A Specific Microdomain (“Glycosynapse 3”) Controls Phenotypic Conversion and Reversion of Bladder Cancer Cells through GM3-mediated Interaction of α3β1 Integrin with CD9

Abstract

Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin ␣3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the ␣3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of ␣3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway

“…Thus, various cell adhesion processes mediated by GSLs and TSP cause extensive phenotypic changes, including reversion from malignant to normal cell phenotype (8,9,25). In analogy, Bissell and colleagues (26,27) showed that functional antibodies directed to ␤1 integrin of breast cancer cells caused reversion, in vitro and in vivo, to normal cellular organization, indicating that malignancy may arise from disorganization of glycosynaptic microdomain rather than from changes in gene structure and expression.…”

“…Cells were grown in six-well culture plates to Ϸ90% confluence, washed, incubated overnight with GSL/nanosphere suspension, washed with PBS containing 1 mM sodium vanadate, and lysed, and levels of P-Src (Tyr-416) or P-p44/42 MAPK (Thr-202/Tyr-204) were measured as described in ref. 8. Blocking effect of mAb 8E11 on p-Src or p-MAPK signaling inhibition by GM2/GM3-coated nanospheres was determined by incubation of HCV29 cells overnight at 37°C with 5% CO 2 and 1 ml of serum-free RPMI medium 1640, containing GM2/GM3-coated silica nanospheres preincubated, as above, with mouse IgG (1 g/ml) or mAb 8E11 (IgG3) (1 g/ml).…”

“…(ii) The GM3/CD9/CD81 complex inhibits tyrosine kinase associated with fibroblast growth factor receptor (FGFR) (6) and blocks functional interaction of integrins with FGFR (7). (iii) Enhancement of GM3 or CD9 level in bladder cancer cell line YTS-1 causes reversion to normal phenotype, whereby Src kinase activity is strongly inhibited (8).…”

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

“…This compound, originally found as a minor component of A431 cells displayed a much stronger inhibitory effect than GM3 on EGFR tyrosine kinase in vitro in isolated membranes, although lyso-GM3 displayed strong cytotoxicity. Lyso-GM3 dimer showed inhibitory effect similar to that of lyso-GM3 but had no cytotoxic effect [18]. Therefore, lyso-GM3 dimer, or its mimetics, are interesting candidates for tumor growth suppression.…”

“…Glycosphingolipids, including gangliosides, have the novel capability to interact with many functional membrane proteins, such as cytoplasmic signal transducers (Src family kinases, small G-proteins) [13,14], tetraspanins (CD9, CD81, CD82) [15][16][17][18], adhesion receptors (integrins) [19,20], and growth factor receptors [1,[21][22][23]. Studies on modulatory effects, initially observed with GM3 on EGFR tyrosine kinase [1], have been extended to effects of GM1, GD1a, GD1b, and GT1b on platelet-derived growth factor receptor [21,24], GM1 and plasmalopsychosine on nerve growth factor receptor [23,25], and GM3 and sialyl-nLc4 on insulin receptors [22,26].…”