BET bromodomain proteins are required for glioblastoma cell proliferation

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.     

The BET Bromodomain Inhibitor I-BET151 Acts Downstream of Smoothened Protein to Abrogate the Growth of Hedgehog Protein-driven Cancers

Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma. To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2 cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity. I-BET151 was also able to inhibit the expression of HH target genes in Sufu^−/− mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of HH-dependent cancers, such as medulloblastoma.     

I-BET151 selectively regulates IL-6 production

Orchestration of the inflammatory response is crucial for clearing pathogens. Although the production of multiple inflammatory cytokines has been thought to be regulated by common mechanisms, recent evidence indicates that the expression of some cytokines is differentially regulated by epigenetic regulatory mechanisms. In this study, we found that IL-6 production is selectively inhibited by the BET bromodomain protein (BRD) inhibitor I-BET151 in RAW264.7 cells stimulated with lipopolysaccharide (LPS), whereas I-BET151 did not alter the production of several other cytokines (TNFα, IL-1β and IL-10) at the concentration of IBET151 used. I-BET151 prevented the binding of CBP to the promoter of IL-6, but I-BET151 did not affect acetylation, phosphorylation, nuclear translocation, or DNA binding of p65-NF-κB. In vivo, I-BET151 treatment in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis decreased the early clinical symptoms, which are thought to be dependent on cytokine production. Altogether, these data suggest that targeting epigenetic-related proteins, such as BET proteins, may provide a strategy to reduce inflammation and the severity of inflammatory diseases, such as multiple sclerosis.     

HOTAIR/miR-125 axis-mediated Hexokinase 2 expression promotes chemoresistance in human glioblastoma

Drug resistance is one of the major obstacles in glioblastoma (GBM) treatments using temozolomide (TMZ) based conventional chemotherapy. Recent studies revealed that Hexokinase 2 (HK2)-mediated glycolysis is one of the sources, as the association of chemoresistance and the expression of HK2 was confirmed in multiple cancers. However, there has been little knowledge of the functional contribution of HK2 to TMZ resistance in GBM. In our study, we found that HK2 expression is crucial for GBM proliferation and chemoresistance. In contrast to the healthy brain, HK2 expression is much higher in human GBM, especially in those patients with GBM recurrence. High HK2 expression is negatively related to the overall survival in GBM patients. HK2 depletion in GBM cells suppressed the GBM cell proliferation and increased sensitivity to TMZ-induced apoptosis. Both HK2-mediated glycolysis and mitochondria permeability transition pore opening (MPTP) were associated with its function in chemoresistance. Furthermore, we also revealed that the abnormal expression of HK2 was modulated by the expression of HOTAIR, a long non-coding RNA (lncRNA). The absence of HOTAIR in GBM cells suppressed the HK2 expression in protein and mRNA level and, therefore, inhibited the cell proliferation and enhanced the cytotoxicity of TMZ both in vivo and in vitro. HOTAIR promoted the expression of HK2 by targeting mir-125, which suppressed the GBM cell proliferation and increased the TMZ-induced apoptosis. These findings shed light on a new therapeutic strategy in modulating HOTAIR/miR-125, which may interfere with the expression of HK2, and enhance the therapeutic sensitivity of GBM to TMZ.     

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.     

PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide

A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells'' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies.     

The RNA helicase DHX33 is required for cancer cell proliferation in human glioblastoma and confers resistance to PI3K/mTOR inhibition

Human Glioblastoma is one deadly disease; the median survival time is reported to be 13.9 months after treatment. In the present study, we discovered that DHX33 is highly expressed in 84% of all Glioblastoma multiforme (GBM). Knockdown of DHX33 led to significant reduced proliferation and migration in glioblastoma cells in vitro and in vivo. Mechanistically, DHX33 regulated a set of critical genes involved in cell cycle and cell migration to promote glioblastoma development. Additionally, DHX33 was found to be induced by inhibitors of PI3K and mTOR whose activation has been detected in 50% of glioblastoma. Overexpression of wild type DHX33 protein, but not the helicase dead mutant, confers resistance to mTOR inhibitors in glioblastoma cells. DHX33 probably functions as a critical regulator to promote GBM development. Our results highlight its therapeutic potential in treating GBM.     

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O⁶-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.     

CDC7-dependent transcriptional regulation of RAD54L is essential for tumorigenicity and radio-resistance of glioblastoma

Accumulating evidence indicates that cell division cycle 7-related protein kinase(CDC7) plays an essential role in tumor cells and it could induces cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CDC7 in GBM still remains unclear. In this study, we identified that CDC7 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and its expression was associated to poor prognosis in GBM patients. Mechanically, CDC7 induced radio resistance in GBM cells and CDC7 knock down increased cell apoptosis when combined with radiotherapy. Moreover, CDC7 regulated The DNA repair/recombination protein 54L (RAD54L) expression via regulation of RAD54L promoter activity. Therapeutically, we found that CDC7 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CDC7 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.     

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.     

Inhibition of Casein Kinase II by CX-4945, But Not Yes-associated protein (YAP) by Verteporfin,Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma

Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O⁶-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone.ImplicationsA combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.     

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.     

Novel cancer stem cell marker MVP enhances temozolomide-resistance in glioblastoma

The resistance of highly aggressive glioblastoma multiforme (GBM) to chemotherapy is a major clinical problem resulting in a poor prognosis. GBM contains a rare population of self-renewing cancer stem cells (CSCs) that proliferate, spurring the growth of new tumors, and evade chemotherapy. In cancer, major vault protein (MVP) is thought to contribute to drug resistance. However, the role of MVP as CSCs marker remains unknown and whether MVP could sensitize GBM cells to Temozolomide (TMZ) also is unclear. We found that sensitivity to TMZ was suppressed by significantly increasing the MVP expression in GBM cells with TMZ resistance. Also, MVP was associated with the expression of other multidrug-resistant proteins in tumorsphere of TMZ-resistant GBM cell, and was highly co-expressed with CSC markers in tumorsphere culture. On the other hands, knockdown of MVP resulted in reduced sphere formation and invasive capacity. Moreover, high expression of MVP was associated with tumor malignancy and survival rate in glioblastoma patients. Our study describes that MVP is a potentially novel maker for glioblastoma stem cells and may be useful as a target for preventing TMZ resistance in GBM patients.     

Serum-derived extracellular vesicles facilitate temozolomide resistance in glioblastoma through a HOTAIR-dependent mechanism

Extracellular vesicle (EV)-mediated transfer of long non-coding RNAs (lncRNAs) has been reported to regulate chemoresistance in various cancers. We herein investigate the therapeutic potential of bioinformatically identified HOTAIR transferred by serum-derived EVs (serum-EVs) in temozolomide (TMZ) resistance of glioblastoma (GBM) and the downstream mechanisms. EVs were isolated from the serum of GBM patients. Expression of HOTAIR was examined in the clinical tissue samples and serum-EVs of GBM patients. The downstream miRNAs of HOTAIR and its target genes were predicted in silico. The effects of the HOTAIR transmitted by serum-EVs in malignant phenotypes, tumor growth, and TMZ resistance were assessed in vitro and in vivo. HOTAIR expression was upregulated in clinical tissues, cells, and serum-EVs of GBM. Co-culture data showed that GBM-serum-EVs facilitated GBM cell proliferative and invasive phenotypes and TMZ resistance by elevating HOTAIR. In GBM cells, HOTAIR competitively bound to miR-526b-3p and weakened miR-526b-3p’s binding ability to EVA1, thus increasing the expression of EVA1. Furthermore, HOTAIR carried by serum-EVs promoted tumor growth and TMZ resistance in vivo by suppressing miR-526b-3p-mediated EVA1 inhibition. GBM-serum-EV-enclosed HOTAIR may augment GBM progression and chemoresistance through miR-526b-3p downregulation and EVA1 upregulation. These results provide a strategy to reduce TMZ resistance in GBM treatment.Subject terms: Neuroscience, Diseases     

Bromodomain protein BRD4 promotes cell proliferation in skin squamous cell carcinoma

The present study examined the expression and biological functions of bromodomain-containing protein 4 (BRD4) in skin squamous cell carcinoma (SCC) cells. Our results show that BRD4 mRNA and protein expression was upregulated in human skin SCC cells, as compared to its level in the normal skin keratinocytes and fibroblasts. Treatment with BRD4 inhibitors, JQ1 and CPI203, resulted in proliferation inhibition, apoptosis and cell cycle arrest in both established (A431 cell line) and primary skin SCC cells. Furthermore, BRD4 knockdown (by targeted shRNAs) or knockout (by CRISPR/Cas9) largely inhibited A431 cell proliferation. Reversely, forced-overexpression of BRD4 in A431 cells facilitated cell proliferation. We show that BRD4 is required for the expression of several oncogenes, including cyclin D1, Bcl-2 and MYC. BRD4 inhibition, knockdown or knockout significantly decreased above oncogene expression in SCC cells. In vivo, CRISPR/Cas9-mediated BRD4 knockout significantly suppressed A431 xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that BRD4 could be a novel and pivotal oncogenic protein of skin SCC.     

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.