Therapeutic Implications of Cellular Heterogeneity and Plasticity in Breast Cancer

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Perspective Therapeutic Implications of Cellular Heterogeneity and Plasticity in Breast Cancer Michael D. Brooks,1,2 Monika L. Burness,1,2 and Max S. Wicha1,* 1Department

of Internal Medicine, University of Michigan Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.08.014 2Co-first

Cellular heterogeneity represents one of the greatest challenges in cancer therapeutics. In many malignancies, this heterogeneity is generated during tumor evolution through a combination of genetic alterations and epigenetic events that recapitulate normal developmental processes including stem cell self-renewal and differentiation. Many, if not most, tumors display similar hierarchal organization, at the apex of which are ‘‘stem-like cells’’ that drive tumor growth, mediate metastasis, and contribute to treatment resistance. Using breast cancer as a model, we discuss how an improved understanding of tumor cellular heterogeneity and plasticity may lead to development of more effective therapeutic strategies. Introduction Generation of Cellular Heterogeneity during Carcinogenesis and Tumor Evolution Several models have been proposed to explain cellular heterogeneity within tumors. The clonal evolution model has focused on random mutation and clonal selection to generate cellular heterogeneity. Elegant mutational analysis has demonstrated the existence of numerous sub-clones within solid tumors including breast cancer (Kandoth et al., 2013). Cells within these sub-clones may interact through paracrine interactions (Zhang et al., 2015; Rajaram et al., 2015; Fillmore et al., 2010). Furthermore, several studies have demonstrated both genetically similar and genetically diverse sub-clones in primary tumors and metastases in individual patients (Ding et al., 2010; Vignot et al., 2013), consistent with clonal evolution models. The cancer stem cell (CSC) model posits that tumors are hierarchically organized and, at the apex of the hierarchy, are cells that display stem cell properties. These properties include self-renewal, as well as the ability to generate more differentiated cells forming the bulk of the tumor. Hierarchical tumor organization was first demonstrated in human leukemia (Bonnet and Dick, 1997). This was followed by the demonstration that solid tumors, including those of the breast (Al-Hajj et al., 2003), colon (O’Brien et al., 2007; Ricci-Vitiani et al., 2007), brain (Singh et al., 2004), ovaries (Zhang et al., 2008), lungs (Eramo et al., 2008), prostate (Collins et al., 2005), and pancreas (Li et al., 2007) display a similar hierarchical organization The existence of CSCs in melanoma remains controversial, since the ability of human melanoma cells to initiate tumors in immunosuppressed mice was shown to be dependent on the mouse model system utilized (Quintana et al., 2008). Nevertheless, the preponderance of evidence suggests that the vast majority of both hematologic and solid tumors contain cells that display stem cell properties (Visvader and Lindeman, 2008). In these tumors, cells displaying protein markers including CD44, CD133, or the enzyme aldehyde dehydrogenase (ALDH) were able to generate tumors when transplanted into immunocompromised mice (Bonnet and Dick, 1997; AlHajj et al., 2003; Ginestier et al., 2007). These transplanted cells generated tumors that recapitulated the phenotypic heterogene260 Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc.

ity of the initial tumor. Although CSC markers have proven useful for the enrichment of CSC populations, their utility is limited by variability of expression and regulation by environmental factors. As a result, definitive identification of CSCs requires functional assays demonstrating the capacity to initiate tumors in mouse models. As has been emphasized by others, stochastic clonal evolution and stem cell models are not mutually exclusive. CSCs may serve as the unit of selection in the genetic evolution of tumors while also being genetically unstable, generating multiple clones consisting of genetically altered CSCs and their differentiated progeny (Figure 1). Furthermore, as discussed below, cancer cells display a remarkable degree of phenotypic plasticity, and there is evidence that more differentiated tumor cells may acquire ‘‘stem cell’’ properties through a ‘‘dedifferentiation’’ process, a phenomenon with significant clinical implications. Interpretations of mouse transplantation studies are limited by the environmental perturbations associated with these approaches. To circumvent these limitations, a number of groups have employed lineage tracing to demonstrate the generation and propagation of CSCs. For example, Parada’s group has demonstrated that a Nestin-driven GFP reporter marks neural stem cells in the brain subventricular zone as well as a corresponding population of glioma stem cells. These Nestin/GFP+ cells repopulate the tumor after treatment with chemotherapy or genetic ablation (Chen et al., 2012). Similar lineage tracing studies utilized stage-specific Cre-recombinase labeling to demonstrate the presence of stem cells in a squamous carcinoma model system (Driessens et al., 2012). Elegant lineage tracing has been reported by the Clevers group who employed this method to follow the fate of colon stem cells during carcinogenesis. Using a confetti reporter, in which Cre randomly activates one of four fluorescent reporters, it was shown that intestinal adenomas originate in cells expressing the stem cell marker Lgr5 (Schepers et al., 2012). These studies and others have provided support to hierarchal models of tumor development in which cells that display stem-like properties drive tumor initiation and propagation. In this Perspective, we will discuss the mechanisms that generate cellular heterogeneity during

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Figure 1. Cellular Heterogeneity and Plasticity in Breast Cancer Recent work has identified two distinct types of CSCs in breast cancer: a mesenchymal, quiescent type marked by being CD44+/CD24 (EMT-CSCs) and an epithelial, proliferative type identified as ALDH+ (MET-CSCs). A double-positive CD44+/CD24 /ALDH+ population with even higher tumorigenicity exists, but it is yet to be determined if this is a stable or transient population. The transition between these states is common in vitro and is likely to be regulated by multiple cell types in the tumor microenvironment through IL-8, Notch, and GH (growth hormone) signaling as well as paracrine interactions between CSCs and their more differentiated progeny. Derived from these two CSC states are two differentiated bulk tumor cell types: an epithelial type produced by MET-CSCs (common in most of the molecular subtypes) and a group of mesenchymal bulk tumor cells derived from EMT-CSCs (rare in the majority of breast cancer cases, but common in the claudin-low molecular subtype). Among the corollaries of this model is the idea that the probability of dedifferentiation is not uniform, but is instead inversely proportional to the number of cellular divisions removed from being a CSC. Furthermore, mutations in CSCs generate new CSCs as well as clones of differentiated progeny generating cellular heterogeneity during tumor evolution.

carcinogenesis. Since the role of genetic clonal heterogeneity in the mediation of therapeutic resistance has been the subject of excellent recent reviews (Bedard et al., 2013; Burrell et al., 2013), we will focus on the role of epigenetically generated heterogeneity and cellular plasticity in generating tumors driven by cells that display stem cell properties. We will also discuss the therapeutic implications of this epigenetic heterogeneity across the molecular subtypes of breast cancer. Breast Cancer Stem Cells: Defining the Cell of Origin, CSCs, and Their Relationships The CSC hypothesis is composed of two important, but separable, components: the first concerns the cellular origin of cancer, and the second addresses the nature of cells that are responsible for tumor maintenance and progression.

Cellular Origin of Breast Cancer Multiple cells within the breast have been proposed as the ‘‘cell of origin’’ in the development of human breast cancer. An understanding of the relationship between cellular hierarchies in the normal breast is critical to elucidating the cells of origin that generate the diverse molecular subtypes of breast cancer. Unfortunately, studies aimed at understanding this hierarchy in normal tissue have been complicated by the choice of markers, plasticity between cell states, and differences between human and mouse tissue. Early studies utilizing the protein marker CD24 suggested that the CD24low population (myoepithelial) had the greatest repopulation capacity compared to CD24hi (luminal) or CD24neg (non-epithelial) cells (Sleeman et al., 2006). Further studies using additional markers, however, revealed that both CD24+/CD29hi (Shackleton et al., 2006) and Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc. 261

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Perspective CD24+/CD49fhi (Stingl et al., 2006) cells retained the greatest capacity to reconstitute mammary glands, containing both lineages when transplanted into the cleared mammary fat pads of syngeneic mice. In addition to the controversies surrounding the selection of stem cell markers, there has been debate concerning the relative importance of bipotent and unipotent luminal and myoepithelial stem cells during normal development and carcinogenesis. Lineage tracing experiments using a series of cytokeratins driving Cre-Recombinase demonstrated that while cytokeratin 14 (K14) marks a multi-potent progenitor capable of generating all of the mammary lineages during development, unipotent basal and luminal stem cells are responsible for lineage maintenance (Van Keymeulen et al., 2011). However, subsequent cytokeratin-5-driven lineage-tracing studies reported that both bipotent mammary stem cells MaSCs and unipotent luminal and myoepithelial progenitor cells are involved in morphogenesis and homeostasis (Rios et al., 2014). Two important recent publications have shed further light on questions concerning the cellular origin of breast cancers as well as the relative roles of particular oncogenic drivers versus the cell of origin in determining their phenotype. Reports by two independent groups using mouse mammary models (Koren et al., 2015; Van Keymeulen et al., 2015) revealed that the introduction of oncogenic PIK3CAH1047R, a common mutation found in human breast cancer, into either luminal or basal restricted mammary progenitor cells resulted in dedifferentiation, endowing the cells with multilineage potential. Furthermore, the phenotype of the transduced cell determined the phenotype of the resulting tumors. These studies suggest the combined importance of particular genetic alterations and the cell of origin in determining molecular characteristics and clinical behavior of breast cancers. Although it remains to be determined if similar mechanisms are involved in the generation of human breast cancer phenotypes, the marked similarities in gene expression profiles in these mouse models and human breast cancers suggest that this may be the case (Van Keymeulen et al., 2015). In parallel to studies in murine mammary development, elucidations of stem cells in the human mammary gland have relied on marker expression coupled with functional assays including mammosphere formation (Dontu et al., 2003) or transplantation into humanized mouse mammary fat pads (Kuperwasser et al., 2004). Quantification of mammary repopulating units in these mouse models demonstrated that the markers CD49f+CD29+CD24low identified a cell population capable of generating human mammary structures composed of luminal and myoepithelial lineages (Eirew et al., 2008). Additional studies utilizing the markers CD49f, EpCAM, and CD10 demonstrated that immature mammary lobules contained the greatest proportion of stem cells with bi-lineage differentiation potential (Arendt et al., 2014). This important spatial distribution was validated by a separate study using a different set of markers including ALDH1A1, SSEA4, CD44, and CK14+/CK19+ (Honeth et al., 2015). However, given the significant differences in anatomy and cellular complexity between human and mouse mammary glands, the exact relationship between stem cells in human and rodent models remains to be definitively determined. 262 Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc.

Guided by the information gathered from the studies on the mouse and human hierarchies, efforts have focused on those cells within normal breast tissue that may be the cell of origin for breast tumors. Although it has been postulated that breast cancers may arise from normal breast stem cells, it is more likely that the various molecular subtypes of breast cancer arise from cells at different levels within the mammary hierarchy. Expression profiling revealed strong similarities between the claudinlow subtype and MaSCs, while the basal subtype aligns with luminal progenitor cells (Shehata et al., 2012). In concordance with these findings, it was shown that breast tumors in women with germline BRCA1 mutations contain enriched populations of luminal progenitor cells. These tumors are most often characterized as the basal molecular subtype (Molyneux et al., 2010). Pre-clinical studies have suggested that BRCA1 plays an important role in mammary stem/progenitor cell differentiation and that the loss of BRCA1 function in mouse models expands the population of cells expressing ALDH1, a marker of both normal and cancer stem-like cells (Molyneux et al., 2010). These and other studies have led to the suggestion that basal breast cancers arise from luminal progenitor cells defined as CD49+/ESA+ (Lim et al., 2009). However, this population itself is heterogeneous and includes a sub-population defined by expression of ALDH, a population recently shown to have properties of epithelial-like multi-potent stem cells (Liu et al., 2014). In addition to being the cell of origin of basal tumors, ALDH1-expressing stem/luminal progenitor cells may also be the cell of origin of other molecular subtypes of breast cancer. A more differentiated luminal progenitor may give rise to the most highly differentiated luminal tumors. The studies designed to elucidate the cell of origin of the different molecular subtypes of breast cancer have largely relied on transduction of activated oncogenes into mammary cells whose differentiation state is defined by marker expression followed by transplantation into the mammary fat pads of immunosuppressed mice. These studies have suggested that both the cell of origin and the specific oncogenic events contribute to the diversity of molecular subtypes of breast cancer. However, these studies are limited by potential artifacts introduced by transplantation. As previously noted, Rios et al. (2014) have demonstrated that normal mouse mammary stem cells maintain the capacity for multiple lineages upon transplantation but also maintain unipotent lineage differentiation in their normal unperturbed microenvironment as accessed by lineage tracing. Application of similar lineage tracing technologies to mammary carcinogenesis may provide a more definitive characterization for the cell of origin of murine mammary cancers. Extrapolation to human disease will necessitate the utilization of transplantation technologies. The elucidation of the cell of origin of human breast cancers is of considerable importance in developing effective cancer prevention strategies. CSC State Plasticity and the Tumor Microenvironment Recent work has demonstrated that breast cancer stem cells (BCSCs) maintain the plasticity to transition between two different phenotypic states: a more proliferative epithelial-like state characterized by expression of the CSC marker ALDH and a more quiescent and invasive, mesenchymal-like state characterized by the expression CD44+/CD24 . These states share the molecular characteristics of both luminal and basal

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Perspective stem cells in the normal breast (Liu et al., 2014). Each of these CSC states is also capable of generating their respective epithelial or mesenchymal bulk cell progeny, which can secrete signals to positively reinforce CSC self-renewal (Zhang et al., 2015; Fillmore et al., 2010). Transition between the two CSC states is mediated by epigenetic alterations regulated by the tumor microenvironment through cytokine and chemokine signaling, transcriptional regulation (including differential expression of microRNAs; Pal et al., 2015), or some combination thereof (Figure 1). These concepts expand upon previous work that suggested an important relationship between CSCs and the process of epithelial mesenchymal transition (EMT) (Mani et al., 2008). In the expanded model, EMT-like CSCs are localized at the tumor invasive edge where they are capable of entering the circulation and forming micro metastases at distant sites. The transition back to the proliferative epithelial state at distant sites is mediated by factors such as ID1, which, through downregulation of TWIST, induce this phenotypic switch (Stankic et al., 2013). A thorough understanding of CSCs requires delineation of how the associated tumor microenvironment mediates the balance between the two CSC states. Unfortunately, while recent experiments using syngeneic mouse models and transplantation into immunocompetent hosts have confirmed the existence of CSCs in the presence of immune cells, most CSC experiments have so far been done in immunocompromised mouse models. These and other studies suggest that CSCs may deploy specific mechanisms to thwart immune surveillance (Pan et al., 2015). Immune cells are critical components of the tumor microenvironment, and rapidly accumulating evidence suggests that the immune system plays a role in carcinogenesis and tumor development. This emphasizes the importance of utilizing immunocompetent models to more fully explore the mechanisms by which the tumor microenvironment regulates CSCs. An important issue in CSC biology is whether bulk tumor cells are capable of ‘‘dedifferentiating’’ into CSCs. If this were a common occurrence, then the concept of ‘‘CSC states’’ rather than fixed populations would be applicable. However, in normal cells, the process of differentiation is very tightly controlled by epigenetic events involving histone and DNA modifications. As a result, the differentiation process is largely unidirectional (Cantone and Fisher, 2013). However, the ability to reprogram fully differentiated cells into pluripotent stem cells through the addition of the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) clearly indicates that dedifferentiation can be driven to occur in normal cells. It is important to note that, in in vitro systems, this process is extremely inefficient. Furthermore, the frequency with which such dedifferentiation events occur during carcinogenesis is a matter of conjecture. Guo et al. demonstrated that differentiated breast cancer cells could be converted back to stem-like cells though combined expression of the transcription factors SLUG and SOX9 (Guo et al., 2012). Dedifferentiation has also been shown in gliomas with loss of NF1 and p53 in astrocytes and neurons (Friedmann-Morvinski et al., 2012). In intestinal tumorigenesis, overexpression of NFkB and b-catenin causes mitotic intestinal epithelial cells to regain stem cell properties (Schwitalla et al., 2013). However, many of these studies suggesting dedifferentiation have been performed in vitro (Guo et al., 2012), and the actual contribution of dedifferentiation during carcinogenesis remains unknown. Lineage

tracing studies involving stem cells as well as differentiationstage-specific promoters in mouse models will be required to definitively demonstrate the importance of dedifferentiation in the generation of CSCs. Once a thorough understanding of how dedifferentiation contributes to cancer initiation and progression has been achieved, subsequent studies can begin to analyze how this information can be used to facilitate better therapeutic treatments. Mathematical modeling has shown that dedifferentiation and plasticity will substantially reduce the effectiveness of CSC-directed therapies and increase rates of resistance across a range of dedifferentiation rates (Leder et al., 2010). An extreme version of cancer cellular plasticity is represented by the phenomenon of ‘‘vascular mimicry,’’ where there is evidence that CSCs from a number of tumor types undergo transdifferentiation to form vascular-like networks (Maniotis et al., 1999; Soda et al., 2011; Ricci-Vitiani et al., 2010). In breast cancer, recent studies have implicated two secretory proteins, SERPINE2 and SLPI, which are involved in this phenomenon (Wagenblast et al., 2015). These vascular mimicry networks have been associated with increased levels of metastasis. However, a more impactful role could be the generation of an expanded niche for CSCs, as endothelial cells have been shown to be important for both normal and CSC regulation. CSCs and the Molecular Subtypes of Breast Cancer Data generated over the past several decades suggest that, rather than being a single disease, breast cancers represent multiple diseases that can be subdivided into clinical subtypes based on cellular expression of estrogen and progesterone receptor (ER and PR, respectively), as well as the growth factor receptor HER2 (Erb-B2). By this classification, tumors are classified as hormone-receptor positive (those that express ER and/or PR), HER2-positive, or triple-negative (TN; those that do not express ER, PR, or HER2). Gene expression profiling has expanded upon this classification, defining tumors as luminallike (further divided into luminal A and luminal B); HER2-positive, characterized by amplification of the HER2 gene; or basal-like (Perou et al., 2000; Sørlie et al., 2001). The development of specific therapeutic strategies for each of the major pathologic subtypes of breast cancer has improved outcomes for many breast cancer patients. In these tailored approaches, ER-positive breast cancers are treated with hormonal therapies, cancers with HER2 gene amplification are treated with HER2-targeting agents and, in the absence of specific targeted therapeutics, the TN collection of breast cancers is treated with cytotoxic chemotherapy. These therapeutic approaches have emphasized genomic and phenotypic differences between the subtypes of breast cancer and have been designed to target bulk tumor cell populations. In addition to having different cells of origin, the different molecular subtypes of breast cancer are characterized by different frequencies of mesenchymal and epithelial CSC types as well as differentiation states of bulk cell populations. These differences are illustrated in Figure 2. The clinically aggressive claudin-low subtype, which is typically a TN breast cancer (TNBC), is characterized by a high proportion of CD44+/CD24low-expressing and ALDH1-expressing CSCs as well as mesenchymal-like bulk tumor cells. In contrast the basal subtype, also commonly TN and often associated with BRCA1 loss of function, contains Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc. 263

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Perspective Figure 2. Model Illustrating Variations in CSCs and Their Differentiated Progeny across the Spectrum of Breast Cancer Subtypes In this model, the different molecular subtypes of breast cancer are characterized by varying proportions of CSCs in mesenchymal (EMT) versus epithelial (MET) states as well as differential blocks in the differentiation hierarchy seen in normal mammary development.

epithelial bulk cells along with a subcomponent of mesenchymal bulk tumor cells and a higher proportion of epithelial ALDH1positive CSCs. The CSCs give rise to bulk tumor cells that are characterized by an epithelial morphology and lack of expression of the steroid hormone receptors ER or PR (Perou, 2011). The HER2-positive subtype of breast cancer is characterized by a high proportion of ALDH1-positive CSCs that give rise to epithelial bulk populations that may or may not express ER and PR (Korkaya and Wicha, 2013). Luminal B breast cancers generally express a lower proportion of cells expressing CSC markers than either HER2-positive or TNBCs. The bulk cells in these tumors are highly epithelial in morphology and a proportion express ER and PR. Luminal A tumors, which have the best prognosis, display the lowest proportion of cells expressing CSC markers. These tumors also display the highest proportion of ER- and PR-expressing cells. Plasticity between cell types is an important part of normal biology, both in development and in adult homeostasis to respond to stimuli and deal with cellular stress; but one hallmark of cancer is its ability to lower the activation barrier and increase 264 Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc.

cellular plasticity. The processes of EMT and mesenchymal-epithelial transition (MET) have been well studied in the context of cancer biology and have profound implications for the plasticity between CSC types and their relation to the molecular subtypes of breast cancer. As previously mentioned, work from the Weinberg lab has shown that EMT, driven by overexpression of mesenchymal transcription factors such as Snail, Twist, Slug, and/or Sox9 (Mani et al., 2008; Guo et al., 2012), can induce an EMT phenotypic shift, resulting in increased numbers of CSCs. This plasticity between cell types results in significant challenges to therapy as EMT increases invasion and dissemination of cells while also decreasing proliferation and avoiding cytotoxic chemotherapy agents (Creighton et al., 2009). Alternatively, MET allows cells to return to a highly proliferative state and mediates tumor relapse at sites of distant metastases after cessation of treatment (Yao et al., 2011). The development of drugs capable of blocking this cellular plasticity, such as inhibitors of c-Met (Sylvester, 2014) and TGFB (Bhola et al., 2013), by preventing transition between CSC states may increase the efficacy of CSC-targeting agents. In addition, as described below, strategies aimed at simultaneous targeting of CSCs and bulk tumor cells may be useful in overcoming the therapeutic challenges posed by tumor cell plasticity. Mutation Profile and CSCs across the Molecular Spectrum of Breast Cancer Genetic analysis of breast cancers has demonstrated different mutational profiles across the subtypes of breast cancer. For example, the most frequent genetic alteration found in luminal breast cancers is mutational activation of PI3K signaling (Creighton et al., 2010). In contrast, TNBCs almost always contain mutations in p53 and also frequently display deletions or epigenetic silencing of the PTEN tumor suppressor gene. In addition to HER2 gene amplification, HER2-positive breast cancers frequently display deletions in PTEN, and indeed this is a likely cause of resistance to HER2-targeted therapies (Dave et al., 2011; Fabi et al., 2010; Park et al., 2014). BRCA1 germline mutations or epigenetic silencing of the Brca1 locus are most frequently associated with TNBCs (Foulkes et al., 2003).

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Perspective Interestingly, all of these molecular alterations have been demonstrated to increase CSC frequency in pre-clinical models as well as in patient samples. The different molecular subtypes of breast cancer are also characterized by distinct profiles of microRNA expression, a number of which regulate CSC states (Pal et al., 2015). In addition to their effects on CSCs, genetic and epigenetic events produce blocks in normal differentiation pathways that differ across the molecular subtypes of breast cancer. It is notable that despite the diversity of genetic changes driving the different molecular subtypes of breast cancer, CSCs within these subtypes have similar patterns of gene expression (Liu et al., 2014), suggestive of common, shared regulatory pathways. For example, the Notch (Al-Hussaini et al., 2011) and Wnt (King et al., 2012) developmental pathways, as well as Her2-Akt (Korkaya et al., 2008) and STAT3-NFkB signal (Iliopoulos et al., 2009) transduction pathways, may play important roles in the regulation of self-renewal and differentiation of CSCs across the molecular subtypes of breast cancer. This observation has important implications for understanding the mechanisms underlying therapeutic resistance and the development of more effective therapeutic strategies capable of producing sustained effects. Therapeutic Implications of CSC Models Approaches involving more specific targeted therapeutics have led to improved outcomes, particularly when they are administered in the adjuvant setting after surgical removal of the primary tumor to lessen the probability of tumor recurrence. However, despite these new options, metastatic breast cancer remains largely incurable (Ruiterkamp et al., 2011), primarily due to the emergence of treatment resistance during therapy. Cellular heterogeneity, generated through genetic and epigenetic mechanisms, contributes to the development of treatment resistance. EZH2, which acts as a transcriptional repressor via control of the repressive histone modification mark H3K27me3, has been linked to aggressiveness in breast cancer (Kleer et al., 2003; Holm et al., 2012). In ER-positive breast cancer, methylation events have been associated with resistance to both tamoxifen and aromatase inhibitors (Magnani et al., 2013), and H3K27me3 marks are correlated with a worse outcome during aromatase inhibitor therapy (Jansen et al., 2013); pre-clinical data suggest that treatment of ER-positive breast tumors with histone deacetylase (HDAC) inhibitors can improve responses to tamoxifen (Hodges-Gallagher et al., 2007; Thomas et al., 2011; Bic¸aku et al., 2008). Epigenetic silencing may also drive tumor cells toward a CSC-like state (Ohm et al., 2007). For example, in basal breast cancers, activation of Zeb1 via epigenetic modulation of its promoter leads to transition of non-CSCs to CSCs (Chaffer et al., 2013). Clinically, however, results of trials utilizing HDAC inhibitors have been mixed. Promising results have been demonstrated in using these agents to reverse endocrine resistance; however, HDAC inhibitors have not shown single-agent efficacy in breast cancer (Munster et al., 2011; Luu et al., 2008). The CSC hypothesis has important clinical implications. CSCs display relative resistance to cytotoxic chemotherapy, both in pre-clinical models and neoadjuvant clinical trials (Creighton et al., 2009). In fact, both chemotherapy and radiation therapy may actually stimulate CSC self-renewal through cytokine production and DNA repair mechanisms (Maugeri-Sacca` et al.,

2012; Li et al., 2008; Phillips et al., 2006). The development of individualized therapies for each molecular subtype of breast cancer was largely based on strategies to target bulk cell populations. However, patients inevitably develop resistance to these therapeutic approaches. The evolution of clones of cells with ‘‘driver’’ mutations undoubtedly contributes to this resistance, providing a substantial therapeutic challenge (McGranahan et al., 2015). Genetic mutations, as well as microenvironmentally driven epigenetic diversity at metastatic sites, further limit the effectiveness of agents targeting genetic lesions of the primary tumor (Oskarsson et al., 2014). Although mutations are shared between CSCs and their clonal progeny (Marusyk et al., 2014), CSCs may be dependent on alternate pathways, many of which are shared across the molecular subtypes of breast cancer. This suggests that the addition of CSC targeting agents to molecular-subtype-specific therapies may improve treatment efficacy. This approach is illustrated in Figure 3 and discussed in more detail below. ER-Positive Breast Cancer Breast cancers that express ER and/or PR have been subdivided into luminal A and luminal B based on molecular profiling. The proliferation marker Ki67 has served as a surrogate for molecular profiling, where a Ki67 index of 14 divides the less proliferative luminal A breast cancers from the more highly proliferative luminal B breast cancers (Goldhirsch et al., 2011). Although both of these luminal breast cancers are treated with hormonal therapy, the clinical outcome is significantly more favorable for patients with luminal A tumors compared to luminal B tumors (Sørlie et al., 2001). It is likely that many luminal A breast cancers may lack a true CSC and instead consist of a relatively uniform population of luminal progenitor/differentiated cells that express steroid hormone receptors; in contrast, luminal B breast cancers contain a proportion of CSCs that do not express ER or PR (Harrison et al., 2013; Horwitz et al., 2008). Of interest, it was recently reported that despite the fact that ALDH-positive CSCs do not express the classical 66 kilodalton ERa (ERa66), they may express ERa36, an alternative ER that is localized in the plasma membrane and is capable of directly activating mitogenic signaling (Zhang et al., 2012a; Deng et al., 2014). In luminal breast cancers, CSCs have been shown to be regulated through paracrine loops involving estrogen-induced FGF 9 (Fillmore et al., 2010) and progesterone-induced RANKL (Schramek et al., 2010). These growth factors, produced by ER/PRexpressing differentiated cells, regulate the self-renewal of CSC populations (Fillmore et al., 2010; Rajaram et al., 2015). In vitro, development of resistance to hormonal therapy is accompanied by an increase in the CSC population (Piva et al., 2014; Wang et al., 2012). The clinical relevance of these findings was evidenced in a study that demonstrated an increase in the proportion of cells expressing the CSC phenotype CD44+/ CD24 following neoadjuvant hormone therapy (Creighton et al., 2009). This suggests that simultaneous targeting of CSC and ERa-expressing differentiated cells might increase the clinical efficacy of hormonal therapy. In addition to direct mitogenic effects of estrogen, luminal subtype CSCs may be regulated by growth factor pathways including PI3K, mTOR, and HER2 (Ithimakin et al., 2013; Korkaya et al., 2009; Zhang et al., 2012b; Zhou et al., 2007). It has been demonstrated that the combination of the mTOR inhibitor Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc. 265

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Figure 3. Strategy for Combining CSC-Targeting Agents with Approved Drugs that Target the Bulk Tumor Populations in Each Molecular Breast Cancer Subtype The diagram on the left illustrates CSC-targeting drugs either approved or in development and, on the right, FDA approved drugs to treat each of the molecular subtypes of breast cancer.

everolimus and hormonal therapy significantly delayed the time to tumor progression in ER-positive breast cancer (Baselga et al., 2012). Similarly in a randomized phase II trial, the addition of the cyclin-dependent kinase (CDK) 4/6 (CDK4/6) inhibitor palbociclib to endocrine therapy resulted in a near doubling of the time to tumor progression (Finn et al., 2015). This clinical benefit has been attributed to the ability of CDK4/6 inhibitors to inhibit the downstream target of estrogen mitogenic signaling, Cyclin D. However, it has recently been reported that the CyclinDCDK4/6 complex also plays a role in the regulation of selfrenewal in breast CSCs and that palbociclib targets this cell population (Finn et al., 2009; Lamb et al., 2013). This raises the intriguing possibility that inhibition of CSCs by targeting mTOR or CDK4/6 may contribute to the clinical benefit of these compounds, supporting the strategy of combining CSC targeting agents with endocrine therapies that target the ER-positive bulk tumor populations as illustrated in Figure 3. HER2-Positive Breast Cancer The development of HER2-targeted therapeutics for the treatment of HER2-positive breast cancer has been one of the greatest advances in breast cancer therapeutics. When used in the adjuvant setting, the HER2-targeting antibody trastuzumab dramatically reduces breast cancer recurrence (Slamon et al., 2011). In addition to trastuzumab, other HER2-targeting agents, including the monoclonal antibody pertuzumab and the immunotoxin conjugate ado-trastuzumab emtansine (TDM-1), have further improved the efficacy of HER2 targeting (Swain et al., 2013; Verma et al., 2012). The importance of HER2 signaling in breast cancer pathogenesis and treatment may relate to the observation that HER2 is an important regulator of BCSC selfrenewal (Giordano et al., 2012; Korkaya et al., 2008). HER2 266 Cell Stem Cell 17, September 3, 2015 ª2015 Elsevier Inc.

overexpression through gene amplification increases the CSC population, whereas HER2-blocking agents such as trastuzumab reduce this cell population. Although the use of HER2targeting agents has been limited to patients whose tumors display HER2 gene amplification, retrospective analyses of large randomized adjuvant trials have suggested that the benefit of adjuvant trastuzumab may extend to patients with tumors that did not display HER2 amplification (Paik et al., 2008). The recent demonstration of HER2 expression in CSCs, in the absence of HER2 gene amplification, may provide a biological explanation for these surprising results (Ithimakin et al., 2013). The utility of HER2 blockade in patients classified as HER2-negative is being tested in a large randomized clinical trial. Despite the clinical efficacy of HER2-targeting agents, the vast majority of patients with metastatic HER2 amplified breast cancer eventually develop resistance to these therapies. The most frequent genetic abnormality associated with this resistance is deletion of the PTEN gene (Dave et al., 2011). It has been demonstrated that PTEN deletion in HER2-overexpressing cells activates cytokine-signaling pathways including IL-6, STAT3, and NFkB (Korkaya et al., 2012). This pathway generates EMT-like CSCs. The existence of CSCs in multiple states, each dependent on alternative signal transduction pathways, may necessitate the development of strategies to target these multiple pathways. A clinical trial adding the IL-6 receptor blocking antibody tocilizumab to HER2-blocking agents is in development (Figure 3). TNBC TNBCs are characterized by lack of expression of ER, PR, and HER2, and these tumors typically have the highest proportion of cells that express CSC markers compared to the other subtypes (Idowu et al., 2012). Recent molecular profiling of TNBC

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Perspective has further demonstrated the heterogeneity of this group of tumors. Unlike the other subtypes of breast cancer, there are currently no targeted therapies that improve patient outcome, and the only established treatments involve the use of cytotoxic chemotherapy. Although these therapies are clearly beneficial, patients invariably develop resistance. This resistance has been shown to be associated with an increase in CSCs. In fact, cytotoxic chemotherapy has been demonstrated to actually increase CSCs through activation of cytokine loops involving IL-6 and IL-8 (Tsavaris et al., 2002; Samanta et al., 2014). It appears that a limited repertoire of signal transduction pathways regulate CSCs of this subtype. Recently, Junankar et al. (2015) reported that the inhibitor of differentiation-4 (ID4) is a key regulator of mammary stem cell renewal and marks a subset of TNBC cases with a particularly poor prognosis. Interestingly, ID4 expression is unrelated to previous molecular TN classifications and was found to control mammary stem cell self-renewal acting upstream of Notch signaling to repress luminal fate commitment. Other pathways that play important roles in the regulation of CSCs within this TN subtype include Wnt (King et al., 2012), Notch (Al-Hussaini et al., 2011), Hedgehog (Liu et al., 2006), and IL-8-CXCR1 (Rody et al., 2011). Early-stage clinical trials utilizing inhibitors of these pathways to target CSCs in women with TNBC are in progress (Figure 3). The increased efficacy of such combinations may also result from the induction of cell differentiation in CSCs by the targeted agents, subsequently rendering the CSCs susceptible to chemotherapy. Recently, it has been demonstrated that defects in DNA repair in TNBC associated with BRCA1 mutation or loss of activity are susceptible to inhibition of poly-ADP-polymerase (PARP; Dent et al., 2013; Shen et al., 2013). The sensitivity of BCSC in these tumors to PARP inhibition remains to be determined. Another approach to treat TNBC utilizes the targeting of tumor angiogenesis using VEGF inhibitors such as bevacizumab. Use of this agent resulted in delayed progression time of patients with TNBC. However, post-treatment monitoring of this patient group revealed that, although treatment delayed tumor progression, once the tumors had progressed they displayed a more aggressive phenotype, resulting in little, if any, difference in ultimate patient survival (Wagner Anna et al., 2012). It has recently been demonstrated that these anti-angiogenic agents stimulate CSCs through generation of tissue hypoxia, with resultant stimulation of HIF-1a, a potent regulator of breast CSCs (Conley et al., 2012). Addition of HIF-1a inhibitors caused blockage of this effect, causing a decrease in CSCs and increasing the efficacy of the anti-angiogenic agents in pre-clinical models (Conley et al., 2015). Most recently, preliminary data suggest that immunotherapy utilizing an immune checkpoint blocker against PD-L1 benefited a subset of women with TNBC (L.A. Emens et al., 2015, Am. Assoc. of Canc. Res. Ann. Meeting, abstract). There is evidence that CSCs may utilize multiple mechanisms to evade immune surveillance. Specific strategies for immune targeting of CSCs are in development. Implications of CSCs for Clinical Trial Design The existence of CSCs in the vast majority of breast cancers has important clinical implications for the development strategies and design of clinical trials to assess treatment efficacy. As described above, pre-clinical models provide strong support

for combining molecular subtype-specific agents with CSCtargeting drugs. This combined approach also applies to molecular targeted therapies, the selection of which is based on molecular profiling of bulk tumor cells. It is critical to consider that, despite their genetic similarities, CSCs and the bulk tumor cells derived from them may be dependent on different signaling pathways. These differential sensitivities, presumably due to epigenetic differences in these cell populations, may have important implications for the selection of the appropriate therapeutic regimen and for the selection of appropriate endpoints to access treatment efficacy. The existence of multiple CSCs in a given tumor presents an additional therapeutic challenge, which may require the combination of multiple CSC-targeting drugs. Since CSCs constitute only a minor fraction of tumor cells, classical clinical endpoints, such as tumor shrinkage as assessed by Response Evaluation Criteria for Solid Tumors (RECIST), are inadequate to access the efficacy of CSC-targeting agents. The assessment of CSC markers in serial tumor biopsies may prove valuable in accessing these effects (Schott et al., 2013). Alternately, circulating tumor cells (CTCs) have been shown to be enriched in CSCs (Aktas et al., 2009) and may provide more readily available liquid biopsies (Pantel and Alix-Panabie`res, 2013). Neoadjuvant clinical trial designs, in which primary therapy is given before surgical removal of tumors, afford the opportunity to directly assess the effects of therapy on both CSC and bulk tumor populations. Indeed, such studies have demonstrated that persistence of CSCs, as assessed by expression of ALDH, is associated with increased development of subsequent metastatic disease (Alamgeer et al., 2014). A number of CSC targeting drugs are now being tested in early stage clinical trials (Figure 3). Following the demonstration of safety, these drugs are being combined with more traditional therapies that target bulk cell populations. Ultimately, randomized clinical trials will be required to ascertain the benefit of these combined approaches for treatment of advanced, metastatic disease. Furthermore, since pre-clinical studies suggest that the benefit of CSC-targeting agents is greatest when they are deployed in the adjuvant setting, use of these agents in earlierstage disease needs to be tested. The use of rational combinations of agents to target both differentiated tumor cells and CSCs holds great promise for improved therapeutics in all subtypes of breast cancer. ACKNOWLEDGMENTS We would like to thank Jill Granger for her help editing the manuscript. The Wicha laboratory is supported, in part, by NIH grants R01-CA129765-06A1 and R01-CA101860-06, The Breast Cancer Research Foundation, Komen For the Cure Foundation, and the Alfred Taubman Research Institute. M.S.W. has financial holdings and is a scientific advisor for OncoMed Pharmaceuticals; is a scientific advisor for Verastem, Paganini, and MedImmune; and receives research support from Dompe Pharmaceuticals, MedImmune, and Cormorant Pharmaceuticals. REFERENCES Aktas, B., Tewes, M., Fehm, T., Hauch, S., Kimmig, R., and Kasimir-Bauer, S. (2009). Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res. 11, R46.

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