Increased HEY1 Expression Contributes to Glioma Progression and Promotes Glioma Cell Growth Kiran Kumar Velpula PhD; Maheedhara R. Guda PhD; Sarah E. Martin MD; Andrew J. Tsung MD

University of Illinois College of Medicine at Peoria; Illinois Neurological Institute, Peoria, IL 61605

Introduction Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by increased necrosis and intense resistance to therapy. Although, little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch, EGFR and pathways, seem to play an important role in the formation/maintenance of GBM.

Methods Notch signaling is aberrantly activated in GBM and orchestrates the malignant traits of GBM, but current HEY1 is overexpressed in hGBM. (A) Whisker knowledge regarding the downstream plots of HEY1 expression catalogued within Potential interaction of HEY1 with the OncomineTM database(B) Immunoblotting players of Notch in GBM remains multiple factors. (A) TF analysis of normal brain and of GBM samples Sodium Butyrate (NaB) inhibits expression -TF array was performed using recombinant limited. Recently we have identified (GS), (n=15) for detecting HEY1 expression (C) of HEY1/p53 (A) qRT-PCR to check the HEY1 and anti-HEY1 antibody as per the that Notch effector HEY1 is highly Immunohistochemical analysis of normal brain expression levels of DNA methyltransferase 1 manufacturer’s instructions (Affymetrix, CA; and GS-16 to detect HEY1 using DAB staining upregulated in hGBM (human) surgical (DNMT1) on the mRNA of 4910, 5310 control #MA1210) and potential HERP2-interacting (Bar = 50µM). Inset images represent biopsies and its overexpression and their treatment of cells with 2.5mM NaB (B) are highlighted. Hematoxylin and Eosin staining of the Immunoblot analysis of HEY1 and p53 in control appears to be correlative with high- respective specimens. and 2.5mM NaB treated cells grade glioma. Our immunoblot Conclusions experiments revealed that HEY1 • Within glioblastoma, HEY-1 is found to be overexpressed when compared to its expression significantly correlated with normal brain counterparts. O6-alkylguanine DNA alkyltransferase • Demethylation of the CpG islands within the (MGMT) levels in hGBM specimens. promoter region of HEY1 occurs in GBM, which is a possible mechanism of overexpression of the gene. Results • Treatment with sodium butyrate (NaB) is The inhibition of HEY1 reduced shown to lower expression of HEY-1 and invasive, migratory and proliferative p53 while diminishing cancer phenotypes. si-HEY1 reduces phenotypes associated • si-HEY1 additionally reduces HEY1 and p53 abilities of GBM cells, in vitro. with cancer in vitro. (A) Immunoblot analysis expression in GBM, also leading to a Furthermore, our experiments with TF of HEY1 and p53 levels in SV and si-HEY1 reversion of proliferative abilities in vitro -TF; TF-DNA arrays conducted using treatments (SV = Scrambled vector; si-HEY1 = and resumed adherence to mitotic small interfering RNA for HEY1) (B & C) qRT- recombinant HEY1 showed its checkpoints. PCR analysis of si-HEY1 treatment to detected CpG islands of the HEY1 gene determine • HEY1 interacts with a number of interaction with multiple transcription the levels of HEY1, p53 and DNMT1 (D) demethylation in GBM. (A) CpG islands on transcription factors and other proteins, factors emphasizing its importance in Clonogenic assay promoter region (left of arrow) of the HEY1 suggesting the involvement of this molecule GBM, highlighting HEY1 as a potential gene(B)sequencing analysis (C) Areas of in a number of signaling cascades that therapeutic target that can be methylation are denoted as as black circles. contribute to the phenotype of GBM. evaluated in GBM.