Poster U12 - Simulated tumor growth-induced mechanical compression induces epigenetic alternations in glioblastoma models in vitro

Introduction: Glioblastoma (GBM) is one of the most challenging brain tumors to treat due to its resistance to current treatment modalities. GBM is known to exhibit physical abnormalities, such as growth-induced mechanical compressive forces (i.e., solid stress). Understanding the impact of these mechanics on tumor progression and treatment resistance will lead to the identification of novel disease mechanisms and therapeutic strategies to improve treatment outcomes. Malignant gliomas are driven by epigenetic aberrations such as overexpression of specific histone deacetylases (HDACs). HDAC inhibitors (HDACIs) are able to exert epigenetic control of tumor growth and apoptosis. In this study, we examined the impact of simulated solid stress on the efficacy HDACIs including Vorinostat (suberoylanilide hydroxamic acid, SAHA), CAY10603 and Valporic acid (VPA). Our in vitro studies include human GBM cells (U87) and their healthy cellular counterpart, immortalized human astrocytes (IHAs).

Materials and

Methods: Mechanical compression was applied to U87 cells, IHAs and U87/IHA co-cultures. Cells were grown to form a monolayer on a transwell insert, with an agarose cushion and weight placed on top of the cells to apply mechanical compression at 0.1 kPa, similar to levels of solid stress we have previously measured in in vivo models of GBM. Control cells were covered with an agarose cushion only, and after 24 hours of compression, cells were collected for downstream analysis. The expression levels of HDAC1, HDAC6 and HSP90 genes were detected with/without compression and before and after treatment with HDAC inhibitors (CAY10603, SAHA, and VPA) using real-time PCR. Protein expression validation is ongoing, and ATAC-sequencing is planned for upcoming studies to examine broad epigenetic changes.

Results, Conclusions, and Discussions: Our results demonstrate that solid stress significantly up-regulated the expression of HDAC6, HDAC1, and HSP90 genes in the U87 cell line, both in mono-culture (3.8-fold, 7.2-fold, and 2.6-fold increases, respectively) and in co-culture with IHAs (5.6-fold, 4.93-fold, and 3.93-fold increases, respectively). However, no significant change was observed in the mRNA expression levels of HDAC1, HDAC6, and HSP90 genes in IHA cells, either in mono-culture or co-culture with U87 cells. Furthermore, we found that under mechanical compression, the CAY10603 drug, a selective inhibitor of HDAC6, down-regulated the expression of the HDAC6 and its downstream target, HSP90, but it was not effective in regulating HDAC1 expression. In contrast, SAHA, a pan-HDAC inhibitor, suppressed HDACs downstream transcriptional activity independent of a reduction in HDAC1 and HDAC6 mRNA levels.
In conclusion, Understanding the impact of GBM mechanical forces on epigenetic profiles and therapy will lead to more rational and novel development of epigenetic-targeting therapies.

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