Document Type

Thesis

Date of Award

5-2022

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

Committee Chairperson

Erica Cassimere

Committee Member 1

Mario Hollomon

Committee Member 2

Audrey Player

Committee Member 3

Mark Harvey

Keywords

Breast cancer, Cancer stem cells, p27KIP1

Abstract

Breast cancer is the second leading cause of cancer death among American women. Traditional therapies, such as chemotherapy and radiation therapy, target proliferative cells within the tumor bulk. However, these treatments do not provide long-term disease survival because they fail to eliminate a small population of long-lived tumor initiating cells known as breast cancer stem cells (CSCs). Two features of CSCs are that they slowly divide and are more resistant to conventional anti-cancer treatments due to altered cellular machinery. It is the breast CSC subpopulation that develops new tumors which have the potential to lead to recurrence. However, the mechanism underlying the dormant nature of breast CSCs that leads to resistance remains poorly defined. p27kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKI) that negatively regulate cell cycle progression. Because p27kip1 levels are highly elevated in quiescent cells and decreased as cells proliferate, we hypothesized that p27kip1 is a potential mediator of stem-like properties in breast CSCs. Using in vitro tumor sphere formation assays and Western blot analysis, we observed that p27kip1 protein levels were markedly enhanced in stem cells isolated from metastatic breast cancer cell lines compared to non-metastatic breast cancer stem cell populations. Moreover, depletion of endogenous p27kip1 using shRNA methodologies dramatically reduced the survival of breast CSCs from metastatic breast cancer cells. p27kip1 levels were unaltered at the transcriptional levels as shown by quantitative real time RT-PCR as well translational levels using expression vectors which lacked internal regulatory sequences. However, p27 protein levels are primarily controlled post-translationally by a series of phosphorylation events which either targets p27 for ubiquitin-dependent degradation or relocalizes p27 to the cytoplasm. Using cycloheximide treatment to inhibit de novo protein synthesis, Western blot analysis revealed that p27kip1 protein levels remain elevated in breast CSCs compared to non-breast CSCs. Based on these data, p27kip1 is a potential regulator of the stem-like nature breast CSCs and may serve as an attractive target in breast CSCs to generate effective therapeutic approaches to eradicate breast cancer.

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