DOI
https://doi.org/10.25772/QB7K-B453
Author ORCID Identifier
https://orcid.org/0009-0002-8102-4639
Defense Date
2025
Document Type
Thesis
Degree Name
Master of Science
Department
Microbiology & Immunology
First Advisor
Dr. Hisashi Harada
Second Advisor
Dr. Molly Bristol
Third Advisor
Dr. David Gewirtz
Abstract
Triple negative breast cancer (TNBC) is an aggressive subset of breast carcinomas that are classified by their lack of estrogen receptor, progesterone receptor, and HER2. Because of this, TNBCs are difficult to treat with conventional hormone therapy and are without available targeted treatment forms. In this study, we utilized the DNA-damaging agent, Doxorubicin (Dox), a drug commonly used to activate the intrinsic apoptotic pathway through disruption of topoisomerase-II mediated DNA-repair. Mediators of this pathway include proteins of the BCL-2 family. This inhibition of DNA-repair also leads to the development of therapy-induced senescence (TIS). Previous research on Dox treatment in human TNBC MDA-MB231 cells has shown increased susceptibility to anti-apoptotic BCL-2 family inhibitors by inducing senescence with Dox. Specifically, their sensitivity to the dual BCL-XL/BCL-2 inhibitor, ABT-263 (Navitoclax) and related senolytic agents was evident. In this project, we examined the effect of senolytics against mouse TNBC cell lines toward the ultimate goal of determining the efficacy and toxicity in syngeneic breast cancer mouse models. To examine the dosage of Dox that could possibly induce apoptosis while maintaining cell viability (IC50), we performed a WST-1 assay in TNBC cell lines. Based on WST-1 assay results, the ideal Dox concentrations were 0.5μM (4T1), 30nM (PyMT-230), and 1 μM (E0771). In order to observe whether this Dox dosage would be sufficient to induce senescence, X-Gal staining was performed in 4T1, E0771, and PyMT-230 using the respective dosages determined by the WST-1 assay. This staining was performed in control and Dox experimental populations, in which blue staining solely occurred in groups that underwent Dox Pirtle 9 treatment. While E0771 showed the most resistance to Dox based on the WST-1 results displayed, it showed a higher level of blue-staining in the Dox treated groups when compared to 4T1 and PyMT-230. To support our data quantitatively, Fluorescence Activated Cell Sorting (FACS) was performed with C12FDG, a substrate that when cleaved by β-galactosidase in the cell, produces a fluorescent stain, as a marker. In 4T1, there was a ~10 percent difference in fluorescence between the control and experimental populations, while E0771 groups showed a difference of approximately 15 percent. In PyMT-230, the difference of C12FDG expression between our control and Dox treated group was approximately 35 percent. To further support that this cell cycle arrest was taking place, quantitative PCR was performed using primers for p21, a marker for cell-cycle arrest, and CXCL10, a chemokine that acts as a SASP. In 4T1 and E0771, there was an upregulation of p21 observed once these cell lines were treated with Dox, displaying the occurrence of cell-cycle arrest when undergoing treatment. However, chemokine CXCL10 was upregulated in 4T1 and downregulated in E0771 once treated with Dox. This data led us to wonder whether different SASPs expression can vary between cell lines. After examining senescence-related data between these cell lines, we wanted to understand the effect that the senolytic agent ABT-263 would have on cell viability once senescence was induced. We compared the effects of ABT-263 against other BCL-2 family inhibitors: AZD-4320 (dual BCLXL/BCL-2 inhibitor), ABT-199 (selective BCL-2 inhibitor), and A1155463 (selective BCL-XL inhibitor). Crystal violet staining revealed that when exposed to the BCL-2 family inhibitors, 4T1 and E0771 were more sensitive to dual inhibitors ABT-263 and AZD-4320. Although PyMT-230 induced more senescence with Dox, the Dox-treated plate showed no difference between the control well and wells treated with BCL-2 family inhibitors. To confirm these results, Fluorescence Activated Cell Sorting was performed in TNBC cell lines using Annexin- Pirtle 10 V, a stain that fluoresces in apoptotic populations, and DAPI, which fluoresces in the presence of necrotic cells. We observed a substantial increase in apoptotic response with combination of Dox and ABT-263 treatments when compared to Dox or ABT-263 alone treatments in 4T1 and E0771. In PyMT-230, there is little to no difference between Dox and combination treated groups, supporting our crystal violet data. To further investigate, expressions of members of the BCL-2 family were compared against TNBC cell lines with and without Dox treatment. The data suggests that high basal levels of anti-apoptotic BCL-2 family member expression in PyMT230 cells may play a role in the cell lines insensitivity to BCL-XL/BCL-2 dual inhibitors, ABT-263 and AZD-4320. There is, however, increased BCL-XL expression in cell lines 4T1 and E0771, which may contribute to sensitivity when exposed to not only dual inhibitors but also A-1155463, a selective inhibitor of BCL-XL. This data led us to inquire whether protein expression of these members could be altered in TNBC cells when not only exposed to Dox alone, but also in the presence of dual-inhibitor ABT-263. In 4T1, E0771, and PyMT-230, the combination group showed decreased expression levels in BCL-XL in comparison to the Dox only group. There was also an increase in pro-apoptotic BAX in the combination groups of these cell lines. This in turn supported the suggestion that the high expression levels of anti-apoptotic BCL-XL, BCL-2, and MCL-1, may play a substantial role in lack of cell death in PyMT-230 when exposed to the selected senolytic agents. Based on the observations from this study, it is evident that Dox can induce senescence in TNBC cell lines. In combination with ABT-263, senolytic treatment increased apoptosis in senescent 4T1 and E0771 cells.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-7-2025