The RB1-E2F complex in Enzalutamide resistant LNCaPs

Enzalutamide (Enz) is an anti-androgen used for treatment of metastatic, advanced prostate cancer. Unfortunately, this treatment will eventually lead to resistance. Understanding the mechanisms behind Enz-resistance will provide useful in predicting Enz-response in patients as well as in defining new therapeutic targets. We generated an in vitro model for Enz-resistance by long-term treatment of LNCaP cells. The resulting cell line was named LNCaP-R. The LNCaP-R cells were investigated for transcriptomic changes via RNAseq analysis. For a large set of androgen-regulated genes in LNCaP and especially for the PSA gene, we observed an attenuated androgen response in the LNCaP-R. This lower AR activity could not be attributed to lower levels of AR protein nor to truncations or other acquired somatic mutations in the AR gene. Striking changes were observed in the expression of cell cycle genes. While there was no prominent change in the mRNA level of RB1, the transcription of many of the E2F target genes was reduced in presence of androgens. At the protein level, the resistant cells demonstrate high levels of phosphorylated RB1 and increased E2F1 levels. This contrasts with the parental LNCaP cells in which the total RB1 levels are much lower and RB1 phosphorylation is regulated by androgens. AR ChIP-seq in LNCaP-R compared to in the parental cells, revealed stronger DHT-induced AR binding on the enhancer and promoter regions of androgen-regulated genes, even when these genes are less androgen responsive in the LNCaP-R. While most AR binding sites in parental and LNCaP-R cells showed a presence of androgen response elements (AREs) and FoxA1 binding sites, further analyses of the ChIP-seq data pointed towards a group of AR binding sites unique for the LNCaP-R cells that do not contain AREs, but are enriched for a.o. E2F binding motifs. Such new binding sites possibly suggest a gain of function of E2F in AR recruitment. We will discuss how these changes in AR, RB1 and E2F behavior might be related to the Enz-resistance.status: publishe