Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Nicholas T. Woods, PhD

Abstract

Genomic instability is one of the enabling characteristics of cancer. DNA damage response pathways are important for genomic integrity and cell cycle progression. Defects in DNA damage repair can often lead to cell cycle arrest, cell death, or tumorigenesis. The activation of the DNA damage response includes tightly regulated signaling cascades that involve kinase phosphorylation and modular domains that scaffold phosphorylated motifs to coordinate recruitment of DNA repair proteins. Modular domains are conserved tertiary structures of a protein that can fold, function, and evolve independently from an intact protein. One of the most common modular domains involved in DNA damage repair is the BRCA1 C-Terminal (BRCT) domain. The BRCT domain is approximately 90-100 amino acids long and functions as a scaffolding domain to help recruit DNA damage-related proteins for DNA repair. Mechanisms in which BRCT domains regulate DNA repair have yet to be fully elucidated. Studying protein-protein interactions mediated by these domains can improve our understanding of how BRCT domains function in DNA repair. The goal of this dissertation is to characterize the scaffolding functions of BRCT domains and how their interactions contribute to DNA damage repair pathways dysregulated in cancer.

BRCA1, the protein in which the BRCT domain was first identified, is the most well-known cancer susceptibility gene, often mutated in cases of breast and ovarian cancers. Our research has identified that BRCA1 BRCT domain-mediated interactions with the mTORC2 complex disrupt the complex and impair Akt activation, which is critical for cancer cell growth, proliferation, and survival. We have sought to identify how mTORC2 activity may regulate BRCA1 function as a DNA damage regulator and a transcriptional co-activator and contribute to the DNA damage response. We have found that rapamycin treatment increases BRCA1 transcriptional coactivation activity. Also, mTORC2 activity prevents cisplatin-induced cell death. Repression of mTOR signaling reduces gH2AX-BRCA1 foci formation. More importantly, this dissertation work highlights a novel chemotherapeutic strategy of targeting the mTOR pathway for breast cancers with BRCA1 mutations or loss of BRCA1 function. BRCA1 loss sensitizes breast cancer cells to mTOR inhibition. Since the PI3K-mTOR-Akt pathway is upregulated in over 70% of breast cancer cases, breast cancer patients with defective BRCA1 may be ideal candidates for mTOR inhibitor therapeutics.

While BRCT domains are largely associated with DNA repair proteins, there are some BRCT domain-containing proteins for which their DNA repair roles are not fully characterized, such as RNA Polymerase II Subunit A C-Terminal Domain Phosphatase 1 (CTDP1). Our research has revealed CTDP1 as a regulator of FANCI in the Fanconi anemia pathway, a pathway important for the repair of interstrand crosslinks (ICLs). CTDP1 influences FANCI chromatin localization, FANDC2 foci formation, sensitivity to ICL-inducing drugs, and homologous recombination repair. In addition, CTDP1 has also been found to be highly expressed in breast cancer cell lines. CTDP1 knockdown in murine mammary orthotopic models prevents tumor formation, thus rendering CTDP1 as a potential target for breast cancer therapeutics.

The findings in this dissertation work contribute to our overall understanding of how BRCT domains use their scaffolding function to regulate the DNA damage response. Elucidating the biological importance of these domains can improve our understanding of cancer susceptibilities, tailor chemotherapeutic strategies, and make better informed decisions in cancer therapies.

Available for download on Wednesday, November 24, 2021

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