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Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM

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Abstract

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells.

Graphical abstract

HuR inhibitor MS-444 arrested embryonic development at 2-cell stage.

Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444.

MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA.

STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.

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Data availability

Low-input RNA sequencing and processed files have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE192494.

Code availability

Not applicable.

References

  • Abe K, Funaya S, Tsukioka D, Kawamura M, Suzuki Y, Suzuki MG, Schultz RM, Aoki F. Minor zygotic gene activation is essential for mouse preimplantation development. P Natl Acad Sci USA. 2018;115:E6780–8.

    Article  CAS  Google Scholar 

  • Bahar Halpern K, Caspi I, Lemze D, Levy M, Landen S, Elinav E, Ulitsky I, Itzkovitz S. Nuclear retention of mRNA in mammalian tissues. Cell Rep. 2015;13:2653–62.

    Article  CAS  Google Scholar 

  • Bates M, Jones SA, Zhuang X. Stochastic optical reconstruction microscopy (STORM): a method for superresolution fluorescence imaging. Cold Spring Harb Protoc. 2013;2013:498–520.

    Article  Google Scholar 

  • Bedoschi G, Navarro PA, Oktay K. Chemotherapy-induced damage to ovary: mechanisms and clinical impact. Future Oncol. 2016;12:2333–44.

    Article  CAS  Google Scholar 

  • Bhasin S, Kerr C, Oktay K, Racowsky C. the implications of reproductive aging for the health, vitality, and economic welfare of human societies. J Clin Endocr Metab. 2019;104:3821–5.

    Article  Google Scholar 

  • Chen JL, Liu TZ, Gao J, Gao L, Zhou LL, Cai MJ, Shi Y, Xiong WY, Jiang JG, Tong T, et al. Variation in carbohydrates between cancer and normal cell membranes revealed by super-resolution fluorescence imaging. Adv Sci. 2016;3(12):1600270.

    Article  Google Scholar 

  • Chu DP, Tian S, Qi L, Hao CJ, Xia HF, Ma X. Abnormality of maternal-to-embryonic transition contributes to MEHP-induced mouse 2-cell block. J Cell Physiol. 2013;228:753–63.

    Article  CAS  Google Scholar 

  • Danilin S, Sourbier C, Thomas L, Lindner V, Rothhut S, Dormoy V, Helwig JJ, Jacqmin D, Lang H, Massfelder T. Role of the RNA-binding protein HuR in human renal cell carcinoma. Carcinogenesis. 2010;31:1018–26.

    Article  CAS  Google Scholar 

  • Denkert C, Koch I, von Keyserlingk N, Noske A, Niesporek S, Dietel M, Weichert W. Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2. Modern Pathol. 2006;19:1261–9.

    Article  CAS  Google Scholar 

  • Galloway SM, McNatty KP, Cambridge LM, Laitinen MPE, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, et al. Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nat Genet. 2000;25:279–83.

    Article  CAS  Google Scholar 

  • Gao J, Wang Y, Cai M, Pan Y, Xu H, Jiang J, Ji H, Wang H. Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging. Nanoscale. 2015;7:2511–9.

    Article  CAS  Google Scholar 

  • Grammatikakis I, Abdelmohsen K, Gorospe M. Posttranslational control of HuR function. Wiley Interdiscip Rev RNA. 2017;8(1):10.1002.

  • Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

    Article  CAS  Google Scholar 

  • Heck KE, Schoendorf KC, Ventura SJ, Kiely JL. Delayed childbearing by education level in the United States, 1969–1994. Matern Child Health J. 1997;1:81–8.

    Article  CAS  Google Scholar 

  • Higgins A, Khan Z, Coddington CC, Hashmi SK, Hefazi M, Alkhateeb H, Litzow MR, Hogan WJ, Cathcart-Rake E, Thompson CA, et al. Utilization and outcomes of fertility preservation techniques in women undergoing allogeneic hematopoietic cell transplant. Biol Blood Marrow Tr. 2019;25:1232–9.

    Article  Google Scholar 

  • Hupalowska A, Jedrusik A, Zhu M, Bedford MT, Glover DM, Zernicka-Goetz M. CARM1 and paraspeckles regulate pre-implantation mouse embryo development. Cell. 2018;175:1902-1916 e1913.

    Article  CAS  Google Scholar 

  • Jemal A, Ward EM, Johnson CJ, Cronin KA, Ma JM, Ryerson AB, Mariotto A, Lake AJ, Wilson R, Sherman RL, et al. Annual report to the nation on the status of cancer, 1975–2014, Featuring survival. Jnci-J Natl Cancer. 2017;I:109.

    Google Scholar 

  • Jing YY, Zhou LL, Chen JL, Xu HJ, Sun JY, Cai MJ, Jiang JG, Gao J, Wang HD. Quantitatively mapping the assembly pattern of EpCAM on cell membranes with peptide probes. Anal Chem. 2020;92:1865–73.

    Article  CAS  Google Scholar 

  • Karim-Kos HE, Kiemeney LA, Louwman MW, Coebergh JW, de Vries E. Progress against cancer in the Netherlands since the late 1980s: an epidemiological evaluation. Int J Cancer. 2012;130:2981–9.

    Article  CAS  Google Scholar 

  • Lai M, Li X, Li J, Hu Y, Czajkowsky DM, Shao Z. Improved clearing of lipid droplet-rich tissues for three-dimensional structural elucidation. Acta Biochim Biophys Sin (shanghai). 2017;49:465–7.

    Article  CAS  Google Scholar 

  • Lang M, Berry D, Passecker K, Mesteri I, Bhuju S, Ebner F, Sedlyarov V, Evstatiev R, Dammann K, Loy A, et al. HuR small-molecule inhibitor elicits differential effects in adenomatosis polyposis and colorectal carcinogenesis. Cancer Res. 2017;77:2424–38.

    Article  CAS  Google Scholar 

  • Levet F, Hosy E, Kechkar A, Butler C, Beghin A, Choquet D, Sibarita JB. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat Methods. 2015;12:1065–71.

    Article  CAS  Google Scholar 

  • Li J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, Duan EK, Hsueh AJW. Activation of dormant ovarian follicles to generate mature eggs. P Natl Acad Sci USA. 2010;107:10280–4.

    Article  CAS  Google Scholar 

  • Li X, Zheng M, Xu B, Li D, Shen Y, Nie Y, Ma L, Wu J. Generation of offspring-producing 3D ovarian organoids derived from female germline stem cells and their application in toxicological detection. Biomaterials. 2021;279: 121213.

    Article  CAS  Google Scholar 

  • Lim SJ, Lee SH, Joo SH, Song JY, Choi SI. Cytoplasmic expression of HuR is related to cyclooxygenase-2 expression in colon cancer. Cancer Res Treat. 2009;41:87–92.

    Article  Google Scholar 

  • Liu HB, Muhammad T, Guo YS, Li MJ, Sha QQ, Zhang CX, Liu H, Zhao SG, Zhao H, Zhang H, et al. RNA-binding protein IGF2BP2/IMP2 is a critical maternal activator in early zygotic genome activation. Adv Sci. 2019;6(15):1900295.

    Article  Google Scholar 

  • Liu B, Xu Q, Wang Q, Feng S, Lai F, Wang P, Zheng F, Xiang Y, Wu J, Nie J, et al. The landscape of RNA Pol II binding reveals a stepwise transition during ZGA. Nature. 2020a;587:139–44.

    Article  CAS  Google Scholar 

  • Liu RP, Wu KY, Li YJ, Sun R, Li XJY. Human antigen R: a potential therapeutic target for liver diseases. Pharmacol Res. 2020b;155:104684.

  • Liu YB, Li XZ, Zhang H, Zhang MM, Wei YL. HuR up-regulates cell surface PD-L1 via stabilizing CMTM6 transcript in cancer. Oncogene. 2021;40(12):2230–42.

  • Luo H, Li X, Tian GG, Li D, Hou C, Ding X, Hou L, Lyu Q, Yang Y, Cooney AJ, et al. Offspring production of ovarian organoids derived from spermatogonial stem cells by defined factors with chromatin reorganization. J Adv Res. 2021;33:81–98.

    Article  CAS  Google Scholar 

  • Meisner NC, Hintersteiner M, Mueller K, Bauer R, Seifert JM, Naegeli HU, Ottl J, Oberer L, Guenat C, Moss S, et al. Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR. Nat Chem Biol. 2007;3:508–15.

    Article  CAS  Google Scholar 

  • Ni J, Hu C, Li H, Li X, Fu Q, Czajkowsky DM, Guo Y, Shao Z. Significant improvement in data quality with simplified SCRB-seq. Acta Biochim Biophys Sin (shanghai). 2020;52:457–9.

    Article  CAS  Google Scholar 

  • Niakan KK, Han JN, Pedersen RA, Simon C, Pera RAR. Human pre-implantation embryo development. Development. 2012;139:829–41.

    Article  CAS  Google Scholar 

  • Nishino M, Kurasawa Y, Evans R, Lin SH, Brinkley BR, Yu-Lee LY. NudC is required for Plk1 targeting to the kinetochore and chromosome congression. Curr Biol. 2006;16:1414–21.

    Article  CAS  Google Scholar 

  • Ohinata Y, Payer B, O’Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature. 2005;436:207–13.

    Article  CAS  Google Scholar 

  • Ovesny M, Krizek P, Borkovec J, Svindrych ZK, Hagen GM. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics. 2014;30:2389–90.

    Article  CAS  Google Scholar 

  • Pabis M, Popowicz GM, Stehle R, Fernandez-Ramos D, Asami S, Warner L, Garcia-Maurino SM, Schlundt A, Martinez-Chantar ML, Diaz-Moreno I, et al. HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs. Nucleic Acids Res. 2019;47:1011–29.

    Article  CAS  Google Scholar 

  • Paz I, Kosti I, Ares M Jr, Cline M, Mandel-Gutfreund Y. RBPmap: a web server for mapping binding sites of RNA-binding proteins. Nucleic Acids Res. 2014;42:W361-367.

    Article  CAS  Google Scholar 

  • Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, Shen Y, Du C, Tang WL, Hamalainen T, Peng SL, et al. Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science. 2008;319:611–3.

    Article  CAS  Google Scholar 

  • Richards NG, Rittenhouse DW, Freydin B, Cozzitorto JA, Grenda D, Rui H, Gonye G, Kennedy EP, Yeo CJ, Brody JR, et al. HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients. Ann Surg. 2010;252:499–506.

    Article  Google Scholar 

  • Ripin N, Boudet J, Duszczyk MM, Hinniger A, Faller M, Krepl M, Gadi A, Schneider RJ, Sponer J, Meisner-Kober NC, et al. Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM. P Natl Acad Sci USA. 2019;116:2935–44.

    Article  CAS  Google Scholar 

  • Romeo C, Weber MC, Zarei M, DeCicco D, Chand SN, Lobo AD, Winter JM, Sawicki JA, Sachs JN, Meisner-Kober N, et al. HuR contributes to TRAIL resistance by restricting death receptor 4 expression in pancreatic cancer cells. Mol Cancer Res. 2016;14:599–611.

    Article  CAS  Google Scholar 

  • Sahab ZJ, Hall MD, Me Sung Y, Dakshanamurthy S, Ji Y, Kumar D, Byers SW. Tumor suppressor RARRES1 interacts with cytoplasmic carboxypeptidase AGBL2 to regulate the alpha-tubulin tyrosination cycle. Cancer Res. 2011;71:1219–28.

    Article  CAS  Google Scholar 

  • Schultz CW, Preet R, Dhir T, Dixon DA, Brody JR. Understanding and targeting the disease-related RNA binding protein human antigen R (HuR). Wiley Interdiscip Rev RNA. 2020;11: e1581.

    Article  CAS  Google Scholar 

  • Shahmoradian SH, Lewis AJ, Genoud C, Hench J, Moors TE, Navarro PP, Castano-Diez D, Schweighauser G, Graff-Meyer A, Godie KN, et al. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nat Neurosci. 2019;22:1099–109.

    Article  CAS  Google Scholar 

  • Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011;61:212–36.

    Article  Google Scholar 

  • So C, Seres KB, Steyer AM, Monnich E, Clift D, Pejkovska A, et al. A liquid-like spindle domain promotes acentrosomal spindle assembly in mammalian oocytes. Science. 2019;364(6447):eaat9557.

  • Soleimani R, Heytens E, Darzynkiewicz Z, Oktay K. Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise. Aging-Us. 2011;3:782–93.

    Article  Google Scholar 

  • Stone MB, Shelby SA, Veatch SL. Super-resolution microscopy: shedding light on the cellular plasma membrane. Chem Rev. 2017;117:7457–77.

    Article  CAS  Google Scholar 

  • Taylan E, Oktay KH. Current state and controversies in fertility preservation in women with breast cancer. World J Clin Oncol. 2017;8:241–8.

    Article  Google Scholar 

  • Tian GG, Zhao X, Hou C, Xie W, Li X, Wang Y, Wang L, Li H, Zhao X, Li J, Ji W. 2022 Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells. Cell Mol Life Sci. 2022;79(1):22.

    Article  CAS  Google Scholar 

  • van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc. 2011;6:991–1009.

    Article  Google Scholar 

  • Wang Q, Sun QY. Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fert Develop. 2007;19:1–12.

    Article  Google Scholar 

  • Wang Y, Gao J, Guo XD, Tong T, Shi XS, Li LY, Qi M, Wang YJ, Cai MJ, Jiang JG, et al. Regulation of EGFR nanocluster formation by ionic protein-lipid interaction. Cell Res. 2014;24:959–76.

    Article  CAS  Google Scholar 

  • Wang J, Wang L, Feng G, Wang Y, Li Y, Li X, Liu C, Jiao G, Huang C, Shi J, et al. Asymmetric expression of LincGET biases cell fate in two-cell mouse embryos. Cell. 2018a;175(1887–1901): e1818.

    Google Scholar 

  • Wang LL, Jin XH, Cai MY, Li HG, Chen JW, Wang FW, Wang CY, Hu WW, Liu F, Xie D. AGBL2 promotes cancer cell growth through IRGM-regulated autophagy and enhanced Aurora A activity in hepatocellular carcinoma. Cancer Lett. 2018b;414:71–80.

    Article  CAS  Google Scholar 

  • Wang YH, Guo Y, Tang CY, Han X, Xu MY, Sun J, Zhao YF, Zhang YW, Wang M, Cao XF, et al. Developmental cytoplasmic-to-nuclear translocation of RNA-binding protein HuR is required for adult neurogenesis. Cell Rep. 2019;29:3101-3117.e7.

    Article  CAS  Google Scholar 

  • Yan QY, Lu YT, Zhou LL, Chen JL, Xu HJ, Cai MJ, Shi Y, Jiang JG, Xiong WY, Gao J, et al. Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging. P Natl Acad Sci USA. 2018;115:7033–8.

    Article  CAS  Google Scholar 

  • Yuan X, Tian GG, Pei X, Hu X, Wu J. Spermidine induces cytoprotective autophagy of female germline stem cells in vitro and ameliorates aging caused by oxidative stress through upregulated sequestosome-1/p62 expression. Cell Biosci. 2021;11:107.

    Article  CAS  Google Scholar 

  • Zhang J, Zhang YL, Zhao LW, Pi SB, Zhang SY, Tong C, Fan HY. The CRL4-DCAF13 ubiquitin E3 ligase supports oocyte meiotic resumption by targeting PTEN degradation. Cell Mol Life Sci. 2020;77:2181–97.

    Article  CAS  Google Scholar 

  • Zhou T, Aumais JP, Liu XQ, Yu-Lee LY, Erikson RL. A role for Plk1 phosphorylation of NudC in cytokinesis. Dev Cell. 2003;5:127–38.

    Article  CAS  Google Scholar 

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Funding

This study was approved by the National Nature Science Foundation of China (81720108017, 32000806), the National Major Scientific Instruments and Equipment Development Project, and National Nature Science Foundation of China (61827814).

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Authors and Affiliations

  1. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China

    Yongqiang Nie, Geng G. Tian, Lin He, Xiaoyong Li & Ji Wu

  2. School of Biomedical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China

    Wei Xu, Xiaowei Li, Yan Guo & Zhifeng Shao

  3. School of Electronic and Optical Engineering, Nanjing University of Science and Technology, Nanjing, 210094, China

    Xuefeng Liu

  4. Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China

    Ji Wu

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  1. Yongqiang Nie
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  2. Wei Xu
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  3. Geng G. Tian
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  4. Xiaowei Li
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  5. Yan Guo
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  9. Xiaoyong Li
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  10. Ji Wu
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Contributions

Yongqiang Nie designed and performed the experiments, analyzed the data, and wrote the draft of the manuscript. Wei Xu performed the experiments of RNA-seq. Geng G. Tian helped to analyze the data. Xiaowei Li, Yan Guo, Xuefeng Liu, Lin He, and Zhifeng Shao gave guidance to the experiment. Xiaoyong Li gave guidance to the experiment, analyzed the data, and revised the manuscript. Ji Wu conceptualized and supervised the entire project, analyzed the data, revised the manuscript, and funded the acquisition.

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Correspondence to Xiaoyong Li or Ji Wu.

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Nie, Y., Xu, W., Tian, G.G. et al. Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM. Cell Biol Toxicol 38, 1175–1197 (2022). https://doi.org/10.1007/s10565-022-09757-7

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