Abstract 2437: Clinical, biological and translational significance in gastric cancer of reprimo-like gene, an uncharacterized member of the reprimo gene family
Authors: Alarcon Maria Alejandra , Olivares Wilda , Rodriguez Andres , Maturana Maria Jose , Bustos Rocío , Polakovicova Iva , Cordova-Delgado Miguel , Muñoz-Medel Matías , Garrido Marcelo , Owen Gareth I. , De Mayo Tomas , Carrasco-Avino Gonzalo , Amigo Julio , Riquelme Arnoldo , Gonzalez Robinson , Barrientos Carlos , Aravena Edmundo , Villarroel-Espíndola Franz , Corvalan Alejandro H. 
Published: September 2020

Background: Gastric cancer (GC) is one of the leading causes of cancer death. Elucidating the molecular bases of GC might contribute to develop opportune diagnostic strategies. Reprimo-like (RPRML) is a poorly characterized member of the Reprimo gene family. The founding member of this family, Reprimo, is reported to be a putative tumor suppresser gene and candidate biomarker for GC diagnosis. We have recently reported the downregulation of RPRML expression in GC tissues compared to paired normal adjacent tissues, and that in vitro expression is regulated by DNA methylation. We hypothesize that RPRML promoter methylation may act as a non-invasive biomarker for GC.

Methods: RPRML expression was evaluated by immunohistochemistry (IHC) in 91 GC cases from The Gastric Cancer Task Force (NCT03158571). Two pathologists scored staining by multiplying the proportion and the intensity of stained cells (range 0-3). Cox proportional hazard ratio was applied to determine an association between RPRML IHC score and global survival, after adjusting by stage. In addition, we investigated the effect of RPRML overexpression in GC cell behavior in vitro through the stable transfection and sorting of RPRML coupled to GFP or GFP alone in the AGS cell line. Functional assays included Ki67 immunofluorescence, colony formation, soft agar, transwell migration and wound healing assays. All experiments were performed in biological triplicate and Kruskal-Wallis statistical analysis applied. The Methylight assay was used to quantify methylated RPRML DNA copies in plasma samples from 25 GC cases (tumor bank repository BTHCUCH) and 25 healthy donors under an endoscopic surveillance program (PREVECAN). ROC curve analysis was performed to determine the cut-off value for the highest sensitivity and specificity.

Results: Among 91 GC cases, RPRML expression showed a median IHC score of 0.024 (range 0-2.45). By using the median as a cut-off point, low RPRML IHC score was associated with poor overall survival of stage III and IV GC patients (HR=2.13, 95%CI:1.1-4.1, p=0.02). In vitro overexpression of RPRML inhibited cell proliferation (p=0.04) and resulted in reduced clonogenic capacity (p=0.001) and anchorage independent growth (p≤0.0001). Furthermore, RPRML overexpression retarded migration and wound healing in AGS cells. Methylation of RPRML DNA in plasma samples discriminated between GC patients and controls with an AUC of 0.66. Utilizing a cut-off value of 0.15 copies/ul, sensitivity was 56% (95%CI: 34.9 - 75.6) and specificity 72% (95%CI: 50.6 - 87.9).

Conclusion: RPRML downregulation associated with poor survival of advanced stage GC patients. In accordance, ectopic overexpression of RPRML in vitro inhibited biological characteristics associated with tumor progression, suggesting that RPRML may have a tumor suppressor role in GC. Future analysis will determine if the methylation of RPRML is a candidate marker for the non-invasive detection of GC.

Grants: CONICYT-FONDAP 15130011 and Fondecyt 1191928

Citation Format: Maria Alejandra Alarcon, Wilda Olivares, Andres Rodriguez, Maria Jose Maturana, Rocío Bustos, Iva Polakovicova, Miguel Cordova-Delgado, Matías Muñoz-Medel, Marcelo Garrido, Gareth I. Owen, Tomas De Mayo, Gonzalo Carrasco-Avino, Julio Amigo, Arnoldo Riquelme, Robinson Gonzalez, Carlos Barrientos, Edmundo Aravena, Franz Villarroel-Espíndola, Alejandro H. Corvalan. Clinical, biological and translational significance in gastric cancer of reprimo-like gene, an uncharacterized member of the reprimo gene family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2437.