In an article published in the journal Nature, scientists described how the accumulation of 7-dehydrocholesterol (7-DHC), a lipid precursor to cholesterol, suppresses cell death by ferroptosis and may favor the survival of cancer cells. Ferroptosis is a form of cell death characterized by the lethal accumulation of lipid peroxidation products catalyzed by iron ions. It has been explored to eliminate tumors resistant to other types of cell death. The researchers identified a pro-ferroptosis activity of 7-dehydrocholesterol reductase (DHCR7), the last enzyme in the cholesterol biosynthesis pathway, and an unexpected pro-survival function of its substrate, 7-DHC.

The study was led by Brazilian scientist José Pedro Friedmann Angeli, currently at the University of Würzburg, in Germany, and had the collaboration of RIDC Redoxoma researchers Sayuri Miyamoto, from the Instituto de Química at Universidade de São Paulo (USP), and Luis E.S. Netto, from the Instituto de Biociências (USP), and their groups, as well as researchers from Germany, the United States, and Canada.

“In this work, we were able to show several important things. One of them is that for cell death to occur, the oxidation of phospholipids is necessary and not of any lipid. As we discovered this protective effect of 7-DHC, we saw that, by stimulating lipid peroxidation, more free radicals go to the membrane, but as long as they don’t reach the phospholipid, there won’t be cell death. This is important as a mechanism. Another aspect is that it was already known that 7-DHC is toxic to neurons, but we showed that this metabolite, when accumulated, generates the most aggressive phenotype in tumors,” Angeli said.

Furthermore, the authors alerted to the fact that there are several FDA-approved drugs able to inhibit the DHCR7 enzyme, such as Trazodone, prescribed over 20 million times a year in the US, sometimes off-label to treat insomnia. Studies have shown that patients taking this medication have increased plasma levels of 7-DHC. According to the authors, epidemiological studies will be required to explore whether any group of patients regularly consume ferroptosis-modulating drugs and whether this has any impact on cancer incidence, metastasis incidence, or other public health-relevant aspects.

Mechanism

Angeli’s group has been investigating for many years the mechanisms of resistance or sensitivity to ferroptosis in genetic and pharmacological models, which converge on the inhibition of the enzyme glutathione peroxidase 4 (GPX4), the only enzyme capable of repairing oxidized phospholipid hydroperoxides (PLOOH). To identify genes that can confer protection against or induce ferroptosis, the researchers used a CRISPR knockout library and subjected a pool of cells with inactivated genes to the ferroptosis process. “By inducing this process in this pool of cells with great genetic variability, some cells will become more resistant to lipid peroxidation, and we identified these cells. In the case of this work, we identified one of the genes, Acls4, which we had described in 2017 and is an essential gene for the incorporation of polyunsaturated lipids. If you do not incorporate polyunsaturated lipids into the membrane, the cells have no substrate to oxidize and are protected. And the other gene that appeared was Dhcr7, which encodes the enzyme DHCR7”.

As inhibition of the DHCR7 enzyme leads to the accumulation of 7-DHC, researchers generated and studied a series of knockout cell lines, in which they modulated the concentration of this intermediate, and demonstrated that 7-DHC has a crucial role in cell protection.

The next step was to understand the impact of this lipid on the oxidation of phospholipids in the cell membrane. “We worked a lot with in vitro models of liposome [vesicles composed of lipid bilayers] oxidation, and doctoral student Alex Inague performed the experiments that showed that 7-DHC protects membrane phospholipids from oxidation induced by free radicals generated by iron ions”, said Miyamoto.

Inague developed the method that allowed researchers to assemble liposomes with different sterols, a class of lipids that includes cholesterol and 7-DHC, and check what happened to the substrates (phospholipids, sterols). “We initially saw that, as 7-DHC is present in the membrane in higher concentration, it oxidizes very quickly, and the phospholipid oxidizes less. We understood that it was sacrificing itself in the membrane”, explains Inague. Using a membrane stability test, the researchers also saw that in the presence of 7-DHC, the membrane ruptured less and released less intramembrane content into the external environment.

Due to its high reactivity with radicals, 7-DHC inhibits phospholipid peroxidation, the formation of truncated phospholipids, and membrane permeabilization. This is one of the mechanisms used by some types of cancer cells to "escape" ferroptosis. – Freitas F.P. et al., Nature 2024 (doi 10.1038/s41586-023-06878-9).

To have a more comprehensive understanding of the role of sterols, the researchers also studied yeasts that accumulate ergosterol, in collaboration with researcher Fernando Gomes, from Luis Netto’s group. “Ergosterol is a sterol with a structure very similar to that of 7-DHC - it has two double bonds in the B ring, just like 7-DHC. We found that when yeast accumulate these sterols with two double bonds, they are better protected against oxidation and, when they are unable to accumulate this type of sterol, they become more sensitive.“

With these results, the researchers proposed that the accumulation of sterols such as 7-DHC and ergosterol is a relevant mechanism for protecting membranes against phospholipid peroxidation at the cellular level. They also defined the differences between lipid and phospholipid peroxidation in cell death processes.

Ferroptosis and cancer

Cancer cells use several strategies to avoid mechanisms that lead to cell death by apoptosis and to become resistant to chemotherapy, leading to the emergence of drug-tolerant cells. In these cases, inducing ferroptosis could be a way to eliminate tumors.

After understanding the molecular basis by which 7-DHC prevents ferroptosis, the researchers decided to investigate whether this protective effect could play a role in tumor growth in conditions in which inhibition of ferroptosis is critical. Rare mutations in the gene encoding the enzyme DHCR7 are found in some types of cancer, such as Burkitt’s lymphoma and neuroblastoma. Based on this information, they created animal models of both types of cancer and observed that the mutations cause loss of enzyme function and consequent accumulation of 7-DHC.

“We used two models, one for lymphoma and one for neuroblastoma. In the lymphoma model, in which the tumor is injected into the circulation, we saw that the cells that accumulate 7-DHC are very aggressive, as they are more resilient. In the neuroblastoma model, we saw that, while they are in the primary tumor, the cells grow similarly, but those that have the mutation and accumulate 7-DHC are more aggressive and more efficient in generating metastasis in distant organs”, says Angeli.

The next step in this work will be to investigate how tumors will evolve if the production of 7-DHC is completely inhibited. “Sometimes during the process of tumor evolution, there may be some modulation that we cannot detect in the models we use. It will be important to see how cells that cannot produce 7-DHC will behave in vivo when injected into these models. Another question is how cells that cannot accumulate 7-DHC respond to normal chemotherapy. These are studies that still need to be performed”, Angeli says.

The article 7-Dehydrocholesterol is an endogenous suppressor of ferroptosis can be accessed here.