Functional characterization of Tribbles pseudokinases in gastric cancer

Introduction: Gastric cancer (GC) is the fifth most common cancer and the fourth leading cause of cancer-related deaths worldwide. It is often diagnosed at advanced stages and is mostly resistant to conventional chemotherapy. To help GC management, there is a need to develop new biomarkers: they are needed to improve diagnosis, as well as novel therapeutic targets that may lead to the development of effective therapies. TRIB2 belongs to the Tribbles family of serine/threonine pseudokinases, scaffold proteins involved in several cellular processes. In cancer, TRIB2 can act either as an oncogene or a tumor suppressor gene, depending on the cellular context. TRIB2 also promotes drug resistance in different cancer types. Since this protein has never been investigated in GC, our study aimed at addressing the significance of TRIB2 in this tumor. Material & Methods: We performed data mining of The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) dataset and of the Broad Institute Cancer Cell Line Encyclopedia (CCLE) GC dataset. Based on in silico results, we then investigated the impact of lentiviral-mediated TRIB2 overexpression (OE) in four GC cell lines (MKN45, KATO-III, MKN74 and NCI-N87) with chromosomal instability phenotype (CIN). The impact of TRIB2 OE was evaluated by different functional assays, including cell proliferation, motility, colony formation and cell cycle analysis. Moreover, we investigated the mitogen-activated protein kinase (MAPK) downstream pathway and the possible involvement of TRIB2 in drug response, following cell treatments with two agents commonly used in GC chemotherapy (5-fluorouracil and doxorubicin). Results: Data mining of the TCGA STAD dataset and of the CCLE dataset revealed a limited number of genetic and genomic alterations and a wide range of mRNA expression levels of TRIBs in GC tissues, and in GC cell lines, respectively. The analysis of TRIBs expression of STAD cases sorted by molecular phenotypes showed that a lower TRIB2 mRNA expression was statistically significantly associated with tumors characterized by chromosomal instability (CIN) compared to tumors characterized by microsatellite instability (MSI). In addition, T4 stage in CIN tumors was statistically significantly associated with low TRIB2 expression. In vitro, TRIB2 OE was able to reduce proliferation and colony formation ability in MKN45 and NCI-N87 cells. Furthermore, TRIB2 OE induced cell cycle arrest in G2/M phase in MKN45 cells and reduced migration ability in NCI-N87 compared to the control cells. On the other side, TRIB2 OE did not affect the MAPK pathway and no differences were detected between TRIB2-overexpressing cells and control cells treated both with 5-fluorouracil and doxorubicin.