Chiara Pastori

Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells

Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA‐directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin‐remodelling factors. Here, we show that a promoter‐targeted siRNA inhibits transcription of the c‐myc gene. Transcriptional silencing of c‐myc did not involve changes of known epigenetic marks. Instead, the c‐myc promoter‐targeted siRNA interfered with transcription initiation blocking the assembly of the pre‐initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter‐associated RNA initiated upstream and overlapping the transcription start site. Silencing of c‐myc led to growth arrest, reduced clonogenic potential and senescence of c‐myc over‐expressing prostate cancer cells with minimal effect on normal cells. RNA‐directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.

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 bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation

Significance Glioblastoma Multiforme (GBM) is the most common and deadliest primary brain tumor in adults. As the median survival is approximately 14 mo there is an urgent need for novel therapies. Epigenetic modulators such as bromodomain and extraterminal (BET) proteins are important therapeutic targets in GBM. Bromodomain inhibitors (including I-BET151) suppress proliferation by repressing oncogenes and inducing tumor suppressor genes through unidentified pathways. Here we demonstrate that HOTAIR (HOX transcript antisense RNA) is overexpressed in GBM, where it is crucial to sustain tumor cell proliferation, and that inhibition of HOTAIR by I-BET151 is necessary to induce cell cycle arrest in GBM cells. Our study outlines the mechanism of action underlying the antiproliferative activity of I-BET151, showing for the first time, to our knowledge, that the oncogenic long noncoding RNA HOTAIR is a major target. 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.

Involvement of long noncoding RNAs in diseases affecting the central nervous system.

DNA sequences associated with protein-coding genes have been the primary focus of most genetic analyses of complex human diseases. Although we are rapidly gaining a comprehensive view of the etiology of certain central nervous system disorders, major gaps in our understanding persist. Recent studies have uncovered that many human genomic sequences are transcribed but not translated, generating an astounding diversity of noncoding RNAs (ncRNAs). This awareness should be taken into account when studying human diseases and may have profound implications on the development of novel biomarkers as well as therapies.

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

Background: Epigenetic regulation plays an important role in cancer-associated signaling pathways. Results: The bromodomain protein inhibitor I-BET151 attenuates Hedgehog signaling downstream of Smoothened. Conclusion: I-BET151 mediated inhibition of Hedgehog-Gli activity acts downstream of Smoothened. Significance: Modulation of bromodomain protein activity attenuates the growth of Hedgehog-driven tumors. 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.

Epigenetic pathways and glioblastoma treatment

Glioblastoma multiforme (GBM) is the most common malignant adult brain tumor. Standard GBM treatment includes maximal safe surgical resection with combination radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. Alarmingly, patient survival at five-years is below 10%. This is in part due to the invasive behavior of the tumor and the resulting inability to resect greater than 98% of some tumors. In fact, recurrence after such treatment may be inevitable, even in cases where gross total resection is achieved. The Cancer Genome Atlas (TCGA) research network performed whole genome sequencing of GBM tumors and found that GBM recurrence is linked to epigenetic mechanisms and pathways. Central to these pathways are epigenetic enzymes, which have recently emerged as possible new drug targets for multiple cancers, including GBM. Here we review GBM treatment, and provide a systems approach to identifying epigenetic drivers of GBM tumor progression based on temporal modeling of putative GBM cells of origin. We also discuss advances in defining epigenetic mechanisms controlling GBM initiation and recurrence and the drug discovery considerations associated with targeting epigenetic enzymes for GBM treatment.

The multiple molecular facets of fragile X-associated tremor/ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset inherited neurodegenerative disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, and cognitive decline. FXTAS is caused by the presence of a long CGG repeat tract in the 5′ UTR of the FMR1 gene. In contrast to Fragile X syndrome, in which the FMR1 gene harbors over 200 CGG repeats but is transcriptionally silent, the clinical features of FXTAS arise from a toxic gain of function of the elevated levels of FMR1 transcript containing the long CGG tract. However, how this RNA leads to neuronal cell dysfunction is unknown. Here, we discuss the latest advances in the current understanding of the possible molecular basis of FXTAS.

Small RNA-directed transcriptional control: New insights into mechanisms and therapeutic applications

The discovery of RNA interference (RNAi) has opened new avenues in biology and medicine. In addition to post-transcriptional gene silencing, new findings are expanding the range of action of small duplex RNAs and broadening the spectrum of the potential applications of RNAi-based therapeutics. In recent years a complex and heterogeneous network of non-protein coding RNAs (ncRNAs) with potential regulatory functions has come into the spotlight providing an unexpected perspective on the mechanisms of transcriptional and epigenetic control of gene expression in human cells. The spread and complexity of these RNA-based transcriptional regulatory networks are still to be explored. However, they are likely to be important mechanisms controlling gene expression in human cells. As we will learn more about these processes, endogenous small RNAs and ncRNAs participating in these transcriptional regulatory networks might become valuable targets to modulate expression of genes involved in human diseases. Thus, understanding these basic processes of gene regulation might be translated in the near future into innovative therapeutic strategies to treat human diseases.

Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells.

CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

A promoter-proximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers

Long noncoding RNAs are emerging players in the epigenetic machinery with key roles in development and diseases. Here we uncover a complex network comprising a promoter-associated noncoding RNA (paRNA), microRNA and epigenetic regulators that controls transcription of the tumour suppressor E-cadherin in epithelial cancers. E-cadherin silencing relies on the formation of a complex between the paRNA and microRNA-guided Argonaute 1 that, together, recruit SUV39H1 and induce repressive chromatin modifications in the gene promoter. A single nucleotide polymorphism (rs16260) linked to increased cancer risk alters the secondary structure of the paRNA, with the risk allele facilitating the assembly of the microRNA-guided Argonaute 1 complex and gene silencing. Collectively, these data demonstrate the role of a paRNA in E-cadherin regulation and the impact of a noncoding genetic variant on its function. Deregulation of paRNA-based epigenetic networks may contribute to cancer and other diseases making them promising targets for drug discovery.