Poly‐aneuploid cancer cells promote evolvability, generating lethal cancer

Abstract Cancer cells utilize the forces of natural selection to evolve evolvability allowing a constant supply of heritable variation that permits a cancer species to evolutionary track changing hazards and opportunities. Over time, the dynamic tumor ecosystem is exposed to extreme, catastrophic changes in the conditions of the tumor—natural (e.g., loss of blood supply) or imposed (therapeutic). While the nature of these catastrophes may be varied or unique, their common property may be to doom the current cancer phenotype unless it evolves rapidly. Poly‐aneuploid cancer cells (PACCs) may serve as efficient sources of heritable variation that allows cancer cells to evolve rapidly, speciate, evolutionarily track their environment, and most critically for patient outcome and survival, permit evolutionary rescue, therapy resistance, and metastasis. As a conditional evolutionary strategy, they permit the cancer cells to accelerate evolution under stress and slow down the generation of heritable variation when conditions are more favorable or when the cancer cells are closer to an evolutionary optimum. We hypothesize that they play a critical and outsized role in lethality by their increased capacity for invasion and motility, for enduring novel and stressful environments, and for generating heritable variation that can be dispensed to their 2N+ aneuploid progeny that make up the bulk of cancer cells within a tumor, providing population rescue in response to therapeutic stress. Targeting PACCs is essential to cancer therapy and patient cure—without the eradication of the resilient PACCs, cancer will recur in treated patients.


| INTRODUC TI ON
All cancer would be curable if it were not for metastases and if metastases were not resistant to cancer therapies. These features result in the death of nearly 10 million people worldwide each year and close to 1,600 deaths every day in the USA (Bray et al., 2018;Siegel, Miller, & Jemal, 2018). These two properties-metastatic success and therapy resistance-occur because cancer cells are under selection to evolve traits that generate heritable variation: evolvability. That is, evolvability describes the capacity to evolve. Heritable variation is the fuel for natural selection. The rate of evolution in response to environmental circumstances is the product of heritable variation and the force of selection.
Cancer cells utilize the forces of natural selection to evolve evolvability. Evolvability describes a system where variation in phenotype is (a) heritable and (b) adaptive. The diverse cancer species inhabiting a patient initiate as a single monophyletic clade arising from a common ancestor in a speciation event where a lineage of normal cells goes from being part of the whole organism's fitness function to becoming its own self-defined evolutionary fitness function (Table 1).
Upon becoming the unit of selection, the initiating species of the cancer cell clade will be far from any evolutionary optimum. Being on the slope rather than the peak of its adaptive landscape means that traits conferring greater heritable variation will be favored. In addition, the heterogeneity of the emerging tumor can select for a diversity of cancer cells. A cancer cell with the capacity to generate heritable variation (evolvability) will be able to diversify more rapidly into different species that specialize on various aspects of tumor heterogeneity. A slower evolving species of cancer cells will be preempted by the faster evolving species. Furthermore, any given region of a tumor is not static. There are constant changes in immune infiltration, blood flow, oxygen, pH, and toxic metabolite buildups (Amend & Pienta, 2015). Any evolutionary optimum, therefore, is constantly shifting, and the constant supply of heritable variation permits a cancer species to evolutionary track changing hazards and opportunities. Over time, the dynamic tumor ecosystem may also be exposed to extreme, catastrophic changes in the conditions of the tumor-natural (e.g., loss of blood supply) or imposed (therapeutic). While the nature of these catastrophes may be varied or unique, their common property may be to doom the current cancer phenotype unless it evolves rapidly to its dire circumstances. In the ecology of threatened species in nature, this is referred to as evolutionary rescue (Carlson, Cunningham, & Westley, 2014;Gomulkiewicz & Holt, 1995;Hammarlund, Von Stedingk, & Påhlman, 2018). While the details of a given catastrophe cannot be anticipated, evolving evolvability permits cancer species to adapt to the unexpected-and often catastrophic-temporal and special events.
Conversely, gene deletions or epigenetic silencing by methylation can downregulate costly or unnecessary metabolic activities that are legacies of the normal cell's whole-organism functions. Lax DNA repair mechanisms or other increases in mutation rates can contribute to greater heritable variation. Chromosomal rearrangements can change gene expression and gene regulation in ways that suppress or uncover new heritable variants. Increased demethylation of arbitrary or specific histones provides ways of creating heritable epigenetic variation that can cause single genes or suites of genes to be unmasked and expressed. Examples from other species suggest that chromosomal rearrangements that may significantly amplify heritable genetic variation could be more common in polyploid cancer cells (James, Usher, Campbell, & Bond, 2008;Selmecki et al., 2015;Yao, Carretero-Paulet, & Van de Peer, 2019). The repeatable evolution of poly-aneuploid forms of cancer species may be the primary culprit and the prime adaptation for cancers' evolvability.

| P OLY-ANEUPLOID C AN CER CELL S A S A CONS TR AINT-B RE AKING ADAP TATION FOR S TRE SS RE S IS TAN CE AND E VOLVAB ILIT Y
Multiple studies have described a minority population of physically large cancer cells within the tumors of patients with metastatic disease. This likely holds for most, if not all, cancer types that have the potential to result in therapeutically resistant metastases.
Virtually, all cancer cells are aneuploid (i.e., 2N+), containing an abnormal number of chromosomes or chromosomal fragments. The Cancer cells with relative high genomic content generally occur at low frequencies and as poly-aneuploids exist as 4N+, 6N+, or greater (Table 2).
It seems that cancer species are able to exist in both 2N+ and poly-aneuploid states, and that cancer cells of a clade shift between these states. As the poly-aneuploids revert back to a 2N+ state, those that retain odd numbers of chromosomes or chromosome fragments might be more fit than those that do not. Thus, by the time the cancer is clinically detectable perhaps all of the observable cancer cells have cycled one or more times between poly-aneuploid and 2N+ states.
We hypothesize that PACCs play a critical and outsized role in lethality by (a) their increased capacity for invasion and motility (high metastatic potential); (b) for enduring novel and stressful environments (successfully metastasize and be intrinsically therapy-resistant); and (c) for generating heritable variation that can be dispensed to their 2N+ aneuploid progeny that make up the bulk of cancer cells within a tumor (providing population rescue) (Amend et al., 2019;Carlson et al., 2014;Lin et al., 2019).
Poly-aneuploid cancer cells have been documented as emerging in response to stress, including therapeutic stress such as chemotherapy ( Figure 1) (Amend et al., 2019;Lin et al., 2019Lin et al., , 2017. These data include tightly controlled in vitro assays as well as clinical data demonstrating increased PACC numbers in ovarian cancer patients following chemotherapy (Niu et al., 2017). Compellingly, published data also show that pressures present in the tumor microenvironment, such as low oxygen, induce the emergence of PACCs in vitro.
PACCs are more common in metastatic lesions than in primary tumors, and they are more common in the primary tumors of patients with metastasis than those who have strictly localized disease (Fei et al., 2015). The presence of PACCs in the diagnostic specimen of prostate cancer predicts a dismal prognosis, with rapid disease progression and reduced overall survival (Alharbi, Marzo, Hicks, Lotan, & Epstein, 2018). Different functional events can produce PACCs including cell-cell fusion, endoreplication, and acytokinesis.
Data suggest that PACCs survive dynamic environments (e.g., sudden onset of hypoxia or nutrient poverty) by exiting the cell cycle and entering quiescence or reversible therapy-induced senescence, therefore protecting their genome and avoiding programmed cell death (Lopez-Sanchez et al., 2014). The formation of a PACC phenotype is also associated with an increased capacity for motility and invasion. Motility allows PACCs to physically move into new environments, a rare feature among largely sessile epithelial cells (Fei et al., 2015). can provide the source of the "rescue effect" associated with the catastrophic event of therapeutic intervention (Carlson et al., 2014).
The formation of PACCs, then, may represent the common convergent evolutionary event across patients that actuates metastasis and therapeutic resistance.
Poly-aneuploid cancer cells provide a notable advantage over other mechanisms for generating heritable variation or evolvability  Cancer cell species that can produce PACCs, therefore, may represent an evolutionary archetype-an entity that has evolved the capacity for evolvability through increased, protected, and mobile genomic content. A central dogma of genetics reflects that the architecture of a genetic system simultaneously permits and constrains the heritable variation available to natural selection. A normal diploid cell, for example, is genetically and epigenetically programmed to perform a tissue-specific set of tasks. A liver cell and a kidney cell each have the same genetic material but perform vastly different functions that are tightly regulated. Due to their increased and disordered genetic content, PACCs circumvent this architecture.

| PREPAREDNE SS FOR THE UNE XPEC TED IS CRITI C AL TO THE E VOLUTION OF E VOLVAB ILIT Y
PACCs evolve swiftly by providing heritable variation at a rate not available within a normal diploid cell (Yang et al., 2019). Similarly, utilizing aneuploidy to enable cross-adaptation to therapeutic agents has been demonstrated in yeast, allowing rapid development of resistance in response to stress (Selmecki et al., 2015;Storchova et al., 2006). Natural selection can only respond to what has happened or is happening.
The certainty that a cancer cell will encounter rare circumstances is why natural selection can evolve evolvability as an adaption. Upon

| CONVERG ENT E VOLUTI ON OF PACC S
The PACC phenotype represents a convergent adaptive response to stress. It appears to happen in all cancer cell lineages across all patients with metastatic disease. PACCs likely allow the cancer cells to produce gene duplications, repurpose redundant genes, generate novel variants from chromosomal rearrangements, and, perhaps most significantly, epigenetically access cellular programs typically restricted to subsets of tissue cell types, for example, macrophages, osteoclasts, and trophoblasts (Brooks et al., 2019;Diaz, Wood, Sibley, & Greenwood, 2014;Pereira et al., 2018;Yang et al., 2019). Notably, PACC characteristics are not restricted to eukaryotic cells, are also observed in other organisms, and emerge as an adaptation to stress, providing evidence for ancestral genetic programs.
Yeast, as noted above, exhibit a near equivalence of PACCs (Selmecki et al., 2015;Storchova et al., 2006). Some yeast form polyploids through meiosis without cytokinesis in response to toxins or adverse physical conditions. This permits rapid evolution of appropriate stress responses and later a return to a euploid state (Selmecki et al., 2015;Storchova et al., 2006;Yang et al., 2019). Tetrahymena vorax, a protist species of ciliate, exhibits two exemplars of stress-contingent strategies (Gronlien, Hagen, & Sand, 2011). When food is generally unavailable, they can duplicate their genome to be essentially polyploid. When favorable times return, they reenter the cell cycle and can sustain several rounds of cell division without having to enter interphase and duplicate their DNA. It is unknown whether diverse heritable variants become possible as a result of this polyploid state. A number of protist species that exist in a haploid state will cease asexual reproduction and engage in sex by fusing into a 2N "polyploid" morph that subsequently undergoes meiosis to produce four 1N offspring. Similarly, some of invertebrate metazoans typically reproduce asexually and only engage in sex (via a "polyploid" state) when the environmental conditions become poor (e.g., water fleas, Daphnia) (Adamowicz, Gregory, Marinone, & Hebert, 2002;Vergilino, Markova, Ventura, Manca, & Dufresne, 2011;Xu et al., 2015). The ancient ameboid protist class Foraminifera alternates between haploid and polyploid states as a means of reproduction, even when conditions are completely devoid of oxygen (Akimoto, Hattori, Uematsu, & Kato, 2001;Pawlowski et al., 2003;Risgaard-Petersen et al., 2006). The resulting propagules can be quiescent for years before starting to grow (Alve & Goldstein, 2010). Through passive suspension transport, these propagules have a remarkable ability to quickly colonize new habitats through opportunistic and pioneering species (Alve & Goldstein, 2003). Overall, versatile solutions to fluctuating ecological conditions are associated with the capacity for alternating genomic contents, resistance, and motility. PACCs seem to be recapitulating this adaptation that has been successful in so many free-living unicellular species that have colonized virtually all places on Earth.

| TARG E TING THE E VOLVAB LE E VOLVAB ILIT Y OF PACC S WILL B E NECE SSARY TO CURE LE THAL C AN CER
We see targeting PACCs as essential to cancer therapy and patient cure. Without the eradication of the resilient PACCs, cancer will recur in treated patients. One strategy to target these critical cells is to turn their capacity for evolvability into a fatal handicap. An evolutionary trap describes a situation in which an organism adopts an adaptive trait in response to an evolutionary environmental pressure that inadvertently makes it vulnerable to another environmental stressor (Basanta, Gatenby, & Anderson, 2012;Gatenby & Brown, 2018;Robertson, Rehage, & Sih, 2013). In essence, the organism is "tricked" into adopting a trait that will soon become extremely maladaptive. Known in evolutionary game theory as an evolutionary double-bind, this strategy can be exploited to treat cancer cells that are otherwise resistant to conventional therapy combinations (Basanta et al., 2012;Gatenby, Zhang, & Brown, 2019;Zhang, Cunningham, Brown, & Gatenby, 2017). Effecting an evolutionary trap requires a two-phased approach, with selection of the first agent or condition to promote a particular targetable adaptive response followed by an agent specifically selected to target the adaptive phenotype (Zhang et al., 2017).
To  Probing the characteristics of the PACCs as ecological strengths may provide novel ways to disrupt them and their role in rendering the metastatic disease lethal and ultimately untreatable.

ACK N OWLED G EM ENTS
The authors dedicate this manuscript to the memory of Donald S.
Coffey, Ph.D., who inspired us all to think out of the box, conduct F I G U R E 2 Using a current standardof-care paradigm, systemic therapies including chemotherapy, radiation, and hormone therapy reduce overall tumor burden, but enrich for PACCs that then eventually give rise to resistant cancer cell population (a). Applying an evolutionary ecology strategy, using standard-of-care systemic therapy to enrich for PACCs and then directly targeting their peculiar vulnerabilities (e.g., requirement for centrosome clustering) or better drug delivery due to decreased tumor bulk opens the door for possible cancer cure in treated patients

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.