Recurrent Phases of Strict Protein Limitation Inhibit Tumor Growth and Restore Lifespan in A Drosophila Intestinal Cancer Model.

Diets that restrict caloric or protein intake offer a variety of benefits, including decreasing the incidence of cancer. However, whether such diets pose a substantial therapeutic benefit as auxiliary cancer treatments remains unclear. We determined the effects of severe protein depletion on tumorigenesis in a Drosophila melanogaster intestinal tumor model, using a human RAF gain-of-function allele. Severe and continuous protein restriction significantly reduced tumor growth but resulted in premature death. Therefore, we developed a diet in which short periods of severe protein restriction alternated cyclically with periods of complete feeding. This nutritional regime reduced tumor mass, restored gut functionality, and rescued the lifespan of oncogene-expressing flies to the levels observed in healthy flies on a continuous, fully nutritious diet. Furthermore, this diet reduced the chemotherapy-induced stem cell activity associated with tumor recurrence. Transcriptome analysis revealed long-lasting changes in the expression of key genes involved in multiple major developmental signaling pathways. Overall, the data suggest that recurrent severe protein depletion effectively mimics the health benefits of continuous protein restriction, without undesired nutritional shortcomings. This provides seminal insights into the mechanisms of the memory effect required to maintain the positive effects of protein restriction throughout the phases of a full diet. Finally, the repetitive form of strict protein restriction is an ideal strategy for adjuvant cancer therapy that is useful in many tumor contexts.

The tracheal immune system of insects - A blueprint for understanding epithelial immunity.

The unique design of respiratory organs in multicellular organisms makes them prone to infection by pathogens. To cope with this vulnerability, highly effective local immune systems evolved that are also operative in the tracheal system of insects. Many pathogens and parasites (including viruses, bacteria, fungi, and metazoan parasites) colonize the trachea or invade the host via this route. Currently, only two modules of the tracheal immune system have been characterized in depth: 1) Immune deficiency pathway-mediated activation of antimicrobial peptide gene expression and 2) local melanization processes that protect the structure from wounding. There is an urgent need to increase our understanding of the architecture of tracheal immune systems, especially regarding those mechanisms that enable the maintenance of immune homeostasis. This need for new studies is particularly exigent for species other than Drosophila.

Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila.

Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.

Airway remodeling: The Drosophila model permits a purely epithelial perspective.

Airway remodeling is an umbrella term for structural changes in the conducting airways that occur in chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). The pathobiology of remodeling involves multiple mesenchymal and lymphoid cell types and finally leads to a variety of hardly reversible changes such as hyperplasia of goblet cells, thickening of the reticular basement membrane, deposition of collagen, peribronchial fibrosis, angiogenesis and hyperplasia of bronchial smooth muscle cells. In order to develop solutions for prevention or innovative therapies, these complex processes must be understood in detail which requires their deconstruction into individual building blocks. In the present manuscript we therefore focus on the role of the airway epithelium and introduce Drosophila melanogaster as a model. The simple architecture of the flies' airways as well as the lack of adaptive immunity allows to focus exclusively on the importance of the epithelium for the remodeling processes. We will review and discuss genetic and environmentally induced changes in epithelial structures and molecular responses and propose an integrated framework of research for the future.

Low-protein diet applied as part of combination therapy or stand-alone normalizes lifespan and tumor proliferation in a model of intestinal cancer.

Tumors of the intestinal tract are among the most common tumor diseases in humans, but, like many other tumor entities, show an unsatisfactory prognosis with a need for effective therapies. To test whether nutritional interventions and a combination with a targeted therapy can effectively cure these cancers, we used the fruit fly Drosophila as a model. In this system, we induced tumors by EGFR overexpression in intestinal stem cells. Limiting the amount of protein in the diet restored life span to that of control animals. In combination with a specific EGFR inhibitor, all major tumor-associated phenotypes could be rescued. This form of treatment was also successful in a real treatment scenario, which means when they started after the full tumor phenotype was expressed. In conclusion, reduced protein administration can be a very promising form of adjuvant cancer therapy.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

Driver mutations in major lung cancer oncogenes can be analyzed in Drosophila models

Lung cancer remains the leading cause of cancer-associated mortality. Despite recent promising achievements, the overall prognosis remains very poor. In order to integrate the advantages of adapted, transgenic animal models with a high-throughput procedure on the one hand and compliance with the 3R principles on the other hand, we have established and evaluated appropriate Drosophila models. To achieve this goal, we ectopically expressed oncogenes representing the most important driver mutations exclusively in the airway system. These oncogenes were either the human oncogenes or the corresponding Drosophila orthologs. We concentrated on two complementary read-out systems, 1) early larval lethality and 2) quantification of concurrently expressed GFP as a proxy for tumor mass. We could show that ectopic expression of EgfrCA, RasV12, Raf, Rolled (MAPK), PI3K92E, Alk, Akt and Arm can induce early lethality. Thus, they can be used in a straight-forward high-throughput screening approach and can replace mouse models to a considerable extent. Moreover, we could also show that measurement of tumor mass by a concurrently expressed marker (GFP) can be used to detect positive treatment results. Our results show that our Drosophila system provides a superb in vivo inver­tebrate screening system amenable to high-throughput approaches and thus effectively complements the toolbox for the development of novel anti-lung cancer treatments, while complying with the 3R principles.

An EGFR-Induced Drosophila Lung Tumor Model Identifies Alternative Combination Treatments.

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.