Thesis presented March 25, 2022

Abstract:
Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine tumor, which is associated with poor prognosis. This cancer is refractory to conventional therapies such as chemotherapy or radiotherapy as well as to targeted therapies. In light of these therapeutic failures, understanding the molecular mechanisms of adrenocortical tumorigenesis will be pivotal for the development of alternative therapies. MicroRNAs (miRNAs) are small non-coding RNAs of around 20 nucleotides-long that repress gene expression at the post-transcriptional level. They play a major regulatory role of the entire genome. Alterations in the expression profile of miRNAs (overexpression of oncogenic miRNAs or repression of tumor suppressor miRNAs) have been identified in all stages of cancer development such as initiation, progression and metastasis. Based on these findings, inhibition of oncogenic miRNAs or restoration of tumor suppressor miRNAs were suggested as relevant therapeutic strategies for cancer. However, the major challenge of these approaches remains the specific and safe delivery of these treatments at the tumor site. The CEA LETI/DTBS laboratory has developed and patented biocompatible and biodegradable solid lipid nanoparticles or Lipidots® (LNP), the components of which are approved by the FDA. Remarkably, these nanoparticles exhibit a marked tropism for the adrenal cortex. Previous studies of our team on miRNA landscape in ACC revealed that two miRNAs, miR-139-5p and miR-483-5p, are overexpressed in aggressive tumors and associated with poor prognosis. The aim of this work was to evaluate miR-139-5p and miR-483-5p as therapeutic targets in ACC, using nanovectorization of their respective antimiRs. In the first part of this study, we show that simultaneous transfection of naked antimiR-139-5p and antimiR-483-5p in the human ACC cell line NCI H295R decreases the expression of several cancer-related proteins, reduces the phosphorylation level of p38 MAP kinase and AKT and negatively modulates the Wnt/beta-catenin signaling pathway. These results suggesting an involvement of miR-139-5p and miR-483-5p in oncogenic signaling pathways led us in a second part of this work to generate and to characterize antimiRs-LNPs complexes using a combination of antimiR-139-5p and antimiR-483-5p. We subsequently show a massive internalization of these complexes in NCI H295R cells, which is accompanied by an efficient and simultaneous inhibition of endogenous miR-483-5p and miR-139-5p expression. In addition, the antimiRs-LNP impede the migration and invasion of NCI H295R cells in culture. Finally, in a third part, we show that the systemic injection of antimiRs-LNP in immunodeficient scid/CB17 mice induces a preferential accumulation of these complexes in the adrenals and ovaries without apparent toxicity. We report in preliminary experiments using subcutaneous xenograft of NCI H295R cells that systemic administration of antimiRs-LNP inhibits tumor growth. This work describes the first use of Lipidots® to vectorize miRNAs for therapeutic purposes and suggests that targeting miRNAs deregulations is a relevant strategy for the treatment of ACC. Although the molecular mechanisms involved in miR-139-5p- and miR-483-5p-mediated adrenocortical tumorigenesis remain to be fully elucidated, these data open new perspectives for the development of innovative therapies for adrenocortical carcinoma.

Keywords:
microRNAs, Nanoparticles, Targeted therapy, Lipidots, Adrenocortical cancer