The BTB-zinc finger transcription factor abrupt acts as an epithelial oncogene in drosophila melanogaster through maintaining a progenitor-like cell state

The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state

ETS-domain transcription factor Elk-1 mediates neuronal survival: SMN as a potential target

AbstractElk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation

BTB-Zinc Finger Oncogenes Are Required for Ras and Notch-Driven Tumorigenesis in <i>Drosophila</i>

<div><p>During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers we used <i>Drosophila</i> epithelial tumor models, which are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, <i>scribbled</i> (<i>scrib</i>). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors we used a comparative microarray analysis to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including <i>chronologically inappropriate morphogenesis</i> (<i>chinmo</i>). <i>chinmo</i> was upregulated by JNK within the tumors, and overexpression of <i>chinmo</i> with either <i>Ras^V12</i> or <i>N^intra</i> was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with <i>Ras^V12</i>, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of <i>scrib</i> mutant tissue, since, although loss of <i>chinmo</i> alone did not significantly impede tumor development, when loss of <i>chinmo</i> was combined with loss of a functionally related BTB-ZF gene, <i>abrupt</i>, tumor overgrowth was significantly reduced. <i>abrupt</i> is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, our work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states.</p></div

<i>fru</i> overexpression cooperates with <i>Ras</i>^<i>ACT</i> or <i>N</i>^<i>ACT</i> to promote JNK-independent tumor overgrowth in the eye-antennal disc.

<p>Larval mosaic eye-antennal discs attached to brain lobes (BL), anterior to the top, at day 5 (A and D) and day 9 (B, C, E and F). Clones are generated with <i>ey-FLP</i>, and are positively marked by GFP (green). Tissue morphology is shown with Phalloidin staining to highlight F-actin (red). The yellow scale bar corresponds to 40μm. (A) Mosaic eye-antennal discs expressing <i>UAS-fru</i>^<i>C</i> in clones are relatively normal in size (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132987#pone.0132987.g003" target="_blank">Fig 3A</a>). (B,C) Mosaic eye-antennal discs co-expressing <i>UAS-fru</i>^<i>C</i> with <i>UAS-Ras</i>^<i>ACT</i> (B) or <i>UAS-fru</i>^<i>C</i> with <i>UAS-N</i>^<i>ACT</i> (C) results in tissue massively overgrowing throughout an extended larval stage of development. (D) Mosaic eye-antennal discs coexpressing <i>UAS-bsk</i>^<i>DN</i> with <i>UAS</i>-<i>fru</i>^<i>C</i> in clones are similar to <i>UAS-fru</i>^<i>C</i> clones alone (A). (E-F) Co-expressing <i>UAS-bsk</i>^<i>DN</i> in <i>fru</i>^<i>C</i> + <i>Ras</i>^<i>ACT</i> (E) or <i>fru</i>^<i>C</i> + <i>N</i>^<i>ACT</i> (F) clones does not restore pupariation to the tumor-bearing larvae, and the mutant tissue overgrows throughout an extended larval stage of development.</p

<i>chinmo</i> overexpression in eye-antennal disc clones cooperates with <i>Ras</i>^<i>ACT</i> or <i>N</i>^<i>ACT</i> to produce massive tumors.

<p>Larval mosaic eye-antennal discs attached to brain lobes (BL), anterior to the top, at day 5 (A-D) and day 9 (E-G). Clones are generated with <i>ey-FLP</i>, and are positively marked by GFP (green). Tissue morphology is shown with Phalloidin staining to highlight F-actin (red). The yellow scale bar corresponds to 40μm. (A-D) Control <i>FRT82B</i> eye-antennal disc clones (A), <i>UAS-N</i>^<i>ACT</i>-expressing clones (B), <i>UAS-Ras</i>^<i>ACT</i>-expressing clones (C) and <i>UAS-chinmo</i>^<i>FL</i>-expressing clones (D) are relatively normal in size at day 5 prior to pupariation. (E-F) Co-expressing <i>UAS-chinmo</i>^<i>FL</i> with <i>UAS-N</i>^<i>ACT</i> (E) or <i>UAS-Ras</i>^<i>ACT</i> (F) in eye-antennal disc clones blocks pupariation, and the clonal tissue massively overgrows throughout an extended larval stage of development. Clones of mutant tissue within the brain lobes also over-proliferate to greatly enlarge the brain lobes (F). (G) Co-expressing <i>UAS-bsk</i>^<i>DN</i> in <i>chinmo</i>^<i>FL</i> + <i>Ras</i>^<i>ACT</i> clones does not restore pupariation to the tumor-bearing larvae, and the mutant tissue overgrows throughout an extended larval stage of development.</p

Ab is expressed in <i>msn-lacZ</i> and <i>chinmo-lacZ</i> expressing tumor cells.

<p>Mosaic eye-antennal discs, anterior to the right (A) and attached to brain lobes (BL) (B-C). Clones are generated with <i>ey-FLP</i>, and are positively marked by GFP (green, or magenta when overlaid with white). Ab (A-C) expression is shown in cyan (red when overlaid with white, dark blue when overlaid with green or yellow when overlaid with white and green in the merges), <i>msn-lacZ</i> (A-B) and <i>chinmo-lacZ</i> (C) expression is shown by antibody detection of β-Galactosidase (white, or red when overlaid with cyan in the merges). Yellow scale bar corresponds to 40μm. (A-B) Ab is endogenously expressed in the anterior progenitor domain of the eye disc (A) and is present in basal and migrating cells (B) of <i>scrib</i>^<i>1</i><i>+ Ras</i>^<i>ACT</i> tumors. These same cells strongly express the JNK reporter <i>msn-lacZ</i> (A’, B’). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132987#pone.0132987.s008" target="_blank">S8A Fig</a> for the endogenous expression of Ab in control eye/antennal discs. (C) The migrating <i>scrib</i>^<i>1</i> + <i>Raf</i>^<i>GOF</i> tumor cells express Ab and are also positive for <i>chinmo-lacZ</i> expression.</p

<i>chinmo</i> overexpression increases <i>esg>GFP</i> cells in the midgut, and cooperates with <i>Ras</i>^<i>ACT</i> to promote midgut tumorigenesis.

<p>Sections of adult midguts expressing transgenes at 29°C under the control of <i>esg-GAL4</i>,<i>tub-GAL80</i>^<i>ts</i>. DNA is stained with Hoechst (white), enteroendocrine cells are identified by Prospero expression (white), and <i>UAS-GFP</i> (green) is expressed in the stem cells and enteroblasts. The overlay of DNA with GFP appears magenta in the merge. Cross sections through the centre of the intestine, stained with Phalloidin to detect F-actin (red) and Hoechst (blue) are shown in B, D, F and H. Yellow scale bar corresponds to 20μM. (A,B) Control midguts expressing <i>UAS-GFP</i> for 5 days at 29°C. (C,D) The expression of <i>UAS-chinmo</i>^<i>FL</i> for 10 days at 29°C greatly increases the number of <i>esg>GFP</i> cells, whilst the number of enteroendocrine cells appears unchanged. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132987#pone.0132987.s005" target="_blank">S5 Fig</a> for quantifications at day 5 and 10. (E,F) Expression of <i>UAS-Ras</i>^<i>ACT</i> for 7 days at 29°C induces changes in the appearance of <i>the esg>GFP</i> cells, but does not lead to the formation of tumors. (G,H) Coexpression of <i>UAS-chinmo</i>^<i>FL</i> with <i>UAS-Ras</i>^<i>ACT</i> for 7 days at 29°C leads to <i>esg>GFP</i> cells overtaking the entire midgut, filling the lumen of the intestine.</p

The ectopic expression of <i>chinmo</i>-<i>lacZ</i> in <i>scrib</i>^<i>1</i> + <i>Raf</i>^<i>gof</i> tumor cells is dependent upon JNK signaling.

<p>Mosaic eye-antennal discs, anterior to the right. Clones are generated with <i>ey-FLP</i>, and are positively marked by GFP (green, or magenta when overlaid with white). <i>chinmo-lacZ</i> expression is shown by antibody detection of β-Galactosidase (white, or magenta when overlaid with GFP in the merges). Brain lobes (BL) remain attached to the eye discs in E, and in E and E’ tissue morphology is shown with Phalloidin staining to highlight F-actin (white). Yellow scale bar corresponds to 40μm. (A) In control <i>FRT82B</i> eye-antennal discs, <i>chinmo-lacZ</i> is expressed in the centre of the antennal disc and in the posterior half of the eye disc. (B) In <i>scrib</i>^<i>1</i> mosaic discs, <i>chinmo-lacZ</i> is ectopically expressed in some mutant antennal disc clones (arrowhead). (C) In <i>scrib</i>^<i>1</i> + <i>Raf</i>^<i>gof</i> mosaic discs, <i>chinmo-lacZ</i> is ectopically expressed in mutant clones in the antennal and eye disc (arrowheads). (D) Expressing <i>UAS-bsk</i>^<i>DN</i> in <i>scrib</i>^<i>1</i> + <i>Raf</i>^<i>gof</i> clones abrogates the ectopic expression of <i>chinmo-lacZ</i> in the mutant clones of tissue (arrowhead). (E) In <i>scrib</i>^<i>1</i> + <i>Raf</i>^<i>gof</i> tumors, <i>chinmo-lacZ</i> is ectopically expressed in the tumor cells that appear to be migrating between the brain lobes (arrowhead, E’ and E’’’).</p

<i>chinmo</i>^<i>FL</i> over-expression in clones blocks differentiation in the eye-antennal disc.

<p>Mosaic eye-antennal discs, anterior to the right. Clones are generated with <i>ey-FLP</i>, and are positively marked by GFP (green, or magenta when overlaid with white). Cell fate is marked by the expression of Elav, Dac and Eya (white, or magenta when overlaid with GFP in the merges). Yellow scale bar corresponds to 40μm. (A-F) Expressing <i>UAS-chinmo</i>^<i>FL</i> in eye-antennal disc clones blocks expression of Elav (A), Dac (C) and Eya (E), and this block is maintained in <i>chinmo</i>^<i>FL</i> + <i>Ras</i>^<i>ACT</i> tumors (B, D, F; arrowheads).</p