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The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Abstract

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth1, we identified grindelwald (grnd), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. Here we show that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib−/− tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth.

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Figure 1: Grnd is a novel member of the TNFR superfamily.
Figure 2: Grnd is required for Egr-induced apoptosis.
Figure 3: Grnd is required for the Egr-induced invasiveness of RasV12/scrib−/− tumours.
Figure 4: grnd is required downstream of the apical Crb polarity complex for JNK-dependent neoplastic growth.

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Acknowledgements

We thank G. Jarretou for technical assistance, the Vienna Drosophila RNAi Centers, the Drosophila Genetics Resource Center, the Bloomington Stock Center, N. Caridi and S. Pasqualato for technical assistance and P.L. laboratory members for comments on the manuscript. This work was supported by the CNRS, INSERM, Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, Association pour la Recherche contre le Cancer (grant no. PJA20131200042 to J.C.), European Research Council (Advanced grant no. 268813 to P.L.), Marie Curie Life Long Training (grant no. 252373 to D.S.A.), the Labex Signalife program (grant ANR-11-LABX-0028-01 to P.L.), the Italian Association for Cancer Research (AIRC IG-12877) and the Italian Ministry of Health (GR-2008-1134103) to M.M.

Author information

Authors and Affiliations

Authors

Contributions

D.S.A., J.C., K.C., A.-O.H., K.B., M.M. and P.L. designed the research; D.S.A., J.C., K.C., V.P., E.B., M.R. and J.T. performed experiments; D.S.A., J.C., K.C., E.B., A.-O.H., K.B., V.P., M.M. and P.L. analysed the data; D.S.A., J.C. and P.L. wrote the manuscript.

Corresponding authors

Correspondence to Julien Colombani or Pierre Léopold.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Identification of Grnd in a genetic screen for molecules implicated in neoplastic growth.

a, Schematic of the genome-wide screen. Ten-thousand one-hundred RNAi lines were screened for their abilities to rescue the delay of the rn>avl-RNAi condition. Of the 121 candidates able to rescue the delay of the rn>avl condition, only 8 also rescued the neoplastic growth. b, Schematic of the five components of the JNK pathway shown to be required for the neoplastic overgrowth in the avl loss of function condition. Although no RNAi line targeting Traf2 was included in the original screen, Traf2 was subsequently shown to be required for neoplastic overgrowth in the avl-RNAi condition using an alternative RNAi line. c–e, Wing discs of the indicated genotypes dissected 5 days (left) or 6 days (right) AED and stained for Wg (green) and Caspase3 (red). f, g, Transverse (f) or xy (g) sections of dissected wing discs expressing grnd RNAi in the patch domain (GFP, green) stained for Grnd (red) and DAPI (g, right, blue) to visualize the nuclei. h, Subcellular fractionation of cleared extract from dissected wing discs shows that Grnd, like the transmembrane receptor Smoothened, localizes to the membrane fraction (PM), whereas Disc-large remains in the cytoplasmic fraction (Cyto). Laminin serves as a control for nuclear fractions.

Extended Data Figure 2 Sequence homology of the extracellular domain of Grnd to CRDs of human TNFRs.

a, Per cent identity matrix and phylogenic data from multiple local sequence alignment of the entire extracellular domains of Grnd and Wgn with extracellular domains (corresponding to a part of CRD2 and all of CRD3) of various human TNFR superfamily members. The extracellular domain of Grnd shares the highest identity with the GBM-harbouring CRD of Fas (26%). A similar result is observed for Wgn, although it exhibits lower identity (22%) and a higher degree of divergence to Fas. b, Sequence alignment of Fas, Grnd and Wgn demonstrates that Grnd shares a higher degree of similarity with the GBM-harbouring CRD of Fas than Wgn. This is particularly apparent in the immediate region encompassing the functionally critical F134 (marked with an asterisk) of the GBM of Fas. Blue indicates residue identity, yellow strong similarity, and grey weak similarity of Fas with Grnd and/or Wgn. c, Schematic representation of Grnd, Wgn, and various members of the human TNFR superfamily showing a high level of diversity in the number and arrangement of CRDs. The colour codes used are: red for signal peptide, green for extracellular domains, orange for transmembrane domains, and blue for intracellular domain. Numbers in each box indicate corresponding amino acid positions. Pro, prodomain; T1–5, TAPE1–5 (threonine-, alanine-, proline- and glutamine-rich repeats). d, Multiple sequence alignment showing the conservation of the extracellular domain of Grnd in other insect species. Whereas global similarity of the extracellular domain of Grnd degenerates outside the melanogaster group, the cysteine residues of the CRD (highlighted in red) and the aromatic residue (F/Y, marked with an asterisk) corresponding to F134 of Fas CRD3 remain highly conserved throughout the Hymenoptera order. Blue indicates residue identity; yellow, strong similarity; and grey, weak similarity to Grnd.

Extended Data Figure 3 Identification of a conserved TRAF6-like binding motif in the intracellular domain of Grnd proteins.

Multiple sequence alignment of the intracellular domain of Grnd and Grnd-like proteins in other species. A binding motif for TRAF6 (PxExx(aromatic/acidic residue)), the closest homologue of Drosophila Traf2, was previously identified in human TNFRs4. The position of a related putative Traf2-binding motif in the intracellular domain of Grnd is indicated. Note that, although sequence similarity of the intracellular domain of Grnd degenerates outside the family Drosophilidae, the core residues of this motif (highlighted in red) remain highly conserved throughout the order Hymenoptera. Blue indicates residue identity; yellow, strong similarity to Grnd.

Extended Data Figure 4 Grnd acts upstream of JNK signalling.

a–d, Wild-type wing discs (a) or discs expressing grnd (b) or grnd-intra (c, d) in the rn domain were dissected 5 days AED and stained for Wg (left; green), Cas3 (middle; red), and Grnd (right; grey). d, e, Wing discs expressing grnd-intra in the sal domain (marked by a truncated line) in a wild-type (d) or hep75 mutant (e) background were dissected 5 days AED and stained for Wg (left; green), Cas3 (middle; red), and Grnd (right; grey).

Extended Data Figure 5 Characterization of the wgnKO mutant allele.

a–e, Light micrographs of Drosophila adult eyes are shown. Original magnification, ×30. The small-eye phenotype caused by GMR-driven expression of egr (a) is rescued by co-expressing puc (c) or reducing the activity of Traf2 (b), but not by reducing Wgn activity (d; also serves as a control to exclude a Gal4 titration effect). f, g, Reducing Grnd activity partially rescues the small-eye phenotype, giving rise to the hanging-eye phenotype (f) and is not further rescued in a wgnKO mutant background (g). h, wgnKO mutant flies do not display any eye phenotype. i, j, Reducing Grnd levels or expressing Grnd-extra in the GMR domain does not affect eye morphology. k, GMR-Gal4-induced expression of a soluble form of Egr lacking the transmembrane and cytoplasmic domain (Ecto-Egr-60) gives rise to a small-eye phenotype10. l, Co-expression of puc2A in a GMR>ecto-egr-60 background partially rescues the small-eye phenotype, giving rise to a hanging-eye phenotype10. m, Sequence analysis of DNA extracted from flies homozygous for the wgnKO mutation showing the two frameshift mutations introduced by cutting the unique intrinsic restriction sites AclI (exon 1) and NotI (exon 2) followed by T4 DNA polymerase-mediated fill-in. n, Western blot of fly extracts from the indicated genotypes probed with an anti-Wgn antibody27. KO, knockout; OE, overexpressing; WT, wild type.

Extended Data Figure 6 Characterization of the grndMinos mutant.

a, Schematic representation of the Minos transposon insertion in the grnd gene region corresponding to the 5′ untranslated region of the transcript. b, Western blot of wing disc extracts from the indicated genotypes probed with anti-Grnd (top) and anti-β-tubulin (bottom) antibodies. Grnd protein migrates at the predicted size of 27 kDa (lane 1, control). Grnd protein levels are decreased below detection level in both grndMinos/Minos and grndMinos/Df mutant flies, indicating that grndMinos/Minos is indistinguishable from a null mutant. c, d, Polarity is not affected in grndMinos/Minos and grndMinos/Df wing discs. Transverse sections of dissected control (c) or grndMinos/Minos (d) wing discs stained for Grnd (top; red), Crb (middle top; blue) and Dlg (middle bottom; green). e–j, Wing discs dissected from third instar larvae after 40 h of Egr expression in the rn domain. Pouch ablation, ubiquitous Wg expression and apoptosis observed in rn>egr, Tub-Gal80ts discs (e) are suppressed to various extents upon co-expression of grnd-RNAi (f) or reduction of JNK activity (g, h) and fully suppressed in grndMinos/Minos (i) and grndMinos/Df (j) mutant backgrounds.

Extended Data Figure 7 Grnd is highly expressed in RasV12/scrib−/− micro-metastases.

a–c, Reducing grnd levels in scrib−/− mutant clones prevent their elimination. Eye discs dissected 5 days AED carrying GFP-labelled MARCM clones of the indicated genotypes stained for Grnd (middle; red) and DAPI (right; grey). d, Reducing Grnd levels does not affect cell viability. Eye discs dissected 5 days AED carrying GFP-labelled grnd RNAi Flip-out clones stained for Grnd (middle; red) and DAPI (right; grey). e–g, Invasive brain tumours and micro-metastases display high levels of Grnd protein. Dissected eye–brain complex (e), gut (f) and fat body (g) displaying GFP-labelled invasive RasV12/scrib−/− clones (e; green) or micro-metastases (f, g; green) stained for Grnd (red) and DAPI (blue).

Extended Data Figure 8 Grnd, but not Wgn, is required for Mmp1 expression both in RasV12/scrib1 and RasV12/Dlg RNAi tumours.

a–c, Eye–brain complexes dissected 7 days AED carrying RasV12/scrib−/− (a), RasV12/scrib−/− + grnd-RNAi (b), or RasV12/scrib−/− + dilp8-RNAi (c; control RNAi to exclude a Gal4 titration effect) clones labelled by GFP and stained for Mmp1 (middle; red). RasV12/scrib−/− and RasV12/scrib−/− + dilp8-RNAi clones invade the ventral nerve cord (a, c; see white arrow). d–g, Eye–brain complexes dissected 7 days AED carrying RasV12/Dlg-RNAi (d) or RasV12/Dlg-RNAi + grnd-RNAi (e, f) clones (labelled by GFP), or RasV12/Dlg-RNAi GFP-labelled clones in a wgn-null mutant background (g) stained for Mmp1 (middle; red) and DAPI (right; blue). RasV12/Dlg RNAi clones invade the ventral nerve cord in wgn-null mutant animals (g; see white arrow).

Extended Data Figure 9 Egr is not required for the rn>avl RNAi-induced neoplastic growth.

a, b, Wing discs of the indicated genotypes dissected 6 days AED and stained for Wg (green). c, d, Transverse sections of dissected wild-type discs stained for Grnd (red) and either E-cad (c; in green), or aPKC (d; in green). e, g, Transverse sections of rn>avl RNAi wing discs dissected 5 days AED and stained for Grnd (top, bottom; red) and either E-cad (e, middle, bottom; green) or Crb (g, middle, bottom; green). The rn expression domain is indicated (e, top). f, Wing discs expressing crb-intra in the rn domain (rn>crb-intra) dissected 6 days AED and stained for Disc-large (Dlg, green) to visualize neoplastic structures and Grnd (red). h, i, Wing discs expressing crb-intra in the rn domain of wild-type (h) or wgn-knockout mutant (i) animals dissected 6 days AED and stained for Wg (green). j–l, Reducing grnd levels does not affect crb-intra-mediated inhibition of Hippo signalling, which causes an upregulation of the Yki transcriptional target Diap1. Control discs (j) or wing discs expressing Crb-intra alone (k) or together with grnd RNAi (l) in the rn domain were dissected from animals carrying a reporter gene for Hippo pathway activity (Diap-GFP, green) and stained for Wg (red).

Extended Data Figure 10 Grnd directly interacts with the PDZ domain of Veli and neither depends on Veli nor Crb for its proper localization.

a, b, Schematic representation of Grnd and Veli (also known as Lin-7) domains and the various truncations used to map the interface of the Grnd–Veli interaction. L27, Lin-2/Lin-7 domain; PDZ, PSD-95/Dlg/ZO-1 domain; TM, transmembrane domain. c, GST–Grnd125–241 immobilized on GSH beads was incubated with full-length Veli (VeliFL) or the complementary Veli fragments L27 (VeliL27) and PDZ domain (VeliPDZ). The PDZ1 domain of the polarity protein Bazooka was included in the pull-down as a specificity control (BazPDZ1). Coomassie-blue stained SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was used to visualize species retained on beads. VeliFL and VeliPDZ bind to Grnd (lanes 10 and 12), but not VeliL27 nor BazPDZ1 (lanes 11 and 9), indicating that Grnd specifically recognizes the PDZ domain of Veli. d, To map the interface between Grnd and VeliPDZ, the same binding assay was performed using a battery of Grnd deletion mutants adsorbed on GSH beads. VeliPDZ forms a complex with Grnd125–241 and Grnd125–168 (lanes 1 and 3), but not with the carboxy-terminal region of Grnd (lanes 2 and 4) or GST alone (lane 5, control). e, To address further the specificity of the binding of Grnd125–241 to the PDZ domain of Veli, we repeated the pull-down assay with the PDZ domains of Baz and its human orthologue PAR3. VeliPDZ is retained on GST–Grnd125–241 beads (lane 9), while PAR3PDZ2, PAR3PDZ3 and BazPDZ1 are not (lanes 10, 11 and 12). f–g, Reducing Crb levels affects the localization of Veli, but not Grnd. f–g, xy (f) or transverse (g) sections of dissected wing discs bearing crb−/− mutant clones (labelled by absence of GFP) stained for Grnd (f, bottom left, g, bottom; white) or Veli (f, bottom right; red). h, Reducing Veli levels does not affect Crb20 or Grnd localization. Transverse sections of dissected wing discs bearing clones expressing veli RNAi (h, bottom; labelled by the presence of GFP) stained for Grnd (top; red) and Crb (middle; blue).

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Andersen, D., Colombani, J., Palmerini, V. et al. The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature 522, 482–486 (2015). https://doi.org/10.1038/nature14298

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