Published OnlineFirst October 4, 2013; DOI: 10.1158/1078-0432.CCR-12-3186 Clinical Cancer Human Cancer Biology Research Contrasting Expression Patterns of Histone mRNA and microRNA 760 in Patients with Gastric Cancer Takeshi Iwaya1,2, Takeo Fukagawa3, Yutaka Suzuki4, Yusuke Takahashi1, Genta Sawada1, Masahisa Ishibashi1, Junji Kurashige1, Tomoya Sudo1, Fumiaki Tanaka1, Kohei Shibata1, Fumitaka Endo2, Hirokatsu Katagiri2, Kaoru Ishida2, Kohei Kume2, Satoshi Nishizuka2, Hisae Iinuma5, Go Wakabayashi2, Masaki Mori6, Mitsuru Sasako7, and Koshi Mimori1 Abstract Purpose: Recent studies revealed that both disseminated tumor cells and noncancerous cells contributed to cancer progression cooperatively in the bone marrow. Here, RNA-seq analysis of bone marrow from gastric cancer patients was performed to identify prognostic markers for gastric cancer. Experimental Design: Bone marrow samples from eight gastric cancer patients (stages I and IV: n ¼ 4 each) were used for RNA-seq analysis. Results were validated through quantitative real-time PCR (qRT-PCR) analysis of HIST1H3D expression in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from gastric cancer patients. miR-760 expression was assayed using qRT-PCR in 105 bone marrow and 96 primary tumor samples. Luciferase reporter assays were performed to confirm whether histone mRNAs were direct targets of miR-760. miR-760 expression was also evaluated in noncancerous cells from gastric cancer patients. Results: RNA-seq analysis of bone marrow samples from gastric cancer patients revealed higher expression of multiple histone mRNAs in stage IV patients. HIST1H3D expression in the bone marrow, peripheral blood, and primary tumor of stage IV patients was higher than that in stage I patients (P ¼ 0.0284, 0.0243, and 0.0006, respectively). In contrast, miR-760 was downregulated in the bone marrow and primary tumor of stage IV patients compared with stage I patients (P ¼ 0.0094 and 0.0018, respectively). Histone mRNA and miR-760 interacted directly. Furthermore, miR-760 was downregulated in noncancerous mucosa in stage IV gastric cancer patients. Conclusion: Histone mRNA was upregulated, whereas miR-760 was downregulated in the bone marrow and primary tumor of advanced gastric cancer patients, suggesting that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer. Clin Cancer Res; 1–12. Ó2013 AACR. Introduction cells (ITC) is an important factor contributing to the metas- The occurrence of distant metastases is the main cause of tasis of solid cancers. In clinical practice, the circulating death in cancer patients. The presence of isolated tumor tumor cell (CTC) detection system (CellSearch System) was first approved by the Food and Drug Administration for metastatic breast cancer and has now been approved for the Authors' Affiliations: 1Department of Surgery, Kyushu University Beppu detection and monitoring of CTCs in the blood from 2 Hospital, Beppu; Department of Surgery, Iwate Medical University, Morioka; patients with metastatic prostate and colorectal cancer. 3Department of Gastric Surgery Division, National Cancer Center Hospital; 4Department of Medical Genome Sciences, Graduate School of Frontier Moreover, recent studies have demonstrated that various Sciences, University of Tokyo, Kashiwa-shi, Chiba; 5Department of Surgery, types of host cells are also involved in cancer development 6 Teikyo University School of Medicine, Tokyo; Department of Gastroenter- and metastasis, including fibroblasts (carcinoma-associated ological Surgery, Graduate School of Medicine, Osaka, University, Suita; and 7Department of Digestive Surgery, Hyogo Medical College, Japan fibroblasts or myofibroblasts), tumor-associated macro- phages, mesenchymal stem cells, platelets, and hematopoi- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). etic progenitor cells (1–8). We previously investigated the presence of ITCs in periph- T. Iwaya, T. Fukagawa, G. Wakabayashi, M. Mori, M. Sasako, and K. Mimori contributed equally to this work. eral blood and bone marrow samples using quantitative real-time PCR (qRT-PCR) analysis of CEA, CK-7, and CK-19 Corresponding Author: Koshi Mimori, Department of Surgery, Kyushu University Beppu Hospital, 4546 Tsurumihara, Beppu 874-0838, Japan. in more than 800 cases of gastric cancer (9). We found that Phone: 81-977-1650; Fax: 81-977-1651; E-mail: ITCs circulated in patients with a range of clinical stages of [email protected] gastric cancer and demonstrated that the simultaneous doi: 10.1158/1078-0432.CCR-12-3186 expression of ITC-associated genes and VEGFR-1, which Ó2013 American Association for Cancer Research. may originate from hematopoietic progenitor cells in www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst October 4, 2013; DOI: 10.1158/1078-0432.CCR-12-3186 Iwaya et al. stage IV patients with liver metastasis, were used for RNA- Translational Relevance seq analysis (Supplementary Table S1). Quantitative real- The presence of isolated tumor cells in the peripheral time PCR (qRT-PCR) was performed to analyze the expres- blood and bone marrow is an important factor contrib- sion of histone mRNA in 175 bone marrow and 92 periph- uting to the metastasis of solid cancers. Moreover, recent eral blood samples and expression of miRNA in105 bone studies have demonstrated that various types of host cells marrow samples. For qRT-PCR analysis of primary tumors, are also involved in cancer development and metastasis. another 127 gastric cancer and corresponding normal tissue We performed RNA-seq analysis of the bone marrow samples were used. For miRNA microarray analysis of 3 from patients with gastric cancer to identify candidate fractions separated from the bone marrow, bone marrow prognostic markers using next-generation sequencing samples were collected from another 4 gastric cancer and demonstrated that histone cluster genes were over- patients. Detailed protocols for sample preparation are expressed in the bone marrow and primary tumor sam- described in the Supplementary Data. ples from stage IV gastric cancer patients compared with those from stage I gastric cancer patients. Furthermore, RNA-seq analysis of bone marrow samples from gastric we proposed the possibility that microRNA-760 (miR- cancer patients by massively parallel sequencing 760) was downregulated in order to degrade upregulated Briefly, a total of 1 mg of extracted RNA was used as a histone mRNA in response to an increase in S-phase cells template to construct RNA-seq libraries. In this step of in the bone marrow and primary tumors of advanced sample preparation, starting with total RNA, the mRNA gastric cancer patients. Our data also suggested that the was poly-A selected, fragmented, and converted into sin- histone mRNA/miR-760 axis had a crucial role in the gle-stranded cDNA using random hexamer priming. development of gastric cancer. Detailed protocols are described in the Supplementary Data. Fold enrichment of the RNA-seq tags between the samples was calculated for each mRNA using the assigned tag counts and was normalized to read per kilobase mRNA peripheral blood and bone marrow, was significantly (RPKM; ref. 11). associated with hematogeneous metastases (9). Therefore, multiple markers are currently needed to predict distant Evaluation of HIST1H3D and miR-760 expression in metastasis and/or prognosis from bone marrow or periph- clinical samples eral blood samples in gastric cancer patients by PCR mRNA and miRNA levels were quantified using a Light- analysis. Disseminated tumor cells (DTC) in the bone Cycler 480 Probes Master Kit (Roche Applied Science) marrow have been detected in all solid tumor types, following the manufacturer’s protocol. HIST1H3D mRNA suggesting that the bone marrow may be a preferred expression levels were measured in 175 bone marrow, 92 reservoir for blood–bone DTCs. The bone marrow envi- peripheral blood, and 115 primary tumor samples from ronment may allow these cells to persist for a prolonged patients with gastric cancer and corresponding noncancer- period and to disseminate into other organs (10). Many ous gastric mucosa samples. miR-760 expression was also cancer-associated host cells are derived from the bone assayed by qRT-PCR in 105 bone marrow, 96 primary marrow. According to these findings, differences in the tumor, and 84 corresponding normal gastric mucosa sam- gene expression status of bone marrow cells may reflect ples from gastric cancer patients. Detailed protocols are different cancer stages or the possibility of distant metas- described in the Supplementary Data. tases. Moreover, the bone marrow is a convenient organ to sample for analysis and is more easily accessible than other Cell lines and cell culture organs that are often sites of metastases, such as the lungs Seven human gastric cancer cell lines (NUGC3, NUGC4, or liver. In this study, we performed RNA-seq analysis of MKN74, AGS, KATOIII, MKN45, and KE39) were provided the bone marrow from patients with gastric cancer in order by the Cell Resource Center of Biomedical Research, Insti- to identify candidate prognostic markers. We demonstrat- tute of Development, Aging, and Cancer, Tohoku Univer- ed that multiple histone cluster genes showed higher sity and the Riken Bioresource Center. Cells were main- expression in the bone marrow of stage IV patients than tained