POU3f2 in human gliomas - Expression pattern and functional role

Gliomas are tumors of the central nervous system (CNS). They are responsible for about 28% of all central nervous system tumors and for 80% of all malignant brain tumors. The most commonly used classification and grading of CNS tumors is the World Health organization (WHO) classification: According to the WHO classification, gliomas are divided into four grades, from grade I to IV. Furthermore, different grade tumors are characterised by different genetic aberrations and phenotypes. Tumor and fetal development display a resemblance due to the fast increase in biomass and extensive cell migration that characterised both processes. Thus the early embryo and tumors may share a common genetic basis. POU domain transcription factors have a critical role in embryo development, and reexpression/overexpression of these transcription factors have been reported in many cancers. POU3f2 is an intronless member of class III POU domain transcription factors. Its expression is confined to the CNS where it is involved in differentiation of neuronal and glial cell lineages in the embryo. Re-expression of POU3f2 has been reported in melanoma cancer by several independent groups. Although POU3f2 expression in gliomas has been sporadically reported, it has not been systematically characterised in a larger panel of gliomas of different grades and histologies. Since melanocytes and the main CNS cell types share a common neuroectodermal origin therefore, we hypothesized that POU3f2 is expressed in human gliomas. In order to assess the expression of POU3f2 in glioma patients, and its possible correlation with malignancy grade, 51 formalin fixed paraffin embedded patient biopsies (grade II-IV) were stained by immunohistochemistry. Secondly, to confirm IHC results, western blotting and qRT-PCR were performed on about 40 human glioma biopsies. In addition, we conducted a series of experiments in vitro and in vivo to investigate how POU3f2 impacted on various aspects of glioma cell behavior. We found that, POU3f2 is expressed in all grades of glioma of both astrocytic and oligodendroglial lineages. Moreover, expression was higher in grade IV than grade II tumors, whereas over-expression of POU3f2 in the U251cell line increased both glioma cell proliferation and colony formation. Conversely, down-regulation of POU3f2 decreased colony formation. Finally, over-expression of POU3f2 in the U251cell line promoted tumor growth in nude mice, compared to U251 glioma cells with down-regulation of POU3f2 or expression of scrambled RNA sequences. My plan for future studies will be to expand the present data to include additional glioma cell lines. In addition, my goal is to establish which signaling pathway mediates the effects of POU3f2.Master i Medisinsk biologiMAMD-MEDBIBMED39

GM-CSF, Flt3-L and IL-4 affect viability and function of conventional dendritic cell types 1 and 2

Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.publishedVersio

The long noncoding RNA NEAT1 and nuclear paraspeckles are up-regulated by the transcription factor HSF1 in the heat shock response

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders

Novel STAT3 Inhibitors Targeting STAT3 Dimerization by Binding to the STAT3 SH2 Domain

Our drug discovery model has identified two novel STAT3 SH2 domain inhibitors 323–1 and 323–2 (delavatine A stereoisomers) in a series of experiments. In silico computational modeling, drug affinity responsive target stability (DARTS), and fluorescence polarization (FP) assays altogether determined that 323–1 and 323–2 directly target the STAT3 SH2 domain and inhibited both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that compound 323s bind to three subpockets of the STAT3 SH2 domain. The 323s inhibition of STAT3 dimerization was more potent than the commercial STAT3 SH2 domain inhibitor S3I-201 in the co-immunoprecipitation assay, correlating with computational docking data. The fluorescence polarization assay further confirmed that the compound 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds downregulated STAT3 target genes MCL1 and cyclin D1. Thus, the two compounds are promising lead compounds for the treatment of cancers with hyper-activated STAT3.publishedVersio

The expression of the long NEAT1_2 isoform is associated with human epidermal growth factor receptor 2-positive breast cancers

The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice

ALYREF, a novel factor involved in breast carcinogenesis, acts through transcriptional and post-transcriptional mechanisms selectively regulating the short NEAT1 isoform

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identifed high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF signifcantly infuenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profling, phenocopy and rescue experiments identifed the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF efects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fne-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifes novel molecular mechanisms of regulation the two isoforms of NEAT1