Abstract 2841: Continuous androgen receptor stimulation in thyroid cancer cells induces irreversible senescence

Abstract

Abstract Cancer of the thyroid gland accounts for 3.8% of all cancer cases in the United States as indicated by the SEER report by the NCI. The incidence of thyroid cancer has increased three-fold over the last thirty years and the American Cancer Society estimates that there will be approximately 62,450 new cases and 1,950 deaths due to the disease in the country in 2015. Papillary Thyroid Cancer (PTC) is the most common endocrine malignancy with a three-fold higher incidence in women than in men. However, PTC exhibits increased aggressiveness with poor prognosis in men diagnosed with the disease. These incongruent observations led us to explore the role of androgen and androgen receptor in this disease. We found an approximately 70% decrease in median AR expression (p<0.0001) in 24 PTC patient tissue samples, compared to matched, normal thyroid tissue. Preliminary data from our lab indicate that androgen receptor (AR) acts as a negative regulator of growth as evidenced by a statistically significant 48% decrease in proliferation over 72 hours upon 5α-dihydrotestosterone (DHT) addition to 8505c anaplastic/PTC cells stably transfected with AR (clone 84E7). Transcriptional profiling using RNAseq and gene ontology analysis, on 48 hour DHT treated 84E7 cells revealed significant changes in gene expression associated with proliferation (474 genes, p = 2.4E-24) and cell cycle progression (129 genes, p = 6.54E-6). Continuous AR activation (3 to 6 days) resulted in G1 growth arrest, not accompanied by cell death, but rather a flattened, vacuolized cell morphology, indicative of senescence. This was substantiated by an increase in SA-βGal positivity from background 2.47% to approximately 65.5%, which was attenuated by the AR antagonist, flutamide. Three to six day DHT exposure resulted in increased total RNA and protein content measured using Acridine Orange and Sulforhodamine B, respectively. Senescent 84E7 cells failed to resume growth when transferred into DHT-free medium for 3 days, suggesting a permanent growth arrest. Reactive oxygen species (ROS), implicated in Rb-mediated senescence, were found to be doubled by day 6 of AR activation, compared to control cells. Additionally, using flow cytometry, we found increased p21, p27, cyclin D1, FOXO1, and phosphorylation of FOXO1 by days 3-6, suggesting that AR-dependent senescence is mediated via p21 and p27 by FOXO1/3 proteins. Furthermore, cytokine profiling of the senescence-associated secretory phenotype (SASP) would aid in defining the pro-tumorigenic or tumor-suppressive potential of androgen-induced senescent thyroid cancer cells. Our study elucidates the induction of senescence as a novel function of AR activation in thyrocytes and may indicate a protective role of AR activation in the decreased incidence of thyroid cancer in men. Citation Format: Anvita Gupta, Melanie Jones, Timmy O’Connell, Dorota Halicka, Jiangwei Li, Hong Zhao, Augustine Moscatello, Zbigniew Darzynkiewicz, Raj Tiwari, Jan Geliebter. Continuous androgen receptor stimulation in thyroid cancer cells induces irreversible senescence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2841.