U18 - H3K27me3 is a regulator of compression induced malignancy in glioblastoma

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

AJ

Allison M. Johnson, MS

Graduate Student
University of Louisville
Louisville, Kentucky, United States

Co-Author(s)

LT

Landon Teer

Graduate Candidate
University of Louisville, United States

Primary Investigator(s)

Joseph Chen, PhD

Assistant Professor
University of Louisville, United States

Introduction: : Glioblastoma (GBM) has an average life expectancy of only 14 months, despite aggressive chemotherapy and radiotherapy. The lack of efficacy of these treatments is due in part to the highly plastic nature of this cancer, which enable therapy evasion through phenotypic switching. In GBM evolution, proneural to mesenchymal transition (PMT), occurs and contributes to increased invasion and therapy resistance, leading to rapid infiltration and successful secondary tumor formation. Recent literature has suggested that biophysical cues in the tumor microenvironment (TME) are sufficient for PMT activation and can drive these transitions independent of biomolecular stimuli. Interrogating this biophysical interaction is important to identify novel TME targeting therapies to disrupt PMT and improve prognosis. As PMT is activated during tumor progression, biophysical alterations in the early-stage TME likely drives this transition. One central biophysical cue during tumor progression is intratumoral compression. Intratumoral compressive stress accumulates as the tumor expands in a confined environment experiencing resistance from the surrounding tissue. This stress increases as the tumor grows, and the application of compressive stress has been shown to increase migration and invasion in breast and pancreatic cancer through nuclear mechanotransduction. Here, we investigate the impact of compression on PMT in GBM using molecular tools and bioengineering platforms. We hypothesize that compressive stress activates PMT in a dose-dependent manner through nuclear deformation dependent heterochromatin formation and histone methylation.

Materials and

Methods: : Glioblastoma cells (U251) were compressed at 0, 400, or 1000 Pascals for 72 hours in a custom in-vitro compression system. After compression, the cells were lifted via Trypsin and reseeded. The cells were either replated either on a fibronectin coverslip at 10,000 cells then fixed with formaldehyde with Triton or replated on a 12 well plate at confluency before being lysed with RIPA containing protease and phosphatase inhibitors. The fixed cells were blocked and stained with γ-H2AX, Lamin, Vimentin, and DAPI before being imaging via Nikon EpiScope. The protein concentrations were normalized via BCA before gel electrophoresis, transfer, blocking, and staining for ZEB1, STAT3, Lamin, H3K27me3, and GAPDH. The cell reseeded on tissue culture plate were observed via Nikon EpiScope for 24 hours at 15 minute intervals and the tracking of each cell was done with ImageJ. For cell inhibition, GSK-343 was solubilized in the media during compression.

Results, Conclusions, and Discussions:: The application of compressive stress showed alterations in the cytoskeletal structure with an increase in overall lamin, as well as reinforced lamin polymerization around the nuclear membrane compared to a more dispersed lamin in the non-treated and high compressed condition (Figure 1A). The application of low compressive stress also increased filamentous vimentin localized to the nucleus in a cage-like structure, whereas the non-treated and high compression condition showed more dispersed non-filamentous vimentin across the cell (Figure 1A). With the increase of compression there was an increase in DNA damage, with the high compressed condition having a dramatic increase compared to that of the non-treated and low compressed condition (Figure 1B). Evaluation of mesenchymal markers STAT3 and ZEB1 showed an increase in the low compressed condition, but a decrease in the high compressed condition, suggesting a dose dependent pro-mesenchymal compressive stress magnitude (Figure 2B,C). The relative expression of H3K27me3 also increases with compression as the low compression condition had an increase but the high compressed condition had a dramatic increase (Figure 2A). The functional behavior of cells after compression was also evaluated through a free motility assay. The cells exposed to low compressive stress had increased migration but those exposed to high compressive stress did not (Figure 2D). This trend was reserved in the motility assessment in both 5 um and 50 um pore sized microchannels with cells exposed to low compressive stress having significant increases in cell speed while no significant increase in the high compressed condition (Figure 2F). Interestingly, the application of a H3K27me3 specific methylation inhibitor significantly reduced the increase in speed seen after the application of low compressive stress suggesting heterochromatin may play a role in regulating cell fate after exposure to compressive stress (Figure 3A). Further investigation into this axis will elucidate a mechanism by which compressive stress promotes malignancy, offering novel insight needed to create therapeutics to inhibit or interrupt this process, improving patient quality of life and life expectancy.

Acknowledgements and/or References (Optional): :