Oncogenic programmes and Notch activity: An ‘organized crime’?

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Review

Oncogenic programmes and Notch activity: An ‘organized crime’? Maria Dominguez ∗ Instituto de Neurociencias, CSIC-UMH, Alicante, Spain

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Drosophila Notch Cancer

a b s t r a c t The inappropriate Notch signalling can influence virtually all aspect of cancer, including tumour–cell growth, survival, apoptosis, angiogenesis, invasion and metastasis, although it does not do this alone. Hence, elucidating the partners of Notch that are active in cancer is now the focus of much intense research activity. The genetic toolkits available, coupled to the small size and short life of the fruit fly Drosophila melanogaster, makes this an inexpensive and effective animal model, suited to large-scale cancer gene discovery studies. The fly eye is not only a non-vital organ but its stereotyped size and disposition also means it is easy to screen for mutations that cause tumours and metastases and provides ample opportunities to test cancer theories and to unravel unanticipated nexus between Notch and other cancer genes, or to discover unforeseen Notch’s partners in cancer. These studies suggest that Notch’s oncogenic capacity is brought about not simply by increasing signal strength but through partnerships, whereby oncogenes gain more by cooperating than acting individually, as in a ring ‘organized crime’. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Overview of Notch signalling cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Notch signalling and organ growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Fringe boundary and the growth promoting ‘organizer’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Notch signalling and its attenuation in endocytotic compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. The ‘initiator’ event: the cytokine upd/JAK/STAT pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. The ‘organizer’ downstream events: four-jointed kinase, as a central regulatory node that integrates the Notch and the Hippo growth controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.5. The Hedgehog signalling, a brake that keeps Notch-induced growth on track? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.6. The turning off the organizer: epigenetic regulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The Notch’s dangerous liaisons: tumour metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Notch signalling is a tightly regulated cell–cell communication network that is used broadly and reiteratively both during development and in adult stem cells [1–3]. In numerous tissues, Notch first promotes proliferation while retaining cells in an undifferentiated state that is unresponsive to signals from other cells. As cells transit from a proliferative to a committed state in order to differentiate, Notch signalling is again employed to control successive cell

∗ Tel.: +34 965919390; fax: +34 965919561. E-mail address: [email protected]

fate choices via lateral inhibition, or lineage cell decisions through inductive signalling [3]. Given these pleiotropic roles in development, it is no wonder that Notch signalling may be either oncogenic or tumour suppressive in distinct human cancers, or that it may even adopt both roles within the same tumour type, albeit at different stages of tumorigenesis [3,4]. When acting as an oncogene, altered Notch signalling can influence tumour growth, angiogenesis, survival and/or the invasive-metastatic cascade [2,5], although the molecular bases as to how Notch influences these multiple process remains incompletely understood. While predictable, the discovery that activated Notch, like most oncogenes, is insufficient for cancer to develop in vivo has motivated researchers to adopt genetic approaches to hunt for Notch’s

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Table 1 Oncogenes and tumour suppressors (‘loss’) that cooperate with Notch signalling to initiate hyperplastic and neoplastic growth in flies.

Tumour metastasis N-ICD N-ICD Delta Delta Delta Delta Delta Delta Delta Delta

Cooperating event

Downstream eventsa

References

Scribbled/SCRIB (loss) Mef2/MEF2 (overexpression) Akt1 (overexpression) Zfh1/ZEB1,2(overexpression) Eyeful (Lola, Psq) (overexpression)

JNK activation Eager/JNK activation

[47,49] [7] [5] [8] [46]

b

Ato/ATOH1 (loss) Cut/CUX1 (loss)b MENA (loss)b dUtx/UTX (loss)b Lid/KDM5A (loss)b

Benign tumour growth mir-7 (overexpression) Delta Delta E(z)/EZH2 (loss)

Decreased H3K4 trimethylation Rbf (inactivation) JNK (inactivation) Akt1 (activation) Elevated Notch protein

ihog and boi (repression) Hedgehog signalling (inactivation)

[58] [60b ,10] [61] [44] [43] [38] [6]

a Genes or pathways regulated by the initiating oncogenic (or loss of tumour suppressor) event and functionally validated to facilitate or suppress tumour growth and/or metastasis. b Tumour metastasis is evaluated in the ‘Delta eyeful’ sensitised genetic background.

partners in tumorigenesis. Truncated, active forms of the Notch receptor, such as those found in paediatric and adult T-cell acute lymphoblastic leukaemia (T-ALL) [1,2], are being used widely to elucidate how Notch participates in cancer, and to define its partners in different cancer cell lines and processes. However, strong activating mutations in the Notch receptor are rare, even in liquid tumours, and in solid tumours, the inappropriate Notch activation arises from an overexpressed Notch receptor or ligands, or through mutations in pathway regulators [2–4]. Elucidating the connections between Notch signalling and other genes that control normal development, including cell proliferation, migration, cell death, cell differentiation and metabolism, is one approach to better understand the mechanisms of Notch signalling and regulation, and by extension, cancer. Cancer genetic screens as those using the Drosophila eye is another approach that is helping to identify genes and pathways that cooperate with Notch in tumour formation in vivo that also operate in human cancers [5–10] (Table 1). Importantly, observations in these fly eye neoplasms suggest that key aspects of tumour initiation differ little from the normal process of early development. At this stage, overall organ growth is organized around a local (maximal) Notch, Hedgehog or Wnt activities (henceforth called ‘organizers’ [11]). The fly eye neoplasms illustrate that Notch similarly orchestrates tumour growth by instructing abnormal behaviour, not only in cells receiving Notch signalling but also by actively perturbing normal surrounding cells and through cooperation with other signalling systems and transcription factors. In this view, I will concentrate on recent advances and knowledge on partnerships in normal growth control and tumorigenesis using the Drosophila eye paradigm. These studies unveil that many partnerships rely on physiological, individual relationships but also highlight that partnerships can change even within the same cellular context; in fact the cooperation and rivalry between oncogenes suggest they act as partners in ‘crime’. By cooperating, the targeting of one of them may fail to stop cancer cells but could be dealt with by breaking up their ‘organization’, as in a ring ‘organized crime’.

and Lag-2 from C. elegans: reviewed in [1]) and (Fig. 1). Notch receptors at the cell surface are heterodimeric molecules with a large extracellular domain (N-ECD) that is non-covalently attached to the membrane carboxyl-terminal transmembrane/intracellular fragment. The heterodimer is generated in the secretory pathway by a “S1” cleavage [1,2]. Drosophila N-ECD includes 36 epidermal growth factor-like repeats (EGFr), of which EGFr11 and EGFr12 are involved in receptor–ligand interactions, and the EGFr24, 25, 27 or 29 (Abruptex mutations) negatively regulate these interactions. N-ECD undergoes extracellular glycosylation. In particular O-fucosylation by O-FucT-1 (Pofut1 in mice) is crucial for all Notchligand interactions. Elongation of the O-fucose monosaccharide by Fringe, an EGF-O-fucose ␤1,3 N-acetylglucosamyltransferase (called Lunatic Fringe, Radical-Fringe, and Maniac-Fringe in mammals) is required for the inductive signalling/boundary formation mode of Notch signalling [3] (Fig. 1). Activation of the Notch receptor upon ligand binding involves several proteolytic steps that trigger the shedding of the N-ECD. This cleavage is performed by members of the ADAM metalloprotease Kuzbanian (Kuz)/TACE family, and it is followed by intramembrane proteolysis (S3 cleavage) by the presenilin (PS)dependent ␥-secretase complex to release a soluble intercellular Notch fragment (N-ICD) (Fig. 1). The soluble, S3-cleaved N-ICD fragment translocates to the nucleus where it associates with a DNA-binding protein called CSL (for human, CBF1; Drosophila, suppressor of hairless; C. elegans, Lag-1) and Master Mind (MAM) to regulate the expression of Notch target genes [1]. Intracellular post-transcriptional modifications like ubiquitinylation, endosome entry and the ionic properties (acidification) of the endosomal lumen play an important role in regulating Notch receptor activity and dampening ligand-independent activation that may result in neoplastic transformation (reviewed in [1,12]: see next sections) (Fig. 1). Other post-transcriptional modifications of the N-ICD, such as acetylation and phosphorylation, further affect Notch signalling intensity or duration [3]. Expression profiling identified members of the Enhancer of split complex and the transcription factor Deadpan as part of Notch’s oncogenic programme in flies [13].

2. Overview of Notch signalling cascade 3. Notch signalling and organ growth In Drosophila, the Notch signalling pathway centres around a single Notch receptor (Notch1-4 in mammals) and two genes that encode Notch ligands: Delta (Delta-like [Dll1], Dll3 and Dll4 in mammals) and Serrate (Jagged [Jag] 1 and Jag2), collectively referred as DSL proteins (for Delta and Serrate from Drosophila

Cancer cells often “hijack” an organ’s physiological growth programme and hence information on how Notch in combination with other genetic systems controls normal eye growth can help to elucidate the mechanism(s) underlying Notch-induced tumorigenesis.

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Fig. 1. Cell–cell signalling by Notch pathway. The ligands are transmembrane proteins that activate Notch receptors in physically adjacent cells. However, Notch signalling can also orchestrate cellular behaviour over a distance of several cell diameters from the signal-receiving cells via relay systems, and refers to as organizers/fringe boundaries’ [3,11]. Only Notch receptor internalization and key endocytic factors linked to neoplasms (in red) are despite [1,12].

3.1. Fringe boundary and the growth promoting ‘organizer’ The overall growth of the developing eye epithelium (called eye imaginal disc) is organized around a local (maximal) concentration of Notch activity at the dorsal–ventral midpoint (called ‘organizer’ [11,14]). This process depends on the fringe gene, the expression of which is negatively regulated by the dorsal transcriptional repressors of the Iroquois complex (Iro-C: called Irx in mammals), thereby restricting fringe to ventral cells (Fig. 2). Fringe inhibits the ability of Serrate to signal Notch and at the same time, potentiates the ability of Delta to activate Notch, thereby generating the local Notch activation that establishes the organizer that promotes global growth. If the boundary fails to form, either by ubiquitous expression of fringe or its loss, the eye growth is arrested resulting in adult fly lacking the eye due to failure to activate Notch [14]. Conversely, ectopic Fringe boundaries or overexpression of ligand Delta results in ectopic boundary cell formation and eye overgrowth [14]. Fringe-mediated modulation of Notch-ligand interactions is also crucial for vascular integrity and tumour (and tissue) perfusion in mice [3] and hence understanding how the differential Notchligand interactions is regulated may identify novel therapeutic points. Thus, tumour neovascularization involves a fine balance between Jagged1, a potent angiogenic regulator, and the Dll4 ligand in endothelial cells expressing Lunatic-Fringe [3]. By antagonizing Lunatic-Fringe-mediated enhancement of Dll4 signalling, elevated Jagged1 in cancer cells promotes Notch signalling resulting in neovascularization and the growth of experimental tumours in mice. Conversely, inhibition of Dll4 leads to non-productive endothelial sprouting, poor perfusion and reduced growth of experimental tumours. A recent work shows that the conserved microRNA miR-200 family (called miR-8 in flies) inhibits angiogenesis in experimental models of ovarian, lung, renal and basal-like breast cancer [15]. Another studies show that this microRNAs directly inhibit JAGGED1 in metastatic prostate, breast, and pancreatic carcinomas [8,16]. In flies, overexpression of mir-8/mir-200c is as a potent suppressor of tumour growth and metastasis by Notch by directly targeting Serrate mRNA by binding to its 3 UTR [8]. It will

be interesting to see whether JAGGED1 is a target of miR-200cmediated normalization of tumour vasculature, thereby supporting the utility of miR-200c as potential therapeutic agent for Notchassociated metastasis. 3.2. Notch signalling and its attenuation in endocytotic compartments What genes are responsible for this local (maximal) activation of Notch that orchestrates global tissue growth? Answers to this question are just beginning to emerge and here I focus on the endocytic trafficking pathway. Observations of fly eye neoplastic tumours occurring upon mutations in steps in the endocytic trafficking pathway has highlighted an important, and complex role for endocytosis of the Notch receptor for signalling and degradation [12]. The full-length Notch protein is continuously internalized into early endosomes and subsequently sorted to other endocytotic compartments, including recycling endosomes, multivesicular bodies/late endosomes and lysosomes (Fig. 1: [12]). Inactivation of genes that act early in the endocytic endosome fusion (e.g. Avalanche/Avt/Syntaxin, Rabenosyn-5, vps45, Rabaptin-5: reviewed in [12] and [17]) induce autonomous tumour growth when wholly mutant accompanied by accumulation of Notch protein in endosomes but there is no evidence of ectopic Notch receptor activation. Conversely, the endocytic tumour suppressors involved in late endosome trafficking, including the endosomal C2 domain-containing protein Lethal (2) giant discs (Lgd) [18–20] or components of the Endosomal Sorting Complex Required for Transport (e.g. mutants in ESCRT-I, II and III) [21–25] and reviewed in [12] (Table 2) all results in nonautonomous neoplastic growth, associated with accumulation of Notch in late endosome/lysosomes, ectopic ligand-independent but ␥-secretase activity-dependent Notch activation and ectopic expression of ‘boundary’ genes. This highlights that these tumours form by mechanisms similar to that of Notch-induced normal growth (Fig. 3). Moreover, concurrently with the heightened Notch activity, these mutants show increased activity of other signalling pathways, including the JAK/STAT, EGFR, Yorkie, and/or the Jun

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Fig. 2. Schematic of the molecular steps leading to the positioning of Fringe boundary (A) and the local Notch activation that instructs global tissue growth (B). In B, dorsal is to the right.

N-terminal kinase (JNK) pathways and the mislocalization of the tumour suppressor Crumbs [12] (Table 2). Thus mutations in later endocytic trafficking factors provide opportunities for signalling systems controlling growth, epithelial polarity, apoptosis and collective cell migration to interact. However, this new appreciation that the nonautonomous neoplatic phenotypes is associated with organizer-like characteristic have implications for how these endocytic steps may differentially effect on Notch signalling strength that deserves further work.

cannot be rescued by activation of the upd/JAK/STAT [27]. It was shown that an activation of the JAK/STAT pathway by upd gene expressed in a ventral domain in early eye development is required for repressing dorsal Iro-C by antagonizing Wingless and Hedgehog signalling [27]. Consistent with this model, upd overexpression induces overgrowth associated with Iro-C inhibition and an ectopic fringe boundary [27]. Thus upd/JAK/STAT facilitates Notch-induced global eye growth by positioning the organizer (Fig. 2), and this interaction may account for their potential cooperation in tumorigenesis.

3.3. The ‘initiator’ event: the cytokine upd/JAK/STAT pathway The activation of the JAK/STAT through the overexpression of it ligand, unpaired (upd) produces eye overgrowth in a nonautonomous fashion (reviewed in [14]). Accordingly, it was initially thought that Notch signalling induces the expression of upd, and that the secreted Upd protein then diffuses throughout the eye disc, promoting global eye growth [26]. However, this attractive model may be incorrect, as Notch deficiency in eye growth

3.4. The ‘organizer’ downstream events: four-jointed kinase, as a central regulatory node that integrates the Notch and the Hippo growth controls The function of organizers in global growth is not fully understood. In the Notch-mediated eye organizer, a central downstream component is the transcriptional repressor Pax6-related Eyegone [26,28], which executes the global growth response in part or large

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Table 2 Tumour suppressor mutations promoting tumorigenesis associated with co-occurring alterations of Notch activity and other signalling pathways. Pathway

Symbol

Effect on Notch

Effect on other pathways

Phenotype

References

Epigenetic pathway PcG

pho

Increased upd, dpp, and decreased hh

Trl/GAF

Nonautonomous neoplastic growth Hyperplastic growth

[39–42]

TrxG

Increased N, Ser, ectopic boundary genes Increased N reporter activity Elevated N protein

Hyperplastic growth

[44]

Hyperplastic growth

[40]

Hyperplastic growth

[40]

Hyperplastic growth

[40]

Hyperplastic growth

[36]

Neoplastic growth Nonautonomous neoplastic growth Nonautonomous neoplastic growth Nonautonomous neoplastic growth

[12] [17–20]

Hyperplastic growth Neoplastic growth

[64,65] [59]

dUtx

[40]

Gycolysis/pyruvate metabolism PGI PDHB DLAT

Mild increased N reporter activity Mild increased N reporter activity Mild increased N reporter activity

Endocytic pathway Ferm-domain proteins

Expanded merlin

Elevated N protein at plasma membrane

Internalization Endosomal C2-domain protein

Shibire (dynamin) Lgd

ESCRT-I

Erupted/TS101 Vps28 Vps2

ESCRT-II

Vps25

Increased N activity Increased N activity ectopic boundary genes Increased N activity ectopic boundary genes Increased N activity ectopic boundary genes

ESCRT-III

Shrub/Vps32 AAA ATPase/vps4

Increased EGFR, Patched, Smoothened, Fat, DE-cadherin, Yorkie, Wnt Increased Wnt activity

Increased JAK/STAT, Yorkie, JNK Increased EGFR, JAK/STAT, Yorkie, JNK and mislocalization of Crumbs

Increased N activity

part through Four-jointed (fj [27]), a Golgi transmembrane type II glycoprotein ([29,30]; and citations therein). The graded expression of fj induced by Notch and upd/JAK/STAT [14] is complementary to the graded expression of the tumour suppressor gene dachsous (Ds) (Fig. 2). Ds and Fat act upstream of the salvador/warts/Hippo tumour suppressor pathway that restrains epithelial imaginal disc growth by inhibiting cell proliferation and promoting apoptosis [30]. Hippo pathway inhibits fj expression and in turn the Golgi, cadherin-domain kinase Fj negatively influences the Hippo pathway by directly phosphorylating the extracellular domains of Fat and Ds as they are trafficked through the Golgi apparatus [29,30], thus Fj acts as a regulatory node integrating

Increased JNK

[12,21] [12,22–25]

Notch and Hippo growth controls. Interestingly, Fj related proteins have also been identified as Notch targets in mammals (e.g. Fjx-1 [31]), suggesting that similar events may occur more generally in Notch-induced tissue growth. Additional upstream components of the Hippo tumour suppressor pathway, including Crumbs [32–34] and the FERM-domain proteins, Expanded and Merlin [30], modulate Notch signalling via regulation of the ␥-secretase activity [35] and the endosomal entry of Notch proteins for degradation [36], respectively. The conserved Salvador/Warts/Hippo kinase cascade inhibits epithelial growth by phosphorylating and inactivating the transcriptional co-activator Yorkie (Drosophila homolog of the human

Fig. 3. Model for how Notch and partners might promote cancer. (A) A prototypical epithelium is shown and the circular arrows represent relay signals that promote growth and cell fate specification at a distance from the organizer region. (B) Oncogenic mutations may result in cell-autonomous phenotypes (left) or confer organizer-like phenotype (right) that orchestrates tumorigenesis by actively co-opting surrounding normal cells (white cells and yellow trachea). Mutations can also occur in the tissue microenvironment (dashed line), enhancing the expression of the neoplastic phenotype. (C) Adult fly with a Delta-induced tumour and metastasis (red masses, arrows). Bellow, a metastatic mass in an open abdomen showing ectopic trachea (arrows) [8], reminiscent to mammalian tumour neovascularization.

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oncogenes Yes-Associated protein (YAP)) and TAZ (transcriptional activator with PDZ-binding motif: reviewed in [30,37]). The precise link between Yorkie and Notch is poorly understood, but observations in fly eye neoplasms lacking ESCRT component vps25 indicate that cell-autonomous activated Notch in vps25− cells induces nonautonomously cell survival and overgrowth by activating Yorkie/YAP [18,21,30]. A similar regulatory event may occur in physiological growth whereby upd-Notch-Eyegone may induces fj expression via Yorkie activation (Fig. 2). Recent work indicates that Yorkie/YAP proteins facilitate tissue growth not only through transcriptional responses but also by decreasing cellular contact inhibition. Importantly, the growth of tumours is characterized by a loss of contact inhibition of proliferation, and activation of YAP/Yorkie by loss of the Hippo pathway releases contact inhibition in cancer cells, which may also facilitate metastasis [37]. 3.5. The Hedgehog signalling, a brake that keeps Notch-induced growth on track? The Hedgehog signalling is expressed in an opposite gradient to that of the ‘organizer’ in the early eye disc [14,38] (Fig. 2). Hedgehog signalling is crucial for maintaining retinal cells competent for differentiation and to promote growth nonautonomously, along the anteriorposterior axis [14]. Hedgehog, like Notch, is a potent oncogene and when co-activated with Notch can trigger tumour growth (M.D. unpublished). Unexpectedly, the converse it is also true: Da Ros et al. [38] have found that dampening Hedgehog signalling also facilitates Delta-induced tumour-like overgrowth. In an unbiased genetic screen, the authors found that overexpression of the conserved microRNA mir-7 converted Delta-induced mild eye overgrowth into tumours. The oncogenic mechanism converged on the silencing of two, functionally redundant Hedgehog receptors: boi (repressed by Notch signalling) and ihog (directly silenced by the microRNA). Knockdown of hedgehog or its core components, smoothened and Ci/Gli, also provoked tumours when combined with Delta overexpression or even invasive tumours [38]. In clones of cells, overexpression of Delta and knockdown of smoothened or making the tissue defective to produce hh, significantly enhanced Delta-induced organizing capacity and nonautonomous organ overgrowth [38]. Given the conservation of miR-7, Notch and Hedgehog, these observations in Drosophila pave the way for similar studies of a tumour suppressor role of Hedgehog in some human tumours. 3.6. The turning off the organizer: epigenetic regulation? The growth and polarity of the eye epithelium that is brought about by the local Notch activity at the dorsal–ventral midpoint (‘organizer’) is turned off as retinal differentiation starts [14]. Tumours occurring upon inactivation of the Polycomb group (PcG) gene polyhomeotic (ph) are accompanied by autonomous activation of ‘organizer’ gene expression, including elevated expression of Serrate, and eyegone genes [39], which results in ectopic Notch activation (as revealed by a Notch reporter construct [40], and the concurrent activation of other growth-promoting genes including the cytokine upd [41]). Importantly, autonomous upd-Notch activation results in nonautonomous overgrowth [42] (Table 2). Thus, it is possible that loss of epigenetic regulation foments tumorigenesis by keeping eye precursor cells ‘locked’ in the ‘early growth programme’ of Notch. There are other epigenetic factors that when altered synergize with gain of Notch signalling to foment hyperplastic and neoplastic growth (Tables 1 and 2). In particular, the PcG component E(z) and its human counterpart E(Z)H2, encoding conserved components of the histone H3 lysine 27 methyltrasferase complex, previously

perceived as exclusively oncogenic, have been shown to act as potent tumour suppressors that facilitate Notch oncogenic programme in T-ALLs and also in flies [6] (Table 1). Reducing the gene dosage of the Drosophila histone H3K4 demethylase Lid, (called KDM5A in humans), a conserved core component of the CSL repressor complex [1,43] also enhances Delta-induced growth and facilitates metastasis in flies ([43], Table 1). An additional link between Notch signalling and epigenetic regulation is dUTX/UTX ([43]: Tables 1 and 2). Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and this residue can be demethylated by the JmjC domain dUtx/UTX. Loss of dUtx results in elevated Notch protein levels and in a tumour paradigm, reducing dUtx activity significantly enhanced tumour growth and metastasis by Notch [44] (Table 1). Further understanding of the molecular underpinning of the extensive crosstalk between epigenetic regulators and Notch-induced growth is therefore of prime importance to understand Notch signalling in tumorigenesis [45].

4. The Notch’s dangerous liaisons: tumour metastasis Notch signalling activation can also result in overt macroscopic metastasis in some flies harbouring the defined gene alterations (Fig. 3). The short life cycle and the short latency (a week) of the evolution of cancer phenotype in flies suggest that tumours that metastasize likely arise in a manner in which the initiating cooperative event drives primary tumour growth and also confers the abilities to form distant metastases. With the data so far, the heterogeneity of the nature of the genes that cooperate with Notch to trigger tumour metastasis may suggest that invasion-metastasis is driven by many diverse mechanisms (Table 1). The epithelial tumours that metastasize in flies display many of the properties of metastatic tumours in mammals [46–48], including cell invasion, migration through the basal membrane and colonization of distant sites. Candidate genes that trigger or facilitate tumour metastasis as the endocytic tumour suppressor and the PcG Ph discussed above can be assayed using allografts, whereby transformed tumour cells are injected into the abdomen of adult flies [50]. However, only tumour metastasis that develops in situ seems to mimic neoplastic initiation and the progression of invasive cancer cells, taking into account the influences of tumour–host interactions (e.g. [47]). A growing list of transcription regulators and signalling pathways has been shown to trigger in situ eye tumours that metastasize throughout the fly (e.g. Fig. 2) when overexpressed (Gal 4-driven overexpression) or inactivated (via endogenous mutations or expression of UAS-RNAi transgenes) in combination with the oncogenic N-ICD or overexpression of Delta (Table 1). These include cooperation between N-ICD and the loss of the epithelial polarity gene scribbled/SCRIB [47,49] and between Delta and two neighbouring epigenetic regulators Lola and Pipsqueak (eyeful [46]) resulting in highly metastatic tumours (Table 1). The cooperation between Delta and PI3K/Akt signalling [5], or Delta and the loss of ci/Gli [38], and the co-expression of N-ICD and the mesodermal factor Mef2/MEF2 [7] are additional cooperative events that result in tumours that metastasize albeit at lower incidence. In mammalian cells, epithelial tumour cells often acquire invasive properties via reprogramming through the EMT [51]. The hallmark of EMT in vertebrate systems is the repression of the cell–cell adhesion molecule E-cadherin, which relaxes the adherence of epithelial and tumour epithelial cells to neighbouring tumour cells or host cells, thereby facilitating tumour cell invasion and the penetration of these cells into extracellular matrices. This paradigm is partially challenged by findings showing that Twist-induced tumour cell dissemination requires E-cadherin [52].

Please cite this article in press as: Dominguez M. Oncogenic programmes and Notch activity: An ‘organized crime’? Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.012

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Loss of epithelial polarity is a common property of some neoplastic tumours in flies though is often been assumed in the absence of experimental evidence that tumour invasion in fly tumours is also associated to EMT-like processes though expression and function of EMT-determinant linked to human cancer cells like Snail, Slug (Snail2), Twist, Zeb1 and 2, and GATA factors, all of which are conserved in flies [51]. In flies, only functional cooperation of the EMT-determinant Zfh1, a ZEB1 homolog, has been reported thus far to be capable to trigger massive metastatic behaviour in flies [8], which likely reflects the existing regulatory interactions between ZEB1/Zfh1 and Notch. In human cells, ZEB1 is a target of the microRNA miR-200c, which as discussed above it is a microRNA that also directly silences the JAGGED1 ([8,16] and citations therein). The underexpression of the conserved miR-200 family in human cells is one important mechanism linking the acquisition of EMT reprogramming to stemness and to drug resistance ([51] and citations therein). Notch-induced cancers are also associated with features like drug resistance, EMT and stemness, as evident in pancreatic cancer, colorectal cancer, breast cancer, and ovarian carcinoma [3,4,45]. Importantly, ZEB1/Zfh1 is a transcriptional target of Notch signalling in metastatic cancer cells [8] and in Drosophila cells [13]. Thus, the aberrant Notch signalling that arises from the underexpression of mir-200c in cancer cells augments the transcriptional activation of ZEB1, the expression of which is also enhanced by the loss of this microRNA. In turn, ZEB1 also represses the expression of mir-200c gene [51] and hence, the initially small changes in JAGGED1 and ZEB1 are further amplified by these regulatory loops, which might result in full EMT and growth of metastatic cells [8]. In mammals, tumour cells in vivo more often disperse as groups of cells (collective cell migration) [53], which may explain that tumour invasion and metastasis in flies is commonly facilitated by the JNK signalling (e.g. [7,12,54–56]) and its target, the matrix metalloproteinase 1 (MMP1) [57] (Tables 1 and 2). However, the single activation of JNK is insufficient for epithelial cells to become tumorous or to disperse and, in some instances, the JNK-dependent apoptosis suppresses rather than enhances tumorigenesis [58,59]. The eyeful paradigm: Ref. [46] has been broadly used to identify genes that further facilitate or limit metastasis [58]. The terminal retinal cell differentiation factor Atonal (called Atoh1 in mammals) was identified as a potent tumour suppressor that when downregulated enhanced tumour growth and metastasis by Notch and eyeful sensitized background [58]. Loss of Atonal causes decreased phospho-JNK, thus inhibiting apoptosis. The authors showed persuasively that overexpressing dominant-negative JNK (bsk) partially mimics ato downregulation in the eyeful cancer model, implying that JNK limits tumour growth and metastatic behaviour [58]. Loss of JNK-dependent apoptosis also facilitates tumorigenesis in cells lacking the AAA ATPase Vps4 [59], a ‘molecular scissors’ that dissembles the ESCRT-III complex. Tumour growth and metastasis is also enhanced by loss of excessive apoptosis due to the homeobox transcription factor Cut [60]. Importantly, recent work has shown that inactivating mutations in the human homologue of Cut, CUX1, are pan-drivers of Notch-associated tumours [10] through activation of the survival PI3K/Akt pathway [10]. Notch and eyeful-induced metastases are also enhanced by knocking down the fly homologue of MENA (ENAH, Mammalian enabled homologue), a gene encoding an actin-regulatory protein that belongs to the ENA/VASP protein family implicated in cellular invasion [61]. Invasive tumours also occur upon overexpression of Vha44, a Drosophila orthologous of the V-ATPase C subunit that causes accumulation of Notch receptors in acidified endolysosomal/lysosomal and heightened enzymatic activity of the ␥-secretase [62]. These events are reminiscent to cell transformation associated with overexpression of mammalian V-ATPases in epithelial carcinomas, such

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as oral squamous cell carcinomas ([62] and citations therein). The ATP6AP2/VhaPRR accessory subunit of the proton pump V-ATPase provides another link between Notch activation, endolysosomal sorting, vesicle acidification and neoplastic phenotypes [63]. In summary, developmental and cancer studies converge to show that enhanced cooperation of existing Notch target pathways and regulators form the basis of Notch-mediated tumorigenic activity. As such, further understanding the dynamics behind the cooperation and the networks they establish may provide deeper insights into Notch-associated cancers. Many of molecular networks discussed also operate in human cancers [5–10], which endorses the use of carrying functional analyses and large-scale genetic screening in flies, which may prove to be authentic treasure troves for preselecting somatic mutations in patient’s tumours as ‘drivers’. Acknowledgements I apologize to all the colleagues whose studies could not be covered due to space limitations and thank members of the laboratory for comments. M.D. is funded by Spanish National Grants (BFU2009-09074, SAF2012-35181 and MEC-CONSOLIDER CSD2007-00023), Generalitat Valenciana Grant (PROMETEO II/2013/001) and Fundación Botin. References [1] Hori K, Sen A, Artavanis-Tsakonas S. Notch signaling at a glance. J Cell Sci 2013;126:2135–40. [2] Kopan R, Ilagan MXG. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2009;137:216–33. [3] Andersson ER, Sandberg R, Lendahl U. Notch signaling: simplicity in design, versatility in function. Development 2011;138:3593–612. [4] Miele L, Espinoza I, Pochampally, Watabe, Xing F. Notch signaling: targeting cancer stem cells and epithelial-to-mesenchymal transition. Ott 2013:1249. [5] Palomero T, Sulis ML, Cortina M, Real PJ, Barnes K, Ciofani M, et al. Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med 2007;13:1203–10. [6] Ntziachristos P, Tsirigos A, Van Vlierberghe P, Nedjic J, Trimarchi T, Flaherty MS, et al. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat Med 2012;18:298–301. [7] Pallavi SK, Ho DM, Hicks C, Miele L, Artavanis-Tsakonas S. Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila. EMBO J 2012;31:2895–907. [8] Vallejo DM, Caparros E, Dominguez M. Targeting Notch signalling by the conserved miR-8/200 microRNA family in development and cancer cells. EMBO J 2011;30:756–69. [9] Roti G, Carlton A, Ross KN, Markstein M, Pajcini K, Su AH, et al. Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer. Cancer Cell 2013;23:390–405. [10] Wong CC, Martincorena I, Rust AG, Rashid M, Alifrangis C, et al. Inactivating CUX1 mutations promotes tumorigenesis. Nat Genet 2014;46:33–8. [11] Irvine KD, Rauskolb C. Boundaries in development: formation and function. Annu Rev Cell Dev Biol 2001;17:189–214. [12] Vaccari T, Bilder D. At the crossroads of polarity, proliferation and apoptosis: the use of Drosophila to unravel the multifaceted role of endocytosis in tumor suppression. Mol Oncol 2009:1–12. [13] Djiane A, Krejˇcí A, Feder B, Fexova S, Millen K, Bray SJ. Dissecting the mechanisms of Notch induced hyperplasia. EMBO J 2012;32:60–71. [14] Roignant J-Y, Treisman JE. Pattern formation in the Drosophila eye disc. Int J Dev Biol 2009;53:795–804. [15] Pecot CV, Rupalmoole R, Yang D, Akbani R, Ivan C, et al. Tumour angiogenesis regulation by the miR200 family. Nat Commun 2013;4:2427. [16] Brabletz S, Bajdak K, Meidhof S, Burk U, Niedermann G, Firat E, et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells. EMBO J 2011:1–13. [17] Thomas, David Strutt, Rabaptin-5. Rabex-5 are neoplastic tumour suppressor genes that interact to modulate Rab5 dynamics in Drosophila melanogaster. Dev Biol 2014;385(January (1)):107–21. [18] Klein T. The tumour suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development. Dev Biol 2003;255:313–33. [19] Childress JL, Acar M, Tao C, Halder G. Lethal giant discs, a novel C2-domain protein, restricts Notch activation during endocytosis. Curr Biol 2006;16:2228–33. [20] Jaekel R, Klein T. The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking. Dev Cell 2006;11:655–69.

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Please cite this article in press as: Dominguez M. Oncogenic programmes and Notch activity: An ‘organized crime’? Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.012