Epigenetics and Cancer Stem Cells: Unleashing, Hijacking, and Restricting Cellular Plasticity
Authors: Elanor N. Wainwright, Paola Scaffidi
Journal: Trends in cancer (2017)

Epigenetic regulators are one of the most commonly mutated classes of genes in cancer. During cancer initiation, mutated epigenetic regulators lead to oncogenic cellular reprogramming and promote the acquisition of uncontrolled self-renewal. The emergence of CSCs requires elaborate reorganization of the epigenome. During cancer growth, epigenetic mechanisms integrate the effect of cell-intrinsic (i.e., subclonal mutations) and cell-extrinsic (i.e., signaling from the microenvironment) changes and establish intratumoral heterogeneity, either promoting or inhibiting the CSC state. 'Loose' epigenetic constraints in cancer cells enhance cellular plasticity and allow reversible transitions between different phenotypic states. Enhanced cellular plasticity favors cancer cell adaptability and resistance to therapy. Modulation of epigenetic processes allows targeting of the most downstream determinants of the CSC state. Epigenetic mechanisms have emerged as key players in cancer development which affect cellular states at multiple stages of the disease. During carcinogenesis, alterations in chromatin and DNA methylation resulting from genetic lesions unleash cellular plasticity and favor oncogenic cellular reprogramming. At later stages, during cancer growth and progression, additional epigenetic changes triggered by interaction with the microenvironment modulate cancer cell phenotypes and properties, and shape tumor architecture. We review here recent advances highlighting the interplay between epigenetics, genetics, and cell-to-cell signaling in cancer, with particular emphasis on mechanisms relevant for cancer stem cell formation (CSC) and function. Epigenetic mechanisms have emerged as key players in cancer development which affect cellular states at multiple stages of the disease. During carcinogenesis, alterations in chromatin and DNA methylation resulting from genetic lesions unleash cellular plasticity and favor oncogenic cellular reprogramming. At later stages, during cancer growth and progression, additional epigenetic changes triggered by interaction with the microenvironment modulate cancer cell phenotypes and properties, and shape tumor architecture. We review here recent advances highlighting the interplay between epigenetics, genetics, and cell-to-cell signaling in cancer, with particular emphasis on mechanisms relevant for cancer stem cell formation (CSC) and function. The molecular makeup of a cancer is the result of multiple changes occurring progressively during its lifetime. Early in the disease, alterations in key genes disrupt normal cell function and endow cells with the ability to initiate a tumor or a hematological malignancy. Subsequently, as a cancer grows, additional changes superimpose onto the initiating events and affect the biological properties of cells, either enhancing or inhibiting their malignant properties. As a result of this constant modulation of cell function, tumors comprise a remarkable collection of distinct cellular phenotypes which differentially contribute to disease progression. Intratumoral functional heterogeneity (see Glossary) has , in part, a genetic basis. Sequencing studies have identified the presence of both clonal mutations, which represent early initiating events, and subclonal mutations that occur at later stages of cancer growth and only affect subsets of cancer cells [1Burrell R.A. et al.The causes and consequences of genetic heterogeneity in cancer evolution.Nature. 2013; 501: 338-345Crossref PubMed Scopus (630) Google Scholar]. While in some cancers distinct subclones coexist and collectively drive tumor growth [2Sottoriva A. et al.A big bang model of human colorectal tumor growth.Nat. Genet. 2015; 47: 209-216Crossref PubMed Scopus (198) Google Scholar], in others subclonal mutations confer a selective advantage and contribute to defining which cells will sustain the disease. Subclonal mutations can also be deleterious and counteract the effects of initiating mutations, resulting in loss of malignant properties in some cells [1Burrell R.A. et al.The causes and consequences of genetic heterogeneity in cancer evolution.Nature. 2013; 501: 338-345Crossref PubMed Scopus (630) Google Scholar]. Similarly, epigenetic mechanisms involving DNA methylation and chromatin play a diverse role in cancer by promoting, sustaining, enhancing, or inhibiting malignant phenotypes at various stages of the disease. Research over the past decade has revealed that both cell-intrinsic (i.e., mutations) and cell-extrinsic (i.e., environmental cues) mechanisms modulate the epigenome of cancer cells, and their combined effect determines which cells preserve the self-renewal capacity acquired during tumorigenesis. These cells, referred to as cancer stem cells (CSCs) or leukemic stem cells (LSCs), are those responsible for driving long-term cancer growth and disease progression [3Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar]. CSCs/LSCs evolve over time as a consequence of genetic heterogeneity, that generates self-renewing subclones with diverse fitness, and environmental changes that modulate their phenotype [3Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar]. In this review we discuss mechanisms and implications of the emerging role of epigenetics in the formation and function of CSCs/LSCs, focusing on how changes in DNA methylation and chromatin affect cellular plasticity at various stages of the disease. The recent identification of driver mutations affecting a wide range of epigenetic regulators in hierarchically organized cancers provides direct evidence for the importance of epigenetic dysregulation in the formation of CSCs. These mutations are typically clonal and promote the acquisition of uncontrolled self-renewal. Leukemias represent a paradigm of hierarchical cancers maintained by LSCs. Numerous studies have identified mutated epigenetic regulators that favor the acquisition of uncontrolled self-renewal ability and initiate the disease. A prominent example is offered by mixed lineage leukemia (MLL)-associated leukemia , which is characterized by chromosomal rearrangements involving the KMT2A/MLL gene. KMT2A/MLL encodes a histone methyltransferase that orchestrates several essential cellular processes through modification of chromatin, mainly regulating accessibility to enhancer regions [4Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (319) Google Scholar]. Oncogenic MLL fusion proteins created by translocations induce LSC formation in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) [4Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (319) Google Scholar, 5Sanjuan-Pla A. et al.Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia.Blood. 2015; 126: 2676-2685Crossref PubMed Scopus (19) Google Scholar]. Importantly, MLL fusion proteins can initiate the oncogenic process both in hematopoietic stem cells and in short-lived progenitors, suggesting that they are able to reprogram committed cells and actively confer de novo self-renewal capacity [6Cozzio A. et al.Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors.Genes Dev. 2003; 17: 3029-3035Crossref PubMed Scopus (488) Google Scholar, 7Somervaille T.C. et al.Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells.Cell Stem Cell. 2009; 4: 129-140Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 8Krivtsov A.V. et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (854) Google Scholar]. Notably, efficient leukemic reprogramming of myeloid progenitors by MLL chimeras requires the action of a MLL antagonist, the Polycomb group (PcG) protein BMI1, to ensure repression of tumor-suppressor genes that would otherwise counteract the effect of the oncogenic fusions [9Yuan J. et al.Bmi1 is essential for leukemic reprogramming of myeloid progenitor cells.Leukemia. 2011; 25: 1335-1343Crossref PubMed Scopus (0) Google Scholar, 10Smith L.L. et al.Functional crosstalk between Bmi1 and MLL/Hoxa9 axis in establishment of normal hematopoietic and leukemic stem cells.Cell Stem Cell. 2011; 8: 649-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar]. 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Recurrent mutations in MLL proteins have also been identified in a variety of solid tumors [14Peifer M. et al.Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer.Nat. Genet. 2012; 44: 1104-1110Crossref PubMed Scopus (471) Google Scholar, 15Parsons D.W. et al.The genetic landscape of the childhood cancer medulloblastoma.Science. 2011; 331: 435-439Crossref PubMed Scopus (462) Google Scholar, 16Fujimoto A. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat. Genet. 2012; 44: 760-764Crossref PubMed Scopus (399) Google Scholar, 17Gui Y. et al.Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder.Nat. Genet. 2011; 43: 875-878Crossref PubMed Scopus (395) Google Scholar], pointing to MLL proteins as general cancer drivers. In addition to histone modifiers, mutated structural proteins regulating the higher-order structure of chromatin, such as cohesins, have been shown to enforce stem cell transcriptional programs and have been implicated in the emergence of LSCs [18Mazumdar C. et al.Leukemia-associated cohesin mutants dominantly enforce stem cell programs and impair human hematopoietic progenitor differentiation.Cell Stem Cell. 2015; 17: 675-688Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. Moreover, inactivating mutations disrupting the function of chromatin-remodeling complexes, which are found at high frequency in various types of cancers, have been linked to aberrant activation of stem cell-related pathways [19Jagani Z. et al.Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog–Gli pathway.Nat. Med. 2010; 16: 1429-1433Crossref PubMed Scopus (0) Google Scholar, 20Wang X. et al.TCR-dependent transformation of mature memory phenotype T cells in mice.J. 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The primary mechanism leading to oncogenesis induced by the K27M mutation is inhibition of the Polycomb repressive complex 2 (PRC2), which results in genome-wide reduction in the repressive histone H3 trimethylated lysine 27 (H3K27me3) mark [24Lewis P.W. et al.Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma.Science. 2013; 340: 857-861Crossref PubMed Scopus (358) Google Scholar] and re-establishment of an earlier developmental program in neural precursor cells and consequent acquisition of oncogenic self-renewal ability [25Funato K. et al.Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation.Science. 2014; 346: 1529-1533Crossref PubMed Scopus (89) Google Scholar]. The identification of a histone protein as a key driver in GBM has particularly important implications because it demonstrates a direct and major role of chromatin in the emergence of CSCs. Interestingly, H3.3 mutations are only found in pediatric GBM, suggesting a different mechanism of CSC formation in adult patients. Nevertheless, chromatin dysregulation is likely to play a crucial role in adult GBM as well, considering that approximately half of adult GBMs harbor mutations in at least one chromatin modifier [26Brennan C.W. et al.The somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar]. Proteins involved in the establishment and maintenance of DNA methylation have also been identified as drivers of CSC formation. The methylation status of CpG dinucleotides depends on the action of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), which apply the methyl-group to cytosines, and methylcytosine dioxygenases (TET1 and TET2), which convert 5-methylcytosine to 5-hydroxymethylcytosine and initiate a demethylation process. DNMT3A is the DNMT most commonly affected by DNA lesions, being mutated in ∼25% of AML patients [27The Cancer Genome Atlas Research Network Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.N. Engl. J. Med. 2013; 368: 2059-2074Crossref PubMed Google Scholar]. Most DNMT3A mutations appear to inhibit the enzyme activity and lead to expansion of pre-LSCs, although the exact mechanism underlying this process is not fully understood and both DNA methylation-dependent and -independent mechanisms have been described [28Lu R. et al.Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene-expression program and acute leukemia development.Cancer Cell. 2016; 30: 92-107Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 29Yang L. et al.DNMT3A loss drives enhancer hypomethylation in FLT3-ITD-associated leukemias.Cancer Cell. 2016; 29: 922-934Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 30Mayle A. et al.Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation.Blood. 2015; 125: 629-638Crossref PubMed Scopus (81) Google Scholar, 31Koya J. et al.DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation.Nat. 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The role of aberrant DNA-methylation patterns in the early stages of tumorigenesis is not limited to leukemia, and mutations in DNMTs and IDHs have been also observed in solid tumors [33Kim M.S. et al.Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers.APMIS. 2013; 121: 85-94Crossref PubMed Scopus (28) Google Scholar, 34Cohen A.L. et al.IDH1 and IDH2 mutations in gliomas.Curr. Neurol. Neurosci. Rep. 2013; 13: 345Crossref PubMed Scopus (82) Google Scholar]. The studies discussed above are only selected examples illustrating how mutations in epigenetic regulators crucially contribute to the formation of the founder CSC in various malignancies. As a group , epigenetic regulators are among the most commonly mutated proteins, both in individual cancer types and in pan-cancer cohorts [35Lawrence M.S. et al.Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Crossref PubMed Scopus (952) Google Scholar, 36Vogelstein B. et al.Cancer genome landscapes.Science. 2013; 339: 1546-1558Crossref PubMed Scopus (2393) Google Scholar]. Some of these genetic lesions lead to oncogenic gain of function such as those generating MLL fusions, while others damage proteins that act as tumor suppressors. Regardless of the type of mutations, a common consequence of such alterations is a global reorganization of the epigenome and consequent disruption of differentiation programs. Mutations can occur in normal stem cells, where they mainly disrupt the balance between self-renewal and differentiation, or in committed cells, where they induce a reprogramming process conferring de novo self-renewal capacity. In both cases, epigenetic constraints imposed during development to keep cellular plasticity under control are disrupted, and cells transform, losing their normal cellular identity (Figure 1). The acquisition of uncontrolled self-renewal is only the first step in the development of a cancer. Transformed cells need to maintain their ability to self-renew while the cancer grows, a task that is evidently not trivial considering that CSCs are often only a small subset of the cancer cell population and that most cells lose self-renewal capacity over time. The presence of at least two functionally distinct subsets of cells (tumorigenic self-renewing CSCs, and non-tumorigenic differentiated cells) in many cancers has been the basic observation supporting the hierarchical model of cancer development over the past two decades. Recent single-cell transcriptomic studies have extended our understanding of the gene expression programs that shape tumor architecture and have confirmed that aberrant differentiation programs establish cellular hierarchies within individual cancers. In a pioneering study, colon cancers were shown to contain multiple cell types with transcriptional profiles resembling those of the cellular lineages making up the normal epithelium. Importantly, single cancer cells could recapitulate the lineage diversity of the primary tumors in transplantation assays, demonstrating that multilineage differentiation represents a key source of intratumoral transcriptional heterogeneity [37Dalerba P. et al.Single-cell dissection of transcriptional heterogeneity in human colon tumors.Nat. Biotechnol. 2011; 29: 1120-1127Crossref PubMed Scopus (307) Google Scholar]. Similar studies in glioblastoma and oligodendroglioma have confirmed this finding, showing that brain tumors contain subpopulations of undifferentiated cells characterized by stem cell and proliferation gene signatures, and subsets of cells that have lost self-renewal potential through neural differentiation [38Patel A.P. et al.Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma.Science. 2014; 344: 1396-1401Crossref PubMed Scopus (634) Google Scholar, 39Tirosh I. et al.Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma.Nature. 2016; 539: 309-313Crossref PubMed Scopus (55) Google Scholar]. The approach used in these studies is powerful because it allows reconstruction of cellular hierarchies from genome-wide expression signatures in primary tumors, avoiding the caveats associated with xenograft assays. However, one limitation of these studies is their descriptive nature, which cannot assign causality to the identified gene signatures in the absence of functional validation. The observation that IDH-induced oligodendrogliomas contain differentiated , non-self-renewing cells underscores the diverse, and at times antithetic, role of epigenetic mechanisms in cancer development. During tumorigenesis, mutations in IDH proteins drive CSC emergence partly through alteration of DNA methylation profiles and epigenetic reprogramming of committed oligodendrocytes. However, during tumor growth, additional epigenetic changes occur and establish developmental hierarchies that restrict the proliferative potential of some cells, counteracting the effect of the initiating mutations (Figure 2). What are the mechanisms that generate distinct epigenetic states within tumors and confer distinct functional properties to CSCs and differentiated cells? Epigenetic regulators belonging to two distinct groups have so far been identified: those that inhibit cancer cell self-renewal and establish differentiation hierarchies, and those that are hijacked by CSCs to avoid differentiation and sustain their phenotype (Table 1). Within the first group, the linker histone H1.0 plays an important role in restricting cancer cells long-term proliferative potential and determines functionally distinct subsets of cells within individual tumors [40Torres C.M. et al.The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity.Science. 2016; 353 (aaf1644)Crossref PubMed Scopus (2) Google Scholar]. In numerous cancer types, H1.0 levels are highly heterogeneous, with low levels in cells expressing CSC markers and high levels in differentiated cells. Functional characterization of the impact of H1.0 expression on cancer cells revealed that only cells able to stably repress H1.0 preserve a chromatin configuration compatible with self-renewal capacity. Re-expression of H1.0 in subsets of cells during tumor growth induces genome-wide silencing of oncogenic and self-renewal genes, and promotes differentiation into non-tumorigenic cells [40Torres C.M. et al.The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity.Science. 2016; 353 (aaf1644)Crossref PubMed Scopus (2) Google Scholar]. Thus, only cells expressing low H1.0 levels can maintain CSC properties. As discussed above for mutations in H3.3, the observation that an integral component of chromatin acts as an important regulator of cancer cell differentiation states highlights the important role of epigenetics in specifying tumor organization and affecting tumor maintenance. Another example supporting this concept comes from MLL-driven AML, in which high levels of the histone demethylase KDM5B revert the histone modification patterns and gene expression programs established by MLL during leukemogenesis, thereby extinguishing LCS potential [41Wong S.H. et al.The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential.Cancer Cell. 2015; 28: 198-209Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. Similarly, in glioma the histone methyltransferase G9a and the related global increase in H3K9me2 inhibit self-renewal of CSCs in vitro [42Tao H. et al.Histone methyltransferase G9a and H3K9 dimethylation inhibit the self-renewal of glioma cancer stem cells.Mol. Cell. Biochem. 2014; 394: 23-30Crossref PubMed Scopus (10) Google Scholar]. Although the presence of cellular hierarchies is recognized in many cancers, our knowledge of how they are established and how cancer cells lose self-renewal capacity during tumor growth is still rudimental. A comprehensive identification of the molecular players that drive cancer cell differentiation is central to understanding, and possibly exploiting, the mechanisms that naturally inhibit tumor maintenance and deprive cancer cells of their malignant properties.Table 1Non-Mutated Epigenetic Regulators Affecting Cancer Cell Self-Renewal and PlasticityaKD, knockdown; KO, knockout, N/A, not available; OE, overexpression. The registered clinical trial identifier (NCT) is indicated (www.clinicaltrials.gov).ProteinCancer typeEvidence for functional roleEffect of inhibitionPreclinical evidence using small moleculesClinical trialRefsProteins inhibiting CSC self-renewalH1.0Breast cancer ,Glioma and glioblastoma ,Melanoma,Kidney renal papillary cell carcinoma ,Liver cancerDifferential expression in CSCs (low levels) and differentiated cells (high levels );KD and OE studies with human cancer cells in graft models ;Patient stratification and prognostic valueIncreased frequency of self-renewing tumor cellsN/AN/A40Torres C.M. et al.The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity.Science. 2016; 353 (aaf1644)Crossref PubMed Scopus (2) Google ScholarKDM5B/JARID1BAcute myeloid leukemiaDifferential H3K4me3 levels in LSCs (high levels) and differentiated cells (low levels );OE and KD studies in vitro and in vivo with mouse and human cells in graft modelsEnhanced LSC self-renewalN/AN/A41Wong S.H. et al.The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential.Cancer Cell. 2015; 28: 198-209Abstract Full Text Full Text PDF PubMed Scopus (28) Google ScholarEHMT2/G9aGliomaLoss of H3K9me2 in CSCs ;OE and pharmacological inhibition studies in vitroEnhanced CSC self-renewalN/AN/A42Tao H. et al.Histone methyltransferase G9a and H3K9 dimethylation inhibit the self-renewal of glioma cancer stem cells.Mol. 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