecutive patients with NMIUCB were measured by immunohistochemical staining.ResultsOf these 13 markers, significant differences in the incidence of intravesical recurrence were noted according to expression levels of E-cadherin, N-cadherin, MMP-2, MMP-9, and TWIST. Univariate analysis also identified expression levels of E-cadherin, N-cadherin, MMP-2, MMP-9 and TWIST, in addition to the tumor size, pathological T category, and concomitant carcinoma in situ, as significant predictors of intravesical recurrence-free survival. Of these significant factors, expression levels of E-cadherin, MMP-9, and TWIST; tumor size; and concomitant carcinoma in situ appeared to be independently associated with intravesical recurrence-free survival on multivariate analysis. Furthermore, there were significant differences in recurrence-free survival according to positive numbers of these 5 independent risk factors (i.e., positive for 0 or 1 factor vs. positive for 2 factors vs. positive for 3 or more factors).ConclusionsConsideration of expression levels of EMT-associated markers in TUR specimens, in addition to conventional prognostic parameters, would contribute to the accurate prediction of intravesical recurrence following TUR for NMIUCB.     

Sirolimus Attenuates Calcineurin Inhibitor-Induced Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma

BackgroundCalcineurin inhibitors (CNIs), which are potent immunosuppressants (ISs), increase the risk for hepatocellular carcinoma (HCC) recurrence after liver transplantation (LTx). Epithelial-mesenchymal transition (EMT) is a key process in which epithelial cancer cells lose their polarity, resulting in cancer progression and metastasis. The aim of this study was to evaluate the effect of sirolimus (SRL) individually and in combination with other ISs to reduce EMT.MethodsHCC SK-Hep1 cells were used and various ISs (SRL, tacrolimus, cyclosporine A, or mycophenolate mofetil) were administered at 2 dosages and in combination therapies. Mice were transplanted with SK-Hep1 cells (in the liver) and were monitored after 2 weeks.ResultsThe in vitro treatment with SRL showed a dose-dependent attenuation of cell proliferation and migration in case of the individual and IS combination treatments; further, decreased levels of pro-EMT proteins, namely, N-cadherin, transforming growth factor-β, ZEB1, Slug, and Snail were observed. In contrast, E-cadherin expression was upregulated after both the individual and IS combination treatments. These results were also observed in the samples from mice transplanted with the SK-Hep1 cells.ConclusionThe present study demonstrated that SRL reduced HCC metastasis by inhibiting EMT. Thus, our findings provide a rationale for the use of SRL in combination with ISs in HCC LTx patients.     

High expression of polo-like kinase 1 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

ObjectivesPolo-like kinase 1 (Plk1) has been widely pursued as an oncology target because it is overexpressed in several human tumor types. To investigate whether Plk1 plays a general role in bladder urothelial carcinoma, we examined the expression of Plk1 protein in bladder urothelial carcinoma and cell lines, and analyzed the relationship among Plk1 protein expression, metastasis, and recurrence of urinary bladder urothelial carcinoma.MethodsImmunohistochemistry was used to detect the expression of Plk1 in 120 bladder urothelial carcinoma. Moreover, the expression of Plk1 was analyzed by Western blot in 60 bladder urothelial carcinoma and 21 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of scytonemin (the inhibitor of Plk1). Statistical analysis was used to discuss the association between Plk1 expression and clinicopathologic parameters, tumor metastasis and recurrence, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells.ResultsThere was a significantly higher Plk1expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Plk1 expression was positively correlated with histologic grade, pT stage, recurrence, and metastasis. With the increasing concentration of scytonemin, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G2/M stage.ConclusionPlk1 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Plk1 played an important function on the bladder cancer cells' proliferation by regulating the cancer cell cycle from G1/S to G2/M and probably promoted the invasion and metastasis of bladder cancer.     

Slug在胰腺癌中的表达及干扰Slug对胰腺癌转移的影响

目的 探讨Slug和E-CADHERIN(E-cadherin)表达与胰腺癌转移的关系以及干扰Slug转录因子对胰腺癌转移的影响.方法 采用免疫组化和RT-PCR技术分别检测36例胰腺癌组织中Slug、E-cadherin和mRNA表达.将肝脏高转移潜能的胰腺癌细胞株SW1990H4,分为3组:对照组、空载体质粒(shRNA-1)和转染干扰S1ug质粒(Slug-shRNA-1),观察Slug对E-cadherin mRNA表达的逆转作用,及对SW1990H4体外侵袭、运动的抑制作用.结果 胰腺癌组织中Slug高表达,而E-cadherin低表达.转移组胰腺癌组织中Slug蛋白和mRNA表达明显高于非转移组,差异分别有统计学意义(P＜0.05),而E-cadherin在转移组胰腺癌中无表达.PCR产物凝胶电泳结果显示,Slug mRNA在空白对照组SW1990H4中表达为0.985±0.016,E-cadherin mRNA表达为0.120±0.001.shRNA-1时转染48 h时,Slug mRNA的表达水平为0.973±0.014,E-cadherin mRNA表达水平分别0.160±0.001,与对照组比差异分别无统计学意义(P＞0.05).转染Slug-shRNA-1组瞬时转染48 h时,Slug mRNA的表达水平为0.554±0.011,E-cadherin mRNA表达水平为0.36±0.002,与对照组和shRNA-1组比差异分别有统计学意义(P＜0.05).SW1990H4稳定转染后,shRNA-1和Slug-shRNA-1组Slug mRNA表达水平分别为0.968±0.015,0.206±0.017,两组比较,差异有统计学意义(P＜0.05),而E-cadherin mRNA表达水平分别为0.18±0.002,0.727±0.006,两组比较,差异有统计学意义(P＜0.05).体外细胞运动实验表明,对照组、转染shRNA-1和SlugshRNA-1组跨膜细胞数393±28、352±24、96±13,差异有统计学意义(P＜0.01).重组细胞基底膜(Matrige1)侵袭实验显示,3组穿透基底膜细胞数分别为223±69、202±64、65±19,差异有统计学意义(P＜0.05).结论 Slug高表达,E-cadherin低表达与胰腺癌的转移相关.胰腺癌细胞中存在Slug mRNA与E-cadherin mRNA表达的逆转关系,抑制Slug mRNA的表达对胰腺癌细胞的侵袭和运动有抑制作用,可能为胰腺癌转移的基因治疗提供新的靶点.

Abstract：

Objective To investigate expression of slug and E-cadherin in pancreatic cancer tissues and determine the inhibitory effects of anti-Slug, an anti-sense plasmid, on the invasion of pancreatic cancer cell lines in vitro. Methods Slug and E-cadherin protein and mRNA was analyzed by IHP and RT-PCR in 36 cases of pancreatic cancer. Then anti-Slug plasmid was transfected into herin and Slug expression. The inhibitory effects of anti-sense Slug were also detected by Transwell motility assay and Matrigel invasion assay. Results The expression of Slug and mRNA in metastatic pancreatic cancer tissue was higher than that in non-metastatic tissue. E-cadherin and mRNA was lower in metastasis tissues(P＜0.05). The inverse relationships were further observed by transient transfection of anti-Slug into SW1990H4 cells. The downregulated expression of Slug and re-expression of E-cadherin were found. The Slug mRNA levels were 0.985±0.016,0.973±0.014, 0. 554±0. 011 after 0, 48 h of transfection of anti-sense Slug, and that of E-cadherin were 0.120±0.001, 0.360±0.002, 0. 727±0. 006, respectively. The diference was significant between different time points (P＜0.05). The Slug mRNA levels were 0. 206±0.017, 0.968±0.015, and that of E-cadherin were 0. 18±0.002,0.727±0.006 after stable transfection of anti-sense Slug, and control plasmid, respectively. The diference was significant (P＜0.05). The motility activity(393±28, 352±24, 96 ±13 )and the invasion activity (223 ± 69, 202 ± 64, 65 ±19) of1 antisense Slug transfectant cells were significantly decreased as compared with those of control cells (P＜0.05). Conclusions Higher expression of slug and lower expression of E-cadherin is related to the invasion and metastasis in pancreatic cancer. A reverse corelation of E-cadherin and Slug expression exists in pancreatic cancer. Slug is possibly a potential target for cancer gene therapy blocking invasion and metastasis in human pancreatic cancer.     

EDARADD silencing suppresses the proliferation and migration of bladder cancer cells

PurposeBladder cancer is a kind of common malignant cancer in the urinary system. The expression of EDARADD (ectodysplasin-A receptor-associated death domain) in bladder cancer is higher than the normal samples. However, its role in bladder cancer remains unknown. In the present study, we analyzed the expression of EDARADD in 81 bladder cancer samples by immunohistochemistry as well as its correlation with clinical characteristics. In addition, the role of EDARADD was also explored through loss of function.Materials and MethodsCell proliferation assay and MTT assay were conducted to assess the proliferation of bladder cancer cells and transwell assay and wound healing assay were conducted to assess the migration of bladder cancer cells. On the other hand, the levels of epithelial-mesenchymal transition (EMT) associated proteins and the key molecules in the MAPK signaling pathway were detected by western blot. In vivo experiments were also conducted to determine the effect of EDARADD silencing on the metastasis of bladder cancer cells and the MAPK signaling pathway.ResultsEDARADD was highly expressed in bladder cancer samples, especially in high-grade bladder cancer samples. The high EDARADD level indicated a poor survival. Interestingly, EDARADD silencing suppressed the proliferation, migration and EMT of bladder cancer cells. Furthermore, the MAPK signaling pathway was repressed by EDARADD silencing. Additionally, silencing EDARADD also inhibited the metastasis of bladder cancer and the MAPK signaling pathway in vivo. It is indicated that silencing EDARADD may suppress the proliferation and metastasis of bladder cancer cells through the MAPK signaling pathway.ConclusionThese results indicate that EDARADD may become a probable target for the treatment of bladder cancer.     

Role of CIP4 in high glucose induced epithelial--mesenchymal transition of rat peritoneal mesothelial cells

Abstract

Background: Peritoneal mesothelial cell (PMC) plays a key role in the process of peritoneal fibrosis. Epithelial–mesenchymal transition (EMT) of PMCs is an important mechanism of peritoneal fibrosis. Prolonged exposure to peritoneal dialysis fluid containing a high concentration of glucose may lead to EMT of PMCs. Cdc42-interacting protein-4 (CIP4) is a critical regulator of cell skeleton and downstream effector of Cdc42 and participates in EMT of tubular epithelial cells. In the present study, we investigate the possible role of CIP4 in EMT of PMC under high glucose (HG) condition in vitro and further explore the potential therapeutic point for peritoneal fibrosis. Methods: Rat peritoneal mesothelial cells (RPMCs) were isolated from the peritonea of rats by enzymatic digestion. Under HG conditions (1.5%, 2.5% and 4.25%), E-cadherin, α-SMA and CIP4 expression were assessed by Western blot. Effect of CIP4-siRNA and pcDNA3.1-CIP4 transfection on E-cadherin, α-SMA and CIP4 expression were also assessed respectively under 2.5% HG concentration. Cells were pretreated for 24?h with PI3K/Akt signaling inhibitor perifosine and effect of perifosine on CIP4 expression were detected by Western blot. Results: EMT induction by HG was confirmed by the prevalence of morphological changes, loss of E-cadherin, increase in α-SMA expression. CIP4-siRNA transfection can reverse EMT of RPMCs. Over-expression of CIP4 promoted characteristics similar to those commonly observed in EMT. Furthermore, the increased CIP4 in response to HG was efficiently inhibited by perifosine. Conclusion: This study shows that CIP4 promotes high glucose-induced EMT through PI3K-Akt signaling pathway in RPMCs.     

Prediction of recurrence and progression in patients with T1G3 bladder cancer by gene expression of circulating tumor cells

ObjectiveTo investigate the role of gene expression of circulating tumor cells (CTCs) as noninvasive prognostic markers in patients with high risk nonmuscle invasive bladder cancer.Materials and methodsWe identified all patients with TIG3 urothelial bladder cancer (UBC) at our institution since 2016.The study included 100 patients with T1G3 UBC and 50 healthy volunteers. CTCs were isolated from blood using immunomagnetic separation and gene expression was performed using 10 bladder cancer associated genes, namely; KRAS, EPCAM, CD133, CD44, mTOR, SURVIVIN, AKT, PI3K, VEGF, and TP53. Gene expression of CTCs was correlated to time to first recurrence and time to progression using Kaplan-Meier curves.ResultsThere was strong negative correlation between CTCs-positive patients and time to first recurrence and time to progression. Significant differences in expression levels of specific genes were observed that can predict recurrence and progression of T1G3 UBC.ConclusionCTCs appear to be noninvasive methods of predicting disease recurrence and progression in patients with high- risk nonmuscle invasive bladder cancer; therefore, studying their molecular profiling may improve prediction of recurrence and progression. Further studies are invited for more in-depth investigation to consolidate our initial results.     

Increase sensitivity in detecting superficial,low grade bladder cancer by combination analysis of hypermethylation of E-cadherin,p16, p14, RASSF1A genes in urine

ObjectivesTo identify a better set of DNA methylation markers to detect superficial, low grade cancer cell in urine sediment for improving cancer treatment, morbidity, and mortality.Materials and MethodsMethylation-specific PCR (MSP) assay was used to detect promoter hypermethylation in 4 genes (E-cadherin, p16, p14, and RASSF1A) to identify reliable biomarkers for bladder cancer diagnosis in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. Urine DNA was compared with 20 healthy controls.ResultsFifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder cancer patients revealed hypermethylation in at least 1 of the 4 analyzed genes, whereas all urine samples from normal controls were negative. The sensitivity of MSP assay for detecting E-cadherin, p16, p14 and RASSF1A in tumor cells in voided urine was 35%, 35%, 33%, and 65%, respectively. Diagnostic sensitivity was 75% for combining RASSF1A and p14, and 83% for RASSF1A, p14 and E-cadherin. Urine cytology, however, detect only 13 (28%) cases of cancer or suspicious cancer. For detecting superficial and invasive bladder tumor, urine cytology revealed a sensitivity of 23% (6/26) and 35% (7/20), respectively. In contrast, MSP detected hypermethylation in the urine of 80% (37/46) bladder cancer patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) of low grade, 75% (15/20) of invasive and 79% (26/33) of high grade bladder cancers. Importantly, hypermethylation was detected in the urine DNA of 90% (18/20) superficial tumors with negative or atypia cytology.ConclusionsHypermethylation of E-cadherin, p14 or RASSF1A in urine sediment DNA is a potential biomarker for detecting superficial, low grade cancer. Besides, hypermethylation of these 3 genes is a valuable adjunct diagnostic marker to urine cytology, which can enhance the diagnostic accuracy and follow-up treatment of bladder cancer patients.