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Sphingadienes show therapeutic efficacy in neuroblastoma in vitro and in vivo by targeting the AKT signaling pathway

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Summary

Neuroblastoma is a childhood malignancy that accounts for approximately 15% of childhood cancer deaths. Only 20–35% of children with metastatic neuroblastoma survive with standard therapy. Identification of more effective therapies is essential to improving the outcome of children with high-stage disease. Sphingadienes (SD) are growth-inhibitory sphingolipids found in natural sources including soy. They exhibit chemopreventive activity in mouse models of colon cancer, where they mediate cytotoxicity by inhibiting key pro-carcinogenic signaling pathways. In this study, the effect of SD on neuroblastoma was analyzed. Low micromolar concentrations of SD were cytotoxic to transformed and primary neuroblastoma cells independently of N-Myc amplification status. SD induced both caspase-dependent apoptosis and autophagy in neuroblastoma cells. However, only inhibition of caspase-dependent apoptosis protected neuroblastoma cells from SD-mediated cytotoxicity. SD also inhibited AKT activation in neuroblastoma cells as shown by reduced phosphorylated AKT levels. Pre-treatment with insulin attenuated SD-mediated cytotoxicity in vitro. SD-loaded nanoparticles (NP) administered parenterally to immunodeficient mice carrying neuroblastoma xenografts resulted in cytotoxic levels of SD in the circulation and significantly reduced tumor growth compared to vehicle-treated controls. Analysis of tumor extracts demonstrated reduced AKT activation in tumors of mice treated with SD-NP compared to controls treated with empty NP. Our findings indicate SD are novel potential chemotherapeutic agents that promote neuroblastoma cell death and reduce tumorigenicity in vivo.

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Change history

  • 29 April 2019

    The authors would like to note an omission of disclosure in this paper. Author JDS is cofounder, equity-holder, and consultant of GILTRx Therapeutics.

  • 29 April 2019

    The authors would like to note an omission of disclosure in this paper. Author JDS is cofounder, equity-holder, and consultant of GILTRx Therapeutics.

References

  1. Matthay KK, George RE, Yu AL (2012) Promising therapeutic targets in neuroblastoma. Clin Cancer Res 18(10):2740–2753

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Ries LA et al (1999) Cancer incidence and survival among children and adolescents: United States SEER Program 1975–1995. National Cancer Institute, SEER Program, NIH Pub. No. 99–4649 192, Bethesda

    Google Scholar 

  3. Young JL Jr, Miller RW (1975) Incidence of malignant tumors in U. S. children. J Pediatr 86(2):254–258

    Article  PubMed  Google Scholar 

  4. Cheung NK, Dyer MA (2013) Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer 13(6):397–411

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Frappaz D et al (2000) LMCE3 treatment strategy: results in 99 consecutively diagnosed stage 4 neuroblastomas in children older than 1 year at diagnosis. J Clin Oncol 18(3):468–476

    Article  PubMed  CAS  Google Scholar 

  6. Speleman F, De Preter K, Vandesompele J (2011) Neuroblastoma genetics and phenotype: a tale of heterogeneity. Semin Cancer Biol 21(4):238–244

    Article  PubMed  CAS  Google Scholar 

  7. Maris JM et al (2007) Neuroblastoma. Lancet 369(9579):2106–2120

    Article  PubMed  CAS  Google Scholar 

  8. Maris JM (2010) Recent advances in neuroblastoma. N Engl J Med 362(23):2202–2211

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Stram DO et al (1996) Consolidation chemoradiotherapy and autologous bone marrow transplantation versus continued chemotherapy for metastatic neuroblastoma: a report of two concurrent Children's Cancer Group studies. J Clin Oncol 14(9):2417–2426

    Article  PubMed  CAS  Google Scholar 

  10. Zheng W et al (2006) Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim Biophys Acta 1758:1864–1884

    Article  PubMed  CAS  Google Scholar 

  11. Durrant LG, Noble P, Spendlove I (2012) Immunology in the clinic review series; focus on cancer: glycolipids as targets for tumour immunotherapy. Clin Exp Immunol 167(2):206–215

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Ogretmen B, Hannun YA (2004) Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 4(8):604–616

    Article  PubMed  CAS  Google Scholar 

  13. Symolon H et al (2004) Dietary soy sphingolipids suppress tumorigenesis and gene expression in 1,2-dimethylhydrazine-treated CF1 mice and ApcMin/+ mice. J Nutr 134(5):1157–1161

    Article  PubMed  CAS  Google Scholar 

  14. Hakomori S (2002) Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci U S A 99(16):10231–10233

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Gilman AL et al (2009) Phase I study of ch14.18 with granulocyte-macrophage colony-stimulating factor and interleukin-2 in children with neuroblastoma after autologous bone marrow transplantation or stem-cell rescue: a report from the Children's Oncology Group. J Clin Oncol 27(1):85–91

    Article  PubMed  CAS  Google Scholar 

  16. Yu AL et al (2010) Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med 363(14):1324–1334

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Barker E et al (1991) Effect of a chimeric anti-ganglioside GD2 antibody on cell-mediated lysis of human neuroblastoma cells. Cancer Res 51(1):144–149

    PubMed  CAS  Google Scholar 

  18. Tettamanti G (2004) Ganglioside/glycosphingolipid turnover: new concepts. Glycoconj J 20(5):301–317

    Article  PubMed  CAS  Google Scholar 

  19. Chun J et al (2002) Synthesis of new trans double bond sphingolipid analogues: D4,6 and D6 ceramides. J Org Chem 67:2600–2605

    Article  PubMed  CAS  Google Scholar 

  20. Struckhoff AP et al (2004) Novel ceramide analogs as potential chemotherapeutic agents in breast cancer. J Pharmacol Exp Ther 309(2):523–532

    Article  PubMed  CAS  Google Scholar 

  21. Fyrst H et al (2009) Natural sphingadienes inhibit Akt-dependent signaling and prevent intestinal tumorigenesis. Cancer Res 69(24):9457–9464

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Kumar A et al (2012) Chemopreventive sphingadienes downregulate Wnt signaling via a PP2A/Akt/GSK3beta pathway in colon cancer. Carcinogenesis 33(9):1726–1735

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2(7):489–501

    Article  PubMed  CAS  Google Scholar 

  24. Bender A et al (2011) PI3K inhibitors prime neuroblastoma cells for chemotherapy by shifting the balance towards pro-apoptotic Bcl-2 proteins and enhanced mitochondrial apoptosis. Oncogene 30(4):494–503

  25. Nguyen TS et al (2008) Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers. Biochim Biophys Acta 1778(1):303–312

    Article  PubMed  CAS  Google Scholar 

  26. Oskouian B et al (2006) Sphingosine-1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is downregulated in colon cancer. Proc Natl Acad Sci U S A 103:17384–17389

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Jiang Q et al (2004) gamma-Tocopherol or combinations of vitamin E forms induce cell death in human prostate cancer cells by interrupting sphingolipid synthesis. Proc Natl Acad Sci U S A 101(51):17825–17830

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Ryan RO, Forte TM, Oda MN (2003) Optimized bacterial expression of human apolipoprotein A-I. Protein Expr Purif 27(1):98–103

    Article  PubMed  CAS  Google Scholar 

  29. Lalefar NR et al (2016) Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells. J Nanobiotechnol 14(1):66

    Article  CAS  Google Scholar 

  30. Suh JH et al (2017) An LC/MS/MS method for quantitation of chemopreventive sphingadienes in food products and biological samples. J Chromatogr B Anal Technol Biomed Life Sci 1061-1062:292–299

    Article  CAS  Google Scholar 

  31. Soubeyran P et al (2001) Homeobox gene Cdx1 regulates Ras, Rho and PI3 kinase pathways leading to transformation and tumorigenesis of intestinal epithelial cells. Oncogene 20(31):4180–4187

    Article  PubMed  CAS  Google Scholar 

  32. Ryan RO (2010) Nanobiotechnology applications of reconstituted high density lipoprotein. J Nanobiotechnol 8:28

    Article  CAS  Google Scholar 

  33. Ghosh M, Ryan RO (2014) ApoE enhances nanodisk-mediated curcumin delivery to glioblastoma multiforme cells. Nanomedicine (London) 9(6):763–771

    Article  CAS  Google Scholar 

  34. Singh AT et al (2011) Curcumin nanodisk-induced apoptosis in mantle cell lymphoma. Leuk Lymphoma 52(8):1537–1543

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Oda MN et al (2006) Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B. J Lipid Res 47(2):260–267

    Article  PubMed  CAS  Google Scholar 

  36. Li MH, Hla T, Ferrer F (2011) Sphingolipid modulation of angiogenic factor expression in neuroblastoma. Cancer Prev Res (Phila) 4(8):1325–1332

    Article  CAS  Google Scholar 

  37. Villullas IR et al (2003) Characterisation of a sphingosine 1-phosphate-activated Ca2+ signalling pathway in human neuroblastoma cells. J Neurosci Res 73(2):215–226

    Article  PubMed  CAS  Google Scholar 

  38. Rahmaniyan M, Qudeimat A, Kraveka JM (2012) Bioactive Sphingolipids in Neuroblastoma. In: Shimada H (ed) Neuroblastoma - Present and Future. InTech, Rijeka, pp 153–184

    Google Scholar 

  39. Li MH, Hla T, Ferrer F (2013) FTY720 inhibits tumor growth and enhances the tumor-suppressive effect of topotecan in neuroblastoma by interfering with the sphingolipid signaling pathway. Pediatr Blood Cancer 60(9):1418–1423

    Article  PubMed  CAS  Google Scholar 

  40. Lee JH et al (2011) Effects of sphingosine-1-phosphate on neural differentiation and neurite outgrowth in neuroblastoma cells. Chonnam Med J 47(1):27–30

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Ponnusamy S et al (2010) Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance. Future Oncol 6(10):1603–1624

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Czubowicz K, Strosznajder R (2014) Ceramide in the Molecular Mechanisms of Neuronal Cell Death. The Role of Sphingosine-1-Phosphate. Mol Neurobiol 50(1):26–37

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Kraveka JM et al (2007) Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells. J Biol Chem 282(23):16718–16728

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Fransson S et al (2014) Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas. Mol Neurobiol 50(1):26–37

  45. Opel D et al (2007) Activation of Akt predicts poor outcome in neuroblastoma. Cancer Res 67(2):735–745

    Article  PubMed  CAS  Google Scholar 

  46. Qiao J et al (2013) Akt2 regulates metastatic potential in neuroblastoma. PLoS One 8(2):e56382

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Foley NH et al (2010) MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2. Mol Cancer 9:83

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Chesler L et al (2006) Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma. Cancer Res 66(16):8139–8146

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Sugawara T et al (2010) Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats. J Lipid Res 51(7):1761–1769

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Fyrst H et al (2008) Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes. J Lipid Res 49(3):597–606

    Article  PubMed  CAS  Google Scholar 

  51. Zhang T et al (2015) Regulation of mitochondrial ceramide distribution by members of the BCL-2 family. J Lipid Res 56(8):1501–1510

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Quehenberger O et al (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51(11):3299–3305

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Serra M, Saba JD (2010) Sphingosine 1-phosphate lyase, a key regulator of sphingosine 1-phosphate signaling and function. Adv Enzym Regul 50(1):349–362

    Article  Google Scholar 

  54. Sugawara T et al (2003) Digestion of maize sphingolipids in rats and uptake of sphingadienine by Caco-2 cells. J Nutr 133(9):2777–2782

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by St. Baldricks Fellowship Grant 245216 (AEA), National Institutes of Health grants R01CA129438, S10OD018070 and Swim Across America Foundation (JDS) and National Institutes of Health grant R37 HL-64159 (ROR).

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Author notes

  1. Ana E. Aguilar

    Present address: Arnold Palmer Hospital for Children, 92 W Miller St MP 318 2nd floor, Orlando, FL, 32806, USA

  2. Latika Puri

    Present address: St Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN, 38105, USA

  3. Ana E. Aguilar and Joanna Y. Lee contributed equally to this work.

Authors and Affiliations

  1. UCSF Benioff Children’s Hospital Oakland, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA, 94609, USA

    Piming Zhao, Ana E. Aguilar, Joanna Y. Lee, Lucy A. Paul, Jung H. Suh, Latika Puri, Meng Zhang, Jennifer Beckstead, Andrzej Witkowski, Robert O. Ryan & Julie D. Saba

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Contributions

All authors contributed substantially to the study. Data were generated and analysed by PZ, AEA, LAP, JYL, JHS, LP, MZ, JB and AW. ROR contributed to the design of some of the studies and interpretation of results. JDS designed the overall study, interpreted all results and wrote the manuscript. All authors reviewed the manuscript for accuracy and contributed to the writing of the manuscript.

Corresponding author

Correspondence to Julie D. Saba.

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Conflicts of interest

Author Piming Zhao declares that he has no conflict of interest. Author Ana E. Aguilar declares that she has no conflict of interest. Author Joanna Y. Lee declares she has no conflict of interest. Author Lucy A. Paul declares she has no conflict of interest. Author Jung H. Suh declares he has no conflict of interest. Author Latika A. Puri declares that she has no conflict of interest. Author Meng Zhang declares he has no conflict of interest. Author Jennifer Beckstead declares she has no conflict of interest. Author Andrzej Witkowski declares he has no conflict of interest. Author Robert O. Ryan declares he has no conflict of interest. Author Julie. D Saba declares she has no conflict of interest.

Ethical approvals

Human tumor and nonmalignant tissues utilized in this study were acquired in accordance with an approved UCSF Benioff Children’s Hospital Oakland Institutional Review Board protocol and with informed consent. All animal experiments conducted in this study were performed in accordance with an approved UCSF Benioff Children’s Hospital Oakland Institutional Animal Care and Use Committee protocol.

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Supplemental Figure 1

SD are cytotoxic to three transformed neuroblastoma cell lines. Kelly, SHSY5Y and LAN-5 neuroblastoma cells were grown to 70% confluence in the preferred medium plus 10% FCS, then changed to serum-free medium and treated with either 10 μM SD (Kelly and SHSY5Y), 15 μM SD (LAN-5) or vehicle (Veh). After 18–24 h, cells were harvested, and viability was determined by MTS assay. Results are presented as arbitrary units (AU). Each condition was analysed in triplicate, and results are representative of at least three experiments. (A) Kelly cells; (B) SHSY5Y cells; (C) LAN-5 cells. * p < 0.05. (GIF 81 kb)

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Supplemental Figure 2

SD are cytotoxic to primary neuroblastoma cells. (A) Immunofluorescence microscopy of primary neuroblastoma cells stained with neuronal markers tyrosine hydroxylase (TH), synaptophysin (SP), and the nuclear marker DAPI. Kelly cells were used as a positive control. (B) Primary neuroblastoma cells were exposed to SD concentrations ranging from 0 to15 μM. After a 24 h incubation, cell viability was assessed by the MTS assay. * p < 0.05 at 5 and 10 μM SD. (GIF 206 kb)

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Supplemental Figure 3

Image quantification results for Fig. 3. (A) Image quantification represents the ratio of P-AKT/T-AKT in the immunoblot autoradiogram shown in (3A). (B) Image quantification represents ratio of cleaved PARP/uncleaved PARP in the immunoblot shown in (3A). (C) Image quantification represents the ratio of P-AKT/T-AKT in the immunoblot autoradiogram shown in (3B). (D) Image quantification represents the ratio of cleaved/uncleaved PARP in the immunoblot autoradiogram shown in (3B). (E) Image quantification represents the ratio of cleaved/uncleaved PARP in the immunoblot autoradiogram shown in (3C). All image quantification results are in relative units, with time zero or untreated condition arbitrarily set at one. These experiments were repeated at least three times with similar results. (GIF 91 kb)

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Zhao, P., Aguilar, A.E., Lee, J.Y. et al. Sphingadienes show therapeutic efficacy in neuroblastoma in vitro and in vivo by targeting the AKT signaling pathway. Invest New Drugs 36, 743–754 (2018). https://doi.org/10.1007/s10637-017-0558-5

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