LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.     

Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice

The Notch signaling pathway is important in regulating formation and anterior-posterior patterning of somites in vertebrate embryos. Here we show that distinct segmentation defects are displayed in embryos mutant for the Notch pathway genes Notch1, Lunatic fringe (Lfng), Delta-like 1 (Dll1), and Delta-like 3 (Dll3). Lfng-deficient mice and Dll3-deficient mice exhibit very similar defects, and marker analysis suggests that progression of the segmentation clock is disrupted in Dll3 mutants. We also show that Radical fringe (Rfng)-deficient mice exhibit no obvious phenotypic defects. To assess whether the absence of a phenotype in Rfng-deficient mice was the result of functional redundancy with the Lfng gene, we generated Lfng/Rfng double homozygous mutant mice. These mice exhibit the skeletal defects normally observed in Lfng-deficient mice, but we detected no obvious synergistic or additive effects in the double mutant animals.     

The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway

The Salvador-Warts-Hippo (SWH) pathway is a complex signaling network that controls both developmental and regenerative tissue growth. Using a genetic screen in Drosophila melanogaster, we identified the sterile 20-like kinase, Tao-1, as an SWH pathway member. Tao-1 controls various biological phenomena, including microtubule dynamics, animal behavior, and brain development. Here we describe a role for Tao-1 as a regulator of epithelial tissue growth that modulates activity of the core SWH pathway kinase cassette. Tao-1 functions together with Hippo to activate Warts-mediated repression of Yorkie. Tao-1's ability to control SWH pathway activity is evolutionarily conserved because human TAO1 can suppress activity of the Yorkie ortholog, YAP. Human TAO1 controls SWH pathway activity by phosphorylating, and activating, the Hippo ortholog, MST2. Given that SWH pathway activity is subverted in many human cancers, our findings identify human TAO kinases as potential tumor suppressor genes.     

l(3)malignant brain tumor and three novel genes are required for Drosophila germ-cell formation

To identify genes involved in the process of germ-cell formation in Drosophila, a maternal-effect screen using the FLP/FRT-ovoD method was performed on chromosome 3R. In addition to expected mutations in the germ-cell determinant oskar and in other genes known to be involved in the process, several novel mutations caused defects in germ-cell formation. Mutations in any of three genes [l(3)malignant brain tumor, shackleton, and out of sync] affect the synchronous mitotic divisions and nuclear migration of the early embryo. The defects in nuclear migration or mitotic synchrony result in a reduction in germ-cell formation. Mutations in another gene identified in this screen, bebra, do not cause mitotic defects, but appear to act upstream of the localization of oskar. Analysis of our mutants demonstrates that two unique and independent processes must occur to form germ cells-germ-plasm formation and nuclear division/migration.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), whilst Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.     

Current review of in vivo GBM rodent models: emphasis on the CNS-1 tumour model

GBM (glioblastoma multiforme) is a highly aggressive brain tumour with very poor prognosis despite multi-modalities of treatment. Furthermore, recent failure of targeted therapy for these tumours highlights the need of appropriate rodent models for preclinical studies. In this review, we highlight the most commonly used rodent models (U251, U86, GL261, C6, 9L and CNS-1) with a focus on the pathological and genetic similarities to the human disease. We end with a comprehensive review of the CNS-1 rodent model.     

UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

Ultraviolet irradiation resistance-associated gene (UVRAG) is a well-known regulator of autophagy by promoting autophagosome formation and maturation. However, little is known about the non-autophagic functions of UVRAG. Here, we present evidence that UVRAG functions as an unusual BCL2-associated X protein (Bax) suppressor to regulate apoptosis. Chemotherapy and radiation induces UVRAG expression and subsequently upregulates autophagy and apoptosis in tumour cells. Depletion of UVRAG expression by RNA interference renders tumour cells more sensitive to chemotherapy- and radiation-induced apoptosis in vitro and in vivo. Moreover, UVRAG interacts with Bax, which inhibits apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. Our findings show that UVRAG has an essential role in the intrinsic mitochondrial pathway of apoptosis by regulating the localization of Bax. This pathway represents a target for clinical intervention against tumours.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), while Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.Key words: Drosophila, tumor suppressor gene, cell polarity, Hippo pathway, Crb, Lgl, aPKC     

Targeting tumour cells with defects in the MHC Class I antigen processing pathway with CD8+ T cells specific for hydrophobic TAP- and Tapasin-independent peptides: the requirement for directed access into the ER

It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells

Notch signalling is important for development and tissue homeostasis and activated in many human cancers. Nevertheless, mutations in Notch pathway components are rare in solid tumours. ZEB1 is an activator of an epithelial-mesenchymal transition (EMT) and has crucial roles in tumour progression towards metastasis. ZEB1 and miR-200 family members repress expression of each other in a reciprocal feedback loop. Since miR-200 members target stem cell factors, ZEB1 indirectly induces stemness maintenance and associated drug resistance. Here, we link ZEB1 and its cancer promoting properties to Notch activation. We show that miR-200 members target Notch pathway components, such as Jagged1 (Jag1) and the mastermind-like coactivators Maml2 and Maml3, thereby mediating enhanced Notch activation by ZEB1. We further detected a coordinated upregulation of Jag1 and ZEB1, associated with reduced miR-200 expression in two aggressive types of human cancer, pancreatic adenocarcinoma and basal type of breast cancer. These findings explain increased Notch signalling in some types of cancers, where mutations in Notch pathway genes are rare. Moreover, they indicate an additional way how ZEB1 exerts its tumour progressing functions.     

The involvement of activated ras genes in determining the transformed phenotype

Activated ras oncogenes have been identified in a wide range of tumours. All examples of ras gene activation in tumours so far result from amino acid substitution at Gly12 or Gln61. To learn more about how mutations in ras genes lead to transformation, we have analysed transforming growth factor production in NIH/3T3 cells transformed by each of the three ras genes. These results show that the transformed phenotype of these cells results from a combination of the presence of the mutant ras protein and TGF alpha production. In a second series of experiments we have shown that the mutation of a ras gene in a tumour cell line can lead to tumour progression towards a more aggressive phenotype.