DHX36 maintains genomic integrity by unwinding G-quadruplexes.

The guanine-rich stretch of single-stranded DNA (ssDNA) forms a G-quadruplex (G4) in a fraction of genic and intergenic chromosomal regions. The probability of G4 formation increases during events causing ssDNA generation, such as transcription and replication. In turn, G4 abrogates these events, leading to DNA damage. DHX36 unwinds G4-DNA in vitro and in human cells. However, its spatial correlation with G4-DNA in vivo and its role in genome maintenance remain unclear. Here, we demonstrate a connection between DHX36 and G4-DNA and its implications for genomic integrity. The nuclear localization of DHX36 overlapped with that of G4-DNA, RNA polymerase II, and a splicing-related factor. Depletion of DHX36 resulted in accumulated DNA damage, slower cell growth, and enhanced cell growth inhibition upon treatment with a G4-stabilizing compound; DHX36 expression reversed these defects. In contrast, the reversal upon expression of DHX36 mutants that could not bind G4 was imperfect. Thus, DHX36 may suppress DNA damage by promoting the clearance of G4-DNA for cell growth and survival. Our findings deepen the understanding of G4 resolution in the maintenance of genomic integrity.

Protective effects of Alda-1, an ALDH2 activator, on alcohol-derived DNA damage in the esophagus of human ALDH2*2 (Glu504Lys) knock-in mice.

Alcohol consumption is the key risk factor for the development of esophageal squamous cell carcinoma (ESCC), and acetaldehyde, a metabolite of alcohol, is an alcohol-derived major carcinogen that causes DNA damage. Aldehyde dehydrogenase2 (ALDH2) is an enzyme that detoxifies acetaldehyde, and its activity is reduced by ALDH2 gene polymorphism. Reduction in ALDH2 activity increases blood, salivary and breath acetaldehyde levels after alcohol intake, and it is deeply associated with the development of ESCC. Heavy alcohol consumption in individuals with ALDH2 gene polymorphism significantly elevates the risk of ESCC; however, effective prevention has not been established yet. In this study, we investigated the protective effects of Alda-1, a small molecule ALDH2 activator, on alcohol-mediated esophageal DNA damage. Here, we generated novel genetically engineered knock-in mice that express the human ALDH2*1 (wild-type allele) or ALDH2*2 gene (mutant allele). Those mice were crossed, and human ALDH2*1/*1, ALDH2*1/*2 and ALDH2*2/*2 knock-in mice were established. They were given 10% ethanol for 7 days in the presence or absence of Alda-1, and we measured the levels of esophageal DNA damage, represented by DNA adduct (N2-ethylidene-2'-deoxyguanosine). Alda-1 significantly increased hepatic ALDH2 activity both in human ALDH2*1/*2 and/or ALDH2*2/*2 knock-in mice and reduced esophageal DNA damage levels after alcohol drinking. Conversely, cyanamide, an ALDH2-inhibitor, significantly exacerbated esophageal DNA adduct level in C57BL/6N mice induced by alcohol drinking. These results indicate the protective effects of ALDH2 activation by Alda-1 on esophageal DNA damage levels in individuals with ALDH2 gene polymorphism, providing a new insight into acetaldehyde-mediated esophageal carcinogenesis and prevention.

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.

Accuracy of Shunt Volume Measured by Three-Dimensional Echocardiography and Cardiac Magnetic Resonance in Patients With an Atrial Septal Defect and a Dilated Right Ventricle.

BACKGROUND: The accuracy of right ventricular (RV) quantification by three-dimensional echocardiography (3DE) has been reported mainly in patients with a normal right ventricle (RV). However, there are no data regarding the accuracy of 3DE in patients with a dilated RV, as in shunt diseases. In this study, we evaluated the accuracy of 3DE and that of volumetric (Vol) cardiac magnetic resonance (CMR) for assessment of RV and left ventricular (LV) stroke volume (SV) and the pulmonary (Qp)/systemic (Qs) blood flow ratio in patients with an atrial septal defect (ASD) using the two-dimensional phase contrast (2DPC) method as the gold standard. METHODS: We retrospectively investigated 83 patients with ASD who underwent transcatheter closure and clinically indicated CMR and 3DE examinations. The ratio Qp/Qs was calculated using RV and LV SV measured by full-volume volumetric 3DE (Vol-3DE) and CMR (Vol-CMR) and by two-dimensional pulsed Doppler quantification (2D-Dop); the parameters were compared using 2DPC-CMR as the gold standard. RESULTS: There was no significant difference in the Qp/Qs value between 2DPC-CMR and Vol-3DE (2.29 ± 0.70 vs 2.21 ± 0.63, P = .79) and 2D-Dop (vs 2.21 ± 0.65, P = 1.00); however, a significant difference was found between 2DPC-CMR and Vol-CMR (P < .001). The Qp/Qs value obtained using Vol-3DE showed the best correlation with 2DPC-CMR (r = 0.93, P < .001). The RV and LV SV values obtained by Vol-3DE showed the best correlation with 2DPC-CMR (RV SV, r = 0.82, P < .001; LV SV, r = 0.73, P < .001), although the absolute values were underestimated. CONCLUSION: Qp/Qs was more accurately evaluated by Vol-3DE than by Vol-CMR or 2D-Dop. Three-dimensional echocardiography assessment was feasible and reproducible even in a dilated RV.

Case series of mobile structures detected vividly by using superb microvascular imaging.

Background: Superb microvascular imaging (SMI) is a new imaging technique that can reveal low-velocity blood flow without use of a contrast agent. SMI is based on an original algorithm and effectively removes tissue motion artifacts (clutter motion) from the background, thereby preserving visibility of low-velocity blood flow. SMI is expected to be useful for the evaluation of heart diseases, as well as blood vessels. Case summary: Here, we report three cases in which a mobile structure in the heart or a blood vessel was detected easily by strong enhancement on SMI. In the heart, the entire mass was strongly enhanced by colour-SMI and had the appearance of 'a fire ball'. In the abdominal aorta and carotid artery, SMI captured a strongly enhanced echo image of a mass and revealed hyperechoic mobile plaque. It was hard to detect with the conventional echocardiography. Discussion: It is important to detect mobile intravascular and intracardiac structures as they are risk factors of thrombosis. Echo images are often strongly affected by the skill of the examiner, the patient's body habitus, and the presence of intestinal gas; thus, it is often difficult to detect a small mass with conventional echocardiography. With the use of SMI, even small mobile structures can be displayed at high intensity in comparison with the surrounding blood flow. Therefore, the non-invasive SMI was useful for the detection of mobile intravascular and intracardiac structures. Our findings of the current report may lead to new developments in SMI for imaging in the cardiac region.

HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function.

Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance. These variants may interact with each other to influence tumor pathophysiology, however, such interactions have not been fully elucidated. Additionally, the effect of target therapy for those variants also unclarified. In this study, we investigated the biological functions of a HER2 mutation (G776S mutation) of unknown pathological significance, which was detected together with APC mutation by cancer genome sequencing of samples from a colorectal cancer (CRC) patient. Transfection of the HER2 G776S mutation alone slightly increased the kinase activity and phosphorylation of HER2 protein, but did not activate HER2 downstream signaling or alter the cell phenotype. On the other hand, the HER2 G776S mutation was shown to have strong oncogenic potential when loss of APC function was accompanied. We revealed that loss of APC function increased Wnt pathway activity but also increased RAS-GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. In addition, afatinib, a pan-HER tyrosine kinase inhibitor, suppressed tumor growth in xenografts derived from HER2 G776S-transfected CRC cells. These findings suggest that this HER2 mutation in CRC may be a potential therapeutic target.

Substitute parameters of exercise-induced pulmonary hypertension and usefulness of low workload exercise stress echocardiography in mitral regurgitation.

In asymptomatic patients with mitral regurgitation (MR), data of exercise-induced pulmonary hypertension (EIPH) are limited, and feasibility of evaluating EIPH is not high. We aimed to investigate prognostic impact of EIPH and its substitute parameters. Exercise stress echocardiography (ESE) were performed in 123 consecutive patients with moderate to severe degenerative MR. The endpoint was a composite of death, hospitalization for heart failure, and worsening of symptoms. EIPH [tricuspid regurgitation peak gradient (TRPG) at peak workload ≥ 50 mmHg] was shown in 57 patients (46%). TRPG at low workload was independently associated with TRPG at peak workload (ß = 0.67, p < 0.001). Early surgical intervention (within 6 months after ESE) was performed in 65 patients. Of the remaining 58 patients with the watchful waiting strategy, the event free survival was lower in patients with EIPH than in patients without EIPH (48.1 vs. 97.0% at 1-year, p < 0.001). TRPG at low workload ≥ 35.0 mmHg as well as EIPH were associated with poor prognosis in patients with the watchful waiting strategy. In conclusion, the importance of ESE and evaluating EIPH in patients with MR was re-acknowledged. TRPG at peak workload can be predicted by TRPG at low workload, and TRPG at low workload may be useful in real-world clinical settings.

The CAM Model for CIC-DUX4 Sarcoma and Its Potential Use for Precision Medicine.

(1) Background: CIC-DUX4 sarcoma is a rare mesenchymal small round cell tumor which belongs to rare cancers that occupy a significant percentage of cancer cases as a whole, despite each being rare. Importantly, each rare cancer type has different features, and thus there is a need to develop a model that mimics the features of each of these cancers. We evaluated the idea that the chicken chorioallantoic membrane assay (CAM), a convenient and versatile animal model, can be established for the CIC-DUX4 sarcoma. (2) Methods: Patient-derived cell lines of CIC-DUX4 were applied. These cells were transplanted onto the CAM membrane and tumor formation was examined by H&E staining, immunohistochemistry and Western blotting. The CAM tumor was transferred onto a fresh CAM and was also used to form organoids. Retention of the fusion gene was examined. (3) Results: H&E staining as well as molecular characterization demonstrated the formation of the CIC-DUX4 tumor on the CAM membrane. Expression of cyclin D2 and ETV4 was identified. The CAM tumor was transferred to a fresh CAM to form the second-generation CAM tumor. In addition, we were successful in forming tumor organoids using the CAM tumor. Retention of the fusion gene CIC-DUX4 in the CAM, second-generation CAM, and in the CAM-derived organoids was confirmed by RT-PCR. (4) Conclusions: The CAM assay provides a promising model for CIC-DUX4 sarcoma.

Experimental model for the irradiation-mediated abscopal effect and factors influencing this effect.

Radiotherapy (RT) is the primary treatment for cancer. Ionizing radiation from RT induces tumor damage at the irradiated site, and, although clinically infrequent, may cause regression of tumors distant from the irradiated site-a phenomenon known as the abscopal effect. Recently, the abscopal effect has been related to prolongation of overall survival time in cancer patients, though the factors that influence the abscopal effect are not well understood. The aim of this study is to clarify the factors influencing on abscopal effect. Here, we established a mouse model in which we induced the abscopal effect. We injected MC38 (mouse colon adenocarcinoma) cells subcutaneously into C57BL/6 mice at two sites. Only one tumor was irradiated and the sizes of both tumors were measured over time. The non-irradiated-site tumor showed regression, demonstrating the abscopal effect. This effect was enhanced by an increase in the irradiated-tumor volume and by administration of anti-PD1 antibody. When the abscopal effect was induced by a combination of RT and anti-PD1 antibody, it was also influenced by radiation dose and irradiated-tumor volume. These phenomena were also verified in other cell line, B16F10 cells (mouse melanoma cells). These findings provide further evidence of the mechanism for, and factors that influence, the abscopal effect in RT.

Patient-derived tumor models of esophageal cancer.

Tumor models are powerful tools to plan preclinical therapeutic strategies that cannot be conducted on humans. Recently, patient-derived tumor models such as patient-derived xenograft (PDX) and/or patient-derived organoid (PDO), are widely used to investigate the antitumor effects of various drugs, because those tumors resemble original tumors at the physiological and molecular level. In this chapter, we describe patient-derived tumor models of esophageal cancers and outline the latest research as well as the molecular pathology of esophageal cancers.

Combination treatment with highly bioavailable curcumin and NQO1 inhibitor exhibits potent antitumor effects on esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most intractable cancers, so the development of novel therapeutics has been required to improve patient outcomes. Curcumin, a polyphenol from Curcuma longa, exhibits various health benefits including antitumor effects, but its clinical utility is limited because of low bioavailability. Theracurmin® (THC) is a highly bioavailable curcumin dispersed with colloidal submicron particles. METHODS: We examined antitumor effects of THC on ESCC cells by cell viability assay, colony and spheroid formation assay, and xenograft models. To reveal its mechanisms, we investigated the levels of reactive oxygen species (ROS) and performed microarray gene expression analysis. According to those analyses, we focused on NQO1, which involved in the removal of ROS, and examined the effects of NQO1-knockdown or overexpression on THC treatment. Moreover, the therapeutic effect of THC and NQO1 inhibitor on ESCC patient-derived xenografts (PDX) was investigated. RESULTS: THC caused cytotoxicity in ESCC cells, and suppressed the growth of xenografted tumors more efficiently than curcumin. THC increased ROS levels and activated the NRF2-NMRAL2P-NQO1 expressions. Inhibition of NQO1 in ESCC cells by shRNA or NQO1 inhibitor resulted in an increased sensitivity of cells to THC, whereas overexpression of NQO1 antagonized it. Notably, NQO1 inhibitor significantly enhanced the antitumor effects of THC in ESCC PDX tumors. CONCLUSIONS: These findings suggest the potential usefulness of THC and its combination with NQO1 inhibitor as a therapeutic option for ESCC.

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.