Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.
Understanding human gene function is fundamental to understanding and treating diseases. Research using the model organism benefits from a wealth of molecular genetic resources and information useful for efficient experimentation. Moreover, offers a balance as a relatively simple organism that nonetheless exhibits complex multicellular activities. Recent examples demonstrate the power and continued promise of research to further our understanding of conserved gene functions.
In humans, cancer-associated cachexia is a complex syndrome that reduces the overall quality of life and survival of cancer patients, particularly for those undergoing chemotherapy. The most easily observable sign of cachexia is organ wasting, the dramatic loss of skeletal muscle and adipose tissue mass. Estimates suggest that 80% of patients in advanced stages of cancer show signs of the syndrome and about 20% of cancer patients die directly of cachexia. Because there is no treatment or drug available to ameliorate organ wasting induced by cancer, cachexia is a relevant clinical problem. However, it is unclear how cachexia is mediated, what factors drive interactions between tumors and host tissues, and which markers of cachexia might be used to allow early detection before the observable signs of organ wasting. In this chapter, we review the current mammalian models of cachexia and the need to use new models of study. We also explain recent developments in Drosophila as a model for studying organ wasting induced by tumors and how fly studies can help unravel important mechanisms that drive cachexia. In particular, we discuss what lessons have been learned from tumor models recently reported to induce systemic organ wasting in Drosophila.
Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S-adenosylmethionine, which, after transferring its methyl group, is converted to S-adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.
Secreted molecules coordinate organ function. In a recent issue of Cell, Hudry et al. (2019) uncover a Drosophila testis-midgut interaction via cytokine and citrate signaling that regulates intestinal metabolism, spermatogenesis, and food intake. This impressive study is a striking example of the role of spatial organization in sex-specific interorgan communication.
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 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.
Metabolic homeostasis is under strict regulation of humoral factors across various taxa. In particular, insulin and glucagon, referred to in Drosophila as Drosophila insulin-like peptides (DILPs) and adipokinetic hormone (AKH), respectively, are key hormones that regulate metabolism in most metazoa. While much is known about the regulation of DILPs, the mechanisms regulating AKH/glucagon production is still poorly understood. In this review, we describe the various factors that regulate the production of DILPs and AKH and emphasize the need for future studies to decipher how energy homeostasis is governed in Drosophila. This article is categorized under: Invertebrate Organogenesis > Flies Signaling Pathways > Global Signaling Mechanisms.
Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis.
Mitochondrial abundance and function are tightly controlled during metabolic adaptation but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5' untranslated region of its gene (PPARGC1A). We find that uORF-mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function.
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 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 . 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.
Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we combine 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation hotspot regions within domain families. Mapping these regions onto structural data reveals that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the ribosomal S11 domain in the yeast ribosomal protein uS11 shows impaired growth and defective cytoplasmic 20S pre-rRNA processing at 16 °C and 20 °C. Altogether, our study identifies phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families.
BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.
Stem cells continuously perceive and respond to various environmental signals during development, tissue homeostasis, and pathological conditions. Mechanical force, one of the fundamental signals in the physical world, plays a vital role in the regulation of multiple functions of stem cells. The importance of cell adhesion to the extracellular matrix (ECM), cell-cell junctions, and a mechanoresponsive cell cytoskeleton has been under intensive study in the fields of stem cell biology and mechanobiology. However, the involvement of mechanosensitive (MS) ion channels in the mechanical regulation of stem cell activity has just begun to be realized. Here, we review the diversity and importance of mechanosensitive channels (MSCs), and discuss recently discovered functions of MSCs in stem cell regulation, especially in the determination of cell fate.
Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, we identified the septate junction (SJ) protein tetraspanin 2A (Tsp2A) as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. We show that when ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, our study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished.
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-yki 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 yki 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.
Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing their stability, interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, most commonly serine, threonine and tyrosine in metazoans. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that any given phosphorylation site might be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for At iProteinDB, scientists can view the PTM landscape for any protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related species. Further, iProteinDB enables comparison of PTM data from to that of orthologous proteins from other model organisms, including human, mouse, rat, , and
Epithelial homeostasis requires the precise balance of epithelial stem/progenitor proliferation and differentiation. While many signaling pathways that regulate epithelial stem cells have been identified, it is probable that other regulators remain unidentified. Here, we use gene-expression profiling by targeted DamID to identify the stem/progenitor-specific transcription and signaling factors in the midgut. Many signaling pathway components, including ligands of most major pathways, exhibit stem/progenitor-specific expression and have regulatory regions bound by both intrinsic and extrinsic transcription factors. In addition to previously identified stem/progenitor-derived ligands, we show that both the insulin-like factor Ilp6 and TNF ligand eiger are specifically expressed in the stem/progenitors and regulate normal tissue homeostasis. We propose that intestinal stem cells not only integrate multiple signals but also contribute to and regulate the homeostatic signaling microenvironmental niche through the expression of autocrine and paracrine factors.
The circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. We tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. Our results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and we find that the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging.
FlyBase (flybase.org) is a knowledge base that supports the community of researchers that use the fruit fly, Drosophila melanogaster, as a model organism. The FlyBase team curates and organizes a diverse array of genetic, molecular, genomic, and developmental information about Drosophila. At the beginning of 2018, 'FlyBase 2.0' was released with a significantly improved user interface and new tools. Among these important changes are a new organization of search results into interactive lists or tables (hitlists), enhanced reference lists, and new protein domain graphics. An important new data class called 'experimental tools' consolidates information on useful fly strains and other resources related to a specific gene, which significantly enhances the ability of the Drosophila researcher to design and carry out experiments. With the release of FlyBase 2.0, there has also been a restructuring of backend architecture and a continued development of application programming interfaces (APIs) for programmatic access to FlyBase data. In this review, we describe these major new features and functionalities of the FlyBase 2.0 site and how they support the use of Drosophila as a model organism for biological discovery and translational research.
Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.