Mammary stem cells, self-renewal pathways, and carcinogenesis
© BioMed Central Ltd 2005
Published: 30 March 2005
The mammary gland epithelial components are thought to arise from stem cells that undergo both self-renewal and differentiation. Self-renewal has been shown to be regulated by the Hedgehog, Notch, and Wnt pathways and the transcription factor B lymphoma Mo-MLV insertion region 1 (Bmi-1). We review data about the existence of stem cells in the mammary gland and the pathways regulating the self-renewal of these cells. We present evidence that deregulation of the self-renewal in stem cells/progenitors might be a key event in mammary carcinogenesis. If 'tumor stem cells' are inherently resistant to current therapies, targeting stem cell self-renewal pathways might provide a novel approach for breast cancer treatment.
The mammary gland in humans and in other mammals is a dynamic organ that undergoes significant developmental changes during pregnancy, lactation, and involution. It is likely that the cellular repertoire of the human mammary gland is generated by a stem cell component. These stem cells have a unique capacity for self-renewal as well as for generating the three lineages that comprise the lobulo-alveolar structure of the adult gland: myoepithelial cells forming the basal layer of ducts and alveoli, ductal epithelial cells lining the lumen of ducts, and alveolar epithelial cells synthesizing milk proteins [1, 2]. Under the regulation of systemic hormones, as well as local stromal epithelial interactions, these cells proliferate extensively, differentiate during each pregnancy and lactation, and undergo apoptosis during mammary involution . It has been shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture, signifying the possible existence of a progenitor cell . Recently, Stingl and colleagues characterized the multipotent epithelial cells in the normal adult breast . In their experimental system, two distinct types of human breast epithelial cell (HBEC) progenitor population could be distinguished on the basis of their differential expression of the MUC-1 glycoprotein CALLA/CD10 and epithelial-specific antigen (ESA). MUC-1+/CALLA-/ESA+ progenitors (luminal restricted progenitor, or alveolar progenitor) expressed typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, and ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, MUC-1-to ±/CALLA± to +/ESA+(bipotent progenitor, or ductal progenitor), generated mixed colonies of both luminal and myoepithelial cells when seeded in two-dimensional and three-dimensional cultures. Furthermore, they suggested that the MUC-1+/CALLA-/ESA+ and the MUC-1-to ±/CALLA± to +/ ESA+ progenitors are candidate in vivo alveolar and ductal progenitors, respectively . HBEC clonal heterogeneity has also been reported by others . Such clonal heterogeneity might be indicative of an underlying stochastic mechanism regulating HBEC differentiation independently of the presence of factors (such as epidermal growth factor and insulin) that might be required to support the viability and/or stimulate the proliferation of these cells .
There is also increasing evidence that stem cells might be the targets of transformation during carcinogenesis. Carcinomas are believed to arise through a series of mutations that occur over many years. Adult stem cells are slowly dividing, long-lived cells, which by their very nature are exposed to damaging agents for long periods. They may therefore accumulate mutations that result in transformation . In favor of the role of stem cells in carcinogenesis comes the observation that normal stem cells and cancer stem cells share several important properties such as the capacity for self-renewal, the ability to differentiate, active telomerase and anti-apoptotic pathways, increased membrane transporter activity, anchorage independence and ability to migrate and form metastasis. The transformation of mammary stem and progenitor cells also contributes to the generation of tumor heterogeneity. There is now evidence for the existence of 'tumor stem cells' in human leukemias, myeloma, and brain tumors, as well as in breast carcinomas [7–12].
A unique property of stem cells is their ability to undergo self-renewal divisions. In normal organogenesis this process is tightly regulated. The deregulation of self-renewal might be one of the key events involved in carcinogenesis. Indeed, pathways involving cell signaling pathways and transcription factors involved in the self-renewal of normal stem cells have all been implicated in carcinogenesis. These pathways include Hedgehog, Notch and Wnt, as well as the transcription factor B lymphoma Mo-MLV insertion region 1 (Bmi-1). In this article we review evidence that these pathways are involved in both stem cell self-renewal and carcinogenesis, which provides support for the concept that breast carcinogenesis results from the deregulation of self-renewal pathways of normal mammary stem cells. We then discuss the implications of these studies for the development of novel therapies that target these self-renewal pathways.
Mammary stem cells
Stem cells are defined by their ability to undergo self-renewal, as well as multi-lineage differentiation. This self-renewal can be either asymmetric or symmetric. Self-renewal is distinguished from other proliferative processes in that at least one of the progeny of self-renewal is identical to the initial stem cell. In all other replicative processes, the progeny of division undergo a series of differentiation events . In asymmetric stem cell self-renewal, one of the two progeny is identical to the initial stem cell, whereas the other cell is a committed progenitor cell, which undergoes cellular differentiation. Because the product of an asymmetric self-renewal division is one stem cell and one differentiated cell, this process maintains stem cell number. In contrast, symmetric self-renewal results in the production of two stem cells; by its very nature this results in stem cell expansion. The processes that regulate the balance between asymmetric and symmetric divisions of stem cells are poorly defined, but recent evidence indicates a role for p53 and inosine monophosphate dehydrogenase . Although stem cells themselves are slowly dividing, progenitor cells derived from them are highly proliferative . This expanding progenitor cell also has the ability to differentiate into the lineages comprising the adult tissue.
The existence of self-renewing multipotent mammary stem cells has been clearly demonstrated by transplantation studies in mice and rats [16–18]. Fragments of mammary epithelium marked with mouse mammary tumor virus were able to regenerate a new gland after transplantation into a mammary fat pad cleared of its epithelial components . Serial transplantation of the clonally derived outgrowth recapitulated the entire functional repertoire of the gland, demonstrating the existence of self-renewing and multipotent mammary stem cells. A recent study in mice combining long-term labeling in vivo using bromodeoxyuridine with immunosorting and transplantation showed that mammary stem cell antigen-1 (SCA-1)-positive population is enriched in progenitor cells able to regenerate the gland in vivo .
Tumor stem cells
There is increasing evidence that both stem and progenitor cells may be the targets of transformation during carcinogenesis. As described above, normal stem cells and cancer cells share several important properties, including the ability to self-renew and undergo differentiation. However, the mutations and/or epigenetic events involved in carcinogenesis may disregulate these pathways. Ensuing aberrant differentiation might in turn contribute to the phenotypic cellular heterogeneity found in tumors. Using different systems, several investigators have demonstrated that only a minority of cells in human cancers are capable of self-renewal. This has been most convincingly demonstrated by examining the ability of subpopulations of tumor cells identified by cell surface markers to form tumors when transplanted into immunosuppressed NOD/SCID mice. This approach was first successfully used to demonstrate the existence of leukemic stem cells .
We have used a similar approach to identify a subpopulation of human mammary cancer cells bearing the phenotype ESA+CD44+CD25-/lowLineage- that have the properties of breast cancer 'stem cells'. As few as 100 of these cells, isolated from primary human breast carcinomas or metastatic lesions, are able to form tumors reproducibly in NOD/SCID mice. In contrast, tens of thousands of cells that do not bear this phenotype are unable to generate tumors in this model. Furthermore, consistent with a stem cell model is the observation that tumor stem cells are able to be serially passaged in NOD/SCID mice, each time generating a stem cell population, as well as the more differentiated non-tumorigenic cells forming the bulk of the tumor . These 'tumor stem cells' thus share the properties of self-renewal and differentiation with their normal stem cell counterparts, although in tumors these processes are deregulated.
Recent studies have provided evidence for the existence of 'tumor stem cells' in human multiple myeloma and brain tumors in addition to acute leukemias and breast cancer [28, 29]. An alternative model to the 'tumor stem cells' model is that cancers arise and evolve through stochastic mutations that are then expanded through clonal selection. Genetic instability and clonal selection undoubtedly do contribute to tumor heterogeneity and progression. However, the tumor stem cell model does not exclude the importance of these stochastic or selective events in tumor evolution. Both may in fact be operative in both tumorigenesis and tumor progression, and contribute to the heterogeneity found in cancer.
There has been some controversy about the nature of the cells that serve as targets of transformation. In a variety of malignancies, evidence for the clonal generation of tumors that display markers of multiple lineages has provided evidence for the stem cell as the cell of origin. However, in other cases, such as acute promyelocitic leukemia and chronic myelogenous leukemia, there is evidence for the transformation of progenitor cells. The transformation of progenitor cells might require mutations that allow them to undergo self-renewal, normally a process limited to stem cells. Indeed, we have recently proposed that the transformation of mammary stem and/or progenitor cells might result in the heterogeneity of breast cancer types between different patients, reflected in molecular profiling data . The molecular profile of tumors might be determined by both the cell of origin as well as the particular mutation profile, in turn determining the differentiation pattern of these cells, which comprise the bulk of the tumor. These categories defined by molecular profiling might have important diagnostic and prognostic implications. Regardless of the cells of origin, the common feature that might be required for transformation is the ability of the target cell to undergo self-renewal and subsequent expansion.
Thus, an understanding of the pathways that govern the self-renewal of normal stem cells, and the ways in which these pathways are deregulated during carcinogenesis, is of utmost importance. Several pathways found to have important roles in development and a transcription factor Bmi-1 have been shown to be involved in the regulation of stem cell self-renewal and differentiation. These pathways include Hedgehog, Notch, and Wnt. We review the role of these signaling pathways in stem cell self-renewal as well as evidence that these same pathways are important in the normal development of the mammary gland. We then discuss evidence that deregulation of these pathways is important in mammary carcinogenesis.
Recent studies have indicated that hedgehog signaling is important in embryonic mammary gland induction, ductal morphogenesis, and alveolar development. A critical role for hedgehog signaling in mediating epithelial stromal interactions during ductal development has been shown by the genetic analysis of two hedgehog signal transduction network genes, Ptch1 and Gli-2. Disruption of either gene leads to similar, yet distinct, defects in ductal morphogenesis that are mainly ductal dysplasias similar to the hyperplasias of the human breast. We have used the mammosphere-based culture system to examine the role of hedgehog signaling in mammary cell fate determination. Our data show that the addition of recombinant Shh can stimulate the formation of primary and secondary mammospheres and can increase mammosphere size, a process that can be blocked by the Smo inhibitor cyclopamine (Liu S et al., manuscript in preparation). These studies suggest that hedgehog signaling is involved in mammary stem cell self-renewal.
The importance of hedgehog signaling in carcinogenesis has been demonstrated by the fact that many of the genes involving hedgehog signaling are known oncogenes, including Smo, Shh, Gli-1, and Gli-2, or that Ptch1 can function as a tumor suppressor. Mutations in these genes have been linked to the development of many common cancers, which were shown to be dependent on activated Hedgehog signaling . Mutations in hedgehog signaling were first described in Gorlin syndrome and basal carcinomas of the skin. More recently, an important role for hedgehog signaling has been shown in medulloblastoma, prostate, and pancreatic carcinomas [33, 34]. Similarities between hedgehog mutation-induced ductal dysplasias and human breast pathologies suggest a role for altered hedgehog signaling in the development of mammary cancer. There is also evidence that altered hedgehog signaling has a direct role in the neoplastic progression of the mammary gland. One study showed Ptch1 mutation in two of seven human breast cancers . Recently, a natural polymorphism in the 3' end of the Ptch1 coding region (C3944T; Pro1315→Leu) has been linked to increased breast cancer risk associated with oral contraceptive use . Evidence for a role in breast cancer also comes from published genetic studies in mice showing hyperplastic defects in the mammary gland of ΔPtch1 + and ΔGli1 mutants . Recently, Kubo and colleagues showed that a specific inhibitor of hedgehog signaling, cyclopamine, is able to inhibit the growth of mammary carcinoma cells in vitro .
We have recently used the mammosphere system described above to study the role of Notch signaling in mammary cell fate determination. Our findings suggested that Notch signaling is active in several distinct developmental stages of the mammary gland and that Notch acts as a regulator of asymmetric cell fate decisions. Notch activation promoted the self-renewal of stem cells, whereas in later stages of development it biased cell fate decisions in mammary progenitor cells toward the adoption of a myoepithelial cell fate versus an epithelial cell fate . Musashi is a positive regulator of Notch signaling through an interaction with Numb mRNA and repression of its translation . More recently, Musashi-1 and Notch1 were shown to be the two key regulators of asymmetric cell division in human breast epithelial stem cells [44, 45]. These findings about the role of Notch in promoting the self-renewal of mammary stem cells, in addition to previous observations that it can function as a proto-oncogene [46, 47], suggest that abnormal Notch signaling might be involved in carcinogenesis, through the deregulation of normal mammary stem cell self-renewal.
The noncanonical Wnt signaling pathway  involves Frizzled receptors and the proteoglycan co-receptor Knypek. A cytoplasmic signal transduction protein Dishevelled (Dsh) localizes to the cell membrane through its DEP domain. Dsh activates Rho through the bridging molecule Daam1. The precise roles of Rho versus other Rho-family small GTPases such as Rac and Cdc42 remain unclear, as is the potential role of the JNK pathway. Dsh can also stimulate calcium flux and sequentially activates the calcium-sensitive kinases protein kinase C and calmodulin-dependent protein kinase II (Fig. 4b).
Recently, several studies have provided evidence for a direct role of Wnt signaling in the self-renewal of hematopoietic, epidermal, and gut stem cells [50, 51]. Retroviral transduction of activated β-catenin results in increased epidermal stem cell self-renewal and decreased differentiation. A direct role for dysfunction of this pathway in cancer was established by experiments in transgenic mice that showed that activation of the Wnt signaling pathway in epidermal stem cells leads to epithelial cancers . Furthermore, in breast cancers, it has been demonstrated that there is upregulation of the uncomplexed transcriptionally active form of β-catenin without mutations afflicting downstream components . A role for Wnt signaling in stem cell self-renewal of mammary stem cells was suggested by recent studies of Alexander and colleagues, who used transgenic mice to show that overexpression of Wnt ligands in mammary stroma or activated β-catenin in mammary epithelium leads to increased numbers of mammary stem cells . Studies linking this process to mammary carcinogenesis include those showing that mammary stem cells and progenitors might be targets for oncogenesis by Wnt 1 signaling elements .
Bmi-1 is a transcriptional repressor belonging to the polycomb (PCG) group of transcription factors. It was first identified in a B-cell lymphoma . Recently, Bmi-1 has been shown to be a key regulator of the self-renewal of both normal and leukemic stem cells [57, 58]. Bmi-1 has also been shown to be important in neuronal stem cell self-renewal . Several recent studies have suggested a link between Bmi-1 and mammary carcinogenesis. Bmi-1 was shown to be overexpressed in several human breast cancer cell lines. Furthermore, it was found that Bmi-1 regulates telomerase expression in mammary epithelial cells. These studies suggest that Bmi-1 might have a role in mammary carcinogenesis . Although the mechanisms by which Bmi-1 regulates stem cell self-renewal remain unclear, one important gene silenced by Bmi-1 might be P-16 . However, P-16 only partly mediated the effects of Bmi-1 proteins in neural stem cells, thereby suggesting that other factors might participate in Bmi-1's effects on stem cell self-renewal. Recent studies by Tlsty and colleagues  have suggested that the epigenetic silencing of P-16 might be an important event in early mammary carcinogenesis. Together, these studies suggest that normal stem cell self-renewal might be regulated through Bmi-1, partly mediated through the repression of P-16. During carcinogenesis, this process might be deregulated by the epigenetic silencing of P-16 through methylation of the P-16 promoter .
Interaction between self-renewal pathways
Although we have described signaling pathways that regulate stem cell self-renewal, individually it is clear that in vivo there are extensive interactions between the pathways. For instance, there is evidence for interaction between Hedgehog signaling and Notch signaling. One study provided evidence that secreted Shh might be involved in reinforcing the cell fate switch executed by Notch . Moreover, a recent study presented intriguing evidence that Notch signaling regulates Gli-2 expression in mouse skin, and inactivation of the Notch-1 gene in epidermis induces sustained expression of Gli-2 resulting in the formation of basal carcinoma-like tumors .
Recently we used our mammosphere-derived culture systems to examine the relationship between the hedgehog pathway and the Notch pathway, and we found that the activation of the Notch pathway resulted in the subsequent activation of the hedgehog pathway, including increased expression of Ptch and Gli. This activation could be blocked by γ-secretase inhibitor, which inhibits Notch signaling (Liu S et al., manuscript in preparation). These studies suggest that hedgehog acts downstream of Notch. In contrast, one study showed that Shh acts upstream of Notch to determine arterial cell fate during arterial endothelial differentiation . Furthermore, we have evidence that activation of hedgehog pathway by the hedgehog ligands (Shh or Ihh) increased the expression of the Notch pathway target, HES1, in the mammospheres, and this effect could be blocked by the hedgehog inhibitor cyclopamine (Liu S et al., manuscript in preparation). Together, these studies indicate that Hedgehog and Notch might form a feedback loop regulating normal development.
Furthermore, deregulation of this loop might be involved in cancer formation. In the skin, the activation of two markers of active Wnt signaling, β-catenin and LEF-1, are associated with Notch-dependent transformation . The activation of Smo might initiate processes during which transcription factors belonging to the Gli family are activated, and modify the transcription of Ptch and Wnt . Wnt regulation has previously been observed in human basal carcinomas, indicating that tumor progression is mediated by interactions of distinct signaling pathways that regulate organ development during embryogenesis. All of these pathways are also intimately involved in the regulation of stem cell self-renewal. Interestingly, Bmi-1 expression was rapidly increased after the addition of Shh or after the overexpression of the Shh target Gli in cerebellar granular cells, which implies that Bmi-1 is a downstream target in the Shh pathway . Overexpression of Bmi-1 correlated with overexpression of Ptch and SuFu, which suggests at least a partial activation of the Hedgehog pathway in Bmi-1 overexpression tumors . In our preliminary data we showed that both the activation of Hedgehog pathway by Shh or Ihh and the activation of the Notch pathway by DSL resulted in the expression of Bmi-1 in the mammosphere culture system, and the induction of Bmi-1 expression could be blocked by the pathway-specific inhibitors cyclopamine in the Hedgehog pathway and γ-secretase inhibitor in the Notch pathway, respectively (Liu S et al., manuscript in preparation).
Conclusions and clinical implications
In this review we have presented evidence that carcinogenesis in the mammary gland, and in other organs, might result from transformation of stem and/or progenitor cells by the deregulation of self-renewal pathways. These pathways include Hedgehog, Notch, Wnt, and the transcription factor Bmi-1. The hypothesis that mammary carcinogenesis results from the deregulation of normal stem cell self-renewal pathways suggests that components of these pathways might provide attractive targets for therapeutic development. This is of great importance because current therapies may be limited in their effectiveness by virtue of the fact that they might selectively target the more differentiated cells in a tumor. Tumor stem cells, by virtue of their slow cell cycle kinetics, transporter proteins, and anti-apoptotic mechanisms, might be resistant to these treatments (reviewed in ). The targeting of self-renewal pathways might provide a more specific approach to the elimination of cancer stem cells. A potential challenge in this regard is the development of therapies that selectively affect cancer stem cells while sparing normal stem cells that may rely on similar mechanisms for self-renewal. Recent studies have shown that inhibitors of hedgehog signaling, such as cyclopamine, can inhibit mammary tumor cells in vitro . Furthermore, a small-molecule inhibitor of the Shh pathway – a Hedgehog antagonist (HhAntag) – has recently been reported to eliminate medulloblastoma in transgenic mice without apparent systemic toxicity . These studies suggest that strategies aimed at targeting cancer stem cell self-renewal might provide a novel therapeutic approach for the treatment of breast and other cancers.
B lymphoma Mo-MLV insertion region 1
glycogen synthase kinase
human breast epithelial cell
suppressor of fused.
Thanks are due to Dr Thomas Giordano for tissue procurement, Dr Michael Clarke for technical advice, and Dr Ilia Mantle and Nam-shik Ahn for critical review of the paper. This work was supported by NIH grants CA66233 and CA101860 and in part by the University of Michigan Cancer Center NIH Support Grant (5 P 30 CA46592).
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