Abstract 5146: GM2 mediates tumor cell migration and induces EMT through involvement of the integrin signaling machinery and targeting YAP, a major component of the HIPPO signaling pathway
Authors: Kundu Manjari , Mahata Barun , Banerjee Avisek , Debnath Shibjyoti , Biswas Kaushik 
Published: July 2016

Background: In the present study we report a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration and anchorage independent growth suggestive of EMT and uncovered its mechanism.

Approach : Specific role of GM2 in cancer cell migration was evaluated by in vitro transwell migration assay using either GM2-synthase silenced or GM2-overexpressed cancer cells or by adding exogenous GM2. Mechanistic details were revealed by studying gene chip profiling and validated by real time PCR, western blot analysis and immunofluorescence. Interaction of GM2 with upstream specific molecules were determined by confocal microscopy, co-IP and surface plasmon resonance (SPR). TALEN mediated targeted genome editing was used to generate stable GM2-synthase knockout mouse tumor cells, to study the role of GM2 in EMT.

Results : siRNA mediated knockdown of GM2-synthase in cancer cells resulted in significant inhibition of tumor cell migration in vitro. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in dramatic increase in migration. TALEN mediated GM2-synthase knockout in Renca-v cells resulted in significant reduction in AIG and increased sensitivity to anoikis, suggesting a plausible role of GM2 in EMT. Gene expression profiling and DNA microarray analysis of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin signaling involving the Erk-MAP kinase pathway, as well as genes targeted by YAP, a major component of the HIPPO signaling pathway which is involved in EMT. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk while, exogenous GM2 treatment caused increased Erk phosphorylation. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Renca-v GM2-synthase knockout cells exhibited significant downregulation of several YAP target genes namely, Ctgf, pdgf C, Cyr61 and LOX while induction of these genes in both Renca as well as 4T1 in response to GM2 treatment suggests that GM2 mediates EMT in cancer cells possibly by targeting YAP. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

Conclusion: Our findings confirm a novel role of GM2 in tumor cell migration involving the integrin signaling pathway and in triggering EMT by targeting YAP, a major component of the HIPPO signaling pathway ultimately leading to enhanced migration and metastasis in tumor cells.

Citation Format: Manjari Kundu, Barun Mahata, Avisek Banerjee, Shibjyoti Debnath, Kaushik Biswas. GM2 mediates tumor cell migration and induces EMT through involvement of the integrin signaling machinery and targeting YAP, a major component of the HIPPO signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5146.