Recently long non-coding RNAs (lncRNA) have emerged as new gene regulators and prognostic markers in several cancers including renal cell carcinoma (RCC). In this study, we investigated the contributions of the lncRNA MALAT1 in RCC with a specific focus on its transcriptional regulation and its interactions with Ezh2 and miR-205. We found that MALAT1 expression was higher in human RCC tissues where it was associated with reduced patient survival. MALAT1 silencing decreased RCC cell proliferation and invasion and increased apoptosis. Mechanistic investigations showed that MALAT1 was transcriptionally activated by c-Fos and that it interacted with Ezh2. After MALAT1 silencing, E-cadherin expression was increased while beta-catenin expression was decreased through Ezh2. Reciprocal interaction between MALAT1 and miR-205 was also observed. Lastly, MALAT1 bound Ezh2 and oncogenesis facilitated by MALAT1 was inhibited by Ezh2 depletion, thereby blocking epithelial-mesenchyme transition via E-cadherin recovery and beta-catenin downregulation. Overall, our findings illuminate how overexpression of MALAT1 confers an oncogenic function in RCC that may offer a novel theranostic marker in this disease.

Background:Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).Methods:Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3′UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.Results:Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3′UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.Conclusion:Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2 0 -deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2 0 -deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.Renal cell carcinoma (RCC) is the third leading cause of death among urologic tumors, accounting for 2% of adult malignancies. 1 Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions are still found at diagnosis in $30% of patients with RCC.2 Wnt/beta-catenin signaling is involved in RCC. Canonical Wnt ligands bind to frizzled (FZD) family receptors and the LRP5/LRP6 coreceptor, which stabilize beta-catenin. Subsequently, beta-catenin interacts with members of the lymphoid enhancer factor 1/T-cell factor (LEF1/ TCF) family, resulting in a functional transcription factor complex and the expression of downstream target genes. 3,4Noncanonical Wnt ligands also bind to FZD family receptors and ROR2 and RYK coreceptors. [4][5][6][7] This signaling is mainly involved in cytoskeletal reorganization during cancer cell invasion and metastasis. 6,7 Among the five Wnt antagonist families [secreted FZD-related protein (sFRP), Wnt inhibitory factor 1 (Wif1), Xenopus cerberus, Wise and Dickkopf (DKK) families], the DKK family consists of four main members (DKK1-4), which contain two distinct cysteine-rich domains. 3,8 Recently, our laboratory reported that Wnt antagonists including Wif1, sFRP1, sFRP2, sFRP4, sFRP5 and DKK3 could be used as epigenetic biomarkers for RCC detection.9 Similar to these Wnt antagonist genes, DKK1 is also silenced...

Purpose: Wnt/β-catenin signaling is involved in renal cancer. DKK2, a Wnt antagonist, is silenced in some cancers, although its function has not been investigated. We hypothesized that DKK2 may be epigenetically silenced and inhibits progression of renal cell carcinoma (RCC). Experimental Design: RCC cell lines and a normal kidney cell line were used for methylation and chromatin immunoprecipitation assays. To assess various functions of DKK2, we established stable DKK2-transfected cells and examined them with regard to cell viability, colony formation, apoptosis, cell cycle, and invasive capability. A total of 52 patients with confirmed conventional RCC were enrolled in this study. Results: RCC cell lines had decreased levels of DKK2, which were significantly increased after treatment with 5-Aza-2′-deoxycytidine alone or 5-Aza-2′-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the levels of acetyl H3, acetyl H4, and dimethylated H3K4 were decreased, whereas the level of dimethylated H3K9 was increased in RCC cell lines compared with HK2 cells. Increased methylation in RCC tissues was associated with higher grades, pathologic stages, and pathologic tumor in RCC. Functional analysis showed that the numbers of viable A498 cells were significantly decreased in DKK2-transfected cells compared with mock cells. The number of apoptotic cells and S/G 2 -M phase cells was significantly increased and decreased after DKK2 transfection, respectively. Corresponding to these results, Bcl2 and cyclin D1 expression were also decreased in DKK2-overexpressing cells. Conclusion: DKK2 is epigenetically silenced by methylation in higher grades and stages of RCC. These results suggest that DKK2 inhibits renal cancer progression through apoptotic and cell cycle pathways. (Clin Cancer Res 2009;15(18):5678-87) Renal cell carcinoma (RCC) is the third leading cause of death among urological tumors, accounting for ∼2% of adult malignancies (1). Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions are still found at diagnosis in ∼30% of RCC patients (2). Wnt/β-catenin signaling is involved in renal cancer. Canonical Wnt ligands bind to frizzled (FZD) family receptors and the LRP5/LRP6 coreceptor, which stabilize β-catenin. Subsequently, β-catenin interacts with members of the lymphoid enhancer factor 1/T-cell factor family, resulting in generation of a functional transcription factor complex and the expression of downstream target genes (3, 4). Noncanonical Wnt ligands also bind to FZD family receptors and ROR2 and RYK coreceptors (4-7). This signaling is mainly involved in cytoskeletal reorganization during cancer cell invasion and metastasis (6, 7). At present, five Wnt antagonist families have been described, namely, secreted FZD-related protein (sFRP), Wnt inhibitory factor 1, Xenopus Cerberus, Wise, and Dickkopf (DKK) families (8).Among Wnt antagonists, the DKK family consists of four main members (DKK1-DKK4), which contain two disti...