miRNA expression profiling of mouse colon cancer stem cells: a tumour-specific signature traceable along colorectal cancer progression with prognostic value in human colon cance

Background: The definition of cancer stem cells (CSC) still lacks conclusive experimental evidence. Colorectal cancer (CRC) EphB2 cells have been correlated to stem-like properties and tumor malignancy. Here, we investigated a panel of miRNA involved in the self-renewal and cell fate during cancer development using murine CRC EphB2 sorted cells and tissue of mouse/human CRC and public dataset (TCGA). Methods: FACS-isolated CD44+ EphB2high cells of the CRC AOM/DSS murine model were analyzed by gene expression and IHC analysis (Lgr5, Ascl2, krt20) to characterize the stem-like/differentiation phenotype. miRNAs expression profiling was performed using TaqMan Low Density Arrays (LifeTechnologies) both on sorted cells and whole tissue samples of AOM/DSS model containing dysplastic ACFs, microadenoma, adenoma, carcinoma. Single real-time PCR for selected miRNAs were performed on 60 FFPE human samples and further validated in The Cancer Genome Atlas (TCGA) stage I–IV CRC (n=130) to determine their potential prognostic role. Results: The analysis of miRNA expression pattern in the EphB2high sorted stem-like cells in comparison to miRNAs of CRC murine whole tissue showed a significant cancer stemness signature in the initiation/progression steps. EphB2 stemness-related miRNAs, which orchestrate cell cycle, apoptosis, tumorigenesis, progression and metastasis, were further analyzed in human samples at different phases (ACF, adenoma, adenocarcinoma) revealing a pattern similar to the murine model. Conclusions: These data provide a comprehensive miRNA signature implicated in the regulation of tumorigenesis, stemness, and cell fate determination that could be exploited for diagnosis and therapeutic design, and could be proposed as novel CRC prognostic biomarkers.