Gastric Cancer (GC) is a major health problem, with 968,350 new cases and 659,853 deaths per year worldwide. GC exhibits a high tendency for recurrence and early-stage metastasis development. Consequently, the prognosis for GC patients is poor, with an average 5-year survival rate of 36%, dropping to only 10% for patients presenting distant metastases. However, the molecular determinants governing GC progression and metastasis are currently unknown.

We took advantage of publicly available GC datasets from TCGA for the identification of matched genes among the 100 genes most associated with overall survival (OS) and disease-free survival (DFS). We confirmed the association of the identified genes with poor prognosis in the ACRG GC cohort and in over 2000 GC cases obtained from the integration of several cohorts using our own analysis pipeline. The Kaplan-Meier method and multivariate Cox regression analyses were used to determine prognostic significance, and linear modelling and correlation analyses were used to assess associations with clinicopathological parameters and biological hallmarks. Moreover, we performed in vitro and in vivo functional studies and transcriptomics (RNA sequencing) in GC cells to assess the role and mechanism of the most promising candidate gene among the initially identified genes.

ANKRD6, ITIH3, SORCS3, NPY1R, and CCDC178 were the matched genes among the 100 genes most associated with overall survival (OS) and disease-free survival (DFS) in the TCGA GC cohort. High expression of these genes, individually and as a signature, was associated with reduced survival, recurrence, and node invasion in different GC cohorts. Moreover, the expression of ANKRD6 and ITIH3 was significantly higher in metastasis, and their levels correlated with markers of Epithelial to Mesenchymal Transition (EMT) and stemness in GC samples. In line with this, RNAseq analysis identified EMT among the pathways significantly altered in ANKRD6-silenced GC cells. Accordingly, ANKRD6 silencing in GC metastatic cells showed abrogation of tumorigenic and metastatic traits in vitro and in vivo.

Our investigation uncovered a novel gene signature implicated in GC malignancy and prognosis and identified, for the first time, the pro-metastatic activity of ANKRD6 in the disease.