Advanced Review
Cell competition in vertebrate organ size regulation Alfredo I. Penzo-Méndez1,2 and Ben Z. Stanger1,2∗ The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that ‘sense’ and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more ‘fit’ cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication. © 2014 Wiley Periodicals, Inc. How to cite this article:
WIREs Dev Biol 2014, 3:419–427. doi: 10.1002/wdev.148
INTRODUCTION
T
he establishment and maintenance of the animal body plan require not only correct specification of different organs and tissue compartments but also tight spatiotemporal coordination of their growth. This critical aspect of animal biology is however still only partially understood. In vertebrates, regulation of organ growth by systemic factors such as the growth hormone (GH) has been known for more than a century. However, the existence of organ-intrinsic size cues was also demonstrated as ∗ Correspondence
to: [email protected]
1 Gastroenterology
Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Conflict of interest: The authors have declared no conflicts of interest for this article.
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early as the 1920s by Harrison’s work on salamander limb development. Harrison’s experiment involved cross-transplantation of limb buds in salamander larvae from two species, Anobium punctatum and A. tigrinum. Punctatum larvae develop their limbs earlier and reach smaller adult sizes than tigrinum larvae. Surprisingly, punctatum limbs grafted on tigrinum larvae grow to be smaller than corresponding donor limbs, while tigrinum buds grafted in punctatum larvae reach gigantic proportions. Based on this result, Harrison proposed that tigrinum limb buds possess a greater intrinsic size ‘potential’, but that actual growth is modulated by a systemic ‘regulator’ more active in punctatum larvae.1 Additional data have since supported the existence of tissue-intrinsic size determinants, but their nature remains largely unknown. Studies in Drosophila development carried out in the 1970s identified cell competition as a potential mechanism by which organs might
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autonomously regulate their size. Cell competition was observed initially in Drosophila in response to a group of mutations designated as Minutes, which result in cell-autonomous growth reduction due to impaired ribosomal biosynthesis. Heterozygote Minute flies display slowed growth but grossly normal morphology, indicating that Minute (M/+) cells are capable of contributing to all tissues. However in mosaic imaginal discs ‘loser’ Minute cells undergo apoptosis and are progressively eliminated, while ‘winner’ wild-type cell proliferation is increased, resulting in normal disc growth.2,3 Subsequent studies reported a similar phenomenon in mosaic discs containing diminutive (dm, d-myc) mutant cells.4,5 Interest for cell competition surged upon the finding that Myc overexpression turns cells into ‘super-competitors’ capable of eliminating their wild-type neighbors. In turn, Myc-expressing super-competitors become losers in the presence of cells with even higher Myc expression levels, demonstrating that the outcome of a competitive cellular interaction is determined by relative rather than absolute properties of a cell, involving a process of active comparison of some ‘fitness’ parameter(s).4,5 The molecular nature of cell fitness and the mechanism by which it is sensed and compared across cells are unclear. But several recent findings, including the discovery that the phenomenon is also conserved in vertebrate cells, have fuelled renewed interest in this field. In this article, we review the ongoing characterization of this phenomenon at the cellular and molecular level and examine the rapidly growing body of evidence supporting a widespread role for cell competition in the control of vertebrate organ size and tumor progression.
CELL COMPETITION IN MAMMALIAN CELLS The first evidence for the existence of an active mechanism of cell competition in vertebrate cells arose from the study of ribosomal protein mutations. Belly spot tail (Bst) is a loss of function mutation of the mouse Rpl24 gene encoding the L24 ribosomal protein. The phenotype of Rpl24Bst/+ mice is similar to that of Minute flies; heterozygous Bst mice exhibit mildly slowed growth and reduced adult body size along with discrete abnormalities of the skeleton and retina. In order to test whether Bts cells display a competitive disadvantage during mouse development, Oliver et al. injected a fixed number of Rosa26-LacZ embryonic stem (ES) into Rpl24Bst/+ or Rpl24+/+ sibling blastocysts, allowing for a comparison of the resulting 420
chimerism. The Rosa26-LacZ ES cell contribution is dramatically increased in the Rpl24Bst/+ chimeras compared to their wild-type siblings, indicating that Rpl24Bst/+ cells are outcompeted by Rpl24+/+ ; Rosa26-LacZ cells during development.6 This result indicated that a phenomenon similar to that observed in the Drosophila imaginal disc was taking place in the early mouse embryo. However, this initial study did not address whether competition in the mouse embryo displays the features that define imaginal disc competition—active loser cell elimination through apoptosis and cell response driven by relative cell fitness levels. Two important studies have recently fulfilled these criteria. In the study by Sancho et al., coculture of wild-type mouse ES cells with ES cells mutant for Bmpr1a, the autophagy gene Atg5, or cells exhibiting polyploidy resulted in elimination of the latter in a caspase-dependent fashion. Conversely, MYC expression turned ES cells into super-competitors, indicating that the ES cell response to cell competition is determined by relative fitness levels, as it is in Drosophila.7 In vivo, the study by Claveria et al. used a transgenic allele that drives either MYC and yellow fluorescent protein (YFP) expression or only cyan fluorescent protein (CFP) expression after a Cre-mediated recombination event. By using this allele on a Myc-null, heterozygote or wild-type background, lineage-traced chimeras with variable Myc genetic dosages can be generated (Figure 1). Mirroring the results from Drosophila studies, Claveria et al. showed that cells with higher Myc genetic dosage expand at the expense of low-Myc cells in the mouse epiblast between embryonic days 6.5 and 9.5 (E6.5–E9.5). Greater differences in genetic dosage resulted in more efficient elimination of the loser compartment, indicating that competition is modulated by differences in cell fitness. Cell competition was blocked by expression of the anti-apoptotic protein P35 in the low-MYC compartment.8 Furthermore, both studies show that endogenous MYC levels correlate with apoptosis in the mouse epiblast, strongly supporting the notion that cell competition plays an important role in early embryonic size regulation by compensating for physiological variation in MYC levels in order to ensure uniform growth.7,8 Both of these studies in mouse ES cells noted that cell competition was tightly associated with cell differentiation. Competition was not observed in ES cells grown under conditions that maintain totipotency, but depended upon conditions that promote an epiblast-like cell state, most notably by withdrawal of leukemia inhibitory factor (LIF) from the culture media.7 In vivo, elimination of loser cells ceased
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FIGURE 1 | Cell competition in the mouse embryo. (a) Schematic representation of the mouse mosaic embryos generated by injection of embryonic stem (ES) cells carrying the inducible randomo mosaic (iMOS) transgene in wild-type or Myc mutant blastocysts. Allele recombination results in expression of either a CFP (IMOSWt ) or a Myc-YFP (IMOST1-Myc ) cassette. The Myc genetic dosages resulting from different recombinant allele and recipient genotype combinations are indicated. (b) Transgenic E9.5 embryos. Contribution of YFP cells is increased in embryos as the Myc dosage difference increases, while embryo size remains unchanged. (Reprinted with permission from Ref 8. © 2014, Nature Publishing Group.)
after E6.5, coinciding with the downregulation of endogenous Myc expression and a marked decrease of endogenous apoptosis in the mouse embryo.8 In addition, several lines of evidence suggest that the capacity for cell competition might be retained at least in some adult stem cells. Hematopoietic stem cells (HSC) deficient for the p53 tumor suppressor display a competitive advantage during reconstitution of the HSC compartment following exposure to ionizing radiation. This form of competition involves loser cells undergoing growth arrest and decreased response to growth factor rather than apoptosis.9 In contrast, a study on liver regeneration showed that embryonic liver precursors grafted in the adult organ expand over time at the expense of recipient tissue and provided evidence of apoptosis in adult recipient hepatocytes in the vicinity of the progenitor grafts. Interestingly, repopulation by embryonic progenitors was more efficient in aged animals, an effect attributed to higher Activin A levels. Activin A drives hepatocyte growth arrest via activation of the Cyclin-dependent kinase inhibitor p15Ink4b, but does not affect embryonic live progenitors.10,11 Finally, active apoptosis-mediated cell competition has also been observed in vitro in Madin-Darby canine kidney (MDCK) cells as a result of RNAi mediated knockdown of cell polarity genes Scribble (Scrib) and Mahjong (Mahj), further supporting the existence of non-cell-autonomous growth and ‘fitness’ sensing mechanisms in differentiated cells.12,13 Volume 3, November/December 2014
MECHANISMS OF CELL COMPETITION Increasing interest in cell competition has resulted in identification of a number of molecular mediators of competitive behavior. While a comprehensive mechanistic model of cell competition is not yet in hand, some recurrent themes have begun to emerge. For the purposes of description, one can roughly divide the process into three molecular components: mechanisms that reflect or report on a cell’s fitness level, mechanisms by which cells measure their own fitness relative to that of their neighbors, and effector mechanisms that mediate a cell’s response to this measurement.
Determinants of Cell Fitness On the basis of the phenotypes observed in Minute and d-myc mutants, cell fitness was initially equated to cell growth. First, d-myc dysregulation effects cell size rather than cell proliferation. Second, d-myc is known to regulate ribosomal activity through the S6 kinase.14 Third, Minute cells overexpressing Myc do not behave as super-competitors, indicating that Myc drives competition by modulating ribosomal output.5 However, further studies have made it clear that not all pathways regulating growth and proliferation are involved in cell competition. Mosaic activation of PI3-kinase signaling or of the cell cycle regulators CyclinD/CDK4 promote growth in the imaginal disc, but do not result in cell competition.4 Three signaling pathways that promote cell growth and survival have been found to be able
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BOX 1
BOX 2
THE HIPPO PATHWAY
CELL POLARITY PROTEIN COMPLEXES
Mutation of the gene encoding for the Hippo kinase results in tissue overgrowth in Drosophila.18 The major mediator of Hippo effects are the proteins encoded by the yorkie (yki) gene in the fly and by YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) in the mouse. Phosphorylation of Yki/YAP/TAZ downstream of Hippo (MST1 and MST2 in the mouse) results in their degradation. Inactivation of Hippo/MST(1,2) downstream of the protocadherins Fats and Dachsous results in stabilization and nuclear translocation of Yki/YAP/TAZ, which then associates with the Scalloped/TEAD(1-4) DNA binding proteins to regulate target gene transcription. The Hippo pathway has been shown to be a key regulator of organ size in mammals.19 Activation of YAP results in increased cell proliferation and decreased apoptosis and many mammalian cell types.20 In vitro, the Hippo pathway has been implicated in the phenomenon of contact inhibition. YAP/TAZ phosphorylation increases in two-dimensional (2D) mammalian cell cultures as cell density increases. Conversely, forced YAP/TAZ activation allows cells to reach higher densities while maintaining contact inhibition.21,22 Expression of a constitutively activated form of YAP in the mouse liver results in hypertrophy and tumor formation; conversely, YAP inactivation results in reduced liver size.23,24 In the gut, YAP activation leads induces overproliferation of enteric crypt cells, while in the pancreas it leads to de-differentiation of acinar cells, suggesting that the Hippo pathway may influence organ size by regulating the size of stem cell niches.22
Establishment of apico-basal polarity in epithelial cells is driven by the regionalized association of specific protein complexes with the plasma membrane. The Par3/Par6/aPKC complex is required for tight junction formation, with Par6/aPKC being more broadly distributed in the apical membrane domain. The Crb/PatJ/Pals1 complex co-localizes with adherens junctions and is required for their assembly. Lastly the Scrib/Dlg/Lgl complex is present throughout the basolateral membrane domain; disruption of this complex results in loss of cell polarity and increased cell proliferation (see text for details).36–38 The complexes interact to define mutually exclusive domains, for instance, aPKC/Par6 phosphorylates Crb and Scrib complex proteins, which in turn inhibits assembly of these complexes. In addition to their role in establishing cell polarity, the Crb and Scrib complexes also influence cell growth and survival through the Hippo pathway. Assembly of the Crb complex drives activation of Yki/YAP/TAZ in a cell-contact dependent fashion in Drosophila epithelial cells and in the mouse blastula.32,39 Assembly of the Scrib complex, on the other hand, drives phosphorylation of Yki/YAP/TAZ in the Drosophila imaginal disc and in mammalian breast cancer cells.40,41 The molecular mechanisms of these interactions are not yet fully understood, but members of both the Crb and the Scrib complexes have been shown to interact physically with Hippo pathway proteins. This suggests that the cell polarity complexes may act as scaffolds facilitating the interaction between Hippo pathway proteins.39,41,42
to induce super-competitor status in Drosophila: the Hippo pathway (Box 1), the canonical WNT pathway and the JAK/STAT pathway (Figure 2). Inactivation of the Hippo kinase, which results in activation of the Yorkie transcription factor, can rescue Minute cells and turn wild-type cells into super-competitors in a Myc-dependent fashion. WNT and JAK/STAT activation, on the other hand, confer super-competitor status independently of Myc and ribosomal output.12,15–17 Further complicating this picture, genes implicated in other cell processes have also been shown to affect cell fitness, most notably apico-basal cell polarity. Mutation of the genes encoding for the proteins
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of the Scrib/Lgl/Dlg basolateral membrane complex (Box 2) results in Yki activation leading to enlarged imaginal disc and tumor formation. Unexpectedly, scrib, discs large (dlg), and lethal giant larvae (lgl) cells behave as losers in mosaic discs and display low levels of Yki activity.25–28 Inactivation of Yki in these cells is mediated by JNK activated in response to the tumor necrosis factor (TNF) Eiger, which is in turn secreted by hemocytes in what has been proposed to be a tumor suppressing response.29,30 In contrast, cell polarity mutant crumbs (crb) cells retain high Yki activity and behave as super-competitors.31,32 Other competition-inducing genes include Vps23, Vps25, and Rab5, which are implicated in endocytosis, and ATG5, which is required for autophagy.7,33–35
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FIGURE 2 | Schematic representation of the signaling pathways associated with cell competition. Results from studies in both Drosophila and mouse are summarized, mouse gene symbols are shown. Dashed lines represent proposed interactions.
This data indicate that cell competition is driven by other cell processes in addition to cell growth through multiple pathways, and raise the question whether they are integrated into one or more fitness sensors.
Competitive Cell Interactions A second, equally intriguing question is how cell fitness is compared across cells. An early model was proposed by Moreno et al. based on their observation that mutation of thickveins (tkv)—which encodes for a receptor of the Decapentaplegic (Dpp) growth factor—results in loser status in imaginal disc cells. Dpp is a BMP family member required for imaginal disc cell survival. Moreno et al. thus proposed that cell competition could be the result of differences in cell capacity to capture trophic ligands.43 Several studies have since produced evidence against this model. While BMP receptor 1 (Bmpr1) deficiency also results in loser status in mouse ES cells, manipulation of BMP levels through expression of BMP ligands and of the BMP sequestering protein NOGGIN did not affect cell competition in these cultures, indicating that loser cell status is not determined by absolute BMP signaling levels (Figure 3).7 Work by Senoo-Matsuda et al. showed that cell competition occurs between wild-type and Myc overexpressing S2 cells cultured in separate compartments of a transwell apparatus, Volume 3, November/December 2014
demonstrating that competition is mediated by diffusing factors. Under the ligand capture hypothesis, exposing wild-type cells to culture medium conditioned by Myc cells should trigger apoptosis since the medium would have been depleted of survival-limiting ligands. Senoo-Matsuda et al. found instead that simple conditioning was insufficient to induce competitive responses in S2 cells,44 indicating that competition is not driven by depletion of medium borne factors. An alternative mechanism was proposed as a result of the study of cell competition in S2 cells. While medium conditioned by Myc overexpressing super-competitor cells did not induce apoptosis in wild-type cells, medium that was successively used on Myc and wild-type cells did induce apoptosis in naïve wild-type cells and increased proliferation in naïve super-competitors. Furthermore, when the order of medium conditioning was reversed, the result was the same.44 This result suggested the existence of separate fitness sensing and response triggering mechanisms mediated by diffusing factors. The flower gene has been identified as a potential component of the sensing mechanism. It encodes for three isoforms of a transmembrane protein. One isoform, FweUbi , is expressed by all disc cells, while the other two, FweLose-A and FweLose-B , are expressed upon competition by loser minute, d-myc, tkv, and scrib cells. Expression of FweLose-A/B or knockdown of FweUbi
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FIGURE 3 | Competition is driven through diffusing factors in Drosophila S2 cells. (a) Cell competition occurs in wild-type and dMyc overexpressing cells separated by a permeable membrane. (b) Medium conditioned by dMyc overexpressing super-competitor cells does not induce apoptosis in wild-type cells. (c) Successive condition by super-competitors and wild-type cells triggers apoptosis of naïve wild-type cells, indicating that cells must ‘sense’ each other before releasing a ‘killing signal’ into the medium.43
expression is sufficient to induce loser status in disc and S2 cells, as long as FweUbi —only expressing cells are also present. It was thus proposed that Fwe isoforms serve as fitness ‘flags’, and that further interaction of the isoforms with other proteins could trigger competitive responses. As changes in fwe isoform expression occur only with competitive coculture, any role played by fwe in cell competition is likely to reside downstream of any cell-intrinsic fitness determinants.45
Response to Cell Competition In contrast to the elusive nature and identity of ‘sensing’ or ‘killing’ signals in cell competition, the events triggered downstream have begun to be elucidated. In Drosophila, expression of the pro-apoptotic proteins encoded by the hid and reaper genes is required for loser cell elimination in the imaginal disc.4,46 Activation of the JNK pathway has also been shown to occur in Drosophila Minute and d-myc loser cells,4,5,43 but its role in cell competition is unclear. One study reported that JNK signaling inactivation prevents elimination of loser cells,4 while another reported that it does not.5 JNK was also found not to be required for cell competition resulting from JAK/STAT pathway downregulation, suggesting that JNK may play a role as a determinant rather than an effector of cell competition.15 Engulfment of loser cells by winner cells has been reported in minute, d-myc, and scrib mutant discs and also in mouse ES cells.47–49 However the role of engulfment in the competition phenomenon 424
is unclear. In one study, Li and Baker found that the draper and wasp genes, which are required for cell engulfment, are also required for elimination of loser cells in M/+mosaic discs. This result has recently been disputed by Lolo et al., who found instead that loser cells are phagocyted by hemocytes, in line with previous studies reporting extrusion of loser cells from the disc epithelium.5,48 The response to competition in winner cells, on the other hand, has recently been shown to involve p53, a major regulator of metabolism, proliferation and survival. Inactivation of p53 does not affect the viability of imaginal disc cells; however, glucose metabolism, proliferation, and survival were reduced in p53-depleted winner cells in the context of hippo/d-myc/minute induced cell competition. Furthermore, medium conditioned by p53-depleted super-competitors and wild-type cells failed to induce a response in naïve cells, indicating that p53 is not only required for compensatory growth but also for the release of the ‘killing signal’.46 These data support the idea that p53 plays a role in interpreting rather than declaring (or determining) differences in cell fitness. In mouse hematopietic stem cells, however, activation of P53 as result of DNA damage-induced stress results in a competitive disadvantage, suggesting that P53 may play roles at both stages of the competition process.9
ROLES OF CELL COMPETITION IN VERTEBRATES The study of animal organ size control has revealed great variation in the degree and mode of regulation across different species, organs, and developmental stages. The tissues where cell competition has been described are all characterized by a high degree of plasticity and tight regulation of their size to a predetermined ‘setpoint’.50,51 There is little doubt that cell competition contributes to this plasticity, as demonstrated by the observation that imaginal discs can maintain their size even when all disc cells overexpress Myc, as long as they do it in a mosaic fashion that results in cell competition.4,5 A physiological role of cell competition in organ size control is further suggested by the fact that cell competition correlates with endogenous heterogeneity in Myc expression levels,4,8 and also by the fact that cell fitness is determined downstream of the Hippo pathway, which has been implicated in organ size control.19 What is less clear is whether cell competition evolved primarily as a size regulating mechanism. Cell competition cannot, for instance, dictate
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Emergence of precancerous super-competitor
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FIGURE 4 | Potential role of cell competition in tumor formation. Cell transformation involves successive mutagenic events promoting unrestrained cell growth. In the presence of active cell competition mechanisms, these events would likely also result in increased cellular fitness. Early precancerous cells would then behave as super-competitors and expand by eliminating the surrounding healthy cells, perceived as less fit. Cell competition would thus result in formation of precancerous fields, increasing the likelihood of cells accumulating additional mutations leading to tumor formation.
an organ’s size ‘setpoint’, as the process equalizes cell growth rates to that of the fastest growing subpopulation (i.e., if this equalization occurs before organ growth is completed, overgrowth will result). In addition, not all of the determinants of ‘loser’ status correlate with impaired size, in fact the opposite is true in the case of cell polarity genes. Furthermore, inhibiting the elimination of loser cells through expression of p35 in the wing imaginal disc results in increased wing size variation; nevertheless, resulting wing size is still within the normal range.4 An alternative interpretation is that cell competition evolved primarily as a mechanism to maintain tissue integrity and viability rather than size. This idea could explain why scrib/lgl/dlg mutant cells behave as losers despite having a high growth potential: they are perceived as unfit because loss of polarity compromises epithelial integrity. This notion is also supported by the observation that liver cells are less fit in aging animals, even though the liver does not actively grow in healthy adult animals.10 Ultimately, addressing the biological significance of cell competition will require that the ‘sensors’ of cell fitness are identified, so that the effects of abolishing cell competition while preserving endogenous cell heterogeneity can be determined in vivo. Independently of the physiological role of cell competition, the confirmation that the process exists in vertebrate cells also raises the interesting possibility Volume 3, November/December 2014
that this mechanism might contribute to the expansion of precancerous and cancerous cells. Many of the genes that have been identified as drivers of cell competition to date are also drivers of tumor formation. It has been proposed that cell competition could drive tumor formation by allowing precancerous cells to expand, increasing the chances of additional mutation leading to transformation and tumorigenesis through a ‘field effect’ phenomenon (Figure 4).52,53 Such precancerous fields have been described in association with several tumor types, most notably squamous carcinomas, and mutational analyses suggest that tumors and precancerous field are derived from the same progenitors.54 This possibility will no doubt receive increased attention by researchers in the near future.
CONCLUSION The phenomenon of cell competition has intrigued the scientific community for 40 years. It bears potential implications to the understanding of fundamental, but little understood areas of biology such as are the evolution of multicellularity, organ size control, and senescence. The recognition that cell competition is conserved from arthropods to vertebrates is an important advance, as it confirms the importance of the process and puts it squarely within the scope of stem cell biology and cancer biology. The most immediate challenge at present is to obtain
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a comprehensive understanding of the signals that cells employ to compare relative rates of growth, proliferation, and other measures of cellular ‘fitness’. Mammalian cells lines, high throughput screening and
next-generation sequencing technologies will be at the forefront of this effort, while genetic models will continue to contribute to elucidate the physiological and pathological roles of cell competition.
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through regulation of the Hippo pathway. Proc Natl Acad Sci USA 2012, 109:484–489. 27. Igaki T, Pagliarini RA, Xu T. Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila. Curr Biol 2006, 16:1139–1146. 28. Pagliarini RA, Xu T. A genetic screen in Drosophila for metastatic behavior. Science 2003, 302:1227–1231. 29. Cordero JB, Macagno JP, Stefanatos RK, Strathdee KE, Cagan RL, Vidal M. Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter. Dev Cell 2010, 18:999–1011.
AR, Poletti A, et al. The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 2011, 147:759–772. 41. Doggett K, Grusche FA, Richardson HE, Brumby AM. Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling. BMC Dev Biol 2011, 11:57. 42. Chatterjee SJ, McCaffrey L. Emerging role of cell polarity proteins in breast cancer progression and metastasis. Breast Cancer (Dove Med Press) 2014, 6:15–27.
30. Igaki T, Pastor-Pareja JC, Aonuma H, Miura M, Xu T. Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila. Dev Cell 2009, 16:458–465.
43. Moreno E, Basler K, Morata G. Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 2002, 416:755–759.
31. Hafezi Y, Bosch JA, Hariharan IK. Differences in levels of the transmembrane protein Crumbs can influence cell survival at clonal boundaries. Dev Biol 2012, 368:358–369.
44. Senoo-Matsuda N, Johnston LA. Soluble factors mediate competitive and cooperative interactions between cells expressing different levels of Drosophila Myc. Proc Natl Acad Sci USA 2007, 104:18543–18548.
32. Robinson BS, Huang J, Hong Y, Moberg KH. Crumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein expanded. Curr Biol 2010, 20:582–590.
45. Rhiner C, López-Gay JM, Soldini D, Casas-Tinto S, Martín FA, Lombardía L, Moreno E. Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila. Dev Cell 2010, 18: 985–998.
33. Ballesteros-Arias L, Saavedra V, Morata G. Cell competition may function either as tumour-suppressing or as tumour-stimulating factor in Drosophila. Oncogene. In press. doi: 10.1038/onc.2013.407. 34. Herz HM, Chen Z, Scherr H, Lackey M, Bolduc C, Bergmann A. vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis. Development 2006, 133:1871–1880. 35. Moberg KH, Schelble S, Burdick SK, Hariharan IK. Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell 2005, 9:699–710. 36. Assémat E, Bazellières E, Pallesi-Pocachard E, Le Bivic A, Massey-Harroche D. Polarity complex proteins. Biochim Biophys Acta 2008, 1778:614–630. 37. Goldstein B, Macara IG. The PAR proteins: fundamental players in animal cell polarization. Dev Cell 2007, 13:609–622. 38. Martin-Belmonte F, Mostov K. Regulation of cell polarity during epithelial morphogenesis. Curr Opin Cell Biol 2008, 20:227–234. 39. Varelas X, Samavarchi-Tehrani P, Narimatsu M, Weiss A, Cockburn K, Larsen BG, Rossant J, Wrana JL. The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-b-SMAD pathway. Dev Cell 2010, 19:831–844. 40. Cordenonsi M, Zanconato F, Azzolin L, Forcato M, Rosato A, Frasson C, Inui M, Montagner M, Parenti
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46. de la Cova C, Senoo-Matsuda N, Ziosi M, Wu DC, Bellosta P, Quinzii CM, Johnston LA. Supercompetitor status of Drosophila Myc cells requires p53 as a fitness sensor to reprogram metabolism and promote viability. Cell Metab 2014, 19:470–483. 47. Li W, Baker NE. Engulfment is required for cell competition. Cell 2007, 129:1215–1225. 48. Lolo FN, Casas-Tintó S, Moreno E. Cell competition time line: winners kill losers, which are extruded and engulfed by hemocytes. Cell Rep 2012, 2:526–539. 49. Ohsawa S, Sugimura K, Takino K, Xu T, Miyawaki A, Igaki T. Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila. Dev Cell 2011, 20:315–328. 50. Rands GF. Size regulation in the mouse embryo. II. The development of half embryos. J Embryol Exp Morphol 1986, 98:209–217. 51. Stanger BZ. The biology of organ size determination. Diabetes Obes Metab 2008, 10(suppl 4):16–22. 52. Baker NE, Li W. Cell competition and its possible relation to cancer. Cancer Res 2008, 68:5505–5507. 53. Moreno E. Is cell competition relevant to cancer? Nat Rev Cancer 2008, 8:141–147. 54. Rubin H. Fields and field cancerization: the preneoplastic origins of cancer: asymptomatic hyperplastic fields are precursors of neoplasia, and their progression to tumors can be tracked by saturation density in culture. Bioessays 2011, 33:224–231.
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Cell competition in vertebrate organ size regulation Alfredo I. Penzo-Méndez1,2 and Ben Z. Stanger1,2∗ The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that ‘sense’ and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more ‘fit’ cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication. © 2014 Wiley Periodicals, Inc. How to cite this article:
WIREs Dev Biol 2014, 3:419–427. doi: 10.1002/wdev.148
INTRODUCTION
T
he establishment and maintenance of the animal body plan require not only correct specification of different organs and tissue compartments but also tight spatiotemporal coordination of their growth. This critical aspect of animal biology is however still only partially understood. In vertebrates, regulation of organ growth by systemic factors such as the growth hormone (GH) has been known for more than a century. However, the existence of organ-intrinsic size cues was also demonstrated as ∗ Correspondence
to: [email protected]
1 Gastroenterology
Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Conflict of interest: The authors have declared no conflicts of interest for this article.
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early as the 1920s by Harrison’s work on salamander limb development. Harrison’s experiment involved cross-transplantation of limb buds in salamander larvae from two species, Anobium punctatum and A. tigrinum. Punctatum larvae develop their limbs earlier and reach smaller adult sizes than tigrinum larvae. Surprisingly, punctatum limbs grafted on tigrinum larvae grow to be smaller than corresponding donor limbs, while tigrinum buds grafted in punctatum larvae reach gigantic proportions. Based on this result, Harrison proposed that tigrinum limb buds possess a greater intrinsic size ‘potential’, but that actual growth is modulated by a systemic ‘regulator’ more active in punctatum larvae.1 Additional data have since supported the existence of tissue-intrinsic size determinants, but their nature remains largely unknown. Studies in Drosophila development carried out in the 1970s identified cell competition as a potential mechanism by which organs might
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autonomously regulate their size. Cell competition was observed initially in Drosophila in response to a group of mutations designated as Minutes, which result in cell-autonomous growth reduction due to impaired ribosomal biosynthesis. Heterozygote Minute flies display slowed growth but grossly normal morphology, indicating that Minute (M/+) cells are capable of contributing to all tissues. However in mosaic imaginal discs ‘loser’ Minute cells undergo apoptosis and are progressively eliminated, while ‘winner’ wild-type cell proliferation is increased, resulting in normal disc growth.2,3 Subsequent studies reported a similar phenomenon in mosaic discs containing diminutive (dm, d-myc) mutant cells.4,5 Interest for cell competition surged upon the finding that Myc overexpression turns cells into ‘super-competitors’ capable of eliminating their wild-type neighbors. In turn, Myc-expressing super-competitors become losers in the presence of cells with even higher Myc expression levels, demonstrating that the outcome of a competitive cellular interaction is determined by relative rather than absolute properties of a cell, involving a process of active comparison of some ‘fitness’ parameter(s).4,5 The molecular nature of cell fitness and the mechanism by which it is sensed and compared across cells are unclear. But several recent findings, including the discovery that the phenomenon is also conserved in vertebrate cells, have fuelled renewed interest in this field. In this article, we review the ongoing characterization of this phenomenon at the cellular and molecular level and examine the rapidly growing body of evidence supporting a widespread role for cell competition in the control of vertebrate organ size and tumor progression.
CELL COMPETITION IN MAMMALIAN CELLS The first evidence for the existence of an active mechanism of cell competition in vertebrate cells arose from the study of ribosomal protein mutations. Belly spot tail (Bst) is a loss of function mutation of the mouse Rpl24 gene encoding the L24 ribosomal protein. The phenotype of Rpl24Bst/+ mice is similar to that of Minute flies; heterozygous Bst mice exhibit mildly slowed growth and reduced adult body size along with discrete abnormalities of the skeleton and retina. In order to test whether Bts cells display a competitive disadvantage during mouse development, Oliver et al. injected a fixed number of Rosa26-LacZ embryonic stem (ES) into Rpl24Bst/+ or Rpl24+/+ sibling blastocysts, allowing for a comparison of the resulting 420
chimerism. The Rosa26-LacZ ES cell contribution is dramatically increased in the Rpl24Bst/+ chimeras compared to their wild-type siblings, indicating that Rpl24Bst/+ cells are outcompeted by Rpl24+/+ ; Rosa26-LacZ cells during development.6 This result indicated that a phenomenon similar to that observed in the Drosophila imaginal disc was taking place in the early mouse embryo. However, this initial study did not address whether competition in the mouse embryo displays the features that define imaginal disc competition—active loser cell elimination through apoptosis and cell response driven by relative cell fitness levels. Two important studies have recently fulfilled these criteria. In the study by Sancho et al., coculture of wild-type mouse ES cells with ES cells mutant for Bmpr1a, the autophagy gene Atg5, or cells exhibiting polyploidy resulted in elimination of the latter in a caspase-dependent fashion. Conversely, MYC expression turned ES cells into super-competitors, indicating that the ES cell response to cell competition is determined by relative fitness levels, as it is in Drosophila.7 In vivo, the study by Claveria et al. used a transgenic allele that drives either MYC and yellow fluorescent protein (YFP) expression or only cyan fluorescent protein (CFP) expression after a Cre-mediated recombination event. By using this allele on a Myc-null, heterozygote or wild-type background, lineage-traced chimeras with variable Myc genetic dosages can be generated (Figure 1). Mirroring the results from Drosophila studies, Claveria et al. showed that cells with higher Myc genetic dosage expand at the expense of low-Myc cells in the mouse epiblast between embryonic days 6.5 and 9.5 (E6.5–E9.5). Greater differences in genetic dosage resulted in more efficient elimination of the loser compartment, indicating that competition is modulated by differences in cell fitness. Cell competition was blocked by expression of the anti-apoptotic protein P35 in the low-MYC compartment.8 Furthermore, both studies show that endogenous MYC levels correlate with apoptosis in the mouse epiblast, strongly supporting the notion that cell competition plays an important role in early embryonic size regulation by compensating for physiological variation in MYC levels in order to ensure uniform growth.7,8 Both of these studies in mouse ES cells noted that cell competition was tightly associated with cell differentiation. Competition was not observed in ES cells grown under conditions that maintain totipotency, but depended upon conditions that promote an epiblast-like cell state, most notably by withdrawal of leukemia inhibitory factor (LIF) from the culture media.7 In vivo, elimination of loser cells ceased
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FIGURE 1 | Cell competition in the mouse embryo. (a) Schematic representation of the mouse mosaic embryos generated by injection of embryonic stem (ES) cells carrying the inducible randomo mosaic (iMOS) transgene in wild-type or Myc mutant blastocysts. Allele recombination results in expression of either a CFP (IMOSWt ) or a Myc-YFP (IMOST1-Myc ) cassette. The Myc genetic dosages resulting from different recombinant allele and recipient genotype combinations are indicated. (b) Transgenic E9.5 embryos. Contribution of YFP cells is increased in embryos as the Myc dosage difference increases, while embryo size remains unchanged. (Reprinted with permission from Ref 8. © 2014, Nature Publishing Group.)
after E6.5, coinciding with the downregulation of endogenous Myc expression and a marked decrease of endogenous apoptosis in the mouse embryo.8 In addition, several lines of evidence suggest that the capacity for cell competition might be retained at least in some adult stem cells. Hematopoietic stem cells (HSC) deficient for the p53 tumor suppressor display a competitive advantage during reconstitution of the HSC compartment following exposure to ionizing radiation. This form of competition involves loser cells undergoing growth arrest and decreased response to growth factor rather than apoptosis.9 In contrast, a study on liver regeneration showed that embryonic liver precursors grafted in the adult organ expand over time at the expense of recipient tissue and provided evidence of apoptosis in adult recipient hepatocytes in the vicinity of the progenitor grafts. Interestingly, repopulation by embryonic progenitors was more efficient in aged animals, an effect attributed to higher Activin A levels. Activin A drives hepatocyte growth arrest via activation of the Cyclin-dependent kinase inhibitor p15Ink4b, but does not affect embryonic live progenitors.10,11 Finally, active apoptosis-mediated cell competition has also been observed in vitro in Madin-Darby canine kidney (MDCK) cells as a result of RNAi mediated knockdown of cell polarity genes Scribble (Scrib) and Mahjong (Mahj), further supporting the existence of non-cell-autonomous growth and ‘fitness’ sensing mechanisms in differentiated cells.12,13 Volume 3, November/December 2014
MECHANISMS OF CELL COMPETITION Increasing interest in cell competition has resulted in identification of a number of molecular mediators of competitive behavior. While a comprehensive mechanistic model of cell competition is not yet in hand, some recurrent themes have begun to emerge. For the purposes of description, one can roughly divide the process into three molecular components: mechanisms that reflect or report on a cell’s fitness level, mechanisms by which cells measure their own fitness relative to that of their neighbors, and effector mechanisms that mediate a cell’s response to this measurement.
Determinants of Cell Fitness On the basis of the phenotypes observed in Minute and d-myc mutants, cell fitness was initially equated to cell growth. First, d-myc dysregulation effects cell size rather than cell proliferation. Second, d-myc is known to regulate ribosomal activity through the S6 kinase.14 Third, Minute cells overexpressing Myc do not behave as super-competitors, indicating that Myc drives competition by modulating ribosomal output.5 However, further studies have made it clear that not all pathways regulating growth and proliferation are involved in cell competition. Mosaic activation of PI3-kinase signaling or of the cell cycle regulators CyclinD/CDK4 promote growth in the imaginal disc, but do not result in cell competition.4 Three signaling pathways that promote cell growth and survival have been found to be able
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BOX 1
BOX 2
THE HIPPO PATHWAY
CELL POLARITY PROTEIN COMPLEXES
Mutation of the gene encoding for the Hippo kinase results in tissue overgrowth in Drosophila.18 The major mediator of Hippo effects are the proteins encoded by the yorkie (yki) gene in the fly and by YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) in the mouse. Phosphorylation of Yki/YAP/TAZ downstream of Hippo (MST1 and MST2 in the mouse) results in their degradation. Inactivation of Hippo/MST(1,2) downstream of the protocadherins Fats and Dachsous results in stabilization and nuclear translocation of Yki/YAP/TAZ, which then associates with the Scalloped/TEAD(1-4) DNA binding proteins to regulate target gene transcription. The Hippo pathway has been shown to be a key regulator of organ size in mammals.19 Activation of YAP results in increased cell proliferation and decreased apoptosis and many mammalian cell types.20 In vitro, the Hippo pathway has been implicated in the phenomenon of contact inhibition. YAP/TAZ phosphorylation increases in two-dimensional (2D) mammalian cell cultures as cell density increases. Conversely, forced YAP/TAZ activation allows cells to reach higher densities while maintaining contact inhibition.21,22 Expression of a constitutively activated form of YAP in the mouse liver results in hypertrophy and tumor formation; conversely, YAP inactivation results in reduced liver size.23,24 In the gut, YAP activation leads induces overproliferation of enteric crypt cells, while in the pancreas it leads to de-differentiation of acinar cells, suggesting that the Hippo pathway may influence organ size by regulating the size of stem cell niches.22
Establishment of apico-basal polarity in epithelial cells is driven by the regionalized association of specific protein complexes with the plasma membrane. The Par3/Par6/aPKC complex is required for tight junction formation, with Par6/aPKC being more broadly distributed in the apical membrane domain. The Crb/PatJ/Pals1 complex co-localizes with adherens junctions and is required for their assembly. Lastly the Scrib/Dlg/Lgl complex is present throughout the basolateral membrane domain; disruption of this complex results in loss of cell polarity and increased cell proliferation (see text for details).36–38 The complexes interact to define mutually exclusive domains, for instance, aPKC/Par6 phosphorylates Crb and Scrib complex proteins, which in turn inhibits assembly of these complexes. In addition to their role in establishing cell polarity, the Crb and Scrib complexes also influence cell growth and survival through the Hippo pathway. Assembly of the Crb complex drives activation of Yki/YAP/TAZ in a cell-contact dependent fashion in Drosophila epithelial cells and in the mouse blastula.32,39 Assembly of the Scrib complex, on the other hand, drives phosphorylation of Yki/YAP/TAZ in the Drosophila imaginal disc and in mammalian breast cancer cells.40,41 The molecular mechanisms of these interactions are not yet fully understood, but members of both the Crb and the Scrib complexes have been shown to interact physically with Hippo pathway proteins. This suggests that the cell polarity complexes may act as scaffolds facilitating the interaction between Hippo pathway proteins.39,41,42
to induce super-competitor status in Drosophila: the Hippo pathway (Box 1), the canonical WNT pathway and the JAK/STAT pathway (Figure 2). Inactivation of the Hippo kinase, which results in activation of the Yorkie transcription factor, can rescue Minute cells and turn wild-type cells into super-competitors in a Myc-dependent fashion. WNT and JAK/STAT activation, on the other hand, confer super-competitor status independently of Myc and ribosomal output.12,15–17 Further complicating this picture, genes implicated in other cell processes have also been shown to affect cell fitness, most notably apico-basal cell polarity. Mutation of the genes encoding for the proteins
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of the Scrib/Lgl/Dlg basolateral membrane complex (Box 2) results in Yki activation leading to enlarged imaginal disc and tumor formation. Unexpectedly, scrib, discs large (dlg), and lethal giant larvae (lgl) cells behave as losers in mosaic discs and display low levels of Yki activity.25–28 Inactivation of Yki in these cells is mediated by JNK activated in response to the tumor necrosis factor (TNF) Eiger, which is in turn secreted by hemocytes in what has been proposed to be a tumor suppressing response.29,30 In contrast, cell polarity mutant crumbs (crb) cells retain high Yki activity and behave as super-competitors.31,32 Other competition-inducing genes include Vps23, Vps25, and Rab5, which are implicated in endocytosis, and ATG5, which is required for autophagy.7,33–35
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This data indicate that cell competition is driven by other cell processes in addition to cell growth through multiple pathways, and raise the question whether they are integrated into one or more fitness sensors.
Competitive Cell Interactions A second, equally intriguing question is how cell fitness is compared across cells. An early model was proposed by Moreno et al. based on their observation that mutation of thickveins (tkv)—which encodes for a receptor of the Decapentaplegic (Dpp) growth factor—results in loser status in imaginal disc cells. Dpp is a BMP family member required for imaginal disc cell survival. Moreno et al. thus proposed that cell competition could be the result of differences in cell capacity to capture trophic ligands.43 Several studies have since produced evidence against this model. While BMP receptor 1 (Bmpr1) deficiency also results in loser status in mouse ES cells, manipulation of BMP levels through expression of BMP ligands and of the BMP sequestering protein NOGGIN did not affect cell competition in these cultures, indicating that loser cell status is not determined by absolute BMP signaling levels (Figure 3).7 Work by Senoo-Matsuda et al. showed that cell competition occurs between wild-type and Myc overexpressing S2 cells cultured in separate compartments of a transwell apparatus, Volume 3, November/December 2014
demonstrating that competition is mediated by diffusing factors. Under the ligand capture hypothesis, exposing wild-type cells to culture medium conditioned by Myc cells should trigger apoptosis since the medium would have been depleted of survival-limiting ligands. Senoo-Matsuda et al. found instead that simple conditioning was insufficient to induce competitive responses in S2 cells,44 indicating that competition is not driven by depletion of medium borne factors. An alternative mechanism was proposed as a result of the study of cell competition in S2 cells. While medium conditioned by Myc overexpressing super-competitor cells did not induce apoptosis in wild-type cells, medium that was successively used on Myc and wild-type cells did induce apoptosis in naïve wild-type cells and increased proliferation in naïve super-competitors. Furthermore, when the order of medium conditioning was reversed, the result was the same.44 This result suggested the existence of separate fitness sensing and response triggering mechanisms mediated by diffusing factors. The flower gene has been identified as a potential component of the sensing mechanism. It encodes for three isoforms of a transmembrane protein. One isoform, FweUbi , is expressed by all disc cells, while the other two, FweLose-A and FweLose-B , are expressed upon competition by loser minute, d-myc, tkv, and scrib cells. Expression of FweLose-A/B or knockdown of FweUbi
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FIGURE 3 | Competition is driven through diffusing factors in Drosophila S2 cells. (a) Cell competition occurs in wild-type and dMyc overexpressing cells separated by a permeable membrane. (b) Medium conditioned by dMyc overexpressing super-competitor cells does not induce apoptosis in wild-type cells. (c) Successive condition by super-competitors and wild-type cells triggers apoptosis of naïve wild-type cells, indicating that cells must ‘sense’ each other before releasing a ‘killing signal’ into the medium.43
expression is sufficient to induce loser status in disc and S2 cells, as long as FweUbi —only expressing cells are also present. It was thus proposed that Fwe isoforms serve as fitness ‘flags’, and that further interaction of the isoforms with other proteins could trigger competitive responses. As changes in fwe isoform expression occur only with competitive coculture, any role played by fwe in cell competition is likely to reside downstream of any cell-intrinsic fitness determinants.45
Response to Cell Competition In contrast to the elusive nature and identity of ‘sensing’ or ‘killing’ signals in cell competition, the events triggered downstream have begun to be elucidated. In Drosophila, expression of the pro-apoptotic proteins encoded by the hid and reaper genes is required for loser cell elimination in the imaginal disc.4,46 Activation of the JNK pathway has also been shown to occur in Drosophila Minute and d-myc loser cells,4,5,43 but its role in cell competition is unclear. One study reported that JNK signaling inactivation prevents elimination of loser cells,4 while another reported that it does not.5 JNK was also found not to be required for cell competition resulting from JAK/STAT pathway downregulation, suggesting that JNK may play a role as a determinant rather than an effector of cell competition.15 Engulfment of loser cells by winner cells has been reported in minute, d-myc, and scrib mutant discs and also in mouse ES cells.47–49 However the role of engulfment in the competition phenomenon 424
is unclear. In one study, Li and Baker found that the draper and wasp genes, which are required for cell engulfment, are also required for elimination of loser cells in M/+mosaic discs. This result has recently been disputed by Lolo et al., who found instead that loser cells are phagocyted by hemocytes, in line with previous studies reporting extrusion of loser cells from the disc epithelium.5,48 The response to competition in winner cells, on the other hand, has recently been shown to involve p53, a major regulator of metabolism, proliferation and survival. Inactivation of p53 does not affect the viability of imaginal disc cells; however, glucose metabolism, proliferation, and survival were reduced in p53-depleted winner cells in the context of hippo/d-myc/minute induced cell competition. Furthermore, medium conditioned by p53-depleted super-competitors and wild-type cells failed to induce a response in naïve cells, indicating that p53 is not only required for compensatory growth but also for the release of the ‘killing signal’.46 These data support the idea that p53 plays a role in interpreting rather than declaring (or determining) differences in cell fitness. In mouse hematopietic stem cells, however, activation of P53 as result of DNA damage-induced stress results in a competitive disadvantage, suggesting that P53 may play roles at both stages of the competition process.9
ROLES OF CELL COMPETITION IN VERTEBRATES The study of animal organ size control has revealed great variation in the degree and mode of regulation across different species, organs, and developmental stages. The tissues where cell competition has been described are all characterized by a high degree of plasticity and tight regulation of their size to a predetermined ‘setpoint’.50,51 There is little doubt that cell competition contributes to this plasticity, as demonstrated by the observation that imaginal discs can maintain their size even when all disc cells overexpress Myc, as long as they do it in a mosaic fashion that results in cell competition.4,5 A physiological role of cell competition in organ size control is further suggested by the fact that cell competition correlates with endogenous heterogeneity in Myc expression levels,4,8 and also by the fact that cell fitness is determined downstream of the Hippo pathway, which has been implicated in organ size control.19 What is less clear is whether cell competition evolved primarily as a size regulating mechanism. Cell competition cannot, for instance, dictate
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FIGURE 4 | Potential role of cell competition in tumor formation. Cell transformation involves successive mutagenic events promoting unrestrained cell growth. In the presence of active cell competition mechanisms, these events would likely also result in increased cellular fitness. Early precancerous cells would then behave as super-competitors and expand by eliminating the surrounding healthy cells, perceived as less fit. Cell competition would thus result in formation of precancerous fields, increasing the likelihood of cells accumulating additional mutations leading to tumor formation.
an organ’s size ‘setpoint’, as the process equalizes cell growth rates to that of the fastest growing subpopulation (i.e., if this equalization occurs before organ growth is completed, overgrowth will result). In addition, not all of the determinants of ‘loser’ status correlate with impaired size, in fact the opposite is true in the case of cell polarity genes. Furthermore, inhibiting the elimination of loser cells through expression of p35 in the wing imaginal disc results in increased wing size variation; nevertheless, resulting wing size is still within the normal range.4 An alternative interpretation is that cell competition evolved primarily as a mechanism to maintain tissue integrity and viability rather than size. This idea could explain why scrib/lgl/dlg mutant cells behave as losers despite having a high growth potential: they are perceived as unfit because loss of polarity compromises epithelial integrity. This notion is also supported by the observation that liver cells are less fit in aging animals, even though the liver does not actively grow in healthy adult animals.10 Ultimately, addressing the biological significance of cell competition will require that the ‘sensors’ of cell fitness are identified, so that the effects of abolishing cell competition while preserving endogenous cell heterogeneity can be determined in vivo. Independently of the physiological role of cell competition, the confirmation that the process exists in vertebrate cells also raises the interesting possibility Volume 3, November/December 2014
that this mechanism might contribute to the expansion of precancerous and cancerous cells. Many of the genes that have been identified as drivers of cell competition to date are also drivers of tumor formation. It has been proposed that cell competition could drive tumor formation by allowing precancerous cells to expand, increasing the chances of additional mutation leading to transformation and tumorigenesis through a ‘field effect’ phenomenon (Figure 4).52,53 Such precancerous fields have been described in association with several tumor types, most notably squamous carcinomas, and mutational analyses suggest that tumors and precancerous field are derived from the same progenitors.54 This possibility will no doubt receive increased attention by researchers in the near future.
CONCLUSION The phenomenon of cell competition has intrigued the scientific community for 40 years. It bears potential implications to the understanding of fundamental, but little understood areas of biology such as are the evolution of multicellularity, organ size control, and senescence. The recognition that cell competition is conserved from arthropods to vertebrates is an important advance, as it confirms the importance of the process and puts it squarely within the scope of stem cell biology and cancer biology. The most immediate challenge at present is to obtain
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a comprehensive understanding of the signals that cells employ to compare relative rates of growth, proliferation, and other measures of cellular ‘fitness’. Mammalian cells lines, high throughput screening and
next-generation sequencing technologies will be at the forefront of this effort, while genetic models will continue to contribute to elucidate the physiological and pathological roles of cell competition.
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