Wilda Olivares

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.

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.

Molecular Pathology of Gastritis

Gastritis is an inflammation of the mucosa of the stomach, and has many etiologies. Gastritis can be classified as being acute or chronic. For the purpose of this chapter, we will focus only on chronic gastritis due to its relevance to gastric cancer. Among the causes of chronic gastritis are chronic bile reflux, stress, certain autoimmune disorders and bacterial infection, primarily Helicobacter pylori. Since 1870, both human and veterinary pathologists have described the presence of tiny curved bacteria within gastric mucosa, but the organisms were dismissed as irrelevant contaminants (1, 2). In 1947, when gastroscopy was first being used, Rudolf Schindler deemed gastritis as “one of the most debated diseases of the human body” and predicted that its significance would be discussed “for some time to come” (3). Schindler himself claimed that the “bacteriological etiology of chronic gastritis has not been convincingly proved in a single case” (3). In 1984, Warren and Marshall proposed that chronic “idiopathic” gastritis had a bacterial cause (i.e., H. pylori) (4). Their hypothesis was met with great skepticism. However, within a few years, the association between H. pylori gastritis, peptic ulcer, and gastric cancer came to be acknowledged and ultimately accepted (4). Subsequently, accurate morphological data were gathered by pathologic examination of autopsy material (5) and, later, of endoscopic biopsy specimens. As a result, distinct types and patterns of gastritis were recognized, which led to the conception, presentation, dismissal, and replacement of many different classification systems.

Combined use of methylated reprimo cell-free DNA and pepsinogens for noninvasive detection of early gastric cancer.

27 Background: Gastric cancer (GC) has been described as a multistep cascade of precursor lesions such as non-atrophic chronic gastritis (NACG), multiphocal atrophic gastritis (MAG), intestinal metaplasia (IM), low grade dysplasia (LGD) and high grade dysplasia (HGD) leading to early stages of GC (EGC). Currently, no non-invasive biomarkers for this progression are clinically available. We have previously identified a potential biomarker based on methylated Reprimo (RPRM) cell-free DNA (cfDNA) (Clin Cancer Res 2008;14:6264-9). In a cross-sectional study of 1,076 patients, we showed a sensitivity of 70.8% (95% CI: 60.3 to 81.3) and specificity of 74.3% (95% CI: 71.5 to 77) for methylated RPRM cfDNA, to distinguish NACG+MAG+IM+LGD vs HGD+EGC+AGC (Digestive Disease Week 2014 #108). However, the crude detection rate of EGC was only 46.6%. Here, we aim to explore the role of the combined use of methylated RPRM cfDNA and well stablished atrophy biomarkers such as pepsinogens, for non-invasive detection of EGC. ...

Su1991 Reactivation of Reprimo by an Artificial Transcription Factor in Gastric Cancer Cell Lines

Introduction: Gastric cancer (GC) is the second cause of cancer-related death worldwide and the leading one in Latin America. It has been proposed that the silencing of Reprimo (RPRM), by epigenetic mechanisms, is a putative biomarker of GC. RPRM is a p53 dependent G2 arrest mediator and is considered to be tumor suppressor gene involved in the pathogenesis of numerous cancers such as gastric, colon, pituitary, among others. We propose that the reactivation of RPRM expression, by means of an activator Artificial Transcription Factor (ATF), modifies the proliferation rate of GC cell lines in vitro. Methods: An activator ATF for RPRM was designed using the method proposed by zincfingertools.org. Thus, an ATF with a unique domain of six zinc-finger capable of binding specifically to 18 consecutive base pairs, located in the promoter region of RPRM, was engineered. This ATF was then inserted into a modified pcDNA3.1+ vector coupled with a potent transcription activator domain (VP64). A luciferase reporter assay was performed using a pGL3 vector (Promega), linked to a 634 base pairs region of the promoter region of RPRM, in order to evaluate the efficiency and specificity of the synthetized ATF. Finally, the ATF was transiently transfected with lipofectamine2000 (Invitrogen). Subsequently, RPRM mRNA expression was evaluated by a RT-qPCR and proliferation rate was evaluated by a MTS assay. The experiments were conducted on AGS and MKN28 GC cell lines. Results: The ATF designed was able to significantly increase the luciferase activity vs controls, both in MKN28 and AGS cell lines. Accordingly, RPRM mRNA was upregulated in the presence of the ATF. These findings were associated with a lower proliferation rate in both cell lines. Since the AGS cell line is highly methylated in the promoter region of RPRM, it might explain a significantly lower response to the presence of ATF, in comparison with that of MKN28. Discussion: Our work suggests that a RPRM specific ATF could be used as a novel tool to reactivate this epigenetically silenced tumor suppressor gene. Moreover, the upregulation of RPRM modifies one of the hallmarks of cancer pathways by reducing the proliferation rate of cancerous cell lines in vitro. Grant Support: Fondecyt #1111014 and Fondef #D09I1137 from the Government of Chile to AHC.

Abstract 1188: Reprimo, a p53-dependent G2 arrest mediator, reduces tumorigenic capacity in human gastric cancer cell lines

Introduction: Gastric cancer (GC) is one of the leading cause of cancer-related deaths worldwide. The progression of GC is related to genetic as well as epigenetic changes, such as DNA methylation, that involves the downregulation of tumor suppressor genes. Recently we have identified Reprimo (RPRM), a p53-dependent G2 arrest mediator candidate as a putative biomarker for early diagnosis of GC (Clin Cancer Res 2008;14:6264-9). However, the role of RPRM in GC is unknown. Methods: The expression and sequence of the promoter and the coding region of RPRM were performed in five GC cell lines (AGS, NCI-N87, KATOIII, MKN28, MKN45). Two expression negative RPRM cell lines were stable transfected with a pCMV6 vector containing the full-length of the coding region of RPRM (pCMV6-RPRM). Cell growth and proliferation were analyzed by MTS, colony formation and soft agar assays. In addition, we analyzed the ability of migration through wound/healing assay. Results: Only two cell lines were negative for RPRM expression (AGS & MKN45). Bisulfite sequence analysis of the promoter region showed a dense CG-rich CpG island suggesting a role for methylation in regulation of gene expression. No mutations in the coding region were found. A significant decreased of cellular proliferation and anchorage independent growth were observed after the transfection with pCMV6-RPRM. The migration ability was also decreased in both cell lines. Conclusion: Our results suggest that RPRM reduces tumorigenic capacity in GC cell lines. Taken together we suggest that downregulation of RPRM is not only a putative biomarker for early diagnosis but also might play a role in the pathogenesis of GC. Grants-in-Aid for Scientific T 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1188. doi:1538-7445.AM2012-1188