Abstract 1822: RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer
Authors: Ghalali Aram , Stopsack Konrad H. , Rice James M. , Wang Liangzhe , Wu Shulin , Wu Chin Lee , Zetter Bruce , Rogers Michael S. 
Published: September 2020

We report here nuclear localization of AZIN1, a protein commonly modified by RNA editing in cancer cells, is associated with significantly increased risk of death in prostate cancer. We further find that this nuclear localization is caused by RNA editing of a single base in the AZIN1 mRNA, which in turn leads to a single Ser to Gly substitution in the AZIN1 protein. This change alone is also sufficient to increase the aggressiveness of prostate cancer cells. Our results reveal that, unexpectedly, this editing event changes the binding repertoire of AZIN1, rather than changing its affinity for known targets.

Using droplet display PCR, we evaluated the presence of edited AZIN1 (edAZIN1) in aggressive prostate cancer using tissues with Gleason &gt; 7, and found that 94% of these samples expressed edAZIN1.

We further measured the expression and localization of AZIN1 in 202 prostate cancer specimens, along with 26 adjacent benign samples and found a negative association between nuclear localization and progression-free survival. Analysis of data from primary prostate cancer patients available via the Cancer Genome Atlas (n = 291 with edAZIN1 calls), the median proportion of edAZIN1 was 6.1% (interquartile range, 4.4 to 8.9). In multivariable models, edAZIN1 was higher with increasing expression of ADAR, a cancer-assoicated RNA editing enzyme, (by 2.8% points per interquartile range increase in ADAR expression; 95% CI, 2.3 to 3.4) and in tumors with higher genomic instability (by 0.6% points per interquartile range increase in copy number alteration burden; 95% CI, 0.3 to 0.9). Downstream, edAZIN1 was associated with higher Gleason grade, with a 2.8%-point difference in edAZIN1 between Gleason 3+3 and Gleason 9-10 tumors (95% CI, 1.2 to 4.5). Together, these results suggest that edAZIN1 is commonly expressed in prostate cancer cells and is associated with increased cellular ADAR expression, nuclear localization, and with increased cancer aggressiveness.

To determine if editing causes increased cellular aggressiveness, we transfected prostate cancer cell lines (PC3, DU145) with constructs coding for wild-type, pseudoedited, and uneditable mRNAs for AZIN1. We found that only constructs capable of coding for edited AZIN1 increased cancer cell aggressiveness. Constructs that were unable to undergo editing, showed no such increase.

The mechanism underlying the increased aggressiveness of cells expressing AZIN1 has been proposed to be caused by higher affinity binding of edAZIN1 to its known substrate, antizyme, relative to wild-type AZIN1, but this has not been shown directly. We therefore measured the affinity of edited and wild-type protein for antizyme, using a novel FRET sensor. Contrary to expectation, we found that editing decreased edAZIN affinity for antizyme, notwithstanding increased complex formation in vivo. We then used tandem affinity purification proteomics to identify selective AZIN1 and edAZIN1 interacting proteins. We identified an edAZIN1-specific complex containing several proteins that may be the driving force behind the nuclear shuttling of edAZIN. Tools developed in this study are now being used to explore the feasibility of developing small molecule drugs that interfere with AZIN1 binding to its preferred substrates and subsequently inhibit with cell growth pathways.

Citation Format: Aram Ghalali, Konrad H. Stopsack, James M. Rice, Liangzhe Wang, Shulin Wu, Chin Lee Wu, Bruce Zetter, Michael S. Rogers. RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1822.