Abstract B19: Modulation of MAPK and PI3K-AKT signaling pathways and poor prognosis are associated with loss of RKIP

Abstract

Abstract RKIP was described as a metastasis suppressor gene, and it loss was associated with several aggressiveness features, such as with higher stage and/or recurrence, presence of vascular invasion and metastasis, and poor overall survival of cancer patients. RKIP is a member of the phosphatidyl-ethanolamine-binding protein (PEBP) family that negatively regulates not only the MAPK signalling, but also the NF-κ β, GPRC-2, and GSK3β pathway. Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumor on the gastrointestinal tract characterized by hotspot mutations in KIT and PDGFRA genes, which are predictive of imatinib-based therapy response. In GIST, it is reported that 9% of tumors have loss of expression in RKIP. Our main goal is to investigate the RKIP protein expression in a series of 72 bona-fide GIST and to correlate with patients' clinicopathologic characteristics. Moreover, we intended to assess in vitro role of RKIP gene depletion. We performed immunohistochemistry in 72 primary GIST previously characterized for CD117, S100, Desmin, and CD34 as well as molecularly for KIT (exons 9, 11, 13, 17), PDGFRA (exons 12, 14, 18), and BRAF (V600E). In a second step, we established a RKIP knockout GIST cell line (GIST-T1) by transfection of a pool of 3 different optimized gRNA constructs, targeting RKIP, cloned into the RKIP CRISPR/Cas9 system. The RKIP knockout was verified by Western blotting. The expression profile of RKIP KO cells was assessed using the nanostring Cancer Pathways platform, which contains a set of 800 cancer-related genes. We found that RKIP is absent or expressed at low levels in 16.7% (12/72) of GIST. RKIP reduction was significantly associated with tumor in small intestine, with tumor size increased, and patient death. The overall five-year survival to RKIP-negative cases was 41.7% versus 73.9% in RKIP-positive patients, yet not significant (p=0.07). The in vitro assays showed that RKIP protein levels were depleted in the RKIP KO cells, comparing with the nontransfected cells and with the negative control. The nanostring expression analysis showed 34 genes downregulated and 9 genes upregulated in RKIP-depleted cells. The downregulated genes are especially involved in PI3K-AKT signaling pathway and the upregulated genes in MAPK signaling pathway. In conclusion, we confirm that the lack of RKIP expression occurs in a subset of GISTs and is associated with poor prognosis. We also showed that in GIST loss of RKIP changes important cancer signaling pathways. Citation Format: Nathália Cristina Campanella, Matias Melendez, Lucas Abrahão-Machado, Cristovam Scapulatempo-Neto, Denise Peixoto Guimarães, Rui Reis. Modulation of MAPK and PI3K-AKT signaling pathways and poor prognosis are associated with loss of RKIP [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B19.