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

The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy

LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Eμ-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death

Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity

Lowe and colleagues explore the senescence-associated secretory phenotype (SASP), the gene expression and protein secretion subroutine of the senescence program. Global proteomic profiling establishes NF-κB as a master regulator of SASP gene expression changes during oncogene-induced senescence, and further in vitro and in vivo analysis finds that NF-κB-mediated SASP influences immune recognition by NK cells, drug resistance, and the outcome of cancer treatment. It is related to the article by Jing et al. (see below)

Identification of Novel Ras-Cooperating Oncogenes in Drosophila melanogaster: A RhoGEF/Rho-Family/JNK Pathway Is a Central Driver of Tumorigenesis

We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (RasACT) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with RasACT in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance RasACT-driven hyperplasia. RasACT-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1ACT), RhoGEF2, and pbl cooperated with RasACT, resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with RasACT upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with RasACT, while in the clonal setting, JNK upregulation was sufficient for RasACT-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of RasV12-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2+ human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with RasACT in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with RasACT

The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in <i>Drosophila melanogaster</i> through Maintaining a Progenitor-like Cell State

<div><p>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 <i>Drosophila melanogaster</i>, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, <i>scribbled</i> (<i>scrib</i>), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with <i>scrib</i> 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 <i>scrib</i> 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 <i>Drosophila</i> can be characterised by the maintenance of a progenitor-like state.</p></div

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models

<i>ab</i> overexpression in <i>scrib</i> mutant clones promotes neoplastic overgrowth of eye/antennal epithelial tissue throughout an extended larval stage.

<p>Mosaic eye/antennal discs (anterior to the left in this and all subsequent figures) generated with <i>ey-FLP</i> and taken from larvae 5 days (A–H) or 9 days (I,J) AEL. Clones are positively marked by GFP (white, or green in merges). Tissue morphology is shown by F-actin (red in merges), and cell fate by Elav and Dll (white, or blue in merges – dark blue when overlaid with GFP). Brain lobes in I,J are marked by BL. GFP (panels A–J), Elav (panels A′,C′,E′,G′,I′), Dll (panel B′,D′,F′H′,J′) and merges (panels A″–J″). (A,B) Control mosaic eye/antennal discs show the normal pattern of Elav expression in developing photoreceptor cells, and Dll expression within the antenna. (C,D) <i>scrib¹</i> cells still express Elav and Dll, although the normal pattern of Elav-expressing photoreceptor cells is disrupted by alterations in tissue morphology. (E,F) <i>ab</i> overexpressing clones still express Elav and Dll, but are often larger than control clones within the antennal region, and in some discs ectopic domains of Dll expression are observed (F, arrowhead). (G,H) <i>scrib¹</i>+<i>ab</i> clones are larger than <i>scrib¹</i> clones, and do not express Elav (G, arrowhead), although Dll expression is maintained (H, arrowhead). (I,J) <i>scrib¹</i>+<i>ab</i> clones at day 9 are massively overgrown and the two eye/antennal discs fuse with each other and with the Elav-expressing brain lobes (I), whilst the Dll-expressing domain in the antennal disc is maintained (J). Yellow scale bar = 50 µm.</p

Potential tumourigenic targets of Ab identified from expression array and ChIP-Seq analysis.

<p>(A) Venn diagram showing the number of differentially expressed probe sets (log base 2 fold change >1, adjusted p value <0.05) within mosaic <i>ab</i> overexpressing eye/antennal discs compared to control mosaic discs, and <i>scrib¹</i>+<i>ab</i> mosaic discs compared to the control mosaic discs. (B) Venn diagram showing the number genes identified as potential Ab targets based upon the occurrence of a significant peak (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#s4" target="_blank">Materials and Methods</a>) either within 500 bp upstream of the transcription start site or within the introns of a gene, in either mosaic <i>ab</i>-expressing eye/antennal discs, or <i>scrib¹</i>+<i>ab</i> mosaic discs, when compared to the respective input DNA controls. (C) Venn diagram that combines the results from the expression array and ChIP-Seq analysis. The six main classes of genes deregulated in <i>scrib¹</i>+<i>ab</i> tumours are shown: Class 1, genes differentially expressed and associated with Ab peaks in both <i>ab</i> and <i>scrib¹</i>+<i>ab</i> samples; Class 2, genes differentially expressed in <i>scrib¹</i>+<i>ab</i> alone, but associated with Ab peaks in both samples; Class 3, genes differentially expressed in both <i>ab</i> and <i>scrib¹</i>+<i>ab</i> samples, but associated with Ab peaks in <i>scrib¹</i>+<i>ab</i> alone; Class 4, genes only differentially expressed and associated with Ab peaks in <i>scrib¹</i>+<i>ab</i> alone; Class 5, genes deregulated in the <i>scrib¹</i>+<i>ab</i> tumours but not associated with Ab peaks; and Class 6, genes deregulated in the <i>scrib¹</i>+<i>ab</i> tumours but only associated with Ab peaks in the non-tumourigenic <i>ab</i>-expressing discs. Note that genes represented by multiple probe sets in the expression array are represented only once amongst the different classes, and assigned to either both genotypes if at least one probe set was significantly deregulated in both genotypes, or assigned to either <i>ab</i> or <i>scrib⁻</i>+<i>ab</i> uniques categories if at least one probe set was deregulated specifically in these genotypes. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s001" target="_blank">Dataset S1</a></b> for the complete gene lists associated with the expression array, ChIP-Seq and Classes 1–6. (D) Selected GO enrichments amongst the deregulated genes and potential Ab targets. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s003" target="_blank">Dataset S3</a></b> for the full listing of significantly enriched GO categories. (E) Heat map highlighting selected functional groups of deregulated genes identified from the expression array (red, upregulated; green, downregulated). For genes represented by multiple probe sets, the following probe sets are shown in the figure: <i>br</i> (1636931_at), <i>chinmo</i> (1636985_s_at), <i>Dll</i> (1636088_at, 1625771_at), <i>dom</i> (1628160_a_at), <i>Eip75B</i> (1635393_s_at), <i>elB</i> (1631207_at), <i>fru</i> (1641338_at, 1632859_a_at), <i>ImpE1</i> (1631375_a_at), <i>lola</i> (1633089_a_at, 1635096_at), <i>Mmp1</i> (1625761_a_at), <i>mod(mdg4)</i> (1627953_at, 1638041_at), <i>toy</i> (1633094_a_at), and <i>Trl</i> (1635305_s_at). See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s002" target="_blank">Dataset S2</a></b> for a full listing of deregulated genes with multiple probe sets, and their relative expression in <i>ab</i> or <i>scrib</i>⁻+<i>ab</i> tissue. A cross (X) denotes genes that were also identified from the ChIP-Seq analysis as potential Ab targets in either the <i>ab</i> alone, and/or <i>scrib¹</i>+<i>ab</i> samples. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s004" target="_blank">Dataset S4</a></b> for ChIP-Seq genome alignments for these genes.</p

Model illustrating the pathways involved in <i>scrib</i>⁻+<i>ab</i> cooperative tumour overgrowth.

<p>Ab cooperates with the loss of <i>scrib</i> to form invasive tumours through modulating the expression of multiple genes involved in all aspects of tumour formation. Potential targets of Ab include genes involved with blocking apoptosis and promoting tumour overgrowth (eg. <i>hid</i>, <i>Buffy</i>, <i>ft</i>, <i>dm</i>, <i>Pten</i>), genes required for eye/antennal disc differentiation (eg. <i>ct</i>, <i>dac</i>, <i>eya</i>, <i>dan</i>), genes involved in promoting cell invasion (eg. <i>Mmp1</i>), and genes involved in the ecdysone-induced pupariation response (eg. <i>Blimp-1</i>, <i>br</i>, <i>Eip75E</i>, <i>Hr39</i>). Whilst not shown on the figure, the steroid hormone receptor coactivator Tai is both required for <i>ab</i>-driven tumour overgrowth and sufficient to cooperate with the loss of <i>scrib</i>, consistent with the possibility that Ab acts in concert with Tai to drive tumour formation. Loss of <i>scrib</i> activates JNK-mediated apoptosis, however, <i>ab</i> overexpression abrogates the apoptotic response, thereby unmasking a key role for JNK in promoting tumour cell migration and invasion through the expression of JNK-induced genes such as <i>Mmp1</i>. Loss of <i>scrib</i> also promotes aPKC-dependent Yki activity that is required and sufficient to cooperate with Ab by impairing differentiation and promoting tumour overgrowth. Other pathways deregulated in <i>scrib</i> mutants may participate in the tumour phenotype and promote the full spectrum of differentiation defects seen in <i>scrib</i>⁻+<i>ab</i> tumours (indicated by the dotted blocking arrow and question mark), such as Dac repression in the antenna. Green = downregulated genes, and red = upregulated genes.</p

Overexpression of <i>ab</i> in <i>scrib</i> mutant clones promotes the retention of a progenitor-like state in the eye and antennal disc.

<p><i>ey-FLP</i> induced eye/antennal disc clones at ∼5 days AEL. Clones are marked by GFP (white, or green in merges), and cell fate is shown by the expression of Dac, Dan and Hth (white, and magenta when overlaid with GFP in the merges) in wild type control clones (A,E,I), <i>scrib¹</i> clones (B,F,J), <i>ab</i> overexpressing clones (C,G,K), and <i>scrib¹</i>+<i>ab</i> clones (D,H,L). GFP (panels A–L), Dac (panels A′–D′), Dan (panels E′–H′), Hth (panels I′–L′) and merges (panels A″–L″). (A–D) Dac expression is only slightly reduced in <i>scrib¹</i> clones (B, yellow arrowhead), and unaffected in <i>ab</i> overexpressing clones in the eye disc, although ectopic Dac expressing antennal-like structures are sometimes observed in the antenna (C, arrowhead). <i>scrib¹</i>+<i>ab</i> clones do not express Dac (D, arrowhead; the magenta staining observed around some clones is derived from GFP bleed-through from underlying sections. (E–H) Dan levels are reduced in <i>scrib¹</i> clones both in the antennal and eye disc (F, arrowheads). <i>ab</i> overexpressing clones do not affect Dan levels in the eye disc (G, arrowhead), although Dan is slightly repressed in the antenna (G, arrow), albeit ectopically expressed in the ectopic antennal-like structures. Dan is repressed in <i>scrib¹</i>+<i>ab</i> clones (H, arrowhead). (I–L) Hth expression is generally unaffected in <i>scrib¹</i> clones (J). In <i>ab</i> overexpressing clones, levels of Hth are slightly reduced in the eye disc (K, arrow), and large clones in the antennal disc do not express Hth (K, arrowhead). In <i>scrib¹</i>+<i>ab</i> clones, Hth is expressed in some clones within the eye disc (L, arrowhead), but not all clones (L, arrow), and is generally reduced in antennal disc clones (L, and data not shown). (M) Diagram summarising the expression of cell fate markers in both wild type eye/antennal discs, as well as in eye/antennal disc <i>scrib</i>⁻+<i>ab</i> tumours (green). In the antenna, proximal refers to the outer circular domains of the tissue, whilst distal refers to the inner, central domains. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s013" target="_blank">Figures S9</a></b> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen.1003627.s014" target="_blank">S10</a></b> for immunohistochemical images of Tsh, Ey, Eya, Ato and Sens; and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003627#pgen-1003627-t002" target="_blank"><b>Table 2</b></a> for a summary of these results. Yellow scale bar = 50 µm.</p