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

Abstract

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.

References

  1. Braga, A. et al. Challenges in the diagnosis and treatment of gestational trophoblastic neoplasia worldwide. World J. Clin. Oncol. 10, 28–37 (2019).

    Article  Google Scholar 

  2. May, T., Goldstein, D. P. & Berkowitz, R. S. Current chemotherapeutic management of patients with gestational trophoblastic neoplasia. Chemother. Res. Pract. 2011, 806256 (2011).

    PubMed  PubMed Central  Google Scholar 

  3. Maesta, I. et al. Effectiveness and toxicity of first-line methotrexate chemotherapy in low-risk postmolar gestational trophoblastic neoplasia: the New England Trophoblastic Disease Center experience. Gynecol. Oncol. 148, 161–167 (2018).

    CAS  Article  Google Scholar 

  4. Bower, M. et al. EMA/CO for high-risk gestational trophoblastic tumors: results from a cohort of 272 patients. J. Clin. Oncol. 15, 2636–2643 (1997).

    CAS  Article  Google Scholar 

  5. Wu, J. & Wang, D. CLIC1 Induces Drug Resistance in Human Choriocarcinoma Through Positive Regulation of MRP1. Oncol. Res. 25, 863–871 (2017).

    Article  Google Scholar 

  6. Neradil, J., Pavlasova, G. & Veselska, R. New mechanisms for an old drug; DHFR- and non-DHFR-mediated effects of methotrexate in cancer cells. Klin. Onkol. 25, 2S87–92S87 (2012). Suppl.

    PubMed  Google Scholar 

  7. Elias, K. M., Harvey, R. A., Hasselblatt, K. T., Seckl, M. J. & Berkowitz, R. S. Type I interferons modulate methotrexate resistance in gestational trophoblastic neoplasia. Am. J. Reprod. Immunol. 77 e12666 (2017).

  8. AlBasher, G. et al. Methotrexate-induced apoptosis in human ovarian adenocarcinoma SKOV-3 cells via ROS-mediated bax/bcl-2-cyt-c release cascading. Onco. Targets Ther. 12, 21–30 (2019).

    CAS  Article  Google Scholar 

  9. Zhao, J. et al. AKR1C3 overexpression mediates methotrexate resistance in choriocarcinoma cells. Int. J. Med. Sci. 11, 1089–1097 (2014).

    Article  Google Scholar 

  10. Shen, Y. et al. The switch from ER stress-induced apoptosis to autophagy via ROS-mediated JNK/p62 signals: a survival mechanism in methotrexate-resistant choriocarcinoma cells. Exp.Cell Res. 334, 207–218 (2015).

    CAS  Article  Google Scholar 

  11. Jun, F., Peng, Z., Zhang, Y. & Shi, D. Quantitative Proteomic Profiling Identifies SOX8 as Novel Regulator of Drug Resistance in Gestational Trophoblastic Neoplasia. Front. Oncol. 10, 557 (2020).

    Article  Google Scholar 

  12. Yang, W., Freeman, M. R. & Kyprianou, N. Personalization of prostate cancer therapy through phosphoproteomics. Nat. Rev. Urol. 15, 483–497 (2018).

    Article  Google Scholar 

  13. Grossi, V., Peserico, A., Tezil, T. & Simone, C. p38alpha MAPK pathway: a key factor in colorectal cancer therapy and chemoresistance. World J. Gastroenterol. 20, 9744–9758 (2014).

    CAS  Article  Google Scholar 

  14. Gao, F. & Liu, W. J. Advance in the study on p38 MAPK mediated drug resistance in leukemia. Eur. Rev. Med. Pharmacol. Sci. 20, 1064–1070 (2016).

    CAS  PubMed  Google Scholar 

  15. Han, X., Zhang, J. J., Han, Z. Q., Zhang, H. B. & Wang, Z. A. Let-7b attenuates cisplatin resistance and tumor growth in gastric cancer by targeting AURKB. Cancer Gene. Ther. 25, 300–308 (2018).

    CAS  Article  Google Scholar 

  16. Jagadeeshan, S. et al. P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer. Ann. Oncol. 27, 1546–1556 (2016).

    CAS  Article  Google Scholar 

  17. Cho, Y. Y. RSK2 and its binding partners in cell proliferation, transformation and cancer development. Arch. Pharm. Res. 40, 291–303 (2017).

    CAS  Article  Google Scholar 

  18. van Jaarsveld, M. T. et al. The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin. Eur. J. Cancer 49, 345–351 (2013).

    Article  Google Scholar 

  19. Jun, F., Peng, Z., Zhang, Y. & Shi, D. Quantitative proteomic analysis identifies novel regulators of methotrexate resistance in choriocarcinoma. Gynecol. Oncol. 157, 268–279 (2020).

    Article  Google Scholar 

  20. Tripathi, S. et al. Meta- and Orthogonal Integration of Influenza “OMICs” Data Defines a Role for UBR4 in Virus Budding. Cell Host Microbe. 18, 723–735 (2015).

    CAS  Article  Google Scholar 

  21. Tan, J. et al. PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell activation, and resistance to mTOR-targeted therapy. Cancer Discov. 3, 1156–1171 (2013).

    CAS  Article  Google Scholar 

  22. Vyse, S. et al. Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib. J. Proteom. 170, 130–140 (2018).

    CAS  Article  Google Scholar 

  23. Adams, J. A. Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry 42, 601–607 (2003).

    CAS  Article  Google Scholar 

  24. Roskoski, R. Jr. Src kinase regulation by phosphorylation and dephosphorylation. Biochem. Biophys. Res. Commun. 331, 1–14 (2005).

    CAS  Article  Google Scholar 

  25. Shimura, Y. et al. RSK2(Ser227) at N-terminal kinase domain is a potential therapeutic target for multiple myeloma. Mol. Cancer Ther. 11, 2600–2609 (2012).

    CAS  Article  Google Scholar 

  26. Somale, D. et al. Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints. Sci. Rep. 10, 591 (2020).

    CAS  Article  Google Scholar 

  27. Shi, X. et al. The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury. J. Cardiovasc. Pharmacol. Ther. 21, 177–186 (2016).

    CAS  Article  Google Scholar 

  28. Kirrane, T. M. et al. Indole RSK inhibitors. Part 2: optimization of cell potency and kinase selectivity. Bioorg. Med. Chem. Lett. 22, 738–742 (2012).

    CAS  Article  Google Scholar 

  29. Fryer, R. M. et al. Mitigation of off-target adrenergic binding and effects on cardiovascular function in the discovery of novel ribosomal S6 kinase 2 inhibitors. J. Pharmacol. Exp. Ther. 340, 492–500 (2012).

    CAS  Article  Google Scholar 

  30. Lee, C. J. et al. RSK2-induced stress tolerance enhances cell survival signals mediated by inhibition of GSK3beta activity. Biochem. Biophys. Res. Commun. 440, 112–118 (2013).

    CAS  Article  Google Scholar 

  31. Sun, H. et al. Aurora-A/SOX8/FOXK1 signaling axis promotes chemoresistance via suppression of cell senescence and induction of glucose metabolism in ovarian cancer organoids and cells. Theranostics 10, 6928–6945 (2020).

    CAS  Article  Google Scholar 

  32. Kang, S. et al. FGFR3 activates RSK2 to mediate hematopoietic transformation through tyrosine phosphorylation of RSK2 and activation of the MEK/ERK pathway. Cancer Cell 12, 201–214 (2007).

    CAS  Article  Google Scholar 

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Funding

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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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). https://doi.org/10.1038/s41374-021-00651-0

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