Cancer Hallmarks and Promiscuous Cell State Plasticity

 

In an earlier Cancer Ecology Commentary post from April, Andrew Fineberg and colleagues described how lineage plasticity, the ability of cells to adopt a range of phenotypic states needed for organ development and injury repair was at the same time providing an additional pathway for neoplastic initiation and development. The epigenome, the set of DNA and chromatin modifications controlling coordinated expression of gene networks was highlighted given its role in determining a cell’s phenotypic state. That regulatory control, however, was subject to stochasticity, random variability and uncertainty, an inevitable consequence of information transfer.

Fineberg’s team, relying on increasingly refined laboratory means of measuring the probability of a cell’s transcriptional state at the single cell level took advantage of an inverse relationship from statistical thermodynamics between the probability of being in a particular state and its (quasi-) potential energy. This permitted the investigators to translate a map of probability of a phenotypic state of a tissue into a field of quasi potential energy. Such a map could define an energy minimum, termed an attractor, associated with the greatest likelihood of a tissue of interest being in a particular phenotypic state. An advantage for employing quasi potential energy allows for measurement of the entropy of that epigenetic regulation, that is the level of unpredictability of that control in maintain stable phenotype. The entropy of the cell state is reflected by the dimensions of the attractor in terms of the attractor’s depth and width. Deep, narrow attractors are associated with low entropy while broad and shallow attractors are a representation of high entropy.  This quantitation in turn allows for prediction of the likelihood of occurrence of an unregulated and potentially deleterious cell state transition.

A logical question that follows asks how it is that increasing entropy actually leads to the formation of cancer. Can we name names of the actual epigenetic modifiers through which this process is thought to occur. An earlier publication from William Flavahan and colleagues from the Massachusetts General Hospital (Flavahan et al., Science 357, 266 (2017)) addresses this issue through a description of epigenetic regulatory balance of active and silenced chromatin. These investigators also chose to use a landscape model of development, first advanced by developmental biologist Conrad Waddington, in describing epigenetic genome regulation using a more qualitative approach. Recall the histologic appearance of nuclear chromatin under light microscopy adopting either a closed, condensed configuration termed heterochromatin associated with genetic silencing and contrasting with an open, relaxed pattern of euchromatin indicative of active transcription, Flavahan examines the molecular determinates which maintain either a restrictive or permissive state allowing gene network transcription. Borrowing from Waddington as seen in Figure 1 and analogous to the depth of an attractor described by Fineberg, here the height of the walls of the developmental channels corresponds with an estimate of the level of restriction for alternate cell state transitions. In an increasingly permissive epigenetic environment marked by lowering of the channel walls, the stochastic variability of cell phenotype increases the chances for state transition. In most situations this will be an uneventful development and the transition will have no consequence. On occasion though cells may ‘sample’ a new phenotypic state which endows an increase in cell fitness for a given environment. This chance increase in fitness will lead to selection favoring the expansion of that group of transitioned cells because of the newly acquired phenotypic states.  Some of these adaptations are referred to as Cancer Hallmarks.

Figure 2 provides an illustration of this variable regulation describing a balance between permissive and restrictive histone modifying enzymes and the resulting chromatin state. In health a state of equipoise exists between the activity of restrictive chromatin modifiers, in this case the chromatin modifying histone methylase, EZH2 (Enhancer of Zeste Homolog 2), a member of the Polycomb family of chromatin modifying enzymes which repress chromatin transcription and MLL (Mixed Lineage Leukemia), a histone trimethylase permitting active chromatin transcription from DNA in a relaxed, open configuration. The balance between these two opposing influences will leave promoters in a poised state receptive to normal cellular or environmental cues typical of homeostasis and permitting controlled phenotypic adjustments. This contrasts to an alternate situation, for example in response to a gain-of-function mutation in EZH2, causing an excessively restrictive chromatin pattern, inhibiting cell state transition and leading to maturation arrest, a potential first step in the development of some leukemias. Conversely, in situations where there is upregulation of MLL, a shift in trimethylation of certain lysine residues along with loss of CpG methylation will lead to an active chromatin state conducive for transcription factor binding to DNA, increase in gene network expression and possible resulting phenotypic divergence described by Flavahan as “promiscuous sampling of alternate phenotypic states”.

Figure 3 diagrams categories the types of influences which are known to affect chromatin regulation and the potential downstream consequences on cell biology and tissue homeostasis which those influences may cause. Importantly the potential influences on epigenetic regulation may stem from either cell intrinsic mechanisms such chromatin modifier mutations as well as cell extrinsic influences from aging, the metabolism, and the environment. In an excessively restrictive environment, such as might occur after promotor hypermethylation, or excess Polycomb repressor activity, impairment may occur in normal apoptotic regulation of tissue growth or alternatively close off differentiation pathways which account for cellular maturation ordinarily coupled with ceasing cell proliferation. As discussed above, permissive chromatin states increase the chances for ‘uncovering’ a proto-oncogene or alternate phenotypic state which may have the effect of endowing fitness and thus leading to enhanced selection.

The Hallmarks of Cancer, initially described by Robert Weinberg and Douglas Hanahan in the year 2000 are a set of consistently observed cancer adaptations which characterize the malignant state. In Figure 4 Flavahan provides a catalog of those Hallmarks and the accompanying epigenetic changes which have come to be associated with those pro-neoplastic and fitness conferring abilities. These include proliferative signaling from oncogene activation through loss of normally sequestered DNA as a result of methylation loss, angiogenesis from promotor hypermethylation of VHL, replicative immortality, resistance to cell death from loss of DNA methyl transferase function, evading growth suppression from p16 silencing from promoter methylation, and invasion and metastasis from an epithelial to a mesenchymal state transition, mediated through epigenetic modifications. This last Hallmark is of special interest as the metastatic capabilities of tumors rest on their ability to alter cell differentiation from an epithelial to a mobile, invasive mesenchymal-like state termed Epithelial-Mesenchymal Transition (EMT). The acquisition of metastatic capability has profound consequences for clinical oncology as metastasis is a leading factor in cancer morbidity and mortality. That this crucial adaptation should occur absent findings of genetic mutation associated with EMT strongly implies likelihood of an underlying epigenetic mechanism to account for the transition.

In an upcoming post the importance of understanding cell state transition will be discussed in a recent set of experiments describing histologic transition in lung cancer. Lung cancer is notorious for displaying multiple histologic subtypes over time raising the question as to what influences are determining such change. The post will examine how sensitivity to oncogene-mediated pro-neoplastic influence is highly context dependent, relying on specific transcriptional states characteristic of certain cell lineages to drive cancer development.

Figure Annotations:

Figure 1 Epigenetic plasticity and the hallmarks of cancer.

(Left) Normal chromatin and associated epigenetic mechanisms stabilize gene expression and cellular states while facilitating appropriate responses to developmental or environmental cues (blue nuclei represent normal cell state). Genetic, environmental, and metabolic insults that disrupt chromatin can lead to either restrictive or overly permissive chromatin states. (Center) Overly permissive chromatin results in epigenetic plasticity; this plasticity permits stochastic activation of alternate gene regulatory programs (red nuclei represent cancer-like cell state). (Right) Some stochastic changes will be inconsequential “passengers” while others will confer fitness and be selected as “drivers”; in this way, chromatin aberrations have the potential to fulfill each hallmark of cancer.

 

Figure 2 Chromatin structure affects cellular identity and state transitions.

(A) Chromatin can adopt active and repressive states. Active states are made accessible to transcription factors and other regulatory factors; they are enriched for histone modifications such as acetylation (H3K27ac) and trimethylation (H3K4me3). Repressive states are compact and are characterized by DNA hypermethylation, chromatin repressors, and specific histone methylation marks (H3K27me3, H3K9me3). CTCF and cohesin partition the genome into discrete regulatory units, termed TADs. (B) Chromatin networks reinforce cell states and affect responsiveness to intrinsic and extrinsic cues. Cells with perturbed chromatin networks fail to respond appropriately to such cues. Overly restrictive chromatin accentuates epigenetic barriers that prevent cell state transitions. Overly permissive chromatin lowers epigenetic barriers, allowing promiscuous sampling of alternate cell states. The opposing activities of the H3K27 methyltransferase EZH2 and the H3K4 methyltransferase MLL are given as an example; however, the concept holds for other regulators such as DNMTs and TET enzymes (see text for details). HDAC, histone deacetylase; DNMTs, DNA methyltransferases; CBP, CREB-binding protein; LSD1, lysine-specific histone demethylase 1A (KDM1A); JMJD2C, JmjC domain–containing histone demethylase 2C (KDM4C); SETDB1, SET domain bifurcated 1; SUV39H1, suppressor of variegation 3-9 homolog 1. Far right: Blue nuclei represent normal cell states; red nuclei represent cancer-like states.

 

Figure 3 Chromatin homeostasis is disrupted in cancer.

Chromatin homeostasis may be disrupted by genetic stimuli (e.g., chromatin regulator mutations or regulatory element translocation) or nongenetic stimuli (e.g., aging, inflammation, hypoxia, etc.). Such stimuli can result in an overly permissive or overly restrictive chromatin network. Permissive states may allow stochastic oncogenic epigenetic changes such as silencing of tumor suppressor genes. Adaptive epigenetic changes that are mitotically heritable will be selected (Fig. 3) and may give rise to hallmarks of cancer (Fig. 4).

 

Figure 4 Genetic and epigenetic mechanisms underlie the hallmarks of cancer.

(A) Epigenetic mechanisms involving aberrant chromatin restriction or plasticity can give rise to each classic hallmark of cancer. [Adapted with permission from (11)] (B to D) Genetic and epigenetic mechanisms are important factors in the development of human cancer, but their relative contribution is dependent on tumor type. Three distinct tumors of the central nervous system illustrate this point, with the potential basis for each hallmark shown in red (genetic) or blue (epigenetic). Most hallmarks can be traced to genetic drivers in glioblastoma, a brain tumor that primarily affects adults; epigenetic factors predominate in pediatric tumors such as ependymoma, which exhibits DNA hypermethylation but lacks recurrent mutations (72, 73). Anaplastic astrocytomas may exhibit examples of both genetic and epigenetic lesions, leading to different hallmarks.