Paraneoplastic renal dysfunction in fly cancer models driven by inflammatory activation of stem cells.

Tumors can induce systemic disturbances in distant organs, leading to physiological changes that enhance host morbidity. In Drosophila cancer models, tumors have been known for decades to cause hypervolemic 'bloating' of the abdominal cavity. Here we use allograft and transgenic tumors to show that hosts display fluid retention associated with autonomously defective secretory capacity of fly renal tubules, which function analogous to those of the human kidney. Excretion from these organs is blocked by abnormal cells that originate from inappropriate activation of normally quiescent renal stem cells (RSCs). Blockage is initiated by IL-6-like oncokines that perturb renal water-transporting cells, and trigger a damage response in RSCs that proceeds pathologically. Thus, a chronic inflammatory state produced by the tumor causes paraneoplastic fluid dysregulation by altering cellular homeostasis of host renal units.

Systemic coagulopathy promotes host lethality in a new Drosophila tumor model.

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak's characterization of tumors as "wounds that never heal."1 Some of these responses lead to profound defects in blood clotting, such as disseminated intravascular coagulopathy (DIC), which correlate with poor prognoses.2,3,4 Here, we demonstrate that a new tumor model in Drosophila provokes phenotypes that resemble coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by exhaustion of host clotting components. We show that rescuing coagulopathy by depleting a tumor-produced clotting factor improves survival of tumor-bearing flies, despite the fact that flies have an open (non-vascular) circulatory system. As clinical studies suggest that lethality in patients with high serum levels of clotting components can be independent of thrombotic events,5,6 our work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality. Moreover, it opens up exploration of other conserved mechanisms of host responses to chronic wounds.

Specialized cells that sense tissue mechanics to regulate Drosophila morphogenesis.

Shaping of developing organs requires dynamic regulation of force and resistance to achieve precise outcomes, but how organs monitor tissue mechanical properties is poorly understood. We show that in developing Drosophila follicles (egg chambers), a single pair of cells performs such monitoring to drive organ shaping. These anterior polar cells secrete a matrix metalloproteinase (MMP) that specifies the appropriate degree of tissue elongation, rather than hyper- or hypo-elongated organs. MMP production is negatively regulated by basement membrane (BM) mechanical properties, which are sensed through focal adhesion signaling and autonomous contractile activity; MMP then reciprocally regulates BM remodeling, particularly at the anterior region. Changing BM properties at remote locations alone is sufficient to induce a remodeling response in polar cells. We propose that this small group of cells senses both local and distant stiffness cues to produce factors that pattern the organ's BM mechanics, ensuring proper tissue shape and reproductive success.

PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones.

Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. We report here experiments to test this requirement in various contexts and find that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. Our experiments used identical stocks with which another group can detect the PTP10D requirement, and our results do not vary under several husbandry conditions including high and low protein food diets. Although we are unable to identify the source of the discrepant results, we suggest that the role of PTP10D in polarity-deficient cell elimination may not be absolute.

Minimal functional domains of the core polarity regulator Dlg.

The compartmentalized domains of polarized epithelial cells arise from mutually antagonistic actions between the apical Par complex and the basolateral Scrib module. In Drosophila, the Scrib module proteins Scribble (Scrib) and Discs-large (Dlg) are required to limit Lgl phosphorylation at the basolateral cortex, but how Scrib and Dlg could carry out such a 'protection' activity is not clear. We tested Protein Phosphatase 1α (PP1) as a potential mediator of this activity, but demonstrate that a significant component of Scrib and Dlg regulation of Lgl is PP1 independent, and found no evidence for a Scrib-Dlg-PP1 protein complex. However, the Dlg SH3 domain plays a role in Lgl protection and, in combination with the N-terminal region of the Dlg HOOK domain, in recruitment of Scrib to the membrane. We identify a 'minimal Dlg' comprised of the SH3 and HOOK domains that is both necessary and sufficient for Scrib localization and epithelial polarity function in vivo. This article has an associated First Person interview with the first author of the paper.

Epithelial monitoring through ligand-receptor segregation ensures malignant cell elimination.

Animals have evolved mechanisms, such as cell competition, to remove dangerous or nonfunctional cells from a tissue. Tumor necrosis factor signaling can eliminate clonal malignancies from Drosophila imaginal epithelia, but why this pathway is activated in tumor cells but not normal tissue is unknown. We show that the ligand that drives elimination is present in basolateral circulation but remains latent because it is spatially segregated from its apically localized receptor. Polarity defects associated with malignant transformation cause receptor mislocalization, allowing ligand binding and subsequent apoptotic signaling. This process occurs irrespective of the neighboring cells' genotype and is thus distinct from cell competition. Related phenomena at epithelial wound sites are required for efficient repair. This mechanism of polarized compartmentalization of ligand and receptor can generally monitor epithelial integrity to promote tissue homeostasis.

Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex.

Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) are basolateral regulators of epithelial polarity and tumor suppressors whose molecular mechanisms of action remain unclear. We used proximity biotinylation to identify proteins localized near Dlg in the Drosophila wing imaginal disc epithelium. In addition to expected membrane- and cytoskeleton-associated protein classes, nuclear proteins were prevalent in the resulting mass spectrometry dataset, including all four members of the nucleosome remodeling factor (NURF) chromatin remodeling complex. Subcellular fractionation demonstrated a nuclear pool of Dlg and proximity ligation confirmed its position near the NURF complex. Genetic analysis showed that NURF activity is also required for the overgrowth of dlg tumors, and this growth suppression correlated with a reduction in Hippo pathway gene expression. Together, these data suggest a nuclear role for Dlg in regulating chromatin and transcription through a more direct mechanism than previously thought.

Tumor-induced disruption of the blood-brain barrier promotes host death.

Cancer patients often die from symptoms that manifest at a distance from any tumor. Mechanisms underlying these systemic physiological perturbations, called paraneoplastic syndromes, may benefit from investigation in non-mammalian systems. Using a non-metastatic Drosophila adult model, we find that malignant-tumor-produced cytokines drive widespread host activation of JAK-STAT signaling and cause premature lethality. STAT activity is particularly high in cells of the blood-brain barrier (BBB), where it induces aberrant BBB permeability. Remarkably, inhibiting STAT in the BBB not only rescues barrier function but also extends the lifespan of tumor-bearing hosts. We identify BBB damage in other pathological conditions that cause elevated inflammatory signaling, including obesity and infection, where BBB permeability also regulates host survival. IL-6-dependent BBB dysfunction is further seen in a mouse tumor model, and it again promotes host morbidity. Therefore, BBB alterations constitute a conserved lethal tumor-host interaction that also underlies other physiological morbidities.

Tumour-host interactions through the lens of Drosophila.

There is a large gap between the deep understanding of mechanisms driving tumour growth and the reasons why patients ultimately die of cancer. It is now appreciated that interactions between the tumour and surrounding non-tumour (sometimes referred to as host) cells play critical roles in mortality as well as tumour progression, but much remains unknown about the underlying molecular mechanisms, especially those that act beyond the tumour microenvironment. Drosophila has a track record of high-impact discoveries about cell-autonomous growth regulation, and is well suited to now probe mysteries of tumour - host interactions. Here, we review current knowledge about how fly tumours interact with microenvironmental stroma, circulating innate immune cells and distant organs to influence disease progression. We also discuss reciprocal regulation between tumours and host physiology, with a particular focus on paraneoplasias. The fly's simplicity along with the ability to study lethality directly provide an opportunity to shed new light on how cancer actually kills.

Distinct activities of Scrib module proteins organize epithelial polarity.

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. Here, we demonstrate dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. Our data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.

Extracellular matrix stiffness cues junctional remodeling for 3D tissue elongation.

Organs are sculpted by extracellular as well as cell-intrinsic forces, but how collective cell dynamics are orchestrated in response to environmental cues is poorly understood. Here we apply advanced image analysis to reveal extracellular matrix-responsive cell behaviors that drive elongation of the Drosophila follicle, a model system in which basement membrane stiffness instructs three-dimensional tissue morphogenesis. Through in toto morphometric analyses of wild type and round egg mutants, we find that neither changes in average cell shape nor oriented cell division are required for appropriate organ shape. Instead, a major element is the reorientation of elongated cells at the follicle anterior. Polarized reorientation is regulated by mechanical cues from the basement membrane, which are transduced by the Src tyrosine kinase to alter junctional E-cadherin trafficking. This mechanosensitive cellular behavior represents a conserved mechanism that can elongate edgeless tubular epithelia in a process distinct from those that elongate bounded, planar epithelia.

A Drosophila Tumor Suppressor Gene Prevents Tonic TNF Signaling through Receptor N-Glycosylation.

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. Here we report that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. We show that ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. Our results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues.

Mechanical stress regulates insulin sensitivity through integrin-dependent control of insulin receptor localization.

Insulin resistance, the failure to activate insulin signaling in the presence of ligand, leads to metabolic diseases, including type 2 diabetes. Physical activity and mechanical stress have been shown to protect against insulin resistance, but the molecular mechanisms remain unclear. Here, we address this relationship in the Drosophila larval fat body, an insulin-sensitive organ analogous to vertebrate adipose tissue and livers. We found that insulin signaling in Drosophila fat body cells is abolished in the absence of physical activity and mechanical stress even when excess insulin is present. Physical movement is required for insulin sensitivity in both intact larvae and fat bodies cultured ex vivo. Interestingly, the insulin receptor and other downstream components are recruited to the plasma membrane in response to mechanical stress, and this membrane localization is rapidly lost upon disruption of larval or tissue movement. Sensing of mechanical stimuli is mediated in part by integrins, whose activation is necessary and sufficient for mechanical stress-dependent insulin signaling. Insulin resistance develops naturally during the transition from the active larval stage to the immotile pupal stage, suggesting that regulation of insulin sensitivity by mechanical stress may help coordinate developmental programming with metabolism.

A Cell Migration Tracking Tool Supports Coupling of Tissue Rotation to Elongation.

Cell migration is indispensable to morphogenesis and homeostasis. Live imaging allows mechanistic insights, but long-term observation can alter normal biology, and tools to track movements in vivo without perturbation are lacking. We develop here a tool called M-TRAIL (matrix-labeling technique for real-time and inferred location), which reveals migration histories in fixed tissues. Using clones that overexpress GFP-tagged extracellular matrix (ECM) components, motility trajectories are mapped based on durable traces deposited onto basement membrane. We applied M-TRAIL to Drosophila follicle rotation, comparing in vivo and ex vivo migratory dynamics. The rate, trajectory, and cessation of rotation in wild-type (WT) follicles measured in vivo and ex vivo were identical, as was rotation failure in fat2 mutants. However, follicles carrying intracellularly truncated Fat2, previously reported to lack rotation ex vivo, in fact rotate in vivo at a reduced speed, thus revalidating the hypothesis that rotation is required for tissue elongation. The M-TRAIL approach could be applied to track and quantitate in vivo cell motility in other tissues and organisms.

Taking Stock of the Drosophila Research Ecosystem.

With a century-old history of fundamental discoveries, the fruit fly has long been a favored experimental organism for a wide range of scientific inquiries. But Drosophila is not a "legacy" model organism; technical and intellectual innovations continue to revitalize fly research and drive advances in our understanding of conserved mechanisms of animal biology. Here, we provide an overview of this "ecosystem" and discuss how to address emerging challenges to ensure its continued productivity. Drosophila researchers are fortunate to have a sophisticated and ever-growing toolkit for the analysis of gene function. Access to these tools depends upon continued support for both physical and informational resources. Uncertainty regarding stable support for bioinformatic databases is a particular concern, at a time when there is the need to make the vast knowledge of functional biology provided by this model animal accessible to scientists studying other organisms. Communication and advocacy efforts will promote appreciation of the value of the fly in delivering biomedically important insights. Well-tended traditions of large-scale tool development, open sharing of reagents, and community engagement provide a strong basis for coordinated and proactive initiatives to improve the fly research ecosystem. Overall, there has never been a better time to be a fly pusher.

Organ sculpting by patterned extracellular matrix stiffness.

How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. We show here that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, we document robust mechanical anisotropy in the ECM-based basement membrane (BM) but not in the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an anterior-posterior (A-P) symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation shows that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. Our results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ.

Microenvironmental autophagy promotes tumour growth.

As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.

The Maturation of Development.

An updated, broader definition of developmental biology is needed to capture the full range of exciting research directions of the field, as current studies of adult homeostasis and physiology are extending developmental biology's "Golden Age."

Symmetry Breaking in an Edgeless Epithelium by Fat2-Regulated Microtubule Polarity.

Planar cell polarity (PCP) information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not. Drosophila egg chambers (follicles) are an acinus-like "edgeless epithelium" and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. Here, we apply an imaging algorithm to "unroll" the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ.

Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting.

Tumors kill patients not only through well-characterized perturbations to their local environment but also through poorly understood pathophysiological interactions with distant tissues. Here, we use a Drosophila tumor model to investigate the elusive mechanisms underlying such long-range interactions. Transplantation of tumors into adults induces robust wasting of adipose, muscle, and gonadal tissues that are distant from the tumor, phenotypes that resemble the cancer cachexia seen in human patients. Notably, malignant, but not benign, tumors induce peripheral wasting. We identify the insulin growth factor binding protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, as a secreted factor mediating wasting. ImpL2 is sufficient to drive tissue loss, and insulin activity is reduced in peripheral tissues of tumor-bearing hosts. Importantly, knocking down ImpL2, specifically in the tumor, ameliorates wasting phenotypes. We propose that the tumor-secreted IGFBP creates insulin resistance in distant tissues, thus driving a systemic wasting response.