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Hindawi Publishing Corporation Journal of Oncology Volume 2011, Article ID 396076, 11 pages doi:10.1155/2011/396076 Review Article Quiescent, Slow-Cycling Stem Cell Populations in Cancer: A Review of the Evidence and Discussion of Signiﬁcance Nathan Moore and Stephen Lyle Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street-LRB 411, Worcester, MA 01605, USA Correspondence should be addressed to Stephen Lyle, firstname.lastname@example.org Received 28 July 2010; Accepted 8 September 2010 Academic Editor: Bo Lu Copyright © 2011 N. Moore and S. Lyle. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Long-lived cancer stem cells (CSCs) with indeﬁnite proliferative potential have been identiﬁed in multiple epithelial cancer types. These cells are likely derived from transformed adult stem cells and are thought to share many characteristics with their parental population, including a quiescent slow-cycling phenotype. Various label-retaining techniques have been used to identify normal slow cycling adult stem cell populations and oﬀer a unique methodology to functionally identify and isolate cancer stem cells. The quiescent nature of CSCs represents an inherent mechanism that at least partially explains chemotherapy resistance and recurrence in posttherapy cancer patients. Isolating and understanding the cell cycle regulatory mechanisms of quiescent cancer cells will be a key component to creation of future therapies that better target CSCs and totally eradicate tumors. Here we review the evidence for quiescent CSC populations and explore potential cell cycle regulators that may serve as future targets for elimination of these cells. 1. Cancer Induction from Adult Stem Cells have been termed cancer stem cells (CSCs), also known as cancer initiating cells, and are deﬁned as the fraction The development of cancer is a complex multistep process of cells within a tumor that are long lived, possess the that requires the accumulation of mutations resulting in a potential to proliferate indeﬁnitely, and can generate all cell acquiring the essential hallmarks of cancer: evasion of heterogeneous lineages of the original tumor in xenograft apoptosis, self-suﬃciency in growth signals, insensitivity to models [6, 8]. CSCs are expected to utilize characteristics antigrowth signals, invasive and metastatic abilities, limitless commonly found in stem cell populations such as diﬀerential replicative potential, and sustained angiogenesis . Given metabolic activity, speciﬁc signaling pathway activity, and that normal adult stem cells already exhibit limitless replica- regulation of cell cycling characteristics, albeit with aberrant regulation [7, 9](Table 1). Importantly, CSCs that survive tive potential, it is hypothesized that transformed stems cells may be the cells of origin for many cancers [2, 3]. In addition treatment could account for tumor recurrence as a result to replicative potential, long-lived stem cells have the oppor- of reactivation of proliferation in surviving CSCs . tunity to accumulate oncogenic mutations over years or Traditional chemotherapy regimens target proliferating cells, decades from common mutagenic sources like inﬂammation, potentially missing slower dividing CSCs that must be radiation, chemicals, or infection, unlike shorter-lived transit eradicated to provide long-term disease-free survival . A amplifying (TA) cells that rapidly proliferate and diﬀerentiate better understanding of CSCs is essential in understanding [4, 5]. Like healthy adult stem cells, transformed stem cells the biological and clinical consequences of existing regimens areexpectedtobeabletogenerateoncogenic TA cells. These and designing new therapies to improve patient outcome . TA cells would be capable of driving tumor formation and Current methods for isolation and study of CSCs rely generating the heterogeneous combination of populations on cell surface markers found to be enriched in populations with stem cell-like properties. This technique was ﬁrst used commonly seen in cancer [6, 7]. Transformed stem cells 2 Journal of Oncology Table 1: Comparison of characteritics between adult and cancer functional implications must be viewed as only tools for stem stem cells. cell enrichment, suggesting the need for a more functionally signiﬁcant means of CSC identiﬁcation . Given the sim- Characteristics Adult Stem Cells Cancer Stem Cells ilarities between normal adult stem cells and CSCs, aberrant Extensive proliferative Extensive proliferative regulation of self-renewal and quiescence is likely central to Replicative capacity with the capacity with the CSC pathology [9, 10]. Targeting pathways that mediate stem Potential potential to exhaust potential to exhaust cell quiescence is therefore an intriguing alternate method for regenerative ability regenerative ability CSC identiﬁcation and use in future therapy. All heterogeneous Diﬀerentiation All lineages of the The primary objectives of this paper are to place quies- lineages within the Ability speciﬁc tissue cent label-retaining studies in the context of what is currently original tumor known about adult stem cells and then review the existing Metabolic Activity Low Unknown evidence for quiescence in cancer stem cells. We will examine Aberrant regulation current evidence for the role of quiescence in CSC resistance Hedghog, Wnt, of Hedghog, Wnt, Signaling Pathway to conventional cancer therapy and recurrence. Finally, we Notch, and BMP Notch, BMP, and will explore current knowledge of quiescence regulation and others how these studies might be considered when developing Cell Cycling Slow cycling, tightly Potentially slow CSC future experiments to develop targeted therapies against Regulation controlled cycling, unknown CSCs. Niche: Compartmentalized Location Unknown or associated with 2. Adult Stem Cells and Quiescence stromal layer Adult stem cells are critical for continued normal tissue Adhesion Tightly Adhesive Unknown homeostasis and response to wounding for many of the Epithelial to Migration epithelial tissues of the body. Adult stem cells are character- No/Slow Migration Mesenchymal Potential ized by their ability to self-renew indeﬁnitely and produce Characteristics progeny capable of diﬀerentiating and repopulating tissue speciﬁc lineages . Populations of adult stem cells have been identiﬁed in tissues throughout the body, including the by Bonnet and Dick in 1997 when they demonstrated that skin [23–25], mammary glands [26, 27], intestine [28, 29], + − only the CD34 CD38 subset of cells were capable of ini- prostate , brain , and the hematopoietic system tiating human acute myeloid leukemia (AML) in immune- [32, 33]. In tissues where cells are frequently lost to the compromised mouse models . Since the work of these environment, like those of the intestine and skin, new cells two pioneers, CSC populations have been identiﬁed in mul- are continuously required to replenish those that are lost. tiple epithelial cancers including the breast , prostate , To facilitate this constant need for new cells, some epithelial pancreas , colon [15–17], ovaries , and brain . tissues are arranged hierarchically with slowly proliferating Unfortunately, the use of CSC markers has not been stem cells that asymmetrically divide to give rise to a new without controversy. One issue centers on uncertainty of stem cell and a rapidly dividing transit amplifying (TA) the functional implications of CSC markers and is best cell . Transit amplifying cells proliferate quickly for a exempliﬁed by the use of CD133 in the identiﬁcation of colon limited number of divisions, allowing for the high degree of CSCs. Shortly after Ricci-Vitiani et al. (2007) demonstrated cell turnover necessary to sustain adult tissues. Infrequent the use of the CD133 to identify CSCs in colon tumor, division or a quiescent nature is not deﬁnitive for adult Shmelkov et al. demonstrated that CD133 expression was stem cell but is suggested to be important for maintenance not restricted to colon CSCs, but that CD133 is expressed on of many adult stem cell pools. Evidence suggests that diﬀerentiated colonic epithelium in both mice and humans quiescence may play an important role in protecting stem [16, 20]. Reasons behind this contradiction of data still cells from exhausting their proliferative capacity, inhibiting remain unclear, but Kemper et al. methodically evaluated diﬀerentiation, and limiting accumulation of mutations the CD133 antibodies used by both groups and reached the during frequent rounds of DNA synthesis [35–37]. conclusion that the AC133 epitope used by Ricci-Vitiani et al. Initial eﬀorts to identify andstudy adultstemcells took recognized a diﬀerentially expressed form of CD133 that is advantage of the slow-cycling nature of stem cell populations not recognized by the antibody used by Shmelkov et al. . in studies employing pulse/chase methodology [23, 28]. In It appears that CD133 is expressed in all colon epithelium, these studies, tritiated thymidine ( H-TdR) or 5-bromo-2- while the AC133 epitope is speciﬁc for the CSC phenotype. deoxy-uridine (BrdU) was repeatedly administered to mice Furthermore, Kemper et al. were unable to determine or cultured cells that were then followed by an extended the functional signiﬁcance of the diﬀerentially expressed period of chase time. During this chase period, rapidly isoforms of CD133, highlighting another drawback to the proliferating TA cells divide the label between daughter cells, use of markers to identify CSCs. Very little is known about consequently diluting the label (Figure 1). In contrast, slow- the function of many of the proposed CSC markers, and cycling stem cells undergo few divisions and retain detectable even less is known about the functional implications they quantities of label for much longer periods of time. Cotsarelis may have for the CSC phenotype. At best, markers without et al. demonstrated that label retaining cells (LRCs) were Journal of Oncology 3 Stem cell i ii iii iv Label cells (a) Stem cell i ii iii iv X X + chemotherapy X X X X XX X X X X XX X X (b) Figure 1: Pulse Chase Labeling and Chemotherapy Survival of Stem Cells. (a) Cell suspensions are labeled with BrdU or other label ((i) and (ii)). As rapidly proliferating transit amplifying cells divide, label is diluted among the daughter cells and eventually becomes undetectable (iii). Slow dividing stem cells retain label occasionally producing a new transit amplifying cell that will quickly dilute out residual label (iv). (b) Heterogeneous tumors are predicted to contain a population of slow cycling label retaining cells (i). Conventional chemotherapies target and kill rapidly proliferating cells, while quiescent cells survive ((ii) and (iii)). Cancer stem cells that survive chemotherapy re-enter the cell cycle and re-establish the tumor. exclusively present in the bulge area of the mouse hair Although likely important for the maintenance of the follicle . These cells were found to be relatively stem-like: stem cell pool, quiescence may not be a requirement for “primitive” in cytoplasmic contents, structurally similar to adultstemcells. Using a lacZ construct under a conditional other putative stem cell populations, and could be stimulated promoter for the stem cell-associated protein leucine-rich G to proliferate. We utilized human skin xenografted onto protein-coupled receptor 5 (Lgr-5), Jaks et al. demonstrated immunodeﬁcient mice to show that LRCs were present in an a distinct nonlabel retaining subpopulation of bulge cells + + analogous bulge region of human skin delineated by keratin that overlap with the CD34 /K15 at telogen but not anagen 15 (K15) expression . Cells present in the bulge region . Lineage tracing techniques conﬁrmed that Lgr-5 have been experimentally shown to be quiescent for up to cells actively cycled during normal homeostasis and had a 1year, and based on the hair growth cycle of scalp multipotent phenotype. The authors of this paper suggest skin can likely remain quiescent for up to 5 years. Using that the Lgr-5 population of cells represents a cycling the K15 promoter to drive expression of EGFP or lacZ, population of stem cells under normal conditions, whereas + + K15positivecells were foundtodiﬀerentiate into all major the label retaining CD34 /K15 stem cells may represent a epithelial lineages of the mouse skin . We demonstrated reserve population that is activated after tissue damage. As that K15+ bulge cells from human skin can diﬀerentiate into yet, a conclusive relationship between these two populations epidermal, sebaceous, and hair follicle lineages in vitro . cannot be ﬁrmly established. Array analysis of the LRC bulge population showed increased Similar label-retaining methods have been used to study activation of Smad and inhibitors of the Wnt pathway, slow-cycling cells in other tissues, such as the small intestine suggesting the ability for LRCs to organize their niche and and colon. Work conducted by Potten and colleagues communicate with neighboring mesenchymal and epithelial identiﬁed slow-cycling LRCs at the +4 position at the base cells, an important characteristic for stem cell function . of the colon crypt. These crypt base cells were found to be The work in our lab and others supports a model in maintained in a steady state of between four and six cells that which the bulge region of hair follicles represents the stem go through division approximately once a week . Upon cell niche in skin. At the onset of the growth phase (anagen) irradiation, these cells demonstrated increased antiapoptotic hair follicle stem cells are activated and produce matrix TA bcl-2 expression, decreased p53 expression and were highly cells that proliferate and diﬀerentiate into the seven diﬀerent activated and involved in clonogenic regeneration of the lineages found within the hair follicle. As matrix TA cells crypt. Detailed biochemical analysis of this population exhaust their proliferative potential they enter a state of has been limited by the absence of reliable markers and destruction (catagen) leading to the loss of the majority of methods capable of suﬃciently isolating these cells. Two the hair follicle excluding the bulge. Catagen is followed by a studies involving the putative stem cell-associated RNA period of rest (telogen) in which the bulge stem cells remain binding protein Musashi-1 (Msi-1) have both demonstrated quiescent until activation into a new anagen stage . colocalization of this protein with colon LRCs, but fell short 4 Journal of Oncology of testing for clonogenicity of this population [44, 45]. did not aﬀect the capacity of quiescent cells to generate Similar to stem cell populations in the skin, β1-integrin spheres in vitro or repopulate the proliferating population was found to be highly expressed in the lower half of the in vivo. The ability to survive and re-enter the cell cycle colonic crypt . When sorted via ﬂow cytometry, β1- suggests a stem cell phenotype for these quiescent cells . integrin showed enrichment for clonogenic cells; however, Prostate slow-cycling LRCs located in the proximal ducts an exact colocalization pattern with LRCs was not evaluated, demonstrated high proliferative potential and the ability to and therefore, the connection remains only speculative. reconstitute the prostate glandular structure in vitro. This From the evidence collected in these studies and others, a ability singles them out as stem cells over more rapidly model has been suggested in which slow-cycling stem cells, cycling TA cells located at the distal region of the ducts . found at the base of the crypt, undergo periodic division Finally in the pancreas, characterization of LRCs around to give rise to TA cells. Transit amplifying cells low in the the acini and ducts suggested a stem cell population by crypt undergo rapid division and slowly progress up the demonstrating increased expression of the putative stem cell crypt, losing replicitative potential and diﬀerentiating as they marker c-Met and activation in response to damage to form increase in crypt height. These cells are ultimately lost to the duct-like structures . environment [6, 43]. Combined, these data indicate an important role for As within the hair follicle, there is convincing evidence quiescent LRCs in maintenance and longevity of multiple for an Lgr-5 nonlabel retaining population of colon stem adult epithelial tissues. cells additionally found at the base of the crypt . While the LRCs reside at the +4 population, Lgr-5 cells are observed 3. Quiescence and CSCs as slender wedge-shaped cells at the +2 position. Again, the exact relationship between the LRCs and the Lgr-5 cells If CSCs do originate from normal adult stem cells, then it is yet to be fully explored, and more data into the lineage is foreseeable that key stem cell regulatory traits are retained potential of both of these cell populations is needed to form through the oncogenic transition; quiescence is potentially acohesivemodel. one of these traits. Little research has been done to address Since the early identiﬁcation of colon and hair follicle how quiescence might play a role in CSC biology, but there slow-cycling stem cell populations, label-retaining tech- are some indications that quiescent stem-like populations niques have been used to identify and validate putative might contribute to at least some tumors. We previously stem cell populations in multiple epithelial tissues. In the identiﬁed a subpopulation of cells in human sebaceous mammary gland, three separate label-retaining populations tumors that expressed the skin stem cell marker keratin 15 have been identiﬁed and proposed as possible stem cells. (Figure 2). These cells appeared to have variable expres- In a study conducted by Welm et al., LRCs were found to sion of the proliferation marker Ki-67, suggesting a low but comprise a subpopulation of stem cell antigen-1 positive higher proliferative rate than normal stem cells. In primary + + + (Sca-1 ) cells . These Sca-1 cells were found to be ovarian tumors, Gao et al. demonstrated that CD24 cells enriched for the ability to form outgrowths, leading the expressing stem cell-associated genes like nestin, oct4,and authors to speculate that the LRCs might represent the both notch1 and notch4 were more slowly proliferating than stem cell population contained within the Sca-1 cells. In the bulk tumor cells suggesting a quiescent phenotype . contrast to this study, Shackleton et al. identiﬁed a long-term Low numbers of slowly proliferating CD24 cells were shown label-retaining population enriched by the marker combi- to produce tumors in a xenograft model where bulk cells hi + nation Lin CD29 CD24 that was able to reconstitute a were foundtobenontumorigenic.Thisdataimplicatesalink functional mammary gland from a single cell . The between quiescence and ovarian tumor CSCs. − hi Lin CD29 CD24 did not enrich for the Sca-1 population, Pece also observed a link between CSCs and quiescence in prompting other groups to suggest a stem cell hierarchy in breast tumors . Using the hNMSC signature generated which multiple layers of stem cells exist within the mammary with normal mammary LRCs, Pece turned his attention gland . Using a slightly diﬀerent methodology, Pece used to the analysis of primary breast tumors, ﬁnding that the the lipophilic ﬂuorescent dye PKH26 to identify a population hNMSC signature was more commonly found in grade 3 of mammary label retaining cells . The use of the PKH26 tumors over that of grade 1. When grade 1 and grade 3 allows for live sorting of LRCs, which is not possible using the mammospheres were analyzed for PKH label retaining cells, nucleotide analogue BrdU and HT-TdR that both require both populations were found to retain label, with grade permeabilization of the cell membrane for antibody labeling. 3 tumors demonstrating a higher percentage. This data Live sorting of PKH26 LRCs demonstrated increased in vitro suggests an increase in stem-like cells as tumors progress. sphere formation eﬃciency and regeneration of cleared fat When evaluated for tumor genicity, breast tumor cells pads over non-LRCs. Pece was also able to conduct tran- positive for the hNMSC signature were more eﬃcient at scriptional analysis of the LRC population, from which he forming in vitro spheres and in vivo xenograft tumors that created a human normal mammary gland stem cell signature those cells lacking the hNMSC signature. (hNMSC) consisting of the markers CD49F/DNER/DLL1. Cultured cancer cell lines are often used to study Unfortunately, the exact relationship between the diﬀerent signaling pathways, invasion, migration, and apoptosis, but populations identiﬁed by these three groups is not yet clear. are rarely thought of as candidates for CSC studies. Many In the brain, high doses of H-TdR kill all but one per- of the most widely used cell lines have been in passage for cent of proliferating subependymal. High dose therapeutics years, are perceived homogeneous, lack interactions with the Journal of Oncology 5 (a) (b) Figure 2: Stem cell markers in Normal Sebaceous Gland and Sebaceous Tumor. Immunohistochemical staining for the skin stem cell marker Keratin 15 (K15). (a) Normal skin sebaceous gland with labeled stem cells (black arrows). (b) Sebaceous tumor with heterogeneous expression of K15. appropriate stromal microenvironment, and change charac- (DiI /SCCs) comprised ∼3% of total cell number. Interest- teristics based on alterations in culture conditions. Therefore, ingly, label retaining cells also exhibited an elongated ﬁbrob- cultured cell line studies assessing CSC characteristics must last shape and an increase in the epithelial-mesenchymal be evaluated critically, with data interpreted within the con- transition markers vimentin, snail, and twist. A ﬁbroblast- text of the experimental parameters, and results conﬁrmed like CSC is consistent with evidence demonstrating an under biologically relevant conditions. Still, interesting work increase in stem-like properties in cells that have undergone in the cultured tumor lines MCF10A, MCF7, SUM149, an epithelial-mesenchymal transition . Furthermore, SUM159, SUM1315, and MDA.MB.231 suggests that these sorted DiI /SCCs demonstrated a 2.5–10-fold increase in lines may not be as homogenous and void of “stem like” cells soft agar colony forming ability, twofold increase in invasive + − + as once thought . CD44 /CD24 /ESA cells within these potential, and more than a tenfold increase in xenograft lines were found to contain the ability to self-renew, recon- formation over nonlabel retaining cells. Combined, these stitute the parental line, and to be up to 90% label retaining. data suggest that DiI /SCCs cells represent an enriched CSC If LRCs are found to retain the CSC phenotype in cultured population. When assessed for common CSC marker status, cell lines, these cell lines may provide an important resource DiI /SCCs were enriched but only partially overlapped with + + + for future delineation of quiescent pathway regulators. CD24 /CD44 and CD133 populations. It is curious to Additional transitive evidence linking quiescence to CSCs consider how these commonly used CSC markers relate to can be found in the work conducted by Roesch et al. in the LRC populations and what role, if any, these markers play melanoma . This group found that primary melanoma in the slow-cycling phenotype? cell lines contained a PKH26 label retaining population that Like the melanoma study by Roesch et al. , Dem- was almost speciﬁcally identiﬁed by the H3K4 demethylase binski and Krauss’s study also indicated the ability for LRCs JARID1B. This population of cells was found to incorporate to produce non-LRCs and surprisingly also for non-LRCs BrdU more slowly but retain it for a longer period of to produce LRCs. Such a dynamic suggests two possibilities time, lack Ki67 staining, and have a doubling time of up (1) that the true unknown CSC population is favored to 4 weeks in vitro. When EGFP was placed under the in the LRCs, but also found in the non-LRCs and can control of the JARID1B promoter, GFP cells demonstrated therefore give rise to both populations, or (2) that there increased sphere forming ability in vitro. Interestingly, GFP exists a dynamic relationship in LRC-CSC populations that cellswereabletoretainBrdU in vivo, but did not show is context dependent and allows for interconversion between increased tumor initiating ability over GFP cell during the two states. The Dembinski and Krauss study argues the time period analyzed. Small hairpin RNA (shRNA) a dynamic population of CSCs that might coincide with knockdown of JARID1B resulted in the in vitro exhaustion of an epithelial-mesenchymal transition (EMT). EMT plays a proliferating cells, demonstrating the need for JARID1B cells central role in embryogenesis and mesoderm diﬀerentiation in maintenance of proliferative capacity but not initiation into multiple tissue types during development . The of tumors. When assessed more fully, both in vitro and in emergence of embryonic stem cell-associated genes like vivo GFP cells gave rise to heterogeneous progeny, including nanog, oct4, sox2,and c-myc in high grade undiﬀerentiated JARID1B GFP+ cells. cancers is suggestive that aberrant regulations of EMT and The most direct evidence to date for quiescence playing a other early development pathways might be playing a role in role in CSCs comes from a study conducted by Dembinski CSC characteristics . This data is a further evidence to and Krauss . In this study Vybrant DiI cell-labeling support a dynamic quiescent slow-cycling model for many solution was used to label pancreatic adenocarcinoma cells types of cancer. Future studies will be important for further and conduct cancer stem studies on ﬂow cytometry sorted development and integration of these observations into the label retaining cells. DiI label retaining slow-cycling cells CSC model for tumor initiation and propagation. 6 Journal of Oncology 4. Quiescence and Resistance to Chemotherapy involved in regulating hair follicle stem cell survival such as caveolin-1 are emerging . At the present time, we have no clear understanding of why Similar mechanisms for survival and self-renewal for some patients recur and which cancers will have resistance CSCs are plausible in instances of tumor recurrence in to conventional types of therapy. Tumors from diﬀerent human patients where cytotoxic agents kill proliferative patients in the same organ are likely to have undergone cancer cells, leaving quiescent slow-cycling . Cancer stem diﬀerent oncogenic transitions, leading to a diversity of pos- cells that survive chemotherapy would have the ability to re- sible regulatory mechanism and pathway activities that might enter the cell cycle and produce highly proliferative-rapidly be contributing to the survival of a speciﬁc cancer. While dividing progenitor cells that can re-establish the tumor. It is broad patterns like the dysregulation of the Wnt pathway even probable that successive cycles of chemotherapy would in colon carcinomas are commonly observed, the secondary intensify a tumor by weakening the normal stem cell pool mutations that may accompany these cancers could be and creating therapy resistant CSCs that give rise to resistant vastly diﬀerent and contribute to survival in diﬀerent ways oﬀ-spring . . Even within the same tumor, diﬀerent CSCs have Slow cycling CSC populations in the colon, breast, the possibility to accumulate unique mutations that may ovaries, and pancreas have been shown to demonstrate both provide added resistance and be passed on to daughter cells. in vivo abilities to survive therapies that kill bulk tumor In context with the vast diﬀerences in tumorigenesis and cells as well as a requirement for doses of up to twice that heterogeneity with a tumor, it is not surprising that the exact which are required to kill rapidly proliferating cells in vitro contributors to chemotherapy resistance and consequently [18, 55, 62, 67]. These data demonstrate how ineﬀective which patients will respond optimally to chemotherapy are conventional therapies can be on quiescent cell populations not well understood. It has been proposed that variations and help to explain why tumors that seem to fully regress in cell cycle control, antiapoptotic proteins, increased DNA during treatment can recur. While large tumor populations damage repair proteins, upregulation of cellular pumps, and may appear to have totally regressed after treatment, single increased metabolic activity may all play important roles in surviving CSCs would not be detectable with current chemotherapy resistance [6, 59–62]. diagnostic technology. Populations of CSCs that are resistant Conventional chemotherapies and radiotherapies target to chemotherapy or radiation are able to re-enter the cell proliferating cells and require active cycling for induction cycle or never fully undergo cell cycle arrest and are primed of apoptosis. The quiescent nature of many adult stem cell to re-establish tumors [53, 68]. Even more devastating to pools is therefore an inherent mechanism for resistance and the survival of patients may be CSC response to stress from cell survival to conventional therapies. In the hematopoietic chemotherapy and radiotherapy. Mouse ovarian tumors system, normal hematopoietic stem cells (HSCs) contain have been demonstrated to undergo accelerated clonogenic cip1/waf1 high levels of the quiescence regulator p21 (p21) production during radiotherapy regimens, expanding the . When treated with the commonly used chemotherapy CSC pool and driving development of a more aggressive agent 5-ﬂuorouracil (5-FU), mice that were p21 deﬁcient secondary tumor . Furthermore, these cells would be had a signiﬁcant decrease in cobblestone area-forming more likely to produce chemotherapy resistant oﬀspring, stem cells (10.8%) than normal p21 expressing wild-type rendering the tumor unaﬀected by later rounds of treatment. mice (60.5%). In the brain, Morshead et al. demonstrated While quiescence is likely to contribute to the survival that high doses of tritiated thymidine ( H-TdR) killed of CSCs in response to chemotherapy and radiation, slow the constitutively proliferating cells in the adult mouse cycling is not the sole mechanism and in all likelihood works forebrain, but had no eﬀect on quiescent stem cell ability to in parallel with other systems to increase survival. Msi-1 generate spheres . This data supports a model in which colon cancer cells have been demonstrated to be less sensitive quiescent mouse forebrain stem cells are able to survive to cytotoxic drugs due to increased IL-4 expression and and re-enter the cell cycle to allow for regeneration of the orchestration of antiapoptotic mechanisms . The expres- damaged tissue. A similar pattern of stem cell survival and sion of other antiapoptotic proteins like c-Flip and Bcl-2 BH- regeneration was observed 72 hours following doxorubicin 3 only family members is frequently seen in stem cell and treatment in mouse intestine. In this experiment, mice CSC populations and has been demonstrated to contribute intestine demonstrated increased amounts of cell death via to cell survival during radiation and chemotherapy [59, 60]. apoptosis in the +3–6 positions and a parallel disappearance Reduced cycling may help to limit cell damage in these cases, of mitotic activity . This period of relatively nonexistent decreasing prodeath signals and increasing the potential for mitotic activity was followed by stem cell re-entry into the CSC survival. cell cycle and tissue regeneration in the +4 position stem Additional mechanisms for CSC survival include incre- cell compartment. Furthermore, colon stem cell survival ased DNA damage repair, upregulation of cell pumps like the during chemotherapy is aided by increased expression of multidrug resistance transporter (MDR1) and the Adeno- BH3-only bcl-2 members that inhibit apoptosis, working in sine triphosphate-binding cassette (ABCB1), and increased parallel with quiescence to increase the likelihood of stem cell metabolic activity through ALDH [61, 62]. Although the survival . In chemotherapy-induced alopecia, the rapidly quiescence contribution to these mechanisms of resistance is dividing TA cells in the hair matrix undergo apoptosis, while unclear, it is likely that reduced proliferative rate only adds the stem cells in the bulge region survive to regenerate the to their eﬀectiveness. Additional time in S or G phase of follicle after chemotherapy is withdrawn. Potential factors the cell cycle coupled with increased DNA repair protein Journal of Oncology 7 activity may aﬀord a survival advantage over bulk cells that CSCs or contribution of other Rb family members like p107 continuously accrue DNA damage and ultimately are forced may be important in CSC maintenance of quiescence. to undergo apoptosis. Reduced cycling speed together with Developing and studying a quiescence signature in increased pumps would facilitate more drug being removed ﬁbroblasts may be important in understanding regulation of from CSCs, limiting overall cytotoxic eﬀects during the the cell cycle, but the exact relevance to quiescent stem cell period of treatment. Additionally, quiescence would allow for populations is not very clear. Primarily, quiescence ﬁbroblast increased metabolic activity of ALDH and other metabolites studies are conducted on large populations of ﬁbroblasts over that of bulk cells with a shorter cell cycle period. under biologically stressful conditions like contact inhibition Importantly, there is no reason why combinations or all of or serum starvation. In contrast, individual stem cells and these resistance mechanisms could not be playing a role in CSCs maintain quiescence while in contact with daughter CSC survival. Future therapies may need to address all these cells and stromal layers and in the presence of normal issues to be successful in complete tumor eradication. mitogenic signals. Additionally, sphere forming assays com- monly used for the identiﬁcation of stem cells and CSCs rely speciﬁcally on proliferation under nonadherent conditions. 5. Regulators of Quiescence If mitogen deprivation, loss of adhesion, and contact inhi- Given the importance of quiescence in the CSC contribu- bition truly activate three diﬀerent transcriptional programs in quiescent ﬁbroblast populations, it is possible that the tion to tumor progression and survival, understanding the mechanisms that govern quiescence will prove important in transcriptional program facilitated by quiescent stem cells the development of future strategies to better target these and CSCs may be very diﬀerent. Quiescence regulation of a stem cell population is most cells. Much of our current understanding of the mechanisms controlling quiescence come from studies using conditional comprehensively understood in the hematopoietic system. induction of quiescence in normal adult ﬁbroblasts. The When compared to diﬀerentiated or cycling HSCs, quiescent induction of quiescence in ﬁbroblasts is generally accom- HSCs were found to have up-regulated genes associated with plished in one of three ways: mitogen deprivation, contact cell cycle regulation, translation and RNA processing, and metabolic process . Down-regulated genes were generally inhibition, or loss of adhesion. Each method of inducing quiescence in ﬁbroblast appears to yield a diﬀerent quies- associated with transcription factors, signaling proteins, cell cent transcriptional program . The three transcription cycle proteins, and inhibitors of cell cycle progression. In line with these ﬁndings, the CKI p21 was found to be necessary programs overlap in diﬀerential expression of 131 genes that Coller et al. have designated a “quiescence signature.” for quiescence and maintenance of the HSC pool . Mice This signature is comprised of genes that regulated cell that are p21 null demonstrate an increase in the number growth and division, suppress apoptosis and diﬀerentiation, of stem cells present and lose the ability to repopulate the and govern intercellular communication. Downregulated bone marrow in serial transplant experiments, suggesting elements in the quiescence signature consist of genes asso- uncontrolled expansion and eventual exhaustion of the stem ciated with cell cycle progression including cyclin B1, cdc20, cell pool. This deregulation of the stem cell pool is likely cul-1, and myc. Up regulated genes included important cell due to p21 downstream eﬀects on Rb family members: Rb, p107, and p130. Rb family members play important roles in cycle regulators like TP53 (p53), cyclin D2, and MXI1. Also up regulated in this signature are regulators of key stem regulating E2F activity and G /S transition. Triple knockout cell-associated pathways including the Wnt pathway (FZD2 of these three family members resulted in hematopoietic progenitor G1/S transition and proliferation, leading to and TCF7L2), the BMP pathway (SMAD1), and the Notch pathway (Hes1). NotchactivationofHes1isofparticular exhaustion of the proliferative potential, similar to that seen interest as it has been shown to control reversibility of in p21 loss . ﬁbroblast quiescence by blocking diﬀerentiation and entry While p21 also appears to play a role in adult neural in irreversible cell cycle arrest . Notch pathway activity stem cell regulation and maintenance, other factors have been shown to be important contributors to quiescent stem is important in mammary gland development as well as the mammary CSC response immediately following irradiation, cell activation . Occasional exit of neural stem cells suggesting that the Notch pathway may be a potential target from the quiescent state is important for proper tissue maintenance and may be controlled though notch signaling in CSCs [5, 71]. Interestingly, there exists a fourth transcriptional pro- via Hes1 oscillations . Down-regulationofHes1in gram in ﬁbroblasts induced by overexpression of cyclin- neural progenitor cells during G phase reduced repression INK4a of cyclinD, ngn2, and Dll1, activating Notch signaling and dependent kinase inhibitors (CKI) like p21 and p16 . The CKI p21 has been found to control entry into qui- driving cell cycle progression and generation of neural escence and maintenance of the quiescent state, allowing cells progenitors. Neural progenitors and neurons continue to to activate a DNA damage-like response . Additionally, retain low levels of Hes1 as they proliferate and diﬀerentiate. maintenance of ﬁbroblast quiescence has also been shown to In neural stem cells, Hes1 expression and control of cyclinD and notch signaling increase until subsequent G entry. be highly regulated by the retinoblastoma family members Rb and p107 . Loss of Rb and p107 did not aﬀect the Interestingly, p21 loss does not appear to play a signiﬁcant ability of ﬁbroblasts to enter G , but these cells were unable role during diﬀerentiation in the brain, suggesting the need for additional means of cell cycle regulation in diﬀerentiated to maintain the quiescent state. While Rb loss is generally associated with the progression of cancer, retention of Rb in senescent cells . 8 Journal of Oncology Signaling pathways with interactions to other CKIs also In colon cancers, mutations in APC or β-catenin are play important roles in quiescent adult stem cell regulation. considered to be a driving force behind transformation . In mammary glands, the Hedgehog pathways components In the presence of Wnt signal, β-catenin is no longer taken up Gli2 and Bmi-1 have been demonstrated to regulate stem by an APC-dependent degradation complex and translocates cell self-renewal . When injected into cleared mammary to the nucleus where it binds TCF/LEF transcriptions factors fat pads, Gli2 or Bmi-1 over expressing mammospheres to control expression of cell cycle target genes. Loss of were able to produce substantially more outgrowths than APC in crypt Lrg5 cells has been demonstrated to be an control mammospheres. Bmi-1 has been demonstrated to important step towards initiation of intestinal adenomas INK4a ARF transcriptionally repress the p16 and p19 , suggest- . Interestingly, cells expressing high Wnt downstream ing a role for Bmi-1 in mammary stem cell cycle con- transcription factors TCF/LEF in primary sphere cultures trol. demonstrated increased clonogenicity and the generation Additional signaling pathways have been demonstrated of both cycling and noncycling cells . In tumors, these to play important roles in stem cell quiescence, speciﬁcally high Wnt expressing cells were located near stromal ﬁbrob- the BMP pathway in skin. BMP and calcineurin signaling lasts that provided signals to activate β-catenin-dependent up-regulate the transcription factor NFAT1c that has been transcription. This data suggests that CSC cell cycle control found to highly colocalize with CD34 cells in the hair follicle may not be entirely cell autonomous and partially regulated . NFAT1c represses transcription of CDK4, stalling cells by microenvironmental signals. Targeting Wnt pathway in G /S phase and maintaining quiescence. Loss of NFAT1c regulators or the ability for CSCs to communicate with their permits entry into the cell cycle, shortening telogen and stromal environment may represent potential mechanism for prompting aberrant entry into anagen. limiting CSC expansion and contribution to recurrence. While signiﬁcant advances are being made in under- There is also mounting evidence for the requirement of standing quiescence control in normal adult stem cell Hedgehog signaling in proliferation and survival of both populations, much less is known about control of quiescent colon and breast tumors. Active Hh-Gli signaling was found CSC populations. Very few studies have been conducted to contribute to the subpopulation of human colon CD133 speciﬁcally addressing control of quiescent CSCs, most likely cellsthatwereabletosurvive andself-renewinxenograft + − due to the diﬃculty of isolating and analyzing pure CSC studies. In breast tumor CD44 /Cd24 cells, the Hh pathway populations. If CSCs are truly derived from adult stem proteins Patch (PTCH1), Gli1, Gli2, and Bmi-1 all demon- INK4a cells, then it is possible that Hes1, p21, p16 ,Rbfamily strated increased expression over bulk tumors cells . Like members, Bmi-1, and NFAT1c play signiﬁcant roles in CSC their adult mammary stem cell counterparts, overexpression regulation. Although rare, there are clues that at least some of Bmi-1 in mammary CSCs suggests a potential role for INK4a ARF of these regulators are important in CSCs. In the colon p16 and p19 in cell cycle regulation and suggests a cancer cell line HCT116, p21 null cells were found to produce potential drug target for improved CSC eradication. tenfold smaller tumors in growth assays when compared While p21, p16, Notch, Wnt, and Hedgehog signaling to normal cells expressing p21 . Under sphere forming may provide tempting targets for the removal of CSCs, tar- conditions, p21 null cells were unable to form spheres, ceased geting of these pathways would require meticulous targeting proliferation, and eventually died. This p21 dependence was of CSC or titration of inhibitors to act on CSCs but not found to be associated with lack of E-cadherin expression normal stem cell populations. Such treatments could severely and suppression of apoptosis signals, suggesting a more weaken patients. Additionally, improper application of cell complex role for p21 in tumor cells than simply regulating cycle inhibitors like p21 may fuel tumor growth and aggres- cell cycle. Small molecule targeting of p21 or downstream siveness. The CDK inhibitor p21 acts as a tumor suppressor p21 targets may therefore prove to be an eﬀective means in dividing cells by protecting against genome instability and of forcing quiescent CSCs to cycle or undergo apoptosis. working with other tumor suppressors to subdue oncogenes Cycling CSCs would be susceptible to chemotherapy and [82, 83]. Loss of p21 combined with chemical induction hopefully eliminated. of carcinogenesis has demonstrated increased induction of Cancers frequently have aberrant signaling in the Wnt, tumors and increased aggressiveness in resulting tumors Hedgehog (Hh), and Notch self-renewal pathways that likely [84, 85]. Combining widespread targeting of p21 with contribute to cell cycle control and diﬀerentiation. Increased chemotherapy may have similar eﬀects of tumors. These data expression of Hes1 has been observed in ovarian, breast, and highlight the necessity to be able to selectively target CSCs nonsmall cell lung carcinomas, suggesting active regulation when using CDK inhibitors and add to the challenges ahead of Notch signaling . In melanoma, the slow cycling in developing treatments to better eradiate CSCs. cells identiﬁed by Rosech et al., repress notch signaling directly though JARID1B interaction with the notch lig- 6. Conclusions and Future Directions and Jagged 1 promoter, consequently reducing intracellular Notch and controlling proliferation . Hes1 and Jagged1 The limited data available on the regulation of quiescence may therefore be potential targets in future cancer treatments equates to a poor understanding for the role of quiescence designed to target CSCs. Targeted reduction of Hes1 would in tumor progression and recurrence. 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