Possible Involvement of RUNX3 Silencing in the Peritoneal Metastases of Gastric Cancers

Abstract

Purpose: Our previous results suggested that a lackof RUNX3 function contributed tohumangastric carcinogenesis, but the role of RUNX3 inprogression and metastasis remains unclear.We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100 %) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-h (TGF-h)^induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of f21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified f28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A11, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in theTGF-h signaling pathway. Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Comparative analysis of DNA methylation between primary and metastatic gastric carcinoma

“…In one study, RUNX3 promoter methylation was detected in 75% of primary gastric carcinomas (n=22) and in 100% of cells from malignant ascites (7). However, in contrast to this previous report, in the present study, RUNX3 methylation was identified in 24.3% of primary gastric carcinomas and was not found to be increased in metastases.…”

“…Frequencies of the methylations of the 11 genes in corresponding non-neoplastic mucosa of the present series were similar to those reported previously (7,13,15,32), although the overall frequencies of CDH1 and TIMP3 methylation were lower than those reported previously. These findings and our findings concerning methylation frequencies in uninvolved lymph nodes, which have not been reported yet, show that CDH1, FHIT and THBS1 are methylated in a cancer-unrelated fashion.…”

“…The metastatic deposits produced were initially believed to have the same molecular profiles as the primary tumors. However, this point of view has recently been challenged, because molecular alterations, such as, DNA methylation and protein expression differences have been demonstrated in primary and metastatic cancers (5)(6)(7)(8)(9).…”

“…In particular, the hypermethylation of specific promoter CpG islands often results in the silencing of important tumor suppressor genes, and thus, promotes the development and progression of human cancers. Differences in the DNA methylation profiles of primary and metastatic cancers have been reported in several studies on breast cancers (5,6) and gastric cancers (7). However, it has not been established whether the DNA methylation profiles of metastatic deposits differ from those of primary tumors.…”

The Epigenetic Aspects of Gastric Cancer

“…Among 45 patients with tumor invasion beyond the MP, 3 of 9 patients with methylation in any of 6 genes were diagnosed as peritoneal recurrence later while only 1 out of 35 patients without methylation in any of 6 genes developed peritoneal metastasis at least 8 months after surgery (Hiraki et al, 2011). Other potential methylation markers of peritoneal metastasis of gastric cancer include RUNX3 which was silenced by DNA methylation in 100% of peritoneal metastases of gastric cancers and 75% of primary gastric cancers but not normal gastric mucosa (Sakakura et al, 2005). In addition to detect methylated DNA in peritoneal fluids, researchers have endeavored to develop sensitive methods to quantify methy l a t e d D N A i n p e r i p h e r a l b l o o d s .…”