The Role of Inflammation and Nitric Oxide Synthase 2 in Triple-Negative Breast Cancer Metastasis

High expression of inflammatory marker nitric oxide synthase 2 (NOS2) expression in tumor cells indicates poor prognosis, poor survival, and chemotherapeutic resistance in triple-negative breast cancer (TNBC) patients. The tumor microenvironment (TME) comprises various cell types that play a role in tumor survival and invasion by providing soluble factors such as pro-inflammatory cytokines IFN-γ, IL-1β, and TNF-α, which are known to drive NOS2 expression. NOS2 is an enzyme that generates nitric oxide (NO), which is suggested to drive tumor progression via increased tumor cell motility and invasion. This dissertation investigates a novel role for NOS2/NO in TNBC, focusing on the impact of NOS2 expression in tumor cells. By optimizing a hemoglobin-based NO detection assay, we determined that TNBC cell line produce NO at levels comparable to that of the RAW 264.7 macrophage cell line, reaching up to 1.3 pmol NO/min/g total cellular protein. We found that CRISPR/Cas9-mediated knockout of the NOS2 gene in the 4T1 parental cell line significantly suppresses lung metastasis from primary 4T1 tumors in Balb/c mice, providing direct evidence that inhibition of NOS2 in tumor cells can suppress metastatic progression in TNBC. We used flow cytometry to immune profile the primary orthotopic 4T1 mammary tumors, confirming the presence of inflammatory cytokine expression in the TME. Our findings suggest that tumoral NOS2/NO modulates the TME by promoting the polarization of tumor-associated macrophages (TAMs) toward a pro-tumorigenic and immunosuppressive M2-like phenotype. We also found that cytokine-induced NOS2/NO enhanced m6A-mRNA methylation, suggesting a potential mechanism by which NOS2/NO dysregulates the epitranscriptomic process by inhibiting demethylase FTO, thereby contributing to the metastatic progression of TNBC. These findings indicate that NOS2/NO is a key regulator of TNBC metastasis, thereby making it a promising therapeutic target for preventing the spread of this aggressive breast cancer subtype.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Graduate College

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2025

Author dc:creator

• Daw, Jennifer

Advisors dc:contributor.advisor

• Montfort, William
• Heimark, Ronald

Committee members dc:contributor.committeemember

• Mouneimne, Ghassan
• Smith, Catharine

• Inducible Nitric Oxide Synthase
• Inflammation
• Metastasis
• Nitric Oxide
• Nitric Oxide Synthase 2
• Triple-negative Breast Cancer

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10150/675779

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/675779