The DNA/RNA helicase DHX9 orchestrates the KDM2B-mediated transcriptional regulation of YAP1 in Ewing sarcoma.

Ewing sarcomas (ES) are aggressive paediatric tumours of bone and soft tissues. Resistance to chemotherapy and high propensity to metastasize remain the main causes of treatment failure. Thus, identifying novel targets for alternative therapeutic approaches is urgently needed. DNA/RNA helicases are emerging as crucial regulators of many cellular processes often deregulated in cancer. Among them, DHX9 is up-regulated in ES and collaborates with EWS-FLI1 in ES transformation. We report that DHX9 silencing profoundly impacts on the oncogenic properties of ES cells. Transcriptome profiling combined to bioinformatic analyses disclosed a gene signature commonly regulated by DHX9 and the Lysine Demethylase KDM2B, with the Hippo pathway regulator YAP1 as a prominent target. Mechanistically, we found that DHX9 enhances H3K9 chromatin demethylation by KDM2B and favours RNA Polymerase II recruitment, thus promoting YAP1 expression. Conversely, EWS-FLI1 binding to the promoter represses YAP1 expression. These findings identify the DHX9/KDM2B complex as a new druggable target to counteract ES malignancy.

RNA Editing in Cancer Progression.

Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Oncogenic Dysregulation of Circulating Noncoding RNAs: Novel Challenges and Opportunities in Sarcoma Diagnosis and Treatment.

Sarcomas comprise a heterogeneous group of rare mesenchymal malignancies. Sarcomas can be grouped into two categories characterized by different prognosis and treatment approaches: soft tissue sarcoma and primary bone sarcoma. In the last years, research on novel diagnostic, prognostic or predictive biomarkers in sarcoma management has been focused on circulating tumor-derived molecules as valuable tools. Liquid biopsies that measure various tumor components, including circulating cell-free DNA and RNA, circulating tumor cells, tumor extracellular vesicles and exosomes, are gaining attention as methods for molecular screening and early diagnosis. Compared with traditional tissue biopsies, liquid biopsies are minimally invasive and blood samples can be collected serially over time to monitor cancer progression. This review will focus on circulating noncoding RNA molecules from liquid biopsies that are dysregulated in sarcoma malignancies and discuss advantages and current limitations of their employment as biomarkers in the management of sarcomas. It will also explore their utility in the evaluation of the clinical response to treatments and of disease relapse. Moreover, it will explore state-of-the-art techniques that allow for the early detection of these circulating biomarkers. Despite the huge potential, current reports highlight poor sensitivity, specificity, and survival benefit of these methods, that are therefore still insufficient for routine screening purposes.

The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer.

BACKGROUND: Prostate cancer (PC) is the most commonly diagnosed male malignancy and an important cause of mortality. Androgen deprivation therapy is the first line treatment but, unfortunately, a large part of patients evolves to a castration-resistant stage, for which no effective cure is currently available. The DNA/RNA helicase DHX9 is emerging as an important regulator of cellular processes that are often deregulated in cancer. METHODS: To investigate whether DHX9 modulates PC cell transcriptome we performed RNA-sequencing analyses upon DHX9 silencing in the androgen-responsive cell line LNCaP. Bioinformatics and functional analyses were carried out to elucidate the mechanism of gene expression regulation by DHX9. Data from The Cancer Genome Atlas were mined to evaluate the potential role of DHX9 in PC. RESULTS: We found that up-regulation of DHX9 correlates with advanced stage and is associated with poor prognosis of PC patients. High-throughput RNA-sequencing analysis revealed that depletion of DHX9 in androgen-sensitive LNCaP cells affects expression of hundreds of genes, which significantly overlap with known targets of the Androgen Receptor (AR). Notably, AR binds to the DHX9 promoter and induces its expression, while Enzalutamide-mediated inhibition of AR activity represses DHX9 expression. Moreover, DHX9 interacts with AR in LNCaP cells and its depletion significantly reduced the recruitment of AR to the promoter region of target genes and the ability of AR to promote their expression in response to 5α-dihydrotestosterone. Consistently, silencing of DXH9 negatively affected androgen-induced PC cell proliferation and migration. CONCLUSIONS: Collectively, our data uncover a new role of DHX9 in the control of the AR transcriptional program and establish the existence of an oncogenic DHX9/AR axis, which may represent a new druggable target to counteract PC progression.

Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK.

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) drives invadopodia by a not fully explored coordinated function of ß-arrestin1 (ß-arr1). Here, we report that ß-arr1 links the integrin-linked kinase (ILK)/ßPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/ß-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.

Dissecting the transcriptional regulatory networks of promoter-associated noncoding RNAs in development and cancer.

In-depth analysis of global RNA sequencing has enabled a comprehensive overview of cellular transcriptomes and revealed the pervasive transcription of divergent RNAs from promoter regions across eukaryotic genomes. These studies disclosed that genomes encode a vast repertoire of RNAs beyond the well-known protein-coding messenger RNAs. Furthermore, they have provided novel insights into the regulation of eukaryotic epigenomes, and transcriptomes, including the identification of novel classes of noncoding transcripts, such as the promoter-associated noncoding RNAs (pancRNAs).PancRNAs are defined as transcripts transcribed within few hundred bases from the transcription start sites (TSSs) of protein-coding or non-coding genes. Unlike the long trans-acting ncRNAs that regulate expression of target genes located in different chromosomal domains and displaying their function both in the nucleus and in the cytoplasm, the pancRNAs operate as cis-acting elements in the transcriptional regulation of neighboring genes. PancRNAs are very recently emerging as key players in the epigenetic regulation of gene expression programs in development and diseases.Herein, we review the complex epigenetic network driven by pancRNAs in eukaryotic cells, their impact on physiological and pathological states, which render them promising targets for novel therapeutic strategies.

Regulation of extracellular matrix degradation and metastatic spread by IQGAP1 through endothelin-1 receptor signalling in ovarian cancer.

The invasive phenotype of serous ovarian cancer (SOC) cells is linked to the formation of actin-based protrusions, invadopodia, operating extracellular matrix (ECM) degradation and metastatic spread. Growth factor receptors might cause engagement of integrin-related proteins, like the polarity protein IQ-domain GTPase-activating protein 1 (IQGAP1), to F-actin core needed for invadopodia functions. Here, we investigated whether IQGAP1 forms a signalosome with endothelin-1 (ET-1)/ß-arrestin1 (ß-arr1) network, as signal-integrating module for adhesion components, cytoskeletal remodelling and ECM degradation. In SOC cells, ET-1 receptor (ET-1R) activation, besides altering IQGAP1 expression and localization, coordinates the binding of IQGAP1 with ß-arr1, representing a "hotspot" for ET-1R-induced invasive signalling. We demonstrated that the molecular interaction of IQGAP1 with ß-arr1 affects relocalization of focal adhesion components, as vinculin, and cytoskeleton dynamics, through the regulation of invadopodia-related pathways. In particular, ET-1R deactivates Rac1 thereby promoting RhoA/C activation for the correct functions of invasive structures. Silencing of either IQGAP1 or ß-arr1, or blocking ET-1R activation with a dual antagonist macitentan, prevents matrix metalloproteinase (MMP) activity, invadopodial function, transendothelial migration and cell invasion. In vivo, targeting ET-1R/ß-arr1 signalling controls the process of SOC metastasis, associated with reduced levels of IQGAP1, as well as other invadopodia effectors, such as vinculin, phospho-cortactin and membrane type 1-MMP. High expression of ETAR/ß-arr1/IQGAP1 positively correlates with poor prognosis, validating the clinical implication of this signature in early prognosis of SOC. These data establish the ET-1R-driven ß-arr1/IQGAP1 interaction as a prerequisite for the dynamic integration of pathways in fostering invadopodia and metastatic process in human SOC.

The oncoprotein Myc controls the phosphorylation of S6 kinase and AKT through protein phosphatase 2A.

This study focuses on the effects of Myc oncoprotein on the translational apparatus of the cell. Translation is an energy consuming process that involves a large number of accessory factors. The production of components of the protein synthesis machinery can be regulated at the transcriptional level by specific factors. It has been shown that the product of the oncogene Myc, a transcription factor frequently activated in cancer, can control translational activity through an increase in the transcription of the eIF4F complex components (eIF4E, eIF4AI, and eIF4GI). However, additional effects at the posttranslational level have also been described. For instance, it has been shown that Myc upregulation can induce mammalian target of rapamycin (mTOR)-dependent 4E-binding protein 1 (4E-BP1) hyperphosphorylation. We induced overexpression or inhibition of Myc through transfection of complementary DNA constructs or specific small interfering RNA in PC3 (prostate carcinoma) and HeLa (cervical carcinoma) cells. We have observed that overexpression of Myc causes an increase in 4E-BP1 phosphorylation and activation of protein synthesis. Unexpectedly, we detected a parallel decrease in the phosphorylation level of S6 kinase (in PC3 and HeLa) and AKT (in HeLa). We report evidence that these changes are mediated by an increase in protein phosphatase 2A activity.

hMENA is a key regulator in endothelin-1/ß-arrestin1-induced invadopodial function and metastatic process.

Aberrant activation of endothelin-1 receptors (ET-1R) elicits pleiotropic effects relevant for tumor progression. The network activated by this receptor might be finely, spatially, and temporarily orchestrated by ß-arrestin1 (ß-arr1)-driven interactome. Here, we identify hMENA, a member of the actin-regulatory protein ENA/VASP family, as an interacting partner of ß-arr1, necessary for invadopodial function downstream of ET-1R in serous ovarian cancer (SOC) progression. ET-1R activation by ET-1 up-regulates expression of hMENA/hMENAΔv6 isoforms through ß-arr1, restricted to mesenchymal-like invasive SOC cells. The interaction of ß-arr1 with hMENA/hMENAΔv6 triggered by ET-1 leads to activation of RhoC and cortactin, recruitment of membrane type 1-matrix metalloprotease, and invadopodia maturation, thereby enhancing cell plasticity, transendothelial migration, and the resulting spread of invasive cells. The treatment with the ET-1R antagonist macitentan impairs the interaction of ß-arr1 with hMENA and inhibits invadopodial maturation and tumor dissemination in SOC orthotopic xenografts. Finally, high ETAR/hMENA/ß-arr1 gene expression signature is associated with a poor prognosis in SOC patients. These data define a pivotal function of hMENA/hMENAΔv6 for ET-1/ß-arr1-induced invadopodial activity and ovarian cancer progression.

Ribosomal stress activates eEF2K-eEF2 pathway causing translation elongation inhibition and recruitment of terminal oligopyrimidine (TOP) mRNAs on polysomes.

The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes.