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Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance., Author summary Cancers develop through the accumulation of multiple oncogenic mutations. Such mutations are different among tumors of different origins and thus identification of a common oncogenesis pathway among different tumors is crucial for developing effective anti-cancer strategies. Here, using fruit fly Drosophila as a model organism, we searched for a common oncogenesis pathway between two malignant tumor models with different oncogenic alterations, namely oncogenic Ras activation with cell polarity defects (RasV12/scrib-/-) and oncogenic microRNA bantam overexpression with endocytic defects (bantam/rab5-/-). Transcriptome analyses followed by an RNAi screen identified Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), as essential for growth and malignancy of these tumor models. Similar to mammalian cancers, JhI-21 was upregulated in these tumors and promoted tumor growth and progression by activating the mTOR-S6 pathway. Administration of LAT1 inhibitors to flies significantly suppressed growth of RasV12/scrib-/-, but not bantam/rab5-/- tumors. Our analyses revealed that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations establish a powerful genetic model for studying cancer progression and drug resistance in Drosophila.

Normal epithelial tissues often put anti‐tumorigenic pressure on newly emerged oncogenic cells through cell–cell communications. In Drosophila epithelium, clones of oncogenic cells mutant for evolutionarily conserved apico‐basal polarity genes such as scribble (scrib) and discs large (dlg) are actively eliminated when surrounded by normal cells. It has been reported that c‐Jun N‐terminal kinase (JNK) signaling in polarity‐deficient cells is crucial for their cell death. However, the mechanism by which normal epithelial tissues exert anti‐tumorigenic effects on polarity‐deficient cells had been elusive. Here, I describe our genetic studies in Drosophila epithelium especially focused on the role of surrounding normal epithelial cells in response to the emergence of polarity‐deficient cells. Furthermore, I also describe recent studies regarding the mechanism by which polarity‐deficient cells are extruded from the tissue, and discuss future perspectives on the study of cell–cell communications in epithelial homeostasis., Normal epithelial tissue eliminates newly emerged oncogenic polarity‐deficient cells through tumor suppressive cell competition. In this review, I summarize recent progress on the mechanism of this tumor suppressive cell competition in Drosophila, especially focused on the role of normal epithelial cells and describe future perspectives.

Heterozygosity of ribosomal protein genes causes a variety of developmental abnormalities in humans, which are collectively known as ribosomopathies, yet the underlying mechanisms remain elusive. Here, we analyzed Drosophila Minute (M)/+ mutants, a group of mutants heterozygous for ribosomal protein genes that exhibit a characteristic thin-bristle phenotype. We found that, although M/+ flies develop essentially normal wings, simultaneous deletion of one copy of the Hippo pathway effector yki resulted in severe wing growth defects. These defects were caused by JNK-mediated cell death in the wing pouch via Eiger/TNF signaling. The JNK activation in M/+, yki/+ wing discs required the caspase Dronc, which is normally blocked by DIAP1. Notably, heterozygosity of yki reduced DIAP1 expression in the wing pouch, leading to elevation of Dronc activity. Dronc and JNK formed a positive-feedback loop that amplifies Dronc activation, leading to apoptosis. Our observations suggest a mechanism of robust tissue growth whereby tissues with reduced ribosomal protein prevent ectopic apoptosis via Yki activity.

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted., Author summary Animal development can be disturbed by various stimuli such as genetic mutations, environmental fluctuations, and physical injuries. However, animals often accomplish normal tissue growth and morphogenesis even in the presence of developmental perturbations. Drosophila Minute mutants, a series of fly mutants for a ribosomal protein gene, show significantly prolonged larval period but develop into essentially normal flies. We found an unexpected massive cell death and subsequent compensatory cell proliferation in developing wing discs of Minute animals. This ‘cell-turnover’ was essential for normal wing morphogenesis in Minute flies. We found that the cell-turnover was induced by elevated Wg expression in the wing pouch and JNK-mediated Dilp8 expression that causes developmental delay. Indeed, cell-turnover was reproduced in non-Minute animals’ wing discs by overexpressing Wg using the wg promoter together with developmental delay caused by ecdysone depletion. Our findings propose a novel paradigm for morphogenetic robustness by cell-turnover, which ensures normal wing growth during the abnormally prolonged larval period, possibly by creating a flexible cell death and proliferation platform to adjust cell numbers in the prospective wing blade.

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. While Yki-induced cell proliferation has extensively been studied, the mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. Here, we show that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. Through a genetic screen in Drosophila, we find that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. Our data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells.

Multicellular organisms repair injured epithelium by evolutionarily conserved biological processes including activation of c-Jun N-terminal kinase (JNK) signaling. Here, we show in Drosophila imaginal epithelium that physical injury leads to the emergence of dying cells, which are extruded from the wounded tissue by JNK-induced Slit-Roundabout2 (Robo2) repulsive signaling. Reducing Slit-Robo2 signaling in the wounded tissue suppresses extrusion of dying cells and generates aberrant cells with highly upregulated growth factors Wingless (Wg) and Decapentaplegic (Dpp). The inappropriately elevated Wg and Dpp impairs wound repair, as halving one of these growth factor genes cancelled wound healing defects caused by Slit-Robo2 downregulation. Our data suggest that JNK-mediated Slit-Robo2 signaling contributes to epithelial wound repair by promoting extrusion of dying cells from the wounded tissue, which facilitates transient and appropriate induction of growth factors for proper wound healing.

The phenomenon of 'cell competition' has been implicated in the normal development and maintenance of organs, such as in the regulation of organ size and suppression of neoplastic development. In cell competition, one group of cells competes with another group through an interaction at their interface. Which cell group "wins" is governed by a certain relative fitness within the cells. However, this idea of cellular fitness has not been clearly defined. We construct two types of mathematical models to describe this phenomenon of cell competition by considering the interaction at the interface as a predator-prey type interaction in a monolayer tissue such as epithelium. Both of these models can reproduce several typical experimental observations involving systems of mutant cells (losers) and normal cells (winners). By analyzing one of the model and defining an index for the degree of fitness in groups of cells, we show that the fate of each group mainly depends on the relative carrying capacities of certain resources and the strength of the predator-prey interaction at the interface. This contradicts the classical hypothesis in which the relative proliferation rate determines the winner.

Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild-type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, we established two assay systems for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3-RNAi-expressing cells) system in which RpS3-RNAi-expressing wing pouch cells are eliminated from wild-type wing disc and (ii) the SURE (supercompetition of RpS3-expressing clones in RpS3/+ tissue) system in which RpS3-over-expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over-expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down-regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, we found that down-regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. Our observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition.

Epithelial tissues robustly respond to internal and external stressors via dynamic cellular rearrangements. Cell extrusion acts as a key regulator of epithelial homeostasis by removing apoptotic cells, orchestrating morphogenesis, and mediating competitive cellular battles during tumorigenesis. Here, we delineate the diverse functions of cell extrusion during development and disease. We emphasize the expanding role for apoptotic cell extrusion in exerting morphogenetic forces, as well as the strong intersection of cell extrusion with cell competition, a homeostatic mechanism that eliminates aberrant or unfit cells. While cell competition and extrusion can exert potent, tumor-suppressive effects, dysregulation of either critical homeostatic program can fuel cancer progression.

During the initial stage of tumor progression, oncogenic cells spread despite spatial confinement imposed by surrounding normal tissue. This spread of oncogenic cells (winners) is thought to be governed by selective killing of surrounding normal cells (losers) through a phenomenon called "cell competition" (i.e., supercompetition). Although the mechanisms underlying loser elimination are increasingly apparent, it is not clear how winner cells selectively occupy the space made available following loser apoptosis. Here, we combined live imaging analyses of two different oncogenic clones (Yki/YAP activation and Ras activation) in the Drosophila epithelium with computer simulation of tissue mechanics to elucidate such a mechanism. Contrary to the previous expectation that cell volume loss after apoptosis of loser cells was simply compensated for by the faster proliferation of winner cells, we found that the lost volume was compensated for by rapid cell expansion of winners. Mechanistically, the rapid winner-dominated cell expansion was driven by apoptosis-induced epithelial junction remodeling, which causes re-connection of local cellular connectivity (cell topology) in a manner that selectively increases winner apical surface area. In silico experiments further confirmed that repetition of loser elimination accelerates tissue-scale winner expansion through topological changes over time. Our proposed mechanism for linking loser death and winner expansion provides a new perspective on how tissue homeostasis disruption can initiate from an oncogenic mutation.

Cancers develop through sequential acquisition of oncogenic mutations, indicating a crucial role of genetic alterations in tumour progression. However, accumulating evidence suggests that cancers also progress towards malignancy through cell-cell interactions within heterogeneous tumour tissue. Recent studies have indicated that tumour heterogeneity not only contributes to drug resistance and tumour recurrence but also plays an active role in promoting tumour progression. Especially, genetic studies in Drosophila have discovered novel types of tumour progression through cell-cell interactions and have dissected the underlying mechanisms. This review focuses on describing recent findings obtained from Drosophila genetics that provide genetic basis of interclonal oncogenic cooperation in heterogeneous tumour tissue.

SummaryA newly emerged oncogenic cell in the epithelial population has to confront antitumor selective pressures in the host tissue. However, the mechanisms by which surrounding normal tissue exerts antitumor effects against oncogenically transformed cells are poorly understood. In Drosophila imaginal epithelia, clones of cells mutant for evolutionarily conserved tumor suppressor genes such as scrib or dlg lose their epithelial integrity and are eliminated from epithelia when surrounded by wild-type tissue. Here, we show that surrounding normal cells activate nonapoptotic JNK signaling in response to the emergence of oncogenic mutant cells. This JNK activation leads to upregulation of PVR, the Drosophila PDGF/VEGF receptor. Genetic and time-lapse imaging analyses reveal that PVR expression in surrounding cells activates the ELMO/Mbc-mediated phagocytic pathway, thereby eliminating oncogenic neighbors by engulfment. Our data indicate that JNK-mediated cell engulfment could be an evolutionarily conserved intrinsic tumor-suppression mechanism that eliminates premalignant cells from epithelia.

Although the apoptotic role of caspases has been largely understood, accumulating evidence in Drosophila suggests that caspases also control other processes than apoptotic cell death. However, how caspases contribute to the development of the mammalian nervous system remains obscure. Here, we provide unique evidence that Apaf-1/caspase-9–mediated caspase signaling regulates the development of olfactory sensory neurons (OSNs), which includes axonal projection, synapse formation, and maturation of these neurons. This caspase signaling leads to a cleavage of Semaphorin 7A, a membrane-anchored semaphorin that is required for the proper axonal projection. Mutant mice deficient for apaf-1 or caspase-9 exhibit misrouted axons, impaired synaptic formation, and defects in the maturation of OSNs without affecting the number of these cells. Our findings suggest that Apaf-1/caspase-9–mediated nonapoptotic caspase signaling is required for the proper neural network formation during olfactory development.

Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. Here, through a genetic screen in Drosophila, we identify Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. Our data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium.

Caspases are essential in multicellular organisms for inducing cell death during normal development and in the immune system. However, caspases can also trigger the degenerative process under certain conditions such as pathophysiological conditions and aging. Here, we identified Semaphorin 7A (Sema7A) as a novel substrate for caspase-9 that can be used to monitor caspase-9 activity in mice, and found nonapoptotic caspase-9 activation in the aged olfactory bulb (OB). Immunostaining of the OB for the caspase-9-cleaved form of Sema7A revealed abundant caspase-9-activated cells in 2-year-old (aged) but not in 2-month-old (young) mice. In fact, various regions of the aged brain, including the OB, exhibited an increased level of caspase-9 activity. However, the number of dying cells in the aged OB was, intriguingly, much lower (

Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Here, through a genetic screen in Drosophila, we identify the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition.

Defects in Mitochondrial respiratory function is frequently observed in human cancers. However, the mechanism by which mitochondrial dysfunction contributes to tumour growth and progression has been unclear. Recent studies in Drosophila epithelium have uncovered that mitochondrial defects induce tumour progression of surrounding tissue by cooperating with oncogenic Ras. Simultaneous Ras activation and mitochondrial dysfunction cause chronic ‘oncogenic inflammation’, which induces overgrowth and metastatic behavior in neighbouring benign tumours via upregulation of an IL-6-like inflammatory cytokine Upd. Further genetic analyses revealed that the nonautonomous tumour progression by oncogenic inflammation is caused through cellular senescence and senescence-associated secretary phenotype (SASP). These findings provide a novel mechanistic basis for tumour progression through cell–cell communication triggered by Ras activation and mitochondrial dysfunction, frequent alterations in human cancers.

Silent information regulator 2 (Sir2) is an NAD(+)-dependent histone deacetylase that establishes repressive chromatin status and extends the life span of both budding yeast and the nematode worm Caenorhabditis elegans. There is growing evidence that its mammalian homologue Sir2alpha protects cells from stress-induced apoptosis. We report here that mammalian Sir2alpha was directly cleaved by both initiator and executioner caspases, and relocated from the nucleus to the cytoplasm in apoptotic cells. These alterations of Sir2alpha were largely inhibited by a caspase-9 dominant-negative mutant or Bcl-xL. Our results indicate that Sir2alpha undergoes dynamic changes in caspase-dependent manner during apoptosis.

The basic helix loop helix-PAS (bHLH-PAS) transcription factors have diverse roles in physiologic responses to the environment and in early development. One bHLH-PAS protein, neuronal PAS domain protein 1 (NPAS1), is reported to be expressed only in the central nervous system beginning at the late embryonic stage, but its function is unknown. Using an anti-NPAS1 antibody, we have shown that NPAS1 expression in cerebral cortex was observed first around embryonic Day 16.5 (E16.5) and was then dispersed throughout the region as cortical development progressed. From the similarity with hypoxia-inducible factor-1α (HIF-1α), we investigated whether NPAS1 regulates transcription of erythropoietin (EPO), which is the target of HIF-1α and is expressed in the brain during the early developmental stage. In the present study, we have shown that NPAS1 binds to the enhancer region of Epo in vivo. A luciferase reporter assay revealed a repressive effect of NPAS1 on hypoxia-responsive element-regulated gene expression. These results raise the possibility that NPAS1 plays a role in late central nervous system development by modulating EPO expression in response to cellular oxygen level. © 2005 Wiley-Liss, Inc.

Cell-cell interactions play important roles in epithelial tumorigenesis. Here we show in Drosophila imaginal epithelium that Ras activation and mitochondrial dysfunction, frequent alterations in cancers, cause cellular senescence and senescence-associated secretory phenotype (SASP), which leads to overgrowth of neighbouring tissue. Ras-activated cells express several hallmarks of cellular senescence such as elevation of senescence-associated β-galactosidase activity, upregulation of the Cdk inhibitor Dacapo, heterochromatinization and cellular hypertrophy. Strikingly, defects in mitochondrial function cause Ras-activated cells to undergo DNA damage response, cell cycle arrest and thereby induce SASP, exhibiting full aspects of cellular senescence. Mechanistically, mitochondrial defects in conjunction with Ras cause production of reactive oxygen species, downregulation of CycE activity and activation of p53, which cooperate together to trigger a cell cycle arrest-Jun N-terminal kinase (JNK) feedback loop that amplifies JNK activation, leading to upregulation of the inflammatory cytokine Unpaired. Our data suggest that mitochondrial defects promote Ras-induced cellular senescence and thereby contribute to tumour progression through SASP.

How the size and shape of organs are determined is one of the most mysterious questions in developmental biology. Organ size and shape are controlled by tissue growth, which is strongly influenced by local homeostatic cell–cell interactions. Studies during the past several years have shown that cells in multicellular communities compete with each other for their existence; cells with higher fitness (“winners”) survive and eliminate cells with lower fitness (“losers”) by inducing cell death. Accumulating evidence, obtained mainly in Drosophila, has revealed that this cellular natural selection, called “cell competition,” could have an important role in organ size and shape control in normal development as well as in some pathophysiological conditions such as stem cell regulation and cancer development. In this chapter, we discuss recent insights in cell competition. Elucidation of the mechanism by which cell competition is controlled could create a new dimension for understanding the basic principle of multicellular systems.

Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs.

Mitochondrial respiratory function is frequently impaired in human cancers. However, the mechanisms by which mitochondrial dysfunction contributes to tumour progression remain elusive. Here we show in Drosophila imaginal epithelium that defects in mitochondrial function potently induce tumour progression of surrounding tissue in conjunction with oncogenic Ras. Our data show that Ras activation and mitochondrial dysfunction cooperatively stimulate production of reactive oxygen species, which causes activation of c-Jun amino (N)-terminal kinase (JNK) signalling. JNK cooperates with oncogenic Ras to inactivate the Hippo pathway, leading to upregulation of its targets Unpaired (an interleukin-6 homologue) and Wingless (a Wnt homologue). Mitochondrial dysfunction in Ras-activated cells further cooperates with Ras signalling in neighbouring cells with normal mitochondrial function, causing benign tumours to exhibit metastatic behaviour. Our findings provide a mechanistic basis for interclonal tumour progression driven by mitochondrial dysfunction and oncogenic Ras.

Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs.

Programmed cell death or apoptosis is required for the patterning and development of multicellular organisms. However, apoptosis is a difficult process to measure because the dead cells are rapidly degraded by their neighbors within a few hours. The post-caspase activation events that determine whether a cell will undergo apoptosis remain elusive. Here we report that apoptosis-specific nuclear events that occur before DNA fragmentation can be distinguished by monitoring the histone H1 status. In both mammals and Drosophila, dying cells failed to be immunolabeled with an anti-H1 monoclonal antibody, AE-4. Real-time imaging of caspase activation and H1 dynamics in mammalian neural cells revealed that H1 changed its location in the nucleus after caspase activation. In addition, the timing of this re-localization was largely dependent on the apoptotic stimulus used. From the staining patterns of AE-4 and anti-active caspase-3 antibodies, cells undergoing the transition from caspase activation to the apoptotic H1 change could be identified as H1-positive caspase-activated cells, providing a novel criterion for early apoptosis and making it possible to characterize caspase-activated cells in tissues. On the basis of these staining patterns, we found that many olfactory sensory neurons in the developing mouse olfactory epithelium showed sustained caspase activity without the H1 change, suggesting a unique caspase function in these neurons.

Programmed cell death (PCD) or apoptosis has crucial roles in the development of the nervous system and in tissue homeostasis. It is generally assumed that about half of the neurons are lost before completion of the neural architecture during development. Neurotrophic cell death in innervating neurons matches the number of neurons to the size of their target field. Damaged and potentially harmful cells are induced to apoptosis. The loss of developing neural cells is involved in the morphogenetic sculpting of the early nervous system and the creation of sexually dimorphic structures. PCD also eliminates pioneer neurons that serve transient functions, such as those that navigate in an environment devoid of other axons. Dysregulation of cell death programmes can feature in developmental and neoplastic disorders of the nervous system, including various neurodegenerative diseases. PCD is ultimately difficult process to measure because of their rapidly degradation by neighbors in a few hours or less. The molecular basis underlying why only certain cells are selected to die at adequate stage and how PCD is integrated with other biological processes are largely unknown. However, recent genetic studies in Drosophila and mice provide new insights into the regulatory system of cell death and survival pathway in vivo. This review describes the current knowledge about the significance and mechanisms of selective cell death in the nervous system.

Apoptosis is associated with morphological changes, including membrane blebbing, cell shrinkage, and chromatin condensation. However, the molecular mechanisms of the dynamic changes in cellular components during apoptosis are largely unknown. Here we developed a new rat monoclonal antibody, 9B1, that specifically immunolabeled dying cells in tissues and in cell cultures. The 9B1 antibody labeled the cytoplasm of apoptotic cells in a caspase-dependent manner. We identified human brain abundant membrane attached signal protein 1 (hBASP1) as the 9B1 antigen using the liquid chromatography with tandem mass spectrometry (LC/MS/MS) method. hBASP1 was present in the nucleus of HeLa cells, but relocated from the nucleus to the cytoplasm after the caspase activation step of apoptosis. Immunostaining analysis revealed that 9B1 preferentially labeled this cytoplasmic form of hBASP1. Labeling by 9B1 to distinguish apoptotic changes could be a novel criterion for determining whether cells with activated caspases are fated for survival or death.

By using cDNA subtraction, we identified an extracellular sulfatase (RsulfFP1) from rat oligodendrocyte progenitor cells (OPCs) whose mRNA expression is down-regulated by tumor necrosis factor-alpha. RsulfFP1 mRNA was expressed specifically in the floor plate and the ventral portion of the rat spinal cord at E15. The expression pattern of RsulfFP1 overlapped with the OPCs, which are also located at the ventral region of the ventricular zone. After this stage, RsulfFP1 expression was attenuated, and the OPCs efficiently migrated throughout the spinal cord. The modification of CG-4 cells, a cell line established from rat O2A cells, by RsulfFP1 activated canonical Wnt signaling. Furthermore, the deletion of RsulfFP1 expression by an antisense oligonucleotide caused impairment of OPC migration in rat spinal cord slice culture. Modification of cells by RsulfFP1 resulted in the increased tyrosine phosphorylation of immunoprecipitated beta-catenin, suggesting that sulfation of the extracellular matrix induced by this sulfatase might be responsible for an increase in Wnt signaling that is involved in the migration of OPCs. Thus, the present study revealed that a sulfatase is responsible for the migration of OPCs and activates intracellular mechanisms that regulate migration.

AhR (aryl hydrocarbon receptor), AhRR (AhR repressor), and Arnt (AhR nuclear translocator) are members of the bHLH (basic-helix-loop-helix)-PAS (Per-AhR/Arnt-Sim homology sequence) transcription factor superfamily. They associate with each other to form heterodimers, AhR/Arnt or AhRR/Arnt, and bind the XRE (xenobiotic responsive element) sequences in the promoter regions of the target genes to regulate their expression. Their basic regions and HLH motifs mediate DNA binding activity and protein dimerization, respectively. The PAS domain includes two incomplete repeats, PAS-A and PAS-B, and is considered to determine the specificity on protein dimerization. However, the three-dimensional structures of PAS folds reported so far are all monomeric, therefore, little is known about the structural basis of protein interaction through PAS domains. In the present study, we prepared heterodimeric bHLH-PAS domains derived from AhR and Arnt, and AhRR and Arnt by co-expressing each pair in E. coli, and showed that the heterodimers formed exhibited full DNA-binding activity, which was not apparently affected by deletion of the highly basic amino acid cluster most N-terminal as to the HLH region of AhR or AhRR. Methylation of the two CpG sites in the XRE core sequence reduced the binding affinity to heterodimeric proteins, with 5-methylcytosine in the AhR recognition site exhibiting a greater inhibitory effect than that in the Arnt recognition site.

Tissue development and homeostasis are regulated by coordination of cell growth, cell proliferation, and cell death within multicellular communities. In Drosophila developing imaginal epithelia, clones of cells mutant for apico-basal polarity genes, such as scribble ( scrib ) and discs large ( dlg ), are eliminated when these mutant clones are surrounded by wild-type tissue. Intriguingly, removal of surrounding wild-type tissue allows these mutant clones to grow aggressively and develop into tumors, suggesting that oncogenic polarity-deficient cells are actively eliminated thorough cell–cell communication between mutant cells and surrounding wild-type cells. We have shown that clones of polarity-deficient cells are eliminated by endocytic activation of TNF-JNK signaling in mutant cells. Furthermore, we have recently found that surrounding wild-type cells activate non-apoptotic JNK signaling, which promotes elimination of polarity-deficient neighbors. To dissect the upstream mechanism of this non-cell autonomous regulation of cell-elimination, we developed a “non-cell autonomous” genetic screen system. In developing eye discs, wild-type cells surrounding oncogenic polarity-deficient cells were mutagenized by EMS or P-element insertions and were screened for mutations that failed to eliminate neighboring mutant cells. We isolated three elimination-defective ( eld ) mutants that failed to eliminate neighboring scrib mutant clones but had no effect on cell growth or cell viability on their own. The mechanism by which eld genes regulate the cell-elimination will be discussed.

Tumor growth and progression is regulated by cooperation of oncogenic alterations that involve both intra- and inter-cellular signal transductions. However, the underlying mechanism of how each oncogenic mutation cooperates with other mutations to progress toward malignancy within multicellular communities is poorly understood. To address this mechanism, we established a genetic screen system in Drosophila to identify factors that drive tumor malignancy. Oncogenic Ras (RasV12), a common oncogenic alteration of human cancers, leads to the development of benign tumors in Drosophila imaginal discs. Using the genetic mosaic technique, we introduced additional mutations in RasV12 benign tumors and screened for mutations that drive tumor progression. We isolated several mutations that cooperate with RasV12 to drive tumor malignancy. Among these mutants, we found a series of genes involved in the endocytic pathway as their responsible genes. Intriguingly, we found that some endocytic gene mutations transformed noninvasive RasV12 tumors into metastatic tumors and simultaneously induced non-cell autonomous overgrowth of surrounding wild-type cells. The mechanism underling tumor progression caused by endocytic gene mutations will be presented.

The c-Jun N-terminal kinase (JNK) pathway is a dual-functional oncogenic signaling that exerts both anti- and pro-tumor activities. However, the mechanism by which JNK switches its oncogenic roles depending on different cellular contexts has been elusive. Here, using the Drosophila genetics, we show that hyperactive Ras acts as a signaling switch that converts JNK's role from anti- to pro-tumor signaling through the regulation of Hippo signaling activity. In the normal epithelium, JNK signaling antagonizes the Hippo pathway effector Yorkie (Yki) through elevation of Warts activity, thereby suppressing tissue growth. In contrast, in the presence of hyperactive Ras, JNK signaling enhances Yki activation by accumulating F-actin through the activity of the LIM domain protein Ajuba, thereby promoting tissue growth. We also find that the epidermal growth factor receptor (EGFR) signaling uses this Ras-mediated conversion of JNK signaling to promote tissue growth. Our observations suggest that Ras-mediated switch of the JNK pathway from anti- to pro-tumor signaling could play crucial roles in tumorigenesis as well as in normal development.