ALEX1, a novel tumor suppressor gene, inhibits gastric cancer metastasis via the PAR-1/Rho GTPase signaling pathway.

ABSTRACT

BACKGROUND: The ALEX is a novel member of the armadillo family and ALEX1 was reported to be reduced or even lost in multiple solid tumors. However, its expression profile and oncogenic role in gastric cancer (GC) remains largely unknown. METHODS: ALEX1 expression was detected in 161 GC samples by immunohistochemistry staining. NCI-N87 cells transfected by ALEX1 lentivirus vectors and MKN28 cells transfected by ALEX1 shRNA were used for biological function investigation. Western blot was applied to explore the molecular mechanism and pull-down assays were applied to measure the activity of Rho GTPases. In vivo tumorigenicity, peritoneal and lung metastasis experiments were performed by tumor cell engraftment into nude mice. Bisulfite genomic sequencing and methylation-specific PCR were applied to check the methylation status of the ALEX1 gene. RESULTS: The expression rate of ALEX1 was significantly reduced in gastric tumor samples compared to non-tumor samples (43.5 vs. 90.2%), and its expression was closely related to the tumor differentiation, TNM staging, and lymph nodes metastasis. ALEX1 overexpression in NCI-N87 cells significantly inhibited cell proliferation, migration, and invasion in vitro, and disrupted the structure of the cytoskeleton. ALEX1 overexpression attenuated xenografts growth, peritoneal, and lung metastasis in nude mice. Mechanistically, the overexpression of ALEX1 inhibits thrombin-induced metastasis and Rho GTPases activation. Bisulfite genomic sequencing and methylation-specific PCR revealed that the promoter of ALEX1 is highly methylated in GC cells and tissues. CONCLUSIONS: ALEX1 expression is reduced in GC and is involved in diverse cellular functions. ALEX1 inhibits metastasis through the PAR-1/Rho GTPase signaling pathway.