Abstract 4346: Novel discovery of miR-30e* regulating Bmi1 expression induced by tumor-associated macrophages in gastrointestinal cancer

Abstract

Abstract Background: Bmi1 is a member of the polycomb-repressive complex 1 with an essential role in maintaining chromatin silencing. Bmi1 plays a function in the self-renewal of neuronal and hematopoietic stem cells through repression of the INK4A/ARF locus. Furthermore, Bmi1 is overexpressed in a variety of human cancers. The expression of Bmi1 is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, solid tumors consist of cancer cells and various types of stromal cells, fibroblasts, endothelial cells and hematopoietic cells, mainly macrophages and lymphocytes. Tumor-associated macrophages (TAMs) contribute to tumor progression by producing various mediators. This study was performed to identify the tumor-associated macrophages (TAMs)-mediated regulation of Bmi1 expression in gastrointestinal cancers. Method: The relationship between the expression of Bmi1 and TAMs was assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and 3D sphere culture. Next, miRNAs microarray in gastric cancer cell co-cultured with macrophages was conducted. To examine the functional relevance of miR-30e* expression, we analyzed the relationship between miR-30e* and Bmi1 expression in high Bmi1 expressing cancer cell lines transfected with miR-30e* mimics, and low Bmi1 expressing cancer cell lines transfected with miR-30e* inhibitors. And we investigated if miR-30e* directly targets the 3′ UTR of Bmi1 using constructs containing the putative miR-30e* target site or a mutated sequence of the 3′ UTR of Bmi1 cloned immediately downstream of a luciferase gene. Furthermore Bmi1 and miR-30e* expression were assessed in cancer tissues. Results: We revealed the positive relationship between with tumor-infiltrating macrophages and Bmi1 expression in cancer cells. We showed co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. Based on microRNA screening analysis and in silico microRNA search, we focused on miR-30e* that could regulate Bmi1 expression. Western blot analysis revealed significantly reduced Bmi1 protein levels in high Bmi1 expressing cancer cell lines transfected with miR-30e* mimics compared with controls, and increased levels in low Bmi1 expressing cancer cell lines cells transfected with miR-30e* inhibitors compared with controls. And Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites. MiR-30e* expression was down-regulated in tumor region compared with in non-tumor one. Furthermore Bmi1 expression was inverse correlation with miR-30e* expression. Conclusions: TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. Citation Format: Hidetaka Sugihara, Takatsugu Ishimoto, Daiauke Izumi, Hiroshi Sawayama, Yu Imamura, Satoshi Ida, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Novel discovery of miR-30e* regulating Bmi1 expression induced by tumor-associated macrophages in gastrointestinal cancer. [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 4346. doi:10.1158/1538-7445.AM2014-4346