Graduation Year
2024
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Program Director
Meredith Protas, PhD
First Reader
Christopher Benz, MD
Second Reader
Maggie Louie, PhD
Abstract
ERBB2 gene-amplified breast cancer is an aggressive form of breast cancer that constitutes 20% of all newly diagnosed cases. Although there are specific treatments for patients with ERBB2-amplified breast cancers, patients often develop therapeutic resistance, underscoring the need for alternative anti-ERBB2 therapeutics. Recently, our lab has shown that inhibiting the histone acetyl transferase (HAT) domain and the bromodomain (Brd, “reader” of lysine acetylation) of the nuclear coactivator proteins EP300 and CBP with drugs A485 and NEO2734, respectively, induces rapid decay of ERBB2 transcripts and thereby impedes the growth of ERBB2-amplified breast cancer cells. In ERBB2-amplified human breast cancer cell line models (SKBr3, BT-474), the overexpressed ERBB2 nuclear mRNA first accumulates in discrete nuclear speckles, and speckle accumulation disappears within several hours of cell exposure to the small molecule inhibitors A485+NEO2734. Newly transcribed mRNA is generally routed either to nuclear speckles for stabilization and cytoplasmic export or to the nuclear RNA exosome system for immediate decay. We hypothesized that A485+NEO2734 blocks ERBB2 mRNA accumulation in nuclear speckles and promotes the accelerated decay of ERBB2 mRNA through the nuclear RNA exosome pathway. To test this hypothesis, we used confocal fluorescence imaging and RT-PCR assays to assess the consequences of siRNA knockdown of the key nuclear RNA exosome components MTREX, RBM7, and the 3' to 5' exonucleases Dis3 and Rrp6. The knockdown of RBM7, MTREX, and Dis3 significantly abrogated ERBB2 mRNA decay (30%-70%), achieving levels usually induced by EP300/CBP inhibition. Additionally, we determined that the ERBB2 transcripts are likely guided by their 3' end through a barrel-like NEXT complex towards the exonuclease Dis3 and away from the Rrp6 exonuclease. A more detailed mechanistic understanding of EP300/CBP-regulated ERBB2 mRNA stability will not only generate additional rationale for further preclinical development of this new anti-ERBB2 treatment strategy but also stimulate a broader exploration of this epigenetic treatment approach designed to cause rapid decay of other oncogenic transcripts similarly regulated by EP300 and CBP.