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Ribosomal S6 protein kinase 4 promotes resistance to EZH2 inhibitors in glioblastoma

Cancer Gene Therapy volume 30, pages 1636–1648 (2023)Cite this article

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Abstract

Glioblastoma (GBM) is a highly malignant type of brain tumor with limited treatment options. Recent research has focused on epigenetic regulatory factors, such as Enhancer of Zeste Homolog 2 (EZH2), which plays a role in gene expression through epigenetic modifications. EZH2 inhibitors have been developed as potential therapeutic agents for GBM, but resistance to these inhibitors remains a considerable challenge. This study aimed to investigate the role of ribosomal S6 protein kinase 4 (RSK4) in GBM and its association with resistance to EZH2 inhibitors. We first induced drug resistance in primary GBM cell lines by treatment with an EZH2 inhibitor and observed increases in the expression of stemness markers associated with glioblastoma stem cells (GSCs) in the drug-resistant cells. We also found high expression of RSK4 in GBM patient samples and identified the correlation of high RSK4 expression with poor prognosis and GSC marker expression. Further experiments showed that knocking down RSK4 in drug-resistant GBM cells restored their sensitivity to EZH2 inhibitors and decreased the expression of GSC markers, thus reducing their self-renewal capacity. From a mechanistic perspective, we discovered that RSK4 directly phosphorylates EZH2, activating the EZH2/STAT3 pathway and promoting resistance to EZH2 inhibitors in GBM. We also found that combining EZH2 inhibitors with an RSK4 inhibitor called BI-D1870 had better inhibitory effects on GBM occurrence and progression in both in vitro and in vivo experiments. In conclusion, this study demonstrates that RSK4 enhances cancer stemness and mediates resistance to EZH2 inhibitors in GBM. Combination treatment with EZH2 inhibitors and RSK4 inhibitors is a promising potential therapeutic strategy for GBM. Collectively, our results strongly demonstrate that RSK4 regulates the EZH2/STAT3 pathway to promote GSC maintenance and EZH2i resistance in a PRC2-independent manner, indicating that RSK4 is a promising therapeutic target for GBM.

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Fig. 1: EZH2i resistance enhances cancer stemness in GBM cell lines.
Fig. 2: RSK4 is highly expressed in GSCs and is associated with poor survival in GBM patients.
Fig. 3: RSK4 is upregulated in EZH2i‑resistant GBM cell lines and promotes GSC maintenance.
Fig. 4: RSK4 directly phosphorylates EZH2 S21 and activates the EZH2/STAT3 pathway in EZH2i‑resistant GBM cell lines.
Fig. 5: Phosphorylation of EZH2 S21 and STAT3 activation are essential for RSK4-mediated EZH2i resistance and GSC maintenance.
Fig. 6: RSK4 inhibition with BI-D1870 increases EZH2i sensitivity in GBM cells in vivo.
Fig. 7: Schematic model.

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Funding

This work was supported by the Key Research and Development Projects of Shaanxi Province (No.2023-YBSF-129, 2022SF-029), the National Natural Science Foundation of China (No.82103522, 82373368), the China Postdoctoral Science Foundation (No.2021TQ0362).

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Author notes

  1. These authors contributed equally: Fangning Pang, Lei Zhang, Mingyang Li.

Authors and Affiliations

  1. The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Institute of Health and Rehabilitation Science, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, China

    Fangning Pang, Yu Wang, Peng Yang, Bin Wen, Jinquan Jiang, Yunpeng Teng & Jin Xu

  2. Department of Neurosurgery, Xijing Hospital, Air Force Medical University, Xi’an, China

    Lei Zhang, Xicai Yi & Li Wang

  3. State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers, Department of Pathology, School of Basic Medicine and Xijing Hospital, Air Force Medical University, Xi’an, China

    Mingyang Li

  4. Department of Neurosurgery, General Hospital of Northern Theater Command, Shenyang, China

    Xinyu Yang & Ligang Chen

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Contributions

FP, LZ, ML: data acquisition; XY, YW, PY: immunohistochemistry and Western blot; BW, JJ, YT, XY: clinical sample collection and data analysis; LC, JX, LW: study design, project administration and revision of manuscript. All authors have reviewed the final version of the manuscript and approved it for publication.

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Correspondence to Ligang Chen, Jin Xu or Li Wang.

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All animal experiments were carried out in compliance with the principles and methods stated in the Guideline for the Care and Use of Laboratory Animals in the Air Force Medical University. The studies involving human participants were reviewed and approved by the Medical Ethical Committee of Xijing Hospital. The patients/participants provided their written informed consent to participate in this study.

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Pang, F., Zhang, L., Li, M. et al. Ribosomal S6 protein kinase 4 promotes resistance to EZH2 inhibitors in glioblastoma. Cancer Gene Ther 30, 1636–1648 (2023). https://doi.org/10.1038/s41417-023-00666-3

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