Nrf2-mediated glutathione pathways protect mesenchymal, metastatic breast cancer cells from ferroptosis

Breast cancer is the second leading cause of death in women worldwide. Although novel therapies have greatly advanced the clinical outcome for cancer patients, metastatic breast cancer still remains a deadly disease due to the capacity of cancer cells to spread throughout the body leading to metastasis formation, particularly in the lung, liver, brain and bones. While molecular strategies employed by metastatic cells to leave the primary tumor, intravasate, and to disseminate to distant organs and grow into colonies are being intensively investigated, little is known about what prompts tumor cells to leave the primary tumor, or what kind of environmental pressure operates on cells bound to metastatic fate. These need to be better understood to enable the development of new therapeutic strategies for metastatic breast cancer patients.
Epithelial-to-mesenchymal transition (EMT), a well conserved cellular program which is critical for embryonic development and wound healing, is known to play a central role in the multiple stages of metastasis formation, where cancer cells dedifferentiate, acquire migratory and invasive capacities by disassembling their cellcell and cell-matrix contacts to reach blood vessels, and spread at a distance. In addition to cancer-intrinsic mutations, malignant tumor progression is also critically influenced by a defined tumor environment, such as hypoxia and inflammation. Notably, hypoxic and inflammatory niches can invariably induce an EMT in cancers, which is coupled to the generation of toxic reactive oxygen species (ROS) and their clearance inside cancer cells. However, the interplay between ROS and the EMT
program is not sufficiently understood. Therefore, I hypothesize that metastasis represents a strategy for cancer cells to avoid oxidative damage and to escape from the excess or ROS in the primary tumor.
In the past years, I have aimed at exploring the crosstalk between ROS and an EMT induced by TGFβ in breast cancer cells. I observed that cells exposed to TGFβ displayed a decrease of ROS levels. To identify the antioxidant pathways activated during EMT, I investigated the effect of long-term oxidative stress, for example by H2O2, on epithelial cells and found that this oxidative treatment promoted both higher
migratory capability and tumorigenicity of epithelial cells. By RNA-sequencing analysis, I identified an activation of the Nrf2 transcription factor in H2O2-resistant cells, as well as in TGFβ-induced mesenchymal cells. Functional experimentation demonstrated an activation of Nrf2 in migrating cells and pinpointed its antioxidant role during EMT. Notably, RNAi-mediated ablation of Nrf2 led to mesenchymal cancer cell death due to ferroptosis, an iron- and oxidative stress-dependent cell death, while differentiated epithelial cancer cells remained unaffected. Moreover, RNA sequencing analysis revealed that several glutathione pathway genes were specifically regulated
by Nrf2, suggesting a role of glutathione-related pathways in mesenchymal cell survival. Additionally, further RNA sequencing analysis highlighted a Nrf2-signature correlating with poor prognosis in breast cancer patients. In the line these observations, I show that the inhibition of glutathione synthesis leads to mesenchymal cell death by ferroptosis in vitro, and decreases primary tumor growth and lung metastasis formation in vivo. Hence, targeting antioxidant pathways may be beneficial for breast cancer patients with metastatic disease.
In summary, my Ph.D. work provides novel insights into mesenchymal and metastatic breast cancer cell sensitivity toward ferroptosis and their addiction for antioxidant pathways via Nrf2-regulated glutathione synthesis and function. This novel understanding pinpoints the importance of antioxidant pathways in metastatic breast cancer and should be considered as a promising choice of novel therapy.