As approximately 70% of human breast tumors are estrogen receptor α (ERα)-positive, estrogen and ERα play essential roles in breast cancer development. By interrupting the ERα signaling pathway, endocrine therapy has been proven to be an effective therapeutic strategy. In this study, we identified a mechanism by which Transcription Start Site (TSS)-associated histone H3K27 acetylation signals the Super Elongation Complex (SEC) to regulate transcriptional elongation of the ESR1 (ERα) gene. SEC interacts with H3K27ac on ESR1 TSS through its scaffold protein AFF4. Depletion of AFF4 by siRNA or CRISPR/Cas9 dramatically reduces expression of ESR1 and its target genes, consequently inhibiting breast cancer cell growth. More importantly, a AFF4 mutant which lacks H3K27ac interaction failed to rescue ESR1 gene expression, suggesting H3K27 acetylation at TSS region is a key mark bridging the transition from transcriptional initiation to elongation, and perturbing SEC function can be an alternative strategy for targeting ERα signaling pathway at chromatin level.

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale‐up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

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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.

Mounting evidence is revealing a granularity within gene regulation that occurs at the level of mRNA translation. Within mammalian cells, canonical cap-dependent mRNA translation is dependent upon the interaction between the m 7 G cap-binding protein eukaryotic initiation factor 4E (eIF4E) and the scaffolding protein eukaryotic initiation factor 4G (eIF4G), the latter of which facilitates pre-translation initiation complex assembly, mRNA circularization, and ultimately ribosomal scanning. In breast epithelial cells, we previously demonstrated that the CELF1 RNA-binding protein promotes the translation of epithelial to mesenchymal transition (EMT) effector mRNAs containing GU-rich elements (GREs) within their 3' untranslated regions (UTRs). Here we show that within this context, CELF1 directly binds to both the eIF4E capbinding protein and Poly(A) binding protein (PABP), promoting translation of GRE-containing mRNAs in mesenchymal cells. Disruption of this CELF1/eIF4E interaction inhibits both EMT induction and experimental metastasis. Our findings illustrate a novel way in which noncanonical mechanisms of translation initiation underlie transitional cellular states within the context of development or human disease.It has now been well-documented that both oncogenes and signaling pathways regulating expression of these oncogenes together converge on the regulation of mRNA translation (2,15).Indeed, within the process of cellular transformation, mechanisms at each stage of translation and the ribosomal machinery may be co-opted to fostering translation that perpetuates the oncogenic program (16)(17)(18). This motivated us to more closely examine how CELF1 regulates translation of GRE-containing EMT effector mRNAs during EMT of breast epithelial cells. Results CELF1 interacts directly with eIF4E and PABP at the m 7 G cap, independent of intact eIF4G1Our previous work suggested that CELF1's control of GU-rich element-containing EMT effector mRNA translation is mediated at the level of 5' m 7 G cap-dependent translation initiation (10). We first confirmed and extended these findings by examining an additional CELF1 regulatory target within this context. We fused the 3' UTRs of a subset of GRE-containing mRNAs (JUNB, CRLF1, SNAI1, and SSBP2) downstream of the Renilla luciferase coding sequence in the pRL-TK-CXCR4-6x (19) reporter plasmid (Figure 1a). Transfection of the GREcontaining 3' UTR reporters into MCF10A cells, either untreated or treated with TGF-β to induce EMT, revealed a significant increase in reporter activity specific to treated cells Figure 1a), independent of any differences in relative mRNA expression (Figure 1b). In parallel, we built a battery of bicistronic constructs in which the same thymidine kinase promoter was utilized to drive expression of the firefly luciferase coding sequence, followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), the Renilla luciferase open reading frame and individual GRE-containing 3' UTRs (pFR-EMCV) (Figure 1c). In contrast to cap-depen...