Paraneoplastic syndromes arise when tumor-derived cytokines reprogram distant organs. Although mediators such as Interleukin-6 have been implicated, how these signals impair host organ function remains incompletely defined. Here, we identify a cytokine-lipid axis that drives hepatic autophagy dysfunction. Specifically, in Drosophila, the gut tumor-derived interleukin-like cytokine Upd3 induces the expression of the triglyceride lipase CG5966, which we named "cancer-associated lipid mobilizer (calm)", and the ceramide synthase schlank in the fat body. This upregulation rewires fat body lipid metabolism, resulting in an autophagic-flux blockade. Genetic reduction of either CG5966 or schlank restores organelle homeostasis and mitigates paraneoplastic phenotypes. This mechanism is conserved in mammals: in mice, IL-6 upregulates the lipoprotein lipase Lpl and ceramide synthases which in turn trigger a hepatic autophagy-flux blockade; in humans, hepatic LPL and ceramide synthases expression correlates with poorer survival in hepatocellular carcinoma. Our findings position hepatic lipid metabolism rewiring, especially ceramide synthesis as a critical, conserved node coupling systemic inflammation to organelle dysfunction, and suggest this pathway as a possible therapeutic entry point for cancer-associated liver disorders.

Stem cells are essential for tissue maintenance and regeneration, balancing self-renewal and differentiation to support homeostasis and repair. Through an RNAi screen in the Drosophila midgut, we identified the epigenetic factor Zrf1 as a critical regulator of intestinal stem cell (ISC) proliferation. Functional analyses reveal that Zrf1 integrates inputs from multiple signaling pathways and interacts with components of the RNA-induced silencing complex (RISC). Our findings suggest that Zrf1 is potentially a key chromatin regulator necessary for maintaining stem cell proliferation, enhancing our understanding of the molecular controls underlying stem cell function and chromatin-associated defects.

Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster . Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the ‘FlyBi’ dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings ( dwg ) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.

Metabolic flexibility of muscle tissue describes the adaptive capacity to use different energy substrates according to their availability. The disruption of this ability associates with metabolic disease. Here, using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, we show that the transcription factor REPTOR is an important regulator of energy metabolism in muscles. We present evidence that REPTOR is activated in muscles of adult flies with gut yorkie-tumors, where it modulates glucose metabolism. Further, in vivo studies indicate that sustained activity of REPTOR is sufficient in wildtype muscles to repress glycolysis and increase tricarboxylic acid (TCA) cycle metabolites. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting oxidative metabolism. Altogether, our results define a conserved function for REPTOR and CREBRF as key regulators of muscle energy metabolism.

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation.

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo . Here, we characterize two short (<15 aa) NanoTag epitopes, 127D01 and VHH05) and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m(6)A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m(6)A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m(6)A RNA methylation and demonstrates their roles in suppressing autophagy.

Significance Methionine metabolism is dramatically affected by age and restricting methionine consumption extends the lifespan across different species. However, we have limited information on the activity of different branches of methionine metabolism during aging. Furthermore, how tissue-specific perturbation of methionine metabolism affects aging is not known. Here, by feeding 13 C5-methionine to Drosophila and tracing its fate in different branches of the methionine metabolism, we were able to identify impairments of methionine flux associated with aging. In addition, to deplete methionine in a tissue-specific manner, we created transgenic flies that express Methioninase . Strikingly, Methioninase expression even in a single tissue can dramatically extend the health- and lifespan.

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1 . We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1 . We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1 , Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1- deficient cells that may be further adapted for the treatment of human cancer.

The authors have requested that this preprint be removed from Research Square.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

yki-induced gut tumors in Drosophila are associated with host wasting, including muscle dysfunction, lipid loss, and hyperglycemia, a condition reminiscent of human cancer cachexia. We previously used this model to identify tumor-derived ligands that contribute to host wasting. To identify additional molecular networks involved in host-tumor interactions, we develop PathON, a web-based tool analyzing the major signaling pathways in Drosophila, and uncover the Upd3/Jak/Stat axis as an important modulator. We find that yki-gut tumors secrete Upd3 to promote self-overproliferation and enhance Jak/Stat signaling in host organs to cause wasting, including muscle dysfunction, lipid loss, and hyperglycemia. We further reveal that Upd3/Jak/Stat signaling in the host organs directly triggers the expression of ImpL2, an antagonistic binding protein for insulin-like peptides, to impair insulin signaling and energy balance. Altogether, our results demonstrate that yki-gut tumors produce a Jak/Stat pathway ligand, Upd3, that regulates both self-growth and host wasting.

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila . In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resemble the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins suggesting that Cysts in an integral component of the apical polarity protein network. Cysts activates Rho at adherens junctions to promote junctional enrichment of myosin II, which requires the RhoGEF domain and the coiled-coil domain containing C-terminal region of Cysts. Cysts recruitment to the apico-lateral cortex depends on Crumbs and Bazooka/Par3 and requires multiple domains within Cysts including the C-terminal region. Together, our findings indicate that Cysts links apical polarity proteins to Rho1 and myosin activation at adherens junctions to support junctional and epithelial integrity in the Drosophila ectoderm.

Interactions between tumors and host tissues play essential roles in tumor-induced systemic wasting and cancer cachexia, including muscle wasting and lipid loss. However, the pathogenic molecular mechanisms of wasting are still poorly understood. Using a fly model of tumor-induced organ wasting, we observed aberrant MEK activation in both tumors and host tissues of flies bearing gut-yki3SA tumors. We found that host MEK activation results in muscle wasting and lipid loss, while tumor MEK activation is required for tumor growth. Strikingly, host MEK suppression alone is sufficient to abolish the wasting phenotypes without affecting tumor growth. We further uncovered that yki3SA tumors produce the vein (vn) ligand to trigger autonomous Egfr/MEK-induced tumor growth and produce the PDGF- and VEGF-related factor 1 (Pvf1) ligand to non-autonomously activate host Pvr/MEK signaling and wasting. Altogether, our results demonstrate the essential roles and molecular mechanisms of differential MEK activation in tumor-induced host wasting.

Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.

[This corrects the article DOI: 10.1371/journal.pone.0152259.].

Aging is a risk factor for many human pathologies and is characterized by extensive metabolic changes. Using targeted high-throughput metabolite profiling in Drosophila melanogaster at different ages, we demonstrate that methionine metabolism changes strikingly during aging. Methionine generates the methyl donor S-adenosyl-methionine (SAM), which is converted via methylation to S-adenosyl-homocysteine (SAH), which accumulates during aging. A targeted RNAi screen against methionine pathway components revealed significant life span extension in response to down-regulation of two noncanonical Drosophila homologs of the SAH hydrolase Ahcy (S-adenosyl-L-homocysteine hydrolase [SAHH[), CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2, which act as dominant-negative regulators of canonical AHCY. Importantly, tissue-specific down-regulation of dAhcyL1/L2 in the brain and intestine extends health and life span. Furthermore, metabolomic analysis of dAhcyL1-deficient flies revealed its effect on age-dependent metabolic reprogramming and H3K4 methylation. Altogether, reprogramming of methionine metabolism in young flies and suppression of age-dependent SAH accumulation lead to increased life span. These studies highlight the role of noncanonical Ahcy enzymes as determinants of healthy aging and longevity.

Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4-8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.