Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Soft selection reduces loss of heterozygosity in asexual reproduction.

The adaptive value of sexual reproduction is still debated in evolutionary theory. It has been proposed that the advantage of sexual reproduction over asexual reproduction is to promote genetic diversity, to prevent the accumulation of harmful mutations or to preserve heterozygosity. Since these hypothetical advantages depend on the type of asexual reproduction, understanding how selection affects the taxonomic distribution of each type could help us discriminate between existing hypotheses. Here, I argue that soft selection, competition among embryos or offspring in selection arenas prior to the hard selection of the adult phase, reduces loss of heterozygosity in certain types of asexual reproduction. Since loss of heterozygosity leads to the unmasking of recessive deleterious mutations in the progeny of asexual individuals, soft selection facilitates the evolution of these types of asexual reproduction. Using a population genetics model, I calculate how loss of heterozygosity affects fitness for different types of apomixis and automixis, and I show that soft selection significantly reduces loss of heterozygosity, hence increases fitness, in apomixis with suppression of the first meiotic division and in automixis with central fusion, the most common types of asexual reproduction. Therefore, if sexual reproduction evolved to preserve heterozygosity, soft selection should be associated with these types of asexual reproduction. I discuss the evidence for this prediction and how this and other observations on the distribution of different types of asexual reproduction in nature is consistent with the heterozygosity hypothesis.

Evidence from automixis with inverted meiosis for the maintenance of sex by loss of complementation.

The adaptive value of sexual reproduction is still debated. A short-term disadvantage of asexual reproduction is loss of heterozygosity, which leads to the unmasking of recessive deleterious mutations. The cost of this loss of complementation is predicted to be higher than the twofold cost of meiosis for most types of asexual reproduction. Automixis with terminal fusion of sister nuclei is especially vulnerable to the effect of loss of complementation. It is found, however, in some taxa including oribatid mites, the most prominent group of ancient asexuals. Here, I show that automixis with terminal fusion is stable if it is associated with inverted meiosis and that this appears to be the case in nature, notably in oribatid mites. The existence of automixis with terminal fusion, and its co-occurrence with inverted meiosis, therefore, is consistent with the hypothesis that loss of complementation is important in the evolution of sexual reproduction.

Delivery of Therapeutic miR-148b Mimic via Poly(ß Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(ß amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.

Polyploidy as an Adaptation against Loss of Heterozygosity in Cancer.

Polyploidy is common in cancer cells and has implications for tumor progression and resistance to therapies, but it is unclear whether it is an adaptation of the tumor or the non-adaptive effect of genomic instability. I discuss the possibility that polyploidy reduces the deleterious effects of loss of heterozygosity, which arises as a consequence of mitotic recombination, and which in diploid cells leads instead to the rapid loss of complementation of recessive deleterious mutations. I use computational predictions of loss of heterozygosity to show that a population of diploid cells dividing by mitosis with recombination can be easily invaded by mutant polyploid cells or cells that divide by endomitosis, which reduces loss of complementation, or by mutant cells that occasionally fuse, which restores heterozygosity. A similar selective advantage of polyploidy has been shown for the evolution of different types of asexual reproduction in nature. This provides an adaptive explanation for cyclical ploidy, mitotic slippage and cell fusion in cancer cells.

A test of the photoprotection hypothesis for the evolution of autumn colours: Chlorophyll resorption, not anthocyanin production, is correlated with nitrogen translocation.

A prominent hypothesis for the adaptive value of anthocyanin production in the autumn leaves of trees and shrubs is that anthocyanins protect leaves from photooxidative stress at low temperatures, allowing a better resorption of nutrients-in particular, nitrogen-before leaf fall. While there is evidence that anthocyanins enable photoprotection, it is not clear whether this translates to improved nitrogen translocation and how this can explain inter-specific variation in autumn colours. A recent comparative analysis showed no correlation between temperature and anthocyanin production across species but did not analyse nitrogen content and nitrogen resorption efficiency. Here, we provide this comparison by analysing the nitrogen content of mature and senescent leaves and their autumn colours in 55 species of trees. We find no correlation between the presence of anthocyanins and the efficiency of nitrogen resorption. We find, instead, that nitrogen resorption is more efficient in species with yellow autumn colours, pointing to chlorophyll resorption, rather than anthocyanin synthesis, as the main determinant of nitrogen translocation efficiency. Hence, our results do not corroborate the photoprotection hypothesis for the evolution of autumn colours.

Inverted meiosis and the evolution of sex by loss of complementation.

Inverted meiosis, in which sister chromatids segregate before homologous chromosomes, is a common aberration of conventional meiosis (in which sister chromatids segregate after homologous chromosomes) and is routinely observed in certain species. This raises an evolutionary mystery: what is the adaptive advantage of the more common, conventional order of segregation in meiosis? I use a population genetic model to show that asexual mutants arising from inverted meiosis are relatively immune from the deleterious effects of loss of complementation (heterozygosity), unlike the asexual mutants arising from conventional meiosis, in which loss of complementation can outweigh the two-fold cost of meiosis. Hence, asexual reproduction can replace sexual reproduction with inverted meiosis, but not with conventional meiosis. The results are in line with analogous considerations on other alternative types of reproduction and support the idea that amphimixis is stable in spite of the two-fold cost of meiosis because loss of complementation in mutant asexuals outweigh the two-fold cost.

A comparative analysis of the photoprotection hypothesis for the evolution of autumn colours.

The adaptive value of autumn colours-the seasonal production of red anthocyanins observed in many species of trees and shrubs-is still debated. According to the photoprotection hypothesis, anthocyanins protect leaves from photo-inhibition and photo-oxidation at low temperatures, enabling the tree to reabsorb nutrients more efficiently before leaf fall. Hence, the hypothesis predicts that autumn colours are more likely to evolve in species growing in colder environments. We tested this prediction by comparing the climatic parameters of 237 North American tree species. We found that, although species with yellow autumn leaves grow under lower minimum temperatures than species with green leaves, there is no significant difference in temperature between species with red autumn leaves and species with green or yellow autumn leaves. We conclude that, although reabsorbing chlorophyll in autumn, and the consequent unmasking of yellow carotenoids, may be an adaptation to cold temperatures, the production of red anthocyanins is not. Hence, our interspecific comparative analysis does not support the photoprotection hypothesis as an explanation for the evolution of autumn colours.

Heterogeneity for IGF-II production maintained by public goods dynamics in neuroendocrine pancreatic cancer.

The extensive intratumor heterogeneity revealed by sequencing cancer genomes is an essential determinant of tumor progression, diagnosis, and treatment. What maintains heterogeneity remains an open question because competition within a tumor leads to a strong selection for the fittest subclone. Cancer cells also cooperate by sharing molecules with paracrine effects, such as growth factors, and heterogeneity can be maintained if subclones depend on each other for survival. Without strict interdependence between subclones, however, nonproducer cells can free-ride on the growth factors produced by neighboring producer cells, a collective action problem known in game theory as the "tragedy of the commons," which has been observed in microbial cell populations. Here, we report that similar dynamics occur in cancer cell populations. Neuroendocrine pancreatic cancer (insulinoma) cells that do not produce insulin-like growth factor II (IGF-II) grow slowly in pure cultures but have a proliferation advantage in mixed cultures, where they can use the IGF-II provided by producer cells. We show that, as predicted by evolutionary game theory, producer cells do not go extinct because IGF-II acts as a nonlinear public good, creating negative frequency-dependent selection that leads to a stable coexistence of the two cell types. Intratumor cell heterogeneity can therefore be maintained even without strict interdependence between cell subclones. Reducing the amount of growth factors available within a tumor may lead to a reduction in growth followed by a new equilibrium, which may explain relapse in therapies that target growth factors.

Cooperation among cancer cells as public goods games on Voronoi networks.

Cancer cells produce growth factors that diffuse and sustain tumour proliferation, a form of cooperation that can be studied using mathematical models of public goods in the framework of evolutionary game theory. Cell populations, however, form heterogeneous networks that cannot be described by regular lattices or scale-free networks, the types of graphs generally used in the study of cooperation. To describe the dynamics of growth factor production in populations of cancer cells, I study public goods games on Voronoi networks, using a range of non-linear benefits that account for the known properties of growth factors, and different types of diffusion gradients. The results are surprisingly similar to those obtained on regular graphs and different from results on scale-free networks, revealing that network heterogeneity per se does not promote cooperation when public goods diffuse beyond one-step neighbours. The exact shape of the diffusion gradient is not crucial, however, whereas the type of non-linear benefit is an essential determinant of the dynamics. Public goods games on Voronoi networks can shed light on intra-tumour heterogeneity, the evolution of resistance to therapies that target growth factors, and new types of cell therapy.

Evolution of optimal Hill coefficients in nonlinear public goods games.

In evolutionary game theory, the effect of public goods like diffusible molecules has been modelled using linear, concave, sigmoid and step functions. The observation that biological systems are often sigmoid input-output functions, as described by the Hill equation, suggests that a sigmoid function is more realistic. The Michaelis-Menten model of enzyme kinetics, however, predicts a concave function, and while mechanistic explanations of sigmoid kinetics exist, we lack an adaptive explanation: what is the evolutionary advantage of a sigmoid benefit function? We analyse public goods games in which the shape of the benefit function can evolve, in order to determine the optimal and evolutionarily stable Hill coefficients. We find that, while the dynamics depends on whether output is controlled at the level of the individual or the population, intermediate or high Hill coefficients often evolve, leading to sigmoid input-output functions that for some parameters are so steep to resemble a step function (an on-off switch). Our results suggest that, even when the shape of the benefit function is unknown, biological public goods should be modelled using a sigmoid or step function rather than a linear or concave function.

Evolutionary dynamics of the Warburg effect: glycolysis as a collective action problem among cancer cells.

The upregulation of glycolysis in cancer cells (the "Warburg effect") is common and has implications for prognosis and treatment. As it is energetically inefficient under adequate oxygen supply, its adaptive value for a tumor remains unclear. It has been suggested that the acidity produced by glycolysis is beneficial for cancer cells because it promotes proliferation against normal cells. Current models of this acid-mediated tumor invasion hypothesis, however, do not account for increased glycolysis under non-limiting oxygen concentrations and therefore do not fully explain the Warburg effect. Here I show that the Warburg effect can be explained as a form of cooperation among cancer cells, in which the products of glycolysis act as a public good, even when oxygen supply is high enough to make glycolysis energetically inefficient. A multiplayer game with non-linear, non-monotonic payoff functions that models the benefits of the acidity induced by glycolysis reveals that clonal selection can stabilize glycolysis even when energetically costly, that is, under non-limiting oxygen concentration. Characterizing the evolutionary dynamics of glycolysis reveals cases in which anti-cancer therapies that rely on the modification of acidity can be effective.

Programming tumor evolution with selection gene drives to proactively combat drug resistance.

Most targeted anticancer therapies fail due to drug resistance evolution. Here we show that tumor evolution can be reproducibly redirected to engineer therapeutic opportunity, regardless of the exact ensemble of pre-existing genetic heterogeneity. We develop a selection gene drive system that is stably introduced into cancer cells and is composed of two genes, or switches, that couple an inducible fitness advantage with a shared fitness cost. Using stochastic models of evolutionary dynamics, we identify the design criteria for selection gene drives. We then build prototypes that harness the selective pressure of multiple approved tyrosine kinase inhibitors and employ therapeutic mechanisms as diverse as prodrug catalysis and immune activity induction. We show that selection gene drives can eradicate diverse forms of genetic resistance in vitro. Finally, we demonstrate that model-informed switch engagement effectively targets pre-existing resistance in mouse models of solid tumors. These results establish selection gene drives as a powerful framework for evolution-guided anticancer therapy.

Evolution of polygamous marriage by maximization of inclusive fitness.

The majority of human societies practice polygynous marriage, in line with the typical mating pattern found in mammals. Polygyny in humans is often associated with the transfer of wealth to a male's sister's offspring, and it has been suggested that this "mother's brother phenomenon" is adaptive when paternity confidence is low. Polyandry, on the other hand, while virtually unknown in mammals, is practiced by a few human societies, and it has been suggested that this is adaptive if the co-husbands are genetically related. The evolution of human marriage strategies, therefore, can be studied in the framework of kin selection and game theory, as strategic transmission of wealth by males and strategic paternity allocation by females can evolve to maximize inclusive fitness. Here I analyse the stability of polygynous and polyandrous marriage using a game theoretical model previously developed to study monogamy. I show that the "mother's brother phenomenon" depends on the degree of resource depletion through division, whereas the paternity threshold commonly discussed in the anthropological literature is largely irrelevant. Resource depletion through division is also the major determinant of the stability of polyandry, whereas relatedness between co-husbands is not essential. Finally, I show that when females control the transfer of their own resources, monogamy is stable under more general conditions than previously believed.

Trading public goods stabilizes interspecific mutualism.

The existence of cooperation between species raises a fundamental problem for evolutionary theory. Why provide costly services to another species if the feedback of this provision also happens to benefit intra-specific competitors that provide no service? Rewarding cooperators and punishing defectors can help maintain mutualism; this is not possible, however, when one can only respond to the collective action of one's partners, which is likely to be the case in many common symbioses. We show how the theory of public goods can explain the stability of mutualism when discrimination between cooperators and defectors is not possible: if two groups of individuals trade goods that are non-linear, increasing functions of the number of contributions, their mutualistic interaction is maintained by the exchange of these public goods, even when it is not possible to punish defectors, which can persist at relatively high frequencies. This provides a theoretical justification and testable predictions for the evolution of mutualism in the absence of discrimination mechanisms.

Review: Game theory of public goods in one-shot social dilemmas without assortment.

We review the theory of public goods in biology. In the N-person prisoner's dilemma, where the public good is a linear function of the individual contributions, cooperation requires some form of assortment, for example due to kin discrimination, population viscosity or repeated interactions. In most social species ranging from bacteria to humans, however, public goods are usually a non-linear function of the contributions, which makes cooperation possible without assortment. More specifically, a polymorphic state can be stable in which cooperators and non-cooperators coexist. The existence of mixed equilibria in public goods games is a fundamental result in the study of cooperation that has been overlooked so far, because of the disproportionate attention given to the two- and N-person prisoner's dilemma. Methods and results from games with pairwise interactions or linear benefits cannot, in general, be extended to the analysis of public goods. Game theory helps explain the production of public goods in one-shot, N-person interactions without assortment, it leads to predictions that can be easily tested and allows a prescriptive approach to cooperation.