Toshio Ota

Transcriptome Analysis of Acetate Metabolism in Corynebacterium glutamicum Using a Newly Developed Metabolic Array

Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a “metabolic array”, was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.

Establishment of a novel monoclonal antibody against LGR5

LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

Overexpression of Chitinase 3-Like 1/YKL-40 in Lung-Specific IL-18-Transgenic Mice, Smokers and COPD

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of −950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV1. Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.

Isolation and identification of the gene of cholesterol oxidase from Brevibacterium sterolicum ATCC 21387, a widely used enzyme in clinical analysis

The gene coding cholesterol oxidase (CHOD) from Brevibacterium sterolicum, which is widely used in clinical analysis, has been selected from pUC19-based gene bank in E. coli MM294 by colony-hybridization using synthetic DNA as probe. The gene was identified to encode the protein having the same amino acid sequence as that determined from amino-acid sequence analysis. The expression of the CHOD gene in E. coli was not observed, probably due to the transcription failure. Attempts are being made to express it in various hosts including Streptomyces lividans, Corynebacterium glutamicum, and B. sterolicum itself.

RhoC and guanine nucleotide exchange factor Net1 in androgen-unresponsive mouse mammary carcinoma SC-4 cells and human prostate cancer after short-term endocrine therapy

Endocrine resistance is a critical issue in managing patients with prostate cancer. This study is undertaken to search for a potential molecular target connected with this process using a model system of androgen‐dependent and androgen‐unresponsive SC‐3 and SC‐4 cells.

Chimeric Mice With Humanized Livers Demonstrate Human-Specific Hepatotoxicity Caused by a Therapeutic Antibody Against TRAIL-Receptor 2/Death Receptor 5

The activation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2)/death receptor 5 (DR5) induces apoptosis in various tumor cells but not in normal human cells. Because some therapeutic antibodies targeting TRAIL-R2 have demonstrated severe hepatotoxicity in clinical applications, novel in vivo models reflecting clinical hepatotoxicity are now required. In this study, we investigated the hepatotoxicity caused by KMTR2, an anti-human TRAIL-R2 monoclonal antibody, in chimeric mice with humanized livers (PXB-mice). PXB-mice were exposed to KMTR2 by single or repeated (weekly for 4 weeks) intravenous administrations, and the analyses of blood chemistry, liver histopathology, hepatic gene expression, and toxicokinetics were performed. Treatment with 1 or 10 mg/kg of KMTR2 increased alanine transaminase (ALT) activity and human ALT1 levels in blood. Histopathological analysis revealed that cell death and degeneration with the infiltration of inflammatory cells in human but not mouse hepatocytes were increased in a time-dependent manner after KMTR2 administration. Furthermore, increases in TdT-mediated dUTP nick end labeling (TUNEL)-positive human hepatocytes and serum concentration of cleaved cytokeratin 18, a human-specific apoptosis marker, were observed. RNA sequence analysis showed that the gene expression profile changed in different manners between human and mouse hepatocytes and the up-regulation of TRAIL-R2-related genes was observed only in human hepatocytes. Taken together, these results indicate that KMTR2-mediated TRAIL-R2 activation induces apoptosis of human hepatocytes and hepatotoxicity in PXB-mice and suggest that chimeric mice with humanized liver can be novel tools for the evaluation of in vivo human-specific hepatotoxicity induced by therapeutic antibodies in pre-clinical studies.

Preclinical and phase I clinical studies of KW-2450, a dual IGF-1R/IR tyrosine kinase inhibitor, in combination with lapatinib and letrozole

Background: KW-2450 is an oral dual insulin-like growth factor-1 receptor/insulin receptor tyrosine kinase inhibitor. We investigated the in vitro and in vivo preclinical activity of KW-2450 plus lapatinib and letrozole and conducted a phase I trial of the triple-drug combination in one male and 10 postmenopausal female patients with advanced/metastatic hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Methods: A series of in vitro and in vivo animal studies was undertaken of KW-2450 in combination with lapatinib and hormonal agents. The phase I trial was conducted to establish the safety, tolerability, and recommended phase II dose (RP2D) of KW-2450 administered in combination with lapatinib and letrozole. Results: Preclinical studies showed KW-2450 and lapatinib act synergistically to induce in vitro apoptosis and inhibit growth of HER2-positive MDA-MB-361 and BT-474 breast cancer cell lines. This combined effect was confirmed in vivo using the MDA-MB-361 xenograft model. KW-2450 showed synergistic in vitro growth inhibition with letrozole and 4-hydroxytamoxifen in ER-positive MCF-7 breast cancer cells and MCF-7-Ac1 aromatase-transfected MCF-7 cells. In the phase I study, dose-limiting toxicity (DLT; grade 3 rash and grade 3 hyperglycemia, respectively) occurred in two of three patients at the dose of KW-2450 25 mg/day plus lapatinib 1500 mg/day and letrozole 2.5 mg/day. The RP2D of the triple-drug combination was established as KW-2450 25 mg/day, lapatinib 1250 mg/day, and letrozole 2.5 mg/day with no DLT at this dose level. Conclusions: The proposed phase II study of the RP2D for the triple-drug combination did not progress because of anticipated difficulty in patient enrollment and further clinical development of KW-2450 was terminated.

Abstract 920: KW-2450, a novel IGF-1R/IR inhibitor, enhances the antitumor effect of lapatinib, letrozole or 4-hydroxy-tamoxifen in breast cancer cells.

[Background] Both the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptors (IR) have been found to be promising targets for breast cancer therapy because their activation has been associated with breast cancer development, progression and drug resistance. KW-2450, a potent inhibitor of both IGF-1R and IR, is currently in clinical development in combination with lapatinib and letrozole for HER2-positive advanced or metastatic breast cancer. To support the rationale for evaluating this combination, we examined the combined effects of KW-2450 and lapatinib, a HER2/EGFR inhibitor, in a HER2/IGF-1R-expressing breast cancer cell line, and the combined effects of KW-2450 and either letrozole or 4-hydroxy-tamoxifen in a hormone-dependent breast cancer cell line. [Results] KW-2450 and lapatinib showed a strong synergistic effect against the HER2/IGF-1R double-positive cell line, MDA-MB-361, probably caused by enhanced caspase-3/7 activation, compared to the treatment with lapatinib alone. In this setting, KW-2450 completely inhibited the phosphorylation of IGF-1R/IR, whereas lapatinib mostly inhibited the phosphorylation of HER2. Interestingly, neither KW-2450 nor lapatinib inhibited Akt phosphorylation, however, the combined treatment showed a remarkable inhibition of Akt phosphorylation. In fact, KW-2450 showed a potent combined anti-tumor effect in vivo. Furthermore, a microarray analysis identified other biomarkers, such as Ki67, survivin and TIMP3, whose expression levels were changed by the combined treatment with KW-2450 and lapatinib. In an aromatase-dependent cell line, MCF-7-Ac1, the combination of KW-2450 and letrozole showed a strong synergistic effect. Moreover, the combination of KW-2450 and 4-hydroxy-tamoxifen showed a synergistic effect in an estrogen-dependent cell line, MCF-7. [Conclusions] Our results suggest that the combination of KW-2450 and lapatinib in HER2/IGF-1R double-positive breast cancers and that of KW-2450 and anti-hormone agents in hormone-dependent breast cancers are highly effective, exhibiting a synergistic anti-cancer activity. Therefore, KW-2450 may be a promising platform for carrying out combination therapy for breast cancer. Citation Format: Hiroshi Umehara, Fumito Koizumi, Yoshimi Maekawa, Makiko Shimizu, Hiroaki Nakamura, Toshio Ota, Shinji Nara, Takeshi Takahashi, Yutaka Kanda, Norihiko Shiraishi, Shiro Akinaga, Yukimasa Shiotsu, Shiro Soga. KW-2450, a novel IGF-1R/IR inhibitor, enhances the antitumor effect of lapatinib, letrozole or 4-hydroxy-tamoxifen in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 920. doi:10.1158/1538-7445.AM2013-920