Role of an E3 ubiquitin ligase, SIAH1 in the adrenocortical physiology and physiopathology – AdSIAH

The adrenal cortex synthetizes steroids that are vital and play a major role in many physiological processes. Therefore, deregulation of steroid secretion causes high morbidities and can be lethal. Adrenocortical adenoma (ACA) are frequent affecting 3 to 7% of the population after 50 years old whereas adrenocortical carcinoma (ACC) are rare (0.5 to 3 new cases/year/million) but aggressive with a 5 years survival inferior to 40%. This poor prognosis may be explained both by the difficulty to predict the malignancy of the tumor and by the resistance to current treatment. Important efforts have then, been made to better understand their etiology. Although “OMICS” analysis performed in the different cohort of adrenocortical tumors (ACT) has enabled the identification of the genetic causes of roughly half of ACT, to date, the origin of the other half remains unknown.
Nevertheless, based on the recent identification of alterations affecting E3 ubiquitin ligases (ZNRF3, Cul3) in ACT, ubiquitination has emerged as an important regulator of adrenocortical homeostasis and tumorigenesis. Here, we will focus more specifically on the role played by a RING finger E3 ubiquitin ligase, SIAH1. Interestingly, we previously identified 2 germline SIAH1 missense variants in the cohort of 81 patients with hormonal hypersecretion. Whereas p.I251L mutation impairs SIAH1 ubiquitin ligase activity in vitro, p.R29I mutation was associated with a somatic loss of heterozygosity. Altogether these results suggest that SIAH1 impairment may induce adrenocortical tumorigenesis and hormonal excess. Accordingly, the whole-body knockout mice for one of the two murine isoforms of SIAH1, Siah1a (Siah1a-/-) develop a primary hyperaldosteronism (PA) but its characterization was limited by their premature death at 1 month of age.
The aim of this project is then, to better understand the molecular mechanism involving SIAH1/Siah1a in the regulation of adrenocortical homeostasis and how its invalidation could participate to adrenocortical tumorigenesis. For this purpose, we will combine in silico, in vitro and in vivo approaches using modified Human adrenocortical cancer cell line, H295R invalidated for SIAH1 and adrenocortical specific Siah1a knockout mice either in all adrenocortical cells or in glomerulosa cells only. We will first identify the importance of SIAH1/Siah1a in the adrenal cortex throughout the characterization of the hormonal, histological and molecular phenotype of our Siah1a knockout models. Furthermore, we will investigate the involvement of SIAH1/Siah1a in the differentiation of the adrenal cortex and study more specifically its potential regulator role of essential signaling pathways such as Wnt/b-catenin pathway. Indeed, as b-catenin known to regulate zona glomerulosa differentiation is also a target of SIAH1 for degradation in others tissues, we would like to determine if the PA previously observed in Siah1a-/- mice might be due to the absence b-catenin inhibition by of Siah1a. Moreover, to get a broader view of SIAH1 role, we will also identify its targets and signaling pathways in adrenocortical cells without a priori using RNAseq and proteomic analysis. These experiments performed both in mouse adrenals and human adrenocortical cells will permit not only to acquire more knowledges on SIAH1 role in the adrenal cortex but might also highlight the importance of actors of the adrenocortical function that are still unknown. One other important aspect of this project is to determine if the loss of SIAH1/Siah1a could at term, leads to adrenocortical tumorigenesis, we will then, analyze if our mouse models develop adrenocortical tumors at 12 and 18 months. Moreover, we will look for potential SIAH1 alterations either, genomic, transcriptomic or in methylation in the data available from ACT to determine if it could participate to the tumorigenesis and if there is association with the group of tumors activated for Wnt/b-catenin pathway.