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Niche appropriation by Drosophila intestinal stem cell tumours

Nature Cell Biology volume 17, pages 1182–1192 (2015)Cite this article

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Abstract

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. We investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch (N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.

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Figure 1: Tissue stress promotes ISC tumour initiation.
Figure 2: Spi/EGFR/MAPK signalling is induced in ISC tumours and the niche.
Figure 3: ISC tumour initiation and outgrowth requires autonomous Spi/EGFR signalling.
Figure 4: Growing ISC tumours induce changes in the niche.
Figure 5: ISC tumour growth induces non-autonomous ISC proliferation.
Figure 6: ISC tumours co-opt niche-derived signals for their growth.
Figure 7: Tumour-induced enterocyte detachment induces JNK and Yki activity and Upd3 expression.
Figure 8: Integrin loss from enterocytes induces JNK activity and promotes ISC tumour growth.

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Acknowledgements

We thank the Bloomington Drosophila Stock Center, Vienna Drosophila RNAi Center (VDRC), National Institute of Genetics (Japan), Developmental Studies Hybridoma Bank, H. Bellen, S. Eaton, G. Halder, S. Hou, Y. N. Jan, H. Jasper, B. Lemaitre, D. McEwen, A. Page-McCaw, N. Perrimon, T. Xu and X. Yang for fly stocks and antibodies. We thank B. Schneider and J. Bousman (FHCRC Electron Microscopy Resource), M. Langlotz (ZMBH Flow Cytometry), D. Ibberson (Deep Sequencing Core Facility, U. Heidelberg), J. Delrow (FHCRC Sequencing Shared Resource) and C. Gläser (ZMBH, Bioinformatics). Financially supported by American Cancer Society Postdoctoral Fellowship (PF-08-040-01-DDC) to P.H.P. and NIH R01 GM051186, DKFZ A220, DFG SFB 873 and ERC Advanced Grant 268515 to B.A.E.

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Authors and Affiliations

  1. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North Seattle, Washington 98109, USA,

    Parthive H. Patel

  2. German Cancer Research Center (DKFZ)-Center for Molecular Biology, University of Heidelberg Alliance, Im Neuenheimer Feld 282 69120 Heidelberg, Germany,

    Parthive H. Patel, Devanjali Dutta & Bruce A. Edgar

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  1. Parthive H. Patel
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Contributions

The project was conceived and developed by P.H.P. and B.A.E. P.H.P. contributed Figs 1–8 and Supplementary Figs 1–8. D.D. contributed Figs 2g, h and 6e, and Supplementary Figs 1g–k, 2a, c, d, 4b, c, 5b and 8b. mRNA-seq data were analysed by both P.H.P. and D.D. P.H.P. and B.A.E. prepared the manuscript.

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Correspondence to Parthive H. Patel or Bruce A. Edgar.

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Integrated supplementary information

Supplementary Figure 1 ISC tumour cells differentiate towards the secretory fate.

(a) Prospero (a, red) in midguts expressing GFP (a, green) with esgts for 3 days. (b) Pdm-1 (b, red) in midguts expressing GFP (b, green) with esgts for 2 days. (c) Delta (c′) and Prospero (Pros, c′′) in midguts expressing GFP and NRNAi (c) with esgts for 3 days. esglowDllow cells expressed high levels of Prospero (c–c′′, arrowheads); esghigh Dlhigh ISC-like cells expressed lower levels of Prospero (c–c′′, arrows). (d) Pdm-1 (d′; d′′, red) in midguts expressing GFP (d; d′′, green) and NRNAi with esgts for 2 days. (e,f) Prospero (e′, f′, red) in midguts expressing GFP (e, green) and NRNAi with esgts for 5 days or in midguts recovering for 7 days at 18 °C after expression of GFP (f, green) and NRNAi for 5 days with esgts. (g–j) Mean normalized expression (NE) value (RPKM, log2) from n = 2 independent experiments of ISC (g) or EE cell (i) associated genes determined by mRNA sequencing of midguts expressing GFP (control, red) or GFP and NRNAi (tumourous, blue) with esgts for 3 days. The adjusted fold change in gene expresson in tumorous midguts (blue), normalized to control midguts (red), is indicated. Benjamini–Hochberg adjusted P-value for Dl, spdo, esg, shg, sc, pros, Rab3 and AstA is 7.71e-322; arm, P = 1.054e-50. The mean NE value (RPKM, log2) from n = 2 independent experiments of ISC (h) and EE (j) associated genes determined by mRNA sequencing of esg+ cells sorted from control midguts expressing GFP (red) or from tumorous midguts expressing GFP and NRNAi (blue) with esgts for 3 days. The adjusted fold change in gene expression in sorted esg+ tumor cells (blue), normalized to esg+ cells from control midguts, is indicated. NS, not significant. Benjamini–Hochberg adjusted P-values for Dl is 2.53e-05; spdo, 1.81e-05; sc, 3.48e-14; pros, 1.26e-14; Rab3, 2.52e-19 and AstA, 1.01e-09. (k) Principal component analysis of transcriptional profiles of ISC tumors, intestinal stem cells (ISCs), esg+ progenitors, SuH+ enteroblasts (EB), enterocytes (EC), enteroendocrine cells (EE) and visceral muscle (VM). DNA is in a′, b′, c′′′, d′′′; e–f′ (blue). Scale bars in a–a′, b–b′, c–c′′′ and d–d′′′, 25 μm; e–f′, 30 μm.

Supplementary Figure 2 ISC tumors induce EGF, insulin, PDGF-VEGF and Wnt signaling in the niche.

(a) Mean normalized expression values (RPKM, log2) of spi determined by mRNA sequencing of sorted midgut cell types from n = 2 independent experiments. (b) Mean fold change (log2) with s.e.m. from n = 4 independent experiments of EGFR ligand mRNA levels determined by qPCR on midguts bearing 7 day neurIF65/− clones. Fold changes were normalized to control midguts bearing 7 day wild-type clones. (c,d) Mean normalized expression (NE) value (RPKM, log2) from n = 2 independent experiments of other midgut signaling factors determined by mRNA sequencing of midguts expressing GFP (control, red) or GFP and NRNAi (tumorous, blue) with esgts for 3 days (c). The adjusted fold change in gene expression in tumorous midguts (blue), normalized to control midguts (red), is indicated. Benjamini–Hochberg adjusted P-value for Ilp3 is 2.47e-35; Pvf2, 4.00e-46 and wg, 0.0016. The mean NE value (RPKM, log2) from n = 2 independent experiments of midgut signaling factors determined by mRNA sequencing of esg+ cells sorted from control midguts expressing GFP (red) or from tumorous midguts expressing GFP and NRNAi (blue) with esgts for 3 days (d). NS, not significant.

Supplementary Figure 3 Tumor frequency and incidence after inhibiting apoptosis, JNK and Yki activity and cytokine expression in enterocytes.

(a) 8 midgut regions used to determine the frequency of neurIF65/− tumor incidence (b) and tumor size (c) along the midgut anterior (A)–posterior (P) axis. (b) Mean percent of total neurIF65/− tumors (n = 45) found in each of 8 midgut regions. (c) Mean number of cells per neurIF65/− tumor (n = 45) with s.e.m. found in each of 8 midgut regions. (d,f,h,j,l) Mean tumors per midgut with s.e.m. 7 or 8 days after tumor induction in midguts expressing GFP (n = 6 midguts) or GFP and p35 (n = 12 midguts) (d), GFP (n = 16 midguts) or GFP and bskK53R (n = 17 midguts), puc (n = 6 midguts), bskRNAi (n = 16 midguts) or hep (n = 28 midguts) (f), GFP (control, n = 16 midguts) or GFP and wts (n = 19 midguts) or ykiRNAi (n = 9 midguts) (h), GFP (control, n = 17 midguts) or GFP and upd2RNAi(1) (n = 11 midguts), upd2RNAi (2) (n = 9 midguts), upd3RNAi(1) (n = 12 midguts) or upd3RNAi (2) (n = 10 midguts) (j), and GFP (control, n = 25 midguts) or GFP and RasV 12 (n = 26 midguts) (l) in ECs with Myo1Ats. (e,g,i,k,m) Mean percent flies with tumors with s.e.m. 7 or 8 days after tumor induction in midguts expressing GFP or GFP and p35 (e), GFP or GFP and bskK53R, puc, bskRNAi or hep (g), GFP or GFP and wts or ykiRNAi (i), GFP or GFP and upd2RNAi(1), upd2RNAi(2), upd3RNAi(1) or upd3RNAi (2) (k), and GFP or GFP and RasV 12 (m) in ECs with Myo1Ats. In b,c, tumors pooled from 2 independent experiments; in d, f, h, j and l, midguts were pooled from 3 independent experiments; in e, g, i, k, and m, mean determined from n = 3 experiments.

Supplementary Figure 4 ISC tumour growth induces enterocyte detachment and loss.

(a) DNA (a, blue) in midguts containing neurIF65/− tumours (yellow dashed line) amongst ECs expressing GFP (a′, green) with Myo1Ats for 8 days. Arrowheads indicate ECs overlying tumours (a,a′). (b,c) Mean normalized expression (NE) value (RPKM, log2) from n = 2 independent experiments of pro-apoptotic factors (hid, rpr, grim and skl) determined by mRNA sequencing of midguts expressing GFP (control, red) or GFP and NRNAi (blue) with esgts for 3 days (b). The adjusted fold change in gene expression in tumorus midguts (blue), normalized to control midguts (red), is indicated. The Benjamini–Hochberg adjusted P-value for grim is 5.86e-127. The mean NE value (RPKM, log2) from two independent experiments of apoptosis factors determined by mRNA sequencing of esg+ cells sorted from control midguts expressing GFP (red) or from tumorous midguts expressing GFP and NRNAi (blue) with esgts for 3 days (c). NS, not significant. (d) Transmission electron micrograph of an apoptotic EC (anterior) from midguts expressing GFP and NRNAi with esgts for 3 days. White arrowheads highlight abnormal EC microvilli. Scale bars in a–a′, 30 μm; in d, 2 μm.

Supplementary Figure 5 Cytokines are induced by ISC tumour growth and not by enteroendocrine cell expansion.

(a) Mean fold change (log2) with s.e.m. from n = 4 independent experiments in Delta and cytokine (upd1, upd2, upd3) mRNA levels determined by quantitative PCR on midguts bearing 7 day old neurIF65/− clones. Fold changes were normalized to control midguts bearing 7 day old wild-type clones. (b) The mean normalized expression (NE) value (RPKM, log2) from n = 2 independent experiments of cytokines (upd1-3) and JAK-STAT target Socs36E determined by mRNA sequencing of esg+ cells sorted from control midguts expressing GFP (red) or from tumorous midguts expressing GFP and NRNAi (blue) with esgts for 3 days. The adjusted fold change in gene expression in sorted esg+ tumour cells (blue), normalized to esg+ cells from control midguts, is indicated. NS, not significant. The Benjamini–Hochberg adjusted P-value for upd2 is 1.36e-10. (c) β-galactosidase (c–c′, red) in midguts of flies carrying upd-lacZ and expressing NRNAi with esgts for 2 days. (d) Mean fold change (log2) with s.e.m. of cytokine (upd 1-3) and EGF ligand (spi, krn, vn) mRNA levels determined by quantitative PCR on midguts expressing NRNAi for 2 days (n = 3 independent experiments), sc for 7 days (n = 3 independent experiments), and SuHRNAi for 5 days (n = 4 independent experiments) with esgts. Fold changes were normalized to control whole midguts overexpressing GFP with esgts for 2 (for NRNAi), 7 (for sc) and 5 days (for SuHRNAi). ND, not determined; NS, not significant. Overexpression of the pro-secretory factor Scute (Sc) expanded the number of Pros+ EEs (Ast+). (e,f) Phospho-histone H3 Ser 10 (red) in control (e) or upd2-3 mutant (Δ2-3) (f) midguts expressing GFP (green) and NRNAi for 14 days with esgts (weak). (g) Delta (g; g′, red) and Prospero (g′′) in midguts expressing GFP (g′, green) and NRNAi with esgts for 4 days. (h) β-galactosidase (red) and D–GFP (green) in midguts of flies carrying Dl-lacZ and 10X STAT-DGFP. (i,j) D–GFP (i, j; i′, j′, green) in midguts of flies carrying 10X STAT-DGFP and expressing NRNAi (i) or NRNAi and domeΔCYT (j) with esgts for 4 days. Rare, large ISC tumour shown in j. DNA in c′, e, f, h, i′, and j′ (blue). Scale bars in c–c′, 35 μm; e, 25 μm; f, 30 μm; g–g′′, 20 μm; h, 4 μm; i–j′, 30 μm.

Supplementary Figure 6 Cytokine induction by ISC tumours is not due to changes in luminal bacteria load.

(a,b) in situ hybridization to upd3 mRNA in midguts from flies infected with Pseudomonas entomophila for 2 days (b; b′, red) and control midguts (no infection; a; a′, red). Cells with high upd3 mRNA are indicated with arrows; with lower upd3mRNA, arrowheads (b–b′). (c) PCR for bacterial 16S rDNA from total genomic DNA isolated from midguts of flies fed normal food or antibiotics for 3 days (undiluted genomic DNA, 1:1). Tumour growth did not increase luminal bacterial load. (d) PCR for the crq gene to detect fly genomic DNA input. (e) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. in flies expressing GFP (control, -antibiotics: n = 20 midguts; + antibiotics:n = 22 midguts) or NRNAi (-antibiotics:n = 26 midguts; + antibiotics:n = 28 midguts) with esgts for 3 days fed normal food or food containing antibiotics. Reducing gut luminal bacteria to negligible levels had no significant effect on tumour growth. Thus Upd2,3 induction after tumour growth was not due to alterations in luminal bacteria but was a response to tumour growth. (f) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. of flies fed food alone (n = 31 midguts), Pseudomonas entomophila (P. e.) containing food (n = 24 midguts; Mann–Whitney: P < 0.0001), sucrose alone (n = 33 midguts) or P. e. containing sucrose (n = 15 midguts; Mann–Whitney: P < 0.0001) for 1 day before tumour induction by expressing NRNAi with esgts (weak) for 3 days (food alone). (g) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. in flies expressing GFP and NRNAi (n = 36 midguts) or GFP, NRNAi, cyclin E (cycE) and cdc25/string (stg) (n = 29 midguts, Mann–Whitney: P < 0.0001) with esgts (weak) for 3 days. In e, midguts pooled from 2 independent experiments; in f,g, from 3 independent experiments. DNA in a′ and b′ (blue). Scale bars in a–a′, 25 μm; b–b′, 35 μm.

Supplementary Figure 7 Oncogenic RasV 12 expression in ECs induces JNK activity and stimulates ISC tumour growth.

(a–c) β-galactosidase (a–c; a′–c′, red) in midguts bearing puc-lacZ and expressing GFP (a′,b′,c′; green) alone for 5 days (a) or GFP and hepAct for 2 days (b) or GFP and RasV 12 for 5 days (c) with Myo1Ats. (d) Mean number of phosphorylated histone H3 Ser10 positive cells per midgut with s.e.m. in flies expressing GFP alone (control, n = 11 midguts), GFP and hep (n = 9 midguts, Mann–Whitney: p = 0.0064) or RasV 12 (n = 15 midguts, Mann–Whitney: P < 0.0001) for 7 days or GFP and hepAct (n = 11 midguts) for 2 days with Myo1Ats. (e) Cells per neurIF65/− tumour with mean (red line) and s.e.m. amongst ECs expressing GFP (control, n = 56 tumours from 25 midguts, skewness = 2.929, kurtosis = 12.39) or GFP and RasV 12 (n = 35 tumours from 26 midguts, Mann–Whitney: p = 0.0004, skewness = 2.049, kurtosis = 4.029) with Myo1Atsfor 7 days. In d, midguts pooled from 2 independent experiments; in e, from 3 independent experiments. DNA in a′, b′ and c′ (blue). Scale bars in a–c′ are 45 μm.

Supplementary Figure 8 Integrin subunit expression and β PS1 basal localization in ECs.

(a) Tumour-induced EC detachment induces graded JNK and Yki activity and upd3 expression. fa, fully attached; pd, partially detached; fd; fully detached. (b) Mean normalized expression (NE) values (RPKM) from two independent experiments of α and β integrin subunits determined by mRNA sequencing of sorted midgut ECs. (c) β-PS1 (c′; c′′ and c′′′′′, red) and Actin (phalloidin) (c′′′–c′′′′′, white) in midguts expressing GFP (c; c′′ and c′′′′, green) with Myo1Ats. Scale bars in c–c′′′′′, 15 μm.

Supplementary information

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Supplementary Information (PDF 3750 kb)

Induction of JNK activity in ECs surrounding ISC tumours (1-Channel).

β-galactosidase (red) in midguts bearing puc-lacZ and expressing NRNAi and GFP with esgts for 3 days. (AVI 4358 kb)

Induction of JNK activity in ECs surrounding ISC tumours (2-Channel).

β-galactosidase (red) in midguts bearing puc-lacZ and expressing NRNAi and GFP (green) with esgts for 3 days. (AVI 8312 kb)

Induction of JNK activity in ECs surrounding ISC tumours (3-Channel).

β-galactosidase (red) in midguts bearing puc-lacZ and expressing NRNAi and GFP (green) with esgts for 3 days. DNA is in blue. (AVI 5815 kb)

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Patel, P., Dutta, D. & Edgar, B. Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17, 1182–1192 (2015). https://doi.org/10.1038/ncb3214

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  • DOI: https://doi.org/10.1038/ncb3214

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