Daniela Grifoni

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.

The human protein Hugl-1 substitutes for Drosophila lethal giant larvae tumour suppressor function in vivo.

Drosophila lethal giant larvae (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and tumorigenesis in humans. Here we investigate the functional conservation between Drosophila lethal giant larvae and its human homologue Hugl-1(Llgl1). We first show that Hugl-1 is lost in human solid malignancies, supporting its role as a tumour suppressor in humans. Hugl-1 expression in homozygous lgl Drosophila mutants is able to rescue larval lethality; imaginal tissues do not show any neoplastic features, with Dlg and Scrib exhibiting the correct localization; animals undergo a complete metamorphosis and hatch as viable adults. These data demonstrate that Hugl-1 can act as a tumour suppressor in Drosophila and thus is the functional homologue of lgl. Furthermore, our data suggest that the genetic pathway including the tumour suppressors lgl, dlg and scrib may be conserved in mammals, since human scrib and mammalian dlg can also rescue their respective Drosophila mutations. Our results highlight the usefulness of fruit fly as a model system for investigating in vivo the mechanisms linking loss of cell polarity and cell proliferation control in human cancers.

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

BackgroundNeoplastic 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 in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.ResultsWe observed that lgl 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 lgl mutant cells elimination was obtained by increasing dMyc levels in lgl 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 lgl 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 lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.ConclusionsOur results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.

Aerobic glycolysis tunes YAP/TAZ transcriptional activity

Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro‐tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth‐promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro‐tumorigenic activities such as YAP/TAZ.

aPKCζ cortical loading is associated with Lgl cytoplasmic release and tumor growth in Drosophila and human epithelia

Atypical protein kinase C (aPKC) and Lethal giant larvae (Lgl) regulate apical–basal polarity in Drosophila and mammalian epithelia. At the apical domain, aPKC phosphorylates and displaces Lgl that, in turn, maintains aPKC inactive at the basolateral region. The mutual exclusion of these two proteins seems to be crucial for the correct epithelial structure and function. Here we show that a cortical aPKC loading induces Lgl cytoplasmic release and massive overgrowth in Drosophila imaginal epithelia, whereas a cytoplasmic expression does not alter proliferation and epithelial overall structure. As two aPKC isoforms (ι and ζ) exist in humans and we previously showed that Drosophila Lgl is the functional homologue of the Human giant larvae-1 (Hugl-1) protein, we argued if the same mechanism of mutual exclusion could be impaired in human epithelial disorders and investigated aPKCι, aPKCζ and Hugl-1 localization in cancers deriving from ovarian surface epithelium. Both in mucinous and serous histotypes, aPKCζ showed an apical-to-cortical redistribution and Hugl-1 showed a membrane-to-cytoplasm release, perfectly recapitulating the Drosophila model. Although several recent works support a causative role for aPKCι overexpression in human carcinomas, our results suggest a key role for aPKCζ in apical–basal polarity loosening, a mechanism that seems to be driven by changes in protein localization rather than in protein abundance.

Comparative analysis of AFLPs and SSRs efficiency in resolving population genetic structure of Mediterranean Solea vulgaris

The performance of different molecular markers in the assessment of population structure was tested using samples of Solea vulgaris collected in the Mediterranean within and outside the hypothetical dispersal ability of the species. A total of 172 individuals belonging to four population samples were analysed using 15 microsatellites [simple sequence repeats (SSRs)] and 153 amplified fragment length polymorphisms (AFLPs). Considering the global qualitative patterns, we found a correlation between SSRs and AFLPs in detecting genetic differentiation among samples. However, on a small geographical scale, AFLPs were able to discriminate individuals from neighbouring populations whereas SSRs were not, and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with SSRs. The high number of loci analysed with the AFLP technique could increase the probability to include outlier loci in the analysis; however, the neutrality test performed on our data set did not show evidence of selection acting on the S. vulgaris samples. Even if the choice of the molecular marker depends mainly on the biological question to be addressed, the higher power of discrimination and the comparative technical ease of obtaining data from AFLPs with respect to SSRs suggest the use of AFLPs for many population genetics studies.

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

BackgroundGenetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways.ResultsOur biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia.ConclusionsOur study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

The tumor suppressor gene fat modulates the EGFR-mediated proliferation control in the imaginal tissues of Drosophila melanogaster

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Here we provide evidence that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following to EGFR oversignalling. We further show that such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, we suggest a model in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore its function is requested downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor.

Identification of Domains Responsible for Ubiquitin-Dependent Degradation of dMyc by Glycogen Synthase Kinase 3β and Casein Kinase 1 Kinases

ABSTRACT In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3β (GSK3β). Using Drosophila S2 cells, we demonstrate that CK1α promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3β in vertebrates. Mutation of ck1α or -ε or sgg/gsk3β in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3β, and members of the CK1 family. We demonstrate that mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1α and GSK3β kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation.

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

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 units, are again able to proliferate and give rise to metastases which migrate to distant sites killing 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 tumorigenesis 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 these later 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 tumorigenesis, giving Drosophila a pivotal role in the study of such a complex genetic disease.