Kaori Kitae

Expression and sub-cellular localization of human ABH family molecules

AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1‐methyladenine and 3‐methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2‐oxoglutarate‐ and Fe(II)‐dependent (2OG‐Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG‐Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4–hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG‐Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG‐Fe(II) oxygenase domain but both an RNA‐binding motif and a methyl‐transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub‐cellular localization of EmGFP‐hABH8 was restricted to the cytoplasm. EmGFP‐hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP‐hABH2 splice variant localized in nucleoplasm with a dot‐like pattern. In some HeLa cells transfected with EmGFP‐hABH5, dot‐like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN.

Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer.

miR-629 Targets TRIM33 to Promote TGFβ/Smad Signaling and Metastatic Phenotypes in ccRCC

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFβ/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFβ-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFβ on the expression of epithelial–mesenchymal transition–related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFβ/Smad signaling pathway via TRIM33 in ccRCC. Implications: This study suggests that miR-629 has biomarker potential through its ability to regulate TGFβ/Smad signaling and accelerate ccRCC cell motility and invasion. Mol Cancer Res; 13(3); 565–74. ©2014 AACR.

AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion.

LOXL2 Status Correlates with Tumor Stage and Regulates Integrin Levels to Promote Tumor Progression in ccRCC

Clear cell renal cell carcinoma (ccRCC) is the most common histologically defined subtype of renal cell carcinoma (RCC). To define the molecular mechanism in the progression of ccRCC, we focused on LOX-like protein 2 (LOXL2), which is critical for the first step in collagen and elastin cross-linking. Using exon array analysis and quantitative validation, LOXL2 was shown to be significantly upregulated in clinical specimens of human ccRCC tumor tissues, compared with adjacent noncancerous renal tissues, and this elevated expression correlated with the pathologic stages of ccRCC. RNAi-mediated knockdown of LOXL2 resulted in marked suppression of stress-fiber and focal adhesion formation in ccRCC cells. Moreover, LOXL2 siRNA knockdown significantly inhibited cell growth, migration, and invasion. Mechanistically, LOXL2 regulated the degradation of both integrins α5 (ITGAV5) and β1 (ITGB1) via protease- and proteasome-dependent systems. In clinical ccRCC specimens, the expression levels of LOXL2 and integrin α5 correlated with the pathologic tumor grades. In conclusion, LOXL2 is a potent regulator of integrin α5 and integrin β1 protein levels and functions in a tumor-promoting capacity in ccRCC. Implications: This is the first report demonstrating that LOXL2 is highly expressed and involved in ccRCC progression by regulating the levels of integrins α5 and β1. Mol Cancer Res; 12(12); 1807–17. ©2014 AACR.

Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03–1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.

Abstract 5197: MiR-629 targets TRIM33 to promote TGF-β/Smad signaling in clear cell renal cell carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents the most common renal histology. To identify a novel biomarker or a candidate target of molecular-targeted drug for ccRCC, we focused on miRNA expression signatures obtained from ccRCC clinical specimens by miRNA-microarray. The expression signatures revealed that miR-629 was significantly up-regulated in ccRCC specimens compared to normal specimens. Inhibition of miR-629 through a hairpin miRNA inhibitor inhibited cell migration and invasion. We found that miR-629 directly targets the Tripartite motif-containing 33 (TRIM33) an inhibitor of TGF-β/Smad signaling pathway. Inhibition of miR-629 significantly suppressed TGF-β -induced Smad activation through up-regulation of TRIM33 expression and subsequent inhibition of Smad2/3 and Smad4 binding. Furthermore, miR-629 inhibition attenuated the effect of TGF-β on expression of EMT-related factors and cell migration and invasion. Finally, in human clinical specimens, TRIM33 was down-regulated where an association with pathological stage and grade. Our findings identify miR-629 as a potent regulator of TGF-β/Smad signaling pathway and functions as an oncomir, which may provide a novel therapeutic strategy for treatment of ccRCC. Citation Format: Kentaro Jingushi, Wataru Nakata, Yuko Ueda, Kaori Kitae, Kazutoshi Fujita, Motohide Uemura, Norio Nonomura, Kazutake Tsujikawa. MiR-629 targets TRIM33 to promote TGF-β/Smad signaling in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5197. doi:10.1158/1538-7445.AM2014-5197

High miR-122 expression promotes malignant phenotypes in ccRCC by targeting occludin

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. Although several studies have reported high expression of miR-122 in ccRCC, its physiological role remains unclear. To clarify the role of miR-122 in ccRCC, we compared miR-122 expression levels in non-cancerous tissue and ccRCC. Significant upregulation of miR-122 was observed in ccRCC specimens. Moreover, ccRCC patients with high miR-122 expression showed poor progression-free survival compared to those with low miR-122 expression. Overexpression of miR-122 using an miRNA mimic promoted proliferation, migration, and invasion activities of ccRCC cells. miR-122 directly targets occludin, a known component of tight junctions. Occludin knockdown promoted the cell migration activity but not proliferation or invasion activities of ccRCC cells. In human clinical specimens, miR-122 expression inversely correlated with occludin protein expression. These findings show that miR-122 is an oncomiR in ccRCC.

Pharmacological regulation of bladder cancer by miR-130 family seed-targeting LNA

Bladder cancer causes an estimated 150,000 deaths per year worldwide. However, no major improvements in clinical outcomes have been achieved in the past several decades; therefore, a promising therapeutic agent is required. Recent studies revealed the existence of multiple subtypes of bladder cancer with distinct molecular signatures. To create novel therapeutics for such heterogeneous cancers, a target molecule should regulate various cancer-related signaling pathways. Here, we focused on the oncogenic miR-130 family (miR-130b, miR-301a, and miR-301b) as a novel therapeutic target for bladder cancer. The pharmacological inhibition of miR-130 family molecules by seed-targeting with an 8-mer tiny locked nucleic acid (LNA) inhibited 5637 bladder cancer cell growth, migration, and invasion by repressing stress fiber formation. Moreover, the miR-130-targeted LNA suppressed the phosphorylation of both FAK and Akt, resulting in the upregulation of two protein phosphatases, phosphatase and tensin homolog and protein tyrosine phosphatase, non-receptor type 11. In addition, administration of miR-130 family-targeted LNA significantly suppressed tumor growth in an in vivo bladder cancer xenograft model. Taken together, the miR-130 family-targeted LNA is expected to be a promising therapeutic agent for bladder cancer. Abbreviations: miR: micro RNA; LNA: locked nucleic acid; FAK: focal adhesion kinase; PTEN: phosphatase and tensin homolog deleted from chromosome 10; PTPN11: Tyrosine-protein phosphatase nonreceptor type 11; UTR: untranslated region