Date of Award
Fall 8-22-2025
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Molecular Biology
First Advisor
Irida Kastrati
Abstract
Triple negative breast cancer (TNBC) makes up only 15-20% of all breast cancer cases, yet it contributes to almost 50% of all breast cancer related mortalities. This highlights the urgent need to develop novel therapeutics for the treatment of TNBC. The thioredoxin (Trx) system is an important cellular antioxidant pathway supported by the enzyme thioredoxin reductase 1, TXNRD1. Upon reduction by TXNRD1, Trx regulates many cellular functions including redox homeostasis and DNA replication and repair through ribonucleotide reductase (RNR), which catalyzes the rate limiting step in the de novo synthesis of dNTPs. Notably, TXNRD1 is elevated in TNBC, thus presents a druggable vulnerability. We identified novel non-covalent inhibitors, TXNRD(i)s, that bind to a unique allosteric site, the “doorstop pocket”, and have previously shown they exhibit anti-cancer activity in TNBC. This study focuses on characterizing the mechanisms of anti-cancer effects elicited by these inhibitors to guide future drug optimization and development in TNBC. To identify major pathways affected, we conducted transcriptomics in TNBC cells treated with two lead TXNRD(i)s or siTXNRD1. Bioinformatic analysis revealed enrichment of oxidative stress (ROS) along with multiple proliferation and cell cycle pathways. Although TXNRD(i) treatment increased intracellular ROS levels, this elevation did not correlate with reduced cell viability. Furthermore, treatment with antioxidant ROS scavengers failed to rescue the TXNRD(i)-induced effects, indicating that elevated ROS is not the primary driver of their cytotoxic activity. We next evaluated the impact of TXNRD(i)s on TNBC cell proliferation and observed a marked reduction in DNA synthesis, accompanied by G1 phase cell cycle arrest. Given that Trx supports ribonucleotide reductase (RNR) activity, we tested whether exogenous supplementation with dNTPs, the products of RNR, could reverse these effects. Indeed, dNTP supplementation fully rescued the cell cycle arrest and significantly restored cell viability while reducing apoptosis and DNA damage. These findings strongly support the conclusion that disruption of RNR function is the key mechanism underlying the anti-cancer effects of TXNRD(i)s in TNBC. Overall, our study challenges the prevailing view that TXNRD1 inhibitors primarily act through pro-oxidant mechanisms and instead highlights a critical role for the TXNRD-Trx-RNR axis in sustaining TNBC cell proliferation and survival.
Recommended Citation
Rullo, Abigail, "Uncovering the Mechanisms of the Anti-Cancer Activity of New Non-Covalent Thioredoxin Reductase inhibitors in Triple Negative Breast Cancer" (2025). Dissertations. 4264.
https://ecommons.luc.edu/luc_diss/4264
