Loss of Expression of Reprimo, a p53-induced Cell Cycle Arrest Gene, Correlates with Invasive Stage of Tumor Progression and p73 Expression in Gastric Cancer

Abstract

Reprimo (RPRM), a downstream effector of p53-induced cell cycle arrest at G2/M, has been proposed as a putative tumor suppressor gene (TSG) and as a potential biomarker for non-invasive detection of gastric cancer (GC). The aim of this study was to evaluate the epigenetic silencing of RPRM gene by promoter methylation and its tumor suppressor function in GC cell lines. Furthermore, clinical significance of RPRM protein product and its association with p53/p73 tumor suppressor protein family was explored. Epigenetic silencing of RPRM gene by promoter methylation was evaluated in four GC cell lines. Protein expression of RPRM was evaluated in 20 tumor and non-tumor matched cases. The clinical significance of RPRM association with p53/p73 tumor suppressor protein family was assessed in 114 GC cases. Tumor suppressor function was examined through functional assays. RPRM gene expression was negatively correlated with promoter methylation (Spearman rank r = -1; p = 0.042). RPRM overexpression inhibited colony formation and anchorage-independent growth. In clinical samples, RPRM gene protein expression was detected in 75% (15/20) of non-tumor adjacent mucosa, but only in 25% (5/20) of gastric tumor tissues (p = 0.001). Clinicopathological correlations of loss of RPRM expression were significantly associated with invasive stage of GC (stage I to II-IV, p = 0.02) and a positive association between RPRM and p73 gene protein product expression was found (p<0.0001 and kappa value = 0.363). In conclusion, epigenetic silencing of RPRM gene by promoter methylation is associated with loss of RPRM expression. Functional assays suggest that RPRM behaves as a TSG. Loss of expression of RPRM gene protein product is associated with the invasive stage of GC. Positive association between RPRM and p73 expression suggest that other members of the p53 gene family may participate in the regulation of RPRM expression.

Fig 1. Regulation of RPRM expression by methylation of its promoter region.

A) RPRM promoter region analyzed by bisulfite quantification. B) DNA Bisulfite sequencing in gastric cancer cell lines. C) Relative expression of RPRM in four gastric cancer cell lines ns: no significative; * p<0.01; ANOVA test One-way. D) Spearman rank correlation between RPRM expression and promoter methylation status of RPRM. E) RPRM methylation in tumor and non-tumor adjacent mucosa (NTAM) tissues, higher methylation levels in tumor tissues are observed in comparison to NTAM in all six paired GC cases.

Fig 2. Immunohistochemical staining for RPRM in gastric cancer.

A) Representative example of non-tumor adjacent mucosa (NTAM) showing positive cytoplasmic staining of RPRM in more than 30% of cells. B) Representative example of tumor (T) gastric cancer sample showing negative staining of RPRM.

Fig 3. Overexpression of RPRM reduces colony formation and anchorage-independent growth in vitro.

A) Western blot of AGS cells with overexpression of RPRM (pCMV6/RPRM) and empty control (pCMV6). B) Overexpression of RPRM in AGS cells (pCMV6/RPRM) resulted in a significant reduction of in vitro colony formation compared with AGS cell line transfected with pCMV6 empty vector (pCMV6). Each experiment was carried out in triplicate. There was a statistically significant reduction of colony formation in overexpression experiments (*p < 0.05). Below each graph are representative plates showing reduction of colonies after gene overexpression Error bars; SD. C) Overexpression of RPRM in AGS cells (pCMV6/RPRM) resulted in a significant reduction of in vitro anchorage-independent colony formation compared with AGS cell line transfected with pCMV6 empty vector (pCMV6). Each experiment was carried out in triplicate. There was a statistically significant reduction of colonies formation in the overexpression experiments (*p < 0.05). Below each graph are representative plates showing reduction of colonies after gene overexpression Error bars; SD