Wilhelm Franziska, Simon Eva, Böger Christine, Behrens Hans-Michael, Krüger Sandra, Röcken Christoph
Department of Pathology, Christian-Albrechts-University, Kiel, Germany.
Mol Cancer Res. 2017 Jun;15(6):776-785. doi: 10.1158/1541-7786.MCR-16-0472. Epub 2017 Feb 20.
Recently, it was shown that leucine-rich repeat-containing receptor 5 (LGR5)-expressing stem cells are the cellular origin of intestinal-type gastric cancer. The aim of our study was to uncover regulatory mechanisms of LGR5 expression in gastric mucosa and their implications for cancer development. Reporter assays identified an LGR5 promoter fragment, which is highly relevant for active LGR5 expression. Chromatin immunoprecipitation verified that SP1 is bound within this region, and reporter activity increased in SP1 transfected cells. Subsequently, the expression of R-spondins (RSPO1 and RSPO2), ligands of LGR5, was explored in neoplastic and nonneoplastic gastric tissue and gastric cancer cell lines. Using IHC, distinct spatial expression patterns of LGR5, RSPO1, and RSPO2 were found in nonneoplastic stomach mucosa and gastric cancer. RSPO expression was lower in gastric cancer compared with nonneoplastic mucosa on both the transcriptional ( = 0.003 for RSPO1 and = 0.000 for RSPO2; = 50) and the translational level. Methylation-specific PCR showed higher methylation levels of RSPO1/2 and reexpression of RSPOs in the gastric cancer cell lines MKN45 and MKN74 were induced by demethylating 5-aza-C treatment. Finally, expression patterns of LGR5 and RSPO were similar in gastric cancer. This report identifies a regulatory mechanism of LGR5 expression in gastric carcinogenesis, with SP1 as an important component of the transcriptional complex and LGR5 activity, which is modulated by its ligands RSPO1 and RSPO2, whose expression is modulated by methylation. http://mcr.aacrjournals.org/content/15/6/776/F1.large.jpg. .
最近的研究表明，表达富含亮氨酸重复序列的受体5（LGR5）的干细胞是肠型胃癌的细胞起源。我们研究的目的是揭示胃黏膜中LGR5表达的调控机制及其对癌症发展的影响。报告基因检测确定了一个与LGR5活跃表达高度相关的启动子片段。染色质免疫沉淀证实SP1结合在该区域内，并且在转染SP1的细胞中报告基因活性增加。随后，在肿瘤性和非肿瘤性胃组织以及胃癌细胞系中研究了LGR5的配体R-spondins（RSPO1和RSPO2）的表达。使用免疫组织化学方法，在非肿瘤性胃黏膜和胃癌中发现了LGR5、RSPO1和RSPO2不同的空间表达模式。与非肿瘤性黏膜相比，胃癌中RSPO的转录水平（RSPO1为 = 0.003，RSPO2为 = 0.000； = 50）和平行水平表达均较低。甲基化特异性PCR显示RSPO1/2的甲基化水平较高，并且通过5-氮杂胞苷去甲基化处理诱导胃癌细胞系MKN45和MKN74中RSPO的重新表达。最后，LGR5和RSPO在胃癌中的表达模式相似。本报告确定了胃癌发生过程中LGR5表达的调控机制，其中SP1是转录复合物和LGR5活性的重要组成部分，LGR5活性受其配体RSPO1和RSPO2调节，而RSPO1和RSPO2的表达受甲基化调节。http://mcr.aacrjournals.org/content/15/6/776/F1.large.jpg。
