Date of Award

4-22-2025

Embargo Period

4-22-2027

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Peggi Angel

Abstract

Breast cancer is the most common type of cancer diagnosed in American women as of 2020. Despite White women (WW) having the highest incidence rates compared to any other racial/ethnic group in the United States, Black women (BW) have the highest overall mortality rates. One major difference between breast cancer diagnosed in BW and WW is the aggressiveness of the malignancy, with BW having over twice the frequency of triple-negative breast cancer (TNBC). TNBC, characterized by the lack of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2), is considered aggressive due to increased proliferation, metastatic potential, recurrence, and ineffective treatment options. Research demonstrates that the disparity in breast cancer mortality between racial groups is not solely attributable to healthcare inequities, but also to distinct tumor characteristics, raising the question of whether biological markers exist within normal breast tissue that may predispose Black women to more aggressive breast cancer subtypes. One important regulator of the tumor microenvironment is the collagen-rich extracellular matrix (ECM), which acts as a dynamic scaffold that influences cell fate and tissue function by mechanical and biochemical signaling. The organization of collagen fibers within the ECM, particularly fibers that project perpendicularly from the tumor, has been associated with worse outcomes in breast cancer due to enhanced tumor cell invasion and metastasis facilitated by this ECM architecture. Although the physical structure of collagen organization has been explored in breast cancer, the molecular composition of collagen proteins, including post-translational changes, has not been well defined for the breast normal or tumor microenvironments (NME/TME). Post-translational modifications (PTMs) within collagen domains, especially proline hydroxylation, have the potential to modulate cellular interactions, function and signaling pathways. Our central hypothesis is that there are specific collagen signatures that characterize increased risk in healthy breast and associate with worse clinical outcomes in TNBC. This work defines collagen signatures within the breast NME from genetic ancestry-defined healthy donors (n = 20 BW and n = 20 WW) and TME from triple-negative breast cancer tumors (n = 78 BW). The combination of ECM and microenvironment spatial evaluation is accomplished through optimized multimodal, multiplexed imaging techniques including ECM-targeted mass spectrometry imaging, antibody-based mass spectrometry imaging – immunohistochemistry, and second harmonic generation microscopy. The assessment of the normal breast, including extracellular and cellular markers, highlighted similarities in the microenvironment based on genetic ancestry alone. However, when clinical risk factors were considered, especially body mass index (BMI), ancestry-dependent differences were observed. These differences include opposing regulation of the physical attributes of collagen fibers, with fiber widths increasing as BMI increases for BW (overweight, OW vs obese, OB, p = 0.008) but decreasing as BMI increases for WW (OW vs OB, p = 0.012). Intensities of collagen peptides within the normal breast widely differed based on BMI; 214 collagen domains showed differential intensities when comparing normal breast from overweight and obese women (FDR p < 0.025). Ancestry-based differences persisted within BMI categories, identifying significantly different peptide intensities when comparing overweight BW and WW (8 peptides, p < 0.05) and obese BW and WW (3 peptides, p < 0.05). Overall, fibrillar collagen proteins, Col11, Col12, and Col31, (37 modified peptides vs 10 unmodified peptides on average), generally expressed decreased intensities in PTM-regulated domains compared to unmodified domains. A similar trend in collagen composition was demonstrated in TNBC samples, with slightly lower intensities of fibrillar collagen domains containing hydroxylated prolines but increases in the number of hydroxylated peptides detected (64 modified domains vs 15 unmodified on average). In a cohort of BW with TNBC, collagen characteristics, including length, width, and curvature varied based on clinical characteristics. Molecular characteristics of collagen within the TNBC TME revealed trends in intensities of collagen domains containing known receptor binding motifs, showing differential expression based on survival status. Further analyses demonstrated that fourteen ECM peptides differentiated the probability of survival, with low intensities relating to better outcomes (log-rank p < 0.05). Novel work considering the immune profiles and the surrounding ECM types as a holistic neighborhood showed distinct microenvironment composition that varied by survival status. This work is one of the first investigating the proteomic profile of the collagen ECM within the ancestry-defined normal breast and TNBC with high molecular detail. The proline hydroxylation PTM variation defined in the normal and TNBC tissue microenvironment provides insight into regulation of the cell-ECM communication that impacts breast tissue homeostasis and pathological development and progression of cancer. Additionally, the identification of novel ECM peptide domains that discriminant survival status in TNBC has the potential for further study as biomarkers or therapeutic targets.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Thursday, April 22, 2027

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