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
|Effective start/end date||1/1/15 → 12/31/16|
- Northwestern Memorial Hospital (Awarded 1/23/15)
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