Abstract 2236: Mechanism of androgen-mediated down-regulation of PD-L1 in thyroid cancer
Authors: Dadafarin Sina , Carnazza Michelle A. , Moscatello Augustine , Tiwari Raj , Geliebter Jan 
Published: September 2020

Abstract
The incidence of thyroid cancer (ThCa) has more than doubled in the last 20 years and the associated healthcare costs of treating ThCa nears $20 billion in the United States alone. An important disparity exists in the incidence of ThCa, with women developing the disease approximately three times more often than men. A potential explanation of this sex disparity is differences in immune elimination of nascent tumor cells between men and women. Previously, our lab investigated the role androgen receptor (AR) activity may play in thyroid cancer development using an androgen-responsive cell culture model. We further studied the effect of androgen on immune checkpoint molecule expression and identified a significant reduction in PD-L1 transcription and surface expression. In this project, we aim to identify the molecular mechanisms linking AR activity to PD-L1 downregulation. First, we generated two new AR-responsive culture models from a papillary thyroid cancer as well as an anaplastic thyroid cancer cell line for all downstream experiments. These cell lines recapitulated the effect of 5α-dihydrotestosterone (DHT) on PD-L1 expression we previously observed, namely a 70% reduction in PD-L1 expression. We then identified AR binding sites from a collection of AR Chromatin Immunoprecipitation (ChIP)-Sequencing studies from the Cistrome Database. Interestingly, members of the interferon gamma (IFNγ) signaling pathway showed significant AR binding to the promoter or gene body, including negative regulators of IFNγ transcription such as PIAS1. Given the well-known role IFNγ signaling in the regulation of PD-L1, we then performed co-treatment with IFNγ and DHT. Surprisingly, DHT effectively eliminated IFNγ-mediated upregulation of PD-L1 in both cell culture models. We followed up the study by examining STAT1/STAT3 phosphorylation and IRF-1 expression using western blots to identify modulation of the JAK/STAT/IRF axis by AR. Next, we performed CRIPSR-Interference (CRISPRi) in cells co-treated with DHT and IFNγ to determine AR-responsive genes in the IFNγ signaling pathway identified by examination of Cistrome ChIP-Seq that mediated the attenuation of PD-L1 expression in response to DHT. Finally, since AR ChIP studies in the Cistrome database were predominately from prostate cancer cell lines and primary prostate cancer tissue, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to map AR binding sites genome-wide in our thyroid cancer cell lines. We plan to expand our investigation beyond cultured cells and are optimizing CUT&RUN to examine AR-binding sites in primary human thyroid cancer tissue. By elucidating the regulatory interactions between interferon, AR, and PD-L1 expression, we can potentially better explain the discrepancy between ThCa incidence among the sexes and identify novel immunotherapeutic targets.
Citation Format: Sina Dadafarin, Michelle A. Carnazza, Augustine Moscatello, Raj Tiwari, Jan Geliebter. Mechanism of androgen-mediated down-regulation of PD-L1 in thyroid 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 2236.