Shizue Ohsawa

Elimination of Oncogenic Neighbors by JNK-Mediated Engulfment in Drosophila

A 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.

Mitochondrial defect drives non-autonomous tumour progression through Hippo signalling in Drosophila

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.

Maturation of the olfactory sensory neurons by Apaf-1/caspase-9–mediated caspase activity

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.

Caspase‐mediated changes in Sir2α during apoptosis

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 Sir2α protects cells from stress‐induced apoptosis. We report here that mammalian Sir2α was directly cleaved by both initiator and executioner caspases, and relocated from the nucleus to the cytoplasm in apoptotic cells. These alterations of Sir2α were largely inhibited by a caspase‐9 dominant‐negative mutant or Bcl‐xL. Our results indicate that Sir2α undergoes dynamic changes in caspase‐dependent manner during apoptosis.

JNK signaling is converted from anti- to pro-tumor pathway by Ras-mediated switch of Warts activity

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 JNKs 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.

Mitochondrial defects trigger proliferation of neighbouring cells via a senescence-associated secretory phenotype in Drosophila

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.

Caspase-mediated changes in histone H1 in early apoptosis: prolonged caspase activation in developing olfactory sensory neurons

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.

The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition

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.

Caspase-9 Activation Revealed by Semaphorin 7A Cleavage Is Independent of Apoptosis in the Aged Olfactory Bulb

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 (<20%) than in the OB of young mice. Furthermore, we found that the lower number dying cells in the aged OB was accompanied by a decreased expression of procaspase-3. These results suggest a survival strategy for aged OB neurons, which can no longer regenerate, in which the central apoptotic machinery downstream of caspase-9 is inactivated.

Novel function of neuronal PAS domain protein 1 in erythropoietin expression in neuronal cells.

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.