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.     

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.     

SNAIL- and SLUG-induced side population phenotype of HCT116 human colorectal cancer cells and its regulation by BET inhibitors

Epithelial-mesenchymal transition (EMT) is associated with cancer malignancies such as invasion, metastasis, and drug resistance. In this study, HCT116 human colorectal cancer cells were transduced with SLUG or SNAIL retroviruses, and EMT cells with mesenchymal morphology were established. The EMT cells showed a high invasive activity and resistance to several anticancer agents such as methotrexate, SN-38, and cisplatin. Furthermore, they contained about 1–10% side population (SP) cells that were not stained by Hoechst 33342. This SP phenotype was not stable; the isolated SP cells generated both SP and non-SP cells, suggesting a potential for differentiation. Gene expression analysis of SP cells suggested the alteration of genes that are involved in epigenetic changes. Therefore, we examined the effect of 74 epigenetic inhibitors, and found that two inhibitors, namely I-BET151 and bromosporine, targeting the bromodomain and extra-terminal motif (BET) proteins, decreased the ratio of SP cells to <50% compared with the control, without affecting the immediate efflux of Hoechst 33342 by transporters. In addition, compared with the parental cells, the EMT cells showed a higher sensitivity to I-BET151 and bromosporine. This study suggests that EMT development and SP phenotype can be independent events but both are regulated by BET inhibitors in SLUG- or SNAIL-transducted HCT116 cells.     

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.     

BET Inhibition Silences Expression of MYCN and BCL2 and Induces Cytotoxicity in Neuroblastoma Tumor Models

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.     

Gli as a Novel Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.     

宫颈鳞癌演进过程P16INK4a及Hh-Gli信号通路相关蛋白表达及其相关性研究

探讨P16^INK4a及Sonic hedgehog(Hh-Gli)信号通路蛋白在宫颈癌及癌前病变(CIN)中的表达相关性及其意义．采用Western-blot方法检测HPV16阳性及HPV18阳性宫颈癌细胞系P16^INK4a及Hh-Gli信号通路蛋白Smo、Ptch及Gli表达．免疫组化检测组织芯片P16^INK4a、Shh、Smo、Ptch及Gli表达,包括20例正常宫颈、18例癌旁组织、54例CIN及28例宫颈鳞癌组织．分析P16^INK4a与Hh-Gli信号通路蛋白间表达相关性及与临床病理因素的关系．结果显示P16^INK4a、Smo、Ptch及Gli蛋白在HPV16及HPV18阳性宫颈癌细胞系中表达无显著差异(P>0.05)．P16^INK4a、Shh、Smo、Ptch及Gli蛋白在宫颈癌中表达强度显著高于癌旁及正常组织(P<0.05),在CINⅠ与正常组织间差异不显著(P>0.05)．P16^INK4a、Shh、Smo及Gli蛋白,在CINⅠ、CINⅡ与CINⅢ之间均有显著性差异(P<0.05)．相关分析显示,CINⅡ-CINⅢ中P16^INK4a与Shh和Smo蛋白表达正相关,浸润癌中P16^INK4a与Shh、Smo和Gli蛋白正相关．结论认为,P16^INK4a及Hh-Gli信号通路异常激活与宫颈癌发生及演进密切相关,且二者间具有相关性．Hh-Gli信号通路的激活可能是Shh配体增高调控Smo高表达而上调Gli蛋白所致．     

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it’s necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.     

The Full-length Unprocessed Hedgehog Protein Is an Active Signaling Molecule

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as ∼45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.     

Analysis of the Sonic Hedgehog Signaling Pathway in Normal and Abnormal Bladder Development

In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb−/−) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb−/− bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb−/− bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb−/− mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.