Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Jill Bargonetti

Committee Members

Frida Kleiman

David Foster

David Jeruzalmi

James Manfredi

Subject Categories

Cancer Biology | Cell Biology | Molecular Biology

Abstract

Cancer cells often lose expression of the p53 protein or express mutant forms of p53. Some of these mutant p53 proteins, called gain-of-function mutant p53, have gained oncogenic functions. Previously, our group observed mutant p53 R273H interacts with replicating DNA and upregulates the chromatin localization of several DNA replication factors including PCNA, MCM2-7, and PARP1 (termed the mtp53-PARP-MCM axis). In this thesis, we explore the contribution of mutant p53 and PARP1 in castration-resistant prostate cancer (mutant p53 P223L and V274F) and triple-negative breast cancer (mutant p53 R273H). In the castration-resistant prostate cancer cell line DU145, we examine two mutant p53 proteins, P223L and V274F, for participation in the mtp53-PARP-MCM axis. We observed that these mutations were not drivers of sensitivity to DNA damage and PARP inhibition.

In the triple-negative breast cancer cell line MDA-MB-468, we examined the role of the R273H C-terminal basic domain, in the mtp53-PARP-MCM axis. We hypothesized that the C-terminal domain in mutant p53 R273H facilitates chromatin recruitment of PARP1. This work compared CRISPR-Cas9-edited MDA-MB-468 cells with truncations in the C-terminal domain (R273H∆381-388), the C-terminal and oligomerization domain (R273H∆347-393) and a frameshift resulting in very low mutant p53 expression (R273Hfs387). Mutant p53 R273H C-terminal domain truncations decreased proliferation rate, as well as chromatin-, PAR-, and PARP1-binding. Interestingly, the cells expressing R273H∆347-393 had the lowest proliferation rate, least chromatin interaction with PARP1 and PAR, had no delay in cell cycle progression, and exhibited the fastest DNA replication speed. As such, increased DNA replication fork speeds have been reported to occur when PARP1 is inhibited, indicating that loss of R273H amino acids 347-393 may result in decreased PARP1 function and a decrease in cancer cell survival. The mutant p53 R273H C-terminal domain participates in gain-of-function activities through interaction with PARP1.

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