Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction.

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.

GPR30 expression and function in breast cancer cells are induced through a cis­acting element targeted by ETS factors.

The capacity that G protein­coupled receptor 30 (GPR30) has demonstrated for triggering estrogen­dependent signaling pathways has attracted the interest of breast cancer researchers; however, the reported expression profiles and functions of GPR30 in breast cancer are inconsistent. The main purpose of the present investigation was to identify transcriptional mechanisms underlying the expression of GPR30 that allow a better understanding of its role in breast cancer progression. In the cell lines used as different polarity models in the present study, it was determined immunologically that GPR30 is expressed in normal mammary gland cells and that this expression decreased considerably during breast cancer development, where cell identity is lost. However, it was also determined that, in spite of low GPR30 expression levels in breast cancer cells with little differentiation, this membrane estrogen receptor (ER) is able to increase cell viability and suppress migration in cells that have acquired metastatic capacity. In addition, through transient expression assays in breast cancer cells, it was revealed that a transcriptional mechanism dependent on protein kinase A and susceptible to retinoic acid in ER­positive cells induces GPR30 expression through a cis­regulatory element for E26 transformation­specific transcription factors, located between ­631 and ­625 bp from the GPR30 translation start codon. Overall, these results suggested that in vitro transcriptional regulation of GPR30 expression in breast cancer cells may serve a relevant role in the conservation of an epithelial phenotype, and also may be important to avoid the transition to metastasis.

Molecular Underpinnings Governing Genetic Complexity of ETS-Fusion-Negative Prostate Cancer.

Inter- and intra-patient molecular heterogeneity of primary and metastatic prostate cancer (PCa) confers variable clinical outcome and poses a formidable challenge in disease management. High-throughput integrative genomics and functional approaches have untangled the complexity involved in this disease and revealed a spectrum of diverse aberrations prevalent in various molecular subtypes, including ETS fusion negative. Emerging evidence indicates that SPINK1 upregulation, mutations in epigenetic regulators or chromatin modifiers, and SPOP are associated with the ETS-fusion negative subtype. Additionally, patients with defects in a DNA-repair pathway respond to poly-(ADP-ribose)-polymerase (PARP) inhibition therapies. Furthermore, a new class of immunogenic subtype defined by CDK12 biallelic loss has also been identified in ETS-fusion-negative cases. This review focuses on the emerging molecular underpinnings driving key oncogenic aberrations and advancements in therapeutic strategies of this disease.

Expression of ELF1, a lymphoid ETS domain-containing transcription factor, is recurrently lost in classical Hodgkin lymphoma.

Loss of B cell-specific transcription factors (TFs) and the resulting loss of B-cell phenotype of Hodgkin and Reed-Sternberg (HRS) cells is a hallmark of classical Hodgkin lymphoma (cHL). Here we have analysed two members of ETS domain containing TFs, ELF1 and ELF2, regarding (epi)genomic changes as well as gene and protein expression. We observed absence or lower levels of ELF1 protein in HRS cells of 31/35 (89%) cases compared to the bystander cells and significant (P < 0·01) downregulation of the gene on mRNA as well as protein level in cHL compared to non-cHL cell lines. However, no recurrent loss of ELF2 protein was observed. Moreover, ELF1 was targeted by heterozygous deletions combined with hypermethylation of the remaining allele(s) in 4/7 (57%) cell lines. Indeed, DNA hypermethylation (range 95-99%, mean 98%) detected in the vicinity of the ELF1 transcription start site was found in all 7/7 (100%) cHL cell lines. Similarly, 5/18 (28%) analysed primary biopsies carried heterozygous deletions of the gene. We demonstrate that expression of ELF1 is impaired in cHL through genetic and epigenetic alterations, and thus, it may represent an additional member of a TF network whose downregulation contributes to the loss of B-cell phenotype of HRS cells.

MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo.

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-ß ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a ß-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a ß-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.

Electrostatic control of DNA intersegmental translocation by the ETS transcription factor ETV6.

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation.

Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.