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Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia


Resistance to chemotherapy is frequently driven by aberrantly activated kinases in cancer. Herein, we characterized the global phosphoproteomic alterations associated with methotrexate (MTX) resistance in gestational trophoblastic neoplastic (GTN) cells. A total of 1111 phosphosites on 713 proteins were significantly changed, with highly elevated Ribosomal S6 Kinase 2 (RSK2) phosphorylation (pS227) observed in MTX-resistant GTN cells. Activation of RSK2 promoted cell proliferation and survival after MTX treatment in GTN cell models. Interestingly, RSK2 might play an important role in the regulation of reactive oxygen species (ROS) homeostasis, as manipulation of RSK2 activation affected ROS accumulation and SOX8 expression in GTN cells. In addition, overexpression of SOX8 partly rescued cell proliferation and survival in RSK2-depleted MTX-resistant GTN cells, suggesting that SOX8 might serve as a downstream effector of RSK2 to promote MTX resistance in GTN cells. Highly activated RSK2/SOX8 signaling was observed in MTX-resistant GTN specimens. Further, the RSK2 inhibitor BIX02565 effectively reduced SOX8 expression, induced ROS accumulation, and enhanced MTX-induced cytotoxicity in vitro and in vivo. Collectively, our findings suggested that RSK2 activation could promote MTX resistance via upregulating SOX8 and attenuating MTX-induced ROS in GTN cells, which may help to develop experimental therapeutics to treat MTX-resistant GTN.

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Fig. 1: Quantitative phosphoproteomic analysis on JAR/MTX and JAR cell models.
Fig. 2: Knockdown of RSK2 attenuates MTX resistance in MTX-resistant GTN cell lines.
Fig. 3: Over-expression of constitutively activated RSK2 promotes MTX resistance in GTN cell lines.
Fig. 4: RSK2 activation reduces ROS generation and induces SOX8 expression in GTN cells.
Fig. 5: Over-expression of SOX8 partly rescues the cellular effect of RSK2 knockdown on MTX- resistant GTN cells.
Fig. 6: RSK2/SOX8 signaling was highly enriched in MTX-resistant GTN samples.
Fig. 7: RSK2 inhibitor BIX02565 downregulates SOX8 expression, induces ROS accumulation, and sensitizes MTX-resistant GTN sublines to MTX.
Fig. 8: RSK2 inhibitor BIX02565 reduces SOX8 expression and suppresses tumor growth in JAR/MTX xenograft model.

Data availability

The original contributions presented in the study are included in the paper/Supplementary material; further inquiries can be directed to the corresponding author.


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This work was supported by The Science and Medicine Joint Project of Hunan Province (Grant Number: 2019JJ80013); National Natural Science Foundation of China (Grant Number: 82000585).

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Authors and Affiliations



D.Z.S. designed the study; S.B.W., M.J.S. and Y.Z. performed the experiments; S.B.W., M.J.S. and Y.Z. analyzed the data; D.Z.S. wrote the paper. All authors have read and approved the final version of the paper.

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Correspondence to Dazun Shi.

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The authors declare no competing interests.

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The studies involving human participants were reviewed and approved by the Institutional Review Board in Xiangya Hospital, Central South University (keshen20203665). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee in Xiangya Hospital, Central South University (dongwulunshen20203665).

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Wu, S., Shao, M., Zhang, Y. et al. Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia. Lab Invest 101, 1494–1504 (2021).

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