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Cell Competition Triggers Suicide by Autophagy
Developmental Cell
Previews Cell Competition Triggers Suicide by Autophagy Caroline Dillard1,2 and Tor Erik Rusten1,2,* 1Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.09.014
Cell competition eradicates weaker cells from an epithelium and is important for organ homeostasis. In this issue of Developmental Cell, Nagata et al. (2019) uncover that eradication of loser cells depends on autophagy-mediated cell death. Originally defined in Drosophila, cell competition entails recognition of relative fitness levels and elimination of the less fit loser cells by winner cells (Morata and Ripoll, 1975). Relative fitness decline like that observed upon loss of one copy of ribosomal components (Minute/+) or relative low levels of Myc, a stimulator of ribosomal biogenesis and protein translation, triggers loser-cell elimination. Cell competition can also be instilled by transformed tumor cells upon expression of Myc, Yorkie (YAP), Wnt/Wg, or JAK-STAT activity that endows these cells the power as ‘‘super competitors’’ over normal neighbors (Amoyel and Bach, 2014). Using a clever strategy, in this issue of Developmental Cell Igaki and colleagues (Nagata et al., 2019) devised a genetic screen to define new cell competition components based on the salient features of cell competition—losing when facing wild-type cells but surviving when without wild-type competition. One such ‘‘hit’’ identified the gene Helicase at 25E (Hel25E) encoding a RNA helicase implicated in mRNA splicing and mRNA transport out of the nucleus. As predicted, Hel25E / mutant cells display reduced protein production compared to wild-type neighbors. Thus, Hel25E / mutant cells appear to conform to related loser-cell situations seen in Myclow or Minute/+ cells that also suffer lower protein production. Indeed, like Myclow and RpL14+/ (Minute) clones, Hel25E / loser-cell death occurs specifically at the immediate interface with winner cells, and blocking Caspase activity partially rescued loser-cell elimination. Thus, Hel25E / cells obey both context and spatial rules of apoptotic cell elimination typical of cell competition.
To unravel the mechanism, Nagata et al. (2019) performed an elegant, dominant modifier genetic screen for genes that suppressed Hel25E / cell elimination. This uncovered several components of the lysosomal vacuolar ATPase multisubunit complex. The V-ATPase proton pump is responsible for acidification of the lysosome and, through this, a myriad of cellular functions such as degradation of endosomal, autophagic cargo and cell-signaling functions. The authors found that the lysosomal marker Lysotracker was elevated in loser cells along the clonal margin and that Caspase activation was lost upon reduced V-ATPase activity. This pointed to a lysosomal function in cell death during cell competition. So, what could this function be? Autophagy (self-eating) captures cytosolic cargo in a double-membrane autophagic vesicle destined for degradation by resident hydrolytic enzymes in the lysosome (Figure 1). Through this process, the cell cleans house by eliminating defective organelles and protein aggregates and recycling amino acids, nucleotides, sugars, and lipids for metabolic and bioenergetic needs upon starvation. Autophagy also engages in developmental cell death, described as autophagic cell death in concert with Caspases (Doherty and Baehrecke, 2018). Igaki and colleagues next investigated whether autophagy was contributing to the functionality of lysosomes during cell elimination. Indeed, the authors found that autophagy activity was high and coincided with Caspase activation in loser cells at the clonal boundary. Furthermore, inhibition of Caspase activity did not affect autophagy markers, but knocking down several autophagy components eliminated Caspase activation. These re-
4 Developmental Cell 51, October 7, 2019 ª 2019 Published by Elsevier Inc.
sults raised further questions, including these: How is autophagy controlled? And how does this stimulate Caspase activity and cell elimination? Previous work has implicated stress signaling through Jun N-terminal kinase (JNK) in loser-cell elimination. Hel25E / clones displayed uniform low levels of JNK activity and transcriptional activation of the Caspase activator head involution defective (hid) at the clonal boundary. hid transcription was unaffected by inhibition of JNK signaling and instead appears to be controlled through autophagy. In agreement with previous findings (Scott et al., 2007), Nagata and colleagues found that direct activation of autophagy by the upstream kinase Atg1, or the transcription factor MITF (TFEB in vertebrates) that stimulates lysosome generation and autophagy, was sufficient to induce hid expression and moderately induce cell death. Experimental coactivation of JNK and autophagy was more effective at inducing cell death than either stimulus alone. The authors provide experimental evidence to suggest that NFkB transcription mediates hid transcription downstream of autophagy, as direct Atg1mediated induction of hid transcription was reduced upon knockdown of either of the three NFkB transcription factors (Dorsal, Dif, or Relish). Autophagy negatively regulates NFkB signaling by degrading Kenny (IKKg) (Tusco et al., 2017). It will be important to resolve the mechanism by which autophagy controls NFkB signaling and to determine if NFkB directly controls hid expression in Hel25E / loser cells. The authors suggest the most parsimonious explanation: independent signals lead to a homogeneous JNK stress response in the Hel25E / population, and only at the
Developmental Cell
Previews of autophagic cell death (Doherty and Baehrecke, 2018). With the recent findings that autophagy is activated in loser cells in response to super competitors in Drosophila, and that Flower-mediated cell competition occurs at tumor-microenvironment boundaries in mammals, the implications of this work are of more than just academic interest (Madan et al., 2019; Katheder et al., 2017). Nibbling away at the microenvironment by coercing loser cells to undergo suicide during tumor expansion through autophagy is a very attractive idea that warrants further investigation.
REFERENCES Amoyel, M., and Bach, E.A. (2014). Cell competition: how to eliminate your neighbours. Development 141, 988–1000. Doherty, J., and Baehrecke, E.H. (2018). Life, death and autophagy. Nat. Cell Biol. 20, 1110–1117.
Figure 1. Cell Competition Triggers Cell Elimination through Autophagy Loss-of-function mutations in Helicase at 25E (Hel25E / ) result in lower protein production and elevated JNK stress signaling. Loser status and Caspase-mediated cell death occur in cells facing neighboring winner cells where transcriptional upregulation of the Caspase activator hid depends on autophagy and lysosomal activity, possibly through regulation of NFkB transcriptional responses. Genetic data suggest a role for Flower-mediated sensing of cell fitness and involvement of innate immune signaling in loser-cell elimination.
clonal boundary, where autophagy is active, does this cooperatively lead to effective cell elimination in a mechanism that likely involves NFkB. Is the role of autophagy in cell competition specific to the elimination of Hel25E / cells? Or does it apply widely to the phenomenon? Both Minute/+ clones and Myclow losers showed upregulation of autophagy and cell elimination that were autophagy dependent. Thus the requirement for autophagy in cellcompetition-induced cell elimination appears to apply generally to at least the instances of cell competition displaying protein production differences. The disparate upstream activation signals of JNK and autophagy in Hel25E / cell elimination remain to be defined. Both Myclow and minute cells display a ‘‘lose’’ signal at the cell surface produced by alternative splicing of the transmembrane protein Flower (Fwe), while the winner cells display a winner isoform. It
is the juxtaposition and computation of winner versus loser Fwe isoforms that set in motion the cell-elimination program in loser cells. Igaki and colleagues demonstrate genetically that elimination of Hel25E / cells does depend on fwe and the downstream factor azot. Double knockdown of Atg1 and fwe or azot was not additive, suggesting that they act in a linear pathway, but this was not further explored. Similarly, innate immune signaling has been implicated in elimination of both Myclow and Rpl14E/+ loser cells (Meyer et al., 2014; Germani et al., 2018). Inhibition of innate immune signaling, specifically in Hel25E / loser cells, also counteracted cell elimination. Again, simultaneous inhibition of autophagy, innate immune signaling components, and fwe were not additive. Nagata et al. (2019) provide compelling genetic evidence that autophagy mediates cell elimination during cell competition, providing another physiological example
Germani, F., Hain, D., Sternlicht, D., Moreno, E., and Basler, K. (2018). The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner. eLife 7, e39939. Katheder, N.S., Khezri, R., O’Farrell, F., Schultz, S.W., Jain, A., Rahman, M.M., Schink, K.O., Theodossiou, T.A., Johansen, T., Juha´sz, G., et al. (2017). Microenvironmental autophagy promotes tumour growth. Nature 541, 417–420. Madan, E., Pelham, C.J., Nagane, M., Parker, T.M., Canas-Marques, R., Fazio, K., Shaik, K., Yuan, Y., Henriques, V., Galzerano, A., et al. (2019). Flower isoforms promote competitive growth in cancer. Nature 572, 260–264. Meyer, S.N., Amoyel, M., Bergantinos, C., de la Cova, C., Schertel, C., Basler, K., and Johnston, L.A. (2014). An ancient defense system eliminates unfit cells from developing tissues during cell competition. Science 346, 1258236. Morata, G., and Ripoll, P. (1975). Minutes: mutants of drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221. Nagata, R., Nakamura, M., Sanaka, Y., and Igaki, T. (2019). Cell competition is driven by autophagy. Dev. Cell 51, this issue, 99–112. Scott, R.C., Juha´sz, G., and Neufeld, T.P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17, 1–11. Tusco, R., Jacomin, A.C., Jain, A., Penman, B.S., Larsen, K.B., Johansen, T., and Nezis, I.P. (2017). Kenny mediates selective autophagic degradation of the IKK complex to control innate immune responses. Nat. Commun. 8, 1264.
Developmental Cell 51, October 7, 2019 5
Previews Cell Competition Triggers Suicide by Autophagy Caroline Dillard1,2 and Tor Erik Rusten1,2,* 1Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.09.014
Cell competition eradicates weaker cells from an epithelium and is important for organ homeostasis. In this issue of Developmental Cell, Nagata et al. (2019) uncover that eradication of loser cells depends on autophagy-mediated cell death. Originally defined in Drosophila, cell competition entails recognition of relative fitness levels and elimination of the less fit loser cells by winner cells (Morata and Ripoll, 1975). Relative fitness decline like that observed upon loss of one copy of ribosomal components (Minute/+) or relative low levels of Myc, a stimulator of ribosomal biogenesis and protein translation, triggers loser-cell elimination. Cell competition can also be instilled by transformed tumor cells upon expression of Myc, Yorkie (YAP), Wnt/Wg, or JAK-STAT activity that endows these cells the power as ‘‘super competitors’’ over normal neighbors (Amoyel and Bach, 2014). Using a clever strategy, in this issue of Developmental Cell Igaki and colleagues (Nagata et al., 2019) devised a genetic screen to define new cell competition components based on the salient features of cell competition—losing when facing wild-type cells but surviving when without wild-type competition. One such ‘‘hit’’ identified the gene Helicase at 25E (Hel25E) encoding a RNA helicase implicated in mRNA splicing and mRNA transport out of the nucleus. As predicted, Hel25E / mutant cells display reduced protein production compared to wild-type neighbors. Thus, Hel25E / mutant cells appear to conform to related loser-cell situations seen in Myclow or Minute/+ cells that also suffer lower protein production. Indeed, like Myclow and RpL14+/ (Minute) clones, Hel25E / loser-cell death occurs specifically at the immediate interface with winner cells, and blocking Caspase activity partially rescued loser-cell elimination. Thus, Hel25E / cells obey both context and spatial rules of apoptotic cell elimination typical of cell competition.
To unravel the mechanism, Nagata et al. (2019) performed an elegant, dominant modifier genetic screen for genes that suppressed Hel25E / cell elimination. This uncovered several components of the lysosomal vacuolar ATPase multisubunit complex. The V-ATPase proton pump is responsible for acidification of the lysosome and, through this, a myriad of cellular functions such as degradation of endosomal, autophagic cargo and cell-signaling functions. The authors found that the lysosomal marker Lysotracker was elevated in loser cells along the clonal margin and that Caspase activation was lost upon reduced V-ATPase activity. This pointed to a lysosomal function in cell death during cell competition. So, what could this function be? Autophagy (self-eating) captures cytosolic cargo in a double-membrane autophagic vesicle destined for degradation by resident hydrolytic enzymes in the lysosome (Figure 1). Through this process, the cell cleans house by eliminating defective organelles and protein aggregates and recycling amino acids, nucleotides, sugars, and lipids for metabolic and bioenergetic needs upon starvation. Autophagy also engages in developmental cell death, described as autophagic cell death in concert with Caspases (Doherty and Baehrecke, 2018). Igaki and colleagues next investigated whether autophagy was contributing to the functionality of lysosomes during cell elimination. Indeed, the authors found that autophagy activity was high and coincided with Caspase activation in loser cells at the clonal boundary. Furthermore, inhibition of Caspase activity did not affect autophagy markers, but knocking down several autophagy components eliminated Caspase activation. These re-
4 Developmental Cell 51, October 7, 2019 ª 2019 Published by Elsevier Inc.
sults raised further questions, including these: How is autophagy controlled? And how does this stimulate Caspase activity and cell elimination? Previous work has implicated stress signaling through Jun N-terminal kinase (JNK) in loser-cell elimination. Hel25E / clones displayed uniform low levels of JNK activity and transcriptional activation of the Caspase activator head involution defective (hid) at the clonal boundary. hid transcription was unaffected by inhibition of JNK signaling and instead appears to be controlled through autophagy. In agreement with previous findings (Scott et al., 2007), Nagata and colleagues found that direct activation of autophagy by the upstream kinase Atg1, or the transcription factor MITF (TFEB in vertebrates) that stimulates lysosome generation and autophagy, was sufficient to induce hid expression and moderately induce cell death. Experimental coactivation of JNK and autophagy was more effective at inducing cell death than either stimulus alone. The authors provide experimental evidence to suggest that NFkB transcription mediates hid transcription downstream of autophagy, as direct Atg1mediated induction of hid transcription was reduced upon knockdown of either of the three NFkB transcription factors (Dorsal, Dif, or Relish). Autophagy negatively regulates NFkB signaling by degrading Kenny (IKKg) (Tusco et al., 2017). It will be important to resolve the mechanism by which autophagy controls NFkB signaling and to determine if NFkB directly controls hid expression in Hel25E / loser cells. The authors suggest the most parsimonious explanation: independent signals lead to a homogeneous JNK stress response in the Hel25E / population, and only at the
Developmental Cell
Previews of autophagic cell death (Doherty and Baehrecke, 2018). With the recent findings that autophagy is activated in loser cells in response to super competitors in Drosophila, and that Flower-mediated cell competition occurs at tumor-microenvironment boundaries in mammals, the implications of this work are of more than just academic interest (Madan et al., 2019; Katheder et al., 2017). Nibbling away at the microenvironment by coercing loser cells to undergo suicide during tumor expansion through autophagy is a very attractive idea that warrants further investigation.
REFERENCES Amoyel, M., and Bach, E.A. (2014). Cell competition: how to eliminate your neighbours. Development 141, 988–1000. Doherty, J., and Baehrecke, E.H. (2018). Life, death and autophagy. Nat. Cell Biol. 20, 1110–1117.
Figure 1. Cell Competition Triggers Cell Elimination through Autophagy Loss-of-function mutations in Helicase at 25E (Hel25E / ) result in lower protein production and elevated JNK stress signaling. Loser status and Caspase-mediated cell death occur in cells facing neighboring winner cells where transcriptional upregulation of the Caspase activator hid depends on autophagy and lysosomal activity, possibly through regulation of NFkB transcriptional responses. Genetic data suggest a role for Flower-mediated sensing of cell fitness and involvement of innate immune signaling in loser-cell elimination.
clonal boundary, where autophagy is active, does this cooperatively lead to effective cell elimination in a mechanism that likely involves NFkB. Is the role of autophagy in cell competition specific to the elimination of Hel25E / cells? Or does it apply widely to the phenomenon? Both Minute/+ clones and Myclow losers showed upregulation of autophagy and cell elimination that were autophagy dependent. Thus the requirement for autophagy in cellcompetition-induced cell elimination appears to apply generally to at least the instances of cell competition displaying protein production differences. The disparate upstream activation signals of JNK and autophagy in Hel25E / cell elimination remain to be defined. Both Myclow and minute cells display a ‘‘lose’’ signal at the cell surface produced by alternative splicing of the transmembrane protein Flower (Fwe), while the winner cells display a winner isoform. It
is the juxtaposition and computation of winner versus loser Fwe isoforms that set in motion the cell-elimination program in loser cells. Igaki and colleagues demonstrate genetically that elimination of Hel25E / cells does depend on fwe and the downstream factor azot. Double knockdown of Atg1 and fwe or azot was not additive, suggesting that they act in a linear pathway, but this was not further explored. Similarly, innate immune signaling has been implicated in elimination of both Myclow and Rpl14E/+ loser cells (Meyer et al., 2014; Germani et al., 2018). Inhibition of innate immune signaling, specifically in Hel25E / loser cells, also counteracted cell elimination. Again, simultaneous inhibition of autophagy, innate immune signaling components, and fwe were not additive. Nagata et al. (2019) provide compelling genetic evidence that autophagy mediates cell elimination during cell competition, providing another physiological example
Germani, F., Hain, D., Sternlicht, D., Moreno, E., and Basler, K. (2018). The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner. eLife 7, e39939. Katheder, N.S., Khezri, R., O’Farrell, F., Schultz, S.W., Jain, A., Rahman, M.M., Schink, K.O., Theodossiou, T.A., Johansen, T., Juha´sz, G., et al. (2017). Microenvironmental autophagy promotes tumour growth. Nature 541, 417–420. Madan, E., Pelham, C.J., Nagane, M., Parker, T.M., Canas-Marques, R., Fazio, K., Shaik, K., Yuan, Y., Henriques, V., Galzerano, A., et al. (2019). Flower isoforms promote competitive growth in cancer. Nature 572, 260–264. Meyer, S.N., Amoyel, M., Bergantinos, C., de la Cova, C., Schertel, C., Basler, K., and Johnston, L.A. (2014). An ancient defense system eliminates unfit cells from developing tissues during cell competition. Science 346, 1258236. Morata, G., and Ripoll, P. (1975). Minutes: mutants of drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221. Nagata, R., Nakamura, M., Sanaka, Y., and Igaki, T. (2019). Cell competition is driven by autophagy. Dev. Cell 51, this issue, 99–112. Scott, R.C., Juha´sz, G., and Neufeld, T.P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17, 1–11. Tusco, R., Jacomin, A.C., Jain, A., Penman, B.S., Larsen, K.B., Johansen, T., and Nezis, I.P. (2017). Kenny mediates selective autophagic degradation of the IKK complex to control innate immune responses. Nat. Commun. 8, 1264.
Developmental Cell 51, October 7, 2019 5