OR02-2 CX-4945 as a Potential Drug for Adrenocortical Carcinoma That Induces Multiple Exon-Skipping and Circular RNA of NR5A1

Orphan nuclear receptor NR5A1 (also called adrenal 4-binding protein/steroidogenic factor-1) is the major regulator of differentiation and function of the adrenal cortex. Adrenocortical carcinoma is rare and deadly with few therapeutic options and is known to have the tendency of increased NR5A1expression along with increased steroidogenesis. Most splicing regulatory compounds are known to have an anti-cancer effect. Thus, we tested these splicing regulatory compounds on the H295R adrenocortical carcinoma cell line and found that CX-4945 had a strong inhibitory effect on growth (IC(50)=6.4 µM, 72h); four times stronger than the conventional drug, mitotane. In H295R cells treated with CX-4945 for 24 hours, enlarged lysosome and autophagy-related apoptosis were observed. NR5A1was suppressed in mRNA and protein level in these cells, and found CX-4945 strongly induced multiple exon-skipping of the gene. We also found a circular RNA comprised of exon 2 to 4 of NR5A1. CX-4945 suppressed cortisol and aldosterone production and expression of steroid metabolic genes. CX-4945 was originally developed as a casein kinase 2 (CK2) inhibitor. However, CK2 subunit alpha suppression by siRNA for CSNK2 did not affect cell proliferation and expression of NR5A1, suggesting that CX-4945 effect on H295R cells is not through CK2 inhibition. These results suggest CX-4945 attenuates NR5A1-mediated proliferation and steroidogenesis via splicing modification. During analysis of circular RNAs, we found peculiar circular RNAs with endoplasmic reticulum (ER) stress response-related IRE1a-binding sites at the joined ends expressed in normal adrenal cortex, not to be expressed but induced by transiently expressing IRE1a in H295R cells. Taken together, CX-4945 has a strong potential in treating adrenocortical carcinoma since it does not show adrenal failure in its on-going clinical trials of other cancers.