Hypoxia-mediated tumour microenvironment in hormone receptor-negative breast cancer

Numprasit, Warapan (2025) Hypoxia-mediated tumour microenvironment in hormone receptor-negative breast cancer. PhD thesis, University of Glasgow.

Abstract

Summary.
Breast cancer is the most common malignancy in women, causing approximately 670,000 deaths annually worldwide. Prognostic and predictive markers largely rely on the expression of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). ER/PR-negative subtypes represent a more aggressive form of the disease, with a higher likelihood of relapse compared to ER/PR-positive cases.

Hypoxia is a hallmark of solid tumours, driving cancer aggressiveness and treatment resistance. It is preferentially associated with ER/PR-negative breast cancer. Carbonic anhydrase IX (CAIX) is a well-established hypoxic marker linked to poor survival outcomes in breast cancer, particularly in ER/PR-negative cases. Currently, a CAIX-targeted inhibitor is under investigation in a phase I clinical trial for advanced solid tumours. This study aimed to explore the CAIX-associated gene signature and identify novel hypoxia-related therapeutic targets in breast cancer.

Bulk RNA sequencing (Tempo-Seq) analysis was conducted on 131 ER/PR-negative breast cancer samples with available CAIX immunohistochemistry (IHC) data from the Glasgow Breast Cancer Cohort. Differential gene expression analysis comparing high versus low CAIX expression identified four significantly upregulated genes: NDRG1, CA9, VEGFA, and PPFIA4. Gene Set Enrichment Analysis (GSEA) revealed that hypoxia and glycolysis were the top two enriched pathways. Additionally, protein-protein interaction analysis using the STRING tool showed moderate interactions between the identified genes with the exception of PPFIA4.

The expression of NDRG1, CA9, VEGFA, and PPFIA4 was validated at both the mRNA and protein levels in vitro using MDA-MB-231, SKBR-3, and MCF-7 breast cancer cell lines. Under hypoxic conditions (1% O₂), all four genes exhibited increased mRNA expression. At the protein level, CAIX and NDRG1 were upregulated in hypoxia, thus NDRG1 warranted further investigation.

Immunohistochemistry analysis of NDRG1 was performed on tissue microarrays (TMA) from the full Glasgow Breast Cancer Cohort. High cytoplasmic NDRG1 expression correlated with significantly poorer overall survival (OS) and cancer-specific survival (CSS). However, when stratified by ER/PR status, NDRG1 expression did not significantly impact survival outcomes. Several limitations should be noted: TMAs might not fully represent whole tumour sections, and there is no standardised cutoff for high versus low NDRG1 expression, which may introduce bias. Additionally, the lack of an independent validation cohort highlights the need for further studies.

To explore the functional role of NDRG1 under hypoxic conditions, it was hypothesised that NDRG1 contributes to aerobic glycolysis, similar to CAIX, as suggested by GSEA. Metabolic assessments were performed using Seahorse assays to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Additionally, lactate and ATP assays were conducted to validate the Seahorse findings. Silencing NDRG1 led to decreased lactate production (a glycolysis byproduct) in MDA-MB-231 and MCF-7 cells. However, its impact on ECAR varied: no effect was observed in MDA-MB-231 cells, while ECAR increased in MCF-7 cells. Silencing NDRG1 increased OCR and ATP production in MCF-7 cells but had no effect in MDA-MB-231 cells. These findings suggest that NDRG1 plays a role in glycolytic pathways under hypoxic conditions, but its function may vary depending on the molecular background of the different breast cancer subtypes. Further studies are needed to investigate alternative metabolic pathways, such as fatty acid oxidation and glutamine metabolism, which may be mediated by NDRG1 under hypoxic conditions, particularly in MDA-MB-231 cells.

This study identified NDRG1, CA9, VEGFA, and PPFIA4 as differentially expressed genes associated with CAIX-driven hypoxia in breast cancer. NDRG1 emerged as a potential hypoxic biomarker, as high expression correlated with worse OS and CSS in the Glasgow Breast Cancer Cohort. Additionally, NDRG1 was upregulated in hypoxia and played a role in aerobic glycolysis. Silencing NDRG1 alleviated glycolysis by reducing lactate production and enhanced mitochondrial respiration by increasing OCR. Further studies are warranted to explore the mechanistic role of NDRG1 in hypoxia-driven metabolism and to validate these findings in independent breast cancer cohorts.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Edwards, Professor Joanne, Quinn, Dr. Jean A. and Thuwajit, Professor Chanitra
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85444
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 Sep 2025 09:01
• Last Modified: 10 Sep 2025 09:03
• Thesis DOI: 10.5525/gla.thesis.85444
• URI: https://theses.gla.ac.uk/id/eprint/85444

Related URLs

• Article DOI