Protein Inhibitor of Activated STAT3 (PIAS3) was originally identified as an endogenous suppressor of STAT3 activity which can specifically interact with activated STAT3 and block its downstream oncogenic effects. Previous studies from our lab have shown that low PIAS3 protein expression in mesothelioma tumors was associated with poor patient survival. Furthermore, low PIAS3 expression in mesothelioma cell lines correlated with higher STAT3 activity. Over-expression of PIAS3 decreased STAT3 activity and cell growth. These findings suggest that low PIAS3 expression may play an important role in mesothelioma tumorigenesis, thus understanding the regulatory mechanism(s) of PIAS3 expression may provide new therapeutic strategies in mesothelioma treatment. Initially, we examined PIAS3 expression by western blotting and RT-qPCR among six mesothelioma cell lines (H28, 211H, H2052, H2452, HAY, YOU) and found that while PIAS3 mRNA levels were relatively constant, PIAS3 protein expression was highly variable. This finding indicated a potential post-transcriptional regulation of PIAS3 expression in mesothelioma. To investigate this further, we first examined PIAS3 protein stability in the presence of proteasome inhibitors and cycloheximide (CHX). We observed no increase in PIAS3 protein levels during proteasome inhibition using either MG132 (10 μM) or bortezemib (1 μM) for up to 8 h in 211H, H2052 and H2452 cells, whereas TP53 protein expression was increased. Blocking translational protein synthesis with 50 or 100 μg/ml CHX for up to 24 h also produced little change in PIAS3 protein expression in mesothelioma cells, indicating that PIAS3 is a very stable protein. We next explored the PIAS3 3’UTR as a potential site for translational regulation. We initially utilized a miR microarray and computational analysis, followed by a PIAS3 3’UTR luciferase reporter assay, to screen miRNAs that are both increased in cells with low PIAS3 and have seed sequences targeting the PIAS3 3’UTR and identified miR-18a as our first candidate. Taken together, our study suggests that variable expression of PIAS3 protein in mesothelioma is regulated by translational control and not by protein stability.

Citation Format: Tian He, Karen McColl, Nneha Sakre, Gary Wildey, Afshin Dowlati. Post-transcriptional regulation on PIAS3 expression in malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2017-4490