Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May 19;12(6):511.
doi: 10.1038/s41419-021-03790-w.

MYCN mediates TFRC-dependent ferroptosis and reveals vulnerabilities in neuroblastoma

Yuxiong Lu #  1   2 , Qing Yang #  3 , Yubin Su #  4 , Yin Ji #  5 , Guobang Li  3 , Xianzhi Yang  3 , Liyan Xu  3 , Zhaoliang Lu  1 , Jiajun Dong  6 , Yi Wu  6 , Jin-Xin Bei  2   7 , Chaoyun Pan  3   8 , Xiaoqiong Gu  9 , Bo Li  2   3   7   8
Affiliations

MYCN mediates TFRC-dependent ferroptosis and reveals vulnerabilities in neuroblastoma

Yuxiong Lu et al. Cell Death Dis. .

Abstract

MYCN amplification is tightly associated with the poor prognosis of pediatric neuroblastoma (NB). The regulation of NB cell death by MYCN represents an important aspect, as it directly contributes to tumor progression and therapeutic resistance. However, the relationship between MYCN and cell death remains elusive. Ferroptosis is a newly identified cell death mode featured by lipid peroxide accumulation that can be attenuated by GPX4, yet whether and how MYCN regulates ferroptosis are not fully understood. Here, we report that MYCN-amplified NB cells are sensitive to GPX4-targeting ferroptosis inducers. Mechanically, MYCN expression reprograms the cellular iron metabolism by upregulating the expression of TFRC, which encodes transferrin receptor 1 as a key iron transporter on the cell membrane. Further, the increased iron uptake promotes the accumulation of labile iron pool, leading to enhanced lipid peroxide production. Consistently, TFRC overexpression in NB cells also induces selective sensitivity to GPX4 inhibition and ferroptosis. Moreover, we found that MYCN fails to alter the general lipid metabolism and the amount of cystine imported by System Xc(-) for glutathione synthesis, both of which contribute to ferroptosis in alternative contexts. In conclusion, NB cells harboring MYCN amplification are prone to undergo ferroptosis conferred by TFRC upregulation, suggesting that GPX4-targeting ferroptosis inducers or TFRC agonists can be potential strategies in treating MYCN-amplified NB.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. MYCN is a regulator of GPX4-dependent ferroptosis.
A Genes selectively dependent on MYCN for NB cell survival are enriched within a variety of GO terms of biological processes, and the top nine processes are shown according to p values. B GSEA assays of ferroptosis-related genes showing enrichment with MYCN amplification based on RNA-seq data of six human NB cell lines. ES, enrichment score. C GPX4 is shared between ferroptosis-related and MYCN-dependent survival genes. D Cell viabilities when different concentrations of the GPX4 inhibitor (1S, 3R)-RSL3 (RSL3) and system Xc(−) inhibitor Erastin were used as ferroptosis inducers in multiple human NB cell lines (MYCN amplified SK-N-BE2, BE(2)-C, NLF, SK-N-DZ, and Kelly are represented in pink; and MYCN non-amplified SHEP, SK-N-AS, and SY5Y are shown in black; n = 4). E Kaplan–Meier survival curves for NB patients based on GPX4 mRNA expression. Gene expression and clinical data were retrieved from GSE45547. F Correlation analysis between GPX4 and MYCN expression based on the data retrieved from GSE45547. *p < 0.05, **p < 0.01.
Fig. 2
Fig. 2. MYCN confers cell sensitivity to ferroptosis upon GPX4 inhibition.
Lipid peroxides were detected by flow cytometry after incubation with C11-BODIPY in SHEP (A) or SK-N-BE2 (B) cells with or without MYCN. DFO, desferrioxamine, n = 3. C Cell viabilities of SHEP cells with or without MYCN overexpression incubated with Erastin (left), RSL3 (middle), or RSL3 combined with either ferroptosis inhibitor (10 μM DFO, 1 μM Ferr-1), apoptosis inhibitor (20 μM z-VAD), or necroptosis inhibitor (2 μM Necrostatin-1) (right). D Cell viabilities of SK-N-BE2 cells with or without MYCN knockdown incubated with Erastin or RSL3. E Metabolic analysis of SHEP cells with or without MYCN. Z-score ±2.75 is corresponding to p = 0.01 and depicted as a dotted line. Data are presented as mean ± SD of three replicates. Two-tailed unpaired t-tests were performed to calculate p values. **p < 0.01.
Fig. 3
Fig. 3. MYCN increases the intracellular iron load.
A Heatmap showing changes in an array of fatty acids in SHEP cells with or without MYCN. Data are shown as relative changes in abundance compared to the PCDH vector. B Heatmap showing the expression profile of ferroptosis-associated metabolic genes. C Expression of MYCN induced iron metabolic genes analyzed by RT-qPCR, and SKP2 is positive control of MYCN targeted genes. D Western blot analysis of SHEP cells with or without MYCN expression using indicated antibodies. E, F TFRC and SLC40A1 expression in NB tumor tissues with or without MYCN amplification. Expression data were retrieved from GSE45547. Cellular iron load is detected with the colorimetric ferrozine-based assay in SHEP G and SK-N-BE2 H cells with or without MYCN, and values are normalized to protein concentration. Data are presented as mean ± SD of three replicates. Two-tailed unpaired t-tests were performed to calculate p values. **p < 0.01.
Fig. 4
Fig. 4. TFRC photocopies MYCN to increase the labile iron pool.
A Iron load and RT-qPCR analysis of SHEP cells with or without TFRC overexpression. B Iron load and western blot analysis of SK-N-BE2 cells with or without TFRC depletion. Cellular labile iron pool (LIP) was detected using the probe calcein acetoxymethyl ester (Cal-AM) in SHEP (C) and SK-N-BE2 (D) cells with or without TFRC. LIP was similarly detected in SHEP (E) and SK-N-BE2 (F) cells with or without MYCN. Data are presented as mean ± SD of three replicates. Two-tailed unpaired t-tests were performed to calculate p values. *p < 0.05, **p < 0.01.
Fig. 5
Fig. 5. TFRC mediates MYCN–induced, GPX4-dependent ferroptosis.
A Cell viability of SHEP cells with or without TFRC overexpression incubated with Erastin (left) or RSL3 (right). Cell viabilities of SHEP (B) or SK-N-BE2 (C) cells with or without TFRC depletion incubated with Erastin (left) or RSL3 (right). Amounts of cellular lipid peroxides measured by C11-BODIPY in SHEP (D) or SK-N-BE2 (E) cells with or without TFRC. Data are presented as mean ± SD of three replicates. Two-tailed unpaired t-tests were performed to calculate p values. **p < 0.01.
Fig. 6
Fig. 6. TFRC is a direct downstream target of MYCN.
A RT-qPCR analysis of SK-N-BE2 cells with or without MYCN depletion. MYCN activation induced by 4-hydroxytamoxifen (4-HT) treatment promotes TFRC expression at both mRNA B and protein C levels. SKP2, DKK3 were used as positive and negative controls, respectively. D Amounts of cellular lipid peroxides measured by C11-BODIPY upon MYCN activation. E Primers designed for ChIP-qPCR analysis, and sequence variants for luciferase assays based on the TFRC promoter region. F ChIP-qPCR analysis of different binding sites of MYCN on the TFRC promoter. MDM2 is a positive control for MYCN binding. G Dual-luciferase assays based on (E). Signal values were normalized to no 4-HT treatment (day 0). Data are presented as mean ± SD of three replicates. Two-tailed unpaired t-tests were performed to calculate p values. **p < 0.01.
See this image and copyright information in PMC

References

    1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369:2106–2120. doi: 10.1016/S0140-6736(07)60983-0. - DOI - PubMed
    1. Matthay KK, et al. Neuroblastoma. Nat. Rev. Dis. Prim. 2016;2:16078. doi: 10.1038/nrdp.2016.78. - DOI - PubMed
    1. Diskin SJ, et al. Common variation at 6q16 within HACE1 and LIN28B influences susceptibility to neuroblastoma. Nat. Genet. 2012;44:1126–1130. doi: 10.1038/ng.2387. - DOI - PMC - PubMed
    1. Rickman DS, Schulte JH, Eilers M. The expanding world of N-MYC-driven tumors. Cancer Discov. 2018;8:150–163. doi: 10.1158/2159-8290.CD-17-0273. - DOI - PubMed
    1. Ruiz-Perez M. V., Henley A. B. Arsenian-Henriksson M. The MYCN protein in health and disease. Genes8, 113 (2017). - PMC - PubMed

Publication types

MeSH terms