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Protein phosphatase 1 regulatory subunit 1A in ewing sarcoma tumorigenesis and metastasis

Oncogene volume 37, pages 798–809 (2018)Cite this article

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Abstract

Protein phosphatase inhibitors are often considered as tumor promoters. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is a potent protein phosphatase 1 (PP1) inhibitor; however, its role in tumor development is largely undefined. Here we characterize, for the first time, the functions of PPP1R1A in Ewing sarcoma (ES) pathogenesis. We found that PPP1R1A is one of the top ranked target genes of EWS/FLI, the master regulator of ES, and is upregulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A resulted in a significant decrease in oncogenic transformation and cell migration in vitro as well as xenograft tumor growth and metastasis in an orthotopic mouse model. RNA-sequencing and functional annotation analyses revealed that PPP1R1A regulates genes associated with various cellular functions including cell junction, adhesion and neurogenesis. Interestingly, we found a significant overlap of PPP1R1A-regulated gene set with that of ZEB2 and EWS, which regulates metastasis and neuronal differentiation in ES, respectively. Further studies for characterization of the molecular mechanisms revealed that activation of PPP1R1A by PKA phosphorylation at Thr35, and subsequent PP1 binding and inhibition, was required for PPP1R1A-mediated tumorigenesis and metastasis, likely by increasing the phosphorylation levels of various PP1 substrates. Furthermore, we found that a PKA inhibitor impaired ES cell proliferation, tumor growth and metastasis, which was rescued by the constitutively active PPP1R1A. Together, these results offered new insights into the role and mechanism of PPP1R1A in tumor development and identified an important kinase and phosphatase pathway, PKA/PPP1R1A/PP1, in ES pathogenesis. Our findings strongly suggest a potential therapeutic value of inhibition of the PKA/PPP1R1A/PP1 pathway in the treatment of primary and metastatic ES.

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References

  1. Ladenstein R, Potschger U, Le Deley MC, Whelan J, Paulussen M, Oberlin O et al. Primary disseminated multifocal Ewing sarcoma: results of the Euro-EWING 99 trial. J Clin Oncol 2010; 28: 3284–3291.

    Article  CAS  Google Scholar 

  2. Ohali A, Avigad S, Zaizov R, Ophir R, Horn-Saban S, Cohen IJ et al. Prediction of high risk Ewing's sarcoma by gene expression profiling. Oncogene 2004; 23: 8997–9006.

    Article  CAS  Google Scholar 

  3. Schaefer KL, Eisenacher M, Braun Y, Brachwitz K, Wai DH, Dirksen U et al. Microarray analysis of Ewing's sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy. Eur J Cancer 2008; 44: 699–709.

    Article  CAS  Google Scholar 

  4. Volchenboum SL, Andrade J, Huang L, Barkauskas DA, Krailo M, Womer RB et al. Gene expression profiling of ewing sarcoma tumors reveals the prognostic importance of tumor-stromal interactions: a report from the Children's Oncology Group. J Pathol Clin Res 2015; 1: 83–94.

    Article  CAS  Google Scholar 

  5. Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 1992; 359: 162–165.

    Article  CAS  Google Scholar 

  6. Crompton BD, Stewart C, Taylor-Weiner A, Alexe G, Kurek KC, Calicchio ML et al. The genomic landscape of pediatric Ewing sarcoma. Cancer Discov 2014; 4: 1326–1341.

    Article  CAS  Google Scholar 

  7. Tirode F, Laud-Duval K, Prieur A, Delorme B, Charbord P, Delattre O . Mesenchymal stem cell features of Ewing tumors. Cancer Cell 2007; 11: 421–429.

    Article  CAS  Google Scholar 

  8. Niedan S, Kauer M, Aryee DN, Kofler R, Schwentner R, Meier A et al. Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma. Oncogene 2014; 33: 3927–3938.

    Article  CAS  Google Scholar 

  9. Sankar S, Gomez NC, Bell R, Patel M, Davis IJ, Lessnick SL et al. EWS and RE1-silencing transcription factor inhibit neuronal phenotype development and oncogenic transformation in ewing sarcoma. Genes Cancer 2013; 4: 213–223.

    Article  Google Scholar 

  10. Smith R, Owen LA, Trem DJ, Wong JS, Whangbo JS, Golub TR et al. Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 2006; 9: 405–416.

    Article  CAS  Google Scholar 

  11. Luo W, Gangwal K, Sankar S, Boucher KM, Thomas D, Lessnick SL . GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance. Oncogene 2009; 28: 4126–4132.

    Article  CAS  Google Scholar 

  12. Sankar S, Tanner JM, Bell R, Chaturvedi A, Randall RL, Beckerle MC et al. A novel role for keratin 17 in coordinating oncogenic transformation and cellular adhesion in Ewing sarcoma. Mol Cell Biol 2013; 33: 4448–4460.

    Article  CAS  Google Scholar 

  13. Sechler M, Parrish JK, Birks DK, Jedlicka P . The histone demethylase KDM3A, and its downstream target MCAM, promote Ewing Sarcoma cell migration and metastasis. Oncogene 2017; 36: 4150–4160.

    Article  CAS  Google Scholar 

  14. Luo W, Peterson A, Garcia BA, Coombs G, Kofahl B, Heinrich R et al. Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex. EMBO J 2007; 26: 1511–1521.

    Article  CAS  Google Scholar 

  15. Virshup DM, Shenolikar S . From promiscuity to precision: protein phosphatases get a makeover. Mol Cell 2009; 33: 537–545.

    Article  CAS  Google Scholar 

  16. Connor JH, Weiser DC, Li S, Hallenbeck JM, Shenolikar S . Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1. Mol Cell Biol 2001; 21: 6841–6850.

    Article  CAS  Google Scholar 

  17. Nicolaou P, Hajjar RJ, Kranias EG . Role of protein phosphatase-1 inhibitor-1 in cardiac physiology and pathophysiology. J Mol Cell Cardiol 2009; 47: 365–371.

    Article  CAS  Google Scholar 

  18. Fleuren ED, Zhang L, Wu J, Daly RJ . The kinome 'at large' in cancer. Nat Rev Cancer 2016; 16: 83–98.

    Article  CAS  Google Scholar 

  19. Kauer M, Ban J, Kofler R, Walker B, Davis S, Meltzer P et al. A molecular function map of Ewing's sarcoma. PLoS One 2009; 4: e5415.

    Article  Google Scholar 

  20. Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suva ML et al. EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell 2014; 26: 668–681.

    Article  CAS  Google Scholar 

  21. Patel M, Simon JM, Iglesia MD, Wu SB, McFadden AW, Lieb JD et al. Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription. Genome Res 2012; 22: 259–270.

    Article  CAS  Google Scholar 

  22. Hurta RA, Wright JA . Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. Biochem Cell Biol 1992; 70: 1081–1087.

    Article  CAS  Google Scholar 

  23. El-Rifai W, Smith MF Jr., Li G, Beckler A, Carl VS, Montgomery E et al. Gastric cancers overexpress DARPP-32 and a novel isoform, t-DARPP. Cancer Res 2002; 62: 4061–4064.

    CAS  PubMed  Google Scholar 

  24. Chaturvedi A, Hoffman LM, Welm AL, Lessnick SL, Beckerle MC . The EWS/FLI oncogene drives changes in cellular morphology, adhesion, and migration in ewing sarcoma. Genes Cancer 2012; 3: 102–116.

    Article  Google Scholar 

  25. Spaderna S, Schmalhofer O, Wahlbuhl M, Dimmler A, Bauer K, Sultan A et al. The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer Res 2008; 68: 537–544.

    Article  CAS  Google Scholar 

  26. Waisberg J, De Souza Viana L, Affonso Junior RJ, Silva SR, Denadai MV, Margeotto FB et al. Overexpression of the ITGAV gene is associated with progression and spread of colorectal cancer. Anticancer Res 2014; 34: 5599–5607.

    PubMed  Google Scholar 

  27. Sainz-Jaspeado M, Lagares-Tena L, Lasheras J, Navid F, Rodriguez-Galindo C, Mateo-Lozano S et al. Caveolin-1 modulates the ability of Ewing's sarcoma to metastasize. Mol Cancer Res 2010; 8: 1489–1500.

    Article  CAS  Google Scholar 

  28. Sand LG, Scotlandi K, Berghuis D, Snaar-Jagalska BE, Picci P, Schmidt T et al. CXCL14, CXCR7 expression and CXCR4 splice variant ratio associate with survival and metastases in Ewing sarcoma patients. Eur J Cancer 2015; 51: 2624–2633.

    Article  CAS  Google Scholar 

  29. Westerlund N, Zdrojewska J, Padzik A, Komulainen E, Bjorkblom B, Rannikko E et al. Phosphorylation of SCG10/stathmin-2 determines multipolar stage exit and neuronal migration rate. Nat Neurosci 2011; 14: 305–313.

    Article  CAS  Google Scholar 

  30. Li F, Tian X, Zhou Y, Zhu L, Wang B, Ding M et al. Dysregulated expression of secretogranin III is involved in neurotoxin-induced dopaminergic neuron apoptosis. J Neurosci Res 2012; 90: 2237–2246.

    Article  CAS  Google Scholar 

  31. Chi LM, Wang X, Nan GX . In silico analyses for molecular genetic mechanism and candidate genes in patients with Alzheimer's disease. Acta Neurol Belg 2016; 116: 543–547.

    Article  Google Scholar 

  32. Wiles ET, Bell R, Thomas D, Beckerle M, Lessnick SL . ZEB2 represses the epithelial phenotype and facilitates metastasis in ewing sarcoma. Genes Cancer 2013; 4: 486–500.

    Article  CAS  Google Scholar 

  33. Cohen PT . Protein phosphatase 1—targeted in many directions. J Cell Sci 2002; 115 (Pt 2): 241–256.

    CAS  PubMed  Google Scholar 

  34. Endo S, Zhou X, Connor J, Wang B, Shenolikar S . Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor. Biochemistry 1996; 35: 5220–5228.

    Article  CAS  Google Scholar 

  35. Croft DR, Olson MF . Conditional regulation of a ROCK-estrogen receptor fusion protein. Methods Enzymol 2006; 406: 541–553.

    Article  CAS  Google Scholar 

  36. Ma D, Zhou P, Harbour JW . Distinct mechanisms for regulating the tumor suppressor and antiapoptotic functions of Rb. J Biol Chem 2003; 278: 19358–19366.

    Article  CAS  Google Scholar 

  37. Mouravlev A, Young D, During MJ . Phosphorylation-dependent degradation of transgenic CREB protein initiated by heterodimerization. Brain Res 2007; 1130: 31–37.

    Article  CAS  Google Scholar 

  38. Naviglio S, Caraglia M, Abbruzzese A, Chiosi E, Di Gesto D, Marra M et al. Protein kinase A as a biological target in cancer therapy. Expert Opin Ther Targets 2009; 13: 83–92.

    Article  CAS  Google Scholar 

  39. Howe AK . Regulation of actin-based cell migration by cAMP/PKA. Biochim Biophys Acta 2004; 1692: 159–174.

    Article  CAS  Google Scholar 

  40. Finger EC, Castellini L, Rankin EB, Vilalta M, Krieg AJ, Jiang D et al. Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells. Proc Natl Acad Sci USA 2015; 112: 4441–4446.

    Article  CAS  Google Scholar 

  41. McKenzie AJ, Campbell SL, Howe AK . Protein kinase A activity and anchoring are required for ovarian cancer cell migration and invasion. PLoS One 2011; 6: e26552.

    Article  CAS  Google Scholar 

  42. Postel-Vinay S, Veron AS, Tirode F, Pierron G, Reynaud S, Kovar H et al. Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma. Nat Genet 2012; 44: 323–327.

    Article  CAS  Google Scholar 

  43. Castro ME, Ferrer I, Cascon A, Guijarro MV, Lleonart M, Ramon Y, Cajal S et al. PPP1CA contributes to the senescence program induced by oncogenic Ras. Carcinogenesis 2008; 29: 491–499.

    Article  CAS  Google Scholar 

  44. Li L, Ren CH, Tahir SA, Ren C, Thompson TC . Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. Mol Cell Biol 2003; 23: 9389–9404.

    Article  CAS  Google Scholar 

  45. Scotlandi K, Benini S, Sarti M, Serra M, Lollini PL, Maurici D et al. Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma/peripheral neuroectodermal tumor: a possible therapeutic target. Cancer Res 1996; 56: 4570–4574.

    CAS  PubMed  Google Scholar 

  46. Potratz J, Tillmanns A, Berning P, Korsching E, Schaefer C, Lechtape B et al. Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease. Mol Oncol 2016; 10: 677–692.

    Article  CAS  Google Scholar 

  47. Huang X, Park H, Greene J, Pao J, Mulvey E, Zhou SX et al. IGF1R- and ROR1-specific CAR T cells as a potential therapy for high risk sarcomas. PLoS One 2015; 10: e0133152.

    Article  Google Scholar 

  48. Franzetti GA, Laud-Duval K, van der Ent W, Brisac A, Irondelle M, Aubert S et al. Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells. Oncogene 2017; 36: 3505–3514.

    Article  CAS  Google Scholar 

  49. von Heyking K, Calzada-Wack J, Gollner S, Neff F, Schmidt O, Hensel T et al. The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma. Mol Oncol 2017; 11: 1288–1301.

    Article  CAS  Google Scholar 

  50. Dela Cruz F, Terry M, Matushansky I . A transgenic, mesodermal specific, Dkk1 mouse model recapitulates a spectrum of human congenital limb reduction defects. Differentiation 2012; 83: 220–230.

    Article  CAS  Google Scholar 

  51. Gautier L, Cope L, Bolstad BM, Irizarry RA . affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 2004; 20: 307–315.

    Article  CAS  Google Scholar 

  52. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015; 43: e47.

    Article  Google Scholar 

  53. Chu Y, Hochberg J, Yahr A, Ayello J, van de Ven C, Barth M et al. Targeting CD20+ aggressive B-cell non-Hodgkin lymphoma by anti-CD20 CAR mRNA-modified expanded natural killer cells in vitro and in NSG mice. Cancer Immunol Res 2015; 3: 333–344.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Drs David Virshup, Shirish Shenolikar, Angus Nairn and James Bibb for helpful discussion. We would also like to thank the members of the Cairo and Lessnick laboratories for technical assistance and helpful discussion, and Erin Morris RN for her administrative assistance in the preparation of this manuscript. This work is supported by funds from Pediatric Cancer Research Foundation (MSC) and Association for Research of Childhood Cancer (WL).

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Authors and Affiliations

  1. Departments of Pediatrics, New York Medical College, Valhalla, NY, USA

    W Luo, J Ayello, J M Rosenblum & M S Cairo

  2. Departments of Pathology, New York Medical College, Valhalla, NY, USA

    W Luo & M S Cairo

  3. James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA

    C Xu

  4. Memorial Sloan Kettering Cancer Center, New York, NY, USA

    F Dela Cruz

  5. Nationwide Children’s Hospital, Columbus, OH, USA

    S L Lessnick

  6. Departments of Medicine, New York Medical College, Valhalla, NY, USA

    M S Cairo

  7. Departments of Immunology and Microbiology, New York Medical College, Valhalla, NY, USA

    M S Cairo

  8. Departments of Cell Biology and Anatomy, New York Medical College, Valhalla, NY, USA

    M S Cairo

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  1. W Luo
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  2. C Xu
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  3. J Ayello
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Correspondence to W Luo or M S Cairo.

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Luo, W., Xu, C., Ayello, J. et al. Protein phosphatase 1 regulatory subunit 1A in ewing sarcoma tumorigenesis and metastasis. Oncogene 37, 798–809 (2018). https://doi.org/10.1038/onc.2017.378

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