ABSTRACT
GM2-synthase produces sialic acid-containing glycosphingolipids called gangliosides, and its mRNA overexpression and the gangliosides it generates are linked to tumor progression, migration, and suppression of tumor-specific host immune responses. However, the mechanism underlying GM2-synthase de-repression in renal cell carcinoma (RCC) is poorly understood. Here, we demonstrate that higher GM2-synthase mRNA expression levels in various cancer cells and in human RCC tumors correlate with higher histone acetylation levels (H3K9, H3K14, or both) at region +38/+187 relative to the transcription start site (TSS) of the GM2-synthase gene than in normal kidney epithelial (NKE) cells or healthy adjacent tissues. An increase in GM2-synthase mRNA expression in cells treated with a histone deacetylase (HDAC) inhibitor was accompanied by increased histone acetylation levels at this promoter region. DNA methylation around the TSS was absent in both RCC cell lines and NKE cells. Of note, both the transcription factor Sp1 and corepressor HDAC1 associated with the +38/+187 region when the GM2-synthase gene was repressed in NKE and tumor-adjacent tissues, indicating plausible site-specific repressive roles of HDAC1 and Sp1 in GM2-synthase mRNA expression. Site-directed mutagenesis of the Sp1-binding site within the +38/+187 region relieved repressed luciferase activity of this region by limiting HDAC1 recruitment. Moreover, Sp1 or HDAC1 knock down increased GM2-synthase transcription, and butyrate-mediated activation of GM2-synthase mRNA expression in SK-RC-45 cells was accompanied by Sp1 and HDAC1 loss from the +38/+187 region. Taken together, we have identified an epigenetic mechanism for the de-repression of the GM2-synthase gene in RCC.
Subject(s)
Carcinoma, Renal Cell/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Histone Deacetylase 1/metabolism , Histones/metabolism , Kidney Neoplasms/metabolism , N-Acetylgalactosaminyltransferases/biosynthesis , Neoplasm Proteins/metabolism , Sp1 Transcription Factor/metabolism , Acetylation , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , HEK293 Cells , Histone Deacetylase 1/genetics , Histones/genetics , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , MCF-7 Cells , N-Acetylgalactosaminyltransferases/genetics , Neoplasm Proteins/genetics , Sp1 Transcription Factor/geneticsABSTRACT
The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. 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. 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.
Subject(s)
Cell Movement , G(M2) Ganglioside/metabolism , Integrin beta1/metabolism , Neoplasms/metabolism , Signal Transduction , Cell Line, Tumor , Cell Movement/drug effects , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , G(M2) Ganglioside/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Immunoprecipitation , Kinetics , Microscopy, Confocal , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation , Protein Binding , Protein Interaction Mapping , RNA Interference , Signal Transduction/drug effects , Surface Plasmon Resonance , Time Factors , Transfection , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.
