Cytoneme-mediated signaling essential for tumorigenesis

Abstract

Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models and tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. We observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. We also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and we determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. Our results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.

Fig 1. Cytonemes in EGFR-Pcn_i tumor and tumor-associated cells.

(A) Cartoon of a 3^rd instar larval wing disc with wing blade primordia (wbp), disc-associated myoblasts (orange), trachea (white, outlined in blue) and air sac primordium (ASP), Dpp expressing cells (green stripe), Bnl expressing cells (blue circle). (B) Control wing disc expressing CD8:GFP in dorsal driven epithelial cells (ap-Gal4; green), and mCherry:CAAX in myoblasts ((15B03-lexA; red). (C) Schematic representation of tumor induction: animals developed for five days at 18°C with Gal4 repressed by Gal80, were transferred to 29°C to induce Gal4 and tumor growth for seven days (unless indicated otherwise). (D-F) Unfixed EGFR-Pcn tumor model wing discs. (D) Wing disc with tumor cells (CD8:GFP; green) and myoblasts (mCherry:CAAX; red). Genotype: ap-Gal4,UAS-psq_RNAi/115B03-lexA,lexO-Cherry-CAAX;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. Scale bars: 100μm. (E) Cytonemes in the tumor epithelial cells (green, arrows); genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. (F-F’) Cytonemes in myoblasts (red, arrows) extend towards epithelial cells (green); genotype: ap-Gal4,UAS-psq_RNAi/115B03-lexA,lexO-Cherry-CAAX;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP; scale bars: 50μm. (G-H) Unfixed wing discs with marked tracheal cells (green, arrows); (G) control, genotype: btl-LHG,lexO-CD2-GFP; (H) EGFR-Pcn tumor, genotype: ap-Gal4,UAS-psq_RNAi/btl-LHG,lexO-CD2-GFP;UAS-EGFR,tub-Gal80^ts/+. Excessive tracheal growth and ectopic branches indicated by arrows; scale bars: 100μm.

Fig 2. Dpp signaling in the EGFR-Pcn tumor model.

(A,A’,B) Unfixed wing discs showing the Dpp distribution. (A) Control disc with Dpp (red, arrow). Genotype: Dpp:mCherry. Scale bar: 100μm. (A’) Disc with EGFR-Pcn tumor induced for 5 days expressing Dpp (red) in tumor cells. Genotype: ap-Gal4,UAS-psq_RNAi/Dpp:mCherry;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. Arrows indicate Dpp up-regulation. (A”-A”’) EGFR-Pcn wing showing epithelial tumor cells (green) fixed and stained with α-phosphorylated MAD (pMad, red) antibody to monitor Dpp signaling and α-Cut (blue) to label myoblasts. Genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. Scale bar: 100μm. (A”’) Higher magnification image of the box area in (A”), scale bar: 50μm. (B-B”) Cytoneme (green) extending from epithelial tumor cell (green) with Dpp:mCherry (red); arrow indicates Dpp:mCherry in cytoneme. Genotype: ap-Gal4,UAS-psq_RNAi/Dpp:mCherry;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. Scale bar: 50μm.

Fig 3. Conditions that ablate cytonemes decrease signaling, reduce myoblast and tumor growth.

(A-I) Fixed wing discs stained with α-phosphorylated MAD (pMad, red) antibody to monitor Dpp signaling and α-Cut (cyan) to label myoblasts. Scale bars: 100μm. (A,A’) Control. (B,B’) EGFR-Pcn tumor, genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-CD8:GFP. (C,C’) Tumor + NrgRNAi, genotype: ap-Gal4,UAS-psq_RNAi/UAS-Nrg_RNAi;UAS-EGFR,tub-Gal80^ts/+. (D) Tumor + Caps^DN, genotype:ap-Gal4,UAS-psq_RNAi/UAS-CAPS^DN;UAS-EGFR,tub-Gal80^ts/+. (E) Tumor + SCARRNAi, genotype:ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-SCAR_RNAi. (F,F’) EGFR-Pcn + diaRNAi in epithelial cells. Genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-dia_RNAi. (G) Tumor + NrgRNAi, diaRNAi, genotype: ap-Gal4,UAS-psq_RNAi/UAS-Nrg_RNAi;UAS-EGFR,tub-Gal80^ts/UAS-dia_RNAi. (I-I’) EGFR-Pcn + diaRNAi expressed in the myoblasts. Genotype: ap-Gal4,UAS-psq_RNAi/115B03-lexA;UAS-EGFR,tub-Gal80^ts/ lexO-dia_RNAi. (H) Quantification of the total wing disc area (blue) and relative area of Cut-expressing cells (orange) of control, EGFR+Pcn tumor, tumor + diaRNAi expressed in epithelial cells, diaRNAi expressed in myoblasts, NrgRNAi, SCARRNAi, Caps^DN, NrgRNAi+diaRNAi, dsRNAi, ftRNAi, fjRNAi and Btl^DN larvae. Data was normalized to control. Student’s t test P values (P >1.10⁻⁹ for all except no significant difference for tumor + dsRNAi, ftRNAi and fjRNAi); n = 15–20 discs for each genotype. (J-J”) Sagittal sections of fixed wing discs stained with α-Dlg antibody (red) to mark the cell’s apical compartments, Scale bar: 50μm. (J) Control. (J’) EGFR-Pcn tumor, genotype: ap-Gal4,UAS-psq_RNAi/UAS-CD8:GFP;UAS-EGFR,tub-Gal80^ts/+) (J”) EGFR-Pcn + diaRNAi expressed in epithelial cells, genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-dia_RNAi.

Fig 4. Conditions that ablate cytonemes promote survival.

(A) Cartoon of a wild type larva depicting cells expressing GFP in the imaginal disc dorsal compartments (ap-Gal4; green, arrow) and of EGFR-Pcn tumor larva with overgrowth and metastasis throughout the larva (green). (B) Survival of EGFR-Pcn tumor, tumor + diaRNAi, NrgRNAi, SCARRNAi and Caps^DN larvae to pupal (blue), pharate adult (orange) and adult stage (gray). Student’s t test P values: (between P<0.05 and P >1.10⁻⁸) with n = 15–30 larvae for each genotype. (C) Control adult wing. (D) EGFR-Pcn + diaRNAi wing, genotype: ap-Gal4,UAS-psq_RNAi/UAS-CD8:GFP;UAS-EGFR,tub-Gal80^ts/UAS-dia_RNAi.

Fig 5. FGF signaling in the EGFR-Pcn tumor model.

(A-B) Unfixed wing discs with Bnl-expressing cells marked with Cherry-CAAX (red, arrows). (A) Control, genotype: bnl-lexA,lexO-mCherry:CAAX/+. Scale bar: 100μm. (B) EGFR-Pcn tumor, genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/bnl-lexA,lexO-mCherry:CAAX. Arrows indicate FGF up-regulation (red). (C-D’) Unfixed wing discs with Btl distribution marked by Btl:mCherry (red, arrows). (C) Control, genotype: Btl:mCherry/+. Btl is only expressed in the tracheal cells. Scale bar: 100μm (C’) Higher magnification image of the boxed area in (C). (D) 5 day EGFR-Pcn tumor disc, genotype: ap-Gal4,UAS-psq_RNAi/UAS-CD4-mIFP;UAS-EGFR,tub-Gal80^ts/Btl:mCherry. Btl expression is upregulated in the tumor cells. (D’) Higher magnification image of the box area in (D). Scale bars: 50μm. (E-F) EGFR-Pcn tumor +Btl^DN fixed wing disc stained with α-phosphorylated MAD (pMad, red) antibody to monitor Dpp signaling (E) and α-Cut (cyan) to label myoblasts (F). Scale bar: 100μm. Genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-Btl^DN. (G) EGFR mRNA measured by quantitative real-time PCR. RNA was extracted from 5 discs of the indicated genotypes. Data were normalized to rp49. The data show mean ± SD from three technical replicates of a representative experiment. Significance was analyzed using Student’s t-test (p<0.001). Comparable results were obtained in 3 independent biological replicates. Genotypes are ap-Gal4,UAS-CD8:GFP (WT), ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/ UAS-CD8:GFP (tumor) and ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-Btl^DN. (H-K) Fixed wing disc stained with α-phosphorylated ERK to monitor EGF signaling (dpERK, red). (H) Control, genotype: ap-Gal4,UAS-CD8:GFP. (I) Control+Btl^DN, genotype: ap-Gal4/+;UAS-Btl^DN/+. (I) EGFR-Pcn tumor disc, genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/ UAS-CD8:GFP. EGFR-Pcn tumor + Btl^DN, genotype: ap-Gal4,UAS-psq_RNAi/+;UAS-EGFR,tub-Gal80^ts/UAS-Btl^DN.

Fig 6. RET-MEN2 tumor growth and survival depends on cytonemes.

(A-B’) Unfixed wing discs expressing CD8:GFP (green) driven by ptc-Gal4. Scale bar: 100μm. (A) Control. (A’) Cytonemes in wild type cells (green, arrows). Scale bar: 50μm. (B) RET tumor, genotype: ptc-Gal4,CD8:GFP;UAS-RET^MEN2/UAS-CD8:GFP. (B’) Cytonemes in RET tumor cells (green, arrows). (C-E) Unfixed wing discs expressing CD8:GFP (green) and either RET + diaRNAi (C); RET + SCARRNAi (D); and RET + Irk2^DN (E). (C) Genotype: ptc-Gal4,CD8:GFP;UAS-RET^MEN2/UAS-dia_RNAi. (D) Genotype: ptc-Gal4,CD8:GFP;UAS-RET^MEN2/UAS-SCAR_RNAi. (E) Genotype: ptc-Gal4,CD8:GFP/Irk2^DN ;UAS-RET^MEN2. (F) Quantification of the area of the disc expressing GFP (% of disc) in control, RET tumor, or RET and either diaRNAi, SCARRNAi or Irk2^DN. Significance was analyzed using Student’s t-test (P<1.10⁻⁵) with 15–18 discs. (G) Survival of RET-tumor and RET and either diaRNAi, SCARRNAi or Irk2^DN to pupa (blue) and adult (orange). Significance using Student’s t-test for adults is (P<0.001), n = 30–40 for each genotype. (H) RET tumor, diaRNAi wing blade; genotype: ptc-Gal4,CD8:GFP;UAS-RET^MEN2/UAS-dia_RNAi. Arrow points to abnormal crossvein.