Hiroko Hanafusa

Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons. We also demonstrated that four mRNA variants were transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.

Reduced expression of the REIC/Dkk-3 gene by promoter-hypermethylation in human tumor cells

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11p15.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in 11 out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased. In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population.

Identification of direct targets for the miR-17-92 cluster by proteomic analysis

MicroRNAs (miRNAs) are small non‐coding RNAs that post‐transcriptionally repress the expression of target genes. Many miRNAs have been implicated in a number of diseases, including cancers. The miR‐17‐92 miRNA cluster is known as a body of oncogenic miRNAs, and has been shown to be overexpressed in several cancers, including lung cancer. Although the overexpression of miR‐17‐92 is clearly implicated in the development of lung cancer, only a few direct targets for the miR‐17‐92 cluster have been identified thus far. In this study, we examined miR‐17‐92 target profiles in SBC‐3 small‐cell lung cancer cells using a quantitative proteomic strategy to identify direct targets of the miR‐17‐92 cluster. By knocking down the expression of endogenous miR‐19a, miR‐20a and miR‐92‐1, which are contained in the cluster, 112 up‐regulated proteins were detected and also identified as potential targets of these miRNAs. Among these candidate targets, we validated one direct target, RAB14. In conclusion, these findings suggest that proteomic approaches are valuable for identifying direct miRNA targets, and we were able to identify a novel direct target for the miR‐92‐1 using our proteomic strategy.

Novel direct targets of miR-19a identified in breast cancer cells by a quantitative proteomic approach.

The miR-17–92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.

Structure of the human retinoblastoma-related p107 gene and its intragenic deletion in a B-cell lymphoma cell line.

The human p107 protein shares many structural and functional features with the retinoblastoma gene product and retinoblastoma-related p130 protein. In this study, we have cloned and elucidated the complete intron-exon organization of the gene encoding the p107 protein. The gene contains 22 exons spanning over 100kilobase pairs of genomic DNA. The length of individual exons ranges from 50 to 840base pairs. The arrays of exons in the p107 gene are rather similar among members of the gene family, especially to those of the p130 gene, while the length of introns is extensively diverse. This study will provide a molecular basis for implementing comprehensive screening for p107 mutations using genomic DNAs from human malignancies. We also show a detailed structure of an intragenic deletion of the p107 gene found in a human B-cell lymphoma cell line, KAL-1, which was shown to occur by homologous recombination between the two directly repeated Alu family sequences.

Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression

Micro RNAs (miRNAs) regulate the expression of target genes posttranscriptionally by pairing incompletely with mRNA in a sequence-specific manner. About 30% of human genes are regulated by miRNAs, and a single miRNA is capable of reducing the production of hundreds of proteins by means of incomplete pairing upon miRNA–mRNA binding. Lately, evidence implicating miRNAs in the development of lung cancers has been emerging. In particular, miR-19a, which is highly expressed in malignant lung cancer cells, is considered the key miRNA for tumorigenesis. However, its direct targets remain underreported. In the present study, we focused on six potential miR-19a target genes selected by miRNA target prediction software. To evaluate these genes as direct miR-19a target genes, we performed luciferase, pull-down, and western blot assays. The luciferase activity of plasmids with each miR-19a–binding site was observed to decrease, while increased luciferase activity was observed in the presence of anti-miR-19a locked nucleic acid (LNA). The pull-down assay showed biotinylated miR-19a to bind to AGO2 protein and to four of six potential target mRNAs. Western blot analysis showed that the expression levels of the four genes changed depending on treatment with miR-19a mimic or anti-miR-19a-LNA. Finally, FOXP1, TP53INP1, TNFAIP3, and TUSC2 were identified as miR-19a targets. To examine the function of these four target genes in lung cancer cells, LK79 (which has high miR-19a expression) and A549 (which has low miR-19a expression) were used. The expression of the four target proteins was higher in A549 than in LK79 cells. The four miR-19a target cDNA expression vectors suppressed cell viability, colony formation, migration, and invasion of A549 and LK79 cells, but LK79 cells transfected with FOXP1 and TP53INP1 cDNAs showed no difference compared to the control cells in the invasion assay.

Abstract LB-246: Identification of novel direct targets of miR-19a in MCF-7 breast cancer cells

Background: MicroRNAs (miRNAs) are small non-cording RNAs (20-23 nucleotides) that negatively regulate gene expression at post-transcriptional level by interacting with 3′UTRs of their target mRNAs. Aberrant expression of miRNAs in cancer indicates that miRNAs play key roles in human cancers acting as either oncogenes or tumor suppressor genes depending on their targets. The miR-17-92 cluster, including miR-17-5p, -17-3p, -18a, -19a, -20a, -19b and -92-1, located on C13orf25 is well known as oncogenic miRNA, which is overexpressed in lung cancer, malignant lymphoma and breast cancer. However, few data have been collected about the targets of miRNAs, including miR-17-92 cluster, and their roles in tumorigenesis. By using a proteomic approach, we identify novel direct targets of miR-19a. Methods and Results: MiR-17-92 expression profiling was performed in a subset of 32 of cancer cell lines by TanqMan Real-time PCR, and MCF-7 breast cancer cell line was identified as a miR-17-92 cluster overexpressed cell line. To detect candidate target proteins for miR-17-92 cluster, we transfected MCF-7 cell with antisense oligonucleotides against miR-19a, -20a and -92-1 and then performed two-dimensional protein electrophoresis (2-DE) and differential display analysis in protein extracts of MCF-7 which was knocked down of endogenous miR-17-92 by antisense oligonucleotides. Of the 1455 defined valid protein spots, 146 distinct proteins changed 1.5-fold their expression. 135 proteins were identified as candidate target proteins for miR-17-92 cluster by LC-MS/MS. To determine direct targets of miR-17-92, bioinformatics analysis, luciferase assay and western blotting analysis were performed as validation studies. Conclusions: Our results of exhaustive analysis revealed significant differences in the proteomic profile between anti-miR-LNA treated and non-treated MCF-7 breast cancer cell line. Among these candidate target proteins of miR17-92 cluster, we identified novel direct target of miR-19a. To better evaluate the role of miR-19a and the target gene, further studies of gene function are being performed now. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-246.