Defining The Reciprocal Regulation Of Abi1 And The Androgen Receptor In Prostate Cancer.

Abstract Background: Prostate cancer (PCa) is a slowly progressing disease that affects nearly 50% of males over the age of 60. Current therapies for patients diagnosed with advanced PCa include Androgen Deprivation Therapy (ADT) and anti-androgen receptor (Anti-AR) treatment. PCa treatment primarily targets the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research has demonstrated that ABI1, acts as a tumor suppressor in PCa. ABI1 is a scaffold protein and an integral member of the WAVE Regulatory Complex (WRC), a nucleation promoting factor. UCSC database indicates there is an AR binding site within the ABI1 gene, suggesting AR plays a role in the transcriptional regulation of ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. The efficiency of CRISPR-Cas9 was evaluated by Western Blotting and genomic alterations confirmed by DNA sequencing. In addition, we have generated ABI1 Isoform-specific rescue cell lines in our LNCaP ABI1 KO cell line with a binding mutation in the SH3 domain (ABI1-W485N) and an SH3 domain deleted (Abi1-ΔSH3) ABI1 protein. To understand AR and ABI1 dependent pathways we performed qPCR, co-immunoprecipitation, Western Blotting, ChIP, in vitro fluorescence spectra assay, immunofluorescence, proximity ligation assays. Results: In vitro binding assays indicate the interaction of AR, NTD poly-proline region, with the ABI1 SH3 domain. Expression of ABI1- W485N in our ABI1 CRISPR KO cell line showed decrease binding, while ABI1-ΔSH3 showed no binding to AR in co-IP assays compared to our control. Consequently, we saw decreased AR nuclear localization compared to our ABI1-WT control. Furthermore, decreased nuclear localization in our ABI1-W485N mutant was associated with decreased mRNA expression of hallmark AR target genes, Prostate-Specific-Antigen (KLK3), and TMPRSS2. ABI1 is an AR responsive gene and was confirmed with ChIP assays in an AR overexpression cell line. Further, the stimulation of AR transcriptional activity increased cell-cell adhesion in an ABI1 dependent system. Conclusions: Our preliminary studies demonstrate that AR and ABI1 have a negative feedback pathway. The loss of ABI1 resulted in a decrease of nuclear AR and a subsequent decrease in mRNA expression of AR target genes. AR can also modulate ABI1 expression on a transcriptional level as needed for this pathway to function. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote EMT through STAT3 activation until AR transcriptional regulation on ABI1 is restored. These findings will allow for novel insights into the mechanisms underlying AR and ABI1 relationship in neoplastic progression. Citation Format: Baylee A. Porter, Alaji Bah, Alfonso Urbanucci, Fan Zhang, Sonia Kung, Ladan Fazli, Martin Gleave, Gennady Bratslavsky, Leszek Kotula. Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2470.