Atsushi Yagishita

Epidermal growth factor receptor (EGFR) signaling regulates global metabolic pathways in EGFR-mutated lung adenocarcinoma.

Background: Genetic mutations in cancer-driver genes induce specific metabolic alterations in cancer cells. Results: EGF receptor signaling has an important role for glycolysis, pentose phosphate pathway, and pyrimidine biosynthesis in EGFR-mutated lung cancer. Conclusion: Our work reveals the relationship between the EGFR signaling axis and key metabolic changes. Significance: These data implicate a possible link between therapeutic response and regulation of metabolism in EGFR-mutated LAD. Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFR-TKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

Effect of novel bright image enhanced endoscopy using blue laser imaging (BLI)

Background and study aims: The novel method of image-enhanced endoscopy (IEE) named blue laser imaging (BLI) can enhance the contrast of surface vessels using lasers for light illumination. BLI has two IEE modes: high contrast mode (BLI-contrast) for use with magnification, and bright mode (BLI-bright), which achieves a brighter image than BLI-contrast and yet maintains the enhanced visualization of vascular contrast that is expected for the detection of tumors from a far field of view. The aim of this study is to clarify the effect of BLI-bright with a far field of view compared to BLI-contrast and commonly available narrow-band imaging (NBI). Patients and methods: Patients with neoplasia, including early cancer in the pharynx, esophagus, stomach, or colorectum, were recruited and underwent tandem endoscopy with BLI and NBI systems. Six sets of images of the lesions were captured with a changing observable distance from 3 to 40 mm. Individual sets of images taken from various observable distances were assessed for visibility among BLI-bright, BLI-contrast, and NBI modes. The brightness and contrast of these images were also analyzed quantitatively. Results: Of 51 patients, 39 were assessed. Image analysis indicated that only BLI-bright maintained adequate brightness and contrast up to 40 mm and had significantly longer observable distances compared to the other methods. Furthermore, BLI-bright enhanced the visualization of serious lesions infiltrating into deeper layers, such as esophageal lamina propria or gastric submucosal cancers. Conclusions: BLI-bright will be a helpful tool for the far-field view with IEE in organs with wider internal spaces such as the stomach.

Endoscopic findings using narrow-band imaging to distinguish between basal cell hyperplasia and carcinoma of the pharynx

Narrow‐band imaging (NBI) has been reported to be useful for detecting superficial‐type esophageal or head and neck squamous cell carcinoma (SCC), and in the present study we have used NBI to detect non‐carcinomatous lesions, such as basal cell hyperplasia (BCH) accompanied by microvascular irregularities; these non‐carcinomatous lesions were pathologically discriminated from squamous cell carcinoma of the pharynx. The aim of the present study was to clarify the endoscopic characteristics of BCH that contribute to the discrimination of superficial‐type head and neck SCC (HNSCC). We examined the key endoscopic findings capable of distinguishing BCH from SCC using 26 BCH and 37 superficial‐type SCC of the pharynx that had been pathologically diagnosed at our institution between January 2008 and July 2012. The clinicopathological factors were also compared. The size of the BCH lesions was significantly smaller (P < 0.001), and their intervascular transparency was more clearly observed (P < 0.001). Intra‐epithelial papillary capillary loop (IPCL) shapes were less variable and monotonous (P < 0.001), and the distribution of the IPCL was more regular with an interval comparable to that of SCC (P < 0.001), although no significant differences in the sharpness of the lesion border, dilatation of IPCL and tortuosity of the IPCL were seen between the BCH and SCC lesions. This study revealed that BCH was an independent entity in terms of not only pathological findings, but also endoscopic findings observed using NBI, such as the regular distribution of IPCL and the preserved intervascular transparency.

Development of Highly Selective Fluorescent Probe Enabling Flow-Cytometric Isolation of ALDH3A1-Positive Viable Cells

Aldehyde dehydrogenase (ALDH) is overexpressed in some subpopulations of stem cells and cancer cells. We have designed and synthesized the first selective fluorescent probe for class 3 ALDH (ALDH3A1). This probe enabled the visualization of ALDH3A1-positive cells by fluorescence microscopy as well as flow-cytometric isolation of ALDH3A1-positive viable cells from a human Caucasian esophageal squamous cell line (OE21) that heterogeneously expresses ALDH3A1.

Abstract 3361: EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma

Recent accumulating evidences suggest that genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancerous cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer metabolism has not yet been clearly elucidated. In this study, we measured extracellular lactate production, glucose consumption and the glucose-induced extracellular acidification rate (ECAR) after the inhibition of EGFR signaling in LAD cell culture. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomic techniques, the major intracellular metabolites in EGFR-mutated LAD cells were quantified in the absence or presence of the tyrosine kinase inhibitor (TKIs) erlotinib. The molecular profile regulating the metabolic enzymes at the transcriptional and post-translational levels was characterized by RT-PCR, Western blotting and flow cytometry. We demonstrated that EGFR signaling was required for lactate production, glucose consumption and glucose-induced ECAR, indicating that EGFR signaling promoted the Warburg effect in LAD cells. Moreover, comprehensive metabolomic analysis revealed that the levels of key intermediate metabolites in glycolysis and the pentose phosphate pathway (PPP) were decreased after treatment with EGFR inhibitors. The glucose transport carried out by GLUT1 and GLUT3 was the most rapid and critically regulated function of glucose metabolism linked to EGFR signaling, although the expression of MYC-dependent glycolytic genes and the phosphorylation of pyruvate kinase muscle (PKM2) were down-regulated by EGFR inhibition at a later time point. A modest down-regulation of both total and membrane-bound GLUT3 was also observed after EGFR inhibition. Despite an equivalent total amount of GLUT1 in LAD cells, the levels of membrane-bound GLUT1 was decreased by EGFR-TKI treatment, suggesting that EGFR signaling controlled GLUT1 function through post-translational modification. We concluded that EGFR signaling regulated aerobic glycolysis in EGFR-mutated LAD cells. Our data suggests that the glycolytic pathway plays a central role in the pathogenesis of EGFR-mutated lung cancer. This regulatory pathway could be an attractive target for the development of targeted therapies to better treat patients with EGFR-mutated LAD. Citation Format: Hideki Makinoshima, Masahiro Takita, Shingo Matsumoto, Atsushi Yagishita, Satoshi Owada, Hiroyasu Esumi, Katsuya Tsuchihara. EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2014-3361

The reduced ph method with indirect plasma for safe and effective disinfection in dentistry and surgery

For the purpose of disinfecting human bodies in dental and surgical therapies, sterilization experiments in solution have been conducted with low-temperature atmospheric-pressure plasmas. For the plasma disinfection in the body fluid, we have successfully developed the reduced pH method that strong bactericidal activity can be achieved when the solution is sufficiently acidic [1]. It is interesting that a critical pH value of about 4.7 exists for the bactericidal activity. This critical value may be associated with pKa of the dissociation equilibrium between superoxide anion radicals (O 2 −•) and hydroperoxy radicals (HOO•), which is known to be approximately 4.8. As the nature without electric charge, HOO• can diffuse into bacterial cytosol via hydrophobic cell membrane, therefore HOO• shows extremely stronger bactericidal activity than negative charged O 2 −•. O 2 −• generated in gas penetrate into liquid via plasma-air-liquid interactions [2, 3]. For the enough long lifetime of O 2 −• in gas as air ion, O 2 −• can be supplied into liquid by non-contact plasma. The non-contact plasma with 400 cm extra tube has e quivalent strong bactericidal activity only with the reduced pH method. In addition, we found that the plasma treated water has strong bactericidal activity for a few minutes with the reduced pH method. This suggests that the disinfection can be done by the plasma treated water which contains short-lived active species (cannot supplied by chemical reagent) and it would bring safety plasma medicine considering usual contact or non-contact plasmas to human body. Currently, animal experiments for root canal therapy in dentistry [4] and surgical site infection prevention in surgery have been done with indirect plasma disinfection techniques of non-contact plasma and plasma treated water.