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.
Highlights
Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). is tumor arises from glial cells and is classified as a grade IV glioma, causing focal or scattered anaplasia and presenting accelerated growth with histological diagnosis based on nuclear atypia and mitotic activity [1]
Since histone deacetylase (HDAC) have been reported to be expressed in GBM cells [28], we first performed a conventional polymerase chain reaction (PCR) to detect the expression pattern of HDACs in commercial U87-MG and GBM011 primary tumor cells in culture
Upon 72 h of iHDAC treatment, there was an increase in the frequency of H4K16ac + U87-MG cells followed by a significant increase in fluorescence intensity per cell (Supplementary Figure S1(B-E)). ese first results show that GBM cells express HDACs from classes I, IIa, and IIb, whose activity blockade leads to a hyperacetylated chromatin status
Summary
Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). is tumor arises from glial cells and is classified as a grade IV glioma, causing focal or scattered anaplasia and presenting accelerated growth with histological diagnosis based on nuclear atypia and mitotic activity [1]. GBM has been shown to be resistant to radiotherapy. One of the reasons for GBM resistance to therapeutic intervention is the complexity of the tumor itself, which presents regions of pseudopalisade necrosis, hemorrhage, pleomorphic nuclei/cells, and microvascular proliferation. Following this line of reasoning, growing evidence indicates that rare populations of tumor cells, called tumor stem cells, play a significant role in GBM resistance mainly contributing to the high degree of phenotypic, cellular, genetic, and epigenetic heterogeneity. Cancer stem cells (CSCs) are crucial to boost invasive tumor growth and subsequent relapse [3]
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