Cancer stem cells and the cell cycle: targeting the drive behind breast cancer

Figure 1. miR-17/20 regulatory loops.

(A) E2F-miR-17/20 regulatory loop. (B) AML1-miR-17/20 regulatory loop. (C) c-Myc-miR-17/20 regulatory loop. (D) Cyclin D1-miR-17/20 regulatory loop^*.

^*From [18].

miR: Micro RNA.

Adult stem cells have the ability to both self renew and provide differentiated progeny. A substantial body of evidence supports a model in which a small subset of cells within a tumor, referred to as cancer stem cells (CSCs), or tumor-initiating cells, contribute to the initiation, maintenance and therapeutic resistance of cancers. CSCs have been identified within hematological and solid tumor types. Initially characterized as slow-cycling, long-lived cells that reside in discrete histological niches, recent studies have identified highly proliferative non-niche multilineage progenitors of several cell types. Genetic-deletion analysis has identified an important role for components of the cell cycle machinery (D-type cyclins, cyclin-dependent kinase inhibitors and pRb protein) in stem cell function. Studies from Drosophila suggest a role for factors regulating asymmetrical cell division, cell shape, protein translational control and miRNA in stem cell division. In turn, the cell cycle control proteins appear to regulate and be regulated by many of these processes. Given the importance of CSCs in therapeutic resistance, approaches targeting either niche components that maintain tumor stem cells or cell cycle components within CSCs represent important new opportunities for intervention.

The ability of a tumor to proliferate and propagate relies on a small population of stem-like cells, known as CSCs. A growing body of evidence suggests that CSCs are the source of primary tumors as well as relapses. CSCs share several properties with normal stem cells, including their capacity for self renewal and their ability to differentiate [1,2]. Candidate CSCs have been identified in leukemias and solid tumors, including glioblastoma, medulloblastomas and carcinomas [3–13]. The hypothesis that tumor growth depends on CSCs is supported by the evidence that:

• • CSCs have been identified in tumors;

• • Purified (or enriched) CSCs have the ability to generate tumors that recapitulate the original tumor heterogeneity when transplanted into animals;

• • CSCs are more resistant to chemo- and radiotherapy than the non-CSC tumor bulk;

• • CSCs seem to increase tumor aggressiveness before and after therapy.

Cell cycle in breast CSC biology

The term ‘stem cell’ was coined by Valentin Haecker in 1890 during experimentation with crustacean primordial germ cells. Stem cells maintain constant cell size over multiple cellular divisions. The length of the G₁ phase in murine embryonic stem cells (MESCs) is shorter than in somatic cells, in the order of approximately 10 h, with a short G₁ and high percentage of S phase. Normal stem cells spend the majority of their time in the G₀ phase of the cell cycle. During differentiation, the proportion of cells in G₁ increases with the loss of pluripotency. Lengthening of the G₁ phase in neural stem cells correlates with a loss of differentiation, suggesting important links between stem cell capacity and cell cycle regulation [14]. Long-term hematopoietic stem cells (HSCs) do not express molecules associated with the initiation of the cell cycle; whereas these molecules are expressed by short-term HSCs and early progenitor cells that are not self-renewing [15].

Mechanistic insights into the cell cycle control mechanisms of stem cells have been produced from studies in Drosophila. In Drosophila, somatic cell expansion is limited by cell cycle factors that directly control the asymmetric cell-division machinery and vice versa[16]. Mutation analysis in Drosophila has identified mutations that trigger progenitor cell overproliferation (e.g., brat, mei-p26, pros, bam, lethal giant larvae and polo) [16]. The cellular polarity machinery can also affect the stem cell cell cycle, since loss of lgl and pins triggers asymmetric self-renewal. The stem cell division rate is regulated by miRNA, suggesting the importance of protein translational control in self-renewal. By limiting biosynthetic capacity and governing asymmetrical cellular division, miRNA plays a key role in coordinating growth with cell cycle control. The germline stem cell division rate is determined by miRNA biogenesis factors (dicer-1 loquaciousness, argonaute family effector proteins [e.g., Ago1, Piwil and miRNA itself] [16]). The miRNA and cell cycle proteins are linked in tightly coordinated feedback loops. In mammalian cells, the miR-290 cluster regulates MESC functions via p21^CIP1[17]. In MESCs, Cdk2 activity, which is elevated throughout the cell cycle without a periodicity, declines during the transition from pluripotency to differentiation associated with increased p21^CIP1. mi-R17/20 represses cyclin D1 through its 3´ untranslated region (UTR) and, in turn, cyclin D1 activates mi-R17/20 through binding to the miRNA17/20 promoter (Figure 1)[18].

Breast CSCs isolated from human tumors are predominantly (75%) in G₀/G₁ phases [4]. Human breast CSCs, characterized by cell surface CD44⁺/CD24^-/low/ESA⁺ and by lineage markers (lack of expression of CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b), form tumors when injected into nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice [4]. The tumors share the histological heterogeneity of the initial tumor. The CD44⁺/CD24^-/low/lineage^- cells form new heterogeneous tumors upon serial passage. Breast CSC have been isolated from patient samples after in vitro propagation [2] and from breast cancer cell lines [19]. Mammospheres enriched with CD44⁺/CD24^-/low-staining cells retain tumor-initiating capability when injected into NOD/SCID mice. Aldehyde dehydrogenase (ALDH), a detoxifying enzyme associated with hematopoietic and neural stem cells [20,21], also serves as a marker of breast CSCs. The expression of ALDH correlates with poor prognosis in human breast carcinomas [22]. In the mouse, a subpopulation of CD24⁺/CD29^high (β1-integrin subunit [CD29]) cells exhibit features of self-renewal and tumorigenicity [23].

The retention of DNA-labeling analogs, such as BrdU or ³H thymidine, is thought to be a characteristic of stem cells. Although epidermal stem cells have been characterized as slow-cycling long-lived cells, recent findings suggest multilineage epidermal progenitors can be isolated from highly proliferative non-niche regions [24]. Mammary stem cells are also cycling with the majority of cells in the G₁ and S/G₂/M phases of the cell cycle [25].

Role of cyclins & cyclin-dependent kinase inhibitors

Analysis of cell cycle control proteins in stem cell function in mice has been revealing. Mice deficient in the D-type cyclins were defective in hematopoietic progenitor cell function, with reduced myeloid progenitors, erythrocytes and megakaryocytes [26]. Defective lymphoid and hematopoietic lineages in these mice indicate the importance of D-type cyclins in stem cell function.

The cyclin-dependant kinase (CDK) inhibitors p21^CIP1/WAF (p21) and p27^KIP1 (p27) act as negative regulators of the cell cycle through the inhibition of cyclin/CDK complexes [27]. CDK inhibitors participate in the cell cycle control of normal stem cells [28] and breast cancer cells [29], suggesting a role of CDK inhibitors in breast CSC fate. p21^CIP1 was initially discovered as a cyclin/CDK inhibitor that functions as a downstream effector of tumor suppressors, including p53, BRCA1, WT1 and TGFβ. p21^CIP1 inhibits cellular growth in tissue culture and tumor xenograft formation [30]. In the absence of p21^CIP1, HSC proliferation and absolute number increase, suggesting that p21^CIP1 governs HSC cell cycle entry [28]. p21^CIP1 mice develop spontaneous tumors [31] and mammary gland-targeted Ha-Ras or c-Myc-induced tumorigenesis were each enhanced by p21^CIP1 deficiency [32]. However, PDGF-induced gliomagenesis and ATM- and p53-mediated tumorigenesis was reduced in the p21^CIP1-deficient background, suggesting a tumor-promoting function of p21^CIP1. p21^CIP1 restricts the entry of HSCs into the cell cycle [28], and is crucial in protecting stem cells under genotoxic stress conditions, such as radiation [33] and 5-fluorouracil exposure [34].

p27^KIP1 functions as a mammary tumor suppressor with haploinsufficiency [35]. A p27^KIP1 allele mutant in the cyclin-binding domain (p27^CK-) resulted in cytoplasmic accumulation of p27^KIP1 and, as a genetic knock-in in the mouse, resulted in the development of spontaneous lung tumors associated with bronchoalveolar stem cell expansion [36]. These studies suggest that p27^KIP1 may play a role in tumor stem cell expansion. As cytoplasmic p27^KIP1 binds and inhibits RhoA [37,38], and inhibition of RhoA in HSCs increases self renewal [39], p27^KIP1 may regulate stem cell function via RhoA. The cytoplasmic cyclin D1-mediated induction of cellular migration requires p27^KIP1[40], and D-type cyclins regulate HSC; thus, the role of D-type cyclins in CSC function will be of interest.

Mitochondrial function has been implicated in cellular differentiation and stem cell function. pRb is required for stress-induced erythropoiesis, and pRb deletion in the erythroid component demonstrated a cell-intrinsic defect in early-to-late erythroblasts [41]. pRb deletion decreases, and cyclin D1 deletion increases, mitochondrial function [41,42], suggesting an important interaction between stem cell function, the cell cycle and mitochondrial metabolism.

Strategies to target the cell cycle in CSCs

Quiescent, noncycling CSCs may contribute to chemotherapeutic resistance, as this therapy acts mainly on cycling cell populations [43]. Strategies to selectively target the cell cycle components have recently been reviewed [44]. An alternative approach to regulate CSCs is to target the CSC niche. The physiologically discrete site or niche in which HSCs reside provides a physical anchoring site for stem cells via adhesion molecules. The niche controls stem cell number, growth and differentiation [45–47]. The niche of CSCs includes fibroblasts, infiltrating cells from the immune system and endothelial cells [39,48], which secrete factors that participate in the regulation of CSCs. Stem cell factor, via its receptor c-Kit, induces HSC migration and expansion, and stem cell factor is induced by factors such as c-jun [49]. It has been hypothesized that the proliferation and/or differentiation of CSCs can be activated by deregulated signaling from the niche [50,51].

CD44 and other adhesion receptors (e.g., cadherins and integrins), mediate the interaction of CSCs with their niche. CD44, for example, interacts with the extracellular matrix component hyaluronic acid to enhance the recruitment of CD44 into a complex with Nanog. In Drosophila, stem cells reside in cellular niches or in noncellular niches on basement membranes [52]. Nanog is a transcription factor that promotes self renewal and maintenance of pluripotency in embryonic stem cells. p53 binds and represses the CD44 promoter [53] and, in p53-null cells, the loss of CD44 repression may allow expansion of the CSC pool. Evidence that integrins modulate CSCs includes the finding, in serial passages of mammospheres, that α6-integrin inactivation with antibodies or siRNA abrogates mammosphere-forming ability and tumorigenicity [54]. The interruption of the signals generated by cells in the CSC niche using antibodies or soluble ligands against adhesion receptors may be useful in blocking cancer progression or improving therapeutic responsiveness [55].

Acknowledgements

We thank Barbara Lupo and Atenssa L Cheek for the preparation of this manuscript.

Financial & competing interests disclosure

Richard Pestell’s work was supported in part by R01CA70896, R01CA75503 and R01CA86072, and the Kimmel Cancer Center is supported by the NIH Cancer Center Core grant P30CA56036. Richard Pestell recieved a generous grant from the Ralph and Marian C Falk Medical Research Trust, and the work was supported in part by a grant from the Pennsylvania Department of Health. The department disclaims responsibility for any analysis, interpretations or conclusions. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.