Joshua M. Nicholson

Is carcinogenesis a form of speciation

Since cancers have individual clonal karyotypes, are immortal and evolve from normal cells treated by carcinogens only after exceedingly long latencies of many months to decades—we deduce that carcinogenesis may be a form of speciation. This theory proposes that carcinogens initiate carcinogenesis by causing aneuploidy, i.e., losses or gains of chromosomes. Aneuploidy destabilizes the karyotype, because it unbalances thousands of collaborating genes including those that synthesize, segregate and repair chromosomes. Driven by this inherent instability aneuploid cells evolve ever-more random karyotypes automatically. Most of these perish, but a very small minority acquires reproductive autonomy—the primary characteristic of cancer cells and species. Selection for autonomy stabilizes new cancer species against the inherent instability of aneuploidy within specific margins of variation. The speciation theory explains five common characteristics of cancers: (1) species-specific autonomy; (2) karyotypic and phenotypic individuality; (3) flexibility by karyotypic variations within stable margins of autonomy; (4) immortality by replacing defective karyotypes from constitutive pools of competent variants or subspecies generated by this flexibility; and (5) long neoplastic latencies by the low probability that random karyotypic alterations generate new autonomous species. Moreover, the theory explains phylogenetic relations between cancers of the same tissue, because carcinogenesis is restricted by tissue-specific transcriptomes. The theory also solves paradoxes of other cancer theories. For example, “aneuploidy” of cancers is now said to be a “paradox” or “cancer’s fatal flaw,” because aneuploidy impairs normal growth and development. But if the “aneuploidies” of cancers are in effect the karyotypes of new species, this paradox is solved.

Cancer-causing karyotypes: chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function

The chromosomes of cancer cells are unstable, because of aneuploidy. Despite chromosomal instability, however, cancer karyotypes are individual and quasi-stable, as is evident especially from clonal chromosome copy numbers and marker chromosomes. This paradox would be resolved if the karyotypes in cancers represent chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function. To test this hypothesis, we analyzed the initial and long-term karyotypes of seven clones of newly transformed human epithelial, mammary, and muscle cells. Approximately 1 in 100,000 such cells generates transformed clones at 2-3 months after introduction of retrovirus-activated cellular genes or the tumor virus SV40. These frequencies are too low for direct transformation, so we postulated that virus-activated genes initiate transformation indirectly, via specific karyotypes. Using multicolor fluorescence in situ hybridization with chromosome-specific DNA probes, we found individual clonal karyotypes that were stable for at least 34 cell generations-within limits, as follows. Depending on the karyotype, average clonal chromosome numbers were stable within +/- 3%, and chromosome-specific copy numbers were stable in 70-100% cells. At any one time, however, relative to clonal means, per-cell chromosome numbers varied +/-18% and chromosome-specific copy numbers varied +/-1 in 0-30% of cells; unstable nonclonal markers were found within karyotype-specific quotas of <1% to 20% of the total chromosome number. For two clones, karyotypic ploidies also varied. With these rates of variation, the karyotypes of transformed clones would randomize in a few generations unless selection occurs. We conclude that individual aneuploid karyotypes initiate and maintain cancers, much like new species. These cancer-causing karyotypes are in flexible equilibrium between destabilizing aneuploidy and stabilizing selection for transforming function. Karyotypes as a whole, rather than specific mutations, explain the individuality, fluidity, and phenotypic complexity of cancers.

The mitotic origin of chromosomal instability

Summary Chromosomal instability (CIN) is a common feature of most human neoplasms and was defined, in a seminal study by Vogelstein and colleagues [1], as persistently elevated rates of whole chromosome mis-segregation. Since then, it was shown that certain errors in mitosis, including defects in the spindle assembly checkpoint [2], sister chromatid cohesion [3], kinetochore-microtubule (kMT) attachments [4,5], and centrosome number [6] can cause chromosome mis-segregation in the form of merotelically attached anaphase lagging chromosomes — chromosomes that lag behind at the spindle equator while all the other chromosomes move toward the spindle poles [7] (Figure 1A). A recent study has suggested that pre-mitotic replication stress generates partially replicated chromosomes during mitosis, and that this results in both numerical and structural chromosome abnormalities through the formation of chromosome bridges and acentric chromosome fragments during anaphase [8]. To determine whether whole chromosome instability in cancer cells is caused by defects originating in mitosis (lagging chromosomes) or from ones originating pre-mitotically (chromatin bridges and acentric fragments), we compared a variety of CIN+ to CIN- cells to determine the types of segregation defects that phenotypically distinguish CIN+ from CIN- cells and whose abrogation can rescue whole chromosomal instability.

Chromosome mis-segregation and cytokinesis failure in trisomic human cells

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome. DOI: http://dx.doi.org/10.7554/eLife.05068.001

How mitotic errors contribute to karyotypic diversity in cancer.

Aneuploidy is a common feature of cancer cells, and is believed to play a critical role in tumorigenesis and cancer progression. Most cancer cells also exhibit high rates of mitotic chromosome mis-segregation, a phenomenon known as chromosomal instability, which leads to high variability of the karyotype. Here, we describe the nature, nuances, and implications of cancer karyotypic diversity. Moreover, we summarize recent studies aimed at identifying the mitotic defects that may be responsible for inducing chromosome mis-segregation in cancer cells. These include kinetochore attachment errors, spindle assembly checkpoint dysfunction, mitotic spindle defects, and other cell division inaccuracies. Finally, we discuss how such mitotic errors generate karyotypic diversity in cancer cells.

Cancer karyotypes: survival of the fittest.

Cancer cells are typically characterized by complex karyotypes including both structural and numerical changes, with aneuploidy being a ubiquitous feature. It is becoming increasingly evident that aneuploidy per se can cause chromosome mis-segregation, which explains the higher rates of chromosome gain/loss observed in aneuploid cancer cells compared to normal diploid cells, a phenotype termed chromosomal instability (CIN). CIN can be caused by various mechanisms and results in extensive karyotypic heterogeneity within a cancer cell population. However, despite such karyotypic heterogeneity, cancer cells also display predominant karyotypic patterns. In this review we discuss the mechanisms of CIN, with particular emphasis on the role of aneuploidy on CIN. Further, we discuss the potential functional role of karyotypic patterns in cancer.

Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.

Selective advantage of trisomic human cells cultured in non-standard conditions.

An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes.

Link between aneuploidy and chromosome instability.

Aneuploidy is widely acknowledged as a leading cause of miscarriage and birth defects in humans, and is generally known to be deleterious to the survival of individual cells. However, aneuploidy is also ubiquitous in cancer and is found to arise as an adaptive response in certain contexts. This dichotomy of aneuploidy has attracted the interest of researchers for over a century, but many studies have reached conflicting conclusions. The emergence of new technology has allowed scientists to revisit the aneuploidy problem and has fueled a number of recent studies aimed at understanding the effects of aneuploidy on cell physiology. Here, we review these studies, in light of previous observations and knowledge, specifically focusing on the effects of aneuploidy on cellular homeostasis, chromosome stability, and adaptation.

Doubling the deck: Tetraploidy induces chromosome shuffling and cancer.

Comment on: Lv L, et al. Cell Cycle 2012; 11:2864-75.