Endocrine Abstracts

Introduction: Adrenocortical Carcinoma (ACC) is a rare and aggressive tumor with poor prognosis. Up to now, CTNNB1 (βcatenin) and TP53 mutations were the most frequent alterations identified in ACC. By a combination of genomic approaches, we have recently analyzed a cohort of 122 ACC (European Network for the Study of Adrenal Tumors, ENSAT). This work confirmed recurrent alterations in CTNNB1 and TP53 and revealed new genes not previously reported in ACC. Strikingly, ZNRF3 (zinc and ring finger 3) was the most frequently altered gene (21%). In a majority of cases, homozygous deletions of ZNRF3 were observed. ZNRF3 had never been associated with other tumor types. This original finding suggests that ZNRF3 could be a novel tumor suppressor gene involved in ACC. Our objective is to demonstrate that ZNRF3 acts as a tumor suppressor gene in the adrenocortical cell line (H295R) and to identify molecular pathways downstream of ZNRF3.

Methods: To investigate the potential function of ZNRF3 on apoptosis and cell proliferation, we performed measurement of Caspase3 activity and MTT assay in H295R cells, transfected with an interfering RNA targeting ZNRF3 or a vector encoding ZNRF3. Then, to screen and identify new ZNRF3 binding partners we performed co-immunoprecipitation (IP)-coupled mass spectrometry (MS) experiments from H295R cells transfected with ZNRF3.

Results: In H295R cells, ZNRF3 overexpression decreases cell proliferation and increases apoptosis, while ZNRF3 silencing confers protection against apoptosis induced by staurosporin. Different proteins partners of ZNRF3 were identified by coIP-MS, involved in various cellular processes. The interaction of ZNRF3 with these proteins is under investigation. Further experiments are required to identify the corresponding signaling pathways.

Conclusion: Our data show that ZNRF3 plays a role of tumor suppressor gene in adrenocortical cells. The results of this research will help to progress toward our understanding of adrenocortical tumorigenesis involving ZNRF3 alterations.