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.

Fra-1 is upregulated in gastric cancer tissues and affects the PI3K/Akt and p53 signaling pathway in gastric cancer

“…Despite an overall decline in incidence over the last several decades, gastric cancer (GC) remains the fourth most common type of cancer and is the second leading cause of cancerrelated death worldwide (26,27). Although some evidence has been reported, the mechanism of GC is not fully understood (28)(29)(30). Thus, identifying molecular aberrations in GC may improve our understanding of gastric carcinogenesis and help us subdivide patients into biologically and clinically relevant subgroups, as well as develop novel therapeutic strategies.…”

Evolutionary history of the reprimo tumor suppressor gene family in vertebrates with a description of a new reprimo gene lineage

“…The RPRM protein is believed to mediate, at least in part, the p53-dependent cell cycle arrest at G2 by the inhibition of Cdc2 activity and nuclear translocation of cyclin B1 (Ohki et al, 2000;Taylor and Stark, 2001). It has also been suggested that RPRM may also be regulated by p73 in an independent manner from p53 in gastric carcinogenesis (Saavedra et al, 2015).The loss of RPRM expression in cancer through abnormal methylation patterns suggests that this gene and its corresponding biochemical pathway might play a role in cancer metabolism and development, and suggests a potential target for treatment against malignancies (Ohki et al, 2000;Wong et al, 2005;Hamilton et al, 2006;Bernal et al, 2008;Luo et al, 2011;Saavedra et al, 2015). To date, efforts to find biomarkers for early stages of cancer have found limited success, and the gene RPRM has emerged as a promising candidate in this area (Bernal et al, 2008).…”

“…The inverse relationship between methylation and transcription has identified RPRM as a target for aberrant methylation in various cancers, including prostate cancer (Ellinger et al, 2008), lung cancer (Nakajima et al, 2009), hepatocellular carcinoma (Nishida et al, 2008), pancreatic cancer (Sato et al, 2008), head and neck carcinomas (Wong et al, 2005), esophageal cancer (Hamilton et al, 2006) and gastric adenocarcinoma (Bernal et al, 2008;Luo et al, 2011;Saavedra et al, 2015). The abnormal regulation of the genome during cancer is thought to be due to atypical methylation processes rather than the presence of somatic mutations.…”

Noncoding Genomics in Gastric Cancer and the Gastric Precancerous Cascade: Pathogenesis and Biomarkers

“…Methylation levels were 5.4- to 303-fold higher in H. pylori -positive than in H. pylori -negative subjects ( p < 0.0001) ( Figure 2 ). Schneider et al [ 62 ] performed a quantitative analysis of the DNA methylation status of the promoter region of Reprimo (RPRM), a putative tumor suppressor gene in gastric cancer [ 63 ], which demonstrated an association of the methylation status of the gene with the presence of virulence factors in the infecting H. pylori strains. Taken together, these findings suggest that H. pylori infection potently induces aberrant DNA methylation.…”

“…An emerging body of evidence suggests that DNA methylation may be used as novel and specific biomarkers in gastric cancer [ 65 , 66 ] through the measurement of circulating DNA in serum (cell-free DNA (cfDNA)). In this scenario, we have discovered that RPRM displays differences in methylation levels between nontumor adjacent mucosa (NTAM) and cancer tissue samples ( Figure 3(a) ) [ 63 ]. Methylation status of this gene may be assessed in plasma samples ( Figure 3(b) ) [ 67 , 68 ], offering the opportunity for noninvasive detection of gastric cancer.…”