Hippo pathway and breast cancer stem cells

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Contents lists available at ScienceDirect

Critical Reviews in Oncology/Hematology journal homepage: www.elsevier.com/locate/critrevonc

Hippo pathway and breast cancer stem cells Marcello Maugeri-Saccà a,b,∗ , Ruggero De Maria b,∗∗ a b

Division of Medical Oncology 2, “Regina Elena” National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy Scientific Direction, “Regina Elena” National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 An overview of the Hippo pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Hippo pathway in mammary gland development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 Hippo pathway in breast cancer stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Role of funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

a r t i c l e

i n f o

Article history: Received 27 October 2015 Received in revised form 16 November 2015 Accepted 14 December 2015 Keywords: Breast cancer Cancer stem Cells Hippo pathway

a b s t r a c t Tumors contain a specialized subset of cells with unique properties, such as self-renewal and tumorigenic potential. These cancer stem-like cells (CSCs) are supposed to be responsible for therapeutic resistance and metastatic spread. Functional characterization of breast CSCs (BCSCs) is beginning to shed light on molecular networks which are specifically activated in this cellular compartment, and that account for the retention/acquisition of stem-like features. The Hippo tumor suppressor pathway has increasingly been tied to breast cancer. Altered Hippo activity, or Hippo-independent mechanisms, mediate the activation of the Hippo transducers TAZ and YAP. When this occurs, cancer cells acquire more aggressive traits. In the realm of BCSCs, improper TAZ/YAP activity sustains self-renewal, resistance to conventional anticancer agents, and metastatic dissemination. In this review, we highlight the involvement of TAZ and YAP in mammary gland development and in BCSCs. We also discuss potential strategies for transferring this information into the clinical setting. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Over the past decade, the idea that only a minority of cells possess tumorigenic potential and are able to generate and repopulate the tumor has gained consensus (Magee et al., 2012). Central to this model is a rare, phenotypically and functionally distinct pool of cancer cells endowed with the ability to self-renew and differentiate. Owing to the similarities that these neoplastic cells share with

∗ Corresponding author at: Division of Medical Oncology 2 and Scientific Direction, “Regina Elena” National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy. Fax +39 6 52665523. ∗∗ Corresponding author. Fax +39 6 52665523. E-mail addresses: [email protected] (M. Maugeri-Saccà), [email protected] (R. De Maria).

tissue-resident stem cells, the definition of cancer stem cells (CSCs) was prevalently adopted. Since 1997, when a stringent hierarchical organization was observed in human acute myeloid leukemia (Bonnet and Dick, 1997), a separation between tumorigenic CSCs and non-tumorigenic cells was also postulated in solid tumors, on the basis of transplantation assays in immunocompromised mice (Al-Hajj et al., 2003; Ricci-Vitiani et al., 2007; Eramo et al., 2008; Singh et al., 2004; Todaro et al., 2010; Prince et al., 2007; Li et al., 2007; Collins et al., 2005; Gao et al., 2010). The CSC model originally posits the existence of a rigid pyramidal organization within a tumor (Magee et al., 2012). Few CSCs placed at the apex of the pyramid give rise to an offspring of more differentiated cells that progressively lose the properties of the ancestors. This oversimplified approach connected CSCs with two key tenets of tumor biology, namely intratumor heterogeneity and

http://dx.doi.org/10.1016/j.critrevonc.2015.12.004 1040-8428/© 2015 Elsevier Ireland Ltd. All rights reserved.

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resistance/adaption to stressful conditions, such as exposure to chemotherapy. Intrinsically plastic CSCs account for the histological and molecular diversity existing within the same tumor entity (Meacham and Morrison, 2015). Moreover, similar to their normal counterparts, CSCs thwart harmful insults through a multiple set of mechanisms, spanning from a slow replication kinetics to an extremely efficient protection of their genome (Maugeri-Saccà et al., 2011). Even though the selective targeting of CSCs was often advocated as the conditio sine qua nonfor the development of more effective anticancer therapies and for pursuing the goal of tumor eradication, therapeutic failures continued to accumulate at a frustrating pace (Kola and Landis, 2004). For instance, putative CSC-directed agents failed to provide hints of antitumor efficacy in clinical trials. A paradigmatic example concerned agents targeting the Hedgehog pathway. Hedgehog signaling activation was extensively connected to CSC biology (Takebe et al., 2015). Nevertheless, outside a background of oncogene addiction, where the inhibition of mutant pathway components lead to deleterious effects on cell fitness, as it is the case of basal cell carcinoma (Sekulic et al., 2012), this class of compounds resulted ineffective in more common tumor types (Takebe et al., 2015). Second, the lesson we have learned in recent years is that neglecting Darwin’s principles of evolution, on which the clonal evolution model was shaped, and replacing clonal dynamics, more complex in nature and yet unpredictable in their trajectories, with the static nature of the hierarchical model might mislead proper interpretation of the CSCs contribution to neoplastic diseases. The possibility of reprogramming mature cells into embryonic stem cells with the introduction of four factors (Oct3/4, Sox2, c-Myc and Klf4) demonstrated that the stem cell state is not immutable (Takahashi and Yamanaka, 2006). More direct evidence provided further ground to this topic, and questioned the incompatibility of hierarchical and clonal evolution models. This was rooted into the observations that, upon exposure to opportune conditions, non-CSCs can be turned into cells with stem-like traits (Mani et al., 2008; Li et al., 2009; Hjelmeland et al., 2011; Todaro et al., 2014; Vermeulen et al., 2010). Nowadays, we know that a variety of conditions, such as epithelial-mesenchymal transition (EMT) (Mani et al., 2008), hypoxia (Li et al., 2009), low pH (Hjelmeland et al., 2011), cytokines (Todaro et al., 2014) and soluble factors (Vermeulen et al., 2010), generate cells harboring CSCs properties. In turn, CSCs can directly manipulate their environment, for instance by generating endothelial-like cells and then contributing to tumor vascularization (Wang et al., 2010; Ricci-Vitiani et al., 2010). The message conveyed is that clonal evolution has profound effects on the CSC compartment, and that microenvironmental cues, a hallmark of evolutionary principles, have a pivotal role in influencing the dynamics controlling CSCs. On the premise that the stem-like state is not a fixed, predetermined condition, but it may be a functional, advantageous trait imposed by the local environment, understanding what pushes cancer cells to become CSCs is of paramount importance. Breast CSCs (BCSCs) were originally isolated on the basis of the CD44high /CD24low /Lin− immunophenotype (Al-Hajj et al., 2003). Over time, various alternative BCSC markers have been proposed in the attempt of enriching subtype-specific BCSCs (Wei and Lewis, 2015). Functional characterization later posed a number of intracellular circuits such as Hedgehog, Notch, Wnt and TGF␤, among others, at the center of the signaling network governing BCSCs (Wei and Lewis, 2015). Recent evidence added the Hippo pathway, a regulator of tissue growth originally discovered in Drosophila melanogaster, to the set of BCSC-associated pathways (Piccolo et al., 2014). The multilevel regulation of Hippo activity, which depends on both intra- and extracellular stimuli, makes it an attractive

candidate for further explaining the oscillations occurring in CSC content. In this article, we first provide an overview on the Hippo pathway and its multifaceted mechanisms of control. We then briefly touch on its involvement in the development of the mammary gland. Next, current knowledge on the Hippo pathway in breast cancer (BC) is summarized, with special emphasis being placed on BCSCs.

2. An overview of the Hippo pathway The Hippo pathway plays important regulatory functions during organ development and regeneration (Piccolo et al., 2014). The first links between Hippo and organogenesis stemmed from evidence that loss-of-function mutations of pathway components in flies led to tissue overgrowth, as a result of increased cell proliferation and decreased cell death (Fernández et al., 2011; Hamaratoglu et al., 2006; Harvey et al., 2003; Justice et al., 1995; Maitra et al., 2006; Sansores-Garcia et al., 2011; Tapon et al., 2002). Hints that the Hippo signaling module is conserved in mammals (Camargo et al., 2007; Dong et al., 2007) prompted a series of studies that attempted to address its contribution to organogenesis and tumorigenesis. However, germline or somatic mutations in Hippo core pathway components are uncommon in BC (Cancer Genome Atlas Cancer Genome Atlas Network, 2012), and more in general in solid tumors, raising the hypothesis that altered Hippo signaling is driven by other deregulated molecular networks that intersect the pathway at various levels Maugeri-Saccà et al., 2015). Core components of the Hippo pathway comprise a regulatory kinase module and a transcriptional module (Cancer Genome Atlas Network, 2012). The first is composed by a set of kinases, namely sterile 20-like kinase 1 (MST1) and 2 (MST2), large tumor suppressor 1 (LATS1) and 2 (LATS2), and the adaptor proteins Salvador homologue 1 (SAV1), MOB kinase activator 1A (MOB1A) and 1B (MOB1B). The transcriptional module encompasses two closely related paralogues: the transcriptional co-activator with PDZ-binding motif (TAZ) and the Yes-associated protein (YAP). The activation of the Hippo kinase cascade results in the phosphorylation of TAZ and YAP, and mediates their nuclear exclusion, cytoplasmic retention and proteasomal degradation. When the pathway is inactive, or its negative regulation on TAZ/YAP is bypassed or suppressed, TAZ and YAP translocate to the nucleus. Here, Hippo transducers interact with a series of partners, such as TEA domain-containing sequence-specific transcription factors (TEAD1 to TEAD4), SMAD and RUNX proteins, ultimately promoting the transcription of target genes, e.g., CTGF, CYR61, ANKRD1, BIRC5, and AXL. Since the activation of the TAZ/YAP transcriptional program feeds tumor-enhancing functions, Hippo was designed as a tumor-suppressor pathway. Fig. 1 illustrates the Hippo cascade. In mammals, the activity of TAZ/YAP is subject to a number of Hippo-dependent and -independent controllers. Mechanisms involved in cell–cell adhesion and apical-basal polarity (Martin-Belmonte and Perez-Moreno, 2011), mechanotransduction through the actin cytoskeleton and Rho GTPase (Dupont et al., 2011), metabolic routes such as the mevalonate pathway and aerobic glycolysis (Sorrentino et al., 2014; Enzo et al., 2015), and stem cell-related signals such as the Wnt pathway (Azzolin et al., 2014), dictate TAZ/YAP nuclear translocation and activation. Briefly, KIBRA and neurofibromin 2 (NF2, also known as Merlin) promote the activation of the core kinase cassette (Genevet et al., 2010; Xiao et al., 2011; Yin et al., 2013; Yu et al., 2010), together with a variety of other regulators that modulate MST kinases, such as TAO (thousand and one amino acid protein) kinases and the cell polarity kinase MAP/microtubule affinity-regulating kinase 1

Please cite this article in press as: Maugeri-Saccà, M., De Maria, R., Hippo pathway and breast cancer stem cells. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.12.004

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Hippo pathway ON MST1/2

P LATS1/2

P

3

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SAV

MST1/2

P

X

MOB1A/B

X

LATS1/2

P

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TAZ/YAP 14-3-3

TAZ/YAP

TAZ/YAP cytoplasmic retenon degradaon Transcripon of target genes TEADs

TAZ/YAP TEADs

Fig. 1. The Hippo pathway. The Hippo core module encompasses kinases (MST1, MST2, LATS1 and LATS2) and adaptors (SAV, MOB1A, MOB1B). When active, the signaling cascade operates an inhibitory phosphorylation of TAZ/YAP, which results in their cytoplasmic retention and proteasomal degradation. When the Hippo cascade is not active, TAZ/YAP translocate to the nucleus. Here, upon interaction with TEAD1-4, TAZ/YAP promote the transcription of target genes.

(MARK1) (Boggiano et al., 2011; Poon et al., 2011; Huang et al., 2013). A second level of regulation concerns TAZ/YAP binding proteins, that include the apical crumbs complex (CRB), which contains the transmembrane protein Crumbs and the cytoplasmic proteins PALS1 and PATJ, and the Angiomotin family (AMOTs) (Varelas et al., 2010; Chen et al., 2010; Chan et al., 2011; Hirate et al., 2013; Zhao et al., 2011; Wang et al., 2011). AMOTs are also able to activate LATS kinases (Paramasivam et al., 2011), even though a recent study has shown that AMOT promotes the nuclear localization of YAP in a context of NF2 deficiencies (Yi et al., 2013). Third, the apicobasal cell polarity (ABCP) proteins, including the key cell polarity determinant Scribble (SCRIB), facilitate the activation of Hippo kinases (Cordenonsi et al., 2011). A fourth branch emanates from adherens junction-associated regulators. In this frame, the E-cadherin/␣-catenin complex operates by regulating MST activity and by sequestering YAP—14-3-3 protein complexes in the cytoplasm (Kim et al., 2011; Schlegelmilch et al., 2011; Silvis et al., 2011). Next, the actin cytoskeleton and Rho GTPase regulate TAZ/YAP independently on the Hippo/LATS cascade, defining a form of regulation known as mechanotransduction (Dupont et al., 2011). In this scenario, TAZ/YAP operates as nuclear relays for changes occurring in the extracellular matrix (ECM), converting physical cues into biochemical stimuli (Dupont et al., 2011). G-protein-coupled receptors (GPCRs), which transduce through Rho GTPases, and the mevalonate pathway, that produces geranylgeranyl pyrophosphate essential for proper membrane tethering of Rho-GTPases, also modulate TAZ/YAP (Sorrentino et al., 2014; Yu et al., 2012). Finally, TAZ and YAP have been placed in the ␤-catenin destruction complex, a set of factors deputed to maintain the Wnt pathway inactive by promoting ␤-catenin degradation (Azzolin et al., 2014). When the Wnt pathway is inactive, TAZ/YAP are critical for ␤-catenin degradation. Upon stimulation by Wnt ligands, the ␤catenin destruction complex is disassembled, and both ␤-catenin

and TAZ/YAP can accumulate in the nucleus. Thus, TAZ/YAP are also deeply integrated in the Wnt cascade. Overall, the existence of a variety of regulatory branches underlies the complexity of the forces governing TAZ/YAP, which span from changes in cell geometry and mechanical forces imposed by the ECM and neighboring cells to metabolic avenues, pathway cross-talks and soluble ligands. Moreover, crosstalk among the different regulatory mechanisms exists, adding a further level of complexity to the forces controlling TAZ/YAP activation. Fig. 2 illustrates various mechanisms controlling the Hippo pathway, together with potential pharmacological strategies proposed for TAZ/YAP inhibition, which are discussed below. For a detailed overview of these mechanisms the reader might refer to (Piccolo et al., 2014).

3. Hippo pathway in mammary gland development TAZ and YAP are often defined as stemness factors. This definition is rooted into the body of evidence documenting that their activation in Hippo mutants or YAP transgenics resulted in embryonic organ overgrowth. Consistently, the developmental role of TAZ/YAP has been established in the liver (Camargo et al., 2007; Dong et al., 2007), heart (von Gise et al., 2012; Xin et al., 2011), intestine (Cai et al., 2010; Lee et al., 2008), skin (Schlegelmilch et al., 2011), and even breast (St John et al., 1999; Chen et al., 2014; Skibinski et al., 2014). In 1999, St John et al. (1999) reported that Lats1-/- female mice were characterized by endocrine dysfunctions, mirrored by decreased levels of prolactin and luteinizing hormone compared with control animals, that resulted in severe fertility defects. The mammary glands of Lats1 knockout female mice displayed decreased epithelial tissue, coupled with complete absence of nipple at macroscopic examination, at least in some cases. At the histological level, the mammary gland often appeared as a fat pad without the epithelial component.

Please cite this article in press as: Maugeri-Saccà, M., De Maria, R., Hippo pathway and breast cancer stem cells. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.12.004

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KIBRA

TAO1-3

MARK1

GPCRs SCRIB

MST1/2

RHO

SAV NF2 LATS1/2

PALS1 CRB

PATJ

HMG-CoAR MOB1A/B

Acn

AMOTs

C) Stans TAZ/YAP

B) Dasanib

WntRs E-cadherin α-catenin

TAZ/YAP TEADs

A) Verteporfin Transcripon of target genes

Fig. 2. Schematic representation of the Hippo pathway network. At least six regulatory branches have been identified: (i) activators of Hippo kinases (black circles), (ii) the apical crumbs complex (green circles), (iii) apicobasal cell polarity proteins such as Scribble (blue circle), (iv) the E-cadherin/␣-catenin complex (purple circles), (v) mechanisms correlated with the activation of Rho GTPase (orange circles), and (vi) the Wnt pathway (brown circle). Putative TAZ/YAP inhibitors (dasatinib, verteporfin and statins) are in the gray box. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Abbreviations: AMOTs: angiomotin family of proteins, CRB: Crumbs, HMG-CoAR: HMG-CoA reductase, KIBRA: kidney and brain protein, MARK1: MAP/microtubule affinityregulating kinase 1, NF2: neurofibromin 2, PALS1: Protein Associated with LIN7, PATJ: PALS1-associated tight junction protein, SCRIB: Scribble, TAO1-3: Thousand and one amino acid protein 1–3, WntRs: Wnt receptors.

Conversely, the analysis of Yap- or Sav1-deficient mammary glands from 6- and 8-week-old virgin mice did not unveil any defects in end bud formation, ductal growth, or ductal branching (Chen et al., 2014). The observation that the Hippo pathway was dispensable in the virgin mammary glands was further supported upon transgenic overexpression of YAP that, again, did not alter the morphology of the gland. Nevertheless, during pregnancy the mammary gland of Yap-deficient mice was hypoplastic and characterized by a significant reduction of alveolar structures, whereas Sav1 mutant glands appeared overall less differentiated. This suggested that Sav1 and Yap are required during pregnancy for mammary cell differentiation and survival, respectively. Next, in a gain-of-function screen carried out to identify transcription factors involved in epithelial lineage plasticity, Skibinski et al. (2014) reported that the forced expression of TAZ in luminal cells caused the acquisition of basal characteristics. This was further confirmed upon the depletion of TAZ in basal cells, that resulted in luminal differentiation. The examination of the mammary glands of TAZ mutant, pubescent (5 and 8 weeks old) mice did not reveal any defects. Conversely, the mammary glands of post-pubertal, TAZ mutant animals were characterized by a decrease of gland cellularity and branching defects. A reduced number of myoepithelial and basal cells accounted for these abnormalities. Indeed, whereas in wild-type epithelia the content of basal cells was comparable to that of luminal cells, in the TAZ-deficient mammary glands authors reported the presence of two to five luminal cells per basal cell. Thus, TAZ is important for the regulation of lineage dynamics in a stage-dependent way, as already observed for SAV1 and YAP. Overall, the central players of the Hippo pathway have been associated with the development of the mammary gland and its post-natal changes, such as those occurring in the post-pubertal period or during pregnancy. In some tissues, such as the intestine and skin, YAP overexpression or activation leads to the expansion

of the stem cell compartment, at least partly through a block in differentiation (Camargo et al., 2007; Zhang et al., 2011). In regard to the mammary gland, phenotypes observed in mice upon manipulation of Hippo pathway components seem to relate to altered cell proliferation and cell survival, even though the lineage imbalance observed when TAZ is lost may reflect a role in maintaining lineage fidelity (Skibinski et al., 2014). Thus, more focused investigations are needed to address the involvement of Hippo pathway at the stem cell level in the mammary gland. Nevertheless, given the crucial role of stem cells during organogenesis and in the maintenance of tissue homeostasis in adults, it is not surprising that developmental signals have been perceived as master controllers of CSC behavior. Coherently, the link existing between the Hippo pathway and BCSCs has been significantly strengthened over the past years, as discussed in next section. 4. Hippo pathway in breast cancer stem cells Expression of TAZ/YAP has been reported, although to a different extent, in various BC subtypes including hormone receptor-positive BC, HER2-positive BC, and triple-negative BC (TNBC) (Bartucci et al., 2015; Vici et al., 2014; Lehn et al., 2014; Min Kim et al., 2015; Kim et al., 2014; Díaz-Martín et al., 2015). Moreover, early hints on the association between TAZ/YAP expression and clinical outcomes have recently been provided (Bartucci et al., 2015; Vici et al., 2014; Lehn et al., 2014; Díaz-Martín et al., 2015). Overwhelming evidence tied TAZ to a variety of oncogenic functions, such as increased migratory and invasive properties, resistance to chemotherapy, and metastatic dissemination (Bartucci et al., 2015; Chan et al., 2008, 2009; Lai et al., 2011; Hiemer et al., 2014; Bendinelli et al., 2013; Yang et al., 2012). Conversely, controversies still exist on the biological outcomes elicited by YAP in BC, considering that both tumor-suppressive

Please cite this article in press as: Maugeri-Saccà, M., De Maria, R., Hippo pathway and breast cancer stem cells. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.12.004

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and tumor-promoting properties were reported upon its activation. This Janus-faced role is rooted into the following observations: (i) the YAP-mediated stabilization of p73 and the subsequent transcription of proapoptotic genes (Strano et al., 2005, 2001; Levy et al., 2007), (ii) a negative regulation of YAP determined by Akt and miR-200a (Basu et al., 2003; Yu et al., 2013), (iii) frequent loss of heterozygosity at 11q22.2 (Yuan et al., 2008; Carter et al., 1994). On the other hand, YAP activation was associated with tumorigenic transformation, EMT, inhibition of apoptosis, generation and maintenance of cancer-associated fibroblasts (CAFs), and metastatic spread (Overholtzer et al., 2006; Wang et al., 2012; Zhang et al., 2009; Lamar et al., 2012; Chen et al., 2012; Calvo et al., 2013). How these conflicting views can be reconciled remains unclear, but we can speculate that YAP acts in opposite manners in different BC subtypes. Consistently, decreased YAP expression was associated with adverse survival outcomes in luminal A BC (Lehn et al., 2014), whereas our unpublished results showed an opposite trend in TNBC, where patients with tumors displaying nuclear YAP expression had significant inferior disease-free survival, especially when TAZ was co-expressed. In the field of stem cells, the Hippo pathway is increasingly being linked to pluripotency (Mo et al., 2014). For instance, in embryonic stem cells (ESCs) TAZ and YAP were found to promote stemness by mediating the activity of some pathways (e.g., TGF-␤/BMP) and by regulating genes that are crucial in maintaining pluripotency, such as those involved in the generation of induced pluripotent stem cells (iPSCs) (Ramalho-Santos et al., 2002; Tamm et al., 2011; Lian et al., 2010; Varelas et al., 2008; Alarcón et al., 2009) Seminal evidence on the connection between the Hippo pathway and BCSCs were provided by Cordenonsi et al. (2011) in 2011. Reasoning that poorly differentiated BC should be enriched for CSCs, the authors interrogated seven BC gene-expression datasets to retrieve differentially represented signatures between G3 (poorly differentiated) and G1 (well-differentiated) tumors. With this approach, a signature registering TAZ/YAP activation was reported as over-represented in G3 BC. Mechanistic studies revealed that TAZ endows self-renewal ability to BC cells, a trait accompanied by an increased activity of multidrug resistanceassociated (MDR) proteins. The molecular process proposed as responsible for the TAZ-mediated induction of stem-like features envisioned that EMT delocalizes the cell polarity determinant Scribble from the cell membrane. This phenomenon relieves the Scribble-mediated inhibition of TAZ by hindering the MSTdependent activation of the LATS kinase. Subsequently, our group expanded the knowledge on the relationship between TAZ and BCSCs by uncovering a crucial role for TAZ in metastatic dissemination (Bartucci et al., 2015). In doing so, a panel of patient-derived BCSCs and their differentiated counterparts were characterized for metastatic potential by taking advantage of an orthotopic mouse model developed in the attempt to simulate the natural history of BC. Importantly, only BCSCs exhibited metastatic ability, and transcriptome analysis of BCSC- and non-BCSC-derived lesions revealed that TAZ was among the topranking differentially expressed genes, having significantly higher levels in tumors generated with BCSCs. Consistently, TAZ depletion in BCSCs hampered their metastatic potential, whereas its overexpression endowed non-BCSCs with metastasis-forming ability. At the functional level, both the WW and transcriptional activation domains of TAZ were found to be crucial for the induction of CSC traits (Li et al., 2015). Similar results in terms of self-renewal and metastatic potential were obtained when TAZ activation was explored within the TNBC framework (Frangou et al., 2014). Importantly, dasatinib, a multi-target kinase inhibitor initially developed as an inhibitor of the Src family kinases and currently used for treating haematological malignancies, was able to selectively kill

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the CSC population in a TAZ-driven model. Indeed, exposure to dasatinib led to the inhibition of anchorage-independent growth, impaired mammosphere-forming ability, and depleted the CD44high /CD24low subpopulation. As discussed above, EMT is linked to the acquisition of CSC properties (Mani et al., 2008), and plays a central role in the TAZ-driven induction of BCSCs (Cordenonsi et al., 2011). Similar outcomes are elicited by hypoxia (Xiang et al., 2014). Hypoxia-inducible factor 1 (HIF-1) was reported to trigger the expression and activation of TAZ (Xiang et al., 2014). Two molecular mechanisms were responsible for this. First, HIF-1 directly binds to WWTR1, the gene encoding for TAZ, promoting its transcription, as documented by the increased levels of TAZ target genes in response to hypoxia. Second, HIF-1 induces the transcription of SIAH1, an hypoxia-induced E3 ubiquitin-protein ligase, which mediates ubiquitination and proteasome-dependent degradation of LATS2. The net results is a defective Hippo-mediated control on TAZ. Later, it was reported on the reciprocal interaction between TAZ and HIF-1. Indeed, the TAZ-HIF-1␣ interaction promotes the expression of HIF-1 target genes, and TAZ inhibition coherently hindered the hypoxia-mediated induction of HIF-1 target genes (Xiang et al., 2015). Importantly, in a variety of cancer cell lines hypoxia was found to promote the expression of stem cell markers, even including the iPSCs factors OCT4, NANOG, SOX2, KLF4, and cMYC (Mathieu et al., 2011). Next, a cooperation between TAZ and the ECM has recently been discovered as a further mechanism responsible for the maintenance of the BCSC pool (Chang et al., 2015). In greater detail, it has been shown that BCSCs produce laminin 511 matrix, which serves as the ligand for the ␣6B␤1 integrin. This interaction promotes self-renewal via TAZ nuclear localization in a Hippo-independent manner. Activation of TAZ, in turn, promoted the transcription of the ␣5 subunit of laminin 511, ultimately feeding a positive feedback loop essential for the maintenance and expansion of the BCSC compartment. Finally, leukemia inhibitory factor receptor (LIFR) was designed as a BC metastasis suppressor owing to its ability to activate the Hippo kinase cascade, with the consequent phosphorylation and inactivation of YAP (Chen et al., 2012). By targeting LIFR, microRNA125a blocked LATS1 and TAZ phosphorylation, and ultimately caused the expansion of the BCSC pool (Nandy et al., 2015). Overall, the studies discussed above pointed to the Hippo transducers as important mediators in BCSCs. Coherently with the intricate molecular network controlling Hippo pathway function, a variety of stimuli, ranging from EMT and hypoxia to the ECM and microRNAs, mediate the recruitment of Hippo transducers and account for their pro-BCSC role.

5. Conclusions and future directions Even though the Hippo pathway within the domain of BCSCs is not as extensively explored as other self-renewal signals, hints collected over the last few years support a rational translation of this knowledge into proof-of-concept clinical studies. These, in turn, may be hypothesis-generating, feeding a virtuous circle where specifically-framed molecular contexts are considered for novel preclinical investigations, while leaving behind settings in which the clinical counterpart does not seem particularly informative. As a general principle, painting a clearer picture on the dynamics through which altered Hippo signaling drives the retention and acquisition of the CSC state has a dual implication. First, the preclinical characterization of BCSC-related pathways may be exploited in search for novel biomarkers or signatures foreseeing therapeutic efficacy and survival outcomes. Second, novel therapeutic entities can be tested with the specific aim of exploring their anti-CSC

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potential, for instance by adopting primary molecular endpoints related to BCSC content or function. Hippo pathway analyses in BC specimens conducted so far mostly focused on the expression pattern of TAZ and/or YAP, without a clear focus on clinical outcomes. Nevertheless, some pilot studies provided some clues. For instance, we reported on the association between elevated TAZ expression and decreased pathological complete response rate in Luminal B/HER2-positive BC patients who received neoadjuvant therapy (Vici et al., 2014). Moreover, nuclear co-expression of TAZ and YAP appeared associated with poorer survival outcomes in a moderately-sized cohort of TNBC patients, without significantly impacting on pathological complete response (personal unpublished results). In evaluating available data, it is worth anticipating that studies with a translational focus were, however, limited both in size and in the number of molecular endpoints considered. TAZ/YAP activation is controlled by a variety of mechanisms, that do not necessarily require Hippo kinases. Thus, more comprehensive studies should envision concomitant assessment of core pathway components (e.g., MST1, MST2, LATS1 and LATS2), endpoints associated with Hippo-independent activation of TAZ/YAP (e.g., HMG-CoA reductase, ␤-catenin), and components of the TAZ/YAP transcriptional program (e.g., AXL and CTGF). Next, the clinical significance of TAZ/YAP, the extent of their expression and intracellular localization, seem to vary according to the intrinsic subtype considered. Intuitively, this heterogeneity calls for larger investigations, that are necessary to define specific criteria for their assessment and scoring. From a therapeutic perspective, and to our knowledge, no direct inhibitors of TAZ/YAP have been developed so far. Nevertheless, at the preclinical level different compounds showed the potential of achieving an effective targeting of TAZ/YAP. Beyond dasatinib (Frangou et al., 2014; Rosenbluh et al., 2012), a variety of additional Food and Drug Administration (FDA)-approved compounds, such as verteporfin (Liu-Chittenden et al., 2012; Yu et al., 2014), dobutamine (Bao et al., 2011), and statins (Sorrentino et al., 2014; Wang et al., 2014), were reported capable of modulating TAZ/YAP in preclinical models. The aforementioned compounds inhibit TAZ/YAP in a Hippo-independent manner. For instance, dobutamine-mediated phosphorylation of YAP, and the consequent cytoplasmic translocation, was unaffected by the knockdown of LATS1 and LATS2 (Bao et al., 2011). Next, the activity of dasatinib on ␤-catenin-active cancer cell lines occurred through dasatinibmediated inhibition of YES1, which in turn hindered the assembly of a YAP-␤-catenin-TBX5 complex essential for the survival of ␤catenin-driven cancer cells (Rosenbluh et al., 2012). Finally, the inhibition of the mevalonate pathway through statins, a class of cholesterol-lowering agents that block the rate-limiting enzyme of the pathway, HMG-CoA reductase, induced cytoplasmic relocalization of TAZ/YAP independently on LATS1/2 kinases (Sorrentino et al., 2014). Rather, the inhibitory effects of statins on TAZ/YAP activity were connected to the reduced geranylgeranylation of Rho GTPases, a process essential for their proper localization at the plasma membrane. Rho GTPases are indeed positive regulators of TAZ/YAP activity (Piccolo et al., 2014) (Fig. 2). Whether the same effects, with the correlated deleterious effects on cancer cell viability, are reproducible in cancer patients is not known. To address this issue, we have recently proposed the administration of atorvastatin in a pre-surgical window-ofopportunity study in early BC patients with elevated levels of Ki-67 (available at ClinicalTrials.gov ID: NCT02416427). Our goal is to obtain information on whether statins are actually able to inhibit TAZ/YAP, and whether their inactivation translates into a mitigation of aggressive molecular features. In conclusion, deregulated Hippo pathway and TAZ/YAP activation have increasingly been tied to BCSC biology. Nevertheless,

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Biographies Marcello Maugeri-Saccà, MD/PhD, is a clinician-scientist at the Division of Medical Oncology, Regina Elena National Cancer Institute. He became a certified medical oncologist in 2008. In 2012, he obtained an international PhD in stem cell research, which focused on searching molecular mechanisms driving cancer stem cell metastasis. After having joined the Regina Elena National Cancer Institute, he engaged in translational and clinical cancer research, with special emphasis being placed on cancer stem cell-related prognostic and predictive biomarkers. Ruggero De Maria, MD, is currently the Scientific Director of the Regina Elena National Cancer Institute (Rome, Italy) who aims to create a productive synergy between basic and clinical research and is also President of the Italian ACC Alliance Against Cancer. His contributions to research include: having defined the major mechanisms responsible for the negative regulation of erythropoiesis and discovering cancer stem cells in colorectal and lung cancers. His current research is devoted to translational oncology, particularly in the development of therapies for advanced solid tumors.

Please cite this article in press as: Maugeri-Saccà, M., De Maria, R., Hippo pathway and breast cancer stem cells. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.12.004