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Targeting ARHGEF12 promotes neuroblastoma differentiation, MYCN degradation, and reduces tumorigenicity

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A Correction to this article was published on 16 October 2023

This article has been updated

Abstract

Purpose

Neuroblastoma arises from developmental block of embryonic neural crest cells and is one of the most common and deadly pediatric tumors. However, the mechanism underlying this block is still unclear. Here, we show that targeting Rho guanine nucleotide exchange factor 12 (ARHGEF12, also named LARG) promotes MYCN degradation and neuroblastoma differentiation, leading to reduced neuroblastoma malignancy.

Methods

The neuroblastoma TARGET dataset was downloaded to assess ARHGEF12 expression. Cell differentiation, proliferation, colony formation and cell migration analyses were performed to investigate the effects of ARHGEF12 knockdown on neuroblastoma cells. Western blotting and immunohistochemistry were employed to determine protein expression. Animal xenograft models were used to investigate antitumor effects after ARHGEF12 knockdown or treatment with the ARHGEF12 inhibitor Y16 in vivo.

Results

We found that the expression level of ARHGEF12 was higher in neuroblastoma than in better-differentiated ganglioneuroblastoma. Knockdown of ARHGEF12 promoted neuroblastoma differentiation, decreased stemness-related gene expression, and increased differentiation-related gene expression. ARHGEF12 knockdown reduced tumor growth, and the resulting tumors showed bigger tumor cells compared to those in control neuroblastoma xenografts. In addition, it was found that ARHGEF12 knockdown promoted MYCN ubiquitination and degradation in MYCN-amplified tumors through RhoA/ROCK/GSK3β signaling. Targeting ARHGEF12 with the small molecular inhibitor Y16 induced cell differentiation and attenuated neuroblastoma tumorigenicity.

Conclusion

Our findings provide new insight into the mechanism by which ARHGEF12 regulates neuroblastoma tumorigenicity and suggest a translatable therapeutic approach by targeting ARHGEF12 with a small molecular inhibitor.

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Data availability

All relevant data and material are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Change history

  • 09 October 2023

    The original version of this article was revised: In Fig. 5f of this article the representative MYCN IHC images of tumor from vehicle-treated mice presented the wrong samples. Fig. 5f has been corrected in the original article. The authors declare that this correction does not affect the description, interpretation, or the original conclusions of the manuscript.

  • 16 October 2023

    A Correction to this paper has been published: https://doi.org/10.1007/s13402-023-00890-x

Abbreviations

GEFs:

Guanine nucleotide exchange factors

NB:

Neuroblastoma

GNB:

Ganglioneuroblastoma

RA:

Retinoic acid

ARHGEF12:

Rho guanine nucleotide exchange factor 12

DMEM:

Dulbecco's modified Eagle medium

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered saline

PI:

Propidium Iodide

PE:

Phycoerythrin

CHX:

Cycloheximide

ARHGEF12 KD:

ARHGEF12 knockdown

H&E:

Hematoxylin-eosin

co-IP:

co-immunoprecipitation

References

  1. E.A. Newman, S. Abdessalam, J.H. Aldrink, M. Austin, T.E. Heaton, J. Bruny, P. Ehrlich, R. Dasgupta, R.M. Baertschiger, T.B. Lautz, D.S. Rhee, M.R. Langham Jr., M.M. Malek, R.L. Meyers, J.D. Nathan, B.R. Weil, S. Polites, M.B. Madonna, Update on neuroblastoma. J Pediatr Surg. 54, 383–389 (2019). https://doi.org/10.1016/j.jpedsurg.2018.09.004

    Article  PubMed  Google Scholar 

  2. T.J. Pugh, O. Morozova, E.F. Attiyeh, S. Asgharzadeh, J.S. Wei, D. Auclair, S.L. Carter, K. Cibulskis, M. Hanna, A. Kiezun, J. Kim, M.S. Lawrence, L. Lichenstein, A. McKenna, C.S. Pedamallu, A.H. Ramos, E. Shefler, A. Sivachenko, C. Sougnez, et al., The genetic landscape of high-risk neuroblastoma. Nat Genet. 45, 279-284 (2013). https://doi.org/10.1038/ng.2529

  3. S. Kiyonari, K. Kadomatsu, Neuroblastoma models for insights into tumorigenesis and new therapies. Expert Opin Drug Discov. 10, 53–62 (2015). https://doi.org/10.1517/17460441.2015.974544

  4. Z. Jin, Y. Lu, Y. Wu, J. Che, X. Dong, Development of differentiation modulators and targeted agents for treating neuroblastoma. Eur J Med Chem. 207, 112818 (2020). https://doi.org/10.1016/j.ejmech.2020.112818

    Article  CAS  PubMed  Google Scholar 

  5. J. Yang, C. Wu, I. Stefanescu, L. Jakobsson, I. Chervoneva, A. Horowitz, RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms. Sci Signal. 9, ra76 (2016). https://doi.org/10.1126/scisignal.aaf0791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. S. Kishida, H. Yamamoto, A. Kikuchi, Wnt-3a and Dvl induce neurite retraction by activating Rhoassociated kinase. Mol Cell Biol. 24, 4487–4501 (2004). https://doi.org/10.1128/mcb.24.10.4487-4501.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. C. Dyberg, S. Fransson, T. Andonova, B. Sveinbjörnsson, J. Lännerholm-Palm, T.K. Olsen, D. Forsberg, E. Herlenius, T. Martinsson, B. Brodin, P. Kogner, J.I. Johnsen, M. Wickström, Rho-associated kinase is a therapeutic target in neuroblastoma. Proc Natl Acad Sci U S A. 114, E6603–e6612 (2017). https://doi.org/10.1073/pnas.1706011114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. C.M. Fife, S.M. Sagnella, W.S. Teo, S.T. Po'uha, F.L. Byrne, Y.Y. Yeap, D.C. Ng, T.P. Davis, J.A. McCarroll, M. Kavallaris, Stathmin mediates neuroblastoma metastasis in a tubulin-independent manner via RhoA/ROCK signaling and enhanced transendothelial migration. Oncogene. 36, 501–511 (2017). https://doi.org/10.1038/onc.2016.220

    Article  CAS  PubMed  Google Scholar 

  9. M. Aittaleb, G. Gao, C.R. Evelyn, R.R. Neubig, J.J. Tesmer, A conserved hydrophobic surface of the LARG pleckstrin homology domain is critical for RhoA activation in cells. Cell Signal. 21, 1569–1578 (2009). https://doi.org/10.1016/j.cellsig.2009.06.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. T.M. Kitzing, A.S. Sahadevan, D.T. Brandt, H. Knieling, S. Hannemann, O.T. Fackler, J. Grosshans, R. Grosse, Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion. Genes Dev. 21, 1478–1483 (2007). https://doi.org/10.1101/gad.424807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. G.X. Shi, W.S. Yang, L. Jin, M.L. Matter, J.W. Ramos, RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases. Proc Natl Acad Sci U S A. 115, E190–e199 (2018). https://doi.org/10.1073/pnas.1708584115

    Article  CAS  PubMed  Google Scholar 

  12. C. Guilluy, V. Swaminathan, R. Garcia-Mata, E.T. O'Brien, R. Superfine, K. Burridge, The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat Cell Biol. 13, 722–727 (2011). https://doi.org/10.1038/ncb2254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. L.D. Osborne, G.Z. Li, T. How, E.T. O'Brien, G.C. Blobe, R. Superfine, K. Mythreye, TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion. Mol Biol Cell. 25, 3528–3540 (2014). https://doi.org/10.1091/mbc.E14-05-1015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. W.R. Thompson, S.S. Yen, G. Uzer, Z. Xie, B. Sen, M. Styner, K. Burridge, J. Rubin, LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage. Bone. 107, 172–180 (2018). https://doi.org/10.1016/j.bone.2017.12.001

    Article  CAS  PubMed  Google Scholar 

  15. M.D. Medlin, D.P. Staus, A.D. Dubash, J.M. Taylor, C.P. Mack, Sphingosine 1-phosphate receptor 2 signals through leukemia-associated RhoGEF (LARG), to promote smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol. 30, 1779–1786 (2010). https://doi.org/10.1161/atvbaha.110.209395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. N. Suzuki, R. Tokita, S. Minami, Involvement of IGF-1/LARG signaling in the differentiation of neural stem cells into oligodendrocytes. J Nippon Med Sch. 74(2–3) (2007). https://doi.org/10.1272/jnms.74.2

  17. D.C. Ong, Y.M. Ho, C. Rudduck, K. Chin, W.L. Kuo, D.K. Lie, C.L. Chua, P.H. Tan, K.W. Eu, F. Seow-Choen, C.Y. Wong, G.S. Hong, J.W. Gray, A.S. Lee, LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast and colorectal cancer. Oncogene. 28, 4189–4200 (2009). https://doi.org/10.1038/onc.2009.266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. S. Gurrapu, E. Pupo, G. Franzolin, L. Lanzetti, L. Tamagnone, Sema4C/PlexinB2 signaling controls breast cancer cell growth, hormonal dependence and tumorigenic potential. Cell Death Differ. 25, 1259–1275 (2018). https://doi.org/10.1038/s41418-018-0097-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. X. Shang, F. Marchioni, C.R. Evelyn, N. Sipes, X. Zhou, W. Seibel, M. Wortman, Y. Zheng, Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors. Proc Natl Acad Sci U S A. 110, 3155–3160 (2013). https://doi.org/10.1073/pnas.1212324110

    Article  PubMed  PubMed Central  Google Scholar 

  20. W.C. Chiu, J.Y. Chiang, F.T. Chiang, Small chemical compounds Y16 and Rhosin can inhibit calcium sensitization pathway in vascular smooth muscle cells of spontaneously hypertensive rats. J Formos Med Assoc. 120, 1863–1868 (2021). https://doi.org/10.1016/j.jfma.2021.03.031

    Article  CAS  PubMed  Google Scholar 

  21. Y. Xie, L. Gao, C. Xu, L. Chu, L. Gao, R. Wu, Y. Liu, T. Liu, X.J. Sun, R. Ren, J. Tang, Y. Zheng, Y. Zhou, S. Shen, ARHGEF12 regulates erythropoiesis and is involved in erythroid regeneration after chemotherapy in acute lymphoblastic leukemia patients. Haematologica. 105, 925–936 (2020). https://doi.org/10.3324/haematol.2018.210286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Y. Yang, L. Song, X. Huang, Y. Feng, Y. Zhang, Y. Liu, S. Li, Z. Zhan, L. Zheng, H. Feng, Y. Li, PRPS1-mediated purine biosynthesis is critical for pluripotent stem cell survival and stemness. Aging (Albany NY) 13, 4063–4078 (2021). https://doi.org/10.18632/aging.202372

    Article  CAS  PubMed  Google Scholar 

  23. Y. Sang, Y. Li, Y. Zhang, A.A. Alvarez, B. Yu, W. Zhang, B. Hu, S.Y. Cheng, H. Feng, CDK5-dependent phosphorylation and nuclear translocation of TRIM59 promotes macroH2A1 ubiquitination and tumorigenicity. Nat Commun. 10, 4013 (2019). https://doi.org/10.1038/s41467-019-12001-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. L. Song, B. Yu, Y. Yang, J. Liang, Y. Zhang, L. Ding, T. Wang, X. Wan, X. Yang, J. Tang, S. Wang, B. Li, Y. Li, H. Feng, Identification of functional cooperative mutations of GNAO1 in human acute lymphoblastic leukemia. Blood. 137, 1181–1191 (2021). https://doi.org/10.1182/blood.2020005622

    Article  CAS  PubMed  Google Scholar 

  25. K.K. Matthay, J.M. Maris, G. Schleiermacher, A. Nakagawara, C.L. Mackall, L. Diller, W.A. Weiss, Neuroblastoma. Nat Rev Dis Primers. 2, 16078 (2016). https://doi.org/10.1038/nrdp.2016.78

  26. L. Chesler, C. Schlieve, D.D. Goldenberg, A. Kenney, G. Kim, A. McMillan, K.K. Matthay, D. Rowitch, W.A. Weiss, Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma. Cancer Res. 66, 8139–8146 (2006). https://doi.org/10.1158/0008-5472.Can-05-2769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. D.J. Duffy, A. Krstic, T. Schwarzl, D.G. Higgins, W. Kolch, GSK3 inhibitors regulate MYCN mRNA levels and reduce neuroblastoma cell viability through multiple mechanisms, including p53 and Wnt signaling. Mol Cancer Ther. 13, 454–467 (2014). https://doi.org/10.1158/1535-7163.Mct-13-0560-t

    Article  CAS  PubMed  Google Scholar 

  28. J.G. Kim, M.J. Kim, W.J. Choi, M.Y. Moon, H.J. Kim, J.Y. Lee, J. Kim, S.C. Kim, S.G. Kang, G.Y. Seo, P.H. Kim, J.B. Park, Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation Through RhoA/ROCK. J Cell Physiol. 232, 1104–1113 (2017). https://doi.org/10.1002/jcp.25572

    Article  CAS  PubMed  Google Scholar 

  29. K.T. Schafernak, J.A. Williams, B.I. Clyde, C. Marcus, B. Decker, R.M. Toydemir, Identification of KMT2AARHGEF12 fusion in a child with a high-grade B-cell lymphoma. Cancer Genet. 258–259, 23–26 (2021). https://doi.org/10.1016/j.cancergen.2021.06.006

    Article  CAS  PubMed  Google Scholar 

  30. I. Panagopoulos, K. Andersen, M. Eilert-Olsen, B. Zeller, M.C. Munthe-Kaas, J. Buechner, L.T.N. Osnes, F. Micci, S. Heim, Therapy-induced deletion in 11q23 leading to fusion of KMT2A with ARHGEF12 and development of B lineage acute lymphoplastic leukemia in a child treated for acute myeloid leukemia caused by t(9;11)(p21;q23)/KMT2A-MLLT3. Cancer Genomics Proteomics 18, 67–81 (2021). https://doi.org/10.21873/cgp.20242

  31. N. Assaf, R. Liévin, F. Merabet, V. Raggueneau, J. Osman, R. Kim, F. Garnache, M. D'Angiò, P. Larghero, C. Meyer, R. Marschalek, P. Rousselot, C. Terré, KMT2A-ARHGEF12, a therapy related fusion with poor prognosis. Mol Biol Rep. 48, 7021–7027 (2021). https://doi.org/10.1007/s11033-021-06621-5

    Article  CAS  PubMed  Google Scholar 

  32. F. Peinemann, E.C. van Dalen, D.A. Tushabe, F. Berthold, Retinoic acid post consolidation therapy for high-risk neuroblastoma patients treated with autologous hematopoietic stem cell transplantation. Cochrane Database Syst Rev. 1, Cd010685 (2015). https://doi.org/10.1002/14651858.CD010685.pub2

    Article  PubMed  Google Scholar 

  33. N. Bayeva, E. Coll, O. Piskareva, Differentiating neuroblastoma: a systematic review of the retinoic acid, its derivatives, and synergistic interactions. J Pers Med. 11 (2021). https://doi.org/10.3390/jpm11030211

  34. K.S. Vrenken, B.M.T. Vervoort, D.S. van Ingen Schenau, Y.H.W. Derks, L. van Emst, P.G. Grytsenko, J.A.J. Middelbeek, F.N. van Leeuwen, The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy. Biochim Biophys Acta Mol Basis Dis. 2020, 165644 (1866). https://doi.org/10.1016/j.bbadis.2019.165644

    Article  CAS  Google Scholar 

  35. G. Ferrari-Amorotti, V. Fragliasso, R. Esteki, Z. Prudente, A.R. Soliera, S. Cattelani, G. Manzotti, G. Grisendi, M. Dominici, M. Pieraccioli, G. Raschellà, C. Chiodoni, M.P. Colombo, B. Calabretta, Inhibiting interactions of lysine demethylase LSD1 with snail/slug blocks cancer cell invasion. Cancer Res. 73, 235–245 (2013). https://doi.org/10.1158/0008-5472.Can-12-1739

    Article  CAS  PubMed  Google Scholar 

  36. G. Ferrari-Amorotti, C. Chiodoni, F. Shen, S. Cattelani, A.R. Soliera, G. Manzotti, G. Grisendi, M. Dominici, F. Rivasi, M.P. Colombo, A. Fatatis, B. Calabretta, Suppression of invasion and metastasis of triple-negative breast cancer lines by pharmacological or genetic inhibition of slug activity. Neoplasia. 16, 1047–1058 (2014). https://doi.org/10.1016/j.neo.2014.10.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported in part by the National Key R&D Program of China (2018YFC1313000/2018YFC1313005) to Y.G. and Y. L; the National Natural Science Foundation of China (81972341 and 32271007) to Y.L.; the Shanghai Municipal Commission of Science and Technology (201409002700 to Y. L., 17411950400 to J. T.); Program of Shanghai Academic/Technology Research Leader (21XD1403100) to H.F.; the Shanghai Jiao Tong University Medical Engineering Cross Fund (No. YG2017MS32); and the Pudong New Area Science & Technology Development Fund (PKJ2018-Y47) to Y. L.; the State Key Laboratory of Oncogenes and Related Genes (KF2115) to F.L.

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Author notes

  1. Yi Yang, Siqi Wang, Jiaoyang Cai, and Jianwei Liang contributed equally to this work

Authors and Affiliations

  1. Pediatric Translational Medicine Institute, Department of Hematology & Oncology, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, National Health Committee Key Laboratory of Pediatric Hematology & Oncology, Shanghai, 200127, China

    Yi Yang, Yingwen Zhang & Yanxin Li

  2. Department of Hematology & Oncology, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, National Health Committee Key Laboratory of Pediatric Hematology & Oncology, Shanghai, 200127, China

    Siqi Wang, Jiaoyang Cai, Jianwei Liang, Yangyang Xie, Jingyan Tang, Yijin Gao & Shuhong Shen

  3. State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China

    Fei Luo & Haizhong Feng

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  1. Yi Yang
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Contributions

Y.L., H.F., S.S. and J.G. designed the experiments. Y.Y., S.W., J.C., J.L. and F.L. performed the experiments. Y.Y., S.W., J.C., Y.W.Z., Y.Y.X. and J.Y.T. analyzed and interpreted the data. Y.Y., Y.L. and H.F. wrote the paper. Y.L. supervised the project.

Corresponding authors

Correspondence to Yijin Gao, Shuhong Shen, Haizhong Feng or Yanxin Li.

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The original version of this article was revised: In Fig. 5f of this article the representative MYCN IHC images of tumor from vehicle-treated mice presented the wrong samples. Fig. 5f has been corrected in the original article. The authors declare that this correction does not affect the description, interpretation, or the original conclusions of the manuscript.

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Yang, Y., Wang, S., Cai, J. et al. Targeting ARHGEF12 promotes neuroblastoma differentiation, MYCN degradation, and reduces tumorigenicity. Cell Oncol. 46, 133–143 (2023). https://doi.org/10.1007/s13402-022-00739-9

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