Live Cell Imaging Supports a Key Role for Histone Deacetylase as a Molecular Target during Glioblastoma Malignancy Downgrade through Tumor Competence Modulation

Abstract

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system, and the identification of the mechanisms underlying the biological basis of GBM aggressiveness is essential to develop new therapies. Due to the low prognosis of GBM treatment, different clinical studies are in course to test the use of histone deacetylase inhibitors (iHDACs) in anticancer cocktails. Here, we seek to investigate the impact of HDAC activity on GBM cell behavior and plasticity by live cell imaging. We pharmacologically knock down HDAC activity using two different inhibitors (TSA and SAHA) in two different tumor cell types: a commercial GBM cell line (U87-MG) and primary tumor (GBM011). Upon 72 hours of in vitro iHDAC treatment, GBM cells presented a very unusual elongated cell shape due to tunneling tube formation and independent on TGF-β signaling epithelial to mesenchymal transition. Live cell imaging revealed that voltage-sensitive Ca++ signaling was disrupted upon HDAC activity blockade. This behavior was coupled to vimentin and connexin 43 gene expression downregulation, suggesting that HDAC activity blockade downgrades GBM aggressiveness mostly due to tumor cell competence and plasticity modulation in vitro. To test this hypothesis and access whether iHDACs would modulate tumor cell behavior and plasticity to properly respond to environmental cues in vivo, we xenografted GBM oncospheres in the chick developing the neural tube. Remarkably, upon 5 days in the developing neural tube, iHDAC-treated GBM cells ectopically expressed HNK-1, a tumor-suppressor marker tightly correlated to increased survivor of patients. These results describe, for the first time in the literature, the relevance of iHDACs for in vivo tumor cell morphology and competence to properly respond to environmental cues. Ultimately, our results highlight the relevance of chromatin remodeling for tumor cell plasticity and shed light on clinical perspectives aiming the epigenome as a relevant therapeutic target for GBM therapy.

Nonredundant, isoform-specific roles of HDAC1 in glioma stem cells

“…While it is known that HDAC1 and HDAC2 (class I HDACs) harbor highly specific and nonoverlapping roles in the developing brain, it is unclear whether these nonredundant functions are retained in glioma cells (2,13). Our current knowledge on the role of HDACs in GBM is primarily based on pan-HDACi studies and isoform-specific knockdown or knockout experiments in serum-grown, long-term cultures of GBM cells and not in GSCs (14)(15)(16)(17)(18)(19)(20). Even the Cancer Dependency Map project, which profiled hundreds of cancer cell lines to identify genetic and pharmacological vulnerabilities, utilized traditionally grown glioma cell lines (20).…”

Obstacles to Glioblastoma Treatment Two Decades after Temozolomide

“…TNTs were first identified by Rustom et al in pheochromocytoma PC12 cells [95]. Since then, a variety of in vitro studies have reported the presence of TNTs in several cell types, including GBM [99][100][101][102]. Moreover, reports have shown that communication between GBM and other central nervous system cells such as astrocytes [102,103] and microglia [100] can occur through TNTs.…”

The function of histone methylation and acetylation regulators in GBM pathophysiology

“…GBM-specific investigations have shown valproic acid treatment in vitro causes increased p21 WAF1/Cip1 expression in multiple cell lines and sensitization to TMZ treatment in GBM cell lines but not in primary GSC cultures derived from human tumors ( 70 , 71 ). Vorinostat (suberoylanilide hydroxamic acid, SAHA), a selective inhibitor of HDACs 1, 2, 3, and 6, has been used to treat certain types of cutaneous T cell lymphoma and has demonstrated similar ability to radiosensitize patient-derived GBM cell cultures in vitro , with this effect acting synergistically with a Bcl-2 pathway inhibitor, obatoclax ( 72 – 75 ). U87 cells treated with vorinostat exhibited cell cycle arrest in G 0 /G 1 and reduced cell motility, whereas other studies have reported conflicting results for these phenotypes in patient-derived GBM cell lines ( 75 , 76 ).…”

“…Vorinostat (suberoylanilide hydroxamic acid, SAHA), a selective inhibitor of HDACs 1, 2, 3, and 6, has been used to treat certain types of cutaneous T cell lymphoma and has demonstrated similar ability to radiosensitize patient-derived GBM cell cultures in vitro, with this effect acting synergistically with a Bcl-2 pathway inhibitor, obatoclax (72)(73)(74)(75). U87 cells treated with vorinostat exhibited cell cycle arrest in G 0 /G 1 and reduced cell motility, whereas other studies have reported conflicting results for these phenotypes in patient-derived GBM cell lines (75,76). In addition to these phenotypic changes, treatment with vorinostat was found to significantly alter the transcriptomic landscape of patient-derived GBM cell lines, with gene expression profiling revealing a shift away from TCGA proneural and classical molecular signatures towards a neural signature, though the existence of this particular molecular subtype has been questioned in more recent work (76,82,83).…”