Abstract 930: Production of functionally active recombinant FOXA1: The first step towards targeted drug discovery

Abstract

Expression of transcription factor estrogen receptor (ER) drives and defines almost three-fourth of all breast cancer (BC). Endocrine therapies such as tamoxifen and aromatase inhibitors form the backbone of therapeutic regimen in treatment of ER-positive (ER+) BC. However, some ER+ BC patients do not respond well to these therapies and develop endocrine resistance. Mechanisms of endocrine resistance is multi-faceted and may require inhibition of multiple pathways. FDA-approved inhibitors of CDK4/6 and mTOR pathways have improved outcomes in ER+ metastatic BC patients. However, additional therapeutic agents targeting novel and essential nodes of endocrine resistance are needed for better management of ER+ BC. Recent research has shown that ER coregulators play a crucial role in endocrine resistance. The DNA interactions and transcriptional potential of ER rely on the pioneer factor Forkhead Box A1 (FOXA1), which plays an essential role in determining tumor growth and progression especially in the endocrine resistance setting. FOXA1 is gene amplified and/or overexpressed in multiple preclinical models and clinical samples of endocrine-resistant BC. Overexpression of FOXA1 promotes metastasis in animal models even during tamoxifen treatment, making it an attractive drug target to overcome endocrine resistance. However, no pharmacologic inhibitors of FOXA1 are currently available. To launch the drug discovery efforts to identify novel FOXA1 inhibitors, we aimed to express and purify functionally active full-length human FOXA1 protein that can be used for high-throughput drug screening in this research. We utilized various protein production strategies that we explored using baculovirus, bacterial, and mammalian expression system to achieve highly stable recombinant FOXA1 in an active form. While the baculovirus system had high protein expression, the yield, stability and purity of the recombinant FOXA1 was substandard. We maximized the yield and purity of FOXA1 with the bacterial expression system, but the protein functionality was lost due to misfolding. The mammalian expression system, which is well-known for its ability to properly recapitulate the protein posttranslational modifications, yielded stable FOXA1 protein. The recombinant FOXA1 expressed and purified in mammalian cells was functionally active as determined by the DNA-binding activity using enzymatic mobility shift assay (EMSA). However, the purity achieved was suboptimal for drug discovery application. Ongoing efforts are focused on optimizing the protein purification strategies that could ensure optimal yield of functional protein with highest purity for downstream applications. Citation Format: Hariprasad Thangavel, Yingmin Zhu, Kurt R. Christensen, Xiaoyong Fu, Dean Edwards, Rachel Schiff, Meghana V. Trivedi. Production of functionally active recombinant FOXA1: The first step towards targeted drug discovery [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 930.