DOI: 10.1111/eci.12415
REVIEW Clinical impact of studying epithelial–mesenchymal plasticity in pluripotent stem cells €ger Boris Kovacic, Margit Rosner, Katharina Schipany, Loredana Ionce and Markus Hengstschla Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria
ABSTRACT Background The ability of cells to travel long distances in order to form tissues and organs is inherently connected to embryogenesis. The process in which epithelial-like embryonic cells become motile and invasive is termed ‘epithelial-to-mesenchymal transition’ (EMT), while the reversion of this programme – yielding differentiated cells and organs – is called ‘mesenchymal-to-epithelial transition’ (MET). Design Here, we review the processes of EMT and MET in development and cancer and combine them with knowledge from pluripotent stem cell research. Results Research has shown that these processes are activated in many cancers leading to dissemination of cancer cells throughout the body and formation of metastasis. While the regulation of EMT during cancer progression has been extensively studied for decades, many fundamental processes that govern normal development are only poorly understood. Recent discoveries, such as reprogramming to pluripotent stem cells and identification of ground and primed states of pluripotent stem cells, have redirected much attention to EMT and MET. Conclusion Findings from pluripotent stem cell research and EMT/MET should be combined in order to design future strategies aimed to improve our understanding of cancer progression and to help develop novel anticancer strategies. Keywords Cancer, embryonic developement, epithelial-to mesenchymal transition, mesenchymal-to epithelial transition, pluripotent stem cells. Eur J Clin Invest 2015; 45 (4): 415–422
Introduction During embryogenesis, cells forming primordial tissues and layers have to be able to reversibly change their phenotype. Two fundamental processes underlying this cellular plasticity are termed epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) – the former to acquire a migratory and the latter to regain a static phenotype. Pluripotent stem cells are at the root of embryonic development. As such, they are able to generate all somatic lineages – which they first accomplish in the embryo by utilizing the processes of EMT and MET – both needed to generate the embryonic germ layers: ectoderm, endoderm and mesoderm. However, EMT and MET programmes are also activated in cancer as the abilities of cancer cells to delaminate from a primary tumour and to form secondary tumours at distant places. In this review, we discuss the fundamental principles of EMT/MET in pluripotent stem cells and during development. We consider the emerging stem cell pluripotency concepts
ranging from the ground state to the primed state. Finally, we focus on novel clinical concepts by combining the knowledge from EMT/MET in development with findings in cancer and with recent advances about distinct functional states of pluripotent stem cells.
Definition and classification of EMT and MET A regulated biological process in which a polarized cell that normally interacts via the basal membrane with its basal surface, undergoes multiple biochemical and morphological changes leading to an enhanced migratory and invasive cell phenotype, is called EMT. Conversely, MET is a reversal of this process leading to a restoration of epithelial characteristics [1– 3]. Instantly, the concept of EMT has been closely intertwined with embryogenesis because of the notion that ultimately all cells of an adult vertebrate have been derived from a single fertilized oocyte through a stepwise differentiation process generating functional epithelia and the mesenchymal tissue
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supporting it [3]. The term EMT applies to three distinct biological settings in which it may occur: type 1 refers to the developmental EMT, type 2 describes such processes in which wound healing, tissue regeneration and organ fibrosis are involved, and type 3 denotes EMT associated with cancer progression and metastasis [3]. While all three EMT types share the main features as stated above, it is noteworthy to stress that developmental EMT lacks inflammatory responses typical of type 2 and type 3 EMTs [3,4] and that the intravasation and extravasation steps occurring during type 3 EMT do not resemble the migratory processes in embryogenesis or during fibrosis [1]. EMT is triggered by various extracellular signals which converge to activate EMT-specific transcription factors, epigenetic regulators and microRNA networks; more detail regarding the biochemical and molecular regulation of EMT are discussed elsewhere [1–5]. In contrast, MET induction is less well characterized in embryonic development and thus the most prominent examples include epithelialization during kidney development. In this review, we focus on pluripotent stem cell research and the developmental processes that rely on EMT and MET and on their similarities with events that occur during cancer progression.
EMT in embryonic development and in pluripotent stem cells So far, three primary EMT events have been described during early vertebrate development: (i) during implantation of the blastocyst/morula into the uterus wall, (ii) during gastrulation of the embryo and (iii) during neural crest formation in amniotes. The term ‘primary’ refers to the fact that these tissues have never undergone a previous EMT process [2]. Central to this concept is the presumption that the developmentally most undifferentiated cells (blastomeres or totipotent embryonic stem cells) are neither of epithelial nor of mesenchymal origin. During early development (Box 1), the totipotency ends by delineation of two lineages: the inner cell mass (ICM), which is the initial pluripotent population, and the trophectoderm (TE), which forms an extraembryonic epithelial cell layer supporting the ICM [6]. At later time points in the blastocyst, ICM differentiates into the pluripotent Nanog-expressing epiblast and the extraembryonic primitive endoderm [7]. Thus, the earliest processes in embryonic development involve the establishment of two extraembryonic and one embryonic epithelial cell layers from presumably unorganized primitive cells. Development of epithelia is the fundament for all subsequent EMTs ending up in the fact that – with exception of the anterior central nervous system and the epidermis, all adult vertebrate tissues have undergone at least one round of EMT [1]. Briefly, primary EMT takes place during embryonic implantation in the endometrium, when a subset of cells in the extraembryonic area (tro-
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phoblast) undergoes EMT to form the future placenta. The first primary EMT event in the embryo occurs when select cells of the epiblast move to the midline to form the primitive streak. The cells that undergo EMT and internalize generate mesoderm and endoderm, while the remaining epiblast cell layer becomes the future ectoderm. Primary EMT also occurs when the epithelial neural plate in the midline of the embryo rolls up to form the neural tube. Some epithelial cells from the dorsal midline of the neural tube migrate away inside the embryo to form ganglia, bone and cartilage of the jaws and glial cells [1]. Another example of EMT in development is represented by epithelial cells of the neuroectoderm that give rise to migratory neural crest cells. As a consequence of EMT, neural crest cells migrate away from the neural folds and disperse throughout the embryo, where they finally undergo further differentiation into distinct cell types, such as melanocytes [8,9]. Historically, embryonic stem cells (ESCs) from mice and humans have been derived from the ICM population of early blastocysts [10,11] and hence represent a pluripotent population giving rise to the future embryo. Accordingly, ESCs from both species are able to differentiate into all three germ layers, thus holding a great promise for regenerative medicine. Differences between the murine and the human ESCs have soon become obvious and have led to the notion that murine ESCs may be more undifferentiated than their human counterparts. This has led to the implementation of terms ‘na€ıve’ and ‘primed’ to describe the distinct cell types [12,13]. In the literature, difference has also been made in the usage of the ‘ground state’ and the ‘naive state’ of pluripotency, both to describe a state prior to the establishment of pluripotency. The ground state corresponds to an unrestricted pluripotent stem cell state acquired in epiblast cells that is free of any developmental constraints and that exhibits a hypomethylated DNA methylome comparable to derestricted ICM or na€ıve epiblast cells at E3.5-E4.5. In contrast, the na€ıve state solely refers to the functional property of ESCs to generate all embryonic lineages upon blastocyst injection [6]. In vitro, ESCs show an altered transcriptional and epigenetic profile compared to cells from preimplantation embryos and hence are considered na€ıve, but not ground state [6]. Interestingly, pluripotency – as a functional property – seems not to be connected to specific molecular and epigenetic signatures or cell culture conditions. In developmental terms, it seems that multiple pluripotent states exist with progressively restricted differentiation potential. Pluripotent cells exhibiting the ground state (and na€ıve functional properties) may thus represent the most uncommitted developmental potential [14]. In contrast, pluripotent cells such as epiblast stem cells (EpiSC) show a lineage-primed functional property towards some developmental lineages and thus have been classified as ‘primed’ pluripotent cells. Murine and human embryonic stem cells have been derived from the inner cell mass of early blastocysts; however, human ESCs (and all subsequently
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Box 1 totipotency A unique property of certain cells to give rise to all embryonic and extraembryonic tissues is called totipotency. During human and mice development, only the zygote and the cells produced during the first 3-4 cleavages (blastomeres) are totipotent. pluripotency It is the ability of cells to differentiate into all embryonic germ layers (ectoderm, endoderm and mesoderm), but not into extraembryonic tissues. Nevertheless, pluripotent stem cells can build up all tissues of the body and thereby represent an inexhaustible source of cells for regenerative medicine and for treatment of a wide range of diseases. Blastocyst Blastocyst formation starts about 5 days after fertilization, when a fluid-filled cavity forms in the morula, a mulberry-like structure comprised of few dozens of cells. Some cells begin to grow inward into the cavity to generate the inner cell mass (ICM), which represents a source of embryonic stem cells. ICM The cells of the ICM are pluripotent and give rise to all three embryonic germ layers, which will eventually develop into the embryo proper and the later foetus. Trophectoderm The outer core of a blastocyst comprises of cells that will develop into extraembryonic tissues. This layer of cells is called trophectoderm and will form the future placenta after implantation into the uterine wall. Embryonic stem cells Embryonic stem cells (ESCs) have been isolated from the ICM region of surplus blastocysts and comprise of a molecularly and epigenetically heterogeneous population able to differentiate into all three embryonic germ layers. ESCs express pluripotent stem cell markers, while lacking expression of proteins corresponding to more differentiated tissues. ESCs have unlimited lifespan and – at least theoretically – can indefinitely self-renew in cell culture. Epiblast stem cells Epiblast stem cells (EpiSCs) comprise of a molecularly and epigenetically heterogeneous population of cells derived from a later stage ICM region, in which a further diversification into epiblast and the primitive endoderm (giving rise to the future yolk sac) has occurred. EpiSCs have a limited pluripotent capacity compared to ESCs and are poised towards some embryonic tissues. Induced pluripotent stem cells Induced pluripotent stem cells (iPSCs) are pluripotent stem cells generated from somatic cells through the process of reprogramming – a yet not fully resolved multistep process involving a forced and simultaneous expression of at least 3 to 4 transcription factors (‘Yamanaka factors’ – historically Oct3/4, Sox2, Klf4 and c-myc). iPSCs have been shown to resemble ESCs on transcriptional and epigenetic level, as well as functionally. However, iPSCs may carry somatic mutation(s) of the adult tissue they have arisen. Migration Any directed movement of a cell, either within tissues in vivo or along basal membranes, ECM fibres and plastic surfaces in vitro, is called migration (for further reading see [52]). Invasion Invasion is a synonym for a penetration of a tissue barrier and an infiltration of the beneath tissues by an invasive (tumour) cell. In vitro, invasion is characterized by a movement through a 3D matrix [52].
generated human induced pluripotent stem cells (iPSCs)) grew flattened, epithelial-like and could never be individualized in culture. This has led to the assumption that human pluripotent stem cells are ‘primed’ and therefore rather correspond to murine EpiSCs. It was only recently that the corresponding na€ıve and
ground state populations could be established from human ESCs and iPSCs [15–19]. There is a rationale that pluripotent stem cells show a spectrum of distinct morphological, functional, transcriptional and epigenetic states as a consequence of plasticity changes upon external developmental cues. This ability to
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reversibly change a cell’s phenotype is a hallmark EMT and MET and represents the major processes regulating embryonic development, as well as cancer. Thus, there is a need to re-evaluate the relationship of pluripotent stem cells and EMT/MET and to combine the knowledge of complex developmental processes with the mechanisms of progression of cancer in order to develop improved therapeutic strategies. To visualize the states with distinct epithelial or mesenchymal morphology, we aligned pluripotent stem cells and their derivatives in a sequence of gradually differentiating stages (Fig. 1). We differentiate between the (I) ‘ground state’ pluripotent stem cell state, (II) the ‘primed’ pluripotent stem cell state, (III) the partially differentiated state and (IV) the terminally differentiated state: (I) the morphology of the most undifferentiated state ground state ESCs and iPSCs resembles dome-
shaped structures composed of small and round cells and cell aggregates with irregular cell shape and invisible cell borders. This stage confers high self-renewal capacity and a high degree of clonogenicity [6,15,20]. The epithelial/mesenchymal identity of this pluripotent state is so far unclear, and thus, it is possible that ground state pluripotent stem cells exhibit distinct EMT marker expression than primed pluripotent stem cells. (II) Primed state ESC cultures form flattened epithelial monolayer colonies have reduced self-renewal capacity and lack of clonogenicity in individualized cells [21]. Yet, it is also unclear whether ground state ESCs are able to differentiate into primed ESCs in vitro and whether this transition requires induction or repression of epithelial and/or mesenchymal markers. Without any doubt, ground state ESCs are supposed to be more undifferentiated than their primed counterparts as shown by gene
Differentiation state “primed“ pluripotent stem cell state
“ground state“ pluripotent stem cell state
Terminally differentiated state
Partially differentiated state
?
EMT
MET
4-6i A/F/T/L
MET
EMT
“sequential“ OSKM Corresponding state in: Embyrogenesis
Inner cell mass (ICM)
Epiblast cells
Gastrulation (mesoendoderm), neural tube formation
Notochord, somites, urogenital system
Cell culture
Naive ESCs/iPSCs
Primed ESCs/iPSCs
Embryoid body
Somatic cells
Cancer
Unknown
Primary cancer
Blood-circulating cancer cells, cancer stem cells
Secondary cancer (metastasis)
Figure 1 Requirements for EMT and MET during embryogenesis, in pluripotent stem cell cultures and cancer. Primed pluripotent stem cells correspond to the epiblast stage during embryogenesis and exist as human ESCs and iPSCs in primary cell culture models. Primed pluripotent stem cells can be converted into ground or na€ıve state using a cocktail of 4-6 distinct inhibitors (4-6i) and combinations of Activin, FGF-2, TGFb and LIF (A/F/T/L). The reverse process (ground state to primed state) has not yet been directly induced in human-derived cells in vitro, but may occur spontaneously. Of note, primed pluripotent stem cells possess an epitheliallike morphology, while ground state pluripotent stem cells form dome-shaped structures. During embryogenesis, epiblast cells induce at least one round of EMT (during gastrulation and neural tube formation) and MET (to form notochord, somites, urogenital system, etc.). In vitro, these stages can be recapitulated during embryoid body (EB) formation and later by differentiation into terminally differentiated cells. Conversely, it has been suggested that during reprogramming by OCT4, Sox2, Klf4 and c-myc (OSKM), somatic cells need to progress in reverse order (EMT, followed by MET) in order to generate primed pluripotent stem cells. Primary cancer has molecular and biological similarities with primed pluripotent stem cells, as both can induce formation of motile and invasive cells by EMT. Metastasis formation is suggested to involve a MET by blood-circulating cancer cells or cancer stem cells. A ‘ground state’ for primary cancers has not yet been described. Transmission light micrographs of na€ıve and primed state pluripotent stem cells can be observed in [12] and [13], the ground state morphology in [17–19] and EBs in [17]. “?” indicates the lack of clarity or knowledge associated with this process.
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expression, chromatin and biochemical analyses [6]. During embryogenesis, cells from the ICM form an epithelial layer, the epiblast, which has a pluripotent capacity. Thus, it remains to be clarified by future studies, whether ground state ESCs represent an ‘ancestor of the primed state’ or a ‘reset state of the metastable primed state’ just before the induction of the germ layer formation in the epiblast. (III) The partially differentiated state is induced by a differentiation of ESCs into three germ layers and can be recapitulated using a well-documented in vitro model termed ‘embryoid body (EB) formation’ which corresponds to gastrulation events observed in vivo. In the embryo, this process is EMT dependent and requires appearance of transiently migratory cells differentiating into mesoderm and endoderm. Both, the migratory phenotype and the induction of EMT, are recapitulated during EB formation in vitro [22]. However, although EBs are already comprised of a large collection of differentiated cell types including neuronal progenitors, cardiac myocytes and red blood cells, the stage of these cells does not represent a terminally differentiated state. (IV) Thus, further lineage restriction is required for somatic cell differentiation often including multiple events of EMT and MET. In developmental terms, this stage corresponds to the development of somatic cells that build up entire organs. Experimental examples utilize highly sophisticated cell culture systems that imitate kidney and eye development in vitro [23,24]. Several lines of evidence support the concept of the possibility to directly interconvert of the before-mentioned states in the reverse direction through a regulated transition between epithelial and mesenchymal stages. Seminal studies aimed to elucidate the mechanism of reprogramming of somatic cells to pluripotent cells have unveiled a process in which exogenous OCT4, Sox2, Klf4 and c-myc (OSKM) expression initiates expression of pluripotency factors leading to a stable phenotype. In most cases, the cells used for reprogramming are genuine mesenchymal cells (e.g. fibroblasts), whereas the induced pluripotent stem cells (iPSCs) are epithelial cells – thus requiring a MET to fulfil this process [25,26]. However, considerable amount of data suggest that reprogramming is induced by an early EMT and that factors known to govern EMT (e.g. TGFb, Slug, Snail or Wnt) actually enhance reprogramming [27–29]. This apparent paradox can be explained by the fact that mouse embryonic fibroblasts are heterogeneous and not fully mesenchymal and thus can be converted into a more homogeneous mesenchymal population by TGFb [27]. This is further underscored by the observation that keratinocytes, an epithelial cell type, require expression of EMT factors to enhance the reprogramming efficiency [29]. The second step of reprogramming, however, is thought to involve a MET [27,29]. This concept involves the existence of a partially differentiated state that, however, may only be transient or even metastable in the setting of reprogramming – although it is
likely to exist in cell cultures [27,29]. Lastly, a combination of 4–6 inhibitors and growth factors such as LIF, Activin and TGFb has been recently reported by several groups to efficiently convert iPSCs to ground state pluripotent stem cells [15–19].
Role of EMT/MET in cancer Evidence for EMT in cancer comes from the notion that bloodcirculating cancer cells and disseminated cancer cells from patients show EMT marker expression and a high degree of plasticity [30–32]. Cancers disseminate through EMT in order to colonize distant organs and to form secondary tumours (metastasis). The EMT phenotype is often observed in cells at the invasive front of primary tumours. It has been suggested that these cells may be involved in subsequent steps of tumour invasion/metastasis formation, that is intravasation, dissemination, extravasation and, finally, formation of micrometastasis [33–35]. Metastasis formation represents the major cause of cancer-associated deaths. However, carcinoma metastasis frequently develops a differentiated epithelial phenotype – a process requiring a reversion of EMT. Although the need for MET during reprogramming is consistent with the epithelial nature of ESCs, it seems paradoxical that the induction of a full EMT phenotype in carcinoma actually increased invasiveness while suppressing its metastatic capacity [36]. Thus, EMTrelated invasion is required but not sufficient to fulfil the formation of metastasis, meaning that cancer cells can be invasive but not metastatic. In the light of (cancer) stemness and epithelial plasticity, the concept of cancer stem cells (CSCs, i.e. the cells that initiate, maintain and propagate cancer) provides another level of complexity in the field, as the EMT is thought to endow CSCs with their tumorigenic properties [5,34,37]. However, to efficiently colonize distant tissues, CSCs must also undergo the reverse process – a MET. Accordingly, CSC properties may actually include the ability to initiate a consecutive round of MET after having executed EMT, in contrast to non-CSCs, which perhaps solely induce EMT. Therefore, CSCs may have a lot in common with ESCs and thus studying early development may help to translate knowledge into potential therapies again cancer.
Clinical impact of EMT/MET on cancer research In patients, invasion and dissemination of cancer cells seem to be concomitant events associated with bad prognosis of cancer. Accordingly, inhibition of EMT may render cancers cells less prone to invasiveness and metastasis formation. However, constitutive activation of EMT may acutely suppress metastasis formation because MET is required for metastasis to form [36]. It may therefore be essential to control the plasticity of cancer by interfering with both, the EMT and MET. Here, we envision
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several potential strategies how therapies may be adjusted to eradicate cancer: I one option is to combine agents that target CSCs with conventional chemotherapy that is effective against nonCSCs. Recent data indicate that cancer stem cells can generate differentiated cancer cells and vice versa [38,39]. This plasticity seems to be a result of distinct chromatin states of the Zeb1 promoter [40]. However, as a targeted therapy against CSCs may not hit non-CSC, this approach should include a combination of the classical chemotherapy together with anti-CSC drugs. II Similar considerations can be employed for the inhibition of EMT to prevent dissemination of cancer cells, while controlling the proliferation of primary tumour via chemotherapy. To hit the EMT, the strategy may include inhibition of its signalling pathways, that is EGFR, PDGFR, IGFR1 and TGFb [41]. However, both approaches may only help in cancers that have not yet disseminated or in which EMT is a late event in tumorigenesis. III Paradoxically, constitutive expression of Snail, pRRX1 or Twist also suppressed metastasis formation of human cancer cell lines in a mouse model in vitro and in vivo [36,42], as EMT factors must be downregulated for metastasis to form [42,43]. In breast or pancreatic cancer, where cancer cells disseminate early [44,45], inhibition of EMT may favour the development of metastasis, thus a prevention of MET should be taken into account rather than of EMT [42,43]. Of note, as there is a potential risk of developing a mesenchymal tumour in a supportive organ, potential strategies against metastasis should preferentially combine anti-EMT and anti-MET blockers. IV An intriguing approach to study cancer progression and its association with distinct developmental states would be to reprogramme patient’s primary cancer cells to pluripotency. Strikingly, despite intensive attempts in the past, only a very limited number of successfully reprogrammed induced pluripotent cell lines have been generated from human cancer so far [46,47]. The low frequency of reprogramming is clearly unexpected, because limiting tumoursuppressor activity of p53, p19ARF, p16INK4a and p21KIP/CIP – a common feature of many cancers – leads to more efficient cellular reprogramming [48]. While the deduction that a reprogrammed cancer cell will never lose its cancer state may actually hold true, it is likewise possible that an ameliorated, epithelial and less malignant phenotype is induced [47]. V Additionally, it remains to be clarified whether induction of ‘ground state pluripotency’ in cancer-derived iPSCs or in primary cancers may enhance or reduce their tumorigenicity. VI Finally, there is a rationale that establishment of drug screening assays may be undertaken to develop novel and
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personalized drug treatment to patients suffering from cancer. Thus, ESCs, iPSCs or cancer-derived iPSCs could be used to identify novel inhibitors or activators of EMT and MET. Examples of potential targets against EMT include recently developed inhibitors for IGFR1 [49], TGFb [50] or EGFR [51], which have already proven effective in several clinical studies.
Conclusion In summary, extending the EMT/MET research to pluripotent stem cell models will help redefine cell plasticity in normal versus malignant development. This knowledge will undoubtedly facilitate the study of cancer progression and help develop novel anticancer strategies.
Conflict of interest All authors declare that they have no conflict of interest. Address Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria (B. Kovacic, M. Rosner, K. Schipany, L. Ionce, M. Hengstschl€ ager). Correspondence to: Boris Kovacic, Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria. Tel.: +43 1 40160 56514; fax: +43 1 40160 956 501; e-mail: [email protected] Received 25 November 2014; accepted 28 January 2015 References 1 Nieto MA. Epithelial plasticity: a common theme in embryonic and cancer cells. Science 2013;342:1234850. 2 Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Investig 2009;119:1438–49. 3 Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig 2009;119:1420–8. 4 Lopez-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med 2009;1:303–14. 5 Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009;9:265–73. 6 Hackett JA, Surani MA. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014;15:416–30. 7 Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O et al. Nanog is the gateway to the pluripotent ground state. Cell 2009;138:722–37. 8 Sauka-Spengler T, Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol 2008;9:557–68.
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44 Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F et al. EMT and dissemination precede pancreatic tumor formation. Cell 2012;148:349–61. 45 Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E et al. Systemic spread is an early step in breast cancer. Cancer Cell 2008;13:58–68. 46 Hu K, Yu J, Suknuntha K, Tian S, Montgomery K, Choi KD et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 2011;117:e109–19. 47 Kim J, Hoffman JP, Alpaugh RK, Rhim AD, Reichert M, Stanger BZ et al. An iPSC line from human pancreatic ductal adenocarcinoma undergoes early to invasive stages of pancreatic cancer progression. Cell Rep 2013;3:2088–99. 48 Banito A, Gil J. Induced pluripotent stem cells and senescence: learning the biology to improve the technology. EMBO Rep 2010;11:353–9.
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49 Reidy DL, Vakiani E, Fakih MG, Saif MW, Hecht JR, GoodmanDavis N et al. Randomized, phase II study of the insulin-like growth factor-1 receptor inhibitor IMC-A12, with or without cetuximab, in patients with cetuximab- or panitumumabrefractory metastatic colorectal cancer. J Clin Oncol 2010;28: 4240–6. 50 Giannelli G, Villa E, Lahn M. Transforming growth factor-beta as a therapeutic target in hepatocellular carcinoma. Cancer Res 2014;74:1890–4. 51 Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 2005;11:8686–98. 52 Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschlager M et al. In vitro cell migration and invasion assays. Mutat Res 2013;752:10–24.
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REVIEW Clinical impact of studying epithelial–mesenchymal plasticity in pluripotent stem cells €ger Boris Kovacic, Margit Rosner, Katharina Schipany, Loredana Ionce and Markus Hengstschla Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria
ABSTRACT Background The ability of cells to travel long distances in order to form tissues and organs is inherently connected to embryogenesis. The process in which epithelial-like embryonic cells become motile and invasive is termed ‘epithelial-to-mesenchymal transition’ (EMT), while the reversion of this programme – yielding differentiated cells and organs – is called ‘mesenchymal-to-epithelial transition’ (MET). Design Here, we review the processes of EMT and MET in development and cancer and combine them with knowledge from pluripotent stem cell research. Results Research has shown that these processes are activated in many cancers leading to dissemination of cancer cells throughout the body and formation of metastasis. While the regulation of EMT during cancer progression has been extensively studied for decades, many fundamental processes that govern normal development are only poorly understood. Recent discoveries, such as reprogramming to pluripotent stem cells and identification of ground and primed states of pluripotent stem cells, have redirected much attention to EMT and MET. Conclusion Findings from pluripotent stem cell research and EMT/MET should be combined in order to design future strategies aimed to improve our understanding of cancer progression and to help develop novel anticancer strategies. Keywords Cancer, embryonic developement, epithelial-to mesenchymal transition, mesenchymal-to epithelial transition, pluripotent stem cells. Eur J Clin Invest 2015; 45 (4): 415–422
Introduction During embryogenesis, cells forming primordial tissues and layers have to be able to reversibly change their phenotype. Two fundamental processes underlying this cellular plasticity are termed epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) – the former to acquire a migratory and the latter to regain a static phenotype. Pluripotent stem cells are at the root of embryonic development. As such, they are able to generate all somatic lineages – which they first accomplish in the embryo by utilizing the processes of EMT and MET – both needed to generate the embryonic germ layers: ectoderm, endoderm and mesoderm. However, EMT and MET programmes are also activated in cancer as the abilities of cancer cells to delaminate from a primary tumour and to form secondary tumours at distant places. In this review, we discuss the fundamental principles of EMT/MET in pluripotent stem cells and during development. We consider the emerging stem cell pluripotency concepts
ranging from the ground state to the primed state. Finally, we focus on novel clinical concepts by combining the knowledge from EMT/MET in development with findings in cancer and with recent advances about distinct functional states of pluripotent stem cells.
Definition and classification of EMT and MET A regulated biological process in which a polarized cell that normally interacts via the basal membrane with its basal surface, undergoes multiple biochemical and morphological changes leading to an enhanced migratory and invasive cell phenotype, is called EMT. Conversely, MET is a reversal of this process leading to a restoration of epithelial characteristics [1– 3]. Instantly, the concept of EMT has been closely intertwined with embryogenesis because of the notion that ultimately all cells of an adult vertebrate have been derived from a single fertilized oocyte through a stepwise differentiation process generating functional epithelia and the mesenchymal tissue
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supporting it [3]. The term EMT applies to three distinct biological settings in which it may occur: type 1 refers to the developmental EMT, type 2 describes such processes in which wound healing, tissue regeneration and organ fibrosis are involved, and type 3 denotes EMT associated with cancer progression and metastasis [3]. While all three EMT types share the main features as stated above, it is noteworthy to stress that developmental EMT lacks inflammatory responses typical of type 2 and type 3 EMTs [3,4] and that the intravasation and extravasation steps occurring during type 3 EMT do not resemble the migratory processes in embryogenesis or during fibrosis [1]. EMT is triggered by various extracellular signals which converge to activate EMT-specific transcription factors, epigenetic regulators and microRNA networks; more detail regarding the biochemical and molecular regulation of EMT are discussed elsewhere [1–5]. In contrast, MET induction is less well characterized in embryonic development and thus the most prominent examples include epithelialization during kidney development. In this review, we focus on pluripotent stem cell research and the developmental processes that rely on EMT and MET and on their similarities with events that occur during cancer progression.
EMT in embryonic development and in pluripotent stem cells So far, three primary EMT events have been described during early vertebrate development: (i) during implantation of the blastocyst/morula into the uterus wall, (ii) during gastrulation of the embryo and (iii) during neural crest formation in amniotes. The term ‘primary’ refers to the fact that these tissues have never undergone a previous EMT process [2]. Central to this concept is the presumption that the developmentally most undifferentiated cells (blastomeres or totipotent embryonic stem cells) are neither of epithelial nor of mesenchymal origin. During early development (Box 1), the totipotency ends by delineation of two lineages: the inner cell mass (ICM), which is the initial pluripotent population, and the trophectoderm (TE), which forms an extraembryonic epithelial cell layer supporting the ICM [6]. At later time points in the blastocyst, ICM differentiates into the pluripotent Nanog-expressing epiblast and the extraembryonic primitive endoderm [7]. Thus, the earliest processes in embryonic development involve the establishment of two extraembryonic and one embryonic epithelial cell layers from presumably unorganized primitive cells. Development of epithelia is the fundament for all subsequent EMTs ending up in the fact that – with exception of the anterior central nervous system and the epidermis, all adult vertebrate tissues have undergone at least one round of EMT [1]. Briefly, primary EMT takes place during embryonic implantation in the endometrium, when a subset of cells in the extraembryonic area (tro-
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phoblast) undergoes EMT to form the future placenta. The first primary EMT event in the embryo occurs when select cells of the epiblast move to the midline to form the primitive streak. The cells that undergo EMT and internalize generate mesoderm and endoderm, while the remaining epiblast cell layer becomes the future ectoderm. Primary EMT also occurs when the epithelial neural plate in the midline of the embryo rolls up to form the neural tube. Some epithelial cells from the dorsal midline of the neural tube migrate away inside the embryo to form ganglia, bone and cartilage of the jaws and glial cells [1]. Another example of EMT in development is represented by epithelial cells of the neuroectoderm that give rise to migratory neural crest cells. As a consequence of EMT, neural crest cells migrate away from the neural folds and disperse throughout the embryo, where they finally undergo further differentiation into distinct cell types, such as melanocytes [8,9]. Historically, embryonic stem cells (ESCs) from mice and humans have been derived from the ICM population of early blastocysts [10,11] and hence represent a pluripotent population giving rise to the future embryo. Accordingly, ESCs from both species are able to differentiate into all three germ layers, thus holding a great promise for regenerative medicine. Differences between the murine and the human ESCs have soon become obvious and have led to the notion that murine ESCs may be more undifferentiated than their human counterparts. This has led to the implementation of terms ‘na€ıve’ and ‘primed’ to describe the distinct cell types [12,13]. In the literature, difference has also been made in the usage of the ‘ground state’ and the ‘naive state’ of pluripotency, both to describe a state prior to the establishment of pluripotency. The ground state corresponds to an unrestricted pluripotent stem cell state acquired in epiblast cells that is free of any developmental constraints and that exhibits a hypomethylated DNA methylome comparable to derestricted ICM or na€ıve epiblast cells at E3.5-E4.5. In contrast, the na€ıve state solely refers to the functional property of ESCs to generate all embryonic lineages upon blastocyst injection [6]. In vitro, ESCs show an altered transcriptional and epigenetic profile compared to cells from preimplantation embryos and hence are considered na€ıve, but not ground state [6]. Interestingly, pluripotency – as a functional property – seems not to be connected to specific molecular and epigenetic signatures or cell culture conditions. In developmental terms, it seems that multiple pluripotent states exist with progressively restricted differentiation potential. Pluripotent cells exhibiting the ground state (and na€ıve functional properties) may thus represent the most uncommitted developmental potential [14]. In contrast, pluripotent cells such as epiblast stem cells (EpiSC) show a lineage-primed functional property towards some developmental lineages and thus have been classified as ‘primed’ pluripotent cells. Murine and human embryonic stem cells have been derived from the inner cell mass of early blastocysts; however, human ESCs (and all subsequently
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Box 1 totipotency A unique property of certain cells to give rise to all embryonic and extraembryonic tissues is called totipotency. During human and mice development, only the zygote and the cells produced during the first 3-4 cleavages (blastomeres) are totipotent. pluripotency It is the ability of cells to differentiate into all embryonic germ layers (ectoderm, endoderm and mesoderm), but not into extraembryonic tissues. Nevertheless, pluripotent stem cells can build up all tissues of the body and thereby represent an inexhaustible source of cells for regenerative medicine and for treatment of a wide range of diseases. Blastocyst Blastocyst formation starts about 5 days after fertilization, when a fluid-filled cavity forms in the morula, a mulberry-like structure comprised of few dozens of cells. Some cells begin to grow inward into the cavity to generate the inner cell mass (ICM), which represents a source of embryonic stem cells. ICM The cells of the ICM are pluripotent and give rise to all three embryonic germ layers, which will eventually develop into the embryo proper and the later foetus. Trophectoderm The outer core of a blastocyst comprises of cells that will develop into extraembryonic tissues. This layer of cells is called trophectoderm and will form the future placenta after implantation into the uterine wall. Embryonic stem cells Embryonic stem cells (ESCs) have been isolated from the ICM region of surplus blastocysts and comprise of a molecularly and epigenetically heterogeneous population able to differentiate into all three embryonic germ layers. ESCs express pluripotent stem cell markers, while lacking expression of proteins corresponding to more differentiated tissues. ESCs have unlimited lifespan and – at least theoretically – can indefinitely self-renew in cell culture. Epiblast stem cells Epiblast stem cells (EpiSCs) comprise of a molecularly and epigenetically heterogeneous population of cells derived from a later stage ICM region, in which a further diversification into epiblast and the primitive endoderm (giving rise to the future yolk sac) has occurred. EpiSCs have a limited pluripotent capacity compared to ESCs and are poised towards some embryonic tissues. Induced pluripotent stem cells Induced pluripotent stem cells (iPSCs) are pluripotent stem cells generated from somatic cells through the process of reprogramming – a yet not fully resolved multistep process involving a forced and simultaneous expression of at least 3 to 4 transcription factors (‘Yamanaka factors’ – historically Oct3/4, Sox2, Klf4 and c-myc). iPSCs have been shown to resemble ESCs on transcriptional and epigenetic level, as well as functionally. However, iPSCs may carry somatic mutation(s) of the adult tissue they have arisen. Migration Any directed movement of a cell, either within tissues in vivo or along basal membranes, ECM fibres and plastic surfaces in vitro, is called migration (for further reading see [52]). Invasion Invasion is a synonym for a penetration of a tissue barrier and an infiltration of the beneath tissues by an invasive (tumour) cell. In vitro, invasion is characterized by a movement through a 3D matrix [52].
generated human induced pluripotent stem cells (iPSCs)) grew flattened, epithelial-like and could never be individualized in culture. This has led to the assumption that human pluripotent stem cells are ‘primed’ and therefore rather correspond to murine EpiSCs. It was only recently that the corresponding na€ıve and
ground state populations could be established from human ESCs and iPSCs [15–19]. There is a rationale that pluripotent stem cells show a spectrum of distinct morphological, functional, transcriptional and epigenetic states as a consequence of plasticity changes upon external developmental cues. This ability to
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reversibly change a cell’s phenotype is a hallmark EMT and MET and represents the major processes regulating embryonic development, as well as cancer. Thus, there is a need to re-evaluate the relationship of pluripotent stem cells and EMT/MET and to combine the knowledge of complex developmental processes with the mechanisms of progression of cancer in order to develop improved therapeutic strategies. To visualize the states with distinct epithelial or mesenchymal morphology, we aligned pluripotent stem cells and their derivatives in a sequence of gradually differentiating stages (Fig. 1). We differentiate between the (I) ‘ground state’ pluripotent stem cell state, (II) the ‘primed’ pluripotent stem cell state, (III) the partially differentiated state and (IV) the terminally differentiated state: (I) the morphology of the most undifferentiated state ground state ESCs and iPSCs resembles dome-
shaped structures composed of small and round cells and cell aggregates with irregular cell shape and invisible cell borders. This stage confers high self-renewal capacity and a high degree of clonogenicity [6,15,20]. The epithelial/mesenchymal identity of this pluripotent state is so far unclear, and thus, it is possible that ground state pluripotent stem cells exhibit distinct EMT marker expression than primed pluripotent stem cells. (II) Primed state ESC cultures form flattened epithelial monolayer colonies have reduced self-renewal capacity and lack of clonogenicity in individualized cells [21]. Yet, it is also unclear whether ground state ESCs are able to differentiate into primed ESCs in vitro and whether this transition requires induction or repression of epithelial and/or mesenchymal markers. Without any doubt, ground state ESCs are supposed to be more undifferentiated than their primed counterparts as shown by gene
Differentiation state “primed“ pluripotent stem cell state
“ground state“ pluripotent stem cell state
Terminally differentiated state
Partially differentiated state
?
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MET
4-6i A/F/T/L
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“sequential“ OSKM Corresponding state in: Embyrogenesis
Inner cell mass (ICM)
Epiblast cells
Gastrulation (mesoendoderm), neural tube formation
Notochord, somites, urogenital system
Cell culture
Naive ESCs/iPSCs
Primed ESCs/iPSCs
Embryoid body
Somatic cells
Cancer
Unknown
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Blood-circulating cancer cells, cancer stem cells
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Figure 1 Requirements for EMT and MET during embryogenesis, in pluripotent stem cell cultures and cancer. Primed pluripotent stem cells correspond to the epiblast stage during embryogenesis and exist as human ESCs and iPSCs in primary cell culture models. Primed pluripotent stem cells can be converted into ground or na€ıve state using a cocktail of 4-6 distinct inhibitors (4-6i) and combinations of Activin, FGF-2, TGFb and LIF (A/F/T/L). The reverse process (ground state to primed state) has not yet been directly induced in human-derived cells in vitro, but may occur spontaneously. Of note, primed pluripotent stem cells possess an epitheliallike morphology, while ground state pluripotent stem cells form dome-shaped structures. During embryogenesis, epiblast cells induce at least one round of EMT (during gastrulation and neural tube formation) and MET (to form notochord, somites, urogenital system, etc.). In vitro, these stages can be recapitulated during embryoid body (EB) formation and later by differentiation into terminally differentiated cells. Conversely, it has been suggested that during reprogramming by OCT4, Sox2, Klf4 and c-myc (OSKM), somatic cells need to progress in reverse order (EMT, followed by MET) in order to generate primed pluripotent stem cells. Primary cancer has molecular and biological similarities with primed pluripotent stem cells, as both can induce formation of motile and invasive cells by EMT. Metastasis formation is suggested to involve a MET by blood-circulating cancer cells or cancer stem cells. A ‘ground state’ for primary cancers has not yet been described. Transmission light micrographs of na€ıve and primed state pluripotent stem cells can be observed in [12] and [13], the ground state morphology in [17–19] and EBs in [17]. “?” indicates the lack of clarity or knowledge associated with this process.
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expression, chromatin and biochemical analyses [6]. During embryogenesis, cells from the ICM form an epithelial layer, the epiblast, which has a pluripotent capacity. Thus, it remains to be clarified by future studies, whether ground state ESCs represent an ‘ancestor of the primed state’ or a ‘reset state of the metastable primed state’ just before the induction of the germ layer formation in the epiblast. (III) The partially differentiated state is induced by a differentiation of ESCs into three germ layers and can be recapitulated using a well-documented in vitro model termed ‘embryoid body (EB) formation’ which corresponds to gastrulation events observed in vivo. In the embryo, this process is EMT dependent and requires appearance of transiently migratory cells differentiating into mesoderm and endoderm. Both, the migratory phenotype and the induction of EMT, are recapitulated during EB formation in vitro [22]. However, although EBs are already comprised of a large collection of differentiated cell types including neuronal progenitors, cardiac myocytes and red blood cells, the stage of these cells does not represent a terminally differentiated state. (IV) Thus, further lineage restriction is required for somatic cell differentiation often including multiple events of EMT and MET. In developmental terms, this stage corresponds to the development of somatic cells that build up entire organs. Experimental examples utilize highly sophisticated cell culture systems that imitate kidney and eye development in vitro [23,24]. Several lines of evidence support the concept of the possibility to directly interconvert of the before-mentioned states in the reverse direction through a regulated transition between epithelial and mesenchymal stages. Seminal studies aimed to elucidate the mechanism of reprogramming of somatic cells to pluripotent cells have unveiled a process in which exogenous OCT4, Sox2, Klf4 and c-myc (OSKM) expression initiates expression of pluripotency factors leading to a stable phenotype. In most cases, the cells used for reprogramming are genuine mesenchymal cells (e.g. fibroblasts), whereas the induced pluripotent stem cells (iPSCs) are epithelial cells – thus requiring a MET to fulfil this process [25,26]. However, considerable amount of data suggest that reprogramming is induced by an early EMT and that factors known to govern EMT (e.g. TGFb, Slug, Snail or Wnt) actually enhance reprogramming [27–29]. This apparent paradox can be explained by the fact that mouse embryonic fibroblasts are heterogeneous and not fully mesenchymal and thus can be converted into a more homogeneous mesenchymal population by TGFb [27]. This is further underscored by the observation that keratinocytes, an epithelial cell type, require expression of EMT factors to enhance the reprogramming efficiency [29]. The second step of reprogramming, however, is thought to involve a MET [27,29]. This concept involves the existence of a partially differentiated state that, however, may only be transient or even metastable in the setting of reprogramming – although it is
likely to exist in cell cultures [27,29]. Lastly, a combination of 4–6 inhibitors and growth factors such as LIF, Activin and TGFb has been recently reported by several groups to efficiently convert iPSCs to ground state pluripotent stem cells [15–19].
Role of EMT/MET in cancer Evidence for EMT in cancer comes from the notion that bloodcirculating cancer cells and disseminated cancer cells from patients show EMT marker expression and a high degree of plasticity [30–32]. Cancers disseminate through EMT in order to colonize distant organs and to form secondary tumours (metastasis). The EMT phenotype is often observed in cells at the invasive front of primary tumours. It has been suggested that these cells may be involved in subsequent steps of tumour invasion/metastasis formation, that is intravasation, dissemination, extravasation and, finally, formation of micrometastasis [33–35]. Metastasis formation represents the major cause of cancer-associated deaths. However, carcinoma metastasis frequently develops a differentiated epithelial phenotype – a process requiring a reversion of EMT. Although the need for MET during reprogramming is consistent with the epithelial nature of ESCs, it seems paradoxical that the induction of a full EMT phenotype in carcinoma actually increased invasiveness while suppressing its metastatic capacity [36]. Thus, EMTrelated invasion is required but not sufficient to fulfil the formation of metastasis, meaning that cancer cells can be invasive but not metastatic. In the light of (cancer) stemness and epithelial plasticity, the concept of cancer stem cells (CSCs, i.e. the cells that initiate, maintain and propagate cancer) provides another level of complexity in the field, as the EMT is thought to endow CSCs with their tumorigenic properties [5,34,37]. However, to efficiently colonize distant tissues, CSCs must also undergo the reverse process – a MET. Accordingly, CSC properties may actually include the ability to initiate a consecutive round of MET after having executed EMT, in contrast to non-CSCs, which perhaps solely induce EMT. Therefore, CSCs may have a lot in common with ESCs and thus studying early development may help to translate knowledge into potential therapies again cancer.
Clinical impact of EMT/MET on cancer research In patients, invasion and dissemination of cancer cells seem to be concomitant events associated with bad prognosis of cancer. Accordingly, inhibition of EMT may render cancers cells less prone to invasiveness and metastasis formation. However, constitutive activation of EMT may acutely suppress metastasis formation because MET is required for metastasis to form [36]. It may therefore be essential to control the plasticity of cancer by interfering with both, the EMT and MET. Here, we envision
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several potential strategies how therapies may be adjusted to eradicate cancer: I one option is to combine agents that target CSCs with conventional chemotherapy that is effective against nonCSCs. Recent data indicate that cancer stem cells can generate differentiated cancer cells and vice versa [38,39]. This plasticity seems to be a result of distinct chromatin states of the Zeb1 promoter [40]. However, as a targeted therapy against CSCs may not hit non-CSC, this approach should include a combination of the classical chemotherapy together with anti-CSC drugs. II Similar considerations can be employed for the inhibition of EMT to prevent dissemination of cancer cells, while controlling the proliferation of primary tumour via chemotherapy. To hit the EMT, the strategy may include inhibition of its signalling pathways, that is EGFR, PDGFR, IGFR1 and TGFb [41]. However, both approaches may only help in cancers that have not yet disseminated or in which EMT is a late event in tumorigenesis. III Paradoxically, constitutive expression of Snail, pRRX1 or Twist also suppressed metastasis formation of human cancer cell lines in a mouse model in vitro and in vivo [36,42], as EMT factors must be downregulated for metastasis to form [42,43]. In breast or pancreatic cancer, where cancer cells disseminate early [44,45], inhibition of EMT may favour the development of metastasis, thus a prevention of MET should be taken into account rather than of EMT [42,43]. Of note, as there is a potential risk of developing a mesenchymal tumour in a supportive organ, potential strategies against metastasis should preferentially combine anti-EMT and anti-MET blockers. IV An intriguing approach to study cancer progression and its association with distinct developmental states would be to reprogramme patient’s primary cancer cells to pluripotency. Strikingly, despite intensive attempts in the past, only a very limited number of successfully reprogrammed induced pluripotent cell lines have been generated from human cancer so far [46,47]. The low frequency of reprogramming is clearly unexpected, because limiting tumoursuppressor activity of p53, p19ARF, p16INK4a and p21KIP/CIP – a common feature of many cancers – leads to more efficient cellular reprogramming [48]. While the deduction that a reprogrammed cancer cell will never lose its cancer state may actually hold true, it is likewise possible that an ameliorated, epithelial and less malignant phenotype is induced [47]. V Additionally, it remains to be clarified whether induction of ‘ground state pluripotency’ in cancer-derived iPSCs or in primary cancers may enhance or reduce their tumorigenicity. VI Finally, there is a rationale that establishment of drug screening assays may be undertaken to develop novel and
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personalized drug treatment to patients suffering from cancer. Thus, ESCs, iPSCs or cancer-derived iPSCs could be used to identify novel inhibitors or activators of EMT and MET. Examples of potential targets against EMT include recently developed inhibitors for IGFR1 [49], TGFb [50] or EGFR [51], which have already proven effective in several clinical studies.
Conclusion In summary, extending the EMT/MET research to pluripotent stem cell models will help redefine cell plasticity in normal versus malignant development. This knowledge will undoubtedly facilitate the study of cancer progression and help develop novel anticancer strategies.
Conflict of interest All authors declare that they have no conflict of interest. Address Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria (B. Kovacic, M. Rosner, K. Schipany, L. Ionce, M. Hengstschl€ ager). Correspondence to: Boris Kovacic, Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria. Tel.: +43 1 40160 56514; fax: +43 1 40160 956 501; e-mail: [email protected] Received 25 November 2014; accepted 28 January 2015 References 1 Nieto MA. Epithelial plasticity: a common theme in embryonic and cancer cells. Science 2013;342:1234850. 2 Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Investig 2009;119:1438–49. 3 Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig 2009;119:1420–8. 4 Lopez-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med 2009;1:303–14. 5 Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009;9:265–73. 6 Hackett JA, Surani MA. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014;15:416–30. 7 Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O et al. Nanog is the gateway to the pluripotent ground state. Cell 2009;138:722–37. 8 Sauka-Spengler T, Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol 2008;9:557–68.
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