Aberrant activation of Wnt/β-Catenin signaling pathway drives the expression of poorprognosis-associated microRNAs in adrenocortical cancer with a major impacton miR-139-5p and its host gene PDE2A

Abstract

Adrenocortical carcinoma (ACC) is a rare malignancy with dismal prognosis. Deregulated microRNA (miRNA) expression has been implicated in ACC aggressiveness. Nevertheless, the mechanisms underlying such deregulations remain unknown. Aberrant Wnt/{beta}-Catenin signaling has been reported in about 40% of ACC and is associated with poor outcome. Here, we investigated the link between constitutive activation of Wnt/{beta}-Catenin pathway and miRNA expression alterations in ACC. Inducible shRNA-mediated gene silencing of {beta}-Catenin ({beta}-Cat) was performed in ACC cells expressing constitutively active {beta}-Catenin. The miRnome of ACC cells was analyzed using RNA-Sequencing. Selected miRNAs and mRNAs were validated using quantitative PCR and functional experiments with an emphasis on miR-139-5p, its host gene phosphodiesterase 2A (PDE2A) and its target gene N-Myc Downstream-Regulated Gene 4 (NDRG4). Prognostic values of Wnt/{beta}-Catenin pathway components or mutational status and their correlations with miRNA/mRNA expressions were determined in COMETE-ENSAT and TCGA cohorts. We carried out the first miRnome analysis in {beta}-Catenin-deficient ({beta}-Cat-) ACC cells. Twelve upregulated miRNAs and 42 downregulated miRNAs among which miR-139-5p and miRNAs of the 14q32 locus were identified in {beta}-Cat- cells. Downregulation of selected poor prognosis-associated miRNAs was confirmed using RT-qPCR. Remarkably, the expression of the intronic miR-139-5p was decreased by 90% in {beta}-Cat- cells with a concomitant repression of its host gene PDE2A and upregulation of its target gene NDRG4. In ACC patients, miR-139-5p levels were highly correlated with the levels of PDE2A and anti-correlated with those of NDRG4. MiR-139-5p and PDE2A expressions were higher in patients with mutations in components of Wnt/{beta}-Catenin signaling pathway or high expression of LEF1, with LEF1 proving a better predictor of prognosis than Wnt/{beta}-Catenin signaling pathway mutational status. Our findings indicate that in addition to inducing protein-coding genes in ACC, constitutively active Wnt/{beta}-Catenin signaling upregulates the expression of a subset of miRNAs involved in tumour aggressiveness and poor clinical outcome.