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

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 9-11 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.

Ganglioside, Disialosyl Globopentaosylceramide (DSGb5), Enhances the Migration of Renal Cell Carcinoma Cells

“…It is noteworthy that knockout of ganglioside synthase, which affects lipid raft composition, reduced migration of normal human fibroblasts (Liu et al 2010), and immunotherapy with anti-GD2 mAbs results in long term survival of patients with neuroblastoma (Ahmed and Cheung 2014). In the present study, we found that knocking down ST6GalNAcVI mRNA expression reduced the effect of DSGb5 on RCC cells; therefore, DSGb5 represents a potential anti-tumor treatment target for RCC patients.…”

A Glycosphingolipid/Caveolin-1 Signaling Complex Inhibits Motility of Human Ovarian Carcinoma Cells

“…Immunoprecipitation Experiments-Aliquots of the detergent-resistant membrane fractions obtained from [1][2][3] H]sphingosine-labeled cells were diluted 10-fold in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM PMSF, 75 milliunits/ml aprotinin, 1% Triton X-100). After preclearing for nonspecific binding, 10 g/ml anti-caveolin-1 rabbit or 4 g/ml anti-c-Src antibody or normal rabbit IgG (as negative control) was added to the supernatants, and the mixtures were put in rotation overnight at 4°C.…”

“…However, gangliosides deeply affect tumor cell adhesion, motility, and migration. Ganglioside depletion by double knock-out of GM3 synthase and GM2 synthase in transformed fibroblasts reduced their migration on fibronectin and reduced their tumorigenic potential in syngeneic immunocompetent mice (3). However, transfection with GM1a/GD1b synthase, leading to the expression of complex gangliosides, inhibited neuroblastoma cell migration by affecting the Rho/Rac1 pathway (4).…”

“…2 Both authors equally contributed to this work. 3 Ganglioside and glycosphingolipid nomenclature is in accordance with the IUPAC-IUBMB recommendations (1).…”

GM1 and GM2 gangliosides: recent developments

“…Depletion of HEXB via siRNA was found to decrease the angiogenic potential. Another study using GM2 and GM3 synthase double knockout oncogenetransformed fibroblasts in a cancer model in mice found that downregulation of GM1 induces strikingly slower tumor growth (52). Although the precise role of GM2 in cancer progression is still not completely understood, it may prove to be an interesting therapeutic target.…”