Contribution of 3q26-29 gene cluster to glioma invasion

Diffuse gliomas are the most common and lethal brain tumors. Cell invasion into the surrounding brain tissue is a hallmark feature of glioma. Understanding the mechanism of glioma invasion could lead to the discovery of novel therapeutic strategies to treat affected patients. Earlier gene expression analyses on human glioma biopsies showed that SOX2 is associated with glioma invasiveness. The gene for transcription factor SOX2 localizes to 3q26.3 in the human genome amid oncogene PIK3CA and genes regulating mitochondrial fusion, MFN1 and OPA1. Increasing evidence points to a role for 3q26-29 genes in tumor invasion. We hypothesized that SOX2 regulates the 3q26-29 candidate genes as effectors of glioma cell invasion.
We used SOX2 expressing human glioma cell lines, LN319 and U373 to test our hypothesis in vitro. Lentiviruses expressing shRNAs against PIK3CA, MFN1, OPA1 or SOX2 were used for genetic knockdown. Engineered cells were assayed for invasion and migration using Boyden chamber and wound healing assays, respectively. Chromatin immuno-precipitation and luciferase assays were used to demonstrate protein-DNA interactions and trans-activation of 3q26-29 genes by SOX2.
Our results show that cells downregulated for 3q26-29 genes exhibited enhanced invasion and migration, while shSOX2 and shPIK3CA cells exhibited reduced proliferation rates compared to sh scramble controls. Furthermore, we show that SOX2 knockdown reduced gene and protein expression of PIK3CA, MFN1 and OPA1 except for PIK3CA at the protein level. Chromatin immuno-precipitation assays suggested that SOX2 binds to the upstream region of 3q26-29 gene promoters in the glioma cells.
Preliminary luciferase assays in HEK293 cells suggested that SOX2 trans-activates PIK3CA and OPA1. Preliminary immunofluorescence analysis showed that cells knocked-down for 3q26-29 genes demonstrated altered mitochondrial morphology compared to sh scramble controls. Overall, our results show that SOX2, PIK3CA, MFN1 and OPA1 contribute to glioma invasion and that SOX2 is a potential regulator of the 3q26-29 genes.