3D chromatin architecture and epigenetic regulation in cancer stem cells.__3D chromatin architecture and epigenetic regulation in cancer stem cells..pdf

REVIEW3D chromatin architecture and epigeneticregulation in cancer stem cellsYuliang Feng1,Xingguo Liu2,3,Siim Pauklin1&1Botnar Research Centre,Nuffield Department of Orthopaedics,Rheumatology and Musculoskeletal Sciences Old Road,University of Oxford,Oxford OX3 7LD,UK2Guangzhou Regenerative Medicine and Health Guangdong Laboratory,CAS Key Laboratory of Regenerative Biology,JointSchool of Life Sciences,Hefei Institute of Stem Cell and Regenerative Medicine,Guangzhou Institutes of Biomedicine andHealth,Chinese Academy of Sciences;Guangzhou Medical University,Guangzhou 510530,China3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine,Institute for Stem Cell and Regeneration,Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences,Guangzhou 510530,China&Correspondence:siim.pauklinndorms.ox.ac.uk(S.Pauklin)Received August 7,2020Accepted December 5,2020ABSTRACTDedifferentiation of cell identity to a progenitor-like orstem cell-like state with increased cellular plasticity isfrequently observed in cancer formation.During thisprocess,a subpopulation of cells in tumours acquires astem cell-like state partially resembling to naturallyoccurring pluripotent stem cells that are temporarilypresent during early embryogenesis.Such characteris-tics allow these cancer stem cells(CSCs)to give rise tothe whole tumour with its entire cellular heterogeneityand thereby support metastases formation while beingresistant to current cancer therapeutics.Cancer devel-opment and progression are demarcated by transcrip-tional dysregulation.In this article,we explore theepigenetic mechanisms shaping gene expression dur-ing tumorigenesis and cancer stem cell formation,withan emphasis on 3D chromatin architecture.Comparingthe pluripotent stem cell state and epigenetic repro-gramming to dedifferentiation in cellular transformationprovides intriguing insight to chromatin dynamics.Wesuggest that the 3D chromatin architecture could beused as a target for re-sensitizing cancer stem cells totherapeutics.KEYWORDSchromatin architecture,3D chromatintopology,epigenetics,tumorigenesis,cancer stem cells,pluripotent stem cellsINTRODUCTIONThe development of many cancers involves a dedifferentia-tion of cellular identity with the acquisition of a stem cell-likestate in a subpopulation of cancer cells.The arising cancerstem cells(CSCs)are exceptionally important because theirdevelopmental plasticity allows them to resist conventionaltherapies,metastasize and give rise to new tumours.Thechanges in cell identity are caused by transcriptional dys-regulation which is a universal feature of tumorigenesis andimpacts all cancer hallmarks(Hanahan and Weinberg,2011).It is increasingly evident that spatiotemporal changesin 3D chromatin architecture have a central function in gov-erning gene transcription and thereby cancer developmentand cellular heterogeneity.This review provides an overview of the roles of 3Dchromatin architecture in cancer development and progres-sion with an emphasis on the processes that regulate thephenotypic plasticity of cancer stem cells.We argue thatearly embryonic development and cancer cell dedifferentia-tion share similar principles in epigenetic regulation and thedynamic changes in 3D chromatin architecture,while rep-resenting the opposite direction of the developmental pro-cesses.Therefore,3D chromatin architecture of embryonicstem cells and early lineage specification provide uniqueinsight to the stem cell-like properties of cancer stem cellsand intra-tumoural heterogeneity.To strengthen this argu-ment,we draw parallels also with induced pluripotent stemcell(iPSC)generation via reprogramming of 3D chromatinarchitecture in differentiated cells.In addition,we compare The Author(s)2021Protein Cell 2021,12(6):440454https:/doi.org/10.1007/s13238-020-00819-2Protein&CellProtein&Cellthe general features of 3D chromatin architecture in pheno-typic plasticity during normal development as well astumorigenesis,which plays an important role in metastaticspread,therapeutic resistance and tumour relapse.Lastly,we propose therapeutic strategies targeting the 3D chro-matin architecture in the maintenance of CSCs stemness forthe intervention of cancer progression.THE HIERARCHY OF 3D CHROMATINARCHITECTURE IN MAMMALIAN CELLSEnhancers are key cis-regulatory elements that mediatetranscription regulation by converging signals from onco-genic and developmental pathways(Hnisz et al.,2015).Theclustering of many enhancers on the same DNA moleculethat span tens of kilobases has been termed as super-en-hancer(Hnisz et al.,2013,Loven et al.,2013).These largeregulatory clusters integrate signals from multiple cell fatepathways,and provide strength and robustness to cell-specific gene transcription(Loven et al.,2013;Hnisz et al.,2015)via tissue-specific transcription factors binding(Spitzand Furlong,2012).Only approximately 7%of enhancers inhuman cells have been estimated to control their closestpromoters while other enhancers can bypass their closestgenes and regulate the target promoters through long-rangephysical communications and thereby impact cellular plas-ticity and differentiation propensity.In most cases,enhan-cers interact with their target gene promoters within the samechromosome.However,increasing evidence suggests theexistence of interchromosomal enhancer-promoter interac-tions(Maass et al.,2019).For example,a recent reportidentified that specific super-enhancers from different chro-mosomes come into proximity and regulate the transcriptionessential for identity of olfactory neurons(Monahan et al.,2019).Mechanistically,enhancer-promotercontactregulatesgene expression via increasing transcriptional burstingfraction(more transcriptional events per time frame)but notbursting size(more RNA molecules per transcriptionalevent)(Bartman et al.,2016).It should be noted thatenhancer-promoter contact may not be necessary for activetranscriptionasseenfromarecentreportshowingdecreased enhancer-promoter proximity in Sonic hedgehog(Shh)gene activation(Benabdallah et al.,2019),implyingthat other mechanisms,such as phase separation may beinvolved in transcription regulation(Misteli,2020).Loop extrusion model proposed that topological struc-tures called insulated neighbourhoods can frame and facili-tate enhancer-promoter interactions.In this model,cohesin-containing extrusion complex loads onto the DNA andextrudes the DNA loop until blocked by convergently ori-entedCTCFmolecules.Thisloopextrusionprocessdepends on the hydrolysis of ATP by cohesins ATPaseactivity(Vian et al.,2018;Davidson et al.,2019;Kim et al.,2019).InhibitionofATPproductionbyoligomycindramatically disrupted establishment of RAD21(a cohesinsubunit)-associated chromatin loops.In addition,encyclo-pedia of DNA elements(ENCODE)consortium generatedRAD21-associated chromatin contact maps by ChIA-PET(chromatin interaction analysis by paired-end tag sequenc-ing)in 24 human cell lines and identified that 28%of allRAD21 loops are cell-type specific and overlapped with cell-type specific enhancers marked by H3K27ac(Grubert et al.,2020),suggesting that specific enhancer-promoter contactsmay be orchestrated by cell-specific insulated neighbour-hoods.In addition,insulated neighbourhoods can furtheraggregate into topologically associating domains(TADs),whose boundaries are demarcated by CTCF binding(Dowenet al.,2014;Hnisz et al.,2016).TADs are clustered intolarger domains called A compartments(expression active)and B compartments(expression inactive)(Lieberman-Ai-den et al.,2009).However,degradation of CTCF by auxinsystem only led to the loss of TADs but compartmentstructures remained largely unchanged,suggesting that theprinciples governing compartment organization are inde-pendent from TADs(Nora et al.,2017).A recent study foundthat compartment shift is correlated with suppression of thestemness program and tumor progression,and related toDNA hypomethylation(Johnstone et al.,2020).Whethermodulation of DNA methylation state can causally alter thecompartment switch should be further examined in thefuture.Based on cell population Hi-C data,previous studies havedemonstrated that TADs remain largely unchanged duringcellular specification(Dixon et al.,2012;Nora et al.,2012).However,by single-cell Hi-C approach,a recent studyshowed that TADs structures vary substantially at single-celllevel(Stevens et al.,2017)and CTCF prevents the interac-tions of inter-TADs(Szabo et al.,2020).Furthermore,bycombining single-cell Hi-C and high-resolution microscopy,the authors revealed that TADs can be subdivided intochromatin nanodomains(CND),which depend on nucleo-some-nucleosome interactions but are depleted of CTCF orcohesin(Szabo et al.,2020).In trichostatin A(an inhibitor ofhistone deacetylases)treated cells,TADs remain unchan-ged but CND organization is largely disrupted,suggestingCND organization relies on the histone acetylation state(Szabo et al.,2020).DEDIFFERENTIATION IN CANCER AND EPIGENETICREPROGRAMMINGCancer stem cells or tumour initiating cells exhibit stem cell-likefeaturessuchasself-renewalanddifferentiationcapacity.CSCs are also resistant to anoikis,a form of pro-grammed cell death when the cells are detached from thesurroundingextracellularmatrix(ECM).Therefore,thetumorigenic process with dedifferentiation of tissue cells to astem cell-like or progenitor state seems to be the oppositeprocess to normal development during early embryogenesis The Author(s)2021441Protein&Cell3D epigenome in cancer stem cellsREVIEWand organogenesis(Fig.1).Dedifferentiation has a similardevelopmentally reversed direction as the generation ofinducible pluripotent stem cells by epigenetic reprogram-ming.During this reprogramming,exogenous expression ofpluripotency factors induces extensive epigenetic remod-elling that leads to the activation of an endogenous genecircuitry that maintains the pluripotent state of cells(Pappand Plath,2013).The resemblance of these two processesdedifferentiation and epigenetic reprogrammingis high-lighted by the effects of tumour suppressors such as p53 andcyclin dependent kinase inhibitors(e.g.,p16)in blockingepigenetic reprogramming,while their inactivation increasesthe epigenetic reprogramming efficiency.p53 is also themost frequently mutated gene across all tumours.Forinstance,in pancreatic ductal adenocarcinoma it is,togetherwith p16 loss of function,a hallmark mutation.There is a further link between reprogramming andoncogenic transformation.Transient expression of repro-gramming factors in vivo in mouse results in tumour devel-opment in various tissues consisting of undifferentiateddysplasticcellsexhibitingglobalchangesinDNAmethylation patterns.This indicates epigenetic regulationassociated with reprogramming in the absence of irre-versible genetic transformations may drive development ofparticular types of cancer(Ohnishi et al.,2014).Further-more,transientexpressionofreprogrammingfactorsin Kras mutant mice is sufficient to induce the robust andpersistent activation of ERK signaling in acinar cells andrapid formation of pancreatic ductal adenocarcinoma(Shi-bata et al.,2018),indicating that reprogramming factorspromote oncogenic transformation if the anti-tumorigenicbarriers have been removed.REGULATION OF CSC PLASTICITY BY CHROMATINTOPOLOGYThe presence of developmentally plastic cell states with self-renewal capacity has been found in many tumour types(Friedmann-Morvinski et al.,2012;Medema,2013;Fried-mann-Morvinski and Verma,2014).These stem cell-likecancer cells,CSCs,make up only a small fraction of thewhole cancer,but they have the potential to initiate stochasticFigure 1.Formation of cancer stem cells and epigenetic reprogramming.Schematic depiction of cell state transitions duringearly development and tumorigenesis indicate dynamic changes according to cell types.Pluripotent stem cells are able todifferentiate to all cell types and lead to fully differentiated cells in adult tissues.Epigenetic reprogramming by expression of variousstem cell factors(e.g.,Oct4,Sox2,KLF4,Myc,Nanog,Lin28)leads to the erasure of the epigenetic barriers and generation ofinduced pluripotent stem cells.Oncogenic transformation has the opposite direction to normal cell differentiation and the epigeneticchanges,expression of stem cell factors and genetic mutations,can facilitate tumorigenesis by lowering the barriers that usuallywould prevent tumorigenesis and cell state changes.Tumorigenesis involves differentiation of the cell state to a dysregulated stemcell-like identity known as cancer stem cells.These cells are developmentally plastic and can self-renew but also differentiate to othercancer cells.REVIEWYuliang Feng et al.442 The Author(s)2021Protein&Cellmaturation processes and transitions between differentiatedcellular phenotypes.Regardless of the initial differentiationstatus,cancer cells can re-establish the heterogeneous cellmix when cultured individually(Gupta et al.,2011).As inESCs,pluripotency factors such as NANOG,OCT4 andSOX2 block cellular differentiation and maturation whenexpressed in progenitor cells.The unscheduled expression ofpluripotency factors can predispose to and drive cancerdevelopment.Many genes involved in regulating stem cellfunctions and stem cell signalling pathways are dysregulatedin cancer cells and therefore promote dedifferentiation withthe emergence of cancer cells with stem cell-like character-istics.Similar to their function in embryonic stem cells,thesegenes act at all stages of tumorigenesis by preventing dif-ferentiation and eroding barriers against dedifferentiation.Forinstance,Oct4 maintains the pluripotent state of embryonicstem cells during preimplantation development,but its acti-vation results in dysplastic growths in epithelial tissues withthe expansion of progenitor cells and inhibition of differentia-tion(Hochedlinger et al.,2005)while blocking Oct4 expres-sion leads to apoptosis of CSC populations in human andmurine cancer cell lines(Hu et al.,2008).In human lungcancer cells,SOX2 can regulate the transcriptional network ofoncogenes and affect lung tumorigenesis(Chen et al.,2012).OCT4 and NANOG enhance malignancy in lung adenocar-cinoma by inducing cancer stem cell-like properties andepithelial-mesenchymaltransdifferentiation(Chiouetal.,2010),and increased metastasis in breast cancer(Lu et al.,2014).Furthermore,inducible expression of SOX2,OCT4and KLF4 in melanoma cells leads to partial reprogrammingof these cancer cells which start exhibiting increased invasionpotentialand lung colonization(Knappe etal.,2016).NANOG-positive cells also exhibit enhanced ability of self-renewal,clonogenicity,and initiation of tumors,which areconsistent with crucial hallmarks in the definition of CSCs(Shan et al.,2012)and its increased expression in cancercells is correlated with a worse clinical outcome in hepato-cellular carcinoma(HCC)(Shan et al.,2012).It is hypothe-sized that OCT4,NANOG,KLF4 and SOX2 form extensivefeed-forward and feedback loops to organize