INHIBITION OF BRD PROTEINS SUPPRESSES THE PHENOTYPE OF UTERINE FIBROIDS VIA REGULATION OF N6-METHYLADENOSINE REGULATORS

Abstract

Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uterine fibroids (UFs), the most common pelvic tumors in women of reproductive age, is unknown. The present study aimed to determine the expression pattern of BRD proteins in UFs and matching myometrium and assess the effect of BRD inhibitors on UF phenotype and epitranscriptomic changes. Human UFs (n=22) and adjacent myometrium tissues (MyoF, n=7) were collected at the time of hysterectomy at the University of Chicago. Western blot (WB) was performed to determine the levels of BRD proteins in UFs and myometrium. Fourteen BRD inhibitors from the Epigenetic Chemical Probe Library provided by the University of Illinois at Chicago were used to determine the cell proliferation by MTT assay. Two BRD inhibitors (I-BRD9 and TP-472) were further used to explore their mechanistic actions. The student’s t-test was used to determine the significant difference. Our studies demonstrated that among 22 UFs analyzed, 96% (21/22, p<0.01), 55% (12/22), 82% (18/22, p<0.01) exhibited the upregulation of BRD2, BRD3, and BRD9 respectively, suggesting the aberrant BRD protein expression may contribute to the pathogenesis of UFs. To examine the biological impact of BRD inhibition on the UFs, 14 BRD inhibitors were screened to identify specific and potent BRD inhibitors in UF cells. 43% (6/14) of these BRD inhibitors selectively inhibited UF, but not myometrial cell proliferation. Furthermore, targeting BRD proteins with BRD inhibitors (I-BRD9 and TP-472) decreased the protein levels of extracellular matrix proteins (Type I Collagen, fibronectin), PCNA (cell proliferation marker), and Bcl-2 (anti-apoptotic marker) in a dose- and time-dependent manner in UF cells. Notably, inhibition of BRD proteins with I-BRD9 and TP-472 downregulated the expression of METTL3, YTHDC2, and YTHDF2, which are the key regulators for N6-methyladenosine (m6A) modification, indicating the tight link between BRD proteins and epitranscriptomic landscape. These results demonstrate for the first time that BRD proteins are aberrantly expressed in UFs. BRD inhibitors suppress the UF phenotype while concomitantly altering the expression of the central m6A regulators.