Yusuke Amanuma

Recent Advances From Basic and Clinical Studies of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and is highly prevalent in Asia. Alcohol and its metabolite, acetaldehyde, are considered definite carcinogens for the esophagus. Polymorphisms in the aldehyde dehydrogenase 2 gene, which encodes an enzyme that eliminates acetaldehyde, have been associated with esophageal carcinogenesis. Studies of the mutagenic and carcinogenic effects of acetaldehyde support this observation. Several recent large-scale comprehensive analyses of the genomic alterations in ESCC have shown a high frequency of mutations in genes such as TP53 and others that regulate the cell cycle or cell differentiation. Moreover, whole genome and whole exome sequencing studies have frequently detected somatic mutations, such as G:C→A:T transitions or G:C→C:G transversions, in ESCC tissues. Genomic instability, caused by abnormalities in the Fanconi anemia DNA repair pathway, is also considered a pathogenic mechanism of ESCC. Advances in diagnostic techniques such as magnifying endoscopy with narrow band imaging or positron emission tomography have increased the accuracy of diagnosis of ESCC. Updated guidelines from the National Comprehensive Cancer Network standardize the practice for the diagnosis and treatment of esophageal cancer. Patients with ESCC are treated endoscopically or with surgery, chemotherapy, or radiotherapy, based on tumor stage. Minimally invasive treatments help improve the quality of life of patients who undergo such treatments. We review recent developments in the diagnosis and treatment of ESCC and advances gained from basic and clinical research.

Serum miR-21, miR-29a, and miR-125b Are Promising Biomarkers for the Early Detection of Colorectal Neoplasia.

Purpose: Circulating microRNAs (miRNA) are emerging as promising diagnostic biomarkers for colorectal cancer, but their usefulness for detecting early colorectal neoplasms remains unclear. This study aimed to identify serum miRNA biomarkers for the identification of patients with early colorectal neoplasms. Experimental Design: A cohort of 237 serum samples from 160 patients with early colorectal neoplasms (148 precancerous lesions and 12 cancers) and 77 healthy subjects was analyzed in a three-step approach that included a comprehensive literature review for published biomarkers, a screening phase, and a validation phase. RNA was extracted from sera, and levels of miRNAs were examined by real-time RT-PCR. Results: Nine miRNAs (miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-24, miR-29a, miR-92, and miR-125b) were selected as candidate biomarkers for initial analysis. In the screening phase, serum levels of miR-21, miR-29a, and miR-125b were significantly higher in patients with early colorectal neoplasm than in healthy controls. Elevated levels of miR-21, miR-29a, and miR-125b were confirmed in the validation phase using an independent set of subjects. Area under the curve (AUC) values for serum miR-21, miR-29a, miR-125b, and their combined score in discriminating patients with early colorectal neoplasm from healthy controls were 0.706, 0.741, 0.806, and 0.827, respectively. Serum levels of miR-29a and miR-125b were significantly higher in patients who had only small colorectal neoplasms (≤5 mm) than in healthy subjects. Conclusions: Because serum levels of miR-21, miR-29a, and miR-125b discriminated patients with early colorectal neoplasm from healthy controls, our data highlight the potential clinical use of these molecular signatures for noninvasive screening of patients with colorectal neoplasia. Clin Cancer Res; 21(18); 4234–42. ©2015 AACR.

Protective role of ALDH2 against acetaldehyde-derived DNA damage in oesophageal squamous epithelium

Acetaldehyde is an ethanol-derived definite carcinogen that causes oesophageal squamous cell carcinoma (ESCC). Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that eliminates acetaldehyde, and impairment of ALDH2 increases the risk of ESCC. ALDH2 is produced in various tissues including the liver, heart, and kidney, but the generation and functional roles of ALDH2 in the oesophagus remain elusive. Here, we report that ethanol drinking increased ALDH2 production in the oesophagus of wild-type mice. Notably, levels of acetaldehyde-derived DNA damage represented by N2-ethylidene-2′-deoxyguanosine were higher in the oesophagus of Aldh2-knockout mice than in wild-type mice upon ethanol consumption. In vitro experiments revealed that acetaldehyde induced ALDH2 production in both mouse and human oesophageal keratinocytes. Furthermore, the N2-ethylidene-2′-deoxyguanosine levels increased in both Aldh2-knockout mouse keratinocytes and ALDH2-knockdown human keratinocytes treated with acetaldehyde. Conversely, forced production of ALDH2 sharply diminished the N2-ethylidene-2′-deoxyguanosine levels. Our findings provide new insight into the preventive role of oesophageal ALDH2 against acetaldehyde-derived DNA damage.

Molecular Mechanisms of Acetaldehyde-Mediated Carcinogenesis in Squamous Epithelium

Acetaldehyde is a highly reactive compound that causes various forms of damage to DNA, including DNA adducts, single- and/or double-strand breaks (DSBs), point mutations, sister chromatid exchanges (SCEs), and DNA–DNA cross-links. Among these, DNA adducts such as N2-ethylidene-2′-deoxyguanosine, N2-ethyl-2′-deoxyguanosine, N2-propano-2′-deoxyguanosine, and N2-etheno-2′-deoxyguanosine are central to acetaldehyde-mediated DNA damage because they are associated with the induction of DNA mutations, DNA–DNA cross-links, DSBs, and SCEs. Acetaldehyde is produced endogenously by alcohol metabolism and is catalyzed by aldehyde dehydrogenase 2 (ALDH2). Alcohol consumption increases blood and salivary acetaldehyde levels, especially in individuals with ALDH2 polymorphisms, which are highly associated with the risk of squamous cell carcinomas in the upper aerodigestive tract. Based on extensive epidemiological evidence, the International Agency for Research on Cancer defined acetaldehyde associated with the consumption of alcoholic beverages as a “group 1 carcinogen” (definite carcinogen) for the esophagus and/or head and neck. In this article, we review recent advances from studies of acetaldehyde-mediated carcinogenesis in the squamous epithelium, focusing especially on acetaldehyde-mediated DNA adducts. We also give attention to research on acetaldehyde-mediated DNA repair pathways such as the Fanconi anemia pathway and refer to our studies on the prevention of acetaldehyde-mediated DNA damage.

Establishment of a Quick and Highly Accurate Breath Test for ALDH2 Genotyping

OBJECTIVES: Acetaldehyde, the first metabolite of ethanol, is a definite carcinogen for the esophagus, head, and neck; and aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that catalyzes the metabolism of acetaldehyde. The ALDH2 genotype exists as ALDH2*1/*1 (active ALDH2), ALDH2*1/*2 (heterozygous inactive ALDH2), and ALDH2*2/*2 (homozygous inactive ALDH2). Many epidemiological studies have reported that ALDH2*2 carriers are at high risk for esophageal or head and neck squamous cell carcinomas by habitual drinking. Therefore, identification of ALDH2*2 carriers would be helpful for the prevention of those cancers, but there have been no methods suitable for mass screening to identify these individuals. METHODS: One hundred and eleven healthy volunteers (ALDH2*1/*1 carriers: 53; ALDH2*1/*2 carriers: 48; and ALDH2*2/*2 carriers: 10) were recruited. Breath samples were collected after drinking 100 ml of 0.5% ethanol using specially designed gas bags, and breath ethanol and acetaldehyde levels were measured by semiconductor gas chromatography. RESULTS: The median (range) breath acetaldehyde levels at 1 min after alcohol ingestion were 96.1 (18.1–399.0) parts per billion (p.p.b.) for the ALDH2*1/*1 genotype, 333.5 (78.4–1218.4) p.p.b. for the ALDH2*1/*2 genotype, and 537.1 (213.2–1353.8) p.p.b. for the ALDH2*2/*2 genotype. The breath acetaldehyde levels in ALDH2*2 carriers were significantly higher than for the ALDH2*1/*1 genotype. Notably, the ratio of breath acetaldehyde level‐to‐breath ethanol level could identify carriers of the ALDH2*2 allele very accurately (whole accuracy; 96.4%). CONCLUSIONS: Our novel breath test is a useful tool for identifying ALDH2*2 carriers, who are at high risk for esophageal and head and neck cancers.

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells

BackgroundEpidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells.MethodsImmortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated.ResultsCells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R (epithelial-like)-derived xenograft tumors but not TE-8 (mesenchymal-like)-derived xenograft tumors.ConclusionsThe factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.

Abstract 164: Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals

Background Esophageal squamous cell carcinoma (ESCC) represents the most common form of esophageal cancer worldwide, especially in East Asia, where alcohol drinking and smoking have been implicated in the field carcinogenesis of ESCC. However, the oncogenic process therein has been poorly understood in terms of gene mutations. Patients & Methods A total of 100 samples, including cancer, dysplastic, and non-dysplastic esophageal tissues, were obtained from 24 individual with (N = 14) or without (N = 10) ESCC (a median of 2.5 samples per case: 1−29) either by endoscopy or surgery and were subjected to whole exome sequencing (WES). An additional paired cancer/non-caner samples from 32 patients was analyzed by targeted sequencing (TS). All samples were analyzed for copy number alterations (CNAs) using SNP array- and/or digital sequencing-based karyotyping. Results In WES, clonal evolution in esophageal epithelia, as determined by the presence of somatic mutations, was detected in 21 of 21 cancer, 12 of 12 dysplastic, and 63 of 67 non-dysplastic samples, where the mean number of mutation per sample showed a significant trend to increase in cancer (65) and dysplastic samples (50) compared to non-dysplastic samples (13) (P = 2.1×10-11). CNAs, especially those involving CDKN2A, CCND1, YAP1, and EGFR, were frequently affected in cancer samples, but rarely so in non-dysplastic samples. Non-dysplastic samples tended to have smaller allelic burden and therefore, clone size, compared to dysplastic and cancer samples (P = 2.2×10-16). Mutations had a predominant age-related signature in non-dysplastic samples but increasing APOBEC3A/3B patterns was observed in cancer and dysplastic samples. Shared mutations were found only within cancer tissues but never among dysplastic or non-dysplastic samples, suggesting the latter lesions are clonally independent from each other. In accordance with previous reports, TP53 mutations were found in 21/21 cancer samples and also found in dysplastic (11/12) and non-dysplastic samples at a lower frequency (26/67). Strikingly, non-dysplastic samples harbored a very high frequency of NOTCH1 mutations (51/67), which were also found in cancer (3/21) and non-dysplastic (8/12) samples but at much lower frequencies (P = 6.6×10-7). TS of validation samples confirmed the trend of higher NOTCH1 (84% vs. 25%) and lower TP53 mutation rates (38% vs. 100%) in non-dysplastic samples compared to cancer samples. The number of mutations in non-dysplastic samples was higher in drinkers than non-drinkers. Multiple NOTCH1 mutations were more common in cancer patients and drinkers than non-drinkers. Conclusion Clonal proliferation in non-cancer esophageal epithelia is common even in non-ESCC cases and extensive in ESCC cases. NOTCH1 and TP53 mutations play major roles in clonal evolution in common but may have differential impacts on esophageal carcinogenesis, which is likely to be shaped by APOBEC-induced mutations and CNAs. Citation Format: Akira Yokoyama, Hiromichi Suzuki, Tetsuichi Yoshizato, Kosuke Aoki, Yusuke Shiozawa, Youichi Fujii, Yusuke Sato, Nobuyuki Kakiuchi, Sugi Kin, Keisuke Kataoka, Kenichi Yoshida, Hideki Makishima, Yusuke Amanuma, Shinya Oohashi, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Brown J.B., Masashi Sanada, Shigeru Tsunoda, Sachiko Minamiguchi, Yoshiharu Sakai, Hironori Haga, Tsutome Chiba, Satoru Miyano, Manabu Muto, Seishi Ogawa. Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals. [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 164.

380 Alcohol Induces ALDH2 As a Novel Cytoprotective Mechanism to Suppress Acetaldehyde-Derived DNA Adduct Formation in Esophageal Epithelial Cells

no statistically significant difference among different cell lines. We also treat CP-A Barretts cell line with acid (pH 6.5) for 24 hours and measured NOTCH signaling molecules by PCR array. We found that acid treatment significantly decreased NOTCH1 (2 fold decrease) and JAG1 (1.6 fold decrease), and increased NOTCH3 (1.7 fold increase) and NOTCH4 (3.9 fold increase) in CP-A Barretts cells. Acid treatment did not cause significant changes of DLL1, DLL2, DLL4, JAG2, NOTCH2 and NUMB. In addition, Western blot analysis showed that acid upregulated NOTCH3 and NOTCH4 protein expression. Acid-induced increase in cell proliferation in CP-A Barretts cells was significantly decreased by knockdown of NOTCH3 (from 285% control to 162% control, p<0.001) and NOTCH4 (from 236% control to 192% control, P<0.001) with their small interfering RNAs. We conclude that downregulation of NOTCH1 and overexpression of NOTCH3 may play an important role in the development of Barretts associated adenocarcinoma. It is possible that acid reflux present in BE patients may downregulate NOTCH1 and upregulate NOTCH3, increasing cell proliferation and thereby contributing to the progression from Barretts esophagus to esophageal adenocarcinoma. Supported by NIH NIDDK R01 DK080703.

381 ALDH2 and Autophagy May Cooperate to Alleviate Acetaldehyde-Mediated DNA Damage and Cytotoxicity in Fission Yeast and Esophageal Epithelial Cells

no statistically significant difference among different cell lines. We also treat CP-A Barretts cell line with acid (pH 6.5) for 24 hours and measured NOTCH signaling molecules by PCR array. We found that acid treatment significantly decreased NOTCH1 (2 fold decrease) and JAG1 (1.6 fold decrease), and increased NOTCH3 (1.7 fold increase) and NOTCH4 (3.9 fold increase) in CP-A Barretts cells. Acid treatment did not cause significant changes of DLL1, DLL2, DLL4, JAG2, NOTCH2 and NUMB. In addition, Western blot analysis showed that acid upregulated NOTCH3 and NOTCH4 protein expression. Acid-induced increase in cell proliferation in CP-A Barretts cells was significantly decreased by knockdown of NOTCH3 (from 285% control to 162% control, p<0.001) and NOTCH4 (from 236% control to 192% control, P<0.001) with their small interfering RNAs. We conclude that downregulation of NOTCH1 and overexpression of NOTCH3 may play an important role in the development of Barretts associated adenocarcinoma. It is possible that acid reflux present in BE patients may downregulate NOTCH1 and upregulate NOTCH3, increasing cell proliferation and thereby contributing to the progression from Barretts esophagus to esophageal adenocarcinoma. Supported by NIH NIDDK R01 DK080703.

Su1962 Induction of Aldehyde Dehydrogenase-2 (Aldh2) Expression in Esophageal Epithelial Cells Suppresses the Acetaldehyde-Mediated DNA Damage

AIM: Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed worldwide and known to induce gastric injury from multiple mechanisms. Aloe vera is known to effectively decrease inflammation and promote ulcer healing but there are still limited data. Therefore, this study performs to evaluate the protective effects of Aloe vera on gastric injury in rats with indomethacin-induced gastropathy. METHODS: Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control group, n = 6) were given orally distilled water (DW) at 0 and 4 hours. Group 2 [indomethacin (IMN) group, n = 6] were given orally IMN (150 mg/kg) dissolved in 5% NaHCO3at time 0 and 4 hours. Group 3 (Aloe vera-treated group, n = 6) were given orally Aloe vera (150 mg/kg) dissolved in DW and IMN at 0 and 4 hours. At 8 hours after the first dose, the rats stomach was removed to determine gastric malondialdehyde (MDA), the number of interleukin-18 (IL-18) positive stained cells (%) by immunohistochemistry method, and histopathological examination. Serum was collected to determine the level of tumor necrosis factorα (TNFα) and Cytokine-induced neutrophil chemoattractant-1 (CINC-1) (by ELISA method). RESULTS: In the IMN group, serum TNF-α, CINC-1and gastric MDA significantly increased when compared to the control group (27.8±1.5 vs. 85.1±49.1 pg/mL, 104.5 ± 45.8 vs. 1054.7 ± 20.4 pg/mL and 1.7±0.2 vs. 9.4±1.1 nmol/mg protein, p<0.05, respectively). The mean level of TNF-α, CINC-1and gastric MDA in the Aloe vera-treated group improved when compared with that of in the IMN group (85.1±49.1 vs. 35.2±1.6 pg/mL, 1054.7 ± 20.4 vs. 813.6 ± 239 pg/mL and 9.4±1.1 vs. 2.7±0.6 nmol/mg protein, p < 0.05, respectively). The number of IL-18 positive stained cells (%) in the gastric epithelial cells of IMN group was significantly higher than that of in the control group. In contrast, the Aloe vera treated group showed decreasing in number of IL-18 positive stained cells (%) significantly when compared with that of in the IMN-treated group. Most rats in the IMN group developed moderate to severe gastric inflammation and erosions. Gastric erosions and neutrophil infiltration scores were significantly lower in the Aloe vera treated group. CONCLUSION: In rat models, Aloe vera attenuated IMN induced gastropathy by the reduction of the oxidative stress, inflammatory cytokines, and the histology confirmed the improvement of gastric injury. Summary of parameters in all groups (n=6, each group)