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NRF2 has a splicing regulatory function involving the survival of motor neuron (SMN) in non-small cell lung cancer

Oncogene volume 42, pages 2751–2763 (2023)Cite this article

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Abstract

The nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) signaling pathway is frequently deregulated in human cancers. The critical functions of NRF2, other than its transcriptional activation, in cancers remain largely unknown. Here, we uncovered a previously unrecognized role of NRF2 in the regulation of RNA splicing. Global splicing analysis revealed that NRF2 knockdown in non-small cell lung cancer (NSCLC) A549 cells altered 839 alternative splicing (AS) events in 485 genes. Mechanistic studies demonstrated that NRF2 transcriptionally regulated SMN mRNA expression by binding to two antioxidant response elements in the SMN1 promoter. Post-transcriptionally, NRF2 was physically associated with the SMN protein. The Neh2 domain of NRF2, as well as the YG box and the region encoded by exon 7 of SMN, were required for their interaction. NRF2 formed a complex with SMN and Gemin2 in nuclear gems and Cajal bodies. Furthermore, the NRF2–SMN interaction regulated RNA splicing by expressing SMN in NRF2-knockout HeLa cells, reverting some of the altered RNA splicing. Moreover, SMN overexpression was significantly associated with alterations in the NRF2 pathway in patients with lung squamous cell carcinoma from The Cancer Genome Atlas. Taken together, our findings suggest a novel therapeutic strategy for cancers involving an aberrant NRF2 pathway.

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Fig. 1: RNA Seq analysis of A549 NSCLC cells with NRF2-knockdown.
Fig. 2: NRF2 regulates SMN expression.
Fig. 3: NRF2 interacts with SMN.
Fig. 4: Co-localization of NRF2 with SMN, Coilin, and Gemin2 in vivo.
Fig. 5: SMN1 mRNA expression strongly increases with NRF2 pathway alteration in squamous cell lung cancer.
Fig. 6: RNA Seq analysis of A549 NSCLC cells with SMN-knockdown.
Fig. 7: Changes of SEs after NRF2-knockdown are associated with those of SMN-knockdown.
Fig. 8: Proposed model for NRF2–SMN interaction mediating alternative splicing in NSCLC lung cancer.

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Data availability

All data generated or analyzed during this study are included in this paper and its supplementary files. The RNA-Seq and ChIP-seq data of raw and aligned files have been deposited in NCBI Gene Expression Omnibus, accession number GSE164720 and 141497, respectively.

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Acknowledgements

We thank Prof. Peter Claus (Institute of Neuroanatomy, Hannover Medical School, Hannover, Germany) for providing the plasmids pEGFP-N2-SMN-1-294 and pET41a- SMN-1-294, and pEGFP-N2-Coilin, Prof. Qiming Sun (Zhejiang University, China) for providing Furipw vector, Prof. Hong-He Zhang and Prof. Yin-Jie Wang (Zhejiang University, China) for comments on the MS. We thank Yanwei Li, Guifeng Xiao, Wei Yin, Zhaoxiaonan Lin and Sanhua Fang from the Core Facilities, Zhejiang University School of Medicine for their technical support. We thank GENERGY BIO-TECHNOLOGY (SHANGHAI) CO., LTD. for RNA-Seq and data analysis. We also thank Lab members Miss Yi-jiao Liao, Miss Yu Tian and Mr. Yemin Shan for technical help. This work was supported by grants from the National Natural Science Foundation of China (31971188 to XT, 91643110 to XJW and 31700690 to YW).

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Author notes

  1. These authors contributed equally: Qinqin Cui, Wei Wang, Akhileshwar Namani.

Authors and Affiliations

  1. Department of Biochemistry and Department of Thoracic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, P. R. China

    Qinqin Cui, Wei Wang, Akhileshwar Namani, Hongyan Wang, Ahmed Hammad, Pu Huang, Yang Gao, Mohamed Elshaer & Xiuwen Tang

  2. Department of Thoracic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, P. R. China

    Yihe Wu

  3. Department of Pharmacology and Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China

    Xiu Jun Wang

  4. Zhejiang Provincial Key Laboratory of Immunity and Inflammatory Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, PR China

    Xiuwen Tang

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Contributions

Conceptualization: XT and XJW; Methodology: QC, WW, AN, HW, AH, PH, YG, ME, YW, XJW and XT; Investigation: QC, WW, AN, HW, AH, YG, ME, XJW and XT; Writing—original draft: XT and XJW; Writing—revision: XT; Funding Acquisition: XT, XJW, PH and YW. Supervision: XT.

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Correspondence to Xiu Jun Wang or Xiuwen Tang.

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Cui, Q., Wang, W., Namani, A. et al. NRF2 has a splicing regulatory function involving the survival of motor neuron (SMN) in non-small cell lung cancer. Oncogene 42, 2751–2763 (2023). https://doi.org/10.1038/s41388-023-02799-z

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