Abstract 1110: Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program
Authors: Mahata Barun , Sarkar Abhisek , Khamrui Elora , Chakraborty Sohini , Ghosh Zhumur , Biswas Kaushik 
Published: August 2018

Purpose: Recent report from our laboratory showed a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration as well invasion. In the present study we report a novel role of GM2 in mediating anchorage independent growth (AIG) and epithelial-mesenchymal transition (EMT) leading to tumor growth, progression and metastasis, through a HIPPO-YAP dependent transcriptional program.

Experimental procedures : Permanent GM2-synthase knockout (KO) cells were generated using targeted genome editing tool, TALEN. TALEN was used to generate Renca-vGM2-syn KO cells (from a GM2-over-expressing variant of a mouse kidney cancer cell line, Renca-v). Renca-vWT as well as Renca-vGM2-syn KO cells were used to define the role of GM2 in AIG, EMT and tumorigenesis both in vitro and in vivo. DNA microarray analysis was used to elucidate the mechanism behind GM2-mediated AIG and EMT. To define involvement of YAP in GM2-mediated EMT, real time PCR was used to validate YAP-target gene expression in GM2-synthase KO or silenced (siRNA) cells as well as in cells treated with exogenous GM2, in the presence or absence of specific inhibitors of the Hippo-YAP signaling pathway. CRISPR-Cas9 mediated YAP/TAZ-double KO (HelaYAP/TAZ KO) cells were used to conclusively prove the involvement of YAP/TAZ in GM2-mediated EMT.

Results : Renca-vGM2-syn KO cells show reduced AIG, higher cell adhesion possibly through increased anoikis sensitivity, indicating a critical role of GM2-synthase and complex gangliosides in GM2-mediated EMT. Furthermore, Renca-vGM2-syn KO cell line shows reduced tumor growth potential, significant reduction in experimental lung metastasis with increased median survival in a syngeneic mice tumor model. Gene expression profiling using DNA microarray with GM2-synthase silenced cells reveal significant modulation of YAP target genes, suggesting a critical role of the Hippo-YAP signaling axis. Molecular insight reveals that GM2-synthase knockout or siRNA mediated knockdown results in significant downregulation of YAP activated genes namely Ctgf, Cyr 61, Pdgf-c, Lox, etc. and upregulation of YAP repressed gene Ddit4, while exogenous addition of GM2 in GM2 deficient cells (Renca, MCF-7 and Hela) show opposite results. Verteporfin mediated disruption of YAP transcriptional program abrogated GM2 mediated modulation of YAP target genes. Finally, significant decrease in YAP-target gene expression in YAP/TAZ-double KO cells, but not in either YAP or TAZ (paralogue of YAP) single KOs confirm the definitive involvement of YAP/TAZ in GM2-mediated EMT.

Conclusion : Our findings confirm a novel role of GM2 in triggering EMT by targeting YAP, ultimately leading to increased tumorigenic potential and metastatic activity in tumor cells.

Citation Format: Barun Mahata, Abhisek Sarkar, Elora Khamrui, Sohini Chakraborty, Zhumur Ghosh, Kaushik Biswas. Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1110.