Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth

The lethal giant larvae tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy

<p>Abstract</p> <p>Background</p> <p>Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells <it>in vivo</it>, we took advantage of the clonal analysis methods that are available in <it>Drosophila </it>for studying the phenotypes due to <it>lethal giant larvae </it>(<it>lgl</it>) neoplastic mutation induced in different backgrounds and tissues.</p> <p>Results</p> <p>We observed that <it>lgl </it>mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of <it>lgl </it>mutant cells elimination was obtained by increasing dMyc levels in <it>lgl </it>mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when <it>lgl </it>mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in <it>lgl</it>-induced tumourigenesis. Furthermore, we show that the <it>eiger</it>-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating <it>lgl </it>mutant cells in the wing pouch; <it>lgl</it>^-/-clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.</p> <p>Conclusions</p> <p>Our results provide the first evidence that dMyc oncoprotein is required in <it>lgl </it>tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside <it>versus </it>outside the mutant clones plays a key role in driving neoplastic overgrowth.</p

dMyc Functions Downstream of Yorkie to Promote the Supercompetitive Behavior of Hippo Pathway Mutant Cells

Genetic analyses in Drosophila epithelia have suggested that the phenomenon of “cell competition” could participate in organ homeostasis. It has been speculated that competition between different cell populations within a growing organ might play a role as either tumor promoter or tumor suppressor, depending on the cellular context. The evolutionarily conserved Hippo (Hpo) signaling pathway regulates organ size and prevents hyperplastic disease from flies to humans by restricting the activity of the transcriptional cofactor Yorkie (yki). Recent data indicate also that mutations in several Hpo pathway members provide cells with a competitive advantage by unknown mechanisms. Here we provide insight into the mechanism by which the Hpo pathway is linked to cell competition, by identifying dMyc as a target gene of the Hpo pathway, transcriptionally upregulated by the activity of Yki with different binding partners. We show that the cell-autonomous upregulation of dMyc is required for the supercompetitive behavior of Yki-expressing cells and Hpo pathway mutant cells, whereas the relative levels of dMyc between Hpo pathway mutant cells and wild-type neighboring cells are critical for determining whether cell competition promotes a tumor-suppressing or tumor-inducing behavior. All together, these data provide a paradigmatic example of cooperation between tumor suppressor genes and oncogenes in tumorigenesis and suggest a dual role for cell competition during tumor progression depending on the output of the genetic interactions occurring between confronted cells

dMyc Functions Downstream of Yorkie to Promote the Supercompetitive Behavior of Hippo Pathway Mutant Cells

Genetic analyses in Drosophila epithelia have suggested that the phenomenon of “cell competition” could participate in organ homeostasis. It has been speculated that competition between different cell populations within a growing organ might play a role as either tumor promoter or tumor suppressor, depending on the cellular context. The evolutionarily conserved Hippo (Hpo) signaling pathway regulates organ size and prevents hyperplastic disease from flies to humans by restricting the activity of the transcriptional cofactor Yorkie (yki). Recent data indicate also that mutations in several Hpo pathway members provide cells with a competitive advantage by unknown mechanisms. Here we provide insight into the mechanism by which the Hpo pathway is linked to cell competition, by identifying dMyc as a target gene of the Hpo pathway, transcriptionally upregulated by the activity of Yki with different binding partners. We show that the cell-autonomous upregulation of dMyc is required for the supercompetitive behavior of Yki-expressing cells and Hpo pathway mutant cells, whereas the relative levels of dMyc between Hpo pathway mutant cells and wild-type neighboring cells are critical for determining whether cell competition promotes a tumor-suppressing or tumor-inducing behavior. All together, these data provide a paradigmatic example of cooperation between tumor suppressor genes and oncogenes in tumorigenesis and suggest a dual role for cell competition during tumor progression depending on the output of the genetic interactions occurring between confronted cells

La cooperazione oncogenica tra lgl- e RasV12 richiede la regolazione trascrizionale e post-trascrizionale di Myc

lethal giant larvae (lgl) \ue8 un gene soppressore di tumore la cui perdita di funzione causa nei tessuti epiteliali compromissione della polarit\ue0 apico-basale, iperproliferazione e perdita delle capacit\ue0 differenziative. Abbiamo dimostrato che l\u2019espansione clonale di cellule mutanti per lgl nel disco imaginale dell\u2019ala richiede l\u2019espressione autonoma di Myc; in contesto selvatico i cloni mutanti mostrano bassi livelli di questa proteina e vengono eliminati per \u2018Competizione Cellulare\u2019, mentre in contesti non competitivi le cellule lgl- sovraesprimono Myc e acquisiscono caratteristiche maligne quali crescita in 3D, perdita della polarit\ue0 apico-basale e capacit\ue0 di degradare la membrana basale. Analoghi tratti maligni sono associati al ben noto modello di cooperazione oncogenica tra la perdita di funzione di lgl e la forma attivata del proto-oncogene Ras, RasV12. I nostri dati indicano che anche in questo contesto la crescita tumorale dipende dall\u2019abilit\ue0 delle cellule mutanti di esprimere alti livelli di Myc. L\u2019attivazione trascrizionale di Myc nelle cellule lgl-; RasV12 dipende dalla deregolazione dell\u2019Hippo pathway, una via di segnalazione molto conservata coinvolta nel controllo della proliferazione dei tessuti epiteliali; affinch\ue9 Myc possa accumularsi e promuovere l\u2019espansione tumorale dei cloni mutanti \ue8 tuttavia necessaria anche la sua stabilizzazione, esercitata dalla via di segnalazione di Ras

The malignant growth of lgl- RasV12 tumours depends on Myc function

Simultaneous loss of function of the tumour suppressor gene lethal giant larvae (lgl) and constitutive activation of Ras signalling (RasV12) represent a powerful model of oncogenic cooperation. In the wing disc we have shown that lgl mutant clones are eliminated by cell competition, while lgl-/- RasV12 cells are able to grow and form tumourous masses in which Yorkie, the transcriptional co-activator downstream of the Hippo pathway, is activated. As previously shown, lgl mutant cells require Myc expression to display malignant behaviour and, since Myc is a Yki transcriptional target and Myc protein is stabilised by the MAPK downstream of Ras signalling, we have investigated Myc contribution to lgl-/- RasV12 -induced tumourous traits. We found that Myc activity is necessary for the growth of lgl-/- RasV12 cells and it is sufficient to drive proliferation and invasive behaviour even in the absence of endogenous Ras signalling. Furthermore, our data suggest that both transcriptional and post-transcriptional regulation of Myc are necessary to induce malignant growth in the wing pouch region. Preliminary data will also be presented about a role of hypoxia in promoting changes in FGF signalling and consequent interactions between lgl-/- RasV12 tumours and the tracheal network

Connecting epithelial polarity, proliferation and cancer in Drosophila: the many faces of lgl loss of function

Loss of cell polarity is a prominent feature of epithelial cancers. Several tumour-suppressor genes are indeed involved in establishing and maintaining a correct apical-basal polarity suggesting that a link exists between disruption of epithelial polarity and the control of cell proliferation. Nevertheless, the molecular basis of this link is only beginning to be unveiled. In Drosophila, the tumour suppressor gene lethal giant larvae (lgl) is widely used as a genetic tool in cancer modelling: its loss of function causes neoplastic growth of the imaginal tissues, larval epithelial organs from which adult structures originate. These mutant epithelia are characterised by loss of cell polarity and tissue architecture as well as hyperproliferation. We observed that in a clonal context, the ability of lgl mutant cells to express their neoplastic potential correlates with the levels of the oncoprotein Myc, a master regulator of cell growth and proliferation. Malignant, polarity-deficient mutant cells upregulate Myc and are able to overcome the tumour-suppressive defences imposed by the surrounding wild-type tissue. How does the loss of lgl function induce an increase in Myc levels? The answer to this question came from the finding that Lgl is an upstream regulator of the Hippo pathway, a highly conserved signalling network that controls proliferation of epithelial cells and organ size. The core of this pathway responds to several upstream regulators and converges on the inhibition of a transcriptional co-factor, Yorkie, which, as we and others have shown, is a direct regulator of the myc promoter. In this review we discuss the key findings that contributed to the identification of this regulatory network that links cell polarity to cell proliferation control

Neural stem cell derived tumourigenesis

In the developing Drosophila CNS, two pools of neural stem cells, the symmetrically dividing progenitors in the neuroepithelium (NE) and the asymmetrically dividing neuroblasts (NBs) generate the majority of the neurons that make up the adult central nervous system (CNS). The generation of a correct sized brain depends on maintaining the fine balance between neural stem cell self-renewal and differentiation, which are regulated by cell-intrinsic and cell-extrinsic cues. In this review, we will discuss our current understanding of how self-renewal and differentiation are regulated in the two neural stem cell pools, and the consequences of the deregulation of these processes

Drosophila Lethal Giant Larvae Neoplastic Mutant as a Genetic Tool for Cancer Modelling

Drosophila lethal giant larvae (lgl) is a tumour suppressor gene whose function in establishing apical-basal cell polarity as well as in exerting proliferation control in epithelial tissues is conserved between flies and mammals. Individuals bearing lgl null mutations show a gradual loss of tissue architecture and an extended larval life in which cell proliferation never ceases and no differentiation occurs, resulting in prepupal lethality. When tissues from those individuals are transplanted into adult normal recipients, a subset of cells, possibly the cancer stem cells, are able to proliferate and migrate to distant sites forming metastases which eventually kill the host. This phenotype closely resembles that of mammalian epithelial cancers, in which loss of cell polarity is one of the hallmarks of a malignant, metastatic behaviour associated with poor prognosis. Lgl protein shares with its human counterpart Human giant larvae-1 (Hugl-1) significant stretches of sequence similarity that we demonstrated to translate into a complete functional conservation, pointing out a role in cell proliferation control and tumourigenesis also for the human homologue. The functional conservation and the power of fly genetics, that allows the researcher to manipulate the fly genome at a level of precision that exceeds that of any other multicellular genetic system, make this Drosophila mutant a very suitable model in which to investigate the mechanisms underlying epithelial tumour formation, progression and metastatisation. In this review, we will summarise the results obtained in the last years using this model for the study of cancer biology. Moreover, we will discuss how recent advances in developmental genetics techniques have succeeded in enhancing the similarities between fly and human tumourigenesis, giving Drosophila a pivotal role in the study of such a complex genetic disease

Cancer cells contribute to distinct malignant traits depending on their MYC and pJNK relative levels

Clonal analysis is common practice in Drosophila. In particular, induction of cell clusters carrying either loss-of-function alleles of neoplastic tumour suppressors or activated forms of oncogenes has successfully been exploited to study cooperative tumourigenesis in different organs. This strategy has allowed collecting a number of morphological and molecular details on the phenotypic traits associated with cancer progression. We previously identified MYC as a target of the Hippo pathway in Drosophila, and showed its expression is sufficient as to rescue the growth deficit of cells mutant for polarity genes, releasing their malignant nature. Here we expand on previous work, showing that cell growth and cell migration are separable traits in Drosophila epithelial cancers. While in situ cancer expansion is supported by MYC, migration depends on the AP-1 protein Fos. These proteins are strategically found at the crossroads of the Hippo, JNK and Ras/MAPK pathways, acknowledged by current literature as central players in cancer progression. Moreover, we show that growth and migration are mutually exclusive behaviours, with cells displaying different MYC and pJNK levels playing distinct roles in cancer evolution