Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biochemistry Program

Abstract

Women diagnosed with triple-negative breast cancer have the worst overall prognosis and frequently present with metastatic tumors. To date, there are no targeted therapies available to combat this aggressive form of breast cancer due to the lack of expression of well-known targets such as ER-alpha, PR, or HER2/neu. Therefore, there is an immediate need to identify novel targets that are responsible for the proliferation, survival, and invasive phenotype. Notch-1 and Notch-4, both potent breast oncogenes, are overexpressed in triple-negative breast cancers-associated with the poorest overall survival. PEA3 (polyomavirus enhancer activator 3), a member of the Ets family of transcription factors, is overexpressed in triple-negative breast cancer and also correlates with aggressive behavior and poor overall survival. Here, we provide new evidence for transcriptional regulation of Notch in triple negative breast cancer and other subtypes of breast cancer. Our results showed that PEA3 is a transcriptional activator of both Notch-1 and Notch-4 genes using a PEA3 siRNA and measuring both transcripts and proteins in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, exogenous overexpression of PEA3 significantly increased Notch-4 transcripts. Chromatin immunoprecipitation confirmed enrichment of PEA3 within the promoter regions of both Notch-1 and Notch-4. Notch- recruitment appears to be AP-1 independent, whereas PEA3 recruitment on the Notch-4 promoter is dependent upon c-JUN. Furthermore, results showed that either c-Jun or Fra-1 are required for Notch-4 transcription while c-FOS was a repressor. Importantly, the combined inhibition of Notch signaling via a gamma-secretase inhibitor (GSI) and knockdown of PEA3 (via siRNA) significantly growth arrested breast cancer cells in G1 and decreased both anchorage dependent and independent growth in vitro. In correlation, a significant decrease in cell viability and tumorigenicity, as well as an increase in apoptotic cells were observed when a GSI was combined with PEA3 siRNA. Interestingly, a combined inhibition of Notch signaling and PEA3 showed no significant changes in invasion and migration. Taken together, results from this study suggest for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. PEA3-mediated Notch-1 transcription is AP-1 independent while Notch-4 transcription requires both PEA3 and AP-1 most probably composed of the c-Jun:Fra-1 complex. Moreover, both PEA3 and Notch signaling are essential for proliferation and survival of MDA-MB-231 breast cancer cells. Thus, dual targeting of both PEA3 and Notch pathways might provide a new therapeutic strategy for triple-negative breast cancer as well as additional therapeutic targeting in other breast cancer subtypes.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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