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Elevated ETV6 expression in glioma promotes an aggressive in vitro phenotype associated with shorter patient survival

ABSTRACT: Purpose It has been postulated that glioblastoma (GBM) has the ability to hijack neural progenitor migration mechanisms to facilitate tumor progression. Our previous data show that ETS variant 6 (ETV6) is highly expressed in human GBM and fetal astrocytes compared to normal mature astrocytes. We hypothesized that ETV6 played a role in GBM tumor progression. Methods Expression of ETV6 was first examined in 2 American and 3 Chinese tissue microarrays. The correlation between ETV6 staining intensity and patient survival was calculated, followed by validation using public databases-TCGA and REMBRANDT. The effect of ETV6 knockdown on glioma cell proliferation (EdU), viability (AnnexinV labeling), clonogenic growth (colony formation), and migration/invasion (transwell assays) in GBM cells was tested. RNA sequencing was performed to ellucidate the underlying molecular mechanisms. Results ETV6 was highly expressed in GBM and associated with an unfavorable prognosis. ETV6 silencing in glioma cells led to increased apoptosis and decreased proliferation, clonogenicity, migration, and invasion. RNA-Seq based gene expression and pathway analyses revealed that ETV6 knockdown in U251 cells led to the upregulation of genes involved in extracellular matrix organization, NF-κB signaling, TNFmediated signaling and downregulation of genes in regulation of cell motility, cell proliferation, PI3KAKT and Ras pathways. Conclusion Our findings suggested that ETV6 was highly expressed in GBM and its high expression correlated with poor survival. ETV6 silencing decreased an aggressive in vitro phenotype. The study encourages further investigation of ETV6 as a potential therapeutic target of GBM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155052 | GEO | 2021/01/01

REPOSITORIES: GEO

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Project description:Treating recurrent GBM is a clinical challenge due to its highly resistant and aggressive nature. In order to develop new therapeutic targets for recurrent GBM a better understanding of its molecular landscape is necessary. Here we used a cellular model, developed in our lab which generates paired primary and recurrent samples from GBM cell lines and primary patient samples hence allowing us to compare the molecular differences between the two populations. Total RNA seq analysis of parent and recurrent population of two cell lines and one patient sample revealed a significant upregulation of Extracellular matrix interaction in recurrent population. Since matrix stiffness plays a pivotal role in cell-ECM interaction and downstream signaling, we developed a system that mimicked the brain like substrate stiffness by using collagen coated polyacrylamide-based substrate whose stiffness can be modified from normal brain (0.5kPa) to tumorigenic (10kPa). Using these substrates, we were able to capture the morphological and physiological differences between parent and recurrent GBM which were not evident on plastic surfaces (~1 GPa). On 0.5kPa, unlike circular parent cells, recurrent GBM cells showed two morphologies (circular and elongated). The recurrent cells growing on 0.5kPa also showed higher proliferation, invasion, migration and in-vivo tumorigenicity in orthotropic GBM mouse model, compared to parent cells. Furthermore, recurrent cells exhibited elevated velocity irrespective of substrate stiffness, which indicated that recurrent cells may possess inherent differential mechanosignalling ability which was reflected by higher expression of ECM proteins like Collagen IVA, MMP2 and MMP9. Moreover, mice brain injected with recurrent cells grown on 0.5kPa substrate showed higher Young’s modulus values suggesting that recurrent cells conditioned on 0.5kPa make the surrounding ECM stiffer. Importantly, inhibition of EGFR signaling, that is amplified with tissue stiffening in GBM resulted in decreased invasion, migration and proliferation in 0.5kPa recurrent cells, but interestingly survival remained unaffected, highlighting the importance of mimicking the physiological stiffness of the brain mimicking clinical scenario. Total RNA seq analysis of parent and recurrent cells grown on plastic and 0.5kPa substrate identified PLEKHA7 as significantly upregulated gene specifically in 0.5kPa recurrent sample. Higher protein expression of PLEKHA7 in recurrent GBM as compared to primary GBM was validated in patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provides a model system that captures the differential mechanosensing signals of primary and recurrent GBM cells and identifies a novel potential target specific for recurrent GBM.

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Elevated ETV6 expression in glioma promotes an aggressive in vitro phenotype associated with shorter patient survival

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