Characterizing the level, pattern, and effect of TNC expression in pediatric high grade glioma (HGG) and brainstem glioma (DIPG)
We previously identified increased Tenascin-C (TNC) protein expression in tumor tissue and cerebrospinal fluid (CSF) in DIPG patients, with highest TNC expression levels observed in K27M mutants. TNC is an extracellular matrix protein that modulates the mitogenic effects of PDGF and NOTCH signaling in oligodendroglial precursor cells (OPCs), the purported cells of origin for DIPG. In adult glioma, TNC expression is associated with NOTCH activation, which is a component of the Hh pathway. TNC overexpression in adult glioma correlates with tumor recurrence, local invasion and poor overall survival. However, the role of TNC expression in pediatric gliomas, and its potential as a therapeutic target, has yet to be explored.
Through a series of in vivo and in vitro studies, we aim to characterize the nature, frequency and variation in level of TNC overexpression in pediatric HGG and DIPG cell lines, tumor tissue, serum and CSF. Cell-specific expression of TNC in DIPG will be explored with FACS sorting to determine whether TNC expression is associated with specific differentiation states or cell stemness. The molecular and effects of modulating TNC expression on tumor cell biology and function will also be investigated in vitro and in vivo.
Exploring Histone H3.3 K27M mutation as a driver TNC expression and DIPG gliomagenesis
To evaluate altered epigenetic regulation due to H3 K27M mutation as a mechanism of observed genetic subgroups of DIPG and TNC overexpression, we are exploring tumor tissue methylation profiles and expression transcripts of molecules belonging to canonical oncogenic pathway genes of interest of interest, including NOTCH, PDGF, Hh, and MYC. Global and specific methylation patterns (including trimethyl K27), will be characterized, as well as expression of miRNA known to regulate pathways and molecules of interest. The effect of transduced K27M mutant Histone H3.3 vs. pharmacologic agent GSKJ4 (a Histone H3 K27 demethylase inhibitor recently found to have anti-tumor activity against K27M DIPG in vivo and in vitro), on tumor biology and expression levels of genes of interest will also be determined.
Investigating TNC as a therapeutic target for pediatric high-grade glioma
Targeted TNC therapies have been investigated for adult high grade glioma (glioblastoma) with favorable results. TNC overexpression has also been observed in pediatric ependymoma, and investigated as a potential therapeutic target. To determine the molecular effects of altered TNC expression in DIPGin vitro, DIPG cell lines with elevated and suppressed TNC expression will be engineered via vector-mediated exogenous gene transfer and shRNA knockdown, respectively. Control modifications will involve use of empty virus or virus expressing scrambled shRNA. Effects of genetic manipulations will be confirmed by western blot, and RT-PCR will be conducted to quantify mRNA expression of transcripts encoding MYCN, NOTCH1, GLI1, PTCH, FGF, PDGF and EGFR. Functional studies will also be performed to compare cell proliferation, neurosphere formation, and survival of modified cells.