Hermann-2018-Pancreatic cancer stem cells_ A s.pdf

Contents lists available at ScienceDirectSeminars in Cancer Biologyjournal homepage: cancer stem cells:A state or an entity?Patrick C.Hermanna,Bruno Sainz Jr.b,c,d,aDepartment of Internal Medicine I,Ulm University,GermanybDepartment of Biochemistry,Universidad Autnoma de Madrid(UAM),Madrid,SpaincDepartment of Cancer Biology,Instituto de Investigaciones Biomdicas“Alberto Sols”(IIBM),CSIC-UAM,Madrid,SpaindChronic Diseases and Cancer,Area 3-Instituto Ramn y Cajal de Investigacin Sanitaria(IRYCIS),Madrid,SpainA R T I C L E I N F OKeywords:Pancreatic ductal adenocarcinomaCancer stem cellsPlasticityTumor microenvironmentCancer stem cell nicheCancer stem cell targeted therapiesA B S T R A C TPancreatic ductal adenocarcinoma(PDAC),the most common type of pancreatic cancer,has a median overallsurvival of 612 months and a 5-year survival of less than 7%.While PDAC currently represents the 4th mostfrequent cause of death due to cancer worldwide,it is expected to become the second leading cause of cancer-related death by 2030.These alarming statistics are primarily due to both the inherent chemoresistant andmetastatic nature of this tumor,and the existence of a subpopulation of highly plastic“stem”-like cells within thetumor,known as cancer stem cells(CSCs).Since their discovery in PDAC in 2007,we have come to realize thatpancreatic CSCs have unique metabolic,autophagic,invasive,and chemoresistance properties that allow them tocontinuously self-renew and escape chemo-therapeutic elimination.More importantly,the concept of the CSC asa fixed entity within the tumor has also evolved,and current data suggest that CSCs are states rather thandefined entities.Consequently,current treatments for the majority of PDAC patients are not effective,and do notsignificantly impact overall patient survival,as they do not adequately target the plastic CSC sub-population northe transient/hybrid cells that can replenish the CSC pool.Thus,it is necessary that we improve our under-standing of the characteristics and signals that maintain and drive the pancreatic CSC population in order todevelop new therapies to target these cells.Herein,we will provide the latest updates and knowledge on theinherent characteristics of pancreatic CSCs and the CSC niche,specifically the cross-talk that exists between CSCsand niche resident cells.Lastly,we will address the question of whether a CSC is a state or an entity and discusshow the answer to this question can impact treatment approaches.1.Pancreatic CancerWhen it comes to cancer,the past decade has seen great progress inthe diagnosis and treatment of many tumor entities;however,the samecannot be said for pancreatic ductal adenocarcinoma(PDAC).Apartfrom being one of the most aggressive tumors,PDAC is currently the 4thmost frequent cause of cancer-related death,and due to its high ag-gressiveness,pronounced resistance to therapy and early metastaticspread,all of which are hallmarks of pancreatic cancer,PDAC is pro-jected to become the second most lethal tumor by the year 2030 1.Less than 20%of PDAC patients present with localized(and thus po-tentially resectable and curable)disease at the time of diagnosis.In1520%of the patients,the tumor is already locally advanced(irre-sectable),and the remaining patients already present with metastaticpancreatic cancer 24.Pancreatic cancer is more common in the el-derly and occurs at similar frequency in females and males.The overall5-year survival rate is dismal at20 packs/year)10,and we have shown that nicotineexposure accelerates tumor formation in the context of KRAS mutations11.Indeed,certain genetic alterations and genetic syndromes havebeen closely associated with pancreatic cancer development.Most im-portantly,up to 90%of PDAC tumors harbor activating mutations inthe KRAS oncogene 12,13 and mutations or deletions in p16(MTS1)14 or Smad4(DPC4)15.Almost as frequent are inactivating mu-tations in p53 13,or the overexpression of receptor tyrosine kinasessuch as EGFR,FGFR,or IGF-1 1618.These receptor kinases can,together with their ligands,influence not only tumor cell growth andinvasion,but also metastatic activity(and thus survival)in PDAC.In-terestingly,a sequence of genetic events comparable to the adenoma-https:/doi.org/10.1016/j.semcancer.2018.08.007Received 5 June 2018;Received in revised form 14 August 2018;Accepted 17 August 2018Corresponding author at:UAM,Calle del Arzobispo Morcillo 4,Madrid,28029,Spain.E-mail address:bruno.sainzuam.es(B.Sainz).Seminars in Cancer Biology 53(2018)223231Available online 18 August 20181044-579X/2018 Elsevier Ltd.All rights reserved.Tcarcinoma progression model proposed for colorectal cancer by Fearonand Vogelstein 19 seems to be applicable to pancreatic cancer:a se-quence of increasingly dangerous precursor lesions(pancreatic in-traepithelial neoplasia,PanIN)occurs with the accumulation of muta-tions such as KRAS,inactivation of CDKN2A and,finally of TP53 andSMAD4 20.Furthermore,the differentiation(or lack thereof)ofcancer cells within pancreatic tumors has been strongly linked to thekey properties of pancreatic cancer in the context of the cancer stem cell(CSC)model of PDAC 21,22,proposing that a specific subpopulationof undifferentiated CSCs mediates not only tumorigenicity,but alsotherapy resistance and metastasis 21.The CSC concept has been ex-tensively reviewed and we refer the reader to the following recentpublications 23 and 24.In this review,however,we will tackle aless explored concept related to CSCs,which is the idea that CSCs arenot a hardwired defined entity but rather represent a plastic state.2.Cancer Stem Cells-hardwired defined entity or a plastic stateOur understanding of cellular plasticity has been based largely onstudies of adult stem cells,and a number of definitions have beenproposed.In general,cellular plasticity can be defined as the ability of acell to move up and down the differentiation lineage or hierarchy.Likewise,plasticity can also refer to a cells phenotypic potential,thatis,the potential of a cell to adopt a new identity or fate in response todifferent conditions or environmental cues.While fundamentally be-lieved to be a trait exclusive of stem cells,we now appreciate thatdaughter cells,progenitor cells,transient cells and even“committed”differentiated cells are plastic and can undergo phenotypic transitionsand/or move throughout the hierarchal continuum.Thus,non-stemcells can reenter the linear and hierarchical process of cellular differ-entiation when necessary,challenging the idea of a fixed unidirectionalhierarchal structure with the stem cell at the top.Using the liver as anexample,it has been shown that terminally differentiated hepatocytesare plastic and upon liver damage can give rise to hepatocytes withoutactually becoming full-fledged stem cells 25.Similar plasticity in non-stem cells has been observed in the mouse intestine.Numerous studieshave shown that“committed”differentiated cells of the secretory orenterocyte lineage can replace leucine-rich repeat-containing hetero-trimeric guanine nucleotide-binding protein-coupled receptor 5 posi-tive(Lgr5+)intestinal stem cells upon their elimination.Thus,daughter cells and even fully differentiated cells can reenter the nicheto replace stem cells when lost,a process known as neutral competition,where stem cell progenies(daughter cells)respond to extrinsic sig-naling factors and compete to occupy the niche 23,2628.With re-spect to the exocrine pancreas,it is uncertain whether neutral compe-tition occurs as a defined stem cell compartment has not beenconclusively identified;however,acinar cells alone are extremelyplastic and can change their lineage fate by undergoing epithelial-to-epithelial transition or EMT and transdifferentiate into ductal or en-docrine cells and even adipocytes in response to appropriate stimuli29,30.Therefore,the existence of cells,including“committed”dif-ferentiated cells,with differing degrees of plasticity may be morecommon than previously believed.In the context of cancer,plasticity has been largely and exclusivelyassociated only to CSCs 31 as it was generally accepted that limitedplasticity existed across non-CSCs.In fact,prior to 2017,it was widelyaccepted that there existed a fixed unidirectional hierarchal structurewithin solid tumors,and at the apex of this hierarchy was a definedsubpopulation of rare“hardwired”stem-like cells with unlimited self-renewal capacity and multi/pluripotent potential.By definition,thesecells are the sole source of tumor initiation,metastasis and cellularheterogeneity,giving rise to intermediate progenitors and terminally-differentiated bulk tumor cells.Likewise,only these cells can undergophenotypic transitions to adapt to stimuli,microenvironmental cues orchemotherapeutic challenge.Evidence to support this unidirectionalhierarchal model first came from intratumor heterogeneity studiesperformed in the late 1950s and early 1960s showing that not all cellsfrom murine cancers had the ability to form de novo tumors followingtransplantation into syngeneic hosts.With the advent of fluorescence-activatedcellsortingtechniquesandtheavailabilityofim-munocompromised mice,Lapidot et al.32 formally showed in 1994that Acute Myeloid Leukemia(AML)is composed of multiple cell types,with CD34+/CD38-cells representing the cells with exclusive tu-morigenic potential.Since then,and for the past 20 years,hundreds ofpapers have been published providing evidence to support the CSCmodel across different tumor types and entities using both mouse andhuman systems.It is important to highlight that the majority of thesestudies have relied on extreme limiting dilution CSC-transplantationassays as evidence for the existence of CSCs in cultured cells,resectedtumors or patient-derived xenografts 21,22,3241.This approachconsists in isolating tumor cells based on the expression of cell surfacemarkers or other functional properties and injecting them into im-munocompromised mice to determine tumor efficiency as a function ofthe number of cells injected.While such transplantation approacheshave supported the existence of CSCs,these assays are not without theirlimitations.For example,the simple transplantation of human cells intoimmunocompromised mice is a selection process,and it is to be ex-pected that human tumor cells of different tumor types and from dif-ferent locations within the same tumor can adapt differently to“growthin a foreign(mouse)milieu”,as stated and shown by Kelly et al.as earlyas 2007 42.In addition,the expression of cell surface markers canvary depending on the source from which the CSCs are isolated(pri-mary tumor versus patient-derived xenograft(PDX)versus primary cellculture)or the tissue digestion or cell suspension protocols used.Likewise,the expression of most markers is neither exclusively norreproducibly linked to a functional CSC phenotype across or evenwithin similar tumor types 39.Indeed,the number of surface markersused and the use of different markers and isolation methodologies hascreated conflicting data in some settings 43,emphasizing the lim-itations associated with isolating pure CSC populations.Fortunately,genetic-based approaches,such as lineage tracing ex-periments,have complemented extreme limiting dilution CSC-trans-plantation assays to support the concept that CSCs are exclusively re-sponsible for tumor formation and tumor heterogeneity.For example,Driessens et al.performed clonal analysis of squamous skin tumorsusing genetic lineage tracing to show that injected cells were nothomogenous,but rather consisted of a large fraction of cells with lim-ited long-term proliferative potential and a smaller fraction of cells withpersistent long-term proliferation and tumor forming capacity 44.Inan elegant study published by Schepers et al.in 2012,the multicolorCre-reporter R26R-Confetti was used to lineage trace Lgr5+cells in amouse model with conditional deletion of the tumor-suppressor geneApc.Using this system,the authors demonstrated that the rare Lgr5+cells(representing about 5 to 10%of the cells in the adenomas)are thedrivers of primary intestinal adenomas and have the capacity to giverise to other Lgr5+cells as well as to all of the other cells present withthe adenomas 45.In a PDAC mouse model,Maddipati and Stangerused multi-color lineage tracing technology to follow the cellular dy-namics of metastasis in vivo 46.While the goal of their study was notto track pancreatic CSCs,they examined the heterogeneity of primarytumors from the first initial stages of tumor development to metastasisdevelopment.Interestingly,the authors showed that not all cells couldsuccessfully extravasate and proliferate at secondary sites.Colonizationof secondary organs was limited to a small subset of cells,and whilemetastatic lesions would begin as polyclonal,they would rapidly losetheir polyclonality and become dominated by a single clonal popula-tion.The conclusion of this study supports the concept of a CSC-like cellalso driving metastatic tumor formation in secondary organs in PDAC.Similar lineage tracing experiments in human cancers have not beenas common due to the complex genetic modifications required for suchexperiments.Nonetheless,with the use of lentiviral infection systems totag human cells,a number of studies have been able to analyze andP.C.Hermann,B.SainzSeminars in Cancer Biology 53(2018)223231224track stem cell hierarchies in human cancers.For example,Dieter et al.used a linear amplification-mediated PCR-based approach for mole-cular tracking of sphere-derived colon cancer tumor initiating cells(TICs)in vivo 47.Using this strategy,the authors identified threetypes of TICs following serial transplantation of cells from primary totertiary mice:extensively self-renewing long-term TICs(LT-TICs),tumor transient amplifying cells(T-TACs)and rare delayed contributingTICs(DC-TICs).Of these three TICs,LT-TICs possessed long-term self-renewing capacity and were responsible for metastasis formation,andDC-TICs represented a silent subpopulation of cells that did not con-tribute quantitatively to tumor formation in primary transplanted micebut could be recruited to the tumor and were active during secondaryand tertiary transplantation.T-TACs,on the other hand,had limitedself-renewal capacity and did not contribute to long-term tumor for-mation.Thus,similar to the mouse lineage tracing experiments detailedabove,tumor formation in human colon cancer was shown to bemediated by a subpopulation of distinct cells with self-renewing capa-city.A recent study by Cortina et al.utilized CRISPR/Cas9 technologyto integrate a lineage-tracing cassette in the LGR5 locus in human CRCorganoids 48.Using this approach,the authors also show thatLGR5+CRC cells self-renew and generate progeny over long periods oftime,and CRC adopts a hierarchical organization reminiscent of normalcolonic epithelium.Taken together,the aforementioned studies provided direct func-tional evidence for the existence of CSCs within primary tu