Received: 22 June 2020 Revised: 11 December 2020

DOI: 10.1002/bies.202000156

PROBLEMS & PARADIGMS

Prospects & Overviews Cancer and the breakdown of multicellularity: What discoideum, a social , can teach us

Sabateeshan Mathavarajah1 Carter VanIderstine1 Graham Dellaire1,2 Robert J. Huber3

1 Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Abstract Scotia, Canada Ancient pathways promoting unicellularity and multicellularity are associated with 2 Department of Biochemistry and Molecular cancer, the former being pro-oncogenic and the latter acting to suppress oncogenesis. Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada However, there are only a limited number of non-vertebrate models for studying these 3 Department of Biology, Trent University, pathways. Here, we review Dictyostelium discoideum and describe how it can be used to Peterborough, Ontario, Canada understand these gene networks. D. discoideum has a unicellular and multicellular life

Correspondence cycle, making it possible to study orthologs of cancer-associated genes in both phases. Robert J. Huber, Department of Biology, Trent During development, differentiated amoebae form a fruiting body composed of a mass University, 1600 West Bank Drive, Peterbor- ough, Ontario K9L 0G2, Canada. of that are supported atop a stalk. A portion of the cells sacrifice themselves Email: [email protected] to become non-reproductive stalk cells. Cheating disrupts the principles of multicellu-

Funding information larity, as cheater cells alter their cell fate to preferentially become spores. Importantly, Killam Pre-Doctoral Award; Nova Scotia Grad- D. discoideum has gene networks and several strategies for maintaining multicellularity. uate Scholarship; President’s Award from Dalhousie University; Natural Sciences and Therefore, D. discoideum can help us better understand how conserved genes and path- Engineering Research Council of Canada, ways involved in multicellularity also influence cancer development, potentially identi- Grant/Award Numbers: RGPIN-2018-04855, RGPIN-2020-04034 fying new therapeutic avenues.

KEYWORDS cancer, cell fate, cheating, Dictyostelium, multicellularity, oncogenesis, social amoeba

INTRODUCTION collectively promote malignancy.[3,4] Both horizontal gene transfer (oncogenes transferred via retroviruses) and vertical gene transfer Many cancer models are based on a framework of evolutionary theo- (evolution of gene networks controlling cellular proliferation and ries that have improved our understanding of neoplastic progression death) contributed to the development of gene networks regulating (the abnormal growth of tumors due to aberrant cell prolifera- these cellular mechanisms in metazoans.[5] Although many of the tion), drug resistance, and clonality.[1] Somatic evolution theory genetic mechanisms driving oncogenesis are well understood, there presents oncogenesis (the development of cancer) as the result of are remaining questions regarding how gene networks are dysreg- the intra-organismal natural selection for malignant cells, facilitated ulated during oncogenesis to promote pathways associated with by mutations in proto-oncogenes and tumor suppressor genes.[2] unicellularity, while downregulating those associated with multicel- These genomic changes alter pathways that control DNA repair, lularity. Therefore, the study of how these gene networks evolved cellular proliferation, cell death, cell invasion, and metastasis, which in unicellular and multicellular organisms represents a previously untapped source of knowledge for gathering novel insights into cancer development and progression that ultimately could be exploited for Abbreviations: CAG, cancer-associated gene; ECM, extracellular matrix; NB, nuclear bodies; PML, promyelocytic leukaemia protein; PTEN, phosphatase and tensin homolog treatment.

BioEssays. 2020;2000156. wileyonlinelibrary.com/journal/bies © 2021 Wiley Periodicals LLC 1of13 https://doi.org/10.1002/bies.202000156 2of13 MATHAVARAJAH ET AL.

HOW DOES THE BREAKDOWN OF The evolutionary history of genes has shaped their MULTICELLULARITY PROMOTE CANCER role(s) in oncogenesis DEVELOPMENT? Evolutionarily conserved genes from pathways associated with unicel- Hallmarks of cancer require dysregulation of the lular (e.g., cell cycle regulation) and multicellular (e.g., maintenance of principles of multicellularity the extracellular matrix) growth states are upregulated and downregu- lated, respectively, in malignant cells.[9,10] For example, transcriptional Recent studies indicate that many of the dysregulated genes associ- changes that promote cancer growth are linked to somatic mutations ated with cancer are divided into two categories; (1) processes that and copy number changes in genes that arose during early metazoan maintain multicellularity and (2) processes that are essential for the evolution, and these genetic changes disrupt regulatory links between survival of unicellular organisms.[6–11] In 2015, Aktipis et al. described unicellular and multicellular gene programs.[11] These findings align the parallels between multicellularity and the well-studied hallmarks of with two key events in the evolutionary history of cancer-associated cancer. For multicellularity to persist, cell proliferation and death must pathways: (1) the genesis of the first cell and (2) the transition to be controlled, metabolic resources and essential cell functions must multicellular life.[8] These two events represent major innovations be allocated, and the extracellular environment is maintained in such a in the cellular strategies of life, with the latter requiring cells to way as to promote tissue homeostasis. In this context, cells that detach develop gene networks for complex cell communication, differen- from the tissue no longer receive the cellular input required to survive, tiation, cell-to-cell adhesion, senescence, and anoikis; and thus unicellularity is inhibited by a form of pathways that together promote multicellularity and act as barriers to known as anoikis.[12] In cancer, these homeostatic processes that pro- oncogenesis.[19] mote multicellularity, such as anoikis, are dysregulated, leading to the Based on evidence linking common somatic mutations in cancer and ability of cancer cells to survive in a unicellular state often through “de- genes involved in the evolution of multicellularity, gene regulatory net- differentiation” or acquisition of stem cell-like properties that promote works that evolved from ancestral species have likely shaped the dif- unicellular growth and survival.[13–15] This de-differentiation occurs ferent roles that unicellular and multicellular genes play in cancer.[10] in a variety of solid tumors (those that do not contain cyst or liquid), From a therapeutic standpoint, it is possible that these gene networks where transcriptional profiles notably shift from multicellular gene sig- could be targeted to suppress or prevent cancer, as pathways such natures of differentiated tissue to that of embryonic stem cells.[9,16] as differentiation and apoptosis are already targeted by cancer ther- This somatic process appears to be under evolutionary pressure within apy. However, much work remains to be done, as these gene networks tumors, as it has been shown in a mouse xenograft model of human also contain genes of unknown function or uncertain significance for breast cancer, that development of metastatic subclones within the carcinogenesis. Trigos et al., 2018 suggested that the incorporation of tumor coincided with the positive selection for loss-of-function muta- gene evolutionary histories can help identify which genes are critical tions in genes associated with multicellularity.[6] for coordinating both unicellular and multicellular processes within the context of cancer.

De-differentiation can occur in normal tissue Cancer and cancer-like behavior is observed in Although de-differentiation and expansion of cells harboring muta- multiple unikont clades tions within a tissue can lead to cancer, such somatic clonal expansion of cells can also occur in normal tissue. For example, during normal Cancer can be viewed as cheating in a cooperative multicellular aging, or following exposure to mutagens, cellular clones within the system, with a population of abnormal cells having malignant cell esophageal epithelium that carry somatic mutations in genes such attributes (e.g., uncontrolled cell proliferation).[13] As a result, can- as NOTCH1 and TP53 that promote clonal expansion.[17,18] With cer and cancer-like behavior is a major destabilizing scenario when middle age humans, NOTCH1 mutations are in fact more common it comes to maintaining multicellularity. Such cancer-like behav- in normal esophageal epithelium than esophageal cancer tissue.[18] ior in unikonts (eukaryotic supergroup consisting of , However, rather than continued growth and metastasis, expansion of Choanozoa, fungi and animals) has been reported in pre-metazoans these mutant cell populations is halted when two well-adapted clones such as Amoebozoa, Basidiomycota,andAscomycota (Figure 1). How- (similar in “fitness”) meet and are forced to share resources. After ever, cancer or cancer-like behavior has not been observed in such an encounter, the proliferative advantage is reduced, and clones early metazoans belonging to Ctenophora, Porifera,andPlacozoa revert back towards balancing proliferation and differentiation. These (Figure 1). It is possible that cancer-like phenomena are controlled findings collectively indicate that tight control of multicellularity, in Amoebozoa, Basidiomycota and Ascomycota by similar genes that which underlies tissue homeostasis in metazoans, forms a substantial prevent cancer in animals. Yet, this has not been examined despite the barrier to cancer development by preventing the clonal expansion and many answers it could provide as to how unicellularity and multicellu- unicellular survival of genetically altered cells. larity associated genes facilitate cancer development and progression. MATHAVARAJAH ET AL. 3of13

FIGURE 2 The asexual life cycle of D. discoideum has various FIGURE 1 Evolution of multicellularity and the occurrence of mechanisms for maintaining integrity. D. discoideum amoebae cancer in unikonts. Phylogenetic tree adapted from Aktipis et al., 2015 are unicellular when in favorable environments with nutrients and/or describing cancer across the tree of life. Phylogenetic relationships (growth phase). When starved, amoebae secrete cAMP to among the described organisms and the extent of multicellularity are induce the aggregation of cells, which leads to the formation of a derived from previously published trees.[20–24] This tree is not mound. During this phase, cells differentiate into pre-stalk or representative of all ancestral states and possible independent origins pre- cells. The multicellular aggregate then undergoes a series of and is more so a general representative of major clades in unikonts. morphological changes to form a finger, slug, and culminant. In the Reported with each clade is the extent of multicellular life and final step of development, a fruiting body forms with a stalk and a mass observations of cancer (which includes uncontrolled proliferation, of viable spores. These spores will be dispersed, and when in favorable invasion and metastasis) or cancer-like phenomena (abnormal conditions, amoebae re-emerge to restart the life cycle. At different differentiation – cheating).[13] In addition, we report the available points of the life cycle (indicated by *), D. discoideum has evolved ways genome resources for studying the species associated with cancer or to maintain genome integrity within the population (described at each cancer-like phenomena across the tree stage). There are unique mechanisms for dealing with DNA damage incurred during growth and spontaneous DNA damage during multicellular development. Abbreviations: HR, homologous recombination; NHEJ, non-homologous end-joining There are two distinct forms of multicellularity in . In the case of social amoebae, their multicellular life cycle is facultative, meaning that a switch between unicellularity and multicellularity nor- USING D. discoideum TO GATHER INSIGHT INTO THE mally occurs. Here, the cancer-like behavior is associated with amoe- MULTICELLULAR FUNCTIONS OF GENES LINKED bae that do not cooperate with the rest of the population during mul- TO CANCER ticellular development (as we will discuss below). In contrast, meta- zoan multicellularity is clonal and obligate where a transition between Social amoebae like D. discoideum emerged prior to the fungi-animal unicellularity and multicellularity does not normally occur. In cancer, split (Figure 1).[25] While most amoebozoan clades are unicellular, pop- related cells (clonal) undergo an abnormal process of de-differentiation ulations of social amoebae species are an exception and are capable to become malignant cells within the tissue or organ. Despite these dif- of aggregating to form complex multicellular structures. Since D. dis- ferences, there are still several similarities between the two forms of coideum belongs to the phylum of amoebozoa, it along with other social multicellularity that we can draw upon to gather insight into the mech- amoebae, represent a separate event of evolution towards multicellu- anisms that cause the breakdown of multicellularity in cancer. lar life shortly after its divergence from unikonts (Figure 1). The emer- In practice, experimentally addressing the connected nature of gence of multicellularity in amoebozoans occurred prior to the expan- these important gene networks poses a significant problem, since most sion of metazoan machinery identified in choanoflagellates (closest well-studied model organisms have conformed to either a single-cell animal relatives) (Figure 1). Over 80% of the genes involved unicellular or multicellular way of life. In addition, many of these pre- in social amoebozoan multicellularity were derived from unicellular metazoan clades with species that must deal with cancer-like behav- ancestors.[26] The initial appearances of multicellularity across unikont ior do not have a well-annotated genome to facilitate gene network branches are thought to converge towards switching between unicel- analysis (Figure 1). This is not the case for Dictyostelium discoideum,a lular and multicellular states through strict control of the cell cycle and with uniquely intersecting unicellular and multicellu- maintenance of the extracellular matrix (ECM).[27] lar stages of life, that has been sequenced (chromosome-level assem- The life cycle of D. discoideum comprises both unicellular and multi- bly). As such, we propose that D. discoideum is an ideal model organism cellular stages, hence the term “social” amoebozoan[28] (Figure 2). Dur- to gather insight into the function of genes associated with cancer. ing the unicellular growth stage, cells divide mitotically as they feed 4of13 MATHAVARAJAH ET AL. on nutrients (e.g., bacteria) in their environment (Figure 2). Although function in animals is difficult because it is essential for embryonic the D. discoideum life cycle encompasses both sexual and asexual mul- development.[45] In D. discoideum, however, it is possible to knockout ticellular phases, here we will focus on the asexual life cycle ini- the PTEN ortholog (pten) and then study its functions during multicellu- tiated by nutrient deprivation. Upon starvation, D. discoideum cells lar development. Such work revealed that the loss of pten completely aggregate to form a multicellular mound that eventually develops abolishes multicellular development.[46] Importantly, multicellularity into a fruiting body (Figure 2). Amoebae aggregate through chemo- was restored by expressing human PTEN in pten– amoebae. This exam- taxis towards a pulsatile source of extracellular cyclic AMP, and after ple demonstrates how D. discoideum can be used to study the impact of reaching a certain cell density, they undergo “streaming” to form a gene mutations on multicellular processes. It also shows that despite mound.[29] The streaming aggregation is mediated by cell adhesion many years of divergence, the fundamental function of PTEN in regu- proteins such as CsaA and CadA that allow amoebae to adhere to lating multicellular development has been conserved between D. dis- one another during aggregation, which marks the beginning of D. coideum and humans. discoideum multicellularity.[29] During the transition to multicellular- ity, cellular processes function to maintain cooperation between cells and promote multicellular pathways (e.g., cell-cell adhesion). Following ASPECTS OF CHEATER CELL BEHAVIOR IN D. aggregation, the multicellular mound develops into a motile pseudo- discoideum MIRROR MALIGNANT CELLS IN CANCER plasmodium (referred to as a slug) (Figure 2). Like human tissue, the cells of the pseudoplasmodium are surrounded by an ECM that reg- Cancer cells promote their own survival and growth at the expense ulates collective and differentiation.[30–32] Within the of other cells in the multicellular tissue; in essence behaving as uni- pseudoplasmodium, roughly 80% of the cells will terminally differenti- cellular organisms to subvert normal tissue homeostasis. In a D. dis- ate into spores (pre-spore cell fate), while the remaining 20% will differ- coideum fruiting body, only the spores pass on their genetic mate- entiate into non-reproductive, vacuolated, dead stalk cells (pre-stalk rial to subsequent generations. As a result, its formation relies on a cell fate)[33,34] (Figure 2). constant pool of altruistic cells to form the stalk. However, like can- Proportioning of cells in the slug determines cell fate, where cells at cer cells, any given population of D. discoideum amoebae will contain the front of the slug become the stalk and cells in the mid and posterior “cheater cells” that do not follow the cooperative nature of the popula- regions become spores.[35] During the slug stage, pre-spore cells pro- tion. Cheaters opt to escape death and become pre-spore cells, which duce and secrete differentiation inducing factor 1 (DIF-1) that induces skews the ratio of pre-stalk and pre-spore cells in the slug[47–49] (Fig- pre-stalk differentiation of other cells.[36] From the slug, an intermedi- ure 3). Thus, they promote unicellularity by dysregulating mechanisms ate structure known as the culminant forms, which contains a polarized involved in maintaining multicellularity (Figure 3).[13] Multicellularity cell layer at the tip that resembles the epithelial organization observed relies on the following criteria: (1) strict control of cell proliferation in metazoans[37] (Figure 2). Finally, terminal differentiation of pre-stalk and death, (2) allocation of resources and labor, and (3) maintenance of and pre-spore cells results in the formation of a fruiting body com- the extracellular environment (e.g., ECM). Pre-stalk cells undergo cell prised of a mass of spores that are supported atop a slender stalk (Fig- death and contribute to the labor of fruiting body formation by form- ure 2); a process mediated by regulatory gene networks that facilitate ing a stalk to physically support the spore mass. Pre-spore cells synthe- the transition from unicellular to multicellular life. size spore coat materials de novo to maintain dormancy in the spore mass. Since cheaters favor pre-spore cell differentiation, they dysreg- ulate the mechanisms that maintain multicellularity in D. discoideum HOW CAN D. discoideum BE USED TO STUDY GENES (Figure 3).[50–52] Therefore, regulatory gene networks and biological ASSOCIATED WITH CANCER? mechanisms must exist to prevent cheaters from reducing cooperation among cells. We propose that there are three unique ways that D. discoideum can Although we describe them as similar, the biological contexts be used to study cancer gene networks: (1) to identify potential func- revolving around cheating and cancer are different. Cancer occurs tions for multicellularity genes dysregulated in cancer, (2) to identify between somatic cell types, while cheating behavior occurs between regulatory genes involved in coordinating unicellularity and multicellu- the somatic-like (stalk cells) and germ-like (spores) cells. Furthermore, larity and (3) by providing novel insights into the molecular evolution cheating does not involve uncontrolled proliferation (a hallmark of can- of characterized cancer gene networks. Despite D. discoideum being an cer) and more so stems from altered cell fate decisions. In addition, evolutionarily distant organism from humans, its genome encodes con- D. discoideum cheaters arise from “germline” mutations, allowing their served orthologs of human proteins associated with cancer.[38–42] Fur- destabilizing effects on multicellularity to be visible in succeeding gen- thermore, D. discoideum presents the opportunity to study the func- erations. This distinction between cheating and cancer is important to tion of cancer genes in the absence of the tumor suppressor p53, as consider when studying D. discoideum. However, owing to their simi- the organism diverged from unikonts prior to the appearance of p53 larities during the breakdown of multicellularity, we and others pro- in choanoflagellates.[43] Perhaps the most well studied tumor suppres- pose that we can gather novel insight on the genes and pathways sor after p53 is phosphatase and tensin homolog (PTEN), which is involved in social amoebae cheating behavior to better understand commonly mutated or deleted in cancer.[44] However, studying PTEN cancer in metazoans. Under the guise of them both acting to destabilize MATHAVARAJAH ET AL. 5of13

FIGURE 3 Cheating behavior is an impediment to the evolution of multicellularity. Cell cycle states determine the fate of amoebae within a population of pre-stalk and pre-spore cells. This is a highly regulated process and follows many of the same principles described by Aktipis et al., 2015. However, cheater cells, which parallel cancer cells, skew this ratio towards pre-spore differentiation, ultimately acting against multicellularity and disrupting the principles. Thus, cheater cells, like cancer cells, do not follow many of the principles required for maintaining multicellularity in D. discoideum multicellularity, we can begin to study the similar approaches taken more closely related individuals within a population to form fruiting by tissues and individual cells to maintain multicellularity. Here, we bodies, while excluding cheaters (Figure 4).[54,59,60] TgrB1 (acts as a focus on the mechanisms that control cheating behavior through receptor) and TgrC1 (acts as a ligand) do not appear to be conserved various biological processes and amoeba regulating the cell cycle in humans but they share many properties with the Major Histocom- and DNA repair, pathways that are also critical for preventing patibility Complex, as both are used for allorecognition. Cheaters can cancer. present a similar TgrB1 and TgrC1 protein (i.e., bound by cooperator cells with no issue) to cooperative cells to reap the benefits of the pop- ulation without incurring the costs associated with stalk differentia- PROCESSES THAT SUPPRESS CHEATING BEHAVIOR tion (e.g., cell death).[61] However, this is thought to lead to an arms IN D. discoideum race between cooperators (population of cells that do not skew dif- ferentiation) and cheaters within a population.[61] As cheater numbers Kin recognition and discrimination promote grow within a population, the cooperators experience selection in the cooperation during D. discoideum development tgrB1 and tgrC1 gene loci to acquire sufficient sequence variation that prevent cheaters in the population from persisting (Figure 4).[61] Thus, Cheating is a complex social interaction that is suppressed by many cooperators select for new tgrB1/tgrC1 allotypes and this limits cheater mechanisms, one of which is kin recognition and discrimination.[53–58] participation during development in the population.[61] Thus, kin dis- In nature, D. discoideum kin recognition is regulated by the polymorphic crimination is one well-studied pathway for excluding cheaters through single-pass transmembrane proteins TgrB1 and TgrC1, which allow for direct cell-cell interactions via TgrB1 and TgrC1. 6of13 MATHAVARAJAH ET AL.

Pleiotropy helps explain the benign effects of non-metastatic tumors

Pleiotropy is not often discussed in the context of cancer but likely con- tributes to differences between benign and malignant tumors. Genes that contribute to motility (key aspect of tumor invasion) can also inhibit proliferation. Thus, inactivating mutations in these genes could drive the tumor growth in benign malignancies. One such example is Tuberous sclerosis, an autosomal dominant disorder caused by muta- tions in TSC1 and TSC2, that is characterized by benign tumors in mul- tiple organs.[65] What is unusual about Tuberous scelerosis is the low frequency of metastases despite the abundance of tumors in affected individuals. While TSC1 inhibits proliferation, it is also required for normal cell migration in part due to its regulation of Rho-GTPases and Focal Adhesion Kinase (FAK).[66,67] Thus, there appears to be a role for functional pleiotropy in explaining the benign outcome of gene mutations in some tumor suppressor genes, a characteristic that FIGURE 4 Kin discrimination helps suppress cheating behavior in could potentially explain the role of putative driver mutations in other D. discoideum. Cheaters (colored green) skew the ratio of pre-spore to benign malignancies. The suppression of cheating to maintain multi- pre-stalk differentiation during development to preferentially become cellularity is multi-faceted in D. discoideum and the continued work a spore. Cooperative cells in a population utilize the TgrB1 and TgrC1 (in purple) proteins to recognize kin. After kin recognition occurs, the in this area may help us better understand the different mechanisms cheaters are not able to develop with the cooperative amoeba. As a behind how human cells also maintain multicellularity and suppress result, kin discrimination limits cheaters from exhibits cheating oncogenesis. behavior and helps maintain multicellularity in amoeba populations

CELL CYCLE REGULATION LIMITS CHEATER MANIPULATION OF CELL FATE Pleiotropy helps prevent cheating behavior in D. discoideum In addition to kin recognition and the other biological processes, other ways to control cheating is through the prevention of cheater cell gene- Kin recognition and discrimination is one mechanism for controlling sis. The appearance of cheaters is limited by (1) DNA repair preventing cheating behavior but there is also noble resistance, high relatedness, mutations that promote cheating behavior and (2) the tight regulation pleiotropy, and the lottery system that all act to control cheating and of cell fate through the cell cycle. Cancer cells often harbor mutations maintain cooperation within the population.[62] These mechanisms in genes that regulate the cell cycle. Recent work in D. discoideum sug- for controlling cheating behavior in a population have been exten- gests there are gene networks that regulate the cell cycle and cell fate sively discussed and reviewed by Strassmann and Queller, 2011.[62] decisions to limit cheating behavior within populations of amoebae. A Pleiotropy is intriguing as a suppressive mechanism from both a cheat- wealth of research has determined that the cell cycle directly influ- ing and cancer standpoint. In D. discoideum, pleiotropy of a gene lim- ences whether a cell will become a spore or a stalk cell.[68–72] Unless its the impact of its mutation on cheating behavior by mutating genes experimentally modified, any given population of D. discoideum amoe- required for cheater genesis by both promoting preferential differenti- bae will be a heterogeneous mixture of cells in different stages of the ation into spores, but also impairing processes in development. Muta- cell cycle.[71,73] Upon starvation, the cell cycle stage will determine tions or loss of a gene with multiple functions (pleiotropy) leads to how amoebae respond to differentiation cues that ultimately dictate a simultaneous gas (pro-cheating) and brake scenario (anti-cheating), their cell fate[34] (Figure 3). Cells in M, S, or early G2 tend to form pre- where cheating is promoted but progression through development is stalk cells, while cells in mid-to-late G2 tend to form pre-spore cells. impeded. Thus, nullifying the ability for a mutant to cheat. The loss of Consequently, the cell cycle facilitates the segregation of cells into sub- cell adhesion protein CsaA illustrates the effect of pleiotropy on cheat- populations of pre-stalk and pre-spore cells. ing behavior.[63,64] Amoeba lacking csaA form more spores when grown Wild isolates of D. discoideum have been identified as cheaters and on agar in chimeras. This is thought to occur by cells with reduced adhe- carry mutations in specific genes.[74,75] Some cheaters harbor muta- sion sloughing off the stalk-forming tip of the mound. However, when tions in orthologs of genes linked to cancer in humans such as the grown on a natural substrate such as soil, the mutant lacking csaA is well-studied fbxA, which encodes an F-box domain-containing protein unable to adhere enough to reach the stage of aggregation. Thus, the A. In humans, the Skp1-cullin-F-box complex facilitates the ubiquiti- pleiotropic nature of csaA and similar pleiotropic genes prevent cheat- nation of proteins involved in cell cycle progression.[76,77] In addition, ing behavior. dysregulation of F-box domain-containing proteins has been linked MATHAVARAJAH ET AL. 7of13 to malignancy by promoting cell proliferation.[78] fbxA is expressed venting damaged from continuing into future populations of only after amoebae begin development and its absence leads to a pre- amoebae protect the integrity of development and reduce the likeli- spore bias in chimeric populations. Both in D. discoideum and humans, hood of cheater mutants in the population. the F-box domain-containing proteins play an integral role in regulat- ing the cell cycle, and when absent or dysregulated, they induce cell cycle changes to promote cheating or cancer, respectively. Therefore, D. discoideum canbeusedtogatherinsightinto FbxA-mediated degradation may bias cell fate through its influence non-homologous end joining repair on developmental progression, akin to cell cycle progression in other eukaryotes.[74,79] FbxA regulates the degradation of RegA in D. dis- Newly germinated D. discoideum amoebae are also vulnerable to coideum, which is a cAMP phosphodiesterase that regulates cell dif- many different environments due to expansive spore dispersal. ferentiation via its hydrolysis of cAMP (a key signaling molecule for Non-homologous end joining (NHEJ), a principle form of DNA double- differentiation).[80] cAMP activates many downstream effectors that stranded break repair, was shown to be essential for viability during regulate development.[81] Furthermore, the wild isolate fbxA mutant spore hatching after cells were exposed to bleomycin (a DNA damaging is an obligatory cheater that relies completely on cheating to persist agent).[86] The impairment of NHEJ was a consequence of ku80 and and cannot form normal fruiting bodies in a clonal population.[74] Thus, dnapkcs loss in freshly germinated amoeba, which are in G1 of the cell much like mutations in human cancer that promote selfish, unicellu- cycle. Interestingly, vegetative cells are not sensitive to bleomycin lar growth of malignant cells, mutations in cell cycle regulatory genes treatment when NHEJ repair is impaired.[86] The reason for this such as fbxA also promote the fitness of multicellularity-destabilizing is that vegetative cells have no discernible G1 phase.[34,71] Rather, cheaters. they become trapped in the G2 phase by a G2/M-like checkpoint and utilize HR-directed repair.[71,84,86] Since freshly germinated amoe- bae are in G1 and sister chromatids are not available as templates, DNA REPAIR MECHANISMS MAINTAIN GENOME they cannot utilize this DNA repair mechanism.[71,86] NHEJ repair STABILITY AND PREVENT CHEATER CELL GENESIS is widely conserved in eukaryotes but there are subtle inter-species differences in NHEJ machinery.[87] In yeast, there appears to be no D. discoideum cells with DNA damage express DNA identifiable ortholog of mammalian DNA-PKc and Artemis. In contrast, repair factors conserved in humans there are orthologs for both DNA-PKc (dnapkcs; DDB_G0281167 – discussed above) and Artemis (dclre1, DDB_G0277755) encoded in the In human cells, DNA repair mechanisms cooperate with the cell cycle to D. discoideum genome.[87] The conservation of these key components prevent genome instability and the propagation of genetic errors that of NHEJ in D. discoideum make it an ideal unicellular model for studying drive oncogenesis. Such DNA damage can arise during normal cell divi- NHEJ repair. Future work describing NHEJ repair in D. discoideum sion and metabolism.[82] Similarly, in D. discoideum, DNA repair path- may provide novel insights into therapy development since DNA ways also prevent the propagation of DNA mutations and thus, these double-stranded break repair is a target for anti-cancer therapy.[88] pathways prevent cheaters from appearing in a population (Figure 2). The full extent to which both HR and NHEJ play in suppressing In both D. discoideum and humans, the RAD51 protein plays a key cheater generation through maintaining genome integrity has not been role in DNA repair by homologous recombination (HR).[83] Geno- examined. However, it is likely that DNA repair directly correlates to toxic stress in D. discoideum is associated with elevated expression of the frequency of cheater mutants that appear within a population of rad51.[84] Cells with high rad51 expression (i.e., experiencing genotoxic amoebae. In cancer, it is well-established that DNA repair suppresses stress) are shed from the multicellular slug and this is used as a strategy the generation of malignant cells.[89] Trigos et al., 2017 found that the by D. discoideum to prevent damaged cells from progressing through dedifferentiation of tumors was facilitated by the downregulation of development. Cell shedding could also represent a terminal strategy double-stranded repair pathway components utilized by multicellular for maintaining genome stability. For example, cells with DNA damage organisms.[9] DNA damage response and repair pathways in D. dis- do not express genes associated with spore differentiation, suggesting coideum share features with the corresponding mammalian pathways, that genes exist to survey genome stability and respond prior to differ- making the organism a promising model for better understanding how entiation. In addition, with their single cell RNA sequencing approach, genome stability helps maintain multicellularity. Miermont et al. (2019) identified genes correlated with rad51 expres- sion (i.e., showing a similar transcriptomic profile). Of the 30 genes identified, 26 are involved in DNA repair and include orthologs of D. discoideum cell shedding shows similarities to human PARP1 (adprt1A and adprt1B), PARP2 (adprt2), Fanconi anemia intestinal epithelium tissue renewal in vertebrates family proteins (fncD2 and fncI), APE1 (apeA) and cell cycle checkpoint proteins (hus1, rad1, rad9,andtopbp2). APE1 is involved in repairing Cell shedding also occurs in certain human tissues like the colon but is DNA base damage that would otherwise lead to point mutations, thus accompanied by a process known as anoikis; a form of programmed cell these results reveal more than one DNA repair pathway is induced dur- death that occurs when epithelial cells detach from the ECM to prevent ing multicellular development.[85] Ultimately, these measures of pre- the growth and spread of detached cells to distant sites in the body.[90] 8of13 MATHAVARAJAH ET AL.

Anoikis occurs in the vertebrate intestinal epithelium, a highly prolif- erative tissue that continuously renews and is composed of a mono- layer of polarized epithelial cells.[91,92] Due to their rapid proliferation, colon cells are highly sensitive to DNA damage. In this context, non- pathogenic cell shedding and anoikis of colorectal epithelium occurs to remove senescent and/or older cells that have accumulated DNA dam- age but not entered apoptosis, thus preventing tumor development and allowing the barrier function of the colon to be maintained.[93] As a result, altered anoikis after cell shedding has been linked to not only colorectal cancer but inflammatory diseases and endotoxemia.[94–97] As such, cell shedding in D. discoideum could provide a model system for understanding evolutionary conserved mechanisms of tissue home- ostasis and tumor suppression in mammals in self-renewing or highly FIGURE 5 proliferative tissues, such as the colonic epithelium, in response to DNA The convergent evolution of cancer-associated gene networks in suppressing cheating. D. discoideum possesses a gene damage. network to prevent cheating behavior during development, comprised of “cooperation” genes. A portion of these genes are orthologous to tumor suppressor proteins in humans (human gene ID (Dictybase ID of GENES INVOLVED IN CHEATING BEHAVIOR ortholog). This indicates that there are shared genes in the gene INCLUDE ORTHOLOGS OF HUMAN TUMOR network that prevent cheating in D. discoideum and suppress cancer in SUPPRESSORS animals, suggesting that there has been convergent evolution in D. discoideum towards maintaining multicellularity via genes involved in tumor suppression in humans The well-established gene networks associated with DNA repair and cell cycle control in the prevention of cancer, likely contribute to the prevention of cheating in D. discoideum. Although fbxA is one clear example of an overlap between the genes involved in cheating and regulation of chromosome stability (BLM and CHFR), the cell cycle their conserved human orthologs in cancer (discussed above), there (CHFR), endoplasmic reticulum stress (SEL1L), and metabolic processes are other similarities. In a genome-wide mutation screen designed (PIK3C2G).[99–103] The PIK3C2G ortholog of D. discoideum is associated to reveal genes that prevent cheating, over 100 mutated genes of with , macropinocytosis, and ; while, orthologs diverse functions were identified (i.e., loss of the gene promotes of BLM, CHFR and SEL1L haveyettobecharacterizedintermsoftheir cheating behavior).[98] The identified genes, termed “cooperation” functions in D. discoideum, including their possible roles in cheating genes, prevent bias and cheating behavior within populations of behavior.[104–106] However, supporting their roles in unicellular behav- cells. Many of the genes encode proteins proposed to function in cell iors that promote malignant transformation in cancer, altered expres- communication, ubiquitin-like protein transferase activity, and cell sion or mutation of BLM, CHFR, and SEL1L is associated with metas- cycle regulation. Importantly, these pathways are associated with the tasis in various human cancers.[107–111] Future work characterizing unicellular and multicellular-promoting pathways that are upregulated these genes will help us better understand the pathways that influence and downregulated, respectively, in human cancers.[9] In addition, genetic conflicts such as cheating. it was noted that obligatory cheaters such as fbxA were rare in the Of the uncharacterized gene orthologs in D. discoideum listed above, wild (only prevalent in ∼1% of screened clones). It is likely that similar CHFR and BLM are associated with promyelocytic leukaemia (PML) gene networks are dysregulated in obligatory cheaters and facultative nuclear bodies (NBs) in mammals.[112–115] PML NBs are key for reg- cheaters, those capable of forming fruiting bodies even in clonal pop- ulating the functions of CHFR and BLM and their evolution likely ulations. However, the extent of the dysregulation may be different, contributed to an expansion of similar regulatory gene networks for where obligatory cheaters display more extreme changes to the gene maintaining chromosome stability in vertebrates (when PML NBs first network. emerged).[116,117] However, to our knowledge D. discoideum does not Among the identified cheater mutants, many of the genes were have a PML gene ortholog. Since the PML protein is the main struc- uncharacterized and not annotated. We examined the uncharacter- tural component of PML NBs, D. discoideum are not expected to form ized genes to determine whether they were orthologous to known these subnuclear domains.[116] Thus, the ancestral functions of CHFR cancer-causing genes. A comprehensive BLAST search of the unchar- and BLM are likely independent of PML and/or do not require PML NBs acterized genes against the human genome identified four orthologs of for their regulatory post-translational modifications or sequestration proteins linked to tumor suppression (DDB_G0272384, BLM ortholog; (functions attributed to these bodies to modulate cheating behaviors in DDB_G0279263, CHFR ortholog; DDB_G0269430, SEL1L ortholog; this organism.[116,118] In total, accumulated findings indicate that there DDB_G0282625; PIK3C2G ortholog) (Nakayama 2002; Sanbhnani and is a degree of convergent evolution of gene networks that regulate Yeong 2012; Semba, et al. 2002) (Figure 5). Each of these genes cheating behavior in D. discoideum and tumor suppression in humans contribute to tumor suppression in different ways, including the (Figure 5). MATHAVARAJAH ET AL. 9of13

ACKNOWLEDGMENTS This review was supported by Discovery Grants from the Natural Science and Engineering Research Council of Canada (RGPIN-2020- 04034 to G.D. and RGPIN-2018-04855 to R.J.H.). S.M. was supported by a Killam Pre-Doctoral Award, a Nova Scotia Graduate Scholarship, and a President’s Award from Dalhousie University. We would like to thank Megan Aoki (Trent University) for allowing us to use their photos of D. discoideum development. We would also like to thank Celine Chedrawe for their critical reading of the manuscript prior to submission

CONFLICT OF INTEREST The authors declare no conflicts of interest. FIGURE 6 D. discoideum development can be utilized as a model to understand multicellularity. D. discoideum developed on agar form fruiting body colonies, marking the last stage of development. (A) AUTHOR CONTRIBUTIONS Development begins with the transition from unicellularity to S.M. conceived of the study, conducted the bioinformatic analyses, and multicellularity as amoeba stream and aggregate to form mounds. (B) prepared the first draft of the manuscript. S.M., C.V., G.D. and R.J.H. Soon after, the aggregated amoebae form a migrating revised and edited the manuscript, and all authors approved the final pseudoplasmodium and (C) eventually form fruiting bodies composed version. of a sorus and a stalk. The entire developmental cycle was visualized within 24 h of plating the amoeba, indicating the ease at which we can study the pathways that maintain multicellularity in the model. Images ORCID show D. discoideum development on agar at different stages. Photos Sabateeshan Mathavarajah https://orcid.org/0000-0003-4871- were taken by Megan Aoki (Trent University) 1373

REFERENCES 1. Casas-Selves, M., & Degregori, J. (2011). How cancer shapes evo- CONCLUSIONS lution, and how evolution shapes cancer. Evolution, 4(4), 624–634. https://doi.org/10.1007/s12052-011-0373-y Understanding the dysregulated gene networks that promote and 2. Little, M. P. (2010). Cancer models, genomic instability and somatic cellular Darwinian evolution. Biol. Direct, 5, 19; discussion 19. https: destabilize multicellularity is a powerful approach for understanding //doi.org/10.1186/1745-6150-5-19 [9,10,119] the cellular and molecular mechanisms underlying cancer. We 3. Hanahan, D., & Weinberg, R. A. (2000). The hallmarks of cancer. Cell, reviewed how D. discoideum has been utilized to study pathways and 100(1), 57–70. orthologous genes associated with cancer to identify novel insights 4. Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: The next generation. Cell, 144(5), 646–674. https://doi.org/10.1016/j.cell. into their functions in human cells. There are also similarities between 2011.02.013 cheating behavior in D. discoideum and cancer, where cheaters disrupt 5. Weiss, R. A. (2017). Exchange of Genetic Sequences Between Viruses and many of the principles that maintain multicellularity like malignant Hosts Viruses, Genes, and Cancer (pp. 1–29). Springer. cells. Furthermore, genes that maintain multicellularity in social 6. Chen, H., Lin, F., Xing, K., & He, X. (2015). The reverse evolution from multicellularity to unicellularity during carcinogenesis. Nat. Commun., amoebae, include cooperation genes that are orthologous to tumor 6, 6367. https://doi.org/10.1038/ncomms7367 suppressors in human cells (BLM, SEL1L, CHFR,andPIK3C2G). There- 7. Chen, W., Li, Y., & Wang, Z. (2018). Evolution of oncogenic signa- fore, D. discoideum presents unique opportunities to study some of tures of mutation hotspots in tyrosine kinases supports the atavis- the mechanisms that regulate cancer cell progression and behavior tic hypothesis of cancer. Sci. Rep., 8(1), 8256. https://doi.org/10.1038/ s41598-018-26653-5 (Figure 6). New insights through an inter-species approach will have 8. Domazet-Loso, T., & Tautz, D. (2010). Phylostratigraphic tracking of implications improve our understanding of cancer and cancer-like cancer genes suggests a link to the emergence of multicellularity in phenomena experienced by eukaryotes. Future work delving into the metazoa. BMC Biol., 8, 66. https://doi.org/10.1186/1741-7007-8-66 similarities in strategies and the evolution of preventative genes will 9. Trigos, A. S., Pearson, R. B., Papenfuss, A. T., & Goode, D. L. (2017). help us better understand how different organisms deal with such Altered interactions between unicellular and multicellular genes drive hallmarks of transformation in a diverse range of solid tumors. issues, ultimately guiding the targeting of therapeutics to novel genes Proc Natl Acad Sci U S A, 114(24), 6406–6411. https://doi.org/10. or unravelling novel mechanisms for preventing cancer in humans. 1073/pnas.1617743114 In conclusion, there are many things we can continue to learn from 10. Trigos, A. S., Pearson, R. B., Papenfuss, A. T., & Goode, D. L. (2018). studying D. discoideum to better understand the similar metazoan How the evolution of multicellularity set the stage for cancer. Br. J. Cancer, 118(2), 145–152. https://doi.org/10.1038/bjc.2017.398 manifestation of shifts from multicellularity to unicellularity seen 11. Trigos, A. S., Pearson, R. B., Papenfuss, A. T., & Goode, D. L. (2019). during cancer development. Somatic mutations in early metazoan genes disrupt regulatory 10 of 13 MATHAVARAJAH ET AL.

links between unicellular and multicellular genes in cancer. Elife, 8, 29. Coates, J. C., & Harwood, A. J. (2001). Cell-cell adhesion and signal e40947. https://doi.org/10.7554/eLife.40947 transduction during Dictyostelium development. J. Cell Sci., 114(24), 12. Taddei, M. L., Giannoni, E., Fiaschi, T., & Chiarugi, P.(2012). Anoikis: An 4349–4358. emerging hallmark in health and diseases. J Pathol, 226(2), 380–393. 30. Huber, R. J., & O’Day, D. H. (2012). A matricellular protein and EGF- https://doi.org/10.1002/path.3000 like repeat signalling in the social amoebozoan Dictyostelium dis- 13. Aktipis, C. A., Boddy, A. M., Jansen, G., Hibner, U., Hochberg, M. E., coideum. Cell. Mol. Life Sci., 69(23), 3989–3997. https://doi.org/10. Maley, C. C., & Wilkinson, G. S. (2015). Cancer across the tree of life: 1007/s00018-012-1068-4 Cooperation and cheating in multicellularity. Philos Trans R Soc Lond 31. Huber, R. J., & O’Day, D. H. (2015). Proteomic profiling of the extra- B Biol Sci, 370(1673), 20140219. https://doi.org/10.1098/rstb.2014. cellular matrix (slime sheath) of Dictyostelium discoideum. Proteomics, 0219 15(19), 3315–3319. https://doi.org/10.1002/pmic.201500143 14. Aktipis, A. (2016). Principles of cooperation across systems: From 32. Huber, R. J. (2017). Loss of Cln3 impacts protein secretion in the human sharing to multicellularity and cancer. Evol Appl, 9(1), 17–36. social amoeba Dictyostelium. Cell Signal, 35, 61–72. https://doi.org/ https://doi.org/10.1111/eva.12303 10.1016/j.cellsig.2017.03.022 15. Soteriou, D., & Fuchs, Y. (2018). A matter of life and death: Stem cell 33. Forman, D., & Garrod, D. (1977). Pattern formation in Dictyostelium survival in tissue regeneration and tumour formation. Nat. Rev. Can- discoideum: I. Development of prespore cells and its relationship cer, 18(3), 187–201. https://doi.org/10.1038/nrc.2017.122 to the pattern of the fruiting body. Development, 40(1), 215– 16. Chen, H., & He, X. (2016). The convergent cancer evolution toward a 228. single cellular destination. Mol. Biol. Evol., 33(1), 4–12. https://doi.org/ 34. Gruenheit, N., Parkinson, K., Brimson, C. A., Kuwana, S., Johnson, E. J., 10.1093/molbev/msv212 Nagayama, K., ... Thompson, C. R. L. (2018). Cell cycle heterogeneity 17. Colom, B., Alcolea, M. P., Piedrafita, G., Hall, M. W. J., Wabik, A., can generate robust cell type proportioning. Dev. Cell, 47(4), 494–508 Dentro, S. C., ... Jones, P. H. (2020). Spatial competition shapes e494. https://doi.org/10.1016/j.devcel.2018.09.023 the dynamic mutational landscape of normal esophageal epithelium. 35. Maruo, T., Sakamoto, H., Iranfar, N., Fuller, D., Morio, T., Urushihara, Nat. Genet. 52(6), 604-614. https://doi.org/10.1038/s41588-020- H., ... Loomis, W. F. (2004). Control of cell type proportioning in 0624-3 Dictyostelium discoideum by differentiation-inducing factor as deter- 18. Martincorena, I., Fowler, J. C., Wabik, A., Lawson, A. R. J., Abascal, F., mined by in situ hybridization. Eukaryot Cell, 3(5), 1241–1248. https: Hall, M. W. J., ... Jones, P. H. (2018). Somatic mutant clones colonize //doi.org/10.1128/EC.3.5.1241-1248.2004 the human esophagus with age. Science, 362(6417), 911–917. https: 36. Kay, R. R. (1992). Cell differentiation and patterning in Dictyostelium. //doi.org/10.1126/science.aau3879 Curr. Opin. Cell Biol., 4(6), 934–938. https://doi.org/10.1016/0955- 19. Grosberg, R. K., & Strathmann, R. R. (2007). The evolution of multicel- 0674(92)90121-r lularity: A minor major transition? Annu. Rev. Ecol. Evol. Syst., 38, 621– 37. Dickinson, D. J., Nelson, W. J., & Weis, W. I. (2012). An epithelial tissue 654. in Dictyostelium challenges the traditional origin of metazoan multi- 20. Niklas, K. J., & Newman, S. A. (2013). The origins of multicellu- cellularity. BioEssays, 34(10), 833–840. https://doi.org/10.1002/bies. lar organisms. Evol Dev, 15(1), 41–52. https://doi.org/10.1111/ede. 201100187 12013 38. Adhikari, N., Kuburich, N. A., & Hadwiger, J. A. (2020). Mitogen- 21. Moroz, L. L., Kocot, K. M., Citarella, M. R., Dosung, S., Norekian, T. P., activated protein kinase regulation of the phosphodiesterase RegA Povolotskaya, I. S., ... Kohn, A. B. (2014). The ctenophore genome and in early Dictyostelium development. Microbiology, 166(2), 129–140. the evolutionary origins of neural systems. Nature, 510(7503), 109– https://doi.org/10.1099/mic.0.000868 114. https://doi.org/10.1038/nature13400 39. Bakthavatsalam, D., White, M. J., Herlihy, S. E., Phillips, J. E., & Gomer, 22. Edgecombe, G. D., Giribet, G., Dunn, C. W., Hejnol, A., Kristensen, R. H. (2014). A retinoblastoma orthologue is required for the sens- R. M., Neves, R. C., ... Sørensen, M. V. (2011). Higher-level meta- ing of a chalone in Dictyostelium discoideum. Eukaryot Cell, 13(3), 376– zoan relationships: Recent progress and remaining questions. Organ- 382. https://doi.org/10.1128/EC.00306-13 isms Diversity & Evolution, 11(2), 151–172. https://doi.org/10.1007/ 40. Funamoto, S., Meili, R., Lee, S., Parry, L., & Firtel, R. A. (2002). Spatial s13127-011-0044-4 and temporal regulation of 3-phosphoinositides by PI 3-kinase and 23. Knoll, A. H. (2011). The multiple origins of complex multicellularity. PTEN mediates chemotaxis. Cell, 109(5), 611–623. https://doi.org/ Annu. Rev. Earth Planet. Sci., 39(1), 217–239. https://doi.org/10.1146/ 10.1016/s0092-8674(02)00755-9 annurev.earth.031208.100209 41. Reymond, C. D., Gomer, R. H., Nellen, W., Theibert, A., Devreotes, 24. Delsuc, F., Brinkmann, H., Chourrout, D., & Philippe, H. (2006). Tuni- P., & Firtel, R. A. (1986). Phenotypic changes induced by a mutated cates and not cephalochordates are the closest living relatives of ras gene during the development of Dictyostelium transformants. vertebrates. Nature, 439(7079), 965–968. https://doi.org/10.1038/ Nature, 323(6086), 340. nature04336 42. Tatischeff, I. (2019). Dictyostelium: A model for studying the extracel- 25. Heidel, A. J., Lawal, H. M., Felder, M., Schilde, C., Helps, N. R., Tunggal, lular vesicle messengers involved in human health and disease. Cells, B., ... Glockner, G. (2011). Phylogeny-wide analysis of social amoeba 8(3), 225. https://doi.org/10.3390/cells8030225 genomes highlights ancient origins for complex intercellular commu- 43. Rutkowski, R., Hofmann, K., & Gartner, A. (2010). Phylogeny and func- nication. Genome Res., 21(11), 1882–1891. https://doi.org/10.1101/ tion of the invertebrate p53 superfamily. Cold Spring Harb Perspect gr.121137.111 Biol, 2(7), a001131. https://doi.org/10.1101/cshperspect.a001131 26. Brunet, T., & King, N. (2017). The origin of animal multicellularity 44. Carracedo, A., Alimonti, A., & Pandolfi, P. P. (2011). PTEN level in and cell differentiation. Dev. Cell, 43(2), 124–140. https://doi.org/10. tumor suppression: How much is too little? Cancer Res., 71(3), 629– 1016/j.devcel.2017.09.016 633. 27. Abedin, M., & King, N. (2010). Diverse evolutionary paths to cell adhe- 45. Di Cristofano, A., Pesce, B., Cordon-Cardo, C., & Pandolfi, P. P. (1998). sion. Trends Cell Biol., 20(12), 734–742. https://doi.org/10.1016/j.tcb. Pten is essential for embryonic development and tumour suppres- 2010.08.002 sion. Nat. Genet., 19(4), 348–355. https://doi.org/10.1038/1235 28. Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E., & Chisholm, R. L. (2007). 46. Nguyen, H. N., Afkari, Y., Senoo, H., Sesaki, H., Devreotes, P. N., & Protocols for growth and development of Dictyostelium discoideum. Iijima, M. (2014). Mechanism of human PTEN localization revealed by Nat. Protoc., 2(6), 1307–1316. https://doi.org/10.1038/nprot.2007. heterologous expression in Dictyostelium. Oncogene, 33(50), 5688– 178 5696. https://doi.org/10.1038/onc.2013.507 MATHAVARAJAH ET AL. 11 of 13

47. Khare, A., & Shaulsky, G. (2010). Cheating by exploitation of devel- suppressor gene product, regulates Rho activation, cell adhesion and opmental prestalk patterning in Dictyostelium discoideum. PLoS Genet., migration. Oncogene, 21(55), 8470–8476. 6(2), e1000854. 67. Goncharova, E., Goncharov, D., & Noonan, D., & Krymskaya, V. 48. Li, S. I., & Purugganan, M. D. (2011). The cooperative amoeba: Dic- P. (2004). TSC2 modulates actin cytoskeleton and focal adhesion tyostelium as a model for social evolution. Trends Genet., 27(2), 48–54. through TSC1-binding domain and the Rac1 GTPase. J. Cell Biol., 49. Santorelli, L. A., Kuspa, A., Shaulsky, G., Queller, D. C., & Strassmann, 167(6), 1171–1182. J. E. (2013). A new social gene in Dictyostelium discoideum,chtB.BMC 68. Araki, T., & Maeda, Y. (1998). Mutual relation between the cell-cycle Evol. Biol., 13(1), 4. progression and prespore differentiation in Dictyostelium develop- 50. Chen, Z. H., & Schaap, P.(2016). Secreted cyclic di-GMP induces stalk ment. Zoological science, 15(1), 77–84. cell differentiation in the eukaryote Dictyostelium discoideum. J. Bac- 69. Gilbert, O. M., Foster, K. R., Mehdiabadi, N. J., Strassmann, J. E., & teriol., 198(1), 27–31. https://doi.org/10.1128/JB.00321-15 Queller, D. C. (2007). High relatedness maintains multicellular coop- 51. Shaulsky, G., & Kessin, R. H. (2007). The cold war of the social eration in a social amoeba by controlling cheater mutants. Proc Natl amoebae. Curr. Biol., 17(16), R684–692. https://doi.org/10.1016/j. Acad Sci U S A, 104(21), 8913–8917. https://doi.org/10.1073/pnas. cub.2007.06.024 0702723104 52. West, C. M., Zhang, P., McGlynn, A. C., & Kaplan, L. (2002). Outside- 70. Maeda, Y., Ohmori, T., Abe, T., Abe, F., & Amagai, A. (1989). Transition in signaling of cellulose synthesis by a spore coat protein in Dic- of starving Dictyostelium cells to differentiation phase at a particular tyostelium. Eukaryot Cell, 1(2), 281–292. https://doi.org/10.1128/ec. position of the cell cycle. Differentiation, 41(3), 169–175. https://doi. 1.2.281-292.2002 org/10.1111/j.1432-0436.1989.tb00744.x 53. Flajnik, M. F. (2018). A cold-blooded view of adaptive immunity. 71. Muramoto, T., & Chubb, J. R. (2008). Live imaging of the Dictyostelium Nat. Rev. Immunol., 18(7), 438–453. https://doi.org/10.1038/s41577- cell cycle reveals widespread S phase during development, a G2 bias 018-0003-9 in spore differentiation and a premitotic checkpoint. Development, 54. Hirose, S., Benabentos, R., Ho, H. I., Kuspa, A., & Shaulsky, G. (2011). 135(9), 1647–1657. https://doi.org/10.1242/dev.020115 Self-recognition in social amoebae is mediated by allelic pairs of 72. Ohmori, T., & Maeda, Y. (1987). The developmental fate of Dic- tiger genes. Science, 333(6041), 467–470. https://doi.org/10.1126/ tyostelium discoideum cells depends greatly on the cell-cycle position science.1203903 at the onset of starvation. Cell Differ, 22(1), 11–18. https://doi.org/10. 55. Marcus, A., Gowen, B. G., Thompson, T. W., Iannello, A., Ardolino, M., 1016/0045-6039(87)90409-x Deng, W., ... Raulet, D. H. (2014). Recognition of tumors by the innate 73. Huber, R. J. (2014). The cyclin-dependent kinase family in the social immune system and natural killer cells. Adv. Immunol., 122, 91–128. amoebozoan Dictyostelium discoideum. Cell. Mol. Life Sci., 71(4), 629– https://doi.org/10.1016/B978-0-12-800267-4.00003-1 639. https://doi.org/10.1007/s00018-013-1449-3 56. Ostrowski, E. A., Katoh, M., Shaulsky, G., Queller, D. C., & Strass- 74. Ennis, H. L., Dao, D. N., Pukatzki, S. U., & Kessin, R. H. (2000). Dic- mann, J. E. (2008). Kin discrimination increases with genetic distance tyostelium amoebae lacking an F-box protein form spores rather than in a social amoeba. PLoS Biol., 6(11), e287. https://doi.org/10.1371/ stalk in chimeras with wild type. Proc Natl Acad Sci U S A, 97(7), 3292– journal.pbio.0060287 3297. https://doi.org/10.1073/pnas.050005097 57. Smith, N. C., Rise, M. L., & Christian, S. L. (2019). A Comparison of the 75. Strassmann, J. E., Zhu, Y., & Queller, D. C. (2000). Altruism and innate and adaptive immune systems in cartilaginous fish, ray-finned social cheating in the social amoeba Dictyostelium discoideum. Nature, fish, and lobe-finned fish. Front Immunol, 10, 2292. https://doi.org/10. 408(6815), 965–967. https://doi.org/10.1038/35050087 3389/fimmu.2019.02292 76. Sheikh, M. O., Xu, Y., van der Wel, H., Walden, P., Hartson, S. D., 58. Strassmann, J. E. (2016). Kin discrimination in Dictyostelium social & West, C. M. (2015). Glycosylation of Skp1 promotes formation amoebae. J Eukaryot Microbiol, 63(3), 378–383. https://doi.org/10. of Skp1-Cullin-1-F-box protein complexes in Dictyostelium. Mol. 1111/jeu.12307 Cell. Proteomics, 14(1), 66–80. https://doi.org/10.1074/mcp.M114. 59. Gruenheit, N., Parkinson, K., Stewart, B., Howie, J. A., Wolf, J. B., & 044560 Thompson, C. R. (2017). A polychromatic ‘greenbeard’ locus deter- 77. Vodermaier, H. C. (2004). APC/C and SCF: Controlling each other and mines patterns of cooperation in a social amoeba. Nat. Commun., 8, the cell cycle. Curr. Biol., 14(18), R787–796. https://doi.org/10.1016/ 14171. https://doi.org/10.1038/ncomms14171 j.cub.2004.09.020 60. Benabentos, R., Hirose, S., Sucgang, R., Curk, T., Katoh, M., Ostrowski, 78. Wang, Z., Liu, P., Inuzuka, H., & Wei, W. (2014). Roles of F-box proteins E. A., ... Kuspa, A. (2009). Polymorphic members of the lag gene fam- in cancer. Nat. Rev. Cancer, 14(4), 233–247. https://doi.org/10.1038/ ily mediate kin discrimination in Dictyostelium. Curr. Biol., 19(7), 567– nrc3700 572. https://doi.org/10.1016/j.cub.2009.02.037 79. Ennis, H. L., Dao, D. N., Wu, M. Y., & Kessin, R. H. (2003). Mutation 61. Ho, H. I., Hirose, S., Kuspa, A., & Shaulsky, G. (2013). Kin recogni- of the Dictyostelium fbxA gene affects cell-fate decisions and spa- tion protects cooperators against cheaters. Curr. Biol., 23(16), 1590– tial patterning. Protist, 154(3-4), 419–429. https://doi.org/10.1078/ 1595. https://doi.org/10.1016/j.cub.2013.06.049 143446103322454158 62. Strassmann, J. E., & Queller, D. C. (2011). Evolution of cooperation 80. Mohanty, S., Lee, S., Yadava, N., Dealy, M. J., Johnson, R. S., & Firtel, R. and control of cheating in a social microbe, Proc Natl Acad Sci U S A. A. (2001). Regulated protein degradation controls PKA function and 108(Supplement 2), 10855–10862. cell-type differentiation in Dictyostelium. Genes Dev., 15(11), 1435– 63. Queller, D. C., Ponte, E., & Bozzaro, S., & Strassmann, J. E. (2003). 1448. https://doi.org/10.1101/gad.871101 Single-gene greenbeard effects in the social amoeba Dictyostelium 81. Loomis, W. F. (2014). Cell signaling during development of Dic- discoideum, Science. 299(5603), 105–106. tyostelium. Developmental Biology, 391(1), 1–16. https://doi.org/10. 64. Ponte, E., Bracco, E., Faix, J., & Bozzaro, S. (1998). Detection of sub- 1016/j.ydbio.2014.04.001 tle phenotypes: The case of the cell adhesion molecule csA in Dic- 82. Lindahl, T., & Barnes, D. E. (2000). Repair of endogenous DNA dam- tyostelium. Proc. Natl. Acad. Sci. USA. 95(16), 9360–9365. age. Cold Spring Harb Symp Quant Biol, 65, 127–133. https://doi.org/ 65. Kwiatkowski, D. J., & Manning, B. D. (2005). Tuberous sclerosis: a GAP 10.1101/sqb.2000.65.127 at the crossroads of multiple signaling pathways. Hum. Mol. Genet. 83. Chapman, J. R., Taylor, M. R., & Boulton, S. J. (2012). Playing 14(suppl_2), R251–R258. the end game: DNA double-strand break repair pathway choice. 66. Astrinidis, A., Cash, T. P., Hunter, D. S., Walker, C. L., Chernoff, J., & Mol. Cell, 47(4), 497–510. https://doi.org/10.1016/j.molcel.2012.07. Henske, E. P.(2002). Tuberin, the tuberous sclerosis complex 2 tumor 029 12 of 13 MATHAVARAJAH ET AL.

84. Miermont, A., Antolovic, V., Lenn, T., Nichols, J. M. E., Millward, of insulin signalling. Nat. Commun., 6, 7400. https://doi.org/10.1038/ L. J., & Chubb, J. R. (2019). The fate of cells undergoing spon- ncomms8400 taneous DNA damage during development. Development. 146(12), 100. Francisco, A. B., Singh, R., Li, S., Vani, A. K., Yang, L., Munroe, R. J., dev174268. https://doi.org/10.1242/dev.174268 ... Long, Q. (2010). Deficiency of suppressor enhancer Lin12 1 like 85. Tell, G., Quadrifoglio, F., Tiribelli, C., & Kelley, M. R. (2009). The (SEL1L) in mice leads to systemic endoplasmic reticulum stress and many functions of APE1/Ref-1: Not only a DNA repair enzyme. embryonic lethality. J. Biol. Chem., 285(18), 13694–13703. https:// Antioxid Redox Signal, 11(3), 601–620. https://doi.org/10.1089/ars. doi.org/10.1074/jbc.M109.085340 2008.2194 101. Kashima, L., Idogawa, M., Mita, H., Shitashige, M., Yamada, T., Ogi, 86. Hudson, J. J., Hsu, D. W., Guo, K., Zhukovskaya, N., Liu, P. H., Williams, K., ... Tokino, T. (2012). CHFR protein regulates mitotic checkpoint J. G., ... Lakin, N. D. (2005). DNA-PKcs-dependent signaling of DNA by targeting PARP-1 protein for ubiquitination and degradation. damage in Dictyostelium discoideum. Curr. Biol., 15(20), 1880–1885. J. Biol. Chem., 287(16), 12975–12984. https://doi.org/10.1074/jbc. https://doi.org/10.1016/j.cub.2005.09.039 M111.321828 87. Hsu, D. W., Gaudet, P., Hudson, J. J., Pears, C. J., & Lakin, N. D. (2006). 102. Maddika, S., Sy, S. M., & Chen, J. (2009). Functional interaction DNA damage signaling and repair in Dictyostelium discoideum. Cell between Chfr and Kif22 controls genomic stability. J. Biol. Chem., Cycle, 5(7), 702–708. https://doi.org/10.4161/cc.5.7.2626 284(19), 12998–13003. https://doi.org/10.1074/jbc.M900333200 88. Srivastava, M., & Raghavan, S. C. (2015). DNA double-strand break 103. Wang, H., Li, S., Zhang, H., Wang, Y., Hao, S., & Wu, X. (2018). BLM repair inhibitors as cancer therapeutics. Chem. Biol., 22(1), 17–29.doi: prevents instability of structure-forming DNA sequences at common https://doi.org/10.1016/j.chembiol.2014.11.013 fragile sites. PLoS Genet., 14(11), e1007816. https://doi.org/10.1371/ 89. Kiwerska, K., & Szyfter, K. (2019). DNA repair in cancer initi- journal.pgen.1007816 ation, progression, and therapy-a double-edged sword. J Appl 104. Williams, T. D., Peak-Chew, S. Y., Paschke, P., & Kay, R. R. (2019). Genet, 60(3-4), 329–334. https://doi.org/10.1007/s13353-019- Akt and SGK protein kinases are required for efficient feeding by 00516-9 macropinocytosis. J. Cell Sci., 132(2), jcs224998. https://doi.org/10. 90. Su, H., Si, X. Y., Tang, W. R., & Luo, Y. (2013). The regulation of anoikis 1242/jcs.224998 in tumor invasion and metastasis. Yi Chuan, 35(1), 10–16. https://doi. 105. Hoeller, O., & Kay, R. R. (2007). Chemotaxis in the absence of PIP3 org/10.3724/sp.j.1005.2013.00010 gradients. Curr. Biol., 17(9), 813–817. https://doi.org/10.1016/j.cub. 91. Sancho, E., Batlle, E., & Clevers, H. (2004). Signaling pathways in 2007.04.004 intestinal development and cancer. Annu. Rev. Cell Dev. Biol., 20, 106. Chen, C. L., Wang, Y., Sesaki, H., & Iijima, M. (2012). Myosin I links 695–723. https://doi.org/10.1146/annurev.cellbio.20.010403. PIP3 signaling to remodeling of the actin cytoskeleton in chemo- 092805 taxis. Sci Signal, 5(209), ra10. https://doi.org/10.1126/scisignal. 92. Miguel, J. C., Maxwell, A. A., Hsieh, J. J., Harnisch, L. C., Al Alam, D., 2002446 Polk, D. B., ... Frey, M. R. (2017). Epidermal growth factor suppresses 107. Yang, S., He, F., Dai, M., Pan, J., Wang, J., & Ye, B. (2019). CHFR intestinal epithelial cell shedding through a MAPK-dependent path- promotes the migration of human gastric cancer cells by inducing way. J. Cell Sci., 130(1), 90–96. https://doi.org/10.1242/jcs.182584 epithelial-to-mesenchymal transition in a HDAC1-dependent man- 93. Andrade, D., & Rosenblatt, J. (2011). Apoptotic regulation of epithe- ner. Onco Targets Ther, 12, 1075–1084. https://doi.org/10.2147/OTT. lial cellular extrusion. Apoptosis, 16(5), 491–501. https://doi.org/10. S191016 1007/s10495-011-0587-z 108. Sun, Z., Liu, J., Jing, H., Dong, S. X., & Wu, J. (2017). The diagnostic 94. Assimakopoulos, S. F., Tsamandas, A. C., Tsiaoussis, G. I., Karatza, E., and prognostic value of CHFR hypermethylation in colorectal can- Triantos, C., Vagianos, C. E., ... Thomopoulos, K. C. (2012). Altered cer, a meta-analysis and literature review. Oncotarget, 8(51), 89142– intestinal tight junctions’ expression in patients with liver cirrhosis: 89148. https://doi.org/10.18632/oncotarget.19408 A pathogenetic mechanism of intestinal hyperpermeability. Eur J Clin 109. Ledet, E. M., Antonarakis, E. S., Isaacs, W. B., Lotan, T. L., Pritchard, Invest, 42(4), 439–446. https://doi.org/10.1111/j.1365-2362.2011. C., & Sartor, A. O. (2020). Germline BLM mutations and metastatic 02609.x prostate cancer. Prostate, 80(2), 235–237. https://doi.org/10.1002/ 95. Kiesslich, R., Duckworth, C. A., Moussata, D., Gloeckner, A., Lim, L. G., pros.23924 Goetz, M., ... Watson, A. J. (2012). Local barrier dysfunction identi- 110. Zhu, X., Chen, H., Yang, Y., Xu, C., Zhou, J., Zhou, J., & Chen, Y. fied by confocal laser endomicroscopy predicts relapse in inflamma- (2018). Distinct prognosis of mRNA expression of the five RecQ tory bowel disease. Gut, 61(8), 1146–1153. https://doi.org/10.1136/ DNA-helicase family members – RECQL, BLM, WRN, RECQL4, and gutjnl-2011-300695 RECQL5 – in patients with breast cancer. Cancer Manag Res, 10, 96. Knodler, L. A., Vallance, B. A., Celli, J., Winfree, S., Hansen, B., Mon- 6649–6668. https://doi.org/10.2147/CMAR.S185769 tero, M., & Steele-Mortimer, O. (2010). Dissemination of invasive 111. Biunno, I., Cattaneo, M., Orlandi, R., Canton, C., Biagiotti, L., Ferrero, Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc S., ... Menard, S. (2006). SEL1L a multifaceted protein playing a role in Natl Acad Sci U S A, 107(41), 17733–17738. https://doi.org/10.1073/ tumor progression. J. Cell. Physiol., 208(1), 23–38. https://doi.org/10. pnas.1006098107 1002/jcp.20574 97. Liu, J. J., Wong, K., Thiesen, A. L., Mah, S. J., Dieleman, L. A., Claggett, 112. Burgess, A., Labbe, J. C., Vigneron, S., Bonneaud, N., Strub, J. M., B., ... Fedorak, R. N. (2011). Increased epithelial gaps in the small Van Dorsselaer, A., ... Castro, A. (2008). Chfr interacts and colocal- intestines of patients with inflammatory bowel disease: Density mat- izes with TCTP to the mitotic spindle. Oncogene, 27(42), 5554–5566. ters. Gastrointest Endosc, 73(6), 1174–1180. https://doi.org/10.1016/ https://doi.org/10.1038/onc.2008.167 j.gie.2011.01.018 113. Daniels, M. J., Marson, A., & Venkitaraman, A. R. (2004). PML bod- 98. Santorelli, L. A., Thompson, C. R., Villegas, E., Svetz, J., Dinh, C., ies control the nuclear dynamics and function of the CHFR mitotic Parikh, A., ... Shaulsky, G. (2008). Facultative cheater mutants reveal checkpoint protein. Nat. Struct. Mol. Biol., 11(11), 1114–1121. https: the genetic complexity of cooperation in social amoebae. Nature, //doi.org/10.1038/nsmb837 451(7182), 1107–1110. https://doi.org/10.1038/nature06558 114. Grierson, P. M., Acharya, S., & Groden, J. (2013). Collaborating func- 99. Braccini, L., Ciraolo, E., Campa, C. C., Perino, A., Longo, D. L., tions of BLM and DNA topoisomerase I in regulating human rDNA Tibolla, G., ... Hirsch, E. (2015). PI3K-C2gamma is a Rab5 effec- transcription. Mutat Res, 743–744, 89–96. https://doi.org/10.1016/j. tor selectively controlling endosomal Akt2 activation downstream mrfmmm.2012.12.002 MATHAVARAJAH ET AL. 13 of 13

115. Min, J., Wright, W. E., & Shay, J. W. (2019). Clustered telomeres in 119. Bussey, K. J., Cisneros, L. H., Lineweaver, C. H., & Davies, P. C. (2017). phase-separated nuclear condensates engage mitotic DNA synthe- Ancestral gene regulatory networks drive cancer. Proc. Natl. Acad. Sci. sis through BLM and RAD52. Genes Dev., 33(13-14), 814–827. https: USA, 114(24), 6160–6162. //doi.org/10.1101/gad.324905.119 116. Dellaire, G., & Bazett-Jones, D. P. (2004). PML nuclear bodies: Dynamic sensors of DNA damage and cellular stress. BioEssays, 26(9), 963–977. https://doi.org/10.1002/bies.20089 How to cite this article: Mathavarajah, S., VanIderstine, C., 117. Zhong, S., Hu, P., Ye, T. Z., Stan, R., Ellis, N. A., & Pandolfi, P. P. (1999). Dellaire, G., Huber, R. J. (2021). Cancer and the breakdown of A role for PML and the nuclear body in genomic stability. Oncogene, 18(56), 7941–7947. https://doi.org/10.1038/sj.onc.1203367 multicellularity: What Dictyostelium discoideum,asocial 118. Bernardi, R., & Pandolfi, P. P. (2007). Structure, dynamics and func- amoeba, can teach us. BioEssays, e2000156. tions of promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell https://doi.org/10.1002/bies.202000156 Biol., 8(12), 1006–1016. https://doi.org/10.1038/nrm2277