Mitsuyo Matsumoto

Estrogen signaling ability in human endometrial cancer through the cancer-stromal interaction.

The estrogen pathway plays an important role in the etiology of human endometrial carcinoma (EC). We examined whether estrogen biosynthesis in the tumor microenvironment promotes endometrial cancer. To examine the contribution of stromal cells to estrogen signaling in EC, we used reporter cells stably transfected with the estrogen response element (ERE) fused to the destabilized green fluorescent protein (GFP) gene. In this system, the endometrial cancer stromal cells from several patients activated the ERE of cancer cells to a variable extent. The GFP expression level increased when testosterone, a substrate for aromatase, was added. The effect was variably inhibited by aromatase inhibitors (AIs), although the response to AIs varied among patients. These results suggest that GFP expression is driven by estrogen synthesized by aromatase in the endometrial cancer stromal cells. In a second experiment, we constructed an adenovirus reporter vector containing the same construct as the reporter cells described above, and visualized endogenous ERE activity in primary culture cancer cells from 15 EC specimens. The GFP expression levels varied among the cases, and in most primary tissues, ERE activities were strongly inhibited by a pure anti-estrogen, fulvestrant. Interestingly, a minority of primary tissues in endometrial cancer showed ERE activity independent of the estrogen-ER pathway. These results suggest that AI may have some therapeutic value in EC; however, the hormonal microenvironment must be assessed prior to initiating therapy.

Methionine Adenosyltransferase II-dependent Histone H3K9 Methylation at the COX-2 Gene Locus

Background: MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. Results: MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. Conclusion: AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. Significance: MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.

Midkine and its clinical significance in endometrial carcinoma

Midkine (MK) is a secreted heparin‐binding growth factor. Several types of human cancer have increased MK expression with elevated serum levels. The purpose of this study was to determine whether MK was expressed in endometrial carcinoma and to evaluate the clinicopathological significance of serum MK in patients with endometrial carcinoma. Immunohistochemical expression of MK was evaluated in 85 endometrial carcinoma samples and 33 controls. MK expression was significantly higher in the carcinomas than in normal endometrium (P < 0.001). Interestingly, MK expression was highest at the margins of invasion and low in the superficial areas of the tumor samples. Using ELISA, we compared serum MK concentration in 120 endometrial carcinoma patients with the concentration in 46 patients with benign gynecologic tumors. Serum MK value in patients with cancer was significantly higher than that in the patients with benign diseases (P = 0.01). Patients with positive lymph node metastasis or recurrence, or cancer death, had a higher serum MK level (P = 0.008, P = 0.009, respectively). In conclusion, MK immunoreactivity in endometrial carcinoma is significantly higher than in normal endometrium. Additionally, preoperative serum MK levels are significantly correlated with prognosis and the presence of lymph node metastasis. Thus, MK may be a useful serum biomarker for identifying high risk patients of endometrial carcinoma. (Cancer Sci 2008; 99: 1125–1130)

Local Biosynthesis of Estrogen in Human Endometrial Carcinoma through Tumor-Stromal Cell Interactions

Purpose: The metabolism and synthesis of intratumoral estrogens are thought to play a very important role in the etiology and progression of endometrial carcinoma. Aromatase is a key enzyme in the conversion of androgens to estrogens, and aromatase localization studies have reported that aromatase immunoreactivity and mRNA were detected mainly in stromal cells. However, the effect of tumor-stromal interactions on local estrogen biosynthesis in endometrial carcinomas remains largely unknown. Experimental Design: The endometrial carcinoma cell lines (Ishikawa and RL95-2) and breast carcinoma cell line (MCF-7) were cocultured with stromal cells isolated from endometrial carcinomas, and aromatization activity was measured using liquid chromatography-tandem mass spectrometry. We then confirmed the local biosynthesis of estrogens and tumor-stromal interactions on aromatase activity in Ishikawa and RL95-2 cells. In addition, we also examined the effects of aromatase inhibitors on cell proliferation. Results: Aromatase activity was significantly higher in cocultures with Ishikawa or RL95-2 than in each monoculture, respectively. Estrone (E1) concentrations were significantly higher than estradiol (E2) concentrations in Ishikawa and RL95-2 cells, whereas E2 was significantly higher than E1 in MCF-7 cells. Cell proliferation was significantly inhibited in Ishikawa and RL95-2 cell cultures treated with aromatase inhibitors compared with control cultures. Conclusions: These results indicate the contribution of not only E2 but also E1 to cancer cell proliferation in endometrial carcinoma. Our study may provide important information on metabolism and synthesis of intratumoral estrogens with regard to the etiology and progression of endometrial carcinoma, thus helping to achieve improved clinical responses in patients with endometrial carcinoma, who are treated with aromatase inhibitors. (Clin Cancer Res 2009;15(19):6028–34)

Expression of retinoic acid receptors in human endometrial carcinoma

The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) β is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RARβ could act as a tumor suppressor. However, the status of RARβ in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RARα, RARβ, and RARγ using AM580 (a RAR‐specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RARβ and RARγ mRNA was significantly induced by AM580, compared with vehicle controls, whereas RARα mRNA was significantly attenuated by AM580, compared with vehicle. RARβ was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RARβ mRNA expression in the Ishikawa cell line, and the expression level of RARβ in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma. (Cancer Sci 2008; 99: 267–271)

A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors

Hematopoietic stem cell and multipotent progenitor (MPP) commitment can be tuned in response to an infection so that their differentiation is biased toward myeloid cells. Here, we find that Bach2, which inhibits myeloid differentiation in common lymphoid progenitors, represses a cohort of myeloid genes and activates those linked to lymphoid function. Bach2 repressed both Cebpb and its target Csf1r, encoding C/EBPβ and macrophage colony-stimulating factor receptor (M-CSFr), respectively, whereas C/EBPβ repressed Bach2 and activated Csf1r. Bach2 and C/EBPβ further bound to overlapping regulatory regions at their myeloid target genes, suggesting the presence of a gene regulatory network (GRN) with mutual repression between these factors and a feedforward loop leading to myeloid gene regulation. Lipopolysaccharide reduced the expression of Bach2, resulting in enhanced myeloid differentiation. The Bach2-C/EBPβ GRN pathway thus tunes MPP commitment to myeloid and lymphoid lineages both under normal conditions and after infection.

Individual transcriptional activity of estrogen receptors in primary breast cancer and its clinical significance

To predict the efficacy of hormonal therapy at the individual‐level, immunohistochemical methods are used to analyze expression of classical molecular biomarkers such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. However, the current diagnostic standard is not perfect for the individualization of diverse cases. Therefore, establishment of more accurate diagnostics is required. Previously, we established a novel method that enables analysis of ER transcriptional activation potential in clinical specimens using an adenovirus estrogen response element–green fluorescence protein (ERE‐GFP) assay system. Using this assay, we assessed the ERE transcriptional activity of 62 primary breast cancer samples. In 40% of samples, we observed that ER protein expression was not consistent with ERE activity. Comparison of ERE activity with clinicopathological information revealed that ERE activity was significantly correlated with the ER target gene, PgR, rather than ER in terms of both protein and mRNA expression. Moreover, subgrouping of Luminal A‐type breast cancer samples according to ERE activity revealed that ERα mRNA expression correlated with ER target gene mRNA expression in the high‐, but not the low‐, ERE‐activity group. On the other hand, the low‐ERE‐activity group showed significantly higher mRNA expression of the malignancy biomarker Ki67 in association with disease recurrence in 5% of patients. Thus, these data suggest that ER expression does not always correlate with ER transcriptional activity. Therefore, in addition to ER protein expression, determination of ERE activity as an ER functional marker will be helpful for analysis of a variety of diverse breast cancer cases and the subsequent course of treatment.

Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population

BackgroundValidation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously.ResultsHere, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%.ConclusionsOur results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls.

Bach1 is critical for the transformation of mouse embryonic fibroblasts by Ras V12 and maintains ERK signaling

Reactive oxygen species (ROS), by-products of aerobic respiration, promote genetic instability and contribute to the malignant transformation of cells. Among the genes related to ROS metabolism, Bach1 is a repressor of the oxidative stress response, and a negative regulator of ROS-induced cellular senescence directed by p53 in higher eukaryotes. While ROS are intimately involved in carcinogenesis, it is not clear whether Bach1 is involved in this process. We found that senescent Bach1-deficient mouse embryonic fibroblasts (MEFs) underwent spontaneous immortalization the same as did the wild-type cells. When transduced with constitutively active Ras (H-RasV12), the proliferation and colony formation of these cells in vitro were markedly reduced. When transplanted into athymic nude mice, the growth and vascularization of tumors derived from Bach1-deficient cells were also decreased. Gene expression profiling of the MEFs revealed a new H-RasV12 signature, which was distinct from the previously reported signatures in epithelial tumors, and was partly dependent on Bach1. The Bach1-deficient cells showed diminished phosphorylation of MEK and ERK1/2 in response to H-RasV12, which was consistent with the alterations in the gene expression profile, including phosphatase genes. Finally, Bach1-deficient mice were less susceptible to 4-nitroquinoline-1-oxidide (4-NQO)-induced tongue carcinoma than wild-type mice. Our data provide evidence for a critical role of Bach1 in cell transformation and tumor growth induced by activated H-RasV12.

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells

Significance Primordial germ cells (PGCs) are the origin of germ cells and are critically important for continuity of multicellular organisms. Although the unique characteristics of mouse PGCs have been described in gene expression and epigenome levels, the metabolomic and proteomic profiles of PGCs and their significance for PGC properties have been unclear. Our findings in this study demonstrate not only distinct energy metabolisms in PGCs and pluripotent stem cells (PSCs), but also the essential contribution of enhanced oxidative phosphorylation and glycolysis in PGC specification from PSCs, and in reprogramming of PGCs into PSCs, respectively. The results uncover the importance of a shift in main energy metabolism for establishment and maintenance of PGC characteristics. Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.