Inflammation linking EMT and cancer stem cells

Oral Oncology 48 (2012) 1068–1075

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Review

Inflammation linking EMT and cancer stem cells Chenchen Zhou a,1, Jeffrey Liu a,b,1, Yaling Tang a,⇑, Xinhua Liang a,c,⇑ a

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu Sichuan 610041, People’s Republic of China Department of Biological Sciences, University of Delaware, Nowak, DE 19716, USA c Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu Sichuan 610041, People’s Republic of China b

a r t i c l e

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Article history: Received 17 April 2012 Received in revised form 5 June 2012 Accepted 8 June 2012 Available online 4 July 2012 Keywords: Epithelial–mesenchymal transition (EMT) Cancer stem cells (CSCs) Inflammation microenvironment

s u m m a r y Similar to actors changing costumes during a performance, cancer cells undergo many rapid changes during the process of tumor metastasis, including epithelial–mesenchymal transition (EMT), acquisition of cancer stem cells (CSCs) properties, and mesenchymal–epithelial transition (MET). Such changes allow the tumor to compete with the normal microenvironment to overcome anti-tumorigenic pressures. Then, once tissue homeostasis is lost, the altered microenvironment, like that accompanying inflammation, can itself become a potent tumor promoter. This review will discuss the changes that cancer cells undergo in converting from EMT to CSCs in an inflammation microenvironment, to understand the mechanisms behind invasion and metastasis and provide insights into prevention of metastasis. Ó 2012 Elsevier Ltd. All rights reserved.

Introduction Metastasis is the leading cause of death by cancer, yet it remains the most poorly understood component of cancer pathogenesis because of its complexity.1 Metastasis involves many steps: local invasion of cancer cells into the surrounding tissue, transport through the microvasculature of the lymph and blood systems, translocation—largely through the bloodstream—to microvessels of distant tissues, exit from the bloodstream, survival and adaptation in the distant microenvironment, and, finally, formation of a secondary tumor.2 Malignant tumors are characterized by an ability to invade and disseminate; indeed, tumors have been referred to as entities that constantly redefine themselves by their everchanging nature.3 However, certain tumor cells are more responsive to the metastatic route, and, of those, an even smaller fraction succeed. Metastasis depends on intrinsic properties of the tumor cells as well as factors derived from the tumor microenvironment.4 The first evidence that non-cancerous tissue elements might affect tumor formation and growth came from the field of inflammation.4 Although the molecular mechanisms involved in inflammation and cancer have remained elusive until recently, a role

for inflammation in tumorigenesis has been generally accepted. In fact, evidence increasingly indicates that an inflammation microenvironment is an essential component of all tumors, as tumors have been observed to arise at the sites of chronic inflammation, and inflammatory cells are present in biopsied tumor samples.4–7 Inflammation originates in an intrinsic pathway, whereby genetic events leading to tumor transformation promote the construction of an inflammatory milieu, as well as an extrinsic pathway established by tumor-infiltrating leukocytes, particularly macrophages.8,9 The inflammatory responses play critical roles at all stages of tumor development: initiation, promotion, malignant conversion, invasion, and metastasis.7,10–15 Recently, epithelial–mesenchymal transition (EMT) has taken center stage as the convergence point between inflammation and cancer.16–19 This review will highlight the changes occurring in cancer cells from EMT to acquisition of cancer stem cells (CSCs) properties in an inflammation microenvironment, to establish an understanding of the mechanisms behind invasion and metastasis. Additionally, this foundation will provide insight into prevention of tumor metastasis.

EMT and inflammation ⇑ Corresponding authors. Address: Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu Sichuan 610041, People’s Republic of China. Tel.: +86 15 884483965; fax: +86 28 85503479 (X. Liang). Tel.: +86 15 008458098; fax: +86 28 85503479 (Y. Tang) E-mail addresses: [email protected] (Y. Tang), [email protected], [email protected] (X. Liang). 1 These authors contributed equally to this work. 1368-8375/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.oraloncology.2012.06.005

EMT is a process through which epithelial cells lose their epithelial traits and acquire instead the attributes of mesenchymal cells, including losing cell–cell adhesion structure, altering their polarity, reorganizing their cytoskeletal system, switching expression from keratin-to-vimentin-type intermediate filaments, and becoming isolated, motile.20 EMT was categorized into three

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distinct subtypes: embryonic and developmental EMT; tissue regeneration and fibrotic EMT; and cancer progression and metastatic EMT.16,21 EMT events in embryonic development have been very well defined22 and EMT in adult functions to promote organ morphogenesis as well as tissue regeneration during wound repair. However, there is an increasing awareness that EMT in cancer can endow cells with migratory and invasive properties.23 EMT program, interconverting epithelial cell types into cells with mesenchymal attributes, is itself accompanied by the acquisition of malignant cellular traits and the ability to complete various steps of the metastatic cascade.22,24–26 On the other hand, this program can be choreographed by a number of transcription factors to initiate the complex transcriptional program to impart malignant traits to cancer cells.24,27–29 Of note, these mechanistic insights into EMT maintenance and metastasis process have been greatly increased by the recent discovery that microenvironmental signals contribute to triggering EMT, including TGF-b, TNF-a, Notch, Hedgehog, Wnt, which imply the critical role of tumor-associated stroma in EMT induction.30–35 As previously mentioned, many of the cellular phenotypes associated with EMT do not arise as purely cell-autonomous processes. Instead, recruited stromal cells in tumor microenvironment seem to play key roles in the acquisition of these traits, and adaptation of cancer cells to these signals, rather than selection, seems to be key in EMT.36 Although further studies concerning the signals and circumstances from tumor microenvironment relevant to EMT induction should be exploited, well-characterized evidence has elucidated the role that inflammation, one of the most important microenvironment, plays in regulating EMT and metastasis. Recently, inflammation is regarded to be likely a key inducer of EMT in the pathological settings of cancer progression and EMT has now been considered to bridge inflammation and cancer.16–19 For example, SNAIL1 itself seemed to upregulate the expression of pro-inflammatory mediators such as several ILs (IL-1, IL-6 and IL-8). And TGF-b1 and HIF-1 in the inflammation microenvironment can induce the expression of TWIST and SIP1. A variety of cell types recruited by islands of cancer cells in advanced primary carcinomas into the surrounding stroma, such as fibroblasts, myofibroblasts, granulocytes, macrophages, myeloid cell-derived suppressor cells (MDSCs), mesenchymal stem cells, and lymphocytes, are thought to create a reactive stroma that appears to result in the release of EMT-inducing signals.37 Co-culture of tumor-associated macrophages (TAMs), one of the most well-characterized types of tumor-infiltrating inflammatory cells, was shown to increase their invasive capacity in an NF-jB-dependent and TNFa-dependent manner.6 Cancer-associated fibroblasts (CAFs), another major cells in inflammation microenvironment, can produce the fibroblast-derived matrix-degrading enzymes and growth factors such as FGF and HGF, all of which have been shown to be important stimuli of EMT.38–40 Breast carcinoma cells incubated with CAF-conditioned media have been characterized by a loss of E-cadherin-dependent adhesion and enhanced motility.41 CAFs depended on NF-jB, cycloxygenase-2 (COX-2) and HIF-1 to exert their propelling role for EMT.42 How do these tumor-infiltrating inflammation cells acquire the ability to promote cancer metastasis? Several mechanisms have been defined. Autocrine and paracrine extracellular signals, as well as genetic and epigenetic alterations, might each contribute to this change. Space constraints do not allow discussion of all the inflammatory components, but we will discuss the most prominence of them, such as NF-jB, TGF-b, TNF-a, STAT3, HIF-1a and inflammation-associated miRNAs, in further details. NF-jB is a key orchestrator of innate immunity and inflammation, and evidences suggest that this transcription factor, a potent EMT inducer, represents a potential molecular bridge between inflammation and cancer.19,43–52 Activation of NF-jB by Akt

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up-regulates SNAIL expression and induces EMT.48,53 SNAIL, in turn, suppresses the expression of the metastasis suppressor gene product Raf kinase inhibitor protein RKIP (inhibits the MAPK and the NF-jB pathways) as well as PTEN (inhibits the PI3K/AKT pathway).54–56 Alternatively, NF-jB directly binds the promoter of miR-448 suppressing its transcription, which induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, increasing EGFR-mediated TWIST1 expression, as well as NF-jB activation. This autoregulation between NF-jB and miR-448 may have a role in the regulation of chemotherapyinduced EMT.57 Whereas NF-jB activation in myeloid cells is associated with tumor promotion and EMT in inflammation-associated cancer models, Gavert et al. reported that L1-mediated metastasis of colorectal cancer cells does not require changes in EMT and operates by activating NF-jb signaling.58 Another master regulator of EMT is TGF-b. The ability of TGF-b to induce EMT is mediated by canonical Smad-dependent59 and non-canonical Smad-independent pathways. TGF-b activates Smad transcription factors and MAPKs to control expression of other regulators of EMT such as SLUG60 and SNAIL61,62 in normal and malignant mammary epithelial cells (MECs).63–66 TGFb-TGFbR-Smad2 signaling axis is needed to maintain epigenetic silencing of critical EMT genes in breast cancer progression.67 Among the non-Smad pathways, increasing evidences are emerging that TGF-b also regulates MECs behavior and the induction of EMT via the stimulation of numerous ‘‘noncanonical’’ effector systems, including small GTP-binding proteins, PI3K, MAPKs and NF-jB.68–74 Increasing findings indicate that many targets are involved in regulating TGF-b-mediated EMT, including Na, K-ATPase,75 IGFBP3,76 ZAG,77 SKIP, TGF-b type I receptor (TbRI),78 Dab2, ROCK and LIMK, PIAS1, nuclear transcription factors including members of SNAIL, SIP1 and TWIST, and Six family of homeobox (Six1).79,80 Then which targets are the most important and the correlation among them awaits further explanation. Secondly, support for the role of TGF-b signaling in EMT is further strengthened by the finding that the members of the miR-30 and/or miR-200 families controlled the process of TGF-b signaling in modulating EMT/MET in anaplastic thyroid carcinomas (ATCs)-derived cells.81 TGF-b abrogates the expression of miR-200, leading to the expression of ZEB1 and ZEB2 and their consequential down-regulation of E-cadherin expression to initiate EMT.82,83 On the other hand, TGF-b stimulation of normal MECs elicits their up-regulated expression of miR-155 via a Smad4dependent pathway.84 And up-regulated expression of miR-21 induced by TGF-b also participates in the initiation of EMT.85,86 Thus, the coupling of TGF-b to the regulation of miRNA expression and activity affords new avenues to potentially manipulate the pathophysiology associated with EMT. In addition, TGF-b signaling was observed to maintain DNA methylation patterns during EMT. For instance, hypermethylation of the E-cadherin promoter marks Ras-transformed MECs that have undergone a stable EMT induced by serum versus a transient EMT induced by TGF-b.87 Similar to NF-jB and TGF-b, TNF-a is also a potent stimulator of EMT by breast cancer cells. TNF-a induced SNAIL1 promoter activity and EMT in cancer cells, and can also stabilize SNAIL1 protein.7,88,89 TNF-a affects, regulates and also augments TGF-b1induced EMT,87,88 and a synergy of TNF-a and TGF-b signaling accelerates EMT dependent on enhanced p38 MAPK activity.33 Through actin remodeling, and the activation of TGF-b signaling, TNF-a promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the dissociation of cell–cell, the augmentation of cellular motility.90 This seemed that TGF-b is another target of TNF-a. In addition, NF-jB activation by TNF-a or expression of constitutively active IKK2 induced an EMT-phenotype.91,92 On the other hand, TGFb-induced EMT was mediated by NF-jB signaling.63,93 The coupling of TGF-b to NF-jB activation induces EMT and metastasis through eliciting the initiation of an autocrine

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Cox-2:PGE2:EP2 receptor signaling cascade.73,94–98 All the observation explained the role of NF-jB as a modulator of TGFb1-induced EMT in addition to the connection among NF-jB, TGF-b and TNF-a in EMT induction. Another mechanism through which pro-inflammatory signal passages can affect EMT is via STAT3-mediated induction of TWIST transcription.51 But recent report suggests that STAT3 is a negative regulator of adenoma–carcinoma transition in colon cancer.99 Thus, these mechanisms remain to be confirmed in vivo. The link between hypoxia and EMT has been recently strengthened by the observed expression of SNAIL, ZEB and TWIST regulated by HIF-1, a key hypoxia effector.19,100,101 Hypoxia or overexpression of HIF-1a can promote EMT and metastatic phenotypes, and hypoxia itself may be an inducer of the EMT.102 Co-expression of HIF-1a, TWIST and SNAIL in primary tumors of patients with head and neck cancers correlated with metastasis and the worst prognosis. Another hypoxia-related gene, lysyl oxydase, was found to interact directly with SNAIL, but the functional and clinical significance of this interaction need to be further investigated.103,104 Support for the connection between cancer EMT and inflammation is further strengthened by studies of the role of inflammationassociated miRNAs in EMT and metastasis. For example, miR-29a induced by TGF-b led to EMT through suppressing tristetraproli.105 And miR-155 expression and promoter activity induced by TGF-b through Smad4 and the knockdown of miR-155 suppressed TGFb-induced EMT and tight junction dissolution, as well as cell migration and invasion.84 Alternatively, miR-9, overexpression in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness.106 TLR4-activated NF-jB rapidly increases the expression of miR-9 that operates a feedback control of the NF-jB-dependent responses.107

EMT confers a cancer stem cell phenotype Our mechanistic insights into the molecular and function features of the mesenchymal cells have been greatly increased by the discovery that sort of them endow the self-renewal trait associated with normal tissue stem cells (SCs) and CSCs.108,109 CSCs, or tumor-initiating cells, are able to self-renew, differentiate, and regenerate a phenotype of the original tumor when implanted into the nonobese diabetic and severe-combined immunodeficient (NOD–SCID) mouse, the gold standard assay for defining the CSCs fraction.110–113 The CD44+ or aldehyde dehydrogenase 1 (ALDH1) population of human head and neck squmaous cancinoma cells (HNSCCs) possess the unique properties of CSCs, the side population (SP).114–119 A link between metastasis and stem cells has also been shown in HNSCC.120–122 The concept of CSCs has sparked excitement and controversy in equal measure. New data emerging from the study of CSCs are forcing the cancer research community to revise basic concepts of cancer biology. The tradition opinion that each cell has the potential to be cancerous has been revised. Instead, only a subset has the competence of initiating cancer.123 On the other hand, the discovery of CSCs raises the conundrum regarding the origin of these cells. Determining the origin and biology of CSCs may reveal strategies for targeting them therapeutically. Although the definitive proof for identifying the origin of CSCs is needed to be provided, evidence exists for the stem cell, the committed progenitor and the fused cells as the origin of CSCs and EMT involving in these processes. Similarity in cell surface markers and morphological and fundamental characteristics suggests that normal tissue stem cells may be the targets of oncogenic transformation and the origin of CSCs.124–128 A potential connection between normal tissue stem cells and EMT has been confirmed by the

observation of EMT at the periphery of human embryonic stem cells (ESCs) clusters grown on matrigels.129 Additionally, Jamieson and his colleagues showed that leukemic granulocyte–macrophage progenitors have been shown to be able to self-renew through the activation of the Wnt/b-catenin signaling pathway,130 the important signal passage of EMT induction and stemness property, which announced the relation between progenitor cells and EMT, and supported the notion that a committed progenitor can be the cancer-initiating cell.130–133 The insight into cell fusion as the third mechanism for the apparent cellular plasticity associated with tissue stem cells is consistent with a proposal that bone marrow-derived cells (BMDCs)-tumor cell fusion expressed mesodermal traits and EMT regulators, and is a potential source of CSCs,134–137 which confirmed the fusion theory, a unifying explanation for metastasis.138 From studies in animal there is little doubt that tumor hybrids are generated in vivo and can be a source of metastases,138 however, it is not clear from previous studies whether stem cells themselves are fused with other cell types in different tissues in vivo. Taken together, these notions imply that CSCs can be formed through EMT. At the same time, the heterogeneity evident in clinical tumor pathology suggests that these induced CSCs cannot represent the only source of cells in the CSC pool within a tumor and intrinsic CSCs are likely to exist from their very inception, long before EMT occurs.37 EMT has been considered to be accompanied by the acquisition of the CSC properties, including tumorigenicity, ability to redifferentiate into an epithelial tumor, and ability to form spheroids.108,109,136 And EMT seems to be a condition to induce CSCs. Major clues as to a link between EMT and the acquisition of stem cell properties came from a recent study by Mani et al.108 who showed that the forced expression of EMT regulators such as SNAIL and TWIST or treatment with TGF-b resulted in cells with a CSC trait. Similarly, Morel and colleagues109 reported that breast cancer cell lines adding active Ras generated a population of CD24 /CD44+ cells that underwent EMT and had CSC attributes. Pawelek et al.136,137 demonstrated that EMT was induced by CD8+ T cells and the resulting tumors had characteristics of CSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In order to integrate the concepts of CSCs and EMT, Brabletz et al.26 proposed the existence of two cancer stem cell populations, a stationary cancer stem cell and a migratory cancer stem cell, in order to model all aspects of tumor progression. This model is further supported by the expression of EMT markers in CSCs from mammary carcinomas and, vice versa, by the activation of stem cell markers in EMT-induced mammary epithelial cells.108 Eastham et al.129 demonstrated that ESCs and mesenchymal stem cells were characterized by a shift from E-cadherin to N-cadherin expression, the expression of SNAIL factors, vimentin, and metalloproteases, but also retained the expression of several totipotent transcription factors, including Oct-4 and Nanog. Recently, Wellner et al.139 showed in pancreatic and colon cancers, ZEB1 linked EMT-activation and stemnessmaintenance by suppressing stemness-inhibiting miR-200 family members. In oral squamous cell carcinoma (OSCC) cell lines, fresh samples of OSCC tumor, and primary and recurrent cutaneous SCC, Biddle et al.140 found a novel CSC population that has undergone EMT (EMT CSC) , and the EMT and non-EMT CSC populations coexist by switching between the two phenotypic states through EMT and MET. In light of the study by Basu et al.141,142 one can speculate that the mesenchymal-like cisplatin-resistant cells represent the CD44+ cancer stem cell population in HNSCC. CD44, a marker associated with EMT and putative cancer stem cells has been identified as a critical target of ERK1/2 in promoting oral cancer aggressiveness.143 ALDH1+ cells within HNSCC cell lines had functional properties like invasion capacity and EMT.144,145 This indicates that stem cells can adopt a mesenchymal phenotype without losing

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their pluripotency, and mesenchymal status seems to be a condition to regain pluripotency.25 Based on the current study, a likely alternative explanation for metastasis progression of cancer is that a few of CSCs within the primary tumor separate from the primary lesion and play a critical role in metastasis because of their potential to migrate into different tissues.146 On the other hand, it is plausible at present that most human carcinoma cells access and exploit the EMT program to acquire malignant cell traits, though hardly proven.146,147 These represent an important conceptual advance; they showed that incoming metastatic cells had CSCs properties, or EMT or the fusion that EMT confers a cancer stem cell phenotype. The idea of CSCs and EMT has recently moved to the forefront of cancer research, however, there is still a lack of a widespread consensus on their description and definition. With the development of significant progress made in characterizing the in vivo architecture and functions of stem cell niches in different model systems including Caenorhabditis elegans, Drosophila and mammals,148–152 the importance of the tumor and the host microenvironment in conditioning the stem cell status itself and the development of therapeutic strategies has been recognized, the mechanisms through which niche factors can modulate stem cell fate decision has been gradually shed light on.

Inflammation and CSCs Although there are some disputes on the role of CSCs and their microenvironment in the stem cell niche, that is whether microenvironment has been considered to be affiliated to stem cells or individual stem cell153,154 was affiliated to environment, the stem cell niche has now been defined as a dynamic microenvironment that responds to local and systemic cues, ultimately influencing stem cell fate and governing stem cell fate.12,150,155–164 Connelly et al.165,166 found that the shape of the extracellular matrix (ECM)-coated surface rather than its ECM composition or density affects the initiation of stem cells and signaling events that mediate the interaction between the basement membrane and the basal epidermal stem cells are required to maintain their stem-like state. For example, stem cells lacking the small GTPase Rac1 fail to undergo shape changes and migrate on the provisional ECM at the wound edge.167 The differentiation of epithelial stem cells in response to extrinsic or intrinsic cellular stress pathways may also act as a protective mechanism against oncogenic perturbation.168 Recently, significant strides have been made in characterizing the in vivo architecture and functions of stem cell niches in different model systems and elucidating the symmetric and asymmetric mechanism through which niche factors can modulate stem cell fate,148–150,169–173 however, an underexplored variable is the inflammation milieu of this highly specialized microenvironment. Certain work has begun to focus on the relevance of inflammation to the stem cell niche. The IL-8/CXCR1 axis secreted by endothelial and stromal cells may regulate mammary stem cell proliferation or self-renewal and play a role in mediating interactions between tumor stem cells and microenvironment.174 The role for IL-6 in breast CSCs and IL-4 in mediating chemoresistance of colon CSCs have also been reported.175–177 Moreover, the metastatic, aggressive behavior of inflammatory breast cancer (IBC) may be mediated by a CSC component that displayed ALDH enzymatic activity.178 In addition, Arwert et al.179 showed that Dexamethasone treatment inhibited tumor formation in InvEE mice. InvEE skin and tumors expressed high levels of IL-1a, and treatment with an IL-1 receptor antagonist delayed tumor onset and reduced incidence. The depletion of cd T cells and macrophages also reduced tumor incidence. These indicated that differentiated epidermal cells can initiate tumor formation without reacquiring the ability

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to divide and that they do so by triggering an inflammatory infiltration. These observations fueled a hypothesis that perhaps inflammation is critical to the milieu of stem cell niches. Furthermore, the molecular mechanisms of inflammation driving CSCs during tumor promotion discussed below continue to drive the excitement behind this hypothesis. TGF-b has been implicated in maintaining hematopoietic stem cells (HSCs) quiescence.180,181 The members of TGF-b superfamily can activate differentiation of vascular progenitor cells derived from mouse ESCs.182,183 In the bone marrow niche, TGF-b signaling can function as a fine-tuning mechanism for cross-talk between hematopoietic stem cells subtypes and myeloid progenitors.184 Another critical signal passage of the inflammation and CSCs is Wnt/b-catenin. The pro-inflammatory cytokine TNF-a promotes nuclear entry of b-catenin during inflammation-associated gastric cancer in the absence of any mutations in Wnt/b-catenin pathway components.185 Activation of b-catenin, downstream to the IKKa, b/NF-jB pathway, may be integral to the hyperproliferative effects of progastrin on proximal colonic crypts.186 Other mechanisms through which pro-inflammatory cytokines can affect CSCs is via STAT3, which is linked to both stem cell reprogramming and stem cell renewal.187 IL-1, TNF-a, and IL-6 can promote MMP expression, invasiveness, and metastasis via NF-jB and STAT3.188 HNSCC cells, HN13 and HN30, were shown to express high levels of IL-6 through NF-jB-dependent transcriptional activation and genetic inhibition of NF-jB reduced IL-6 levels resulting in a decreased in STAT3 phosphorylation and activation.189–191 PGE2 played a crucial role in HSCs growth and development not only in embryonic, but also in adult stem cell homeostasis in both simple and complex vertebrate systems.192 By expressing one or more of the same cytokines such as IL-6, activated prostate epithelial stem cells acquired a survival advantage.193 IL1a-dependent NF-jB signaling can activate both skin stem cell proliferation and cutaneous inflammation.194 The establishment of one or more autocrine signalling loops results in an expansion of these stem cells in the absence of inflammation, as a potential first stage in the development of the tumor. Subsequently, bioimaging approaches that allow the continuous long-term observation of individual differentiating mouse hematopoietic progenitor cells (HPCs) demonstrate that the physiological cytokines, macrophage colony-stimulating factor and granulocyte colony-stimulating factor, can instruct hematopoietic lineage choice.195 On the other hand, inflammation cells are also involved. Self-renewal and pluripotent properties of human ESCs depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblastlike cells, which define the stem cell niche of pluripotent human stem cells through insulin-like growth factor (IGF)-II/IGF1R axis.196 While the recognition that inflammation as well as growth factors and cytokines produced by inflammation may confer a stem cell-like phenotype upon tumor progenitors or stimulate stem cell expansion, thereby enlarging the cell pool that is targeted by environmental mutagens,10 is being increasingly documented and accepted, the association of inflammation molecular pathways with CSCs and the role of EMT during this process are still matter of discussion.

EMT, inflammation and CSCs There are increasing evidences that pointed to inflammationinduced EMT as critical to the connection of inflammation and CSCs, just as EMT as a common link between inflammation and cancer. One of the best explanations is provided by the model of inflammation cells. Bone marrow-derived cells (BMDCs)-tumor cell fusion, one of CSCs origins, can explain EMT in cancer through EMT regulators (TWIST, SPARC, and others).134,136 CAFs induce EMT

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and stemness through a pro-inflammatory signature, which exploits reactive oxygen species to drive a migratory and aggressive phenotype of prostate carcinoma cells.42 In an epithelial breast cancer CD8+ T cells can induce EMT and the resulting tumors had characteristics of breast cancer stem cell (BCSCs), a CD24( / lo)CD44(+) phenotype.137 These indicate inflammation cells may play a critical role in EMT conferring to CSCs. The direct and strong evidence of the association of inflammation, EMT and the CSCs property to adapt to a particular target tissue microenvironment came from the results that the TGF-b-inducedEMT in immortalized human mammary epithelial cells resulted in the acquisition of mesenchymal traits and the expression of stem-cell markers, and stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas expressed EMT markers.108,109 The TGF-b pathway was specifically active in CD44+ cancer cells197 and TGF-b-induced-EMT associated with the selection and expansion of breast cancer stem cells,198 which suggested that TGFb-induced-EMT might represent a common molecular mechanism underlying the anti-cancer stem cells and anti-fibrotic actions of metformin.199 Tetracycline-regulated expression of SNAIL induced the differentiation of mouse embryonic stem cell (MESECs) into mural cells, whereas knockdown of SNAIL expression abrogated TGFb2-induced mural differentiation of MESECs.186 In addition, Activin-Nodal signaling acted through SIP1 to regulate the cell-fate decision between neuroectoderm and mesendoderm in the progression from pluripotency to primary germ layer differentiation.200 TWIST1 was responsible for the regulation of the IKKb/NF-jB and PTEN/AKT pathways and its association of ovarian cancer stem cell differentiation through the expression of hsa-miR-199a/hsa-miR-214, which suggested EMT-associated inflammation and CSCs were regulated by miRNAs.201,202 The above and many other studies have shown that inflammation-EMT and CSCs are not merely spectators in the process of tumor spread but often have a position at center stage, orchestrating and actively participating in the transformation process. Taken together, these lines of evidence challenge the unidirectional view of inflammation only as inducers of carcinogenesis and tumor progression, provocatively suggesting that inflammation may under some circumstances help tumor cell to spread through EMT and CSCs, and warning against previously underestimated dangers and pitfalls of inflammation. Conclusion It is unlikely that inflammation teaches us how to fight or prevent metastasis, however, they can give us a hint of why metastasis occurs. No doubts exist that the more unfavorable life in the primary tumor is for cancer cells, the higher the risk of metastatic spread is.203 Given that inflammation microenvironment, and EMT and CSCs in particular, are now recognized as important determinants of tumor dissemination, a more thorough understanding of their functional role in tumor spread will contribute to the translation from bench to bedside. It is early days to anticipate the successful clinical translation of any of the CSCs-targeted strategies to completely kill cancer cells, however, one should be optimistic about the future of the multitude of therapeutic options offered by increasingly understanding of stem cell biology in cancer metastasis. Conflict of interest statement None declared. Acknowledgements We apologize to those colleagues whose work we could not reference directly due to space constraints. This work was

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