Takafumi Nakagaki

CRKL oncogene is downregulated by p53 through miR-200s.

Tumor suppressive miRNAs that target oncogenes are frequently downregulated in cancers, and this downregulation leads to oncogene pathway activation. Thus, tumor suppressive miRNAs and their target oncogenes have been proposed as useful targets in cancer treatment. miR‐200 family downregulation has been reported in cancer progression and metastasis. The miR‐200 family consists of two gene clusters, miR‐200b/200a/429 and miR‐200c/141, which are located on human chromosomes 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of the miR‐200 family and confirmed that miR‐200s are transcriptional targets of the p53 family. In silico analyses of miRNA targets established the CRKL oncogene as a potential target for miR‐200b/200c/429. Moreover, miR‐200b/200c/429 inhibited CRKL mRNA and protein expression by directly targeting its 3′‐UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells through the introduction of p53 family and endogenous p53 activation. Moreover, the downregulation of CRKL by siRNA inhibited cancer cell growth. The Oncomine database demonstrates that CRKL is overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that the p53 target miR‐200b/200c/429 miRNAs are negative regulators of the CRKL oncogene.

Genomic characterization of esophageal squamous cell carcinoma: Insights from next-generation sequencing

Two major types of cancer occur in the esophagus: squamous cell carcinoma, which is associated with chronic smoking and alcohol consumption, and adenocarcinoma, which typically arises in gastric reflux-associated Barretts esophagus. Although there is increasing incidence of esophageal adenocarcinoma in Western counties, esophageal squamous cell carcinoma (ESCC) accounts for most esophageal malignancies in East Asia, including China and Japan. Technological advances allowing for massively parallel, high-throughput next-generation sequencing (NGS) of DNA have enabled comprehensive characterization of somatic mutations in large numbers of tumor samples. Recently, several studies were published in which whole exome or whole genome sequencing was performed in ESCC tumors and compared with matched normal DNA. Mutations were validated in several genes, including in TP53, CDKN2A, FAT1, NOTCH1, PIK3CA, KMT2D and NFE2L2, which had been previously implicated in ESCC. Several new recurrent alterations have also been identified in ESCC. Combining the clinicopathological characteristics of patients with information obtained from NGS studies may lead to the development of effective diagnostic and therapeutic approaches for ESCC. As this research becomes more prominent, it is important that gastroenterologist become familiar with the various NGS technologies and the results generated using these methods. In the present study, we describe recent research approaches using NGS in ESCC.

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.

Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 target

The p53 tumor suppressor inhibits cell growth through the activation of both cell cycle arrest and apoptosis, which maintain genome stability and prevent cancer development. Here, we report that intercellular adhesion molecule-2 (ICAM2) is transcriptionally activated by p53. Specifically, ICAM2 is induced by the p53 family and DNA damage in a p53-dependent manner. We identified a p53 binding sequence located within the ICAM2 gene that is responsive to wild-type p53, TAp73, and TAp63. In terms of function, we found that the ectopic expression of ICAM2 inhibited cancer cell migration and invasion. In addition, we demonstrated that silencing endogenous ICAM2 in cancer cells caused a marked increase in extracellular signal-regulated kinase (ERK) phosphorylation levels, suggesting that ICAM2 inhibits migration and invasion of cancer cells by suppressing ERK signaling. Moreover, ICAM2 is underexpressed in human cancer tissues containing mutant p53 as compared to those with wild-type p53. Notably, the decreased expression of ICAM2 is associated with poor survival in patients with various cancers. Our findings demonstrate that ICAM2 induction by p53 has a key role in inhibiting migration and invasion.

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 3649: Targeted sequencing of 409 cancer-related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers

Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. In this study, we describe a next-generation semiconductor sequencing protocol for rapid (2 days), standardized, and cost-effective gene analysis for human cancer specimens including FFPE samples. DNA was extracted from 19 human cancer cell lines and 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas and multiple myelomas. 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). Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 461x, and >95% of targeted bases were represented by at least 20 reads. We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of cancer patients. Pathway assessment has shown that somatic aberrations within myeloma genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify human cancer mutations. The 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: Yasushi Sasaki, Ryota Koyama, Takafumi Nakagaki, Miyuki Tamura, Masashi Idogawa, Takashi Tokino. Targeted sequencing of 409 cancer-related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers. [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 3649.

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