A2B5 and ST8sia3 in glioblastoma

Cancer stem-like cells fullfill specific features such as extensive cell-renewal ability (sphere generation), cancer-initiating ability upon orthotopic implantation, aberrant differentiation properties and karyotypic and genetic alterations. In glioblastoma (GB), the most malignant primary intracranial tumor, there is a need to identify specific and exclusive markers to facilitate our understanding of the glioblastoma stem cells as well as therapeutic development. This remains a major therapeutic challenge.

The project:
Experimental evidences suggested that A2B5+ cells isolated from GB might represent GB initiating cells [1, 2]. These cells express polysialogangliosides recognized by A2B5. As genetic manipulation of gangliosides is not possible, we focused on sialyltransferases involved in the synthesis of polysialogangliosides recognized by A2B5. To this end, we manipulated the expression of the sialyltransferase ST8sia3 and we developed glioblastoma cell lines with various levels of A2B5 reactivity. The overexpression of ST8sia3 resulted in a dramatic increase of A2B5 immunoreactivity resulting in greater ability of cells to proliferate and migrate in vitro. Conversely, lentiviral ST8sia3 inactivation resulted in a dramatic decrease of A2B5 imunoreactivity and reduced proliferation and migration abilities. Moreover, when the ST8sia3-overexpressing cells were orthotopically injected in nude mice, the overall survival was reduced as compared to control cell line-injected mice. The mechanisms by which high A2B5 expression in GB cell lines induces increased cell proliferation, migration and tumorigenicity are not fully understood. Pan-transcriptomic analysis of the modified cells has shown several changes in the expression of key genes involved in gliomagenesis [3]. Other genes that had not been previously related to pathogenesis of GB have been highlighted and we need to confirm their differential expression in a large panel of cell lines that have been already obtained as well as in fresh samples of GB and find their relationship with ST8sia3 and A2B5 in GB.

[1] Tchoghandjian A, Baeza N, Colin C, Cayre M, Metellus P, Beclin C, et al. A2B5 cells from human glioblastoma have cancer stem cell properties. Brain Pathol 2010;20(1):211-21.
[2] Tchoghandjian A, Baeza-Kallee N, Beclin C, Metellus P, Colin C, Ducray F, et al. Cortical and subventricular zone glioblastoma-derived stem-like cells display different molecular profiles and differential in vitro and in vivo properties. Ann Surg Oncol 2012;19 Suppl 3:S608-19.
[3] Baeza-Kallee N, Denicolai E, Appay R, Souberan A, Colin C et al. Deciphering the role of A2B5 trisialogangliosides in glioblastomas. Oncogene, under review.

Our proposals for this project:
– Analysis of mRNA and protein expression of differentially expressed genes in A2B5+ and A2B5- fractions isolated from various GB cancer stem cell lines and from fresh GB samples.
– When relevant, corresponding siRNA will be performed in GB cell lines followed by functional analysis (proliferation and migration) of the produced cell clones.

Techniques and skills to be developed :
– Immunomagnetic cell sorting and FACS
– Cell culture
– RT-qPCR, western blot and flow cytometry
– Microscopy
– Use of databases and bioinformatics

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