gsk1210151a and Glioblastoma

A significant roadblock in treatment of GBM multiforme (GBM) is resistance to temozolomide (TMZ). In this study, we investigated whether I-BET151, a specific BET inhibitor, could sensitize GBM cells to TMZ. Our findings showed that the action of I-BET151 could augment the effect of TMZ on cancer cells U251 and U87 cells. In U251 cells, administration of I-BET151 increased the TMZ-induced apoptosis GBM cells. I-BET151 remarkably enhanced the activities of caspase-3. In addition, I-BET151 promoted TMZ-induced migration and invasion in GBM cells. Moreover, I-BET151 increased the amount of reactive oxygen species as well as superoxide anions with a decrease of activity of SOD and the anti-oxidative properties of GBM cells. I-BET151 also induced increased PUMA expression, which is required for the functions of I-BET151 and regulates the synergistic cytotoxic effects of i-BET151 and TMZ in GBM cells. I-BET151 with TMZ also showed synergistic cytotoxic effects in vivo. These point out to an approach to tackle GBM using TMZ along with BET inhibitors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Brain Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Female; Gene Knockout Techniques; Glioblastoma; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Proto-Oncogene Proteins; Temozolomide; Transcription Factors; Xenograft Model Antitumor Assays

Glioblastoma multiforme, a type of deadly brain cancer, originates most commonly from astrocytes found in the brain. Current multimodal treatments for glioblastoma minimally increase life expectancy, but significant advancements in prognosis have not been made in the past few decades. Here we investigate cellular reprogramming for inhibiting the aggressive proliferation of glioblastoma cells. Cellular reprogramming converts one differentiated cell type into another type based on the principles of regenerative medicine. In this study, we used cellular reprogramming to investigate whether small molecule mediated reprogramming could convert glioblastoma cells into neurons. We investigated a novel method for reprogramming U87MG human glioblastoma cells into terminally differentiated neurons using a small molecule cocktail consisting of forskolin, ISX9, CHIR99021 I-BET 151, and DAPT. Treating U87MG glioblastoma cells with this cocktail successfully reprogrammed the malignant cells into early neurons over 13 days. The reprogrammed cells displayed morphological and immunofluorescent characteristics associated with neuronal phenotypes. Genetic analysis revealed that the chemical cocktail upregulates the Ngn2, Ascl1, Brn2, and MAP2 genes, resulting in neuronal reprogramming. Furthermore, these cells displayed decreased viability and lacked the ability to form high numbers of tumor-like spheroids. Overall, this study validates the use of a novel small molecule cocktail for reprogramming glioblastoma into nonproliferating neurons.

Topics: Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cellular Reprogramming; Cellular Reprogramming Techniques; Colforsin; Diamines; Glioblastoma; Heterocyclic Compounds, 4 or More Rings; Homeodomain Proteins; Humans; Isoxazoles; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neurons; POU Domain Factors; Pyridines; Pyrimidines; Thiazoles; Thiophenes; Up-Regulation

Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Heterocyclic Compounds, 4 or More Rings; Humans; Mice, Nude; Microscopy, Fluorescence; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Long Noncoding; Transcription Factors; Xenograft Model Antitumor Assays

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Case-Control Studies; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Heterocyclic Compounds, 4 or More Rings; Heterografts; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Nuclear Proteins; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Transcription Factors