Ignacio A. Wichmann

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

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death, whose patterns vary among geographical regions and ethnicities. It is a multifactorial disease, and its development depends on infection by Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV), host genetic factors, and environmental factors. The heterogeneity of the disease has begun to be unraveled by a comprehensive mutational evaluation of primary tumors. The low-abundance of mutations suggests that other mechanisms participate in the evolution of the disease, such as those found through analyses of noncoding genomics. Noncoding genomics includes single nucleotide polymorphisms (SNPs), regulation of gene expression through DNA methylation of promoter sites, miRNAs, other noncoding RNAs in regulatory regions, and other topics. These processes and molecules ultimately control gene expression. Potential biomarkers are appearing from analyses of noncoding genomics. This review focuses on noncoding genomics and potential biomarkers in the context of gastric cancer and the gastric precancerous cascade.

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

Genes related to human diseases should be natural targets for evolutionary studies, since they could provide clues regarding the genetic bases of pathologies and potential treatments. Here we studied the evolution of the reprimo gene family, a group of tumor-suppressor genes that are implicated in p53-mediated cell cycle arrest. These genes, especially the reprimo duplicate located on human chromosome 2, have been associated with epigenetic modifications correlated with transcriptional silencing and cancer progression. We demonstrate the presence of a third reprimo lineage that, together with the reprimo and reprimo-like genes, appears to have been differentially retained during the evolutionary history of vertebrates. We present evidence that these reprimo lineages originated early in vertebrate evolution and expanded as a result of the two rounds of whole genome duplications that occurred in the last common ancestor of vertebrates. The reprimo gene has been lost in birds, and the third reprimo gene lineage has been retained in only a few distantly related species, such as coelacanth and gar. Expression analyses revealed that the reprimo paralogs are mainly expressed in the nervous system. Different vertebrate lineages have retained different reprimo paralogs, and even in species that have retained multiple copies, only one of them is heavily expressed.

Reprimo tissue-specific expression pattern is conserved between zebrafish and human

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

Inverse expression of survivin and reprimo correlates with poor patient prognosis in gastric cancer

BACKGROUND The objective of the study was to determine the relationship between Survivin and Reprimo transcript/protein expression levels, and gastric cancer outcome. METHODS In silico correlations between an agnostic set of twelve p53-dependent apoptosis and cell-cycle genes were explored in the gastric adenocarcinoma TCGA database, using cBioPortal. Findings were validated by regression analysis of RNAseq data. Separate regression analyses were performed to assess the impact of p53 status on Survivin and Reprimo. Quantitative reverse-transcription PCR (RT-qPCR) and immunohistochemistry confirmed in silico findings on fresh-frozen and paraffin-embedded gastric cancer tissues, respectively. Wild-type (AGS, SNU-1) and mutated p53 (NCI-N87) cell lines transfected with pEGFP-Survivin or pCMV6-Reprimo were evaluated by RT-qPCR and Western blotting. Kaplan-Meier method and Long-Rank test were used to assess differences in patient outcome. RESULTS cBioPortal analysis revealed an inverse correlation between Survivin and Reprimo expression (Pearson’s r= −0.3, Spearman’s ρ= −0.55). RNAseq analyses confirmed these findings (Spearman’s ρ= −0.37, p<4.2e-09) and revealed p53 dependence in linear regression models (p<0.05). mRNA and protein levels validated these observations in clinical samples (p<0.001). In vitro analysis in cell lines demonstrated that increasing Survivin reduced Reprimo, while increasing Reprimo reduced Survivin expression, but only did so in p53 wild-type gastric cells (p<0.05). Survivin-positive but Reprimo-negative patients displayed shorter overall survival rates (p=0.047, Long Rank Test) (HR=0.32; 95%IC: 0.11-0.97; p=0.044). CONCLUSIONS TCGA RNAseq data analysis, evaluation of clinical samples and studies in cell lines identified an inverse relationship between Survivin and Reprimo. Elevated Survivin and reduced Reprimo protein expression correlated with poor patient prognosis in gastric cancer.

The Reprimo Gene Family: A Novel Gene Lineage in Gastric Cancer with Tumor Suppressive Properties

The reprimo (RPRM) gene family is a group of single exon genes present exclusively within the vertebrate lineage. Two out of three members of this family are present in humans: RPRM and RPRM-Like (RPRML). RPRM induces cell cycle arrest at G2/M in response to p53 expression. Loss-of-expression of RPRM is related to increased cell proliferation and growth in gastric cancer. This evidence suggests that RPRM has tumor suppressive properties. However, the molecular mechanisms and signaling partners by which RPRM exerts its functions remain unknown. Moreover, scarce studies have attempted to characterize RPRML, and its functionality is unclear. Herein, we highlight the role of the RPRM gene family in gastric carcinogenesis, as well as its potential applications in clinical settings. In addition, we summarize the current knowledge on the phylogeny and expression patterns of this family of genes in embryonic zebrafish and adult humans. Strikingly, in both species, RPRM is expressed primarily in the digestive tract, blood vessels and central nervous system, supporting the use of zebrafish for further functional characterization of RPRM. Finally, drawing on embryonic and adult expression patterns, we address the potential relevance of RPRM and RPRML in cancer. Active investigation or analytical research in the coming years should contribute to novel translational applications of this poorly understood gene family as potential biomarkers and development of novel cancer therapies.

Expression of RPRM/rprm in the Olfactory System of Embryonic Zebrafish (Danio rerio)

The Reprimo (RPRM) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (Danio rerio) embryogenesis, and primarily locates in the nervous system. Its most characterized member, RPRM, which duplicated to give rise rprma and rprmb in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three rprm genes (rprma, rprmb, and rprml) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish, rprma mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE), rprmb is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas rprml is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of RPRM has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of RPRM gene products during zebrafish and mouse development as a first step to approach the physiological role of the RPRM gene family.

Role of microRNAs and Exosomes in Helicobacter pylori and Epstein-Barr Virus Associated Gastric Cancers

Emerging evidence suggests that chronic inflammation caused by pathogen infection is connected to the development of various types of cancer. It is estimated that up to 20% of all cancer deaths is linked to infections and inflammation. In gastric cancer, such triggers can be infection of the gastric epithelium by either Helicobacter pylori (H. pylori), a bacterium present in half of the world population; or by Epstein-Barr virus (EBV), a double-stranded DNA virus which has recently been associated with gastric cancer. Both agents can establish lifelong inflammation by evolving to escape immune surveillance and, under certain conditions, contribute to the development of gastric cancer. Non-coding RNAs, mainly microRNAs (miRNAs), influence the host innate and adaptive immune responses, though long non-coding RNAs and viral miRNAs also alter these processes. Reports suggest that chronic infection results in altered expression of host miRNAs. In turn, dysregulated miRNAs modulate the host inflammatory immune response, favoring bacterial survival and persistence within the gastric mucosa. Given the established roles of miRNAs in tumorigenesis and innate immunity, they may serve as an important link between H. pylori- and EBV-associated inflammation and carcinogenesis. Example of this is up-regulation of miR-155 in H. pylori and EBV infection. The tumor environment contains a variety of cells that need to communicate with each other. Extracellular vesicles, especially exosomes, allow these cells to deliver certain type of information to other cells promoting cancer growth and metastasis. Exosomes have been shown to deliver not only various types of genetic information, mainly miRNAs, but also cytotoxin-associated gene A (CagA), a major H. pylori virulence factor. In addition, a growing body of evidence demonstrates that exosomes contain genetic material of viruses and viral miRNAs and proteins such as EBV latent membrane protein 1 (LMP1) which are delivered into recipient cells. In this review, we focus on the dysregulated H. pylori- and EBV-associated miRNAs while trying to unveil possible causal mechanisms. Moreover, we discuss the role of exosomes as vehicles for miRNA delivery in H. pylori- and EBV-related carcinogenesis.

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. ...