Paola Bellosta

Drosophila myc regulates organ size by inducing cell competition.

Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and led to the idea that competition is a mechanism of regulating organ and tissue size. We have assessed competitive interactions between cells in a developing organ and examined their effect on its final size. We show that local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc protooncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. We demonstrate that cell competition is executed via induction of the proapoptotic gene hid and that both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, we provide evidence that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size.

Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor

Axl is a tyrosine kinase receptor and although it is expressed in malignancy such as leukemia, colon cancer, melanoma, endometrial, prostate and thyroid cancers, its role has not been completely elucidated yet and appears to be complex. The ligand of Axl, Gas6, is a 75 KDa multimodular protein with an N‐terminal gamma‐carboxy‐glutamic acid that is essential for binding. Gas6 has a mitogenic effect on several normal cell lines. The receptor Axl is expressed in primary prostate carcinoma and in prostate cancer cell lines as such as PC‐3 and DU 145. We demonstrated a mitogenic activity determined by Gas6/Axl interaction in these undifferentiated metastatic human prostatic cancer cell lines. This effect is proportional to Axl expression, not due to inhibition of apoptosis, and induces AKT and MAPK phosphorylation. However, only MEK phosphorylation seems to be essential for growth signaling. Our results suggest that Axl overexpression and activation by Gas6 could be involved in progression of prostate neoplastic disease.

Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation

ARK (AXL) is the prototype of a distinctive family of receptor tyrosine kinases which contain in their extracellular domains features reminiscent of cell adhesion molecules. ARK is capable of homophilic binding, which results in a degree of receptor activation, but can also be activated by a heterophilic ligand, Gas6, a member of the family of vitamin K dependent proteins that is preferentially expressed in quiescent cells. Since a number of tissues and cell lines express both ARK and Gas6, we studied the effect of endogenous and exogenous Gas6 on the phenotype of ARK expressing cells. Here we show that constitutive expression of Gas6 in an NIH3T3 cell line that does not spontaneously express this protein does not result in cell transformation or uncontrolled growth, but protects from apoptosis induced by serum deprivation. Recombinant exogenous Gas6 was also capable of protecting cells from apoptosis at concentrations that did not result in significant induction of DNA synthesis. Activation of ARK phosphorylation and a weak but significant induction of MAP kinase activity accompanied the increased survival of cells treated with Gas6. The antiapoptotic effect of ARK signaling was confirmed by studies using fibroblasts from ARK knock-out mice, that showed that the absence of ARK resulted in higher levels of serum deprivation-induced apoptosis, that could not be rescued by the addition of Gas6. Interestingly ARK signaling protects from apoptosis induced by serum deprivation, myc overexpression, or by TNFα but not from u.v. irradiation or Staurosporine. These results suggest that a major function of Gas6 – ARK signaling is that of increasing cell survival under conditions which do not allow cell proliferation.

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.

A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a ‘promiscuous liaison’ between both receptors that enables interleukin (IL)‐15 to transactivate the signaling pathway of a tyrosine kinase. IL‐15 protects murine L929 fibroblasts from tumor necrosis factor α (TNFα)‐induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl‐overexpressing fibroblasts are TNFα‐resistant. IL‐15Rα and Axl colocalize on the cell membrane and co‐immunoprecipitate even in the absence of IL‐15, whereby the extracellular part of Axl proved to be essential for Axl/IL‐15Rα interaction. Most strikingly, IL‐15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL‐15Rα, and activation of the phosphatidylinositol 3‐kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild‐type but not Axl−/− or IL‐15Rα−/− mice. Thus, IL‐15‐induced protection from TNFα‐mediated cell death involves a hitherto unknown IL‐15 receptor complex, consisting of IL‐15Rα and Axl RTK, and requires their reciprocal activation initiated by ligand‐induced IL‐15Rα.

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.

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis.

ABSTRACT Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-Å resolution. FGF4 adopts a β-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the βC′-βE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.

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.

Whole-genome analysis reveals a strong positional bias of conserved dMyc-dependent E-boxes.

ABSTRACT Myc is a transcription factor with diverse biological effects ranging from the control of cellular proliferation and growth to the induction of apoptosis. Here we present a comprehensive analysis of the transcriptional targets of the sole Myc ortholog in Drosophila melanogaster, dMyc. We show that the genes that are down-regulated in response to dmyc inhibition are largely identical to those that are up-regulated after dMyc overexpression and that many of them play a role in growth control. The promoter regions of these targets are characterized by the presence of the E-box sequence CACGTG, a known dMyc binding site. Surprisingly, a large subgroup of (functionally related) dMyc targets contains a single E-box located within the first 100 nucleotides after the transcription start site. The relevance of this E-box and its position was confirmed by a mutational analysis of a selected dMyc target and by the observation of its evolutionary conservation in a different Drosophila species, Drosophila pseudoobscura. These observations raise the possibility that a subset of Myc targets share a distinct regulatory mechanism.

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.