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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Feb 1, 2013.
Published in final edited form as:
PMCID: PMC3271803
NIHMSID: NIHMS336374

Cancer Stem Cells: Distinct Entities or Dynamically Regulated Phenotypes?

Yunqing Li1,2 and John Laterra1,2,3,4

Abstract

The origins of tumor propagating neoplastic stem-like cells (cancer stem cells, CSCs) and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features(i.e. transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors co-exist in dynamic equilibrium and are subject to bidirectional conversion. This mini-review discusses emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical to the development of therapeutic strategies for depleting tumors of their tumor-propagating and treatment-resistant cell subpopulations.

Background

Most solid malignancies consist of phenotypically heterogeneous neoplastic cells among which only a subset displays the capacity to efficiently propagate tumor xenografts that recapitulate with high fidelity the heterogeneity and pathological features of the original clinical cancer. These uniquely tumorigenic cells are commonly referred to as cancer stem cells (CSCs) since they exhibit a spectrum of biological, biochemical, and molecular features consistent with a stem-like phenotype. These features include growth as nonadherent spheres in defined “stem cell” medium, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. These features and evidence that CSCs are particularly resistant to cytotoxic therapeutics support the hypothesis that CSCs are the cell sub-population most likely responsible for treatment failure and cancer recurrence. Understanding the origins of CSCs, mechanisms that support CSCs, and their relationship to the bulk population of tumor cells that display low self-renewal capacity and higher probability of terminal differentiation (i.e. transit-amplifying cancer progenitor cells) is obviously of considerable relevance for improving cancer therapeutics.

The cancer stem cell hypothesis, in its most prevailing form, currently mirrors normal stem cell biology by emphasizing a hierarchical unidirectional path through which CSCs self-renew and generate more differentiated neoplastic progenitor cells through asymmetric replication. Emerging evidence is beginning to support the existence of a contextually regulated equilibrium between CSCs and transit-amplifying neoplastic progenitors, including the capacity of progenitor cell dedifferentiation to form CSCs. This evidence from multiple laboratories includes our recent work identifying a reprogramming-like mechanism through which the c-Met receptor tyrosine kinase via downstream transcription factors (i.e. Sox2, c-Myc, Klf4, Oct4, and Nanog) induces glioblastoma cells to express a stem-like phenotype (1). This mini-review discusses the microenvironmental influences, signaling pathways, molecular circuitries including transcriptional networks, microRNAs, and epigenetic modifications currently implicated in the de novo generation of CSCs from cancer progenitors (Figure1).

Figure 1
Transit-amplifying cancer progenitor cells acquire stem-like phenotypes by dedifferentiating mechanisms. A schematic model depicting the functional connections between microenvironmental signals, signal transduction pathways, and molecular circuitries ...

Key Findings

Transcription factor networks

Specific transcriptional networks play an essential role in sustaining the growth and self-renewal of embryonic stem (ES) cells and neoplastic stem-like cells. Expressing a defined and limited set of transcription factors (TFs) (e.g. Sox2, c-Myc, Klf4, Oct4, Lin28) can reprogram mouse and human somatic cells to embryonic-like cells called induced pluripotent stem cells (iPSCs), which efficiently self-renew and display the capacity to differentiate to all tissue types (2).These reprogramming TFs are frequently overexpressed in human cancers and their expression levels often correlate with tumor progression and poor prognosis. Furthermore, the transcriptomes of high grade tumors and embryonic stem cells are remarkably similar (3). This suggests “dedifferentiation mechanisms” through which the expression and function of reprogramming transcription factors influence the malignant phenotype by supporting the formation and/or maintenance of neoplastic stem-like cells. Induced cancer cell reprogramming has recently been recapitulated in several types of malignant cells. By expressing defined transcription factors, including Oct4, Sox2, c-Myc, and Klf4, gastrointestinal cancer cells have been induced to express an embryonic stem-like state (4). Ectopic co-expression of Oct4 and Nanog in human A549 lung cells also induced a cancer stem–like phenotype characterized by CD133-expression, sphere formation, enhanced tumor-propagating capacity, and drug resistance (5). Enforced Lin28 expression in human colon cancer cells increased expression of the colonic stem cell markers LGR5 and PROM1, and enhanced metastatic potential (6).An alternative transcriptional network that drives epithelial-mesenchymal transition (e.g. Snail, Twist and Zeb) has also been found to induce stem cell properties in human mammary carcinoma cells (7).Critical unanswered questions include how these multiple reprogramming transcription factors function cooperatively and which transcriptional events are essential for reprogramming neoplastic progenitors to a stem-like tumor-propagating state. Oct4 alone appears to be sufficient to directly reprogram human and mouse neural stem cells to iPS cells(8), suggesting that Oct4 may initiate the transcription network. Consistent with this, our recent unpublished findings show that Oct4 is the most upstream transcription factor in the reprogramming network driven by c-Met signaling in human glioblastoma cells. While exogenous Nanog is not necessary for iPS cell generation, the induction of endogenous Nanog by Oct4 and other reprogramming factors appears to be necessary to accomplish full somatic cell reprogramming. Consistent with this role in non-neoplastic cells, we found that silencing Nanog inhibits the reprogramming capacity of c-Met signaling in glioblastoma cells(1). Nanog is emerging as a critical transcription factor through which several signaling pathways including c-Met, hedgehog (Hh) and TGF-β sustain non-neoplastic and neoplastic cell stemness(1, 9, 10). Interestingly, the tumor suppressor p53 reduces the efficiency of iPS cell generation from non-neoplastic somatic cells by repressing Nanog induction (11) and loss of p53 enables mammary cancer cells to acquire a stem cell-like transcriptional signature (12).

Stem cell miRNAs

MicroRNAs (miRNA) represent a class of small non-coding RNAs(20–25 nucleotides) that regulate a diverse array of biological processes through posttranscriptional silencing of specific target genes. Recent studies demonstrate that specific miRNAs called stem cell miRNAs such as miR-302 cluster, miR-372/373, let-7 and miR-200 family, play a critical role in controlling pluripotency by targeting multiple genes involved in cell cycle regulation, epigenetic modifications, and epithelial-mesenchymal transition (EMT). iPS cells have been successfully generated from human and mouse somatic cells by expressing specific stem cell miRNA(13, 14). Lin et al showed that expressing miR-302a alone promotes the dedifferentiation of human skin cancer cells to an iPS-like state (15), demonstrating that miRNAs are also capable of reprogramming cancer cells to a pluripotent embryonic stem cell-like state. While the complete spectrum of “reprogramming” miRNAs and their molecular mechanism of action remain to be fully identified, stem cell miRNAs appear to regulate the stem cell state at least in part by modulating the expression of reprogramming transcriptional networks(6, 16).Several lines of evidence show that stem cell miRNAs such as miR-145, miR-302 cluster, miR-200 family and miR-134/296/470 regulate ESCs pluripotency by targeting Oct4, Nanog and Sox2(1618).Wellner et al. found that miR-200 family members target Klf4 and Sox2 and that repressing miR-200 miRNAs with the zinc finger E-box-binding homeobox protein ZEB1 promotes a stem-like tumor initiating phenotype in pancreatic and colorectal cancer cells(16). Li et al found that miRNA-93 and miR-106b enhance iPSC induction by targeting Tgfbr2 and p21 (14). Stem cell miRNAs act through multiple targets and pathways to regulate non-neoplastic and neoplastic stemness and cell differentiation. How and in what context these miRNAs regulate the acquisition and maintenance of the stem cell state in cancer cells needs to be established.

Microenvironmental signals

Cancer cell subsets localize to specific tumor microenvironments and evidence increasingly points to the influence of microenvironmental-specific factors on the plasticity and differentiation of cancer cells including the subset of cancer stem cells. A broadly relevant example is tumor hypoxia, which through hypoxia-inducible factors (HIFs) induces the stem-like phenotype in cancer progenitors isolated from multiple solid tumor subtypes. Mechanisms involve the induction of an embryonic stem cell-like transcriptional program consisting of specific reprogramming factors (Oct4, Nanog, Sox2, KLF4) and miRNA-302 (19, 20). HIF in combination with the iPSC inducers Oct4, Nanog and Lin28 reprogrammed differentiated lung cancer cells into stem-like cells that efficiently propagated aggressive tumors in mice (20). Hypoxia also enhances the efficiency of non-neoplastic iPSC formation from mouse embryonic fibroblasts and human dermal fibroblasts (21).In addition to hypoxia, tumor cell stemness is influenced by microenvironmental inflammation. For example, tumor infiltrating macrophages increase the tumor-initiating capacity and anticancer drug resistance of neoplastic stem-like cell populations through the secretion of IL-6 and macrophage–derived milk-fat globule EGF8 (MFG-E8) that cooperatively induce tumor cell Stat3 and Hh signaling (22). Notably, interleukin(IL)-6 was found to enhance the conversion of breast cancer progenitor cells to a more stem-like phenotype via a positive feedback loop involving NF-κB, Lin28, and Let-7 microRNA (23). The neoplastic stem-like phenotype is also influenced by paracrine signals derived from neighboring cells. For example, Charles et al. found that the perivascular niche promotes glioma cell conversion to a more stem-like state through endothelia-derived nitric oxide dependent induction of glioma cell Notch signaling(24). Similarly, hepatocyte growth factor (HGF) secreted by stromal myofibroblasts was found to activate colorectal cancer cell c-Met and beta-catenin transcription and thereby turn nontumorigenic cancer cells into highly tumorigenic stem-like cells (25).

Autocrine/paracrine oncogenic signaling pathways such as WNT, Notch, Hedgehog (Hh), TGF-β, and receptor tyrosine kinases (RTK) (e.g. c-Met, EGF, PDGF) are strongly implicated in regulating the CSC pool and pathways involving endogenous reprogramming mechanisms. Several studies support functional interactions between oncogenic signaling pathways and critical stem cell reprogramming transcription factors. For example, Wnt signaling promotes the reprogramming of murine fibroblasts to pluripotency (26) and maintains the pluripotency of human ESCs and CSCs through the downstream target gene Tcf3, which occupies and regulates the promoters for Oct4, Sox2, c-Myc and Nanog (27). Hedgehog(Hh)-Gli signaling was found to have an essential role for GBM stem cell self-renewal via downstream effectors Gli1 and Gli2, which bind to the Nanog promoter and directly regulate Nanog expression (9). EGFR activation promotes Sox2 expression, which in turn binds to the EGFR promoter and directly up-regulates EGFR expression (28). Autocrine TGF-beta signaling plays an essential role in the retention of glioblastoma cell stemness by inducing Sox4 and Sox2 expression (29). Our recent findings also show that c-Met signaling induces the GBM stem-like phenotype through a reprogramming network involving Sox2, c-Myc, Klf4, Oct4 and Nanog Overall, these and similar findings from a diverse array of experimental models along with correlative clinical data strongly support the hypothesis that the CSC phenotype is maintained by dedifferentiating mechanisms activated through oncogenic signaling pathways.

Epigenetic modification

Transcriptional networks that determine cell identity and fate are regulated by chromatin architecture and promoter DNA modification. The ectopic expression of reprogramming transcription factors Oct4, Sox2, c-Myc and Klf4 triggers a sequence of epigenetic events including DNA demethylation and histone acetylation/methylation that eventually results in heritable induced pluripotency. Promoter DNA demethylation and histone H4 and H3 hyperacetylation during iPS cell generation leads to activation of endogenous pluripotency genes includingOct4 and Nanog (30, 31). DNA methyltransferase 1 (DNMt1) inhibitors such as 5-aza-cytidine and RG108 or histone deacetylase inhibitors such as valproic acid, trichostatin A, and suberoylanilide hydroxamic acid (SAHA) accelerates the reprogramming of mouse and human fibroblasts (32). Interestingly, these pharmacologic epigenetic modulators also have cell differentiating and anti-neoplastic properties emphasizing the need to better understand the dynamic context-dependent genome-wide epigenetic mechanisms regulating cancer cell stemness and tumor propagating potential.

Implications and future directions

The identification of a dynamic contextually-regulated bidirectional interchange between tumor-propagating CSCs and the considerably larger population of more differentiated transit-amplifying cancer progenitor cells in solid malignancies has considerable conceptual and practical implications. Current dominant models of the cancer stem cell hypothesis depict the tumor propagating CSC as a distinct minority cell population from which all other neoplastic tumor cells derive through asymmetric replication. In contrast, the capacity to dedifferentiate transit-amplifying neoplastic cells into new CSCs through autocrine/paracrine signals and endogenous transcriptional reprogramming networks points alternatively to a more dynamically regulated CSC phenotype and possibly to variable degrees of cancer cell “stemness”. It is currently unknown if “reprogrammed” CSCs are of variable stability determined dynamically by the relative balance of differentiating and de-differentiating/reprogramming signals or, alternatively, if they are relatively stable and maintained through heritable epigenetic mechanisms, as are non-neoplastic iPSCs. Relatively inefficient autocrine/paracrine reprogramming stimuli acting on large populations of transit-amplifying cancer progenitors could be sufficient to generate a sufficient number of therapy resistant tumor-propagating cells to substantially impact tumor growth and recurrence. The capacity for endogenous reprogramming impacts therapeutic strategies designed to target the CSC pool within tumors since any successful strategy will have to target pre-existing CSCs and also inactivate endogenous dedifferentiation mechanisms through which additional CSCs are generated. Understanding the contributions of the diverse molecular circuitries including transcription factor networks, stem cell miRNA expression, and epigenetic modification will be crucial. Recent discoveries pointing to the critical roles for Oct4, Nanog, and their target genes provide an early road map toward this end. In addition, elucidating mechanisms of CSC formation by induced dedifferentiation opens the possibility to generate in the laboratory unlimited numbers of patient-specific induced CSCs, differentiated neoplastic progenitors, and tumor xenografts for mechanistic science and cancer drug discovery.

Acknowledgements

The authors thank Charles G. Eberhart (Johns Hopkins School of Medicine)and Amy M. Fulton (University of Maryland School of Medicine) for their helpful discussions. This work was supported by the American Brain Tumor Association Discovery Grant (YL), NIH grants NS43987 (JL) and CA129192 (JL) and the Brain Tumor Funders Collaborative & James S. McDonald Foundation (JL)

References

1. Li Y, Li A, Glas M, Lal B, Ying M, Sang Y, et al. c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jun 14;108(24):9951–9956. [PMC free article] [PubMed]
2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663–676. [PubMed]
3. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nature genetics. 2008 May;40(5):499–507. [PMC free article] [PubMed]
4. Miyoshi N, Ishii H, Nagai K, Hoshino H, Mimori K, Tanaka F, et al. Defined factors induce reprogramming of gastrointestinal cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2010 Jan 5;107(1):40–45. [PMC free article] [PubMed]
5. Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hsieh WJ, et al. Coexpression of oct4 and nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer research. 2010 Dec 15;70(24):10433–10444. [PubMed]
6. King CE, Cuatrecasas M, Castells A, Sepulveda AR, Lee JS, Rustgi AK. LIN28B promotes colon cancer progression and metastasis. Cancer research. 2011 Jun 15;71(12):4260–4268. [PMC free article] [PubMed]
7. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008 May 16;133(4):704–715. [PMC free article] [PubMed]
8. Kim JB, Greber B, Arauzo-Bravo MJ, Meyer J, Park KI, Zaehres H, et al. Direct reprogramming of human neural stem cells by OCT4. Nature. 2009 Oct 1;461(7264):649–643. [PubMed]
9. Po A, Ferretti E, Miele E, De Smaele E, Paganelli A, Canettieri G, et al. Hedgehog controls neural stem cells through p53-independent regulation of Nanog. EMBO J. 2010 Aug 4;29(15):2646–2658. [PMC free article] [PubMed]
10. Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, et al. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell stem cell. 2008 Aug 7;3(2):196–206. [PMC free article] [PubMed]
11. Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, Appella E, et al. p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol. 2005 Feb;7(2):165–171. [PubMed]
12. Mizuno H, Spike BT, Wahl GM, Levine AJ. Inactivation of p53 in breast cancers correlates with stem cell transcriptional signatures. Proceedings of the National Academy of Sciences of the United States of America. 2010 Dec 28;107(52):22745–22750. [PMC free article] [PubMed]
13. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell stem cell. 2011 Jun 3;8(6):633–638. [PubMed]
14. Li Z, Yang CS, Nakashima K, Rana TM. Small RNA-mediated regulation of iPS cell generation. EMBO J. 2011 Mar 2;30(5):823–834. [PMC free article] [PubMed]
15. Lin SL, Chang DC, Chang-Lin S, Lin CH, Wu DT, Chen DT, et al. Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA. 2008 Oct;14(10):2115–2124. [PMC free article] [PubMed]
16. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol. 2009 Dec;11(12):1487–1495. [PubMed]
17. Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell. 2009 May 15;137(4):647–658. [PubMed]
18. Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 2008 Oct 23;455(7216):1124–1128. [PubMed]
19. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle. 2009 Oct 15;8(20):3274–3284. [PMC free article] [PubMed]
20. Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM, Pinna CM, et al. HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells. Cancer research. 2011 Jul 1;71(13):4640–4652. [PMC free article] [PubMed]
21. Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. Hypoxia enhances the generation of induced pluripotent stem cells. Cell stem cell. 2009 Sep 4;5(3):237–241. [PubMed]
22. Jinushi M, Chiba S, Yoshiyama H, Masutomi K, Kinoshita I, Dosaka-Akita H, et al. Tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jul 26;108(30):12425–12430. [PMC free article] [PubMed]
23. Iliopoulos D, Hirsch HA, Wang G, Struhl K. Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jan 25;108(4):1397–1402. [PMC free article] [PubMed]
24. Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, et al. Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell stem cell. 2010 Feb 5;6(2):141–152. [PMC free article] [PubMed]
25. Vermeulen L, De Sousa EMF, van der Heijden M, Cameron K, de Jong JH, Borovski T, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol. 2010 May;12(5):468–476. [PubMed]
26. Marson A, Foreman R, Chevalier B, Bilodeau S, Kahn M, Young RA, et al. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell stem cell. 2008 Aug 7;3(2):132–135. [PMC free article] [PubMed]
27. Cole MF, Johnstone SE, Newman JJ, Kagey MH, Young RA. Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. Genes Dev. 2008 Mar 15;22(6):746–755. [PMC free article] [PubMed]
28. Hu Q, Zhang L, Wen J, Wang S, Li M, Feng R, et al. The EGF receptor-sox2-EGF receptor feedback loop positively regulates the self-renewal of neural precursor cells. Stem cells (Dayton Ohio) 2010 Feb;28(2):279–286. [PubMed]
29. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell stem cell. 2009 Nov 6;5(5):504–514. [PubMed]
30. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell stem cell. 2007 Jun 7;1(1):55–70. [PubMed]
31. Yamanaka S. Elite and stochastic models for induced pluripotent stem cell generation. Nature. 2009 Jul 2;460(7251):49–52. [PubMed]
32. Feng B, Ng JH, Heng JC, Ng HH. Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells. Cell stem cell. 2009 Apr 3;4(4):301–312. [PubMed]

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