Tenascin-C as a Therapeutic Target in Pediatric Brainstem Glioma

Project: Research project

Project Details

Description

Objective 1. To characterize the pattern of TNC expression in DIPG
TNC expression in DIPG will be quantified in vitro using DIPG wild type (n = 1) and
K27M mutant (n = 2) cell lines29, and compared to commercially available glioma cell
lines known to overexpress TNC. FACS sorting will be performed to isolate TNC+ and
TNC- cell populations, and co-expression of OPC markers will be evaluated to
characterize cell types expressing TNC. Functional studies to measure cell proliferation,
survival and migration will be performed on TNC+ and TNC- populations. Western blot
and immunohistochemical staining using DIPG cell lysates and tissue specimens,
respectively, with normal (post-mortem) brainstem tissue specimens as controls, will be
performed to confirm and quantify level of TNC expression (tissue specimens in hand).
Correlative molecular profiling, including Histone H3 mutation status via sequencing of
H3F3A and HIST1H3B, RT-PCR for TNC mRNA expression quantification, and
characterization of TNC gene promoter methylation status, will be performed using
extracts from DIPG tumor and normal brainstem tissue. Serum collected from mice with
DIPG brainstem xenografts will be tested for TNC by western blot and Elisa, and will be
compared with serum from mice without tumor. TNC levels will also be measured in
CSF and serum specimens from children with glioma, including DIPG, and will be
compared to controls (specimens in hand).
Objective 2: To explore the relationship of SHH signaling, Histone 3 mutation
status, and TNC expression in DIPG tumorigenesis
The role of Histone H3 mutation and SHH signaling in DIPG tumorigenesis will be
investigated using SHH inhibitor cyclopamine and Histone H3 K27 demethylase inhibitor
GSKJ4, which we have recently determined as having in vitro and in vivo anti-tumor
activity against K27M DIPG. Inhibitor effects on proliferation, survival and migration in
K27M and wild type cell lines will be measured. RT-PCR of resultant cell lysates will be
conducted to compare expression levels of canonical SHH pathway genes (GLI1, HHIP,
PTCH), as well as on TNC expression. We have identified gene expression alterations
associated with GSKJ4 treatment of DIPG cells, which include suppression of PDGFR
expression, and these, including effects of treatment on TNC expression, will also be
similarly examined. Biologic property effects from GSKJ4 treatment, as described above
for cyclopamine inhibition, will also be determined.
Objective 3. To characterize the molecular mechanism and downstream effects of
TNC upregulation in DIPG
DIPG cell lines with elevated and suppressed TNC expression will be developed via
vector-mediated exogenous gene transfer and siRNA knockdown, respectively. RTPCR
will be conducted to quantify mRNA expression of regulatory and related
transcripts, including for integrins, MMPs, SOX4, NOTCH2, GLI1, PTCH, FGF, PDGF
and EGFR, in genetically modified DIPG cells. SHH pathway activation (NOTCH2, GLI1)
will be explored as a potential mechanism of increased TNC expression in DIPG.
Results from molecular profiling will be used to correlate the level of SHH signaling and
Histone H3 mutation status with control of TNC expression.
Objective 4. To exploit TNC as a potential therapeutic target for DIPG
The effects of targeting TNC expression on DIPG will be investigated in vivo using an
animal model29. Local brainstem injection of genetically-modified DIPG cells, in which
endogenous TNC expression has been either upregulated or suppressed, will be
performed. Subsequent tumor growth
StatusFinished
Effective start/end date1/1/1512/31/16

Funding

  • Northwestern Memorial Hospital (Awarded 1/23/15)

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