NeuroD1-GPX4 signaling leads to ferroptosis resistance in hepatocellular carcinoma

Abstract

Cell death resistance is a hallmark of tumor cells that drives tumorigenesis and drug resistance. Targeting cell death resistance-related genes to sensitize tumor cells and decrease their cell death threshold has attracted attention as a potential antitumor therapeutic strategy. However, the underlying mechanism is not fully understood. Recent studies have reported that NeuroD1, first discovered as a neurodifferentiation factor, is upregulated in various tumor cells and plays a crucial role in tumorigenesis. However, its involvement in tumor cell death resistance remains unknown. Here, we found that NeuroD1 was highly expressed in hepatocellular carcinoma (HCC) cells and was associated with tumor cell death resistance. We revealed that NeuroD1 enhanced HCC cell resistance to ferroptosis, a type of cell death caused by aberrant redox homeostasis that induces lipid peroxide accumulation, leading to increased HCC cell viability. NeuroD1 binds to the promoter of glutathione peroxidase 4 (GPX4), a key reductant that suppresses ferroptosis by reducing lipid peroxide, and activates its transcriptional activity, resulting in decreased lipid peroxide and ferroptosis. 

Introduction

Cell death resistance is one of the hallmarks of cancer [1]. The balance of cell death plays a vital role in regulating cell population size and tumorigenesis. Cell death can be classified as accidental cell death (ACD), a biologically uncontrolled process, and regulated cell death (RCD), which is characterized by controlled signaling pathways [2]. RCD includes apoptosis, necroptosis, autophagy, and ferroptosis, which can occur in the presence or absence of exogenous environmental or intracellular perturbations [3]. Tumor cells can escape the RCD route by evolving various mechanisms that lead to increased cell death thresholds [4]. Aberrant expression of RCD-related genes, such as the caspase and Bcl-2 families, due to mutations or impaired regulatory mechanisms is frequently observed in tumor cells, leading to an elevation in the cell death threshold [5–8]. Cell death resistance is involved in every step of tumorigenesis. At the tumor initiation stage, mutations in tumor suppressor genes such as TP53 and BRCA1/2 block cell death induced by DNA damage, leading to the initiation of tumor formation [9]. With an increase in tumor mass, the tumor microenvironment becomes more depleted of nutrients and oxygen; however, cell death resistance helps tumor cells endure such a severe microenvironment [10]. 

Material and methods

Ethics statement

Animal study was carried out in the Chongqing University Cancer Hospital, and was approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Cancer Hospital. All animal experiments conformed to the approved guidelines of the Animal Care and Use Committee of the Chongqing University Cancer Hospital. All efforts were made to minimize suffering. For the clinical HCC samples, prior patients’ written informed consents were obtained. The study was approved by the Institutional Research Ethics Committee of Chongqing University Cancer Hospital, and conducted in accordance with Declaration of Helsinki.

Results

NeuroD1 knockdown induces cell death

To investigate the role of NeuroD1 in hepatocellular carcinoma (HCC), we first analyzed its expression levels in clinical HCC samples. As shown in Fig 1A, NeuroD1 expression was significantly higher in tumor lesions than in adjacent tissues, and was localized in both cytoplasm and nucleus. Next, we confirmed the efficacy of the two shRNA expression vectors targeting NeuroD1 in HCC-LM3 cells by assessing NeuroD1 mRNA expression levels (S1A Fig). These results were further confirmed by the NeuroD1 protein expression level in HCC-LM3 and MHCC-97H cells (S1B Fig). The results showed that shND1-1 had a stronger suppressive effect; thus, we used this vector in further experiments. Furthermore, we also confirmed the efficacy of NeuroD1 overexpression vector in HCC-LM3 and MHCC-97H cells (S1C Fig). We examined the effects of altering NeuroD1 expression on the viability and colony potential of these cell lines. Knocking down NeuroD1 significantly suppressed the viability (Figs 1B and S2A) and colony formation potential (S2B and S2C Fig) of HCC-LM3 and MHCC-97H cells, while NeuroD1 overexpression robustly increased them (S2D–S2G Fig). These results suggest that NeuroD1 enhances HCC cell viability.

Discussion

Cell death is a fundamental physiological process occurring in almost all human cells. Different types of cell death such as apoptosis, necroptosis, and ferroptosis trigger different cellular reactions and affect disease progression via distinct mechanisms [52]. Ferroptosis is an iron-dependent cell death process mediated by lipid peroxides. The main mechanism of ferroptosis involves bivalent iron and lipoxygenase, which catalyzes the peroxidation of PUFAs in the cell membrane, leading to cell death [53]. Aberrant iron metabolism leads to the Fenton reaction and enhances oxidative stress, resulting in ROS accumulation and lipid peroxidation. This, in turn, damages the mitochondria, causing shrunken mitochondria with decreased crista, condensed membrane, and ruptured outer membrane [50]. Furthermore, disruption of redox homeostasis due to aberrant amino acid metabolism, such as glutamate-cysteine exchange and GSH synthesis, and by the impaired GPX4 expression, leads to defects in the ability to reduce lipid peroxidation, and thus is a crucial factor that triggers ferroptosis [54]. Meanwhile, an increase in ferroptosis resistance has been observed in various tumors, including HCC as well as esophageal, gastric, colorectal, and breast cancers [55–59]; thus, inducing ferroptosis has attracted attention as a potential strategy to overcome tumor drug resistance [60]. 

Acknowledgments

We thank Professor Makoto Miyagishi (Life Science Innovation, School of Integrative and Global Majors, University of Tsukuba, Japan) for his technical support.

Citation: Huang P, Duan W, Ruan C, Wang L, Hosea R, Wu Z, et al. (2023) NeuroD1-GPX4 signaling leads to ferroptosis resistance in hepatocellular carcinoma. PLoS Genet 19(12): e1011098. https://doi.org/10.1371/journal.pgen.1011098

Editor: Carmen Priolo, Brigham and Women’s Hospital, UNITED STATES

Received: June 13, 2023; Accepted: December 7, 2023; Published: December 22, 2023

Copyright: © 2023 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: 2. This work was supported by grants from the National Natural Science Foundation of China (32070715, 32270778 to VK, and 82173029, 81872273 to SW); and the Natural Science Foundation of Chongqing (CSTB2022NSCQ-MSX0611 to SW, and CSTB2022NSCQ-MSX0612 to VK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011098#abstract0