Thesis presented November 10, 2022

Abstract:
Adrenocortical carcinoma (ACC) is a rare and aggressive cancer with a poor prognosis. Treatment options are limited and chemotherapies, targeted therapies and immunotherapies do not improve the overall survival of patients. A better understanding of the molecular mechanisms underlying the pathophysiology of ACC is therefore essential to develop therapies that are more effective. Two large integrated genomic studies of ACC have shown that the aberrant activation of the Wnt/ß-Catenin pathway was present in about 40% of ACC, due to somatic mutations and homozygous deletions of two key genes: CTNNB1 (ß-Catenin) and ZNRF3. In addition, they allowed the identification of microRNA signatures specific to ACC with good or poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs of about 20 nucleotides which play a major role is the post-transcriptional regulation of gene expression. Our team has identified two miRNAs overexpressed in aggressive ACC and associated with poor prognosis: miR-139-5p and miR-483-5p.
The objective of my thesis was to determine the impact of the aberrant activation of the Wnt/ß-Catenin signaling pathway on miRNA expression, in particular on miR-139-5p and miR-483-5p.
The ACC H295R cell line exhibits a constitutive activation of the Wnt/ß-Catenin pathway due to an activating mutation of ß-Catenin. We showed that silencing ß-Catenin expression in H295R cells changes the expression of 54 miRNAs, by decreasing the expression of 42 of them (77.8%). Among the miRNAs whose expression was impacted by the repression of ß-Catenin, we identified miR-139-5p, as well as several miRNAs from the chromosomal imprinted locus 14q32 which is known to be involved in carcinogenesis in other cancers. We subsequently showed that the decrease in miR-139-5p was correlated with that of its host gene, PDE2A, a recently identified target gene of ß-Catenin. Furthermore, the decrease in miR-139-5p was accompanied by an increase in NDRG4, one of the target genes of miR-139-5p identified by our team.
We then analysed publicly available transcriptome and miRnome data from the COMETE-ENSAT and TCGA patient cohorts. We found that patients with somatic mutations or molecular alterations in CTNNB1 or ZNRF3 genes had significantly higher miR-139-5p and PDE2A levels and significantly lower NDRG4 expression levels as compared to the other patients. We also showed that the expression level of miR-139-5p was highly correlated with that of its host gene PDE2A in patients. However, by analysing a subgroup of patients with high miR-139-5p levels without identified genomic alterations in CTNNB1 or ZNRF3, we found that the expression level of the transcription factor LEF1, a partner of ß-Catenin, was associated with miR-139-5p expression, independently of the mutational status of the Wnt/ß-Catenin pathway. Furthermore, patients with high levels of LEF1 had worse overall survival than those with low levels of LEF1. Interestingly, LEF1 expression level was a better predictor of overall survival than the presence of CTNNB1 or ZNRF3 molecular alterations.
In conclusion, my thesis work represents the first global analysis of Wnt/ß-Catenin signaling-mediated regulation of the miRnome in ACC and in cancer in general. The expression of miR-139-5p, a miRNA associated with poor prognosis in ACC, is tightly controlled by Wnt/ß-Catenin signaling, in correlation with its host gene PDE2A. Furthermore, we identified LEF1, a major transcription factor of the pathway, as a new prognostic marker for ACC patient survival.

Keywords:
adrenocortical carcinoma, Wnt/β-Cat signaling pathway, microRNAs, prognosis, miR-139-5p

On-line thesis. ​