Oxidative stress and its end-products, such as 4-hydroxynonenal (HNE), initiate activation of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)/Kelch Like ECH Associated Protein 1 (KEAP1) signaling pathway that plays a crucial role in the maintenance of cellular redox homeostasis. However, an involvement of 4-HNE and NRF2 in processes associated with the initiation of cancer, its progression, and response to therapy includes numerous, highly complex events. They occur through interactions between cancer and stromal cells. These events are dependent on many cell-type specific features. They start with the extent of NRF2 binding to its cytoplasmic repressor, KEAP1, and extend to the permissiveness of chromatin for transcription of Antioxidant Response Element (ARE)-containing genes that are NRF2 targets. This review will explore epigenetic molecular mechanisms of NRF2 transcription through the specific molecular anatomy of its promoter. It will explain the role of NRF2 in cancer stem cells, with respect to cancer therapy resistance. Additionally, it also discusses NRF2 involvement at the cross-roads of communication between tumor associated inflammatory and stromal cells, which is also an important factor involved in the response to therapy.
It has been proposed that excessive production of reactive oxygen species (ROS) and numerous cellular redox adaptation responses are involved in cancer initiation, progression, and drug resistance. Nevertheless, persistent oxidative stress renders tumor cells increasingly vulnerable to additional stressors and reverses resistance to treatment. Accordingly, redox perturbation could be instrumental in the selective elimination of cancer cells.
ROS (Reactive Oxygen Species) are oxygen-containing molecules formed by reduction/oxidation reactions (redox reactions) or electronic excitation. Key ROS molecules include hydroxyl and superoxide free radicals and nonradical molecules, such as hydrogen peroxide. When ROS production increases or their scavenging by antioxidants decreases, cells undergo a process of oxidative stress. Several growth factors and cytokines regulate ROS production in the mitochondria. This regulation is mainly via the electron transport chain, where oxygen is reduced to form superoxide anion, peroxisomes (through the β-oxidation of fatty acids), and endoplasmic reticulum (through the oxidation of proteins). Exposure to exogenous agents, including radiation, heavy metals (especially transition metals such as iron, or metal complexes), atmospheric pollutants, and various chemicals (including xenobiotics and especially chemotherapeutic agents), leads to increased production of ROS.
Although potentially very harmful, even cytotoxic, ROS are crucial for cellular life. Namely, if present in moderate concentrations, ROS act as second messengers in the transduction of extracellular signals and in the control of gene expression related to cellular proliferation, differentiation, and survival. At higher levels, ROS are also produced by cells as defense agents against pathogens. Excessively high cellular levels of ROS can cause damage to proteins, nucleic acids, lipids, membranes, and organelles, which may lead to the activation of such cell death processes as apoptosis.
Several lines of evidence prove that ROS can cause DNA damage and contribute to occurrence of oncogenic mutations. Cancer cells, through their aberrant energy metabolism, commonly produce higher levels of ROS than normal cells. An increased level of ROS is associated with the activation of oncogenes, the inactivation of tumor suppressor genes, and mitochondrial malfunction. Genotoxic stress has recently been shown to be a trigger of an inflammatory signaling cascade which results in the release of pro-inflammatory factors and an increase in the amount of infiltrating immune cells. These events additionally contribute to ROS production and lead to the occurrence of a vicious circle of carcinogenic oxidative stress.
Under such circumstances, ROS serve cancer as pro-growth signaling molecules, also triggering a self-catalyzed chain reaction process of lipid peroxidation of polyunsaturated fatty acids (PUFAs), in particular. The strongly induced peroxidation of PUFAs generates reactive aldehydes, molecules which are much more stable than ROS themselves. Therefore, they are considered to be “second messengers” of ROS. Among such reactive aldehydes, 4-hydroxynonenal (HNE), the end-product of n6-polyunsaturated fatty acid peroxidation is considered to be one of the most bioactive aldehydes. HNE has the ability to modify various cellular signaling pathways and processes. It exerts its activity by binding to the cysteine, histidine, arginine, and lysine moieties of proteins changing their activity. Additionally, HNE can bind to DNA, thereby causing mutations but also to lipids. The effects of HNE are concentration- and cell type-dependent.
The type of response may be dependent on the metabolism of HNE. It is primarily detoxified through conjugation with glutathione (GSH). Thereafter, this complex is exported through RBP1 (RalA-Binding Protein 1). In low concentrations, HNE binds to proteins involved in signaling pathways or modulates proteins involved in the previously mentioned biological processes.
Interactions of HNE with cellular proteins, lipids and DNA. HNE is an end-product of n6-polyunsaturated fatty acids (n6-PUFA) peroxidation which acts in a concentration dependent manner. Low concentrations: interaction with DNA (black arrow) results in forming exocyclic guanine adducts. High concentrations: occurrence of sister chromatid exchange, DNA fragmentation, and inhibition of nucleotide excision repair. HNE also binds to membrane lipids (purple arrow). Interactions with proteins are much more complex, as HNE directly or indirectly causes an increase (green arrows) or decrease (red arrow) in the activity or expression of Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB), Cyclooxygenase 2 (COX2), Hypoxia-Inducible Factor (HIF1) Vascular Endothelial Growth Factor (VEGF), TP53 and Epidermal Growth Factor Receptor (EGFR). Interactions with NRF2 will be reviewed separately.
For maintaining redox homeostasis and limiting cellular damage, eukaryotic cells have developed mechanisms for the tight regulation of ROS levels. They are based on a complex scavenging system containing superoxide dismutases (SODs), catalases, thioredoxins, peroxiredoxins, and glutathione peroxidases. Non-enzymatic antioxidants, such as glutathione (GSH), vitamin C (ascorbate), vitamin E (tocopherols), and polyphenols also act directly on ROS and other pro-oxidative agents.
However, some clinical trials and experimental models suggest that dietary supplementation with antioxidants, especially carotenoids and vitamin E, could increase cancer incidence and cancer-related deaths in humans. On the one hand, this may be due to the abuse of these antioxidants, followed by an increase of their concentrations above the physiological level, and their conversion into metabolites which interfere with cellular metabolism. On the other hand, cancer cells, in parallel with an increase of ROS, also increase their unique antioxidative capacities. In this way, cancer cells optimize ROS-driven proliferation and avoid ROS thresholds that would otherwise trigger cellular death.
In response to an excessive ROS production, cancer cells develop several transcriptional programs which rely on transcription factors/their binding partners that contain redox-responsive cysteines. These programs include members of the Forkhead Box Protein O3 (FOXO) family, Hypoxia Inducible Factors (HIFs), Kelch-Like ECH-Associated Protein 1 (KEAP1) with NRF2 and the Tumori protein P53 (TP53) tumor suppressor-related transcriptional program.
In healthy tissues, a transient activation of NRF2 has been long recognized as a
