Genistein Represses HOTAIR/Chromatin Remodeling Pathways to Suppress Kidney Cancer.

BACKGROUND/AIMS: Genistein, a soy isoflavone, has been shown to have anti-cancer effects in various cancers including renal cancer. Long non-coding RNA, HOX transcript antisense RNA (HOTAIR), is involved in cancer progression and metastasis, such as renal cancer. Our aim was to investigate the effects of genistein on HOTAIR chromatin remodeling functions. METHODS: We used MTS assays and Transwell migration assays to study the effects of genistein on cell proliferation and migration respectively in human renal cell carcinoma (RCC) cell lines. We used Western blots to analyze SNAIL and ZO-1 expression. We performed chromatin immunoprecipitation (ChIP) assays to study recruitment of the polycomb repressive complex 2 (PRC2) to the ZO-1 promoter. We performed RNA immunoprecipitation (RIP) assays to study interaction between HOTAIR and PRC2, SMARCB1 or ARID1A. We also performed transfection experiments to overexpress EED, HOTAIR and knockdown SMARCB1. RESULTS: Genistein reduced cell proliferation and migration of human renal cell carcinoma cell lines. ChIP assays indicated that genistein reduces recruitment of the PRC2 to the ZO-1 promoter and increased its expression. RIP assays showed that genistein inhibits HOTAIR interaction with PRC2, leading to tumor suppression. Immunoprecipitation also revealed that genistein reduced EED levels in PRC2, suggesting that decreased EED levels suppress HOTAIR interaction with PRC2. EED overexpression in the presence of genistein restored PRC2 interaction with HOTAIR and reduced ZO-1 transcription, suggesting genistein activates ZO-1 by inhibiting HOTAIR/PRC2 functions. RIP assays also showed that HOTAIR interacts with SMARCB1 and ARID1A, subunits of the human SWI/SNF chromatin remodeling complex and genistein reduces this interaction. Combination of HOTAIR overexpression and SMARCB1 knockdown in the presence of genistein revealed that genistein inhibits SNAIL transcription via the HOTAIR/SMARCB1 pathway. CONCLUSION: Genistein suppresses EED levels in PRC2 and inhibits HOTAIR/PRC2 interaction. Genistein suppresses HOTAIR/PRC2 recruitment to the ZO-1 promoter and enhances ZO-1 transcription. Genistein also inhibits SNAIL transcription via reducing HOTAIR/SMARCB1 interaction. We demonstrate that the reduction of HOTAIR interaction with chromatin remodeling factors by genistein represses HOTAIR/chromatin remodeling pathways to suppress RCC malignancy.

Role of the PI3K/Akt pathway in cadmium induced malignant transformation of normal prostate epithelial cells.

This study investigated the role of the PI3K/Akt pathway in cadmium (Cd) induced malignant transformation of normal prostate epithelial (PWR1E and RWPE1) cells. Both PWR1E and RWPE1 cells were exposed to 10 µM Cd for one year and designated as Cd-PWR1E and Cd-RWPE1. Cd-RWPE1 cells robustly formed tumors in athymic nude mice. Functionally, Cd-exposure induced tumorigenic attributes indicated by increased wound healing, migration and invasion capabilities in both cell lines. RT2-array analysis revealed many oncogenes including P110α, Akt, mTOR, NFKB1 and RAF were induced whereas tumor suppressor (TS) genes were attenuated in Cd-RWPE1. This was validated by individual quantitative-real-time-PCR at transcriptional and by immunoblot at translational levels. These results were consistent in Cd-PWR1E vs parental PWR1E cells. Gene Set Enrichment Analysis revealed that five prostate cancer (PCa) related pathways were enriched in Cd-exposed cells compared to their normal controls. These pathways include the KEGG- Pathways in cancer, Prostate Cancer Pathway, ERBB, Apoptosis and MAPK pathways. We selected up- and down-regulated genes randomly from the PI3K/Akt pathway array and profiled these in the TCGA/GDC prostate-adenocarcinoma (PRAD) patient cohort. An upregulation of oncogenes and downregulation of TS genes was observed in PCa compared to their normal controls. Taken together, our study reveals that the PI3K/Akt signaling is one of the main molecular pathways involved in Cd-driven transformation of normal prostate epithelial cells to malignant form. Understanding the molecular mechanisms involved in the Cd-driven malignant transformation of normal prostate cells will provide a significant insight to develop better therapeutic strategies for Cd-induced prostate cancer.

Activation of the Erk/MAPK signaling pathway is a driver for cadmium induced prostate cancer.

PURPOSE: Cadmium (Cd) is reported to be associated with carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer (PCa) remain elusive. MATERIALS AND METHODS: RWPE1, PWR1E and DU 145 cells were used. RT2 Profiler Array, real-time-quantitative-PCR, immunofluorescence, cell cycle, apoptosis, proliferation and colony formation assays along with Gene Set Enrichment Analysis (GSEA) were performed. RESULT: Chronic Cd exposure of non-malignant RWPE1 and PWR1E cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. Even a two-week Cd exposure of PCa cell line (DU 145) significantly increased the proliferation and decreased apoptosis. RT2 profiler array of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis in both Cd-RWPE1 and Cd-PWR1E cell lines. GSEA showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from Erk/MAPK pathway and performed profile analysis in a PCa data set from the TCGA/GDC data base. We observed upregulation of these genes in PCa compared to normal samples. An increase in phosphorylation of the Erk1/2 and Mek1/2 was observed in Cd-RWPE1 and Cd-PWR1E cells compared to parental cells, confirming that Cd-exposure induces activation of the Erk/MAPK pathway. CONCLUSION: This study demonstrates that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate cells. Understanding these dominant oncogenic pathways may help develop optimal therapeutic strategies for PCa.

Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression.

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

BPTF transduces MITF-driven prosurvival signals in melanoma cells.

Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

Antitumor activity of miR-1280 in melanoma by regulation of Src.

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.

Long non-coding RNA HOTAIR is targeted and regulated by miR-141 in human cancer cells.

HOTAIR is a long non-coding RNA that interacts with the polycomb repressive complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition process, and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in human cancer cells. miR-141 binds to HOTAIR in a sequence-specific manner and suppresses HOTAIR expression and functions, including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Ago2 (Argonaute2) complex, and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner.

CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma.

BACKGROUND: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. METHODS: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses. RESULTS: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples. CONCLUSIONS: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

Cytochrome P450 1B1 polymorphisms and risk of renal cell carcinoma in men.

The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C → G, Arg → Gly, rs10012), 119 (G → T, Ala → Ser, rs1056827), 432 (C → G, Leu → Val, rs1056836), 449 (C → T, Asp, rs1056837), and 453 (A → G, Asn → Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.

MicroRNA-34a suppresses malignant transformation by targeting c-Myc transcriptional complexes in human renal cell carcinoma.

We investigated the functional effects of microRNA-34a (miR-34a) on c-Myc transcriptional complexes in renal cell carcinoma. miR-34a down-regulated expression of multiple oncogenes including c-Myc by targeting its 3' untranslated region, which was revealed by luciferase reporter assays. miR-34a was also found to repress RhoA expression by suppressing the c-Myc-Skp2-Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression and inhibition of cell invasion, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress the c-Myc-P-TEFb transcription elongation complex, indicating one of the mechanisms by which miR-34a has profound effects on cellular functions. Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that activates or elongates transcription, indicating a novel role of miR-34a in the regulation of transcription by c-Myc.

Catechol-O-methyltransferase-mediated metabolism of 4-hydroxyestradiol inhibits the growth of human renal cancer cells through the apoptotic pathway.

Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.

miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein.

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.

Wnt signaling pathways in urological cancers: past decades and still growing.

The Wnt signaling pathway is involved in a wide range of embryonic patterning events and maintenance of homeostasis in adult tissues. The pathological role of the Wnt pathway has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. Constitutive activation of the Wnt signaling pathway is a common feature of solid tumors and contributes to tumor development, progression and metastasis in various cancers. In this review, the Wnt pathway will be covered from the perspective of urological cancers with emphasis placed on the recent published literature. Regulation of the Wnt signaling pathway by microRNAs (miRNA), small RNA sequences that modify gene expression profiles will also be discussed. An improved understanding of the basic genetics and biology of Wnt signaling pathway will provide insights into the development of novel chemopreventive and therapeutic strategies for urological cancers.

IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

The Wnt/ß-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ ß-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and ß-catenin expression was increased. Also the ß-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis.

Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer.

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma.

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'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2'-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.

Wnt antagonist DICKKOPF-3 (Dkk-3) induces apoptosis in human renal cell carcinoma.

The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. However, the functional significance and mechanisms of inactivation of Wnt antagonist Dkk-3 gene in renal cell carcinoma (RCC) has not been reported. In this study, we examined potential epigenetic mechanisms regulating Dkk-3 expression in RCC cells and whether Dkk-3 expression affects cell growth and apoptosis. The expression of Dkk-3 is regulated by histone modification rather than CpG island DNA methylation in renal cancer cells. Renal cancer cell proliferation was significantly inhibited and apoptosis was promoted in Dkk-3 transfected renal cancer cells. Dkk-3 did not inhibit the Wnt/beta-catenin signaling pathway but induced apoptosis via the noncanonical JNK pathway in renal cancer cells. Expression of p21, MDM-2, and Puma genes were increased after transfecting RCC cell lines with a Dkk-3 expression plasmid. Overexpression of Dkk-3 induced G(0)/G(1) arrest together with an increase in p21 expression. Growth of stable Dkk-3 transfected cells in nude mice was decreased compared to controls. Our data show for the first time that mRNA expression of Dkk-3 is regulated by histone modification and that Dkk-3 inhibits renal cancer growth through modulation of cell cycle and apoptotic pathways.

A lncRNA TCL6-miR-155 Interaction Regulates the Src-Akt-EMT Network to Mediate Kidney Cancer Progression and Metastasis.

Metastasis is the leading cause of mortality from kidney cancer, and understanding the underlying mechanism of this event will provide better strategies for its management. Here we investigated the biological, functional, and clinical significance of lncTCL6 and its interacting miR-155 in clear cell renal cell carcinoma (ccRCC). We employed a comprehensive approach to investigate the lncTCL6-miR-155-Src/Akt-mediated epithelial-to-mesenchymal transition (EMT) pathway as a novel regulatory mechanism in ccRCC progression. Expression analyses revealed that lncTCL6 is downregulated in ccRCC compared with normal tissues. Overexpression of lncTCL6 in ccRCC cell lines impaired their oncogenic functions, such as cell proliferation and migration/invasion, and induced cell-cycle arrest and apoptosis; conversely, depletion of lncTCL6 rescued these phenotypic effects. Furthermore, lncTCL6 directly interacted with miR-155. Unlike lncTCL6, miR-155 was overexpressed in ccRCC. Stable knockdown of miR-155 phenocopied the effects of lncTCL6 overexpression. Conversely, reconstitution of miR-155 and suppression of lncTCL6 in noncancerous renal cell HK2 induced tumorigenic characteristics. Patients with higher expression of lncTCL6 and lower expression of miR-155 had better survival probability. When overexpressed, lncTCL6 recruited STAU1 and mediated decay of Src mRNA, followed by a marked downregulation of an integrated network of Src target genes involved in migration, invasion, and EMT. However, the interaction between miR-155 and lncTCL6 attenuated the regulatory role of lncTCL6 on Src-mediated EMT. In conclusion, this study is the first report documenting the lncTCL6-miR155-Src/Akt/EMT network as a novel regulatory mechanism in aggressive ccRCC and a promising therapeutic target to inhibit renal cancer. SIGNIFICANCE: This study's investigation of noncoding RNA interactions in renal cell carcinoma identify miRNA-155-lncRNA TCL6-mediated regulation of the Src-Akt-EMT network as a novel mechanism of disease progression and metastasis.