Date of Graduation

8-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Du, Yuchun

Committee Member

Pinto, Ines

Second Committee Member

Erf, Gisela F.

Third Committee Member

Lessner, Daniel J.

Keywords

Breast cancer; Cancer; Carcinomas; Metastasis; Proteins; RNAs

Abstract

Carcinomas are malignancies of epithelial cells and account for >80% of cancer-related deaths. Approximately 75% of breast cancers express estrogen receptor alpha (ERα). ERα promotes breast cancer progression by enhancing cancer cell proliferation. Tamoxifen is commonly used to treat ERα-positive breast cancers but can result in endocrine therapy resistance. The mechanisms underlying endocrine therapy resistance remain elusive. The Du lab recently found that ERα is an RNA-binding protein. However, the significance of the ERα-RNA interaction is unknown. We fractionated ERα-positive MCF7 cells into cytoplasmic and nuclear fractions, followed by immunoprecipitation of ERα with associated RNAs and RNA-sequencing. We identified many potential ERα-interacting RNAs in the cell’s cytoplasm and nucleus. A bioinformatics analysis of the identified RNAs resulted in 15 strong RNA candidates highly likely to associate with ERα in breast cancer cells. When 3 identified/selected RNAs were tested for their interactions with ERα by an in-vitro binding assay, all were found to bind specifically to ERα. Exosomes are nanoparticles released by cells containing various molecules and participate in cellular processes and diseases. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are proteins anchored to cellular membranes by GPI modifications. GPAA1 catalyzes the GPI anchor and is upregulated in breast cancer. Using a quantitative proteomics approach, we characterized the exosomal proteins from breast cancer MDA-MB-231, noncancer MCF-10A, and GPAA1-knockout MDA-MB-231 cells to identify the exosomal GPI-APs whose GPI modifications are controlled by GPAA1. We found that exosomal proteins from MDA-MB-231 and MCF-10A cells were completely different, with 37 and 12 potential GPI-APs controlled by GPAA1, respectively. This highlights the role of GPAA1 in breast cancer progression. Subsequent analysis of several GPI-APs exclusively localized in exosomes from MDA-MB-231 cells revealed altered expressions in clinical samples of breast and/or other cancer types. Further analysis of these 37 proteins is warranted as they may serve as biomarkers for breast cancer detection and potential therapeutical targets for treating breast and other types of cancers. The E-cadherin (E-cad) protein participates in adherens junctions and is essential for the integrity of epithelial tissues. A hallmark of carcinoma metastasis is the loss of E-cad expression, resulting in the epithelial-mesenchymal transition (EMT). However, this assertion is controversial. Many carcinomas express E-cad and metastasize successfully. We analyzed large bioinformatics databases of clinical cancer samples and cell lines. We found that CDH1 mRNA (which codes for E-cad) and E-cad protein levels were not downregulated in most carcinomas analyzed, disputing textbook knowledge of E-cad in EMT. Interestingly, the CDH1 mRNA was upregulated during the early stages of several carcinomas, implying a potential diagnostic value of CDH1 mRNA for early detection of these carcinomas. In this project, we explored two novel aspects of breast cancer: ERα-interacting RNAs and GPAA1-controlled GPI-APs in exosomes from breast cancer cells. Additionally, we employed a systematic bioinformatics approach to understand the contentious role of E-cadherin in carcinoma metastasis, including breast cancer. Our conclusions offer insights into the mechanisms underlying breast cancer formation and progression. Moreover, the findings may lead to innovative approaches for breast cancer diagnosis and treatment.

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