EAF2 Associates with FOXA1 and EAF2 Alleviates FOXA1-Mediated Repression of Androgen Receptor Transactivation

Prostate Cancer (PCa) is the most commonly diagnosed cancer and the second most common cause of cancer death in men in the United States. During the development of PCa, several androgen receptor (AR) regulated tumor-suppressors are down regulated, including ELL-associated factor 2 (EAF2), also known as androgen up-regulated 19 (U19), an RNA polymerase II transcription elongation factor. In order to determine how EAF2 is regulated and find other genes that interact with EAF2, an RNAi screen was performed in C. elegans. When eaf-1, the C. elegans ortholog to EAF2 was knocked out, the resulting worms had reduced fertility and the screen was designed to detect phenotypic enhancers, RNAis that increased the eaf-1KO reduced fertility phenotype. Knockdown of the gene pha-4, the C. elegans ortholog to Forkhead Box A1 (FOXA1), was found to cause egg degeneration rendering the eaf-1KO worms sterile. FOXA1, a pioneer factor for AR, is required for normal differentiation of prostatic epithelial cells and up-regulation of FOXA1 is associated with a worse prostate cancer prognosis in advanced prostate cancer. We hypothesized that EAF2 associates with FOXA1 to modulate AR transactivation. Transfected FOXA1 associated with EAF2 when co-immunoprecipitated and FOXA1 associated with both the N-terminus and C-terminus of EAF2. Co-expression of EAF2 and FOXA1 reduced EAF2 and FOXA1 protein levels relative to EAF2 or FOXA1 alone. FOXA1 protein level increased and PSA protein and mRNA levels decreased when EAF2 was knocked down in human PCa cell line LNCaP by siRNA. Similarly, EAF2 knockout also increased FOXA1 protein level in the mouse prostate. In a luciferase assay, FOXA1 reduced PSA-promoter expression in C4-2 prostate cancer cells, and EAF2 transfection alleviated FOXA1-mediated repression of PSA. In a colony formation assay performed in LNCaP cells, over-expression of EAF2 reduced the number of colonies, over-expression of FOXA1 increased the number of colonies, and over-expression of FOXA1 and EAF2 produced an intermediary number of colonies. These findings suggest a potential functional and physical interaction of EAF2 and FOXA1 that may represent a new pathway and could potentially play a role in controlling growth in the normal prostate and prostate cancer