Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Abstract

The risk of specific cancers increases in patients with metabolic dysfunction, including obesity and diabetes. Here, we use Drosophila as a model to explore the effects of diet on tumor progression. Feeding Drosophila a diet high in carbohydrates was previously demonstrated to direct metabolic dysfunction, including hyperglycemia, hyperinsulinemia, and insulin resistance. We demonstrate that high dietary sugar also converts Ras/Src-transformed tissue from localized growths to aggressive tumors with emergent metastases. Whereas most tissues displayed insulin resistance, Ras/Src tumors retained insulin pathway sensitivity, increased the ability to import glucose, and resisted apoptosis. High dietary sugar increased canonical Wingless/Wnt pathway activity, which upregulated insulin receptor gene expression to promote insulin sensitivity. The result is a feed-forward circuit that amplified diet-mediated malignant phenotypes within Ras/Src-transformed tumors. By targeting multiple steps in this circuit with rationally applied drug combinations, we demonstrate the potential of combinatorial drug intervention to treat diet-enhanced malignant tumors.

Figure 1. HDS diverts Ras/Src-activated cells into aggressive tumors

(A–I) Developmental stage matched third instar larvae fed control diet or HDS. (A and B) lacZ control in control diet (day 7 AEL) and HDS (day 11 AEL) (C and D) csk^−/− in control diet (day 7 AEL) and HDS (day 11 AEL), (E and F) ras1^G12V in control diet (day 8 AEL) and HDS (day 12 AEL), (G, H and I) ras1^G12V;csk^−/− in control diet (day 9 AEL) and HDS (day 13 AEL). The latter demonstrated secondary tumors in a subset of animals (arrowhead in I). (A′–H′), Matching dissected eye epithelial tissue stained with DAPI (red). (J) Quantitation of the observed phenotypes. Blue bar: eye discs without overgrowth (e.g.; Fig. 1G). Red bar: eye discs with tumor overgrowth and overall enlarged tissue size (e.g.; Fig. 1H). Green bar: animals with secondary tumors (e.g.; Fig. 1I). Results are shown as mean ± SEM. (K–R) Chronological age matched larvae fed control diet or HDS. ras1^G12V;csk^−/− animals shown at day 7 AEL (K and L), day 9 AEL (M and N), day 11 AEL (O and P), and day 13 AEL (Q and R). (K′, M′, N′, P′ and R′), Matching dissected eye epithelial tissue stained with DAPI (red). (S) Quantitation of the percentage of GFP-positive clone area percentage relative to total eye tissue area. Results are shown as mean ± SEM of individual eye discs. * Not assessed due to early (small discs, Day 7) or late (everted pupal discs) stages. See also Figure S1.

Figure 2. Ras/Src-tumors in HDS spread into hemolymph and colonized near trachea to form secondary tumors

(A–D) Laminin A staining (red) of developmental stage matched ras1^G12V;csk^−/− or inR^CA,ras1^G12V;csk^−/− eye discs raised on indicated diets. (E–H) MMP1 staining (red) of developmental stage matched ras1^G12V;csk^−/− or inR^CA,ras1^G12V;csk^−/− eye discs raised on indicated diets. (I–K) Ventral view of ras1^G12V;csk^−/− animals raised on HDS (I). Zoom up of the inset is shown in (J), and the extracted hemolymph on mineral oil is shown in (K). (L–O) ras1^G12V;csk^−/− animals raised on HDS with growing secondary tumor (arrow). GFP (M), bright field (BF)(N) and merged image (O) of the dissected secondary tumor is shown. (P) phospho-Histone H3 staining (p-HH3; red) of ras1^G12V;csk^−/− secondary tumor. (Q) Chitin-binding probe staining (red) of ras1^G12V;csk^−/− secondary tumor attached to trachea. Zoom up of the inset is shown in the middle panel. (R) Branchless staining (bnl; red) of a ras1^G12V;csk^−/− secondary tumor attached to trachea. (S) Phenotype quantitation. Blue bar: % animals with loss of Laminin staining in the eye discs. Red bar: % animals with GFP-positive cells in the hemolymph. Green bar: % animals with secondary tumors. Results are shown as mean ± SEM. See also Figure S2.

Figure 3. Ras/Src-activated cells evade insulin resistance

(A and B) Developmental stage matched animals raised on HDS with the genotype, (A) ras1^G12V;csk^−/−, (B) ras1^G12V;csk^−/−,akt^hypo/hypo, (C and D) Developmental stage matched animals raised on control diet with the genotype, (C) ras1^G12V;csk^−/−, (D) inR^CA,ras1^G12V;csk^−/−. Note that inR^CA;ras1^G12V;csk^−/− animals fed control diet led to overgrowth but not secondary tumor formation. (E) inR^CA,ras1^G12V;csk^−/− animals fed HDS. (A′–E′) Matching dissected eye epithelial tissue stained with DAPI (red). (F) Quantitation of the observed phenotypes. The three color bars are explained in the legend of Fig. 1J. Results are shown as mean ± SEM. (G and H) Dissected eye tissue of lacZ (G) or ras1^G12V;csk^−/− (H) animals fed control diet or HDS were treated with or without Insulin, and total Akt, phospho-Akt (p-Akt), and Syntaxin (Syt) levels were examined by immunoblotting. The results of immunoblots were quantitated using Image J software and p-Akt/Syt values relative to subject without Insulin stimulation were determined. Results are shown as mean ± SEM. (I) Dissected eye tissue from ras1^G12V;csk^−/− animals fed HDS were treated with Insulin, and immunostained with anti-phospho-Akt (p-Akt) antibody. (J–O) Glucose uptake was examined by uptake of 2-NBDG of the dissected eye tissue from inR^CA,ras1^G12V;csk^−/− (J), ras1^G12V;csk^−/− (K–M), lacZ (N), or ras1^G12V;csk^−/−,akt^hypo/hypo (O) animals fed control diet or HDS, with or without Insulin stimulation. See also Figure S3.

Figure 4. Ras/Src-tumors are resistant to apoptosis in high sucrose diet

(A–C) Cleaved Caspase-3 staining (red) of ras1^G12V;csk^−/− (A and B) or inR^CA,ras1^G12V;csk^−/− (C) eye discs raised on indicated diets. (D–F) TUNEL assay (red) was used to label apoptotic cell death of ras1^G12V;csk^−/− (D and E) or inR^CA,ras1^G12V;csk^−/− (F) eye discs raised on indicated diets. (G–J) β-galactosidase (β-gal) staining (red) of eye discs from ras1^G12V;csk^−/−,diap1-lacZ animals in a control diet (G), HDS at day 13 AEL (H), HDS at day 11 AEL (I), and inR^CA,ras1^G12V;csk^−/−,diap1-lacZ animals in a control diet (J). (K–N) Wingless (Wg) staining (red) of eye discs from ras1^G12V;csk^−/− animals in a control diet (K), HDS at day 13 AEL (L) HDS at day 11 AEL (M), and inR^CA,ras1^G12V;csk^−/−,diap1-lacZ in a control diet (N). See also Figure S4.

Figure 5. Wg mediates Ras/Src-tumorigenesis in HDS

(A–D) Animals raised on HDS with the genotype, (A) ras1^G12V;csk^−/−, (B) ras1^G12V;csk^−/−wg, ^RNAi, (C) ras1^G12V;csk^−/−,tcf^DN, (D) ras1^G12V;csk^−/−,bsk^DN. (A′–D′) Matching dissected eye epithelial tissue stained with DAPI (red). (E and F) Wingless (Wg) staining (red) of ras1^G12V;csk^−/−,bsk^DN eye discs raised on HDS (E) and inR^CA,ras1^G12V;csk^−/−,bsk^DN eye discs raised on a control diet (F). (G) Dissected eye tissue of LacZ control or HA-Wg animals fed HDS were treated with or without Insulin, and total Akt, phospho-Akt (p-Akt), and Syntaxin (Syt) levels were examined by immunoblotting. The results of immunoblots were quantitated using Image J software. Results are shown as mean ± SEM. (H and I) Histogram showing the levels of rpL32, inR, chico, and lnk mRNAs measured by quantitative RT-PCR. Total RNA was isolated from LacZ-expressing control eye discs (H) or ras1^G12V;csk^−/− eye discs (I) raised on a control diet (Black bar) or HDS (White bar) or ras1^G12V;csk^−/−,tcf^DN eye discs raised on HDS (Grey bar). Results are shown as mean ± SEM. Asterisks indicate statistically significant difference (*, p<0.01; **, p<0.05). (J) Dissected eye tissue of ras1^G12V;csk^−/− animals fed control diet or HDS were treated with or without Insulin, and phospho-Insulin Receptor (p-InR) and Syntaxin (Syt) levels were examined by immunoblotting. (K) Model of diet-mediated tumorigenesis of Ras/Src-activated cells. See also Figure S5.

Figure 6. Combinatorial multi-node drug treatment for Ras/Src-tumors in HDS

(A–H) ras1^G12V;csk^−/− animals fed HDS containing (A) 0.05% DMSO, (B) 20 μM acarbose, (C) 25 μM pyrvinium, (D) 50 μM AD81, (E) 20 μM acarbose plus 25 μM pyrvinium, (F) 20 μM acarbose plus 50 μM AD81, (G) 25 μM pyrvinium plus 50 μM AD81, (H) 20 μM acarbose plus 25 μM pyrvinium plus 50 μM AD81. All phenotypes were assessed at day 17 AEL. (I) Cell extracts from dissected eye tissue of ras1^G12V;csk^−/− animals fed HDS supplemented with DMSO, AD80 or AD81 were examined by immunoblotting. (J) Percent pupariation of DMSO- or drug-treated ras1^G12V;csk^−/− animals was determined at day 17 AEL. Results are shown as mean ± SEM. See also Figure S6.