Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Charles Chalfant, Ph.D.

Co-Major Professor

Margaret Park, Ph.D.

Committee Member

Sandy Westerheide, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Committee Member

Jason Carlyon, Ph.D.

Keywords

Breast Cancer, hnRNPR, IRES, SRSF3

Abstract

Breast cancer is the second leading cause of cancer-related deaths for women in the U.S. Although the overall 5-year survival rate for breast cancer is 90%, this rate drops substantially for triple-negative breast cancer (TNBC) due to its high metastatic potential. Furthermore, there is a lack of targeted therapeutics for TNBC, and clinical trials have been largely unsuccessful. These characteristics validate the need for identifying novel therapeutic targets for the treatment of TNBC. The study of alternative splicing (AS) has emerged as a powerful tool to elucidate the molecular underpinnings driving cancer.

Our lab has identified cytoplasmic polyadenylation element-binding protein 2 (CPEB2), which has two main isoforms, CPEB2A and CPEB2B, which differ via the inclusion/exclusion of exon four in the mature mRNA transcript. These two isoforms have opposing functions as translational regulators of mRNA species implicated in metastatic progression. A shift in the spicing ratio favoring an increase in CPEB2B and a reduction in CPEB2A resulted in increased translation of HIF1α and TWIST1 mRNA, transcription factors important in the regulation of the hypoxic response and epithelial-to-mesenchymal (EMT) pathways and contributed to the acquisition of anoikis resistance (AnR) and metastasis in TNBC cells in vivo.

Increased levels of serine/arginine-rich factor 3 (SRSF3), an AS regulator, were identified in TNBC AnR cells. SRSF3 was also determined to be the trans-splicing factor responsible for regulating the inclusion/exclusion of exon four of CPEB2 by binding to a consensus sequence within exon four. Mutation of the SRSF3 consensus sequence in exon four of CPEB2 ablated SRSF3 binding resulting in decreased inclusion of exon four. A minigene construct investigation of the SRSF3/CPEB2 exon four alternative splicing axis indicated that downregulation of SRSF3 via siRNA resulted in a CPEB2 alternative splicing ratio shift favoring the production of CPEB2A (exon for exclusion). However, in the siSRSF3 CPEB2 mutant minigene, the ratio shift was ablated. Furthermore, siRNA targeting SRSF3 decreased CPEB2B (exon four inclusion) and reduced AnR and survival in TNBC, which was “rescued” by the ectopic expression CPEB2B. Ultimately, these studies demonstrate the importance of CPEB2 AS via the trans-splicing factor SRSF3 in the acquisition of AnR and metastasis in TNBC.

We also propose a mechanism of HIF1[U+F061] and TWIST1 translational regulation via CPEB2A and CPEB2B. We determined that the CPEB2A isoform bound to the CPE sites located in HIF1[U+F061] and TWIST1 3'UTR mRNA and interacted with known polyadenylation complex proteins. Similarly, we found CPEB2B associated with polyadenylation complex proteins, albeit a weaker interaction, but did not bind CPE sites in HIF1[U+F061] and TWIST1 3'UTR mRNA. We also found that CPEB2A and CPEB2B localize to both the nucleus and cytoplasm. Furthermore, we identified novel protein interactions for CPEB2B, specifically exon four, which is absent in the CPEB2A transcript. CPEB2B was shown to interact with proteins, specifically, the translation invitation factor eIF3H and heteronuclear-ribonuclear proteins hnRNPR and hnRNPF/H, which have been identified as IRES trans-acting factors (ITAFs) important in cap-independent translational activation of mRNAs in cellular stress events. Interestingly, we also identified a strong interaction between CPEB2A and CPEB2B proteins suggesting a potential connection between the CPEB2 alternative splicing regulation and translational activation/inhibition of HIF1[U+F061] and TWIST1. These novel interactions have never been described and provide evidence that alternative splicing inducing a ratio shift of CPEB2A to CPEB2B proteins results in translational activation of HIF1[U+F061] and TWIST1 mRNA through an IRES-mediated cap-independent translational mechanism promoting the acquisition AnR and the metastatic phenotype in TNBC.

Download

Share

COinS