Ceruloplasmin overexpression is associated with oncogenic pathways and poorer survival rates in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most prevalent renal malignancy. The pathogenesis of the disease is currently poorly understood, and the prognosis is poor. Therefore, in this study, we focused on exploring and identifying genes and signal transduction pathways that are closely related to ccRCC. Differentially expressed genes (DEGs) were analyzed using the renal cell oncogene expression profiles GSE100666 and GSE68417. DAVID evaluation of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses was used. We constructed a protein-protein interaction (PPI) network of DEGS using Cytoscape software and analyzed the submodules with the CytoHubba plugin. Finally, we performed western blot, immunohistochemistry, and PCR validation by collecting tissues, and also utilized cells for in vitro functional analysis of ceruloplasmin (CP). In total, 202 DEGs (52 upregulated and 150 downregulated genes) were identified. Upregulated DEGs are significantly rich in angiogenesis, cell adhesion, and response to hypoxia, whereas downregulated DEGs are involved in intracellular pH regulation, excretion, coagulation, and chloride transmembrane transport. We selected the interactions of the top 20 hub genes provided by the PPI network, all of which are involved in important physiological pathways in vivo, such as complement and coagulation cascades. Tissue protein assays demonstrated that renal cancer highly expressed CP, while in vitro experiments showed that CP could promote the invasion of renal cancer cells. Our study suggests that ALB, C3, LOX, HRG, CXCR4, GPC3, SLC12A3, CP, and CASR may be involved in the development of ccRCC, and is expected to provide theoretical support for future studies on the diagnosis and targeted therapy of ccRCC.

Dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibits human renal cell carcinoma cell growth.

CC-115 is a dual inhibitor of DNA-PKcs and mTOR, both are valuable therapeutic targets for renal cell carcinoma (RCC). Our results showed that CC-115 inhibited survival and proliferation of established RCC cell lines (786-O and A489) and primary human RCC cells. The dual inhibitor induced selective apoptosis activation in RCC cells, as compared to no cytotoxicity nor apoptotic effects toward normal renal epithelial cells. CC-115 inhibited DNA-PKcs and mTORC1/2 activation in RCC cells. It was however ineffective in DNA-PKcs-mTOR double knockout (DKO) 786-O cells. CC-115 induced feedback autophagy activation in RCC cells. Autophagy inhibitors or Beclin-1/Light chain 3 (LC3) silencing potentiated CC-115-induced anti-RCC cell activity. Conversely, ectopic overexpression of Beclin-1 inhibited CC-115-induced cytotoxicity. At last CC-115 oral administration inhibited 786-O subcutaneous xenograft growth in nude mice. Taken together, dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibited RCC cell growth.

Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth.

Bromodomain-containing protein 4 (BRD4) and PI3K-AKT are both important for renal cell carcinoma (RCC) development and progression. SF2523 is a BRD4 and PI3K-AKT dual inhibitor. The present study demonstrated that SF2523 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. SF2523 induced activation of caspase and apoptosis in RCC cells. Further, SF2523 disrupted RCC cell cycle progression and inhibited cell migration in vitro. At the signaling level, SF2523 in-activated PI3K-AKT-mTOR, and downregulated BRD4-dependent proteins, Bcl-2 and Myc, in RCC cells. Remarkably, SF2523 was more efficient than Wortmannin (the PI3K inhibitor) and JQ1 (the BRD4 specific inhibitor) in killing RCC cells. In vivo, SF2523 administration at well-tolerated doses suppressed 786-O xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that concurrent blockage of BRD4 and PI3K-AKT signalings by SF2523 efficiently inhibits RCC cell growth in vitro and in vivo.

microRNA-302c-3p inhibits renal cell carcinoma cell proliferation by targeting Grb2-associated binding 2 (Gab2).

The expression and biological function of Grb2-associated binding 2 (Gab2) in renal cell carcinoma (RCC) cells was tested here. We showed that Gab2 expression was significantly elevated in human RCC tissues and RCC cells. It was correlated with over-activation of Akt and downregulation of microRNA-302c-3p ("miR-302c-3p"), a putative Gab2-targeting microRNA. Knockdown of Gab2 inhibited Akt activation and 786-O RCC cell proliferation. Reversely, forced over-expression of Gab2 led to Akt hyper-activation to facilitate 786-O cell proliferation. Exogenous expression of miR-302c caused Gab2 downregulation, Akt inhibition and 786-O cell proliferation inhibition. On the other hand, miR-302c-3p depletion by expressing its anti-sense ("antagomiR-302c") led to Gab2 upregulation, Akt activation and increased 786-O cell proliferation. Significantly, miR-302c-3p failed to affect the proliferation of 786-O cells with shRNA-depleted Gab2. Together, we suggest that miR-302c-3p depletion in human RCC cells leads to Gab2 over-expression, Akt hyper-activation and cell proliferation.

Concurrent inhibition of mTORC1 and mTORC2 by WYE-687 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Mammalian target of rapamycin (mTOR)in renal cell carcinoma (RCC) represents a valuable oncotarget for treatment. We here tested the potential anti-RCC activity by a novel mTOR kinase inhibitor WYE-687in vitro and in vivo.WYE-687 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. Yet, it was non-cytotoxic toHK-2 tubular epithelial cells.WYE-687 provoked caspase-dependent apoptosis in the RCC cells. At the molecular level, WYE-687 almost completely blocked mTORC1 (p-S6K1 and p-S6) and mTORC2 (p-Akt Ser 473) activation in both 786-Ocells and primary human RCC cells, where it downregulated both hypoxia-inducible factor (HIF)-1α and HIF-2α expression. Significantly, oral administration of WYE-687 potently suppressed786-O tumor xenograft growth in nude mice. mTORC1/2 activation and HIF-1α/2α expression were also remarkably downregulated in WYE-687-treated tumor tissues. Thus, our preclinical results imply that WYE-687 may have important translational value for the treatment of RCC.

Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation.

Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation.

[Establishment of a scoring system for predicting the positive rate of prostatic biopsy for prostate cancer].

OBJECTIVE: To identify the predictors of the positive results of transrectal ultrasound (TRUS)-guided biopsy for prostate cancer. METHODS: We performed univariate and multivariate logistic regression analyses on the relevant data on 385 male patients that underwent TRUS-guided biopsy for prostate cancer, including such potential predictors as age, body mass index (BMI), symptoms, results of digital rectal examination (DRE), tPSA, fPSA, free/total PSA ratio (f/tPSA), prostate volume (PV), and PSA density (PSAD) for identification of the risk factors related to the positive rate of biopsy. Then we constructed a scoring system as a tool for predicting prostate cancer in repeat biopsies and determined the sensitivity of the system by calculating the false positive rate using the receiver operating characteristic curve. RESULTS: Among the 385 patients, 139 (36.1%) were diagnosed with prostate cancer. On multivariate analysis, age (P < 0.01), DRE (P < 0.01), tPSA (P < 0.01), fPSA (P < 0.01), f/tPSA (P < 0.01), PV (P < 0.01), and PSAD (P < 0.01) were all significant predictors of prostate cancer. Multivariate logistic regression analysis showed age, tPSA, f/tPSA, PV, and PSAD to be independent predictors, with ORs and 95% CIs of 1.07 (1.05-1.16), 1.05 (1.02-1.15), 0.97 (0.86-0.99), 0.98 (0.87-0.96), and 1.79 (1.48-2.06), respectively. Moreover, patients with the risk score of 3-5 had a significantly higher rate of prostate cancer than those with 0-2 (64% vs 11%, P < 0.001). CONCLUSION: The scoring system on the key predictors of prostate cancer can help urologists to identify the men in need of prostatic biopsy.

Pre-clinical evaluation of AZD-2014, a novel mTORC1/2 dual inhibitor, against renal cell carcinoma.

Here we found that dual mTORC1/2 inhibitor AZD-2014 significantly inhibited RCC cell survival and growth, with higher efficiency than conventional mTORC1 inhibitors rapamycin and RAD001. RCC cell apoptosis was also induced by AZD-2014. AZD-2014 disrupted mTORC1/2 assembly and activation, while downregulating HIF-1α/2α and cyclin D1 expressions in RCC cells. Meanwhile, AZD-2014 activated autophagy, detected by p62 degradation, Beclin-1/ATG-5 upregulation and light LC3B-I/-II conversion. Autophagy inhibition by pharmacologic or siRNA-based means increased AZD-2014 activity in vitro, causing substantial RCC cell apoptosis. In vivo, AZD-2014 was more efficient than RAD001 in inhibiting 786-0 xenografts and downregulating HIF-1α/2α or p-AKT (Ser-473). Finally, AZD-2014's activity in vivo was further enhanced by co-administration of the autophagy inhibitor 3-methyaldenine. We provide evidence for clinical trials of using AZD-2014 in RCC treatment.

Diagnostic value of interleukin-8 in colorectal cancer: a case-control study and meta-analysis.

AIM: To assess the diagnostic value of serum interleukin-8 (IL-8) in the detection of colorectal cancer (CRC). METHODS: An original study was conducted to explore the potential value of IL-8 in CRC diagnosis. Receiver operating characteristic (ROC) analysis was performed and the area under the curve (AUC) value was calculated. PUBMED and EMBASE were searched (to October, 2013), supplemented with manual screening for relevant publications. Meta-analysis methods were applied to pool sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratios and to construct a summary receiver-operating characteristic (sROC) curve. Heterogeneity across studies was checked by the I(2) test and Deek's funnel plot method was applied to test publication bias. RESULTS: In our original study, serum IL-8 yielded an AUC of 0.742 [95%CI: 0.635-0.849]. The sensitivity and specificity were 85.42% and 54.05%, respectively, at a cut-off value of 5.39. In this meta-analysis, we included five studies with 668 CRC patients and 374 controls and one study in our own center with 48 CRC patients and 37 controls. The pooled sensitivity and specificity of IL-8 were 0.69 (95%CI: 0.42-0.87) and 0.85 (95%CI: 0.68-0.94) for CRC detection. Besides, the area under the sROC curve was 0.86 (95%CI: 0.82-0.88). Subgroup analysis suggested that there was no heterogeneity in the Asian subgroup but some in the European subgroup. In addition, no publication bias was found according to the Deek's funnel plot asymmetry test. CONCLUSION: Serum IL-8 is a promising biomarker for CRC detection and may become a clinically useful tool to identify high-risk patients.

[Correlation between androgen receptor expression and hepatitis B virus X protein and its clinical significance in hepatocellular carcinoma].

OBJECTIVE: To investigate the expression of androgen receptor (AR) and hepatitis B virus X protein (HBx) in hepatocellular carcinoma (HCC), and analyze the relationship between AR and HBx expressions. METHODS: Tumor tissues and peritumoral tissues of 83 HBV-associated HCC cases were investigated in this study. Fourteen cases of HBV-negative HCC and 13 cases of hemangioma peritumoral tissues were considered as control. AR and HBx mRNA levels were determined by quantitative fluorescence real-time RT-PCR and their protein levels were assayed by Western blot. The expression of AR and HBx proteins in tissues were examined with EnVision immunohistochemical staining. The methylation status of AR promoter was determined using methylation-specific PCR (MSP). RESULTS: Both expression levels of AR mRNA and protein of the peritumoral tissues were significantly higher (0.17) than that of tumor tissues (0.09) in HBV-associated HCC (P < 0.01), but such a difference was not found in HBV-negative HCC (0.06 vs. 0.07, P > 0.05). The level of AR expression in peritumoral tissues was associated with tumor differentiation in HBV-associated HCC. AR mRNA and protein levels of peritumoral tissues in HBV-associated HCC were significantly higher than that in HBV-negative HCC and hemangioma (all P < 0.05). In the tumor tissues, HBV-associated HCC had significantly higher AR expression than HBV-negative HCC at mRNA level (P < 0.05), but not at protein level. Spearman rank correlation analysis showed that the AR mRNA or AR protein levels were positively correlated with HBx in both tumor and peritumoral tissues in HBV-associated HCC, but the expressions of AR and HBx were not associated with AR promoter methylation status. The relative expression levels of AR mRNA and protein in the HBV-associated peritumoral tissues were negatively correlated with tumor differentiation (r = -0.213, P < 0.05; r = -0.313, P < 0.05), the higher the AR expression, the poorer differentiation. But this correlation of AR mRNA and protein was not shown in the hepatocellular carcinoma tissues. CONCLUSIONS: HBx may enhance AR expression in HBV-associated HCC, but AR promoter demethylation maybe not been involved in its main mechanism. An increased AR expression is probably an early event during the development and progression of HBV-associated HCC, and AR expression in the peritumoral tissue is correlated with HBV-associated HCC differentiation. AR may play different roles in HBV-associated HCC and HBV-negative HCC.

[Effects of caveolin-1 on biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line GU].

OBJECTIVE: To investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro. METHODS: Eukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy. RESULTS: The expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2. CONCLUSION: Overexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.

[Inhibited effects of exogenous caveolin-1 on the growth of laryngeal squamous cell carcinoma cell line Hep-2, experiments in vitro and in vivo].

OBJECTIVE: To study the effect of exogenous caveolin-1 on the growth of laryngeal squamous cell carcinoma and its mechanisms. METHODS: Eukaryotic expression vectors containing human caveolin-1 gene were transfected into Hep-2 cell line, the positive clones with high expression of caveolin-1 were identified by fluorescence quantitative real time reverse transcriptase-polymerase chain reaction and Western Blot. Cell proliferation viability was tested by methyl thiazolyl tetrazolium assay, the protein expression of epidermal growth factor receptor (EGFR), P-EGFR, extracellular signal-regulated kinase 1, 2 (Erk1, 2), P-Erkl, 2 and caveolin-1 were detected by Western Blot. The combination of caveolin-1 and EGFR were studied by immunoprecipitation and Western Blot. The in vivo antitumor activity of caveolin-1 was tested in Hep-2 xenograft tumor models in athymic nude mice, and the protein expressions of P-EGFR, P-Erk1, 2 and caveolin-1 were examined by immunohistochemistry. RESULTS: Three of caveolin-1 stably transfected Hep-2 cell clones were established. MTT assay showed that the proliferation of caveolin-1 overexpression Hep-2 cell clones decreased significantly comparing with the control. Immunoprecipitation and western Blot showed that caveolin-1 and EGFR were combined in Hep-2 cell line. Comparing with the parental cell line and cells transfected with control vector, there were the lower phosphorylation of EGFR and Erk1, 2 in the caveolin-1 overexpression Hep-2 cell clones. In the xenograft tumor models in nude mice, caveolin-1 overexpression Hep-2 cell clones showed the slower growth, smaller tumor size and the lower phosphorylation of EGFR and Erk1, 2. CONCLUSIONS: Overexpression of caveolin-1 inhibits growth of Hep-2 cell line in vitro and in vivo, arresting EGFR-MAPK signal pathway may involve in its mechanism.

[Expression and significance of hTERT mRNA in breast carcinoma and its relation to p53].

OBJECTIVE: This study was designed to investigate the significance of hTERT mRNA in breast carcinogenesis and to explore the diagnostic efficacy, and to study the effect of tumor suppressor gene p53 on the expression of hTERT mRNA. METHODS: The expression of hTERT mRNA was examined by in situ hybridization in 12 cases of normal breast tissue nearby cancer, 7 of simple ductal hyperplasia, 20 of atypical hyperplasia, 18 of ductal carcinoma in situ and 25 with invasive ductal carcinoma. The expression of p53 protein were examined by immunohistochemistry in 43 carcinomas. RESULTS: hTERT was not detected in normal breast tissue nearby cancer and simple ductal hyperplasia. The positive rate of hTERT mRNA in atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 25.0%, 83.3% and 88.0%, respectively. The prevalence and intensity of hTERT mRNA expression were much greater in carcinoma than those in simple or atypical hyperplasia and normal breast tissue nearby cancer (P < 0.05). The expression of hTERT was not correlated with tumor size and lymph node metastasis (P > 0.05). The positive correlation between hTERT mRNA and p53 was found in breast carcinoma (r = 0.5540, P < 0.01). CONCLUSION: hTERT mRNA expression is closely related to the malignant transformation of breast tissue. Semi-quantitative detection of hTERT mRNA expression in situ is helpful in differentiated diagnosis of carcinoma in situ and atypical hyperplasia. Inactivation of p53 may play a role in the transcriptive activation of hTERT gene in breast carcinoma.