Document Type

Dissertation - Open Access

Award Date

2023

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Rachel Willand-Charnley

Abstract

Multidrug resistance (MDR) remains a major challenge in cancer treatment, accounting for over 90% of chemotherapeutic failures. Cancers utilize sugar residues to engage in multidrug resistance. The underlying mechanism of action involving glycans, specifically the glycan sialic acid (Sia) and its various functional group alterations, has not been explored. ATP-binding cassette (ABC) transporter proteins, key proteins utilized by cancers to engage in MDR pathways, contain Sias in their extracellular domains. Modulating the expression of acetylated-Sias on Breast Cancer Resistance Protein (BCRP), a significant ABC transporter implicated in MDR, in lung and colon cancer cells directly impacted the ability of cancer cells to either retain or efflux chemotherapeutics. Acetylation was modulated by the removal of CAS1 Domain-containing protein (CASD1) and Sialate O-Acetyl esterase (SIAE) genes via CRISPR-Cas 9 gene editing. Using a variety of cell and molecular based approaches, we confirmed that deacetylated Sias regulated a MDR pathway in colon and lung cancer in early in vitro models. When deacetylated Sias were expressed on BCRP, colon and lung cancer were able to export high levels of BCRP to the cell’s surface, resulting in an increased BCRP efflux activity, reduced sensitivity to the anticancer drug Mitoxantrone, and high proliferation relative to control cells. These observations correlated with increased levels of cell survival proteins, BcL-2 and PARP1. Further studies also implicated the lysosomal pathway for the observed variation in BCRP levels among the cell variants. RNAseq data analysis of clinical samples revealed higher CASD1 expression as a favorable marker of survival in lung adenocarcinoma. Using same in vitro models, we further explored the role deacetylated Sia plays in the EGFR targeted therapy. Specifically, we investigated how deacetylated Sia modulated the activity of three characterized novel Cucurbitacin-Inspired Estrone analogs (CIEAs: MMA 294, MMA 321, and MMA 320) with potent anti-proliferative and dual inhibitory activities, targeting the EGFR and MAPK pathways. Compared to control cells, the CIEAs elicited a ˜2 to 17 -fold sensitivity, induced a G2/M cell cycle arrest and apoptosis in deacetylated Sia-expressing knockout cancer cells. Further studies implicated overexpression of CIEAs’ cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that deacetylated Sia is utilized by colon and lung cancers to engage in MDR via overexpression and efflux action of BCRP and renders lung and colon cancer cells susceptible to EGFR therapeutics. This study provides great insights for future therapeutic interventions.

Library of Congress Subject Headings

Breast -- Cancer -- Treatment.
Sialic acids.
Drug resistance in cancer cells.
Protein-tyrosine kinase -- Inhibitors -- Therapeutic use.

Publisher

South Dakota State University

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Rights Statement

In Copyright