Enhancing the efficacy of tumor immunotherapy - research on the mechanism of ferroptosis by Zhu Xiao-Feng and Deng Rong's team published in Nature Cell Biology

January 24, 2022

On January 13, Nature Cell biology, the top international journal of cell biology, published the latest achievement of Professors Zhu Xiao-feng and Deng Rong’s team from Sun Yat-sen University Cancer Center - "PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis". Prof. Zhu Xiao-Feng and Prof. Deng Rong, from the State Key Laboratory of Oncology in South China, are the corresponding authors and Dr. Zhang Hai-Liang from the State Key Laboratory of Oncology in South China is the first author.

What is ferroptosis?
Ferroptosis, is a non-apoptotic form of regulated cell death induced by the excessive accumulation of lipid-peroxidation products in an iron-dependent manner. There are at least three major scavenging enzymes that inhibit ferroptosis through lipid-peroxide clearance: glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein 1 and GTP cyclohydrolase-1. Several enzymes—including ACSL4, NADPH-cytochrome P450 reductase and lipoxygenases—have been reported to participate in lipid-peroxide generation. In eukaryotic cells, ACSL4 activates polyunsaturated fatty acids (PUFAs)—especially arachidonic acid (C20:4) and adrenic acid (C22:4)—to acyl-CoA, which are inserted into the phospholipids of the plasma membrane by lysophosphatidylcholine acyltransferase 3 (LPCAT3). Arachidonic acid (C20:4) and adrenic acid (C22:4)-containing phospholipids might be the primary lipid-peroxidation substrates that contribute to ferroptosis. P450 reductase generates H2O2 and this in turn generates radicals in the presence of ferrous iron. Some lipoxygenases that contain non-haem iron can directly oxygenate esterified PUFAs, which require oxidation of non-haem iron to Fe3+.

Research on the core mechanism of ferroptosis will lay the foundation for applications in tumor therapy
Ferroptosis is associated with many pathological processes, such as ischemia-reperfusion injury, neurodegenerative diseases, and tumors. Studies have reported that cancer immunotherapy and radiation therapy can inhibit tumor growth by inducing ferroptosis. In addition, tumor cells change the composition of the cell membrane to escape ferroptosis and promote metastasis by absorbing peripheral oleic acid in the process of lymphatic metastasis. 

Considering the important role of ferroptosis in the malignant progression and treatment of tumors, the elucidation of the core mechanism of ferroptosis will lay the foundation for the clinical application of ferroptosis. Although the function of these enzymes in lipidperoxidation-product generation and scavenging has been confirmed, the sensors and amplifying process of lipid peroxidation linked to ferroptosis remain elusive.

Zhu Xiao-feng and Deng Rong's team found key inducers in the process of ferroptosis
In this study, through independent genome-wide CRISPR–Cas9 and kinase inhibitor library screening, PKCβII activation was identified as essential for the execution of ferroptosis. The authors also identified PKCβII as a lipid peroxide sensor.

In order to clarify the molecular mechanism of PKCβII-induced ferroptosis in tumor cells, the authors confirmed that PKCβII phosphorylates 328 threonine of ACSL4 by means of co-immunoprecipitation, phosphorylation site prediction, in vitro kinase, specific phosphorylated antibody preparation and lipidomics. Phosphorylation of Thr328 promotes ACSL4 activation. Activated ACSL4 significantly promoted the biosynthesis of PUFA-containing lipids and then promotes the generation of lipid-peroxidation products. Finally, the lipid peroxidation–PKCβII–ACSL4 positive-feedback loop launches ferroptosis by facilitating the amplification of lipid peroxidation to lethal levels. 

The authors further confirmed the role of PKCβII phosphorylation of ACSL4 in rapidly amplifying lipid peroxidation in ferroptosis-related animal models. It was found that ACSL4-T328A (phospho-inactive mutant ACSL4) tumors were less sensitive to PD-1 antibodies compared with ACSL4-WT (wild-type ACSL4). Similarly, knockout of PKCβII significantly inhibited tumor sensitivity to PD-1 antibody, overexpression of PKCβII could restored tumor immunotherapy efficacy, and ferroptosis inhibitors significantly inhibited PKCβII-mediated immunotherapy efficacy enhancement. This indicates that PKCβII-ACSL4 enhances the efficacy of immunotherapy by promoting ferroptosis, suggesting that PKCβII and ACSL4 could be used for clinical applications, such as a predictor for responses to cancer immunotherapy.

Figure: PKCβII-ACSL4 positive feedback axis initiates the amplification process of lipid peroxidation to induce ferroptosis
PKCβII activation is essential for the execution of ferroptosis. We demonstrate that the PKCβII–ACSL4 axis actively facilitates ferroptosis through rapid amplification of lipid peroxidation to lethal levels. Our findings also confirm that attenuation of this pathway impairs the efficacy of cancer immunotherapy by inhibiting ferroptosis, suggesting that in-depth study of this pathway may offer potential targets and strategies for ferroptosis-associated cancer therapy.

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