Jinhuan Wei

Protein Expression of ZEB2 in Renal Cell Carcinoma and Its Prognostic Significance in Patient Survival

Background ZEB2 has been reportedly shown to mediate the epithelial-to-mesenchymal transition (EMT) and disease aggressiveness in human tumors. However, the expression status of ZEB2 in renal cell carcinoma (RCC) and ZEB2’s clinicopathologic/prognostic significance are poorly understood. Methodology/Principal Findings In this study, tissue microarray, immunohistochemistry (IHC) and western blot analyses were utilized to investigate the ZEB2 expression status in RCC and adjacent renal tissue samples. In our study, samples from 116 RCC patients treated with radical nephrectomy were used as a training set to generate a ZEB2 optimal cut-point for patient outcome by receiver operating characteristic (ROC) analysis. For validation, the correlation of ZEB2 expression with the clinical characteristics and patient outcomes in another set (including 113 patients) was analyzed to validate the obtained cut-point. In the training and validation sets, high expression of ZEB2, defined by ROC analysis, predicted a poorer overall survival and progression-free survival, as evidenced by the univariate and multivariate analyses. In different subsets of overall patients, ZEB2 expression was also a prognostic indicator in patients with stage I/II, stage III/IV, grade 1/2 and grade 3/4 disease (P<0.05). Downregulation of ZEB2 by shRNA decreased the migration and invasion ability of 769-P cells in vitro. Furthermore, high ZEB2 expression was positively correlated with vimentin expression and inversely linked to E-cadherin expression in RCC. Conclusions/Significance Our findings provide a basis for the concept that high ZEB2 expression in RCC may be important in the acquisition of an aggressive phenotype. This evidence suggests that ZEB2 overexpression (examined by IHC) is an independent biomarker for the poor prognosis of patients with RCC.

Overexpression of Rab25 contributes to metastasis of bladder cancer through induction of epithelial–mesenchymal transition and activation of Akt/GSK-3β/Snail signaling

Rab25, an epithelial-specific member of the Rab family of small guanosine triphosphatases, is associated with several human cancers. The goal of this study was to determine its function in bladder cancer (BC). We examined the Rab25 expression pattern in two different cohorts of BC patients treated with radical cystectomy by quantitative PCR, western blotting and immunohistochemical staining. A series of in vitro and in vivo assays were performed to elucidate the function of Rab25 in BC and its underlying mechanisms. Rab25 expression was significantly elevated at both the messenger RNA and protein levels in BCs compared with normal bladder tissues. High Rab25 expression was closely associated with lymph node (LN) metastasis and was an independent predictor for poor disease-free survival in BC patients. Downregulation of Rab25 in BC cells markedly inhibited invasive motility in vitro and metastatic potential in vivo. In addition, downregulation of Rab25 in BC EJ and T24 cells increased the expression levels of epithelial markers (E-cadherin and α-catenin) and decreased the levels of mechamechy markers (vimentin and fibronectin). Simultaneously, downregulation of Rab25 in EJ and T24 cells resulted in the inactivation of downstream phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-β (p-GSK-3β) and snail signaling. This study demonstrates that Rab25 can promote BC metastasis through induction of epithelial-mesenchymal transition process and activation of Akt/GSK-3β/Snail signaling pathway; Rab25 expression level can predict LN metastasis and inferior clinical outcome in BC patients.

The inhibition of human bladder cancer growth by calcium carbonate/CaIP6 nanocomposite particles delivering AIB1 siRNA.

Previously, we reported that inorganic amorphous calcium carbonate (ACC) hybrid nanospheres functionalized with Ca(II)-IP6 compound (CaIP6) is a promising gene vector in vitro. Here, nonviral gene carrier, ACC/CaIP6 nanocomposite particles (NPACC/CaIP6), was evaluated for efficient in vitro and in vivo delivery of small interfering RNA (siRNA) targeting human Amplified in breast cancer 1 (AIB1). The nanoparticle is capable of forming ACC/CaIP6 nanoparticle-siRNA complexes and transferring siRNA into targeted cells with high transfection efficiency. Meanwhile the ACC/CaIP6 nanoparticle-siRNA complexes have no obvious cytotoxicity for human bladder cancer T24 cells. Furthermore, NPACC/CaIP6 effectively protected the encapsulated siRNA from degradation, AIB1 knockdown mediated by ACC/CaIP6/siRNA complexes transfection resulted in cells proliferation inhibition, apoptosis induction and cell cycle arrest in vitro. NPACC/CaIP6 exhibited well tissues penetrability in localized siRNA delivering, intratumoral injection of NPACC/CaIP6/siAIB1 could attenuate tumor growth and downregulation of PI3K/Akt signaling pathway in vivo. We conclude that ACC/CaIP6 nanoparticle is a promising system for effective delivery of siRNA for cancer gene therapy.

EIF5A2 predicts outcome in localised invasive bladder cancer and promotes bladder cancer cell aggressiveness in vitro and in vivo.

Background:EIF5A2, eukaryotic translation initiation factor 5A2, is associated with several human cancers. In this study, we investigated the role of EIF5A2 in the metastatic potential of localised invasive bladder cancer (BC) and its underlying molecular mechanisms were explored.Methods:The expression pattern of EIF5A2 in localised invasive BC was determined by immunohistochemistry. In addition, the function of EIF5A2 in BC and its underlying mechanisms were elucidated with a series of in vitro and in vivo assays.Results:Overexpression of EIF5A2 was an independent predictor for poor metastasis-free survival of localised invasive BC patients treated with radical cystectomy. Knockdown of EIF5A2 inhibited BC cell migratory and invasive capacities in vitro and metastatic potential in vivo and reversed epithelial–mesenchymal transition (EMT), whereas overexpression of EIF5A2 promoted BC cells motility and invasiveness in vitro and metastatic potential in vivo and induced EMT. In addition, we found that EIF5A2 might activate TGF-β1 expression to induce EMT and drive aggressiveness in BC cells. EIF5A2 stabilized STAT3 and stimulated nuclear localisation of STAT3, which resulted in increasing enrichment of STAT3 onto TGF-β1 promoter to enhance the transcription of TGF-β1.Conclusions:EIF5A2 overexpression predicts tumour metastatic potential in patients with localised invasive BC treated with radical cystectomy. Furthermore, EIF5A2 elevated TGF-β1 expression through STAT3 to induce EMT and promotes aggressiveness in BC.

AIB1 predicts bladder cancer outcome and promotes bladder cancer cell proliferation through AKT and E2F1.

Background:We previously demonstrated that AIB1 overexpression is an independent molecular marker for shortened survival of bladder cancer (BC) patients. In this study, we characterised the role and molecular mechanisms of AIB1 in BC tumorigenicity.Methods:AIB1 expression was measured by immunohistochemistry in non-muscle-invasive BC tissue and adjacent normal bladder tissue. In addition, the tumorigenicity of AIB1 was assessed with in vitro and in vivo functional assays.Results:Overexpression of AIB1 was observed in tissues from 46 out of 146 patients with non-muscle-invasive BC and was an independent predictor for poor progression-free survival. Lentivirus-mediated AIB1 knockdown inhibited cell proliferation both in vitro and in vivo, whereas AIB1 overexpression promoted cell proliferation in vitro. The growth-inhibitory effect induced by AIB1 knockdown was mediated by G1 arrest, which was caused by reduced expression of key cell-cycle regulatory proteins through the AKT pathway and E2F1.Conclusion:Our results suggest that AIB1 promotes BC cell proliferation through the AKT pathway and E2F1. Furthermore, AIB1 overexpression predicts tumour progression in patients with non-muscle-invasive BC.

CaCO3/CaIP6 composite nanoparticles effectively deliver AKT1 small interfering RNA to inhibit human breast cancer growth

Background Small interfering RNA (siRNA)-mediated gene therapy is a promising strategy to temporarily inhibit the expression of genes involved in development of breast cancer. The lack of a safe and efficient gene delivery system has become a major hurdle for siRNA-mediated gene therapy in breast cancer. Our previous studies have demonstrated that inorganic amorphous calcium carbonate (ACC) hybrid nanospheres functionalized with CaIP6 (ACC/CaIP6) nanoparticles are an efficient nucleic acid delivery tool. The present study aimed to evaluate the safety and efficiency of ACC/CaIP6 in delivering siRNA targeting AKT1 (siAKT1) for the treatment of breast cancer. Methods The cytotoxicity of the ACC/CaIP6 nanoparticles was evaluated using a tetrazolium assay. The transfection efficiency and intracellular distribution of ACC/siAKT1 were analyzed by flow cytometry and confocal laser scanning microscopy, respectively. A series of in vitro and in vivo assays was performed to evaluate the effects of ACC/CaIP6/siAKT1 on growth of breast cancer cells. Results ACC/CaIP6 nanoparticles effectively transfected cells with little or no toxicity. AKT1 knockdown by ACC/CaIP6/siAKT1 inhibited cell cycle progression and promoted apoptosis of MCF-7 cells. Intratumoral injection of ACC/CaIP6/siAKT1 significantly suppressed the growth of breast cancer in mice. Conclusion ACC/CaIP6 nanoparticles are a safe and efficient method of delivering siRNA for gene therapy in breast cancer.

The putative tumor suppressor microRNA-30a-5p modulates clear cell renal cell carcinoma aggressiveness through repression of ZEB2

Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma, can easily invade local tissues and metastasize, and is resistant to currently available treatments. Recent studies profiling microRNA expression in ccRCC have suggested miR-30a-5p may be deregulated in these cancer cells. To determine its role and mechanism of action in ccRCC, miR-30-5p expression levels were quantified and functions were analyzed using in vitro and in vivo experiments and bioinformatics. A decrease in miR-30a-5p expression was frequently noted in ccRCC cells and tissues. Importantly, low miR-30a-5p levels were significantly associated with a poor ccRCC patient prognosis. Stable overexpression of miR-30a-5p in 769-P cells was sufficient to prevent cellular proliferation and invasion in vitro and in vivo. Upon further examination, it was found that miR-30a-5p directly targeted the 3′-UTR of ZEB2 and suppressed ccRCC cell epithelial–mesenchymal transition. In addition, miR-30a-5p may be downregulated by the long non-coding RNA DLEU2. Taken together, these data reveal an important role for miR-30a-5p in the regulation of ccRCC proliferation and invasion, and indicate the potential for miR-30a-5p in applications furthering ccRCC prognostics and therapeutics.

Impact of age on the cancer-specific survival of patients with localized renal cell carcinoma: martingale residual and competing risks analysis.

Background Age at diagnosis has been shown to be an independent prognostic factor of localized renal cell carcinoma (RCC) in several studies. We used contemporary statistical methods to reevaluate the effect of age on the cancer-specific survival (CSS) of localized RCC. Methods and Findings 1,147 patients with localized RCC who underwent radical nephrectomy between 1993 and 2009 were identified in our four institutions. The association between age and CSS was estimated, and the potential threshold was identified by a univariate Cox model and by martingale residual analysis. Competing risks regression was used to identify the independent impact of age on CSS. The median age was 52 years (range, 19–84 years). The median follow-up was 61 months (range, 6–144 months) for survivors. A steep increasing smoothed martingale residual plot indicated an adverse prognostic effect of age on CSS. The age cut-off of 45 years was most predictive of CSS on univariate Cox analysis and martingale residual analysis (p = 0.005). Age ≤45 years was independently associated with a higher CSS rate in the multivariate Cox regression model (HR = 1.59, 95% CI = 1.05–2.40, p = 0.027) as well as in competing risks regression (HR = 3.60, 95% CI = 1.93–6.71, p = 0.001). Conclusions Increasing age was associated with a higher incidence of cancer-specific mortality of localized RCC. Age dichotomized at 45 years would maximize the predictive value of age on CSS, and independently predict the CSS of patients with localized RCC.

miR-106b-5p promotes renal cell carcinoma aggressiveness and stem-cell-like phenotype by activating Wnt/β-catenin signalling

Purpose To examine the role of miR-106b-5p in regulating the cancer stem-cell-like phenotype in clear cell renal cell carcinomas (ccRCC). Experimental Design Real-time PCR was performed to evaluate miR-106b-5p levels in ccRCC cell lines and patients specimens. A series of in vivo and in vitro assays were performed to confirm the effect of miR-106b-5p on ccRCC stemness phenotype. Results ccRCC cells and tissues expressed more miR-106b-5p than normal controls. Gain- and loss-of-function studies demonstrated that overexpression of miR-106b-5p in ccRCC cells increased the spheres formation ability and the proportion of side population cells. Ectopic expression of miR-106b-5p in ccRCC cells increased tumour growth rates and the number of metastatic colonies in the lungs by using an orthotopic kidney cancer model and a tail vein injection model, respectively. Mechanistic studies revealed that, miR-106b-5p has an activating effect on Wnt/β-catenin signalling. miR-106p-5p overexpression simultaneously targets multiple negative regulators of the Wnt/β-catenin pathway, namely, LZTFL1, SFRP1 and DKK2. In addition, we also confirmed that miR-106b-5p and its targets expression correlates with the overall-survival of ccRCC patients from TCGA. Conclusions These findings suggest that miR-106b-5p mediates the constitutive activation of Wnt/β-catenin signalling, likely serving as a potential therapeutic target for ccRCC.

RIN1 promotes renal cell carcinoma malignancy by activating EGFR signaling through Rab25

We previously identified the important role of RIN1 expression in the prognosis of clear cell renal cell carcinoma (ccRCC). The role of RIN1 in ccRCC malignancy and underlying molecular mechanisms remain unclear. Here we report that ccRCC cells and tissues expressed more RIN1 than normal controls. Gain‐of‐function and loss‐of‐function studies demonstrated that RIN1 enhanced ccRCC cell growth, migration and invasion abilities in vitro and promoted tumor growth and metastasis in vivo. Mechanistic studies revealed that RIN1 has an activating effect on EGFR signaling in ccRCC. In addition, we unveil Rab25, a critical GTPase in ccRCC malignancy, as a functional RIN1 interacting partner. Knockdown of Rab25 eliminated the augmentation of carcinoma cell proliferation, migration and invasion by ectopic RIN1. We also confirmed that RIN1 and Rab25 expression correlates with the overall‐survival of ccRCC patients from TCGA. These findings suggest that RIN1 plays an important oncogenic role in ccRCC malignancy by activation of EGFR signaling through interacting with Rab25, and RIN1 could be employed as an effective therapeutic target for ccRCC.