Fig. 1: AA downregulates pRb-regulated genes and upregulates HDAC pathways of cellular senescence.

Transcriptome sequencing was performed with the castration-resistant C4-2 cells treated with AA. a The retinoblastoma tumour suppressor, pRb, is a transcriptional repressor. GO gene sets identified from RNA-seq analysis of treated C4-2 cells with AA indicates that AA downregulates significantly pRb-regulated genes. b Western blot experiments reveal hypophosphorylation of pRb at serines 780 and 807/811 by AA. β-Actin was used as loading control. c Treatment with AA induces cellular senescence indicated by SA β-Gal activity staining. d AA induces cellular senescence in a concentration-dependent manner. e GO gene set analysis indicates HDAC1, 2 and 3 pathways are significantly induced by AA. f Activity prediction (by IPA software) of upstream regulators upon AA treatment compared to control. g RNA-seq analysis of GO gene sets suggests that AA treatment of C4-2 cells inhibits cell proliferation and h tumour metastasis pathways. For all the comparisons, Wilcoxon signed-rank test 2-sided was used. i AA reduces the androgen-mediated transactivation of AR mutants that are resistance to antagonist therapy. Expression vectors for mutant ARs AR-T877A, AR-W741C, and AR-F876L that mediate therapy resistance to clinically used AR antagonists, Flutamide (F), Bicalutamide (Bic) and Enzalutamide (Enz), respectively, were analysed in reporter assays. CV1 cells lacking AR were treated with the AR antagonists F, Bic, Enz (each 10 µM), AA (100 µM) and androgen DHT (10 nM), or (R1881 1 nM) in charcoal-treated 5% FBS. Results represent luciferase activity normalised to β-galactosidase activity derived from the co-transfected pCMV-lacZ vector. Error bars show standard deviation of the mean (SD). These results were verified by at least two additional replicates.