Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth

Oncogene volume 29, pages 3297–3306 (2010)Cite this article

Subjects

Abstract

Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-MycT58A+H-RasG12V). The transformed cells, WTt and DKOt respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKOt cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/107 cells from 9 to 11 nmol/107 WTt or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKOt cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • Allende ML, Proia RL . (2002). Lubricating cell signaling pathways with gangliosides. Curr Opin Struct Biol 12: 587–592.

    Article  CAS  Google Scholar 

  • Buggins AG, Milojkovic D, Arno MJ, Lea NC, Mufti GJ, Thomas NS et al. (2001). Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways. J Immunol 167: 6021–6030.

    Article  CAS  Google Scholar 

  • Cazet A, Groux-Degroote S, Teylaert B, Kwon KM, Lehoux S, Slomianny C et al. (2009). G(D3) synthase overexpression enhances proliferation and migration of MDA-MB-231 breast cancer cells. Biol Chem 390: 601–609.

    Article  CAS  Google Scholar 

  • Deng W, Li R, Guerrera M, Liu Y, Ladisch S . (2002). Transfection of glucosylceramide synthase antisense inhibits mouse melanoma formation. Glycobiology 12: 145–152.

    Article  CAS  Google Scholar 

  • Floutsis G, Ulsh L, Ladisch S . (1989). Immunosuppressive activity of human neuroblastoma tumor gangliosides. Int J Cancer 43: 6–9.

    Article  CAS  Google Scholar 

  • Hakomori S . (1973). Glycolipids of tumor cell membrane. Adv Cancer Res 18: 265–315.

    Article  CAS  Google Scholar 

  • Hakomori S . (1996). Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res 56: 5309–5318.

    CAS  Google Scholar 

  • Hyuga S, Yamagata S, Takatsu Y, Hyuga M, Nakanishi H, Furukawa K et al. (1999). Suppression by ganglioside GD1A of migration capability, adhesion to vitronectin and metastatic potential of highly metastatic FBJ-LL cells. Int J Cancer 83: 685–691.

    Article  CAS  Google Scholar 

  • Inokuchi J, Momosaki K, Shimeno H, Nagamatsu A, Radin NS . (1989). Effects of D-threo-PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells. J Cell Physiol 141: 573–583.

    Article  CAS  Google Scholar 

  • Jeyakumar M, Butters TD, Dwek RA, Platt FM . (2002). Glycosphingolipid lysosomal storage diseases: therapy and pathogenesis. Neuropathol Appl Neurobiol 28: 343–357.

    Article  CAS  Google Scholar 

  • Kendall SD, Linardic CM, Adam SJ, Counter CM . (2005). A network of genetic events sufficient to convert normal human cells to a tumorigenic state. Cancer Res 65: 9824–9828.

    Article  CAS  Google Scholar 

  • Ladisch S . (2003). The role of tumor gangliosides in the immune dysfunction of cancer. In: Finke JH and Bukowski RM (eds) Current Clinical Oncology: Cancer Immunotherapy at the Crossroads: How Tumors Evade Immunity and What Can Be Done. Humana Press, Totowa, NJ, pp 145–156.

    Google Scholar 

  • Ladisch S, Gillard B . (1985). A solvent partition method for microscale ganglioside purification. Anal Biochem 146: 220–231.

    Article  CAS  Google Scholar 

  • Ladisch S, Gillard B, Wong C, Ulsh L . (1983). Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides. Cancer Res 43: 3808–3813.

    CAS  Google Scholar 

  • Ladisch S, Kitada S, Hays EF . (1987). Gangliosides shed by tumor cells enhance tumor formation in mice. J Clin Invest 79: 1879–1882.

    Article  CAS  Google Scholar 

  • Lavie Y, Cao H, Bursten SL, Giuliano AE, Cabot MC . (1996). Accumulation of glucosylceramides in multidrug-resistant cancer cells. J Biol Chem 271: 19530–19536.

    Article  CAS  Google Scholar 

  • Ledeen RW . (1978). Ganglioside structures and distribution: are they localized at the nerve ending? J Supramol Struct 8: 1–17.

    Article  CAS  Google Scholar 

  • Li R, Liu Y, Ladisch S . (2001). Enhancement of epidermal growth factor signaling and activation of SRC kinase by gangliosides. J Biol Chem 276: 42782–42792.

    Article  CAS  Google Scholar 

  • Li RX, Ladisch S . (1991). Shedding of human neuroblastoma gangliosides. Biochim Biophys Acta 1083: 57–64.

    Article  CAS  Google Scholar 

  • Liu Y, Li R, Ladisch S . (2004). Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization. J Biol Chem 279: 36481–36489.

    Article  CAS  Google Scholar 

  • Liu Y, McCarthy J, Ladisch S . (2006). Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling. Cancer Res 66: 10408–10414.

    Article  CAS  Google Scholar 

  • Liu Y, Su Y, Wiznitzer M, Epifano O, Ladisch S . (2008). Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts. Glycobiology 18: 593–601.

    Article  CAS  Google Scholar 

  • Mitsuzuka K, Handa K, Satoh M, Arai Y, Hakomori S . (2005). A specific microdomain (‘glycosynapse 3’) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9. J Biol Chem 280: 35545–35553.

    Article  CAS  Google Scholar 

  • Rani CS, Abe A, Chang Y, Rosenzweig N, Saltiel AR, Radin NS et al. (1995). Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases. J Biol Chem 270: 2859–2867.

    Article  CAS  Google Scholar 

  • Regina Todeschini A, Hakomori SI . (2008). Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains. Biochim Biophys Acta 1780: 421–433.

    Article  CAS  Google Scholar 

  • Ronnov-Jessen L, Villadsen R, Edwards JC, Petersen OW . (2002). Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts. Am J Pathol 161: 471–480.

    Article  CAS  Google Scholar 

  • Saha S, Mohanty KC, Mallick P . (2005). Gangliosides enhance migration of mouse B16-melanoma cells through artificial basement membrane alone or in presence of laminin or fibronectin. Indian J Exp Biol 43: 1130–1138.

    CAS  Google Scholar 

  • Schenck M, Carpinteiro A, Grassme H, Lang F, Gulbins E . (2007). Ceramide: physiological and pathophysiological aspects. Arch Biochem Biophys 462: 171–175.

    Article  CAS  Google Scholar 

  • Shevchuk NA, Hathout Y, Epifano O, Su Y, Liu Y, Sutherland M et al. (2007). Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts. Biochim Biophys Acta 1771: 1226–1234.

    Article  CAS  Google Scholar 

  • Shurin GV, Shurin MR, Bykovskaia S, Shogan J, Lotze MT, Barksdale Jr EM . (2001). Neuroblastoma-derived gangliosides inhibit dendritic cell generation and function. Cancer Res 61: 363–369.

    CAS  Google Scholar 

  • Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G et al. (2004). Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet 36: 1225–1229.

    Article  CAS  Google Scholar 

  • Thompson TC, Southgate J, Kitchener G, Land H . (1989). Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ. Cell 56: 917–930.

    Article  CAS  Google Scholar 

  • Uzzo RG, Rayman P, Kolenko V, Clark PE, Cathcart MK, Bloom T et al. (1999). Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells. J Clin Invest 104: 769–776.

    Article  CAS  Google Scholar 

  • Valentino L, Moss T, Olson E, Wang HJ, Elashoff R, Ladisch S . (1990). Shed tumor gangliosides and progression of human neuroblastoma. Blood 75: 1564–1567.

    CAS  Google Scholar 

  • van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP . (1996). A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24: 131–139.

    Article  CAS  Google Scholar 

  • Villar J, Arenas MI, MacCarthy CM, Blanquez MJ, Tirado OM, Notario V . (2007). PCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C delta-dependent mechanism. Cancer Res 67: 10859–10868.

    Article  CAS  Google Scholar 

  • Yamashita T, Hashiramoto A, Haluzik M, Mizukami H, Beck S, Norton A et al. (2003). Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci USA 100: 3445–3449.

    Article  CAS  Google Scholar 

  • Yamashita T, Wu YP, Sandhoff R, Werth N, Mizukami H, Ellis JM et al. (2005). Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions. Proc Natl Acad Sci USA 102: 2725–2730.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Rick Proia for the GM3S mice. This work was supported by NIH grants CA61010 and CA42361 to SL, CA64472 to VN, and GM62116 to the Consortium for Functional Glycomics.

Author information

Authors and Affiliations

  1. Center for Cancer and Immunology Research, Children's National Medical Center, NW, Washington, DC, USA,

    Y Liu, S Yan, A Wondimu, D Bob, M Weiss, K Sliwinski, M Sutherland, A M Colberg-Poley & S Ladisch

  2. Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC, USA

    Y Liu, A M Colberg-Poley & S Ladisch

  3. Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, NW, Washington, DC, USA,

    J Villar & V Notario

  4. Department of Biochemistry and Molecular Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA

    A M Colberg-Poley & S Ladisch

Authors
  1. Y Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A Wondimu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Bob
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K Sliwinski
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J Villar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. V Notario
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M Sutherland
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A M Colberg-Poley
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. S Ladisch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S Ladisch.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Y., Yan, S., Wondimu, A. et al. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth. Oncogene 29, 3297–3306 (2010). https://doi.org/10.1038/onc.2010.85

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.85

Keywords

This article is cited by

Search

Advanced search

Quick links