Lung cancer is the leading cause of cancer-associated mortality. Mutations in the EGFR gene are among the most important inducers of lung tumor development, but success of personalized therapies is still limited because of toxicity or developing resistances. We expressed constitutively active EGFR (EGFRCA) exclusively in the airway system of Drosophila melanogaster and performed comprehensive phenotyping. Ectopic expression of EGFRCA induced massive hyper- and metaplasia, leading to early death. We used the lethal phenotype as a readout and screened a library of FDA-approved compounds and found that among the 1,000 compounds, only the tyrosine kinase inhibitors (TKI) afatinib, gefitinib, and ibrutinib rescued lethality in a whole-animal screening approach. Furthermore, we screened the library in the presence of a subtherapeutic afatinib dose and identified bazedoxifene as a synergistically acting compound that rescues EGFR-induced lethality. Our findings highlight the potential of Drosophila-based whole-animal screening approaches not only to identify specific EGFR inhibitors but also to discover compounds that act synergistically with known TKIs. Moreover, we showed that targeting the EGFR together with STAT-signaling is a promising strategy for lung tumor treatment.

Lung cancer is the leading cause of cancer-related deaths, with more than 1 million newly diagnosed cases worldwide each year (1). Non–small cell lung cancers (NSCLC), and in particular adenocarcinomas, account for the majority of all lung cancers. A relatively small number of oncogenic driver mutations are responsible for both the initiation and maintenance of NSCLCs (2–4). One of the most important driver oncogenes in lung cancer is the EGFR. Somatic mutations in the EGFR gene occur in 10% of all NSCLCs (5). Although, targeted therapy for patients that carry a mutation in the EGFR gene has been successful, the risk to develop drug resistance during this treatment is still around 50% (6) and the occurrence of novel mutations in resistant tumors demanded for the development of third and fourth-generation EGFR inhibitors. Therefore, novel compounds or, more importantly, synergistically acting combinations of compounds are urgently needed as alternative therapeutic strategies. They hold the potential to interfere with simultaneously activated pathways and thereby eventually improve the effectivity of a treatment. To identify such compounds, whole-animal screens, especially those based on simple model organisms that are amenable to high-throughput screening such as the fruit fly Drosophila melanogaster could develop into invaluable tools to complement the drug discovery pipeline. The human lung and the fly's tracheal system share a large number of structural and physiologic similarities (7, 8). Moreover, homologous molecular factors shape the lung and the tracheal system during development (9). These similarities also apply for airway epithelial cells. In addition, the organization of the epithelial innate immune systems of Drosophila and mammals is surprisingly similar (10, 11). These commonalities, in combination with the unrivaled freedom of genetic manipulation possible in Drosophila (12), gave rise to highly useful Drosophila models for chronic inflammatory diseases of the lung, including asthma, COPD, and lung cancer (7, 13–15).

Here, we developed and used a Drosophila lung tumor model by targeted ectopic expression of a constitutively active isoform of the EGFR in the airway epithelium of flies. The excessive tissue growth that was seen upon this treatment resulted in early larval death, which was used as read-out for a high-throughput screening approach based on rescuing this lethality. We identified the small compounds afatinib, gefitinib, and ibrutinib that rescued this lethal phenotype. A screening designed to find compounds that act synergistically with afatinib, identified bazedoxifene, a compound that specifically interferes with JAK/STAT signaling. This finding implies that the combined targeting of tyrosine kinases and JAK/STAT signaling could be a potent strategy for future cancer therapies.

Experimental setup and Drosophila husbandry

For ectopic expression specifically in the airway epithelium, the Gal4/UAS system was used (16). Experiments were performed on concentrated medium (CM) [5% (w/v) yeast extract (Becton Dickinson), 5% (w/v) sucrose, 8.6% cornmeal, 0.5% (w/v) agar, 0.03% (v/v) propionic acid, 0.3% (v/v) methyl-4-hydroxybenzoate], drug experiments were performed on low-melt CM medium (CM with low-melt agarose). Transgenic fly strains used for crosses: control line w1118 (BDSC_5905), tracheal driver lines for trachea ppk4-Gal4 (10, 11) and btl-Gal4, UAS-gfp; Gal80ts (Maria Leptin Lab), UAS-EGFRCA (BDSC_9533). This mutation is characterized by the A887T substitution leading to constitutive activation of the EGFR (17, 18). The here described EGFR-induced tumor model was generated by the crossing: ppk4-Gal4>UAS-EGFRCA (EGFRCA), the corresponding crossing with the wild-type (ppk4-Gal4>w1118) served as control. Embryos from these crossings were used for pharmacologic tests, rescue experiments, RT-PCR, and RNA-seq. All animal studies were performed according to local animal protection rules of the state Schleswig-Holstein.

Pharmacologic tests and rescue experiments

Compounds from a FDA-approved library (Selleck Chemicals, L1300), dissolved in DMSO were used for drug screening. The stock solutions were diluted with ethanol and used at a final concentration of 50 μmol/L in three replicates. DMSO (0.5%)/ethanol (0.5%) served as a negative control. Constitutively active EGFR (EGFRCA) embryos were placed on drug containing low-melt CM medium in a 96-well format and sealed with an air-permeable membrane. The number of pupae was counted after 7 days at 25°C. Positive hits were replicated. In a second screening approach, the compounds (50 μmol/L) from the library were supplemented with a low concentration of afatinib (2 μmol/L). For rescue experiments, different afatinib and gefitinib concentrations were used and the pupae and eclosed adults were counted every day.

Quantification of epithelial thickness and nuclei number

The tracheae of early 3rd instar larvae were dissected and the dorsal trunk of Tr8 was selected for microscopy and quantification using Z-stack analysis (see above). Z-stack images were documented in DIC and DAPI channels with the Axio Imager.Z1 with Apo Tome (Zeiss). Measurements were performed using the AxioVision SE64 Rel. 4.9 Software (Zeiss). Statistical analyses were performed with GraphPad Prism7 (GraphPad).

IHC

Tracheae of early 3rd instar were dissected in HL3 buffer, fixed with 4% PFA, and blocked with 10% normal goat serum (Sigma-Aldrich). Incubation of antibodies was carried out overnight at 4°C. Staining was performed with anti-Drosophila Coracle C2 (DSHB, 1:500) antibody in combination with Alexa Fluor 488-conjugated anti-mouse IgG (Jackson ImmunoResearch, 1:300) and with anti–phospho-histone H3 (Ser10; Cell Signaling Technology, 1:100) antibodies. Chitin staining was carried out with 505 Star-conjugated Chitin-binding Probe (New England BioLabs, 1:200).

Hypoxia

Embryos from a 10X STAT-GFP reporter line [25] were treated with 100 μmol/L bazedoxifene as described above; 0.1% DMSO served as control. Hypoxia was applied as described earlier (13).

qRT-PCR

Trachea from control and EGFRCA 3rd instar larvae were manually dissected and the qRT-PCR experiment was performed as described previously (19). rpl32 served as control.

RNA-sequencing and data evaluation

RNA isolation was performed as described above (section qRT-PCR). RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs) as per the manufacturer's recommendations. Size and quality of the libraries were visualized on a Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies). Diluted libraries were multiplex-sequenced on the Illumina HiSeq 2500 Instrument (Illumina) by single-read sequencing (1 × 50 bp). Gene ontology analyses were performed with the FlyMine program package (20) and analysis of transcription factor–binding sites were done with pscan (21). The RNAseq data had been deposited at the European Nucleotide Archive with the accession number PRJEB19963.

Targeted expression of an EGFRCA induced larval death and malformation of the tracheal epithelium

We focused on EGFR as an oncogenic driver to initiate a tumor phenotype and induced ectopic expression of a constitutively active EGFR isoform (17) exclusively in the airway epithelium of Drosophila larvae. To address the larval tracheal system, we used two different airway-specific Gal4 driver lines, ppk4-Gal4 and btl-Gal4 (11, 13, 14). Ectopic expression of EGFRCA by the ppk4-Gal4 driver line for a moderate EGFRCA expression induced death not before the early 3rd instar stage (Fig. 1A and B). It is reasonable to assume that the lethality was caused by severe undersupply of oxygen. This interpretation is supported by the observation that, prior to death, these larvae left the lawn (day 4), a typical sign of oxygen deprivation (Fig. 1B; ref. 13). The control larvae left the lawn later to develop into pupae, a time point at which EGFRCA larvae were already dead (day 6; Fig. 1A). Use of the strong tracheal driver btl-Gal4 led to death in the embryonic stage. By using the Gal4 inhibitor, Gal80, we are able to tightly regulate target gene expression to a later time point in larval development (22). By this, early death was prevented but a significant increase in tracheal mass was observed in these larvae (Fig. 1C, c′).

Figure 1.

Expression of an EGFRCA leads to malformation of the tracheal epithelium and larval death. Drosophila culture tubes containing larvae after 4 or 6 days. A, Normal development and behavior of control larvae (ppk4-Gal4>w1118). B, EGFRCA larvae (ppk4-Gal4>UAS-EGFRCA) left the lawn at earlier developmental stages and died. C, Heat induced EGFRCA expression (c′, btl-Gal4, UAS-gfp; Gal80ts>UAS-EGFRCA) in the tracheal system is shown in the whole larvae after 5 days at 30°C, compared with the control (C, btl-Gal4, UAS-gfp; Gal80ts>UAS-EGFRCA). Scale bars, 100 μm. D–F, Tracheae isolated from larval controls were compared with those isolated from EGFRCA animals (d′–f′). D, Transmitted light images of the dissected tracheal system. Scale bars, 200 μm. E, Chitin layer of tracheal tubes, visualized by a conjugated chitin-binding probe. F, Cell membrane of epithelial cell membrane visualized by coracle staining. Transmitted light images of ppk4-Gal4>w1118 (G), ppk4-Gal4>UAS-EGFRCA (H, I), and btl-Gal4>UAS-hEGFR-dEGFR (J). Scale bars, 50 μm.

Figure 1.

Expression of an EGFRCA leads to malformation of the tracheal epithelium and larval death. Drosophila culture tubes containing larvae after 4 or 6 days. A, Normal development and behavior of control larvae (ppk4-Gal4>w1118). B, EGFRCA larvae (ppk4-Gal4>UAS-EGFRCA) left the lawn at earlier developmental stages and died. C, Heat induced EGFRCA expression (c′, btl-Gal4, UAS-gfp; Gal80ts>UAS-EGFRCA) in the tracheal system is shown in the whole larvae after 5 days at 30°C, compared with the control (C, btl-Gal4, UAS-gfp; Gal80ts>UAS-EGFRCA). Scale bars, 100 μm. D–F, Tracheae isolated from larval controls were compared with those isolated from EGFRCA animals (d′–f′). D, Transmitted light images of the dissected tracheal system. Scale bars, 200 μm. E, Chitin layer of tracheal tubes, visualized by a conjugated chitin-binding probe. F, Cell membrane of epithelial cell membrane visualized by coracle staining. Transmitted light images of ppk4-Gal4>w1118 (G), ppk4-Gal4>UAS-EGFRCA (H, I), and btl-Gal4>UAS-hEGFR-dEGFR (J). Scale bars, 50 μm.

Close modal

Dissection of the tissue from ppk4-Gal4>UAS-EGFRCA (EGFRCA) animals revealed a strong malformation of the whole airway structure, including thickened and shortened dorsal trunks with partially blocked airways depending on the progression of the phenotype (Fig. 1D, d′). It can be assumed that the larval death occurred because of these substantial structural changes of the airway system that prevented normal gas exchange (Fig. 1).

The chitinous inner tracheal lining, which is important for maintaining the tubular structure, was highly compressed in EGFRCA animals (Fig. 1E, e′). Staining of the septate junction protein, Coracle, illustrated that the cell–cell borders became irregular and frayed, implying that the tightness of the septate junctions was reduced and the tissue organization was impaired because of EGFRCA expression (Fig. 1F, f′). The epithelial layer of the airway trunks was thickened and almost completely destroyed in EGFRCA-expressing animals (Fig. 1H), compared with the thin epithelial layer in the control animals (Fig. 1G). Moreover, the air-conducting tubes were compressed and no longer air-filled, compared with the control trachea (Fig. 1I). Expression of a chimer of the extracellular human EGFR and the cytoplasmic domain of the Drosophila EGFR led to a similar set of phenotypes with the substantial epithelial thickening as the most distinctive one (Fig. 1J).

Meta- and hyperplasia of the tracheal epithelium

To quantify this hyperplasia of targeted EGFRCA expression in the tracheal system, we measured and quantified two parameters–the epithelial thickness and the number of nuclei per section of the dorsal trunk as a value that shows if cell divisions occurred (Fig. 2A–E). Trachea from larvae that experienced EGFRCA-expression had a significantly increased epithelial thickness that was seen throughout the entire tracheal system (8 ± 2.7 μm; Fig. 2A, a′, D), and an increased number of enlarged nuclei (22.5 ± 6.6; Fig. 2B, b′, E) observed in well-defined sections of the dorsal trunk, compared with matching control (5.9 ± 2.6 μm and 18.2 ± 2.5). In addition to these phenotypes, staining of the trachea with anti–phospho-histone H3 revealed substantial DNA synthesis in almost all airway epithelial cells (Fig. 2C, c′). To understand the molecular alterations that underlie these structural changes, we performed a RNA-seq analysis of manually isolated control trachea and those that express the EGFRCA. We identified 965 genes with a statistically significant increased abundance in transcript levels and 474 genes with a reduced abundance in EGFRCA-expressing trachea (Supplementary Table S1). An analysis of the differentially regulated genes revealed enriched pathways that include various metabolic ones including general metabolic pathways (P = 5.4E-6), sphingolipid metabolism (P = 1.6E-4), lysosome (P = 3.5E-3), or metabolism of xenobiotics (P = 3.8E-3) indicative for a hyperplastic transformation of these epithelial cells (Supplementary Table S2). Furthermore, we performed a Gene Ontology (GO) analysis with these differentially transcribed genes and found that molecular functions of epithelial cells, especially those devoted to chitin synthesis are enriched in the group of differentially transcribed genes (Fig. 2F). This cohort of differentially transcribed genes was also analyzed regarding their transcription factor–binding sites present in their presumptive promoter sequences. With pscan (focused on −450 to +50 bp relative to the transcription start site), we found a small set of significantly enriched transcription factor–binding sites (Fig. 2G). Most interestingly, Mad, a transcription factor of the TGFβ signaling pathway was the statistically best-supported, enriched binding site (Fig. 2G).

Figure 2.

Expression of EGFRCA leads to meta- and hyperplasia of the tracheal epithelium. Tracheae isolated from larval controls (ppk4-Gal4>w1118) were compared with those isolated from animals ectopically expressing EGFRCA (ppk4-Gal4>UAS-EGFRCA). A, Transmitted light images of the tracheal epithelium in the dorsal trunks. B, DAPI staining of cell nuclei. C, Mitotic activity of the nuclei, visualized by anti-pH3 staining. Quantitative evaluation of epithelial thickness (D) and number of nuclei (E). Statistical significance was evaluated using the Mann–Whitney test. n = 25–29, **, P < 0.01. Whiskers show mean values along with SD. Scale bars, 50 μm. F and G, RNA-seq analysis of differentially regulated genes in control and EGFRCA. F, Enriched molecular function Gene Ontology terms. G, Enriched transcription factor–binding sites, ordered by significance.

Figure 2.

Expression of EGFRCA leads to meta- and hyperplasia of the tracheal epithelium. Tracheae isolated from larval controls (ppk4-Gal4>w1118) were compared with those isolated from animals ectopically expressing EGFRCA (ppk4-Gal4>UAS-EGFRCA). A, Transmitted light images of the tracheal epithelium in the dorsal trunks. B, DAPI staining of cell nuclei. C, Mitotic activity of the nuclei, visualized by anti-pH3 staining. Quantitative evaluation of epithelial thickness (D) and number of nuclei (E). Statistical significance was evaluated using the Mann–Whitney test. n = 25–29, **, P < 0.01. Whiskers show mean values along with SD. Scale bars, 50 μm. F and G, RNA-seq analysis of differentially regulated genes in control and EGFRCA. F, Enriched molecular function Gene Ontology terms. G, Enriched transcription factor–binding sites, ordered by significance.

Close modal

Afatinib and gefitinib rescued larval death and malformation of the tracheal epithelium

Larvae of this specific Drosophila lung tumor model only develop to early larval stages and do not complete the development into pupal stage and adulthood (Fig. 3A). We took advantage of this lethal phenotype to screen large numbers of small compounds. Positive hits were identified by the occurrence of later developmental stages and survival of EGFRCA-expressing animals (Fig. 3A). The pharmacologic tests were performed in 96-well format (Fig. 3B).

Figure 3.

Schematic description of the large-scale drug screening approach and rescue effect of the lead hits afatinib and gefitinib. A, The untreated EGFRCA animals (ppk4-Gal4>UAS-EGFRCA) die on day 4 (top); positive hits show a rescue effect in survival and development to pupae and adults (bottom). The number of pupae on day 7 was used as final read out. B, Description of the experimental approach. The substances to be tested, the medium, as well as the experimental fly embryos are introduced into wells of a 96-well plate and incubated for 7 days. EGFRCA larvae leave the lawn, show the typical sign of oxygen deprivation and die = untreated animals or negative hits. EGFRCA larvae were able to develop into pupae = positive hit. C–F, Control (ppk4-Gal4>w1118) and EGFRCA (ppk4-Gal4>UAS-EGFRCA) embryos were treated with 100 μmol/L afatinib or gefitinib. The number of formed pupae after 7 days (C) or eclosed adults after 14 days (D) are expressed as a ratio relative to the initial number of embryos. Control animals (E) and EGFRCA animals (F) were treated with different gefitinib concentrations. G, Control and EGFRCA animals were treated with different afatinib or gefitinib concentrations from 0.1 to 100 μmol/L. Dashed lines represent controls. DMSO served as control. Statistical significance was tested using Mann–Whitney test. n = 4–6. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Whiskers show mean values along with SD (C–F) or SEM (G).

Figure 3.

Schematic description of the large-scale drug screening approach and rescue effect of the lead hits afatinib and gefitinib. A, The untreated EGFRCA animals (ppk4-Gal4>UAS-EGFRCA) die on day 4 (top); positive hits show a rescue effect in survival and development to pupae and adults (bottom). The number of pupae on day 7 was used as final read out. B, Description of the experimental approach. The substances to be tested, the medium, as well as the experimental fly embryos are introduced into wells of a 96-well plate and incubated for 7 days. EGFRCA larvae leave the lawn, show the typical sign of oxygen deprivation and die = untreated animals or negative hits. EGFRCA larvae were able to develop into pupae = positive hit. C–F, Control (ppk4-Gal4>w1118) and EGFRCA (ppk4-Gal4>UAS-EGFRCA) embryos were treated with 100 μmol/L afatinib or gefitinib. The number of formed pupae after 7 days (C) or eclosed adults after 14 days (D) are expressed as a ratio relative to the initial number of embryos. Control animals (E) and EGFRCA animals (F) were treated with different gefitinib concentrations. G, Control and EGFRCA animals were treated with different afatinib or gefitinib concentrations from 0.1 to 100 μmol/L. Dashed lines represent controls. DMSO served as control. Statistical significance was tested using Mann–Whitney test. n = 4–6. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Whiskers show mean values along with SD (C–F) or SEM (G).

Close modal

We screened a library containing 978 FDA-approved drugs at 50 μmol/L (Supplementary Table S3). The number of formed pupae was used as read out after 7 days of incubation (Fig. 3B). Three compounds out of the 978 (afatinib, gefitinib, and ibrutinib) were able to rescue larval lethality. Especially afatinib and gefitinib yielded impressive results in survival rate and formation of pupae and adult flies (Fig. 3E–G). The lethal phenotype induced by ectopic EGFRCA expression in the tracheal system was completely rescued (Fig. 3C–F). The majority of the animals not only survived to the last larval stage (3rd instar), but also completed their larval life, started pupation and developed into adult flies (Fig. 3C and D). Although gefitinib treatment rescued larval lethality, the percentage of adult offspring was significantly reduced in healthy control flies, implying that the compound itself had a negative effect on development during the pupal stage (Fig. 3E and F). Afatinib, on the other hand, induced an almost complete rescue, with normal percentages of adult offspring. Higher gefitinib concentrations led to a decreased number of adult flies not only in EGFRCA (Fig. 3F) but also in controls (Fig. 3E). The rescue effect became apparent at lower concentrations of 10 and 25 μmol/L (Fig. 3F). We used different doses of both compounds to identify the concentration ideal for rescuing the EGFR-induced larval lethality. Concentrations as low as 10 μmol/L were as effective as 100 μmol/L in rescuing development until the pupal stage (gefitinib), whereas the efficacy was reduced substantially if the concentration was reduced to 1 μmol/L or below (Fig. 3G).

Both afatinib and gefitinib could also completely reverse the structural phenotype observed in the trachea of the animals (Fig. 4), including malformation of the chitinous layer (Fig. 4A–D), cellular structure (Fig. 4a′–d′), epithelial thickness (Fig. 4a″–d″), and the nucleus number (Fig. 4a″′–d″′). With this, both meta- and hyperplasia were rescued in the treated animals, reflected by reduced epithelial thickness (gefitinib) and decreased numbers of nuclei (afatinib and gefitinib) down to a control level (Fig. 4E and F).

Figure 4.

Afatinib and gefitinib treatments rescue structural impairment and meta- and hyperplasia of the tracheal epithelium. DMSO-treated control larvae (ppk4-Gal4>w1118; A) were compared with DMSO-treated EGFRCA (ppk4-Gal4>UAS-EGFRCA; BD) and EGFRCA larvae treated with 100 μmol/L afatinib (C) or gefitinib (D). A–D, Transmitted light images of the epithelial thicknesses in the dorsal trunks (arrows). (a′-d′) DAPI staining of the nuclei. (a″-d″) Coracle staining of the cell membrane. (a″′-d″′) Chitin staining of the inner chitin layer of the tube. Scale bars, 50 μm. Quantification of the rescued epithelial thickness (E) and decreased nucleus number (F) after treatment with afatinib or gefitinib. Statistical significance was evaluated with Mann–Whitney test. n = 25–34. *, P < 0.05; ***, P < 0.001. Whiskers show mean values along with SD.

Figure 4.

Afatinib and gefitinib treatments rescue structural impairment and meta- and hyperplasia of the tracheal epithelium. DMSO-treated control larvae (ppk4-Gal4>w1118; A) were compared with DMSO-treated EGFRCA (ppk4-Gal4>UAS-EGFRCA; BD) and EGFRCA larvae treated with 100 μmol/L afatinib (C) or gefitinib (D). A–D, Transmitted light images of the epithelial thicknesses in the dorsal trunks (arrows). (a′-d′) DAPI staining of the nuclei. (a″-d″) Coracle staining of the cell membrane. (a″′-d″′) Chitin staining of the inner chitin layer of the tube. Scale bars, 50 μm. Quantification of the rescued epithelial thickness (E) and decreased nucleus number (F) after treatment with afatinib or gefitinib. Statistical significance was evaluated with Mann–Whitney test. n = 25–34. *, P < 0.05; ***, P < 0.001. Whiskers show mean values along with SD.

Close modal

Screening with a subtherapeutic afatinib dose identified bazedoxifene as a synergistically acting compound interfering in JAK/STAT signaling

To identify synergistically acting compounds that can rescue the lethal phenotype together with a tyrosine kinase inhibitor (TKI) such as afatinib, a secondary pharmacologic screening of the FDA-approved library compounds was performed. A concentration of 2 μmol/L afatinib, that is too low to show a rescue by itself, was combined with each of the 978 compounds from the library. Interestingly, seven compounds, which did not show any rescue effects in the primary screening, were newly identified as positive hits. A replication of the experiment with these seven compounds revealed bazedoxifene as being significantly potent in this combination (Fig. 5A). Although the effectiveness of bazedoxifene is not as high as from the specific EGFR inhibitors, it shows an unequivocal rescue effect in combination with low dosages of the specific EGFR inhibitor afatinib (25.6 ± 17.9%) compared with the control EGFRCA animals (2 μmol/L: 7.8 ± 3.9%; Fig. 5A).

Figure 5.

Low afatinib concentrations in a drug combination approach uncover synergistic operating drugs. Embryos of controls (ppk4-Gal4>w1118) and EGFRCA animals (ppk4-Gal4>UAS-EGFRCA) were treated with different drugs in different concentrations. The number of pupae, normalized against the initial number of embryos, was determined after 7 days. A, Embryos were treated with different compounds (specified below). The compounds (50 μmol/L) were supplemented with 2 μmol/L afatinib. DMSO (0.05%) served as negative control; 100 μmol/L afatinib served as positive control. The dashed line represents the basic pupation level by treatment with 2 μmol/L afatinib alone. Whiskers show minimum and maximum values. B, qRT-PCR experiment to show JAK/STAT pathway activation by constitutive EGFRCA expression in the tracheal system. Bars show mean values along with SD. Statistical significance was evaluated with Mann–Whitney test. n = 4–5, **, P < 0.01. C, Hypoxia-induced JAK/STAT activity in trachea from early 3rd instar larvae after treatment with bazedoxifene. Scale bars, 50 μm.

Figure 5.

Low afatinib concentrations in a drug combination approach uncover synergistic operating drugs. Embryos of controls (ppk4-Gal4>w1118) and EGFRCA animals (ppk4-Gal4>UAS-EGFRCA) were treated with different drugs in different concentrations. The number of pupae, normalized against the initial number of embryos, was determined after 7 days. A, Embryos were treated with different compounds (specified below). The compounds (50 μmol/L) were supplemented with 2 μmol/L afatinib. DMSO (0.05%) served as negative control; 100 μmol/L afatinib served as positive control. The dashed line represents the basic pupation level by treatment with 2 μmol/L afatinib alone. Whiskers show minimum and maximum values. B, qRT-PCR experiment to show JAK/STAT pathway activation by constitutive EGFRCA expression in the tracheal system. Bars show mean values along with SD. Statistical significance was evaluated with Mann–Whitney test. n = 4–5, **, P < 0.01. C, Hypoxia-induced JAK/STAT activity in trachea from early 3rd instar larvae after treatment with bazedoxifene. Scale bars, 50 μm.

Close modal

In the fly's airways, ectopic expression of EGFRCA was able to induce expression of the JAK/STAT ligands upd2 and upd3 (Fig. 5B), which seems to be part of a positive feedback loop (Fig. 5B). Thus, we evaluated whether bazedoxifene is able to inhibit hypoxia-induced activation of JAK/STAT signaling in the trachea of Drosophila larvae, using the 10X STAT reporter line (23). Indeed, treatment with bazedoxifene reduced this hypoxia-induced signal substantially (Fig. 5C).

In this study, we designed and evaluated an EGFRCA-induced Drosophila model for human lung cancer that is amenable to high throughput, whole-animal compound screening approaches. This strategy is in line with other successful high-throughput-screening (HTS) assays for specific cancer subtypes based on in vivo analysis of tailored Drosophila strains (24–27). A recent study by Levine and Cagan (15) demonstrated the previously unrealized potential of personalized Drosophila models to serve as whole-animal compound screening platforms to dramatically accelerate the identification of novel lead compounds for treatment of various types of lung cancer.

We could show ectopic activation of EGFR can induce lung tumor–like phenotypes in Drosophila. The observed phenotypes, primarily hyper- and metaplasia of airway epithelial cells, are not exact copies of the histologic features of human lung cancer. The tumor-like phenotype observed in the Drosophila airway system represents a very early stage of tumor development; therefore, a direct assignment to specific human lung tumors is not possible. As the induction of this phenotype is organ-wide, lethality in larval stages results from these early steps toward tumor formation. This highly appreciated phenotype that allows to set up straight-forward screening approaches thus permits at the same time a more sophisticated characterization of the tumor type. Upon oncogene induction, the normally differentiated epithelial cells of the dorsal trunks of the airway system started to proliferate, which resulted in the death of the larvae. Furthermore, the nuclei were positive for the mitosis marker pH3 and showed striking morphologic changes. The RNA-seq analysis of airway epithelia experiencing constitutive EGFR activation supported this interpretation. The gene ontologies that were enriched in differentially regulated genes imply that cells lost their epithelial cell identity. Furthermore, Mad binding sites were enriched in promoters of differentially regulated genes, which implies that TGFβ signaling was operative. TGFβ signaling is often causally related with a hyperplastic transformation from epithelial cells into other cell identities (28, 29). The observation that EGFR pathway activation induces structural changes in the fly's airway epithelium affirms that our model could also be adapted to other oncogenes downstream of the receptor, raising the possibility of generating panels of personalized fly models for human lung cancers with different driver mutations. EGFR signaling has previously been reported to be involved in maintaining organ homeostasis during development. This highly important signaling system contributes to epithelial integrity (30) in the tracheal system and it takes a central role in controlling tube elongation during development (31).

The fast life cycle and development of the fly is beneficial and provides a fast and feasible opportunity to test drugs in a whole-animal approach. Because we started treatment with the compounds of choice during the first larval stages, full rescue is seen only if the compounds are nontoxic and do not interfere with the complex development of a multicellular organism. Moreover, animals must pass through the pupal stage, which is highly susceptible to chemically induced impairment, to give rise to normal, fully functional adults. This requirement excludes compounds that nonspecifically interfere with major developmental processes. This scenario demonstrates the superiority of whole-animal screens over cell-based assays. Moreover, the design of the screen focuses only on those compounds rescuing lethality while being well tolerated even at high concentrations. Thus it allows false negative results to occur, which is taken into account, especially as these false positive hits would be due to undesirable properties of these compounds (e.g., high toxicity).

Mutations leading to elevated activity of EGFR signaling even in the absence of cognate ligands have been detected in the corresponding cancer subtypes (5, 32, 33). These mutations induce chronically activated signaling via the EGFR pathway, leading to hyper- and metaplasia of the corresponding cell, and ultimately to cancer development. In the screen described here, we tested the compounds of a comprehensive library containing FDA-approved drugs. The mutation A887T of the Drosophila EGFR is located in the N-lobe of the tyrosine kinase domain and thus corresponds to activating mutations of the human EGFR in exons 18 and 19, such as the deletions in the 747-752 region or the G719S substitution (34). Three small compounds, which are already commonly used, were effective in rescuing the lethal phenotype and therefore validated our screening system. These lead hits belong to the class of specific TKIs. Besides the specific EGFR inhibitors, the Brutons tyrosine kinase (Btk) inhibitor, ibrutinib, turned out as positive hit. This finding is in line with recent studies showing that ibrutinib is able to interact with EGFR at higher compound concentrations (35). Despite their shared ability to rescue EGFRCA-induced lethality and structural epithelial changes, detailed characterization of afatinib and gefitinib exhibited striking differences. Treatment with the second-generation TKI, afatinib, induced complete rescue of the phenotype, because seemingly normal adults emerged after treatment. In contrast, treatment with the first-generation TKI, gefitinib, rescued the larval lethality, but very few adults emerged. The monitored phenotype was consistent with earlier described adverse effects on adult structures (36). Differing activities of afatinib and gefitinib are also observed in human patients with lung cancer, with the former conferring longer median survival relative to the latter (37). However, the improved progression-free survival in patients, treated with afatinib, is accompanied with side effects in a higher extent.

In the second screening approach, we combined a subtherapeutic afatinib concentration with the compounds from the library and could identify additional positive hits. Bazedoxifene was confirmed to rescue the lethal phenotype in our EGFR-induced tumor model in combination with this low afatinib concentration. Bazedoxifene is approved as a selective estrogen receptor modulator and is commonly used in preventing osteoporosis. Recently, it was discovered as a novel promising GP130/STAT3 pathway signaling inhibitor (38) and considered for treatment in pancreatic cancer (39). Although the combined inhibition of EGFR- and JAK/STAT signaling has already been shown to represent a valuable strategy for a number of different cancer entities (40, 41), its application for therapies is still limited. Such a cross-talk was found to be operative in an epithelial tumor model (42) and studies employing Drosophila found a robust interaction between both signaling systems (43). The interaction between EGFR and STAT signaling often employs cytokines of the JAK/STAT signaling pathway that are released upon EGFR pathway activation (44). A similar situation is found in our model, where we observed a massive upregulation of upd2 and upd3, which both code for ligands of the JAK/STAT signaling pathways. Synergistically targeting EGFR and JAK/STAT pathway for cancer therapy is intriguing in two ways: (i) because the IL6, or more generally the gp130 signaling pathway correlated with metastasis and epithelial–mesenchymal transition in lung cancer (45), this metastatic behavior might be dampened, and (ii) IL6-mediated signaling plays an important role in the development of treatment resistance, which can be specifically targeted by a combined approach (44, 46, 47). Thus, the combination of a TKI and a JAK/STAT inhibitor could be a promising and potential drug in treating lung cancer (48). The observation that EGFR pathway activation exerts some of its effects via the JAK/STAT signaling pathway is in line with the positive effects of the afatinib/bazedoxifene combination observed in this study. Thus, targeting both, the EGFR and the JAK/STAT pathway at the same time would be a valuable strategy for the treatment of NSCLC with an EGFR mutation. With the here established EGFRCA-induced tumor model, we offer another possibility to use Drosophila in a HTS approach to identify unknown compounds with inhibiting effect on EGFR. Furthermore, it holds the promising potential to newly identify FDA-approved compounds that act synergistically with TKIs. Thereby, TKIs could be used in low concentrations with fewer side effects while exerting the full inhibitory potential by intervening with other pathways to eventually improve clinical outcome and success for patients with lung cancer.

No potential conflicts of interest were disclosed.

Conception and design: J. Bossen, R. Pfefferkorn, C. Fink, H. Heine, T. Roeder

Development of methodology: J. Bossen, K. Uliczka, R. Pfefferkorn, Mandy M.-Q. Mai, L. Burkhardt, H. Heine, T. Roeder

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Bossen, K. Uliczka, L. Steen, L. Burkhardt

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Bossen, K. Uliczka, Mandy M.-Q. Mai, M. Spohn, H. Heine

Writing, review, and/or revision of the manuscript: J. Bossen, K. Uliczka, R. Pfefferkorn, I. Bruchhaus, H. Heine, T. Roeder

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Bossen, R. Pfefferkorn

Study supervision: T. Roeder

The work was funded by grants of the Bundesministerium für Forschung und Bildung as part of program “Alternativen zum Tierversuch” (031L0110A - DroLuCa) and the Deutsche Forschungsgemeinschaft (DFG) as part of the Exc Inflammation at Interfaces to T. Roeder. We would like to thank Britta Laubenstein and Christiane Sandberg for excellent technical assistance as well as the Bloomington Stock Center, Mike Welsh (Iowa), and Maria Leptin (Heidelberg, Germany) for flies.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Stewart
BW
,
Wild
CP
,
editors
.
World Cancer Report
2014
.
Lyon, France: WHO Press
; 
2014
.
2.
Ding
L
,
Getz
G
,
Wheeler
DA
,
Mardis
ER
,
McLellan
MD
,
Cibulskis
K
, et al
Somatic mutations affect key pathways in lung adenocarcinoma
.
Nature
2008
;
455
:
1069
75
.
3.
Kandoth
C
,
McLellan
MD
,
Vandin
F
,
Ye
K
,
Niu
B
,
Lu
C
, et al
Mutational landscape and significance across 12 major cancer types
.
Nature
2013
;
502
:
333
9
.
4.
Rotow
J
,
Bivona
TG
. 
Understanding and targeting resistance mechanisms in NSCLC
.
Nat Rev Cancer
2017
;
17
:
637
58
.
5.
Sharma
SV
,
Bell
DW
,
Settleman
J
,
Haber
DA
. 
Epidermal growth factor receptor mutations in lung cancer
.
Nat Rev Cancer
2007
;
7
:
169
81
.
6.
Wu
SG
,
Shih
JY
. 
Management of acquired resistance to EGFR TKI-targeted therapy in advanced non-small cell lung cancer
.
Mol Cancer
2018
;
17
:
38
.
7.
Roeder
T
,
Isermann
K
,
Kallsen
K
,
Uliczka
K
,
Wagner
C
. 
A Drosophila asthma model - what the fly tells us about inflammatory diseases of the lung
.
Adv Exp Med Biol
2012
;
710
:
37
47
.
8.
Behr
M
. 
Molecular aspects of respiratory and vascular tube development
.
Respir Physiol Neurobiol
2010
;
173
:
S33
6
.
9.
Ghabrial
A
,
Luschnig
S
,
Metzstein
MM
,
Krasnow
MA
. 
Branching morphogenesis of the Drosophila tracheal system
.
Annu Rev Cell Dev Biol
2003
;
19
:
623
47
.
10.
Wagner
C
,
Isermann
K
,
Fehrenbach
H
,
Roeder
T
. 
Molecular architecture of the fruit fly's airway epithelial immune system
.
BMC Genomics
2008
;
9
:
446
.
11.
Wagner
C
,
Isermann
K
,
Roeder
T
. 
Infection induces a survival program and local remodeling in the airway epithelium of the fly
.
FASEB J
2009
;
23
:
2045
54
.
12.
Pfeiffer
BD
,
Ngo
TT
,
Hibbard
KL
,
Murphy
C
,
Jenett
A
,
Truman
JW
, et al
Refinement of tools for targeted gene expression in Drosophila
.
Genetics
2010
;
186
:
735
55
.
13.
Kallsen
K
,
Zehethofer
N
,
Abdelsadik
A
,
Lindner
B
,
Kabesch
M
,
Heine
H
, et al
ORMDL deregulation increases stress responses and modulates repair pathways in Drosophila airways
.
J Allergy Clin Immunol
2015
;
136
:
1105
8
.
14.
Roeder
T
,
Isermann
K
,
Kabesch
M
. 
Drosophila in asthma research
.
Am J Respir Crit Care Med
2009
;
179
:
979
83
.
15.
Levine
BD
,
Cagan
RL
. 
Drosophila lung cancer models identify trametinib plus statin as candidate therapeutic
.
Cell Rep
2016
;
14
:
1477
87
.
16.
Brand
AH
,
Perrimon
N
. 
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes
.
Development
1993
;
118
:
401
15
.
17.
Lesokhin
AM
,
Yu
SY
,
Katz
J
,
Baker
NE
. 
Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila
.
Dev Biol
1999
;
205
:
129
44
.
18.
Read
RD
,
Cavenee
WK
,
Furnari
FB
,
Thomas
JB
. 
A drosophila model for EGFR-Ras and PI3K-dependent human glioma
.
PLoS Genet
2009
;
5
:
e1000374
.
19.
Prange
R
,
Thiedmann
M
,
Bhandari
A
,
Mishra
N
,
Sinha
A
,
Hasler
R
, et al
A Drosophila model of cigarette smoke induced COPD identifies Nrf2 signaling as an expedient target for intervention
.
Aging
2018
;
10
:
2122
35
.
20.
Lyne
R
,
Smith
R
,
Rutherford
K
,
Wakeling
M
,
Varley
A
,
Guillier
F
, et al
FlyMine: an integrated database for Drosophila and Anopheles genomics
.
Genome Biol
2007
;
8
:
R129
.
21.
Zambelli
F
,
Pesole
G
,
Pavesi
G
. 
Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes
.
Nucleic Acids Res
2009
;
37
:
W247
52
.
22.
McGuire
SE
,
Mao
Z
,
Davis
RL
. 
Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila
.
Sci STKE
2004
;
2004
:
pl6
.
23.
Bach
EA
,
Ekas
LA
,
Ayala-Camargo
A
,
Flaherty
MS
,
Lee
H
,
Perrimon
N
, et al
GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo
.
Gene Expr Patterns
2007
;
7
:
323
31
.
24.
Levinson
S
,
Cagan
RL
. 
Drosophila cancer models identify functional differences between ret fusions
.
Cell Rep
2016
;
16
:
3052
61
.
25.
Das
T
,
Cagan
R
. 
Drosophila as a novel therapeutic discovery tool for thyroid cancer
.
Thyroid
2010
;
20
:
689
95
.
26.
Willoughby
LF
,
Schlosser
T
,
Manning
SA
,
Parisot
JP
,
Street
IP
,
Richardson
HE
, et al
An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery
.
Dis Model Mech
2013
;
6
:
521
9
.
27.
Markstein
M
,
Dettorre
S
,
Cho
J
,
Neumuller
RA
,
Craig-Muller
S
,
Perrimon
N
. 
Systematic screen of chemotherapeutics in Drosophila stem cell tumors
.
Proc Natl Acad Sci U S A
2014
;
111
:
4530
5
.
28.
Buonato
JM
,
Lan
IS
,
Lazzara
MJ
. 
EGF augments TGFbeta-induced epithelial-mesenchymal transition by promoting SHP2 binding to GAB1
.
J Cell Sci
2015
;
128
:
3898
909
.
29.
Massague
J
. 
TGFbeta in Cancer
.
Cell
2008
;
134
:
215
30
.
30.
Cela
C
,
Llimargas
M
. 
Egfr is essential for maintaining epithelial integrity during tracheal remodelling in Drosophila
.
Development
2006
;
133
:
3115
25
.
31.
Olivares-Castineira
I
,
Llimargas
M
. 
EGFR controls Drosophila tracheal tube elongation by intracellular trafficking regulation
.
PLoS Genet
2017
;
13
:
e1006882
.
32.
Rosell
R
,
Carcereny
E
,
Gervais
R
,
Vergnenegre
A
,
Massuti
B
,
Felip
E
, et al
Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial
.
Lancet Oncol
2012
;
13
:
239
46
.
33.
Siegelin
MD
,
Borczuk
AC
. 
Epidermal growth factor receptor mutations in lung adenocarcinoma
.
Lab Invest
2014
;
94
:
129
37
.
34.
Irmer
D
,
Funk
JO
,
Blaukat
A
. 
EGFR kinase domain mutations - functional impact and relevance for lung cancer therapy
.
Oncogene
2007
;
26
:
5693
701
.
35.
Wang
A
,
Yan
XE
,
Wu
H
,
Wang
W
,
Hu
C
,
Chen
C
, et al
Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation
.
Oncotarget
2016
;
7
:
69760
9
.
36.
Aritakula
A
,
Ramasamy
A
. 
Drosophila-based in vivo assay for the validation of inhibitors of the epidermal growth factor receptor/Ras pathway
.
J Biosci
2008
;
33
:
731
42
.
37.
Park
K
,
Tan
EH
,
O'Byrne
K
,
Zhang
L
,
Boyer
M
,
Mok
T
, et al
Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial
.
Lancet Oncol
2016
;
17
:
577
89
.
38.
Li
H
,
Xiao
H
,
Lin
L
,
Jou
D
,
Kumari
V
,
Lin
J
, et al
Drug design targeting protein-protein interactions (PPIs) using multiple ligand simultaneous docking (MLSD) and drug repositioning: discovery of raloxifene and bazedoxifene as novel inhibitors of IL-6/GP130 interface
.
J Med Chem
2014
;
57
:
632
41
.
39.
Chen
X
,
Tian
J
,
Su
GH
,
Lin
J
. 
Blocking IL-6/GP130 signaling inhibits cell viability/proliferation, glycolysis, and colony forming activity in human pancreatic cancer cells
.
Curr Cancer Drug Targets
2018
;
19
:
417
27
.
40.
Quesnelle
KM
,
Boehm
AL
,
Grandis
JR
. 
STAT-mediated EGFR signaling in cancer
.
J Cell Biochem
2007
;
102
:
311
9
.
41.
Wen
W
,
Wu
J
,
Liu
L
,
Tian
Y
,
Buettner
R
,
Hsieh
MY
, et al
Synergistic anti-tumor effect of combined inhibition of EGFR and JAK/STAT3 pathways in human ovarian cancer
.
Mol Cancer
2015
;
14
:
100
.
42.
Herranz
H
,
Hong
X
,
Hung
NT
,
Voorhoeve
PM
,
Cohen
SM
. 
Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model
.
Genes Dev
2012
;
26
:
1602
11
.
43.
Heigwer
F
,
Scheeder
C
,
Miersch
T
,
Schmitt
B
,
Blass
C
,
Pour Jamnani
MV
, et al
Time-resolved mapping of genetic interactions to model rewiring of signaling pathways
.
Elife
2018
;
7
.
doi: 10.7554/eLife.40174
.
44.
Ray
K
,
Ujvari
B
,
Ramana
V
,
Donald
J
. 
Cross-talk between EGFR and IL-6 drives oncogenic signaling and offers therapeutic opportunities in cancer
.
Cytokine Growth Factor Rev
2018
;
41
:
18
27
.
45.
Shang
GS
,
Liu
L
,
Qin
YW
. 
IL-6 and TNF-alpha promote metastasis of lung cancer by inducing epithelial-mesenchymal transition
.
Oncol Lett
2017
;
13
:
4657
60
.
46.
Kim
SM
,
Kwon
OJ
,
Hong
YK
,
Kim
JH
,
Solca
F
,
Ha
SJ
, et al
Activation of IL-6R/JAK1/STAT3 signaling induces de novo resistance to irreversible EGFR inhibitors in non-small cell lung cancer with T790M resistance mutation
.
Mol Cancer Ther
2012
;
11
:
2254
64
.
47.
Gao
SP
,
Chang
Q
,
Mao
N
,
Daly
LA
,
Vogel
R
,
Chan
T
, et al
JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors
.
Sci Signal
2016
;
9
:
ra33
.
48.
Codony-Servat
C
,
Codony-Servat
J
,
Karachaliou
N
,
Molina
MA
,
Chaib
I
,
Ramirez
JL
, et al
Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC)
.
Oncotarget
2017
;
8
:
47305
16
.