The Effect of Chronic Intermittent Hypoxia on Breast Cancer Cell Gene Expression and Malignant Properties

Abstract

Breast cancer is the most common cancer in women worldwide and the second most common cancer overall. The most aggressive and most lethal form of breast cancer is inflammatory breast cancer (IBC). The mechanisms underlying the aggressiveness of IBC are still poorly understood. A key feature in the development and progression of breast cancer is the tumor microenvironment that is, amongst others, characterized by a specific composition of the extracellular matrix (ECM) and by the occurrence of tissue hypoxia and oxidative stress. Due to vascular remodeling, leading to structurally and functionally abnormal tumor vessels, tumor cells are exposed to alternating periods of hypoxia and reoxygenation, called chronic intermittent hypoxia (CIH). CIH is a cause of increased reactive oxygen species (ROS) production and has been identified as an important factor in promoting tumor progression and metastasis. However, the molecular mechanisms through which CIH increases the aggressiveness of breast cancer still need to be elucidated. Here, we aimed to assess the impact of CIH on gene expression and cancer cell malignant properties in IBC and non-IBC cell lines. The triple-negative IBC cell line SUM149PT and the estrogen receptor-positive cell line T47D were exposed to 20 cycles of intermittent hypoxia (24 h 0.2% O2, 48 h 21% O2). The effects of CIH on the transcriptomic profile were analyzed in order to obtain insights into signaling pathways, which might be involved in the repeatedly reported enhancement of tumor aggressiveness upon CIH. CIH caused distinct changes in gene expression in both cell lines, with a much higher number of differentially expressed genes in SUM149PT cells compared to T47D cells. In T47D cells, we positively validated the differential gene expression of two ECM proteins which have been linked to tumor progression. In SUM149PT cells, CIH caused a strong upregulation of pro-metastatic genes encoding ECM proteins and inflammatory mediators. We positively validated the CIHmediated increased mRNA and protein expression of the ECM protein tenascin-C (TNC), a key factor in tumor progression. Additionally, for the first time we identified an oxidative stress mediated regulation of TNC in IBC cells, which was dependent on activation of the NF-κB pathway but not on activation of other redox signaling pathways. The observed changes in gene expression suggest an enhanced metastatic potential of SUM149PT cells. Performing in vitro assays to analyze the cancer cell malignant properties, we observed a trend towards a differential attachment behavior of SUM149PT cells following CIH. In summary, this thesis identifies CIH in mediating tumor promotive gene expression changes. Furthermore, CIH and oxidative stress may play an important role in the constitutive activation of NF-κB in IBC, which has repeatedly beenv described in the literature