Tomoko Ohashi

Identification and analysis of large intergenic non-coding RNAs regulated by p53 family members through a genome-wide analysis of p53-binding sites

p53 is one of the most important known tumor suppressor genes, and it is inactivated in approximately half of human cancers. p53 family members execute various functions, such as apoptosis induction and cell cycle arrest, by modulating transcriptional regulation. Therefore, the direct transcriptional targets of the p53 family must be explored to elucidate the functional mechanisms of family members. To identify the direct transcriptional targets of p53 family members, we performed chromatin immunoprecipitation together with next-generation sequencing (ChIP-seq) and searched for p53-binding motifs across the entire human genome. Among the identified ChIP-seq peaks, approximately half were located in an intergenic region. Therefore, we assumed large intergenic non-coding RNAs (lincRNAs) to be major targets of the p53 family. Recent reports have revealed that lincRNAs play an important role in various biological and pathological processes, such as development, differentiation, stemness and carcinogenesis. Through a combination of ChIP-seq and in silico analyses, we found 23 lincRNAs that are upregulated by the p53 family. Additionally, knockdown of specific lincRNAs modulated p53-induced apoptosis and promoted the transcription of a gene cluster. Our results suggest that p53 family members, and lincRNAs constitute a complex transcriptional network involved in various biological functions and tumor suppression.

Long non‐coding RNA NEAT1 is a transcriptional target of p53 and modulates p53‐induced transactivation and tumor‐suppressor function

p53 is one of the most important tumor suppressor genes, and the direct transcriptional targets of p53 must be explored to elucidate its functional mechanisms. Thus far, the p53 targets that have been primarily studied are protein‐coding genes. Our previous study revealed that several long non‐coding RNAs (lncRNAs) are direct transcriptional targets of p53, and knockdown of specific lncRNAs modulates p53‐induced apoptosis. In this study, analysis of next‐generation chromatin immunoprecipitation‐sequencing (ChIP‐seq) data for p53 revealed that the lncRNA NEAT1 is a direct transcriptional target of p53. The suppression of NEAT1 induction by p53 attenuates the inhibitory effect of p53 on cancer cell growth and also modulates gene transactivation, including that of many lncRNAs. Furthermore, low expression of NEAT1 is related to poor prognosis in several cancers. These results indicate that the induction of NEAT1 expression contributes to the tumor‐suppressor function of p53 and suggest that p53 and NEAT1 constitute a transcriptional network contributing to various biological functions and tumor suppression.

AKR1B10, a Transcriptional Target of p53, Is Downregulated in Colorectal Cancers Associated with Poor Prognosis

p53 is one of the most important tumor suppressor genes, and it is frequently inactivated in various cancers. p53 modulates various cellular functions, such as apoptosis and cell-cycle arrest via transcriptional regulation. Recently, p53 has been reported to be involved in a wide range of cellular metabolic pathways, including glycolysis, oxidative phosphorylation, glutaminolysis, and the antioxidant response. To understand the functional mechanism of p53, it is important to find out the direct transcriptional targets of p53. In this study, aldo-keto reductase family 1, member B10 (AKR1B10) was identified as a direct target of the p53 family by cDNA microarray analysis after comparing the mRNA expression of control and H1299 cells that overexpressed with p53 family members. In addition, we found that the expression of AKR1B10 was significantly decreased in colorectal cancers and adenomas as compared with normal colon tissues. Knockdown of AKR1B10 significantly inhibited p53-induced apoptosis in colorectal cancer cells, whereas the overexpression of AKR1B10 enhanced p53-induced apoptosis and inhibited tumor proliferation in vivo. Furthermore, low expression of AKR1B10 in colon cancer patients was correlated with decreased survival and poor prognosis. These results suggest that decreased expression of AKR1B10 could disrupt the tumor suppressive function of p53, which result in decreased survival in colorectal cancer patients. In summary, AKR1B10 may be a novel prognostic predictor and a novel therapeutic target for colorectal cancer. Implications: AKR1B10, a transcriptional target of p53, is also a novel prognostic and therapeutic molecule in colorectal cancer. Mol Cancer Res; 11(12); 1554–63. ©2013 AACR.

p53 mediates the suppression of cancer cell invasion by inducing LIMA1/EPLIN

The tumor suppressor gene p53 is frequently mutated in human cancer. p53 executes various functions, such as apoptosis induction and cell cycle arrest, by modulating transcriptional regulation. In this study, LIM domain and Actin-binding protein 1 (LIMA1) was identified as a target of the p53 family using a cDNA microarray. We also evaluated genome-wide occupancy of the p53 protein by performing chromatin immunoprecipitation-sequencing (ChIP-seq) and identified two p53 response elements in the LIMA1 gene. LIMA1 protein levels were increased by treatment with nutlin-3a, a small molecule that activates endogenous p53. In addition, LIMA1 expression was significantly downregulated in cancers compared with normal tissues. Knockdown of LIMA1 significantly enhanced cancer cell invasion and partially inhibited p53-induced suppression of cell invasion. Furthermore, low expression of LIMA1 in cancer patients correlated with decreased survival and poor prognosis. Thus, p53-induced LIMA1 inhibits cell invasion, and the downregulation of LIMA1 caused by p53 mutation results in decreased survival in cancer patients. Collectively, this study reveals the molecular mechanism of LIMA1 downregulation in various cancers and suggests that LIMA1 may be a novel prognostic predictor and a therapeutic target for cancer.

Identification and characterization of a metastatic suppressor BRMS1L as a target gene of p53

The tumor suppressor p53 and its family members, p63 and p73, play a pivotal role in the cell fate determination in response to diverse upstream signals. As transcription factors, p53 family proteins regulate a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 family proteins are important for the regulation of cell invasion and migration. Microarray analysis showed that breast cancer metastasis suppressor 1‐like (BRMS1L) is upregulated by p53 family proteins, specifically p53, TAp63γ, and TAp73β. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly indicate that BRMS1L is one of the mediators downstream of the p53 pathway, and that it inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results indicate that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer.

Profiling cancer-related gene mutations in oral squamous cell carcinoma from Japanese patients by targeted amplicon sequencing

Somatic mutation analysis is a standard practice in the study of human cancers to identify mutations that cause therapeutic sensitization and resistance. We performed comprehensive genomic analyses that used PCR target enrichment and next-generation sequencing on Ion Proton semiconductor sequencers. Forty-seven oral squamous cell carcinoma (OSCC) samples and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverage of the entire coding regions of 409 cancer-related genes (covered regions: 95.4% of total, 1.69 megabases of target sequence). The number of somatic mutations in 47 patients with OSCC ranged from 1 to 20 with a mean of 7.60. The most frequent mutations were in TP53 (61.7%), NOTCH1 (25.5%), CDKN2A (19.1%), SYNE1 (14.9%), PIK3CA (10.6%), ROS1 (10.6%), and TAF1L (10.6%). We also detected copy number variations (CNVs) in the segments of the genome that could be duplicated or deleted from deep sequencing data. Pathway assessment showed that the somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation and RTK-MAPK-PI3K. This study may enable better selection of therapies and deliver improved outcomes for OSCC patients when combined with clinical diagnostics.Somatic mutation analysis is a standard practice in the study of human cancers to identify mutations that cause therapeutic sensitization and resistance. We performed comprehensive genomic analyses that used PCR target enrichment and next-generation sequencing on Ion Proton semiconductor sequencers. Forty-seven oral squamous cell carcinoma (OSCC) samples and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverage of the entire coding regions of 409 cancer-related genes (covered regions: 95.4% of total, 1.69 megabases of target sequence). The number of somatic mutations in 47 patients with OSCC ranged from 1 to 20 with a mean of 7.60. The most frequent mutations were in TP53 (61.7%), NOTCH1 (25.5%), CDKN2A (19.1%), SYNE1 (14.9%), PIK3CA (10.6%), ROS1 (10.6%), and TAF1L (10.6%). We also detected copy number variations (CNVs) in the segments of the genome that could be duplicated or deleted from deep sequencing data. Pathway assessment showed that the somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation and RTK–MAPK-PI3K. This study may enable better selection of therapies and deliver improved outcomes for OSCC patients when combined with clinical diagnostics.

Abstract 1938: The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data

p53 is inactivated in approximately half of human cancers. p53 execute various functions by modulating transcriptional regulation. To identify direct p53 transcriptional targets including non-coding RNAs, we performed gene expression analysis with next-generation sequencing (RNA-seq) in osteosarcoma U2OS cells treated with Nutlin-3a which activates endogenous p53. We identified 261 genes increased more than 4-fold and 373 long non-coding RNAs (lncRNAs) increased more than 2-fold. Next, we analyzed the data of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) and then searched for p53 binding sites in the whole human genome. p53 binding sites were located in the neighborhood of 126/261 (48.3%) genes including 28 known target genes and 122/373 (32.7%) lncRNAs including 7 lncRNAs we already identified in previous study. Recent reports have revealed that lncRNAs play an important role in various biological and pathological processes such as development, differentiation, stemness and carcinogenesis. Although all functions of lncRNAs have not been elucidated, many lncRNAs are involved in transcriptional regulation like transcriptional factors. Therefore, we performed a gene network analysis of cancer transcriptome including lncRNAs using public RNA-seq data of cancer tissues. As a result, we identified several hub lncRNAs which regulate the transcription of many downstream genes. Our results suggest that p53 and lncRNAs comprise a complex transcriptional network for various biological functions and tumor suppression. Citation Format: Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Takashi Tokino. The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1938.

Abstract 3707: Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma

Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. To better understand the molecular pathogenesis of oral squamous cell carcinoma (OSCC) patients, we performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent semiconductor sequencers (PGM and Proton). DNA (40 ng) was extracted from 45 human OSCC specimens and their corresponding non-cancerous tissues including FFPE samples. Using the Ion Ampliseq Comprehensive Cancer Panel, we sequenced 15992 loci from 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers (covered regions = 95.4% of total, 1.5 megabases of target sequence). We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data. Each sample underwent on average 5.9 million sequencing reads after quality filtering. The mean read depths were 367.8 x, and >95% of targeted bases were represented .The number of somatic mutations (SNVs and indels) in 45 patients with OSCC ranged from 1 to 36 with a median of 7.33 (6.40/Mb). The most frequent mutations were detected on TP53 (28 of 45; 62.2%). Many of the mutations on TP53 were detected in the DNA-binding domain (23 of 28; 82.1%). NOTCH1 mutations were identified in 10 cases (8 missense, 1 coding frameshift, and 1 essential splice site mutations). CDKN2A mutations were observed in 8 cases; and PIK3CA were mutated 3 cases. Although the most common mutations in OSCC were C/G>T/A transitions, which are consistent with previous reports on head and neck, lung and oesophageal SCCs, the second most frequent mutations were C/G>A/T transversions. We also identified a median of 100 significant CNVs (range of 0-481) per sample. Pathway assessment has shown that somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation (p53 pathway, 84.4%) and RTK-MAPK-PI3K (62.2%). This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings. Citation Format: Takafumi Nakagaki, Yasushi Sasaki, Masashi Idogawa, Ryota Koyama, Kenta Kobashi, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Hiroyoshi Hiratsuka, Takashi Tokino. Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3707.

Abstract 4905: Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing

Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. We performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM). We first validated a multigene sequencing screen interrogating 2855 mutational hotspots in 50 cancer-related genes using the Ion Torrent AmpliSeq cancer panel v2. Ten nanogram of DNA was used as template to amplify 207 regions in oral squamous cell carcinoma (OSCC). Approximately 1000 average coverage was obtained with more than 90% of target bases having at least 100 sequence reads. We detected mutations of the TP53 gene in 96% (22 of 23) of OSCC cell lines. Of the 62 OSCC specimens, 42 presented at least one somatic mutation among the 50 investigated genes, and 21 of these showed multiple gene somatic mutations. The most frequent mutations were detected on TP53 (26 of 62; 41.9%). CDKN2A mutations were identified in 9 cases; STK11 mutations were observed in 8 cases; and PIK3CA were mutated 5 cases. In addition, we sequenced 409 cancer-related genes in matched tumor and normal DNA from OSCC patients using the Ion Ampliseq Comprehensive Cancer Panel. This panel targets all exons of 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated (covered regions: 95.4% of total), and 15,992 amplicons amplify more than 1.2 megabases of target sequence. We piloted the use of this platform to identify somatic mutations in 37 OSCC specimens including formalin-fixed paraffin-embedded (FFPE) samples. We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data. This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings. Citation Format: Takafumi Nakagaki, Yasushi Sasaki, Kenta Kobashi, Kousuke Takeda, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Masashi Idogawa, Hiroyoshi Hiratsuka, Takashi Tokino. Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4905. doi:10.1158/1538-7445.AM2015-4905