The Multifaceted and Sometimes Paradoxical Nature of Reactive Oxygen Species in Cancer

Reactive oxygen species (ROS) are a feature of all living eukaryotic cells, normal or cancerous. Maintaining redox homeostasis is a complex and delicate balance involving numerous biochemical production and defense systems. Often mentally relegated to ‘basal’ or ‘housekeeping’ status, the chemistry and biology of ROS are actually subtle, intricate, and not limited to metabolism but also involved in signaling in a variety of ways. ROS are, by their nature, toxic to cells because they are highly reactive and damaging to biological macromolecules, and therefore, appropriate balance is essential. Regulation and disruption of redox balance are also powerful and growing venues of anti-cancer medicine and research.

Where Do ROS Come From?

There are three main sources of production of ROS within living cells (Wu et al., 2024) – see Figure 1.

• Mitochondrial ATP production, via oxidative metabolism and the citric acid cycle (TCA) produces superoxide radical (O2•^–).
• Peroxisomes, while fulfilling their roles in lipid metabolism, are another highly oxidative organelle and produce hydrogen peroxide (H₂0₂).
• Various NADPH oxidases (NOX) localized to specific membrane locations produce O2•^– and some H₂0₂.

Most cellular O2•^– are transformed into H₂0₂ by the action of superoxide dismutase (SOD) enzymes specific to cellular compartments. Besides these main sources, the endoplasmic reticulum produces some ROS in support of oxidative protein folding. Though not an initiator of ROS production, the Fenton reaction can use Fe2+ to produce hydroxyl radicals (HO•^–) from H₂0₂. All of these ROS species are capable of damaging cellular structures (Cheung and Vousden, 2022). O2•^– can damage iron cluster proteins. HO•^– radicals can irreversibly damage proteins, DNA, and lipids. In fact, HO•^– damage to polyunsaturated fatty acids (PUFA) is the initiator step in the self-sustaining chain reaction of lipid hydroperoxidation that ultimately leads to ferroptosis and cell death (Wu et al., 2024).

Figure 1: Generation and Metabolism of ROS (Source: Wu et al., 2024).

What Systems Control the Levels of ROS?

There are multiple redox systems that cells use as antioxidant defense against ROS. The more prevalent player is the glutathione system centered around the simple and ubiquitous glutathione molecule (GSH in its reduced form). In addition, there are a series of interconnected and complementary antioxidant systems, including thioredoxin (TXN), peroxiredoxin (PRDX), and sulfiredoxin (SRX), that regulate redox balance in a cooperative way with GSH. Also, the ubiquitous enzyme catalase (CAT) can reduce H₂0₂ to 0₂ and water. The molecular currency of reductive potential in the cell is the cofactor Nicotinamide Adenine Dinucelotide Phosphate (NADPH in its reduced form). A number of sources in cellular metabolism contribute NADPH to the redox balance, including the oxidative pentose phosphate pathway (oxPPP). Additionally, isocitrate dehydrogenases (IDH) and malic enzymes (ME), as well as one-carbon metabolism (centered on the folate cycle), all contribute reductive capacity (Wu et al., 2024).

Hallmarks of Cancer Connections to ROS

The hallmarks of cancer structures were first laid out more than 20 years ago and have been updated, as recently as 2022 by Hanahan. Within this framework, ROS plays roles in many of the hallmarks (Wu et al., 2024). In particular, ROS contributes to the original core hallmarks, sustaining proliferative signaling, resisting cell death, inducing or accessing vasculature, and activating invasion and metastasis. ROS is also involved in the later defined core hallmarks, deregulation of cellular metabolism and avoiding immune destruction. In addition, ROS is involved in the so-termed ‘enabling’ hallmark of genomic instability and mutation.

Balance is Everything

As stated above, ROS production is obligatory for living cells and can participate in processes integral to cancer. Interestingly, some ROS production can be cancer-promoting and some can be cancer-limiting. ROS can be supportive in early phases of tumorigenesis due to its damaging effects on DNA and follow-on genomic instability (Wu et al., 2024). Counter to this is the fact that excessive ROS production can result in triggering regulated cell death or senescence, in particular via ferroptoisis (Cheung and Vousden, 2022).

ROS molecules can activate signaling cascades that promote growth and proliferation, thus contributing to tumorigenesis. In particular, the nuclear factor erythroid 2–related factor 2 (NRF2), as well as BTB and CNC homology 1 (BACH1) signaling pathways, are important regulatory circuits for maintaining redox homeostasis but also play roles in tumorigenesis (Wu et al., 2024).

Some ROS production is required for cell migration signaling, involving selected NOX activity, and cancer cell invasion potential is dependent on this same signaling cascade. However, there are cases where ROS production can suppress invasion contextually, so spatiotemporal regulation is key. ROS can promote epithelial-to-mesenchymal transition (EMT) and also cancer stem cell (CSC) trait acquisition, thus promoting survival, progression, and invasive potential of cancer. Counter to this are cases of CSC selectively surviving drug treatment via being able to limit ROS levels. ROS overproduction can limit growth and survival of cells in blood circulation due to the highly oxidative environment, but this is less true in static tumor environments (TME). Cancer cells, able to migrate through the lymphatic system, are relatively protected due to anti-oxidant environmental factors, like lower free iron, elevated GSH, and oleic acid content (Cheung and Vousden, 2022).

Ferroptosis: a Significant Pathway in Cancer Biology

Of the multiple regulated cell death pathways that can be influenced by ROS, ferroptotis has been a hot topic recently. Ferroptosis is an iron-dependent form of cell death and is characterized by the accumulation of lipid hydroperoxides. Ferroptosis is limited by the GSH system, specifically glutathione peroxidase 4 (GPX4), which reduces lipid hydroperoxides, and many cancers show increased reliance on GSH, as well as sensitivity to GPX4 inhibitors (Cheung and Vousden, 2022). When not controlled by cellular defense mechanisms, lipid hydroperoxides trigger ferroptotic cell death signaling, which is in part regulated by NRF2 and ferroptosis suppressor protein 1 (FSP1). Other factors, such as elevated intracellular iron, enhanced PUFA synthesis, GSH depletion, and SLC7A11 inhibition, predispose cancer cells to ferroptosis succeptibility (Wu et al., 2024). Besides ferroptotis, apoptosis, and other regulated cell death cascades such as necroptosis, pyroptosis, paraptosis, parthanatos, and oxeiptosis are all influenced by ROS to varying degrees and in particular cell contexts (An et al., 2024).

Anti-Cancer Therapies Targeting Aspects of Redox Biology

As cancer cells frequently present with elevated ROS and higher activity of anti-ROS defense systems, such as GSH, it is unsurprising that cancer therapies that attempt to alter ROS balance have been put forward. On the simple hypothesis that ROS production is strictly cancer-promoting, antioxidants such as N-acetyl-L-cysteine (NAC), selenium, β-carotene, and vitamin E have been tested for effectiveness and paradoxically can be tumor-promoting (Cheung and Vousden, 2022; Wu et al., 2024). However, this apparent defeat does not mean that redox biology is not a useful target but that the path is more complex. Going forward, targeting redox biology will need to be more specifically modulated in both time and context. A number of cancer therapy areas are being investigated for the impact of ROS and potential therapeutic additions by way of redox manipulation, including:

1. Radiotherapy – Radiotherapy itself is known to be ROS-generating. Radiotherapy treatments are known to upregulate ferroptotic markers and regulators, including GPX4 (Wu et al., 2024).
2. GSH activity – The elevated GSH seen in many cancers has led to the targeting of members of the GSH biosynthesis and regeneration chain. Buthionene sulfide targets GCL and is used to treat several cancers. As glutamine and cysteine are essential for GSH synthesis, depletion of cysteine levels by exogenous cysteinase can deplete GSH (Wu et al., 2024).
3. Glutamine metabolism – Not solely for its role in GSH but also for central involvement in TCA cycle, nucleotide, and amino acid biosynthesis, glutamine is a critical nutrient for many cancer types. Therefore, many agents are being evaluated that target different aspects of glutamine metabolism, including inhibition of SLC1A5 (a glutamine transporter), inhibition of glutaminase (GLS, which regulates the glutamine/glutamate balance), and inhibition of SLC7A11 (an essential part of the cystine-glutamine antiporter) (Wu et al., 2024).
4. NADPH metabolism – Inhibition of the PPP can lead to NADPH depletion, increased oxidative stress, and heightened sensitivity to radiotherapy. Glucose-6-phosphate dehydrogenase (G6PD) is essential to NAPDH production; therefore, targeting this enzyme in combination therapies can be tumor-suppressive (Wu et al., 2024).
5. Ferroptosis – Some SLC7A11 and GPX4 inhibitors are inducers of ferroptosis. Some inhibitors of tetrahydrobiopterin metabolism also trigger ferroptotsis. More direct pharmacological inhibition of FSP1 to induce ferroptosis is now being investigated as well (Wu et al., 2024).

Company profiles in Redox Cancer Biology

Shown here is a list of example companies and the products they are developing.

• Dracen Pharma: DRP-104 (sirpiglenastat), a glutamine antagonist.
• Solasia Pharma: Darinaparsin (formerly ZIO-101) (Darvias^®), an organoarsenic compound that drives ROS generation, and which is approved in Japan for refractory peripheral T-cell lymphoma.
• Biogen: Omaveloxolone was recently approved for Friedreich’s ataxia, and which reduces ROS and RNS and consequently augments T-cell anticancer activity.
• Tharimmune, Inc (formerly Hillstream BioPharma): HSB-1216 a ferreoptosis inducer.
• Prometheus Laboratories: RIDAURA® (auranofin) approved in 1985 for rheumatoid arthritis (RA), and which inhibits TXNRD1/2. RIDAURA is currently in clinical trial for cancer applications.

References

Wu et al., The pleiotropic functions of reactive oxygen species in cancer. (2024) Nat Cancer, Mar;5(3):384-399.

Hanahan D., Hallmarks of Cancer: New Dimensions. (2022) Cancer Discov, Jan;12(1):31-46.

Cheung and Vousden, The role of ROS in tumour development and progression. (2022) Nat Rev Cancer, May;22(5):280-297.

An et al., Oxidative cell death in cancer: mechanisms and therapeutic opportunities. (2024) Cell Death Dis, Aug 1;15(8):556.