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 a LGR5 promoter fragment, which is highly relevant for active LGR5 expression. Chromatin immunoprecipitation (ChIP) 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, were explored in neoplastic and non-neoplastic gastric tissue and gastric cancer cell lines. Using immunohistochemistry (IHC), distinct spatial expression patterns of LGR5, RSPO1 and RSPO2 were found in non-neoplastic stomach mucosa and gastric cancer. RSPO expression was lower in gastric cancer compared with non-neoplastic mucosa on both the transcriptional (p=0.003 for RSPO1 and p=0.000 for RSPO2; n=50) and the translational level. Methylation-specific PCR showed higher methylation levels of RSPO1/2 and re-expression of RSPOs in the gastric cancer cell lines MKN45 and MKN74 was induced by demethylating 5-azaC treatment. Finally, expression patterns of LGR5 and RSPO were similar in gastric cancer. Implications: 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.
- Received December 19, 2016.
- Revision received January 25, 2017.
- Accepted February 4, 2017.
- Copyright ©2017, American Association for Cancer Research.