Abstract 909: Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9

Abstract

Abstract Background: Androgen receptor (AR) is the key drug target in prostate cancer. AR splice variant-7 (AR-V7) is the most relevant AR variants implicated in castration-resistant prostate cancer (CRPC). Detection of AR-V7 in both liquid biopsies and tissue biopsies predicts poorer response to novel AR-targeting drugs. However, because AR-V7 always coexists with the full-length AR (AR-FL), the functional importance of AR-V7 needs to be dissected in the context of AR-FL. Lack of relevant cell line models presented a hurdle for detailed dissection of the role for AR-V7. It is essential to develop cell lines that express endogenous AR-V7 at a level comparable to clinical specimens, with or without AR-FL, to facilitate functional studies. The goal of this study is to develop and characterize LNCaP95 lines following specific and complete knock-out of AR molecules by CRISPR-Cas9 system. Methods: Different AR isoforms were knocked-out in parental LNCaP95 cells by CRISPR-Cas9. AR-FL was knocked-out through sgRNAs targeting AR exon5 or exon6. AR isoforms with functional N-terminal domain (total AR) were knocked-out through sgRNA targeting AR exon1. Complete knockout of AR-FL and total AR was achieved in LNCaP95 cells. The on-target editing of AR genomic regions were verified by Sanger Sequencing. Results: Complete knockout of AR-FL protein and total AR protein was confirmed in two clones carrying homologous indels, ARfl-KO#1/2 and AR-KO#1/2. Antibodies against different AR domains were used to differentiate expression of different AR isoforms, by Western blot and immunohistochemistry (IHC). Cell proliferation was not affected by androgen in AR-FL or total AR knockout cells, as shown in cell growth and clonogenic assays. The expression of AR-target genes such as prostate-specific antigen (PSA) were analyzed by realtime-PCR. PSA was expressed at similar level compared to parental cells in AR-FL knockout cells, which was totally lost in total AR knockout cells. Interestingly, total AR knockout cells are more resistant to ionizing radiation and PARP inhibitor olaparib compared to parental cell line. Conclusion: We have successfully developed the first prostate cancer cell line expressing endogenous AR-V7 at a level comparable to clinical specimens without AR-FL, allowing dissection of the functions mediated by AR-FL and AR-V7. Initial functional characterization suggested AR-V7 maintained the baseline level of AR signaling activity, which was not dependent on androgen. The chromatin-binding pattern of AR-V7 in the absence of AR-FL is under investigation. We have also developed a novel total AR knockout cell model from the AR-positive parental cell line. This model suggested prostate cancer cells were able to survive in an AR-null manner, although loss of AR significantly inhibited cell proliferation. Preliminary studies have found loss of total AR confers resistance to ionizing radiation and PARP inhibition. Citation Format: Yezi Zhu, Pei Zhao, Changxue Lu, Shihua Chen, Jun Luo. Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 909.