Abstract A40: Emergence of CD47- high expression cells confers enhanced tumorigenicity upon KDM6B suppression in pancreatic cancer

Abstract

Abstract The role of genetic mutations in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) is well established. However, it is still unclear if epigenetic aberrations contribute to PDAC progression. We previously reported a novel role for the H3K27 demethylase KDM6B/JMJD3 in regulating PDAC progression (Carcinogenesis 2014;35(11):2404-14). KDM6B was downregulated in high grade PDACs and knockdown (KD) of KDM6B in PDAC cells increased the tumorigenicity and enhanced the aggressive phenotypes of these cells in vivo. Furthermore, CCAAT enhancer binding protein alpha gene (CEBPA) was identified as a direct target of KDM6B, and reduced KDM6B- C/EBPα axis was resulted in increased aggressiveness in PDAC cells. To dissect further the pathological effects caused by loss of KDM6B- C/EBPα function in PDAC cells, we tried to identify a surrogate molecular marker of the cells lacking of KDM6B- function. For the purpose, we used a cDNA microarray to compare the expression profiles of KDM6B- KD and control BxPC3 PDAC cells. 906 genes were upregulated and 639 downregulated in KDM6B- KD BxPC3 cells compared to the control cells. We focused on 58 genes encoding cell-surface molecules that were upregulated in KDM6B- KD BxPC3 cells and validated the expression of 9 surface marker candidates, including 3 that have already been reported to be expressed on PDAC tumor-initiating cells, namely, CD24, CD44, and CD133. Only CD47 was significantly upregulated in the KDM6B- KD BxPC3 cells as confirmed by both quantitative RT-PCR and flow cytometric analysis. CD47 was also upregulated in other PDAC cell lines following KDM6B knockdown. It has recently been reported that CD47 is upregulated in various malignancies and that an increase in CD47 expression is correlated with a poor prognosis. In line with the previous reports, CD47high cells formed about 4-fold more spheres than non-CD47high cells. The close relationship between CD47 expression and the sphere-forming ability was supported by the finding that CD47low cells formed even fewer spheres. To confirm these results in vivo, the sorted CD47high and non-CD47high cells were subcutaneously xenotransplanted into nude mice. All CD47high cells formed tumors more efficiently than the unfractionated KDM6B- KD cells, while the tumor-forming rate of non-CD47high cells was comparable to that of the Ctrl cells. In addition, when the cells were injected into the spleens of nude mice, CD47high cells demonstrated higher liver metastatic potential than the non-CD47high population. These data suggested that the increased tumor-initiating potential of KDM6B- KD cells was attributable to this induced CD47high population. Consistently, the expression of KDM6B and C/EBPα inversely correlated with CD47 expression and tumor grade in human PDAC tumors. Collectively, our data provides a link between epigenetic change and PDAC progression, thus offering a novel strategy to target PDAC aggressiveness by intervening in the dynamics of epigenetic process. Citation Format: Keisuke Yamamoto, Keisuke Tateishi, Yotaro Kudo, Koji Miyabayashi, Ryota Takahashi, Takuma Nakatsuka, Hiroaki Fujiwara, Yousuke Nakai, Yasuo Tanaka, Hideaki Ijichi, Hiroyuki Isayama, Kazuhiko Koike. Emergence of CD47- high expression cells confers enhanced tumorigenicity upon KDM6B suppression in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A40.