Biochimica et Biophysica Acta 1856 (2015) 121–129

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Review

The Hippo signal transduction pathway in soft tissue sarcomas Abdalla D. Mohamed a, Annie M. Tremblay c,d,e, Graeme I. Murray b, Henning Wackerhage a,⁎ a

School of Medical Sciences, University of Aberdeen, AB25 2ZD Scotland, UK School of Medicine and Dentistry, University of Aberdeen, AB25 2ZD Scotland, UK c Stem Cell Program, Children's Hospital, Boston, MA 02115, USA d Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA e Harvard Stem Cell Institute, Cambridge, MA 02138, USA b

a r t i c l e

i n f o

Article history: Received 10 April 2015 Received in revised form 27 May 2015 Accepted 28 May 2015 Available online 4 June 2015 Keywords: Sarcoma Rhabdomyosarcoma Hippo pathway YAP TAZ Fusion genes

a b s t r a c t Sarcomas are rare cancers (≈1% of all solid tumours) usually of mesenchymal origin. Here, we review evidence implicating the Hippo pathway in soft tissue sarcomas. Several transgenic mouse models of Hippo pathway members (Nf2, Mob1, LATS1 and YAP1 mutants) develop various types of sarcoma. Despite that, Hippo member genes are rarely point mutated in human sarcomas. Instead, WWTR1-CAMTA1 and YAP1-TFE3 fusion genes are found in almost all cases of epithelioid haemangioendothelioma. Also copy number gains of YAP1 and other Hippo members occur at low frequencies but the most likely cause of perturbed Hippo signalling in sarcoma is the cross-talk with commonly mutated cancer genes such as KRAS, PIK3CA, CTNNB1 or FBXW7. Current Hippo pathway-targeting drugs include compounds that target the interaction between YAP and TEAD G proteincoupled receptors (GPCR) and the mevalonate pathway (e.g. statins). Given that many Hippo pathwaymodulating drugs are already used in patients, this could lead to early clinical trials testing their efficacy in different types of sarcoma. Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology of soft tissue sarcomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hippo pathway & cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hippo mouse models that develop sarcomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics of soft tissue sarcoma and the Hippo pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Hippo fusion genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Hippo copy number alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Epigenetic regulation of Hippo genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Hippo signalling and the genomic landscape of sarcoma . . . . . . . . . . . . . . . . . . . . . . . 6. Hippo-targeted therapies in soft tissue sarcoma?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Drugs that target YAP-TEAD interaction or TEADs: verteporfin, cyclic peptides, VGLL4-mimicking peptides 6.2. G protein-coupled receptors (GPCRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Statins and bisphosphonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Summary and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transparency document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The Hippo pathway was discovered as a result of tumour suppressor screens in the fruit fly (Drosophila melanogaster) and was later found to ⁎ Corresponding author. E-mail address: [email protected] (H. Wackerhage).

http://dx.doi.org/10.1016/j.bbcan.2015.05.006 0304-419X/Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.

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be conserved in mammals. In flies, inactivation of Hippo members resulted in an overgrowth that resembles the skin of a Hippopotamus, thereby naming the pathway [1]. Hippo pathway members are rarely point mutated in cancer [2,3] but the experimental mutation of Hippo members usually causes overgrowth in fruit flies [4] and tumours in mice, including sarcomas [2,3,5]. Here, we review the role of the Hippo pathway specifically in soft tissue sarcomas excluding osteosarcomas

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and viral-mediated sarcomas such as Kaposi's sarcoma. After introducing sarcomas and the Hippo pathway, we review animal models where Hippo pathway-related transgenesis results in sarcomas. We then discuss findings and hypotheses implicating the Hippo pathway in sarcoma signal transduction, development and pathology. Finally, we review the current possibilities for Hippo pathway-targeting treatments in sarcoma and highlight areas for future research.

2. Pathology of soft tissue sarcomas Soft tissue sarcomas (from Greek sarx flesh) represent a biologically, clinically, pathologically, and genetically [6] diverse array of malignant tumours arising mostly from mesenchyme-derived tissues. Sarcomas can occur in both adults and children. They are rare cancers and account for about 1% of solid tumours in adults and for a significantly higher proportion of cases in children (Cancer Research UK, 2015). In 2014, it was estimated that 790 new cases of rhabdomyosarcoma and bone tumours, including osteosarcomas and Ewing sarcomas, would be diagnosed in children 0–14 years old, representing 7% of all childhood cancers (American Cancer Society, 2014). The most common types of soft tissue sarcomas include leiomyosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and angiosarcoma (see Fig. 1 [7]). The current classification of soft tissue sarcomas is based on a combination of tumour morphology, immunophenotype and molecular pathology. Genetically, sarcomas can show either aberrant, chimeric transcription regulators as a consequence of fusion genes such as PAX3/7-FOXO1 [6,8], somatic point mutations of well-known cancer genes such as oncogenic RAS isoforms, PIK3CA or TP53, or DNA copy number gains or losses [6,9–12]. As we will show later, there is no evidence for recurrent Hippo gene point mutations in sarcomas, while there is abundant evidence for mutations of typical cancer genes [13, 14] that can cross-talk to the main members of the Hippo pathway. Additionally, fusion genes involving WWTR1 or to a lesser extent YAP1 are found in nearly all cases of epithelioid haemangioendothelioma [15,16], whereas YAP1 and VGLL3 copy number gains have been reported for some types of sarcoma, especially rhabdomyosarcoma [17–19]. It is unclear to this date whether VGLL3, which is associated with tumour suppression in ovarian cancer [20], is a bona fide Hippo pathway member. The increasing use of immunohistochemistry and the application of sophisticated molecular techniques combined with a better understanding of soft tissue sarcoma biology have led to a continued refinement of the classification of soft tissue sarcomas. Some previously recognised types of soft tissue sarcoma, most notably malignant fibrous histiocytoma, are now being reclassified. Most tumours previously classified as malignant fibrous histiocytomas but showing no specific immunophenotype would now be regarded as undifferentiated pleomorphic sarcomas. The majority of soft tissue sarcomas arise in the limbs of older people although rhabdomyosarcoma, especially the Dedifferentiated Pleomorphic Myxoid

Well differentiated

Biphasic

Liposarcoma Gastrointestinal (adipocytes) stromal tumour (GIST)

Leiomyosarcoma (smooth muscle)

Synovial sarcoma

Soft tissue sarcoma

Angiosarcoma Kaposi’s sarcoma

Rhabdomyosarcoma (myoblasts)

Embryonal Alveolar

Vascular

Spindle cell

Epithelioid haemangioendothelioma

Fibrosarcoma (fibroblasts) Spindle cell

Pleomorphic

Low-grade fibromyxoid sarcoma

Myxofibrosaroma

Fig. 1. Classification of soft tissue sarcomas on the basis of their differentiation.

embryonal subtype, tends to occur predominantly in young children [21]. Most soft tissue sarcomas arise sporadically but muscle injury, most likely by increasing the number of activated satellite cells, enhances the penetrance and shortens the latency of rhabdomyosarcoma phenotypes in mice [18,22]. In addition, known risk factors for the development of specific types of sarcoma in humans include exposure to radiation (e.g. post-radiation angiosarcoma) or exposure to environmental/occupational carcinogens (e.g. vinyl chloride-associated angiosarcoma). Sarcomas, in contrast to carcinomas, have a predilection for showing a vascular pattern of spread with the lungs generally being one of the predominant sites of metastasis. A combination of surgery, cytotoxic chemotherapy and radiotherapy are the mainstays of current treatment with some types of sarcoma being treated with neoadjuvant chemo-radiotherapy prior to definitive surgery [23–25]. Immunotherapy and targeted, novel biologic therapies are also now being evaluated in the treatment of specific types of sarcoma [26–29] and as we show below, targeting the Hippo pathway in sarcoma should already be possible with existing drugs. The prognosis and outcome of soft tissue sarcomas depend on a range of factors including the particular subtype of tumour, the anatomical location, size and grade as well as tumour stage at time of diagnosis [7,30,31]. The overall 5-year survival rate for soft tissue sarcomas has gradually been improving, especially in children, but the outcome varies with the specific type of soft tissue sarcoma and depends on the prognostic factors that have been outlined above. Overall the 5-year survival rate for adults with soft tissue sarcomas is approximately 60% while it is higher in children, reaching approximately 70% (Cancer Research UK, 2015). 3. Hippo pathway & cancer The fruit fly (D. melanogaster) has been used over many decades as a model organism to identify genes whose knockout results in cancerous growth [32]. This research has led to the discovery of a set of genes that encode two interacting kinases and auxiliary proteins, now defined as the core Hippo pathway (see Fig. 2). The main function of the Hippo pathway is to inhibit proliferation and to promote apoptosis, thereby limiting organ growth [4]. In the conserved mammalian Hippo pathway, the STE20-like protein kinases 1 and 2 (MST1 and MST2, gene symbols: STK4 and STK3) regulate the large tumour suppressor kinases 1 and 2 (protein name and gene symbol: LATS1 and LATS2), through phosphorylation [33]. Active LATS1 and LATS2 then interact through their PPxY motifs with the WW domains of the transcriptional co-factors YAP (gene symbol YAP1) or TAZ (gene symbol WWTR1; note that the gene TAZ encodes a protein termed Tafazzin which is not part of the pathway) [34]. This physical contact allows LATS1 and LATS2 to inhibit YAP [35] and TAZ [36] through the phosphorylation of multiple HXRXXS amino acid motifs. The phosphorylation of these motifs promotes the inactivation of YAP and TAZ through translocation from the nucleus into the cytosol and degradation. Additionally, YAP can be phosphorylated at Tyr357 by the tyrosine kinase YES1, which has resulted in its name Yes-associated protein (YAP) [37]. Nuclear and active YAP, which was first discovered by Marius Sudol [38,39], and its paralogue TAZ is believed to exert their tumourigenic functions mainly via the TEAD transcription factors [40]. Specifically, YAP and presumably TAZ de-repress and activate the TEAD transcription factors that otherwise recruit transcriptional repressors [41]. Additionally, YAP and TAZ are capable of co-regulating other transcription factors including those belonging to the Smad family [42] and Tbx5 in some contexts [37,43]. In addition to the Hippo kinases, extensive cross-talk mechanisms also regulate the activity of YAP and TAZ, notably mechanotransduction [44], WNT signalling [45,46], and G protein-coupled receptors [47]. The early studies in fruit flies and subsequent studies in mammals demonstrate that the upstream proliferation-inhibiting Hippo proteins and the proliferation-promoting Hippo transcriptional regulators act as potent tumour suppressors and oncogenes, respectively. For

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Fig. 2. Schematic drawing depicting proteins associated with the Hippo pathway in sarcoma. A The Hippo kinases MST1/2 and LATS1/2 together with the auxiliary proteins SAV1 and Mob1 inhibit the transcriptional co-factors YAP and TAZ by phosphorylation of HXRXXS motifs such as Ser127 on YAP. B YAP and TAZ dephosphorylation by phosphatases results in their nuclear translocation, allowing the de-repression or activation of TEAD1-4 transcription factors. In some contexts, VGLL4 acts as a co-repressor of TEAD1-4 [41] and other VGLL isoforms are likely to be capable of interaction with TEADs [105]. C–G Links between the Hippo pathway and proteins whose genes are recurrently mutated in sarcoma (shown in red) C In epithelioid haemangioendothelioma, mostly the WWTR1-CAMTA1 [16] but also YAP1-TFE3 [15] fusion genes (encoding TAZ-CAMTA1 and YAP-TFE3 fusion proteins) presumably regulate the TEAD transcription factors. D The Wnt-regulator β-CATENIN (CTNNB1) can also interact with YAP and TAZ. It has been proposed that β-CATENIN, YAP, the tyrosine kinase YES1 and the transcription factor TBX5 form complexes of functional importance in cancer. In this context, the tyrosine kinase YES1 can activate YAP by Tyr357 phosphorylation [37]. E KRAS and PI3K can enhance the co-activator function of YAP, or their activity can be substituted by YAP to overcome oncogene addiction, identifying YAP as a modulator of drug resistance. F In alveolar rhabdomyosarcoma the PAX3-FOXO1 fusion proteins/genes drive the expression of RASSF4 which inhibits MST1 [84]. Not shown: Others suggest that YAP and TAZ are included in the Wnt destruction complex [46]. G The SCF (βTRCP) ubiquitin ligase regulates YAP [35] and especially TAZ [94] degradation via phosphodegron-mediated mechanisms. Recently another ubiquitin ligase named FBXW7, which is frequently mutated in rhabdomyosarcoma [73], was identified as part of a complex that mediates ubiquitin-dependent destruction of YAP and TAZ and its loss was associated with increased YAP abundance [95]. Drugs and drug targets that modulate the activity of the Hippo pathway are shown as red text. Verteporfin [97] and cyclic peptides [106] inhibit the binding of YAP to TEAD transcription factors. VGLL4-mimicking peptides repress TEAD transcription factors [79]. Specific G protein-coupled receptors (GPCRs) signal to YAP [47]. Given that GPCR-targeting drugs are the largest class of currently used drugs some might be applicable for Hippo activity modulation in sarcomas. The activity of YAP and TAZ is regulated by the mevalonate pathway [102,103], which can itself be targeted for example by statins. For more Hippo pathway related drugs see [107].

example, YAP hyperactivity as a consequence of the sole expression of the constitutively active YAP1 S127A mutant in mice alone is sufficient to initiate hyperplasia and over time cause cancer in the liver [48,49] and skin [50]. Also, along with injury in the skeletal muscle, YAP1 S127A expression in satellite cells alone is sufficient to causes rhabdomyosarcoma [18]. These studies identify YAP as an unusually potent oncogene. Thus it would be intuitive that YAP-activating mutations such as YAP1 S127A occur by chance and be a driver of tumourigenesis. However, this is not the case. So far, even if the LATS kinases appear to be mutated in some rare cases [51], recurrent mutations of Hippo pathway genes have not been reported [2,3,5,13]. It is even more surprising that not a single YAP1 S127A mutation was found in 21,441 tumour DNA samples from 91 cancer studies that are compiled in the cBIOPortal database [52,53]. From this conundrum two questions arise: First, why are Hippo genes, unlike other well established cancer-associated genes such as TP53, PIK3CA or KRAS [13], rarely point mutated in cancer? Second, if point mutations are not major the source of tumourigenic Hippo signalling, what else is causing the high YAP activity frequently seen in cancers? A potential answer to the first question is that the somatic (i.e. cancer cell) mutation frequency differs at least 100-fold inbetween different types of cancer and also varies across the genome from ≈ 0.1 to ≈ 10 mutations per 100,000,000 bases depending on DNA replication timing and transcriptional activity [54,55]. There is evidence that copy number gains and other rearrangements are especially

found in early replicating chromatin [55]. Thus, Hippo genes may lie in chromatin with a low mutation rate perhaps due to their functional importance. Regarding the second question, alternative mechanisms of YAP and TAZ activation in cancer include Hippo fusion genes, copy number alterations of Hippo genes, hypermethylation of Hippo kinase gene promoters and cross-talk from significantly mutated non-Hippo cancer genes. Most prominently, Hippo fusion genes have been identified for almost all cases of epithelioid haemangioendothelioma [15,16], a vascular sarcoma that will be discussed below. Copy number gains have been reported for YAP1 and VGLL3 [17,18,56] and frequently mutated cancer genes such as CTNNB1 (protein: β-catenin [37] or KRAS [57–59] have been shown to affect YAP activity and require YAP for some aspects of tumourigenesis. 4. Hippo mouse models that develop sarcomas Important direct evidence for a role of the Hippo pathway in sarcoma stems from transgenic Hippo mouse models that develop sarcoma. Such models are summarised in Table 1 and discussed in greater detail below. As discussed earlier, the Hippo pathway kinase Lats1 inhibits YAP and TAZ by phosphorylation and restricts their nuclear localization and transcriptional activity with the TEAD factors. Most Lats−/− mice die in utero, and only ~8% survive. The surviving mice display a strong growth retardation phenotype, severe fertility defects and pituitary

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Table 1 Hippo transgenic animal models that lead to the development of sarcomas. Genotype

Sarcoma type

Incidence

Latency

Reference

Lats1−/− Lats1−/− and mutagen (DMBA+ UVB exposure) Nf2/Merlin +/−

Pax7CreER TetO-YAP1 S127A and cardiotoxin injury

Embryonal rhabdomyosarcoma

14% 83% 63% 9% Low frequency Low frequency 77% 32% 74% 8% 24% 22% 100%

4–10 months 10–14 weeks after birth and treatment 10–30 months 10–30 months Not determined Not determined b5 months b5 months 4–21 months 4–21 months 25–70 weeks 25–70 weeks 10–11 weeks after cardiotoxin injury

[60] [60] [61]

Mob1aΔ/+1btr/tr or Mob1aΔ/Δ1btr/+

Skin fibrosarcoma Skin fibrosarcoma Osteosarcoma*** Fibrosarcoma* Chondrosarcoma Uterine sarcoma Osteosarcoma*** Fibrosarcoma* Osteosarcoma*** Fibrosarcoma* Extraskeletal osteosarcoma, fibrosarcoma†

Nf2/Merlin +/− p53 +/− (cis)** Nf2/Merlin +/− p53 +/− (trans)**

[61] [61] [62] [18]

*Of note: In this study, the tumours were reported to also contain areas presenting features of rhabdomyosarcoma within the same tumours, but were classified predominantly as fibrosarcomas. **p53 mutations were introduced on the same allele as the NF2/Merlin mutation (in cis), or on the other allele (in trans). ***In nearly all sarcomas, loss of the other wild-type NF2/Merlin allele was observed in the tumours (Loss of Heterozygosity, LOH); † or myofibrosarcoma.

hyperplasia leading to hormonal impairment. Indeed, Lats1−/− females have reduced levels of luteinizing hormone, prolactin and growth hormone. All Lats1−/− females have underdeveloped mammary glands and develop ovarian stromal cell tumours by 3 months of age. Large skin fibrosarcomas develop in 14% of all Lats1−/− females between 4-10 months of age. The incidence of skin fibrosarcomas increases to 83% when LATS1−/− mice are subjected to a mutagen treatment consisting of a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) followed by repeated exposure to UVB, whereas all Lats1+/− animals remained tumour-free [60]. Neurofibromatosis type II is a dominantly inherited disorder caused by mutations in the neurofibromin 2 (gene symbol Nf2) gene, encoding a protein called Merlin, also known as schwannomin. In both drosophila and mammals, Nf2/Merlin acts upstream of the Hippo kinases to control the transcriptional activity of Yap [63,64]. Mutations in the Nf2 gene identified in Neurofibromatosis type II patients lead to the production of shorter protein products that have lost the tumour suppressor function associated with full-length Merlin. Consequently, neurofibromatosis type II patients are predisposed to developing tumours affecting the nervous system, primarily schwannomas, meningiomas and ependymomas [61]. However, mice expressing a mutated Nf2 allele, originally intended to mimic human Neurofibromatosis type II in mice, do not develop schwannomas, meningiomas or ependymomas. Instead, they principally develop osteosarcomas (63%), fibrosarcomas (9%) and hepatocellular carcinomas (9%) occurring between 10 and 30 months of age [61]. Notably, human neurofibromatosis type II patients do not develop osteosarcomas, fibrosarcomas or hepatocellular carcinomas at higher incidence than the normal population [61]. Also, chondrosarcomas and uterine sarcomas arose at low frequency in Nf2+/− mice, and in some cases loss of the wild-type Nf2 allele was detected [61]. However, nearly all of the osteosarcomas and fibrosarcomas displayed a loss of the wild-type Nf2 allele (LOH), supporting a role for the complete loss of Merlin function in the aetiology of those tumour types. These tumours also displayed an enhanced metastatic potential [61]. An additional hemizygous inactivation of p53 produced a similar tumour spectrum but enhanced the incidence of sarcomas (osteosarcomas, 77%; fibrosarcomas, 32%) and significantly reduced tumour-free survival [61]. Mob1 proteins are core components of the Hippo tumour suppressor pathway that are required for activation of the LATS kinases by the MST kinases [65]. Mice bearing a null mutation of the Mps one binder (Mob) kinase activator 1A (Mob1aΔ/Δ) or a trapped mutation of the Mob1b gene (Mob1btr/tr) show no abnormalities in morphology, body weight, histology, or life span [62]. While the complete loss of both genes (Mob1a and Mob1b; Mob1aΔ/Δ1btr/tr) is embryonic lethal, doublemutant mice retaining one allele of either Mob1a (Mob1aΔ/+1btr/tr) or

Mob1b (Mob1aΔ/Δ1btr/+) survive. Of these heterozygotes, 52% develop dental malocclusion, 25% have a disorganization of the inner ear hair bundles in the cochlear organ of Corti resulting in a disequilibrium and 31% display increased trabeculae in femurs [62]. In addition, 100% of the Mob1aΔ/+1btr/tr and Mob1aΔ/Δ1btr/+ heterozygotes mice spontaneously develop various types of tumours, while only 4% of the control mice (heterozygotes for both genes; Mob1aΔ/+1btr/+) developed tumours [62]. All single heterozygote mice developed skin cancer (100%), 92% developed benign bony overgrowths called exostoses, 24% developed extraskeletal osteosarcomas and 22% developed subcutaneous fibrosarcomas or myofibrosarcomas. These mice also developed other tumour types at lower incidence, such as breast (16%), lung (5%) and salivary gland (5%) tumours. Interestingly, while 50% of the Mob1aΔ/Δ1btr/+ mice developed liver tumours, not a single liver tumour was found in the Mob1aΔ/+1btr/tr mice [62]. YAP, along with its paralogue TAZ, is the main transducer of the Hippo pathway activity via its de-repression and activation of TEAD transcription factors. Specifically in activated but not quiescent satellite cells, the overexpression of YAP1 S127A alone is sufficient to cause embryonal rhabdomyosarcomas in 100% of the mice [18], in line with the pro-proliferative and anti-differentiation roles of YAP in myoblasts and primary satellite cells in culture [66,67]. The mouse tumours match human ERMS well as judged by their pathology and gene expression profile. Conversely, YAP1 knockdown in the human RD ERMS cell line reduces their proliferation, soft agar growth and xenotransplant burden as well as promotes myogenic differentiation [18]. The fact that YAP1 S127A drives tumourigenesis only in activated but not quiescent satellite cells matches clinical observations. First, ERMS occurs especially in infants and young children where activated satellite cells support longitudinal muscle growth [21]. Additionally, mdx mice, where many satellite cells are activated to regenerate damaged muscle [68] spontaneously develop rhabdomyosarcomas [69]. Dystrophin and dysferlin double-mutants are also emerging as potential models for rhabdomyosarcoma [70]. 5. Genetics of soft tissue sarcoma and the Hippo pathway In this section we will review Hippo-related genetic changes in sarcomas. Specifically, we discuss Hippo fusion genes, Hippo copy number changes, epigenetic alterations of Hippo genes and mutations that may affect Hippo members through cross-talk in sarcomas. 5.1. Hippo fusion genes Fusion products involving Hippo genes have been associated especially with epithelioid haemangioma, a vascular sarcoma. In this

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tumour, the WWTR1-CAMTA (WWTR1 is the gene symbol for TAZ) fusion product was found in 17 cases independent of location [16]. A follow-up analysis on the 10% of epithelioid haemangiomas that were negative for the WWTR1-CAMTA identified those as a pathologically distinct subset. In eight of these cases a YAP1-TFE3 fusion product was found [15]. These findings were confirmed by a study on 35 epithelioid haemangioendotheliomas in which WWTR1-CAMTA fusions were found in 33 cases and YAP1-TFE3 fusion genes in the remaining 2 cases [71]. In this type of tumour, fusion products involving Hippo genes appear to be as specific as the PAX3/7-FOXO1 fusions are for alveolar rhabdomyosarcomas [72,73]. Apart from epithelioid haemangioma, a single case of spindle cell rhabdomyosarcoma was associated with a TEAD1-NCOA1 fusion protein [74]. How these Hippo fusion genes contribute to tumourigenesis and why epithelioid haemangioendotheliomas appear to depend almost in every case on WWTR1-CAMTA or YAP1-TFE3 fusion genes is unknown. Recently, the WWTR1-CAMTA fusion was shown to inhibit the tumour suppressive function of the CAMTA transcriptional regulator and to confer a TAZ-like transcriptional programme in cells expressing the fusion, thereby favouring oncogenic transformation and resistance to anoikis [75]. 5.2. Hippo copy number alterations Generally, specific types of cancer are either dominated by high levels of somatic point mutations (termed M type) or by copy number gains with TP53 mutations (termed C type), or by intermediate but not high levels of both [76]. This may reflect different causes of mutagenesis and also be related to chromatin features, as point mutations and copy number losses are associated with late replicating chromatin whereas copy number gains and rearrangements are associated with early replicating chromatin [54,55]. Generally, Hippo genes appear far more affected by copy number gains and rearrangements than by point mutations as evidenced from Hippo genes analyses using the cBioPortal. This supports the idea that Hippo genes are possibly located in early replicating chromatin, consistent with the elevated copy number alterations found in C type cancers and the existence of YAP1 and WWTR1 fusion genes. Indeed, sporadic Hippo copy number gains are found in different types of sarcoma (see Fig. S1 for a re-analysis of the data of Barretina et al. 2010 using the cBIOPortal platform [77]). Copy number gains of YAP1 (11q) were reported in 22 cases and copy number gains of VGLL3 (3p) were found in 19 cases of soft tissue sarcoma subtypes [17]. YAP1 copy number gains were additionally observed in embryonal but not alveolar rhabdomyosarcoma [18] and high level VGLL3 amplifications were found in 10 out of 12 cases of myxoinflammatory, fibroblastic sarcoma and hemosiderotic, fibrolipomatous tumours [78]. Additionally the re-analysis of cBioPortal-deposited sequencing data for different types of sarcoma [77] reveals YAP1, WWTR1, VGLL1-4 or TEAD1-4 copy number gains but no losses in 17% of the cases (YAP1 3%, WWTR1 2%, VGLL4 2%, VGLL3 5%; Fig. S1). However, it is currently unknown, although seemingly unlikely, whether all of these copy number gains in Hippo genes are bona fide cancer drivers and whether their effect is a major one. Indeed, copy number gains of YAP1, VGLL3, VGLL4 or TEAD1-4 appear to co-exist even though VGLL4 has been associated with a disruption of TEAD/YAP transcriptional activity and tumour suppression [41,79]. Second, high YAP activity appears to result in the activation of a negative feedback loop [67]. In line with this, a higher concentration of YAP (or elevated YAP nuclear localization and transcriptional activity) is generally associated with an increase in the levels of inhibitory YAP Ser127 phosphorylation because of the increased expression of several members of the Hippo kinase cascade that inhibits YAP through phosphorylation [67]. This is functionally supported by the fact that the expression of a wild-type YAP1 does not prevent myogenic differentiation whereas the expression of constitutively active YAP1 S127A does prevent differentiation [66]. Collectively, this suggests that recurrent copy number alterations, especially gains, in the TEAD co-regulator genes YAP1, WWTR1, VGLL3 and VGLL4 in a subset

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of sarcomas can only partially explain why the balance is tipped towards tumourigenesis. 5.3. Epigenetic regulation of Hippo genes Epigenetically mediated loss of the expression of the Hippo kinases or other Hippo pathway components has been associated with certain cancers [80,81]. In primary soft tissue sarcomas, the CpG island in of MST1 and MST2 kinase promoters were found to be significantly hypermethylated. MST1 was methylated in liposarcoma (25%), leiomyosarcoma (50%), malignant fibrous histiocytoma and myxofibrosarcoma (17%) as well as in rhabdomyosarcoma (80%). In contrast MST2 was found methylated principally in liposarcoma (15%), malignant fibrous histiocytoma and myxofibrosarcoma (31%) as well as synovial sarcoma (50%). MST1 methylation was associated with a decreased expression of the MST1 mRNA, and surprisingly, a significantly increased risk for tumour-related death was found for patients with an unmethylated MST1 promoter. The tumour suppressor kinase LATS1 was also methylated in 7% of all sarcomas tested and the LATS2 and WW45/Salvador genes were not found methylated in sarcomas. The promoter of the tumour suppressor gene RASSF1A has been shown to be methylated in liposarcoma (18%), leiomyosarcoma (39%), malignant fibrous histiocytoma and myxofibrosarcoma (6%) and in neurogenic sarcomas (50%), which is associated with an unfavourable prognosis for cancer patients [82]. These results, obtained with a small sample size, are a proof-of-principle for the epigenetic regulation of Hippo genes. It remains to be determined if the methylation status of the MST kinases is linked with YAP activity in sarcomas. Indeed, the MST kinases have been shown to be dispensable for Hippo pathway activity in some cell types, notably in mouse embryonic fibroblasts and skin [83]. 5.4. Hippo signalling and the genomic landscape of sarcoma So far two studies have reported the sequencing of multiple types of sarcoma [77] and of rhabdomyosarcoma, a sarcoma where skeletal muscle-related genes are expressed [73]. Additionally, preliminary data from a TCGA sarcoma study are accessible through cBIO but these data are under publication embargo until 2016. In the earliest largescale sequencing study 207 high grade, seven types of soft-tissue sarcomas were analysed for the expression of 722 protein-coding and microRNA genes [77]. A limitation of this study was the subjective selection of the genes sequenced and the relatively small sample number for such an analysis. However, the mutated genes CTNNB1, PIK3CA, NF1 and PIK3CA identified in this study were also identified later in a larger unbiased study involving next generation sequencing of rhabdomyosarcomas [73]. In rhabdomyosarcoma, the most distinct subgroup is alveolar rhabdomyosarcoma (ARMS). This subtype is in most cases marked by PAX3-FOXO1 or PAX7-FOXO1 fusion genes with rare alternative PAX fusions [73]. A recent study has linked PAX3-FOXO1 to the increased expression of RASSF4 which inhibits the Hippo kinase MST1 [84]. Such an inhibition of a Hippo kinase should result in an increased transcriptional output downstream of the Hippo signalling cascade, but the exact mechanism could not be identified. Nevertheless, this suggest cross-talk between the ARMS PAX3/7-FOXO1 fusions and Hippo signalling. Conversely, the rhabdomyosarcomas that are negative for PAX3/ 7-FOXO1 fusions, which represent mostly embryonal rhabdomyosarcomas and 20% of the alveolar RMS cases [85], carry point mutations of cancer genes that are frequently mutated pan-cancer [13] including KRAS, HRAS, NRAS, NF1, PIK3CA, CTNNB1 and FBXW7 [73]. Many of these mutated cancer genes are likely to cross-talk to Hippo members (described in more detail below). Additionally, the genomes of ERMS are complex and are aneuploid (i.e. changed chromosome numbers) and display segmental aberrations whereas the genomes of ARMS only show few of such changes [73,86]. While the genes affected in ERMS vary greatly, 171 out of 196 ERMS tumours were positive for

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YAP by immunohistochemistry and in 143 out of these 171 cases YAP was nuclear [18]. Additionally, the expression of constitutively active YAP1 S127A in mice resulted in the formation of ERMS-like tumours with short latency in every case [18]. This suggests that several of the mutated ERMS genes [73], together with the YAP1 copy number gains [17,18], converge to towards increasing YAP activity in ERMS, which is likely driving the disease. There is evidence that several of the somatically point mutated ERMS and sarcoma genes can cross-talk to YAP. Several recent reports have linked KRAS to YAP in cancer. To identify genes that can substitute for oncogenic RAS, 15,294 genes were expressed in KRAS-dependent colon cancer cells. This non-biased analysis revealed that YAP could rescue the viability of KRAS-addicted cells and that YAP was required for KRAS-induced cell transformation [57]. In another study, the authors observed in a mouse model of pancreatic ductal adenocarcinoma that some KRAS G12D-driven tumours relapsed after KRAS inhibition. The resultant tumours exhibited an amplification and overexpression of YAP1 [58]. YAP1 was also identified in a non-biased screen using 27,500 shRNAs to knockdown 5046 signalling genes as the top hit for a gene responsible for resistance to RAF or MEK inhibitor therapy [87]. Also, in pancreatic ductal adenocarcinoma, it was found that YAP1 expression was increased and that the deletion of YAP1 prevented the progression of early lesions [59]. Taken together, it seems likely that similar mechanisms operate in ERMS where oncogenic RAS mutations (NRAS, KRAS, HRAS or mutations of the RAS inhibitor NF1) are relatively common [73]. In line with this, the knockdown of YAP in the RD ERMS cell line, which carries a NRAS Q61H mutation (accompanied by MYC amplification and TP53 mutation with a karyotype of 51-hyperdiploidy [88]), reduces proliferation, soft agar growth, and xenotransplant tumour burden while promoting the differentiation of these cells [18]. Somatic mutations of PIK3CA, which encodes phosphoinositide 3kinase (PI3K), have been reported in 14% of all cases in a large-scale pan-cancer study [13] showing that PI3K is a frequently mutated gene pan-cancer. It has been demonstrated that epidermal growth factor (EGF) inhibits the Hippo pathway through PI3K, resulting in YAP nuclear localisation transcriptional activity [89]. In this study, it has been suggested that this effect is independent of PKB/AKT, which is a key kinase downstream of PI3K. However, several studies have demonstrated cross-talk between Hippo signalling and AKT1, which can phosphorylate MST1 at Thr120 and Thr387 leading to the inhibition of Mst1 activity [90–92]. Thus, AKT1-MST1 cross-talk could be an alternative mechanism by which PIK3CA mutations affect the activity of YAP or TAZ. Mutations of the Wnt gene CTNNB1 (β-catenin) occur in some ERMS cases [73]. We have already mentioned the close integration of Wnt and Hippo signalling. Specifically, it was demonstrated that YAP was crucial for the proliferation and soft agar growth of cancer cell lines with high β-catenin activity [37]. It was shown that β-catenin can form complexes with YES1, YAP and TBX5 [37]. Intriguingly, a loss-of-function screen implicated YES1 in the survival of rhabdomyosarcoma cells [93], perhaps suggesting that similar mechanisms operate in sarcomas. The SCF (β-TrCP) ubiquitin ligase regulates YAP [35] and TAZ [94] degradation via well-defined phosphodegron-mediated mechanisms. FBXW7 is another E3 ubiquitin ligase that was suggested to regulate the degradation of YAP [95]. Indeed, an inverse correlation was reported between FBXW7 and YAP protein abundance in hepatocellular carcinoma. Furthermore, YAP overexpression could counteract the apoptosis and growth arrest caused by FBXW7 [95]. This effect of FBXW7 on YAP levels is consistent with the notion that a loss of FBXW7 in ERMS could favour a higher level of YAP activity. Collectively, this also implies that Hippo signalling plays an important but different role in both ARMS [84] and ERMS downstream of the mutated genes [18]. 6. Hippo-targeted therapies in soft tissue sarcoma? Before 2012, the Hippo pathway was considered un-druggable, but since 2012 several compounds and treatment strategies have emerged.

Generally, these drugs can be sub-divided into either drugs that target the binding of YAP to TEAD transcription factors or into drugs that modulate other members of the wider Hippo signal transduction network, such as G protein-coupled receptors or the mevalonate pathway. Several potential Hippo drugs such as verteporfin or statins are already widely used in patients to treat other conditions and this can pave the way for early clinical trials involving such compounds. Key obstacles for systemic and long-term Hippo-targeting treatments for sarcoma, and for cancer in general, are that the Hippo pathway is ubiquitously expressed (i.e. the drugs may perturb signalling in other tissues and cause side effects), although, in many tissues, the loss-of-function of YAP is inconspicuous under homeostatic conditions until tissue regeneration is required. Here we discuss the different Hippo treatment options that might become useful for the treatment of sarcomas that result from dysregulated Hippo signalling. 6.1. Drugs that target YAP-TEAD interaction or TEADs: verteporfin, cyclic peptides, VGLL4-mimicking peptides Verteporfin, a member of the porphyrin family, contains aromatic heterocyclic molecules formed of four modified pyrrole units interconnected at their carbon atoms by methane bridges. Verteporfin is already a clinically approved photosensitizer for vascular macular degeneration [96]. Verteporfin binds to YAP and changes its conformation thereby inhibiting its interaction with TEAD2 and, presumably, with other TEAD isoforms. Furthermore, verteporfin decreased liver overgrowth and blocked liver tumourigenesis induced by YAP overexpression or endogenous YAP activation [97]. Moreover, uveal melanoma is a cancer resulting from gain-of-function mutations in the GNAQ and GNA11 oncogenes encoding the heterotrimeric Gαq family that stimulate YAP activity. Verteporfin has also been shown to reduce the tumour formation potential of GNAQ/GNA11 mutated uveal melanoma cells in xenograft assays [98,99]. 6.2. G protein-coupled receptors (GPCRs) G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Deep sequencing studies have shown an association between aberrant expression and activity of GPCRs and tumourigenesis in nearly 20% of human cancers carrying mutations in GPCRs [100]. Recent studies have revealed that the Hippo pathway can be broadly regulated by different GPCRs. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) inhibit the Hippo kinases LATS1/2 by acting through G12/G13 coupled receptors and Rho leading to induction of YAP/TAZ transcription activity [47]. Therefore, the drugs targeting the GPCR isoforms affecting Hippo signalling represent a potential therapeutic approach to treat sarcomas driven by high activity of YAP or TAZ. 6.3. Statins and bisphosphonates Aberrant activity of the mevalonate pathway can promote tumour progression in vivo and anchorage independent growth of cells in vitro. Also, high mRNA levels of hydroxymethylglutaryl coenzyme A reductase (HMGCR) and mevalonate pathway genes are associated with poor prognosis and reduced survival in breast cancer patients [101]. Two recent studies have shown that YAP/TAZ activity can be regulated by the mevalonate pathway [102,103]. Mechanistically, the geranylgeranyl phosphate produced by the mevalonate cascade activates Rho GTPases, which in turn increases YAP/TAZ activity by inhibiting their phosphorylation and enhancing their nuclear accumulation. Interestingly, statins and bisphosphonates that inhibit distinct enzymes of the mevalonate pathway have a marked effect on YAP/TAZ transcriptional activity. Taken together these findings suggest that YAP/TAZ are presumably mediating the oncogenic responses associated with the dysregulation of the mevalonate pathway. Moreover, statins

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and bisphosphonates are potential treatments to target the malignant effects of YAP and TAZ in cancer cells [102,103]. In accordance with this, a number of recent studies have reported a possible preventive or therapeutic role of statins in cancer, and further studies are ongoing to validate and characterize the anticancer effects of statins in various contexts [104]. 7. Summary and perspectives In summary, the Hippo pathway comprises potent tumour suppressors and oncogenes that are rarely somatically point mutated in sarcomas or in other types of human cancer. The involvement of Hippo members, especially YAP and TAZ, in human epithelioid haemangioma occurs in the form of WWTR1-CAMTA and YAP1-TFE3 fusion genes, whereas copy number alterations of YAP1 and other Hippo genes occur generally at a low frequency in other types of sarcomas. However, it is unclear whether these copy number alterations are important or even essential for sarcomagenesis in the presence of functional Hippo kinases. Thus, the majority of the changes in the activity of Hippo genes, and specifically YAP1, in sarcoma probably result from the cross-talk with several genes that are frequently mutated in sarcoma and across cancers. For instance, the PAX3-FOXO1 fusion gene driving alveolar RMS can interplay with the Hippo pathway via the upregulation of RASSF4, a negative regulator of the MST1 kinase, and perhaps also by modulating YAP1 activity [84]. In other sarcoma subtypes like embryonal RMS, somatic gainof-function mutations in KRAS, PIK3CA or CTNNB1 and loss-of-function mutations in FBXW7 could affect Hippo pathway activity. Perturbed Hippo signalling can clearly drive sarcomagenesis as evidenced from mouse models expressing YAP1 S127A (gain-of-function) or from mice harbouring LATS1, Nf2 or Mob1a/b loss-of-function mutations. Also, the near 100% penetrance of WWTR1-CAMTA or YAP1-TFE3 fusion genes in epithelioid haemangioendotheliomas suggests that these Hippo fusion genes are driving sarcomagenesis. Given the apparent importance of Hippo signal transduction pathway members in sarcoma it is welcome news that many different validated or putative Hippo treatments have emerged since 2012. They can be subdivided into drugs that directly target the Hippo transcriptional regulators and drugs that target other members of the wider Hippo signal transduction network. Some of these drugs are already widely used for the treatment of other diseases and this could facilitate the progression towards preclinical studies aimed at inhibiting the activity of YAP or TAZ in sarcomas. Several lines of evidence now support the notion that the Hippo pathway might be a major player in sarcomagenesis, but they also raise a number of questions yet to be answered: – Can the expression, abundance or transcriptional activity of YAP (as assessed by gene expression signatures [18]) be used in the clinic for classification of sarcomas and prediction of clinical outcome? – Does the Hippo pathway mostly regulate generic functions such as proliferation and apoptosis or does it consistently interfere with lineage-specific fate determination gene expression as in ERMS where it mainly suppresses the expression of differentiated muscle genes [18]? – Why is there a nearly 100% penetrance of WWTR1-CAMTA or YAP1TFE3 fusion genes in epithelioid haemangioendothelioma? What do these fusion genes do and why are they so important in this type of sarcoma?

Even if the role of the Hippo pathway in cancer is now well established, Hippo research is still in its infancy compared with other tumour suppressor and oncogenic proteins and pathways such as P53, MYC or oncogenic RAS, and there is still much left to understand before achieving full control over this intriguing pathway to specifically target sarcomas and other cancers.

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbcan.2015.05.006. Transparency document The Transparency document associated with this article can be found, in the online version. Funding Work in the University of Aberdeen laboratories is supported by grants from Sarcoma UK (reference 004.2012), Friends of Anchor (references 14004 and 14005), and the Medical Research Council (grant number 99477). Annie M. Tremblay is recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR). References [1] R.S. Udan, M. Kango-Singh, R. Nolo, C. Tao, G. Halder, Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway, Nat. Cell Biol. 5 (2003) 914–920. [2] K.F. Harvey, X. Zhang, D.M. Thomas, The Hippo pathway and human cancer, Nat. Rev. Cancer 13 (2013) 246–257. [3] T. Moroishi, C.G. Hansen, K.L. Guan, The emerging roles of YAP and TAZ in cancer, nature reviews, Cancer 15 (2015) 73–79. [4] K. Harvey, N. Tapon, The Salvador–Warts–Hippo pathway — an emerging tumoursuppressor network, Nat. Rev. Cancer 7 (2007) 182–191. [5] D. Pan, The hippo signaling pathway in development and cancer, Dev. Cell 19 (2010) 491–505. [6] B.S. Taylor, J. Barretina, R.G. Maki, C.R. Antonescu, S. Singer, M. Ladanyi, Advances in sarcoma genomics and new therapeutic targets, nature reviews, Cancer 11 (2011) 541–557. [7] J.R.F. Goldblum, A.L., S. Weiss, Enzinger and Weiss's Soft Tissue Tumors, 6th ed. Elsevier, 2014. [8] J.L. Anderson, C.T. Denny, W.D. Tap, N. Federman, Pediatric sarcomas: translating molecular pathogenesis of disease to novel therapeutic possibilities, Pediatr. Res. 72 (2012) 112–121. [9] B.S. Fletcher, J.A. Bridge, P.C.D. Hogendoorn, F. Mertens, World Health Organisation, Classification of tumours: Tumours of Soft Tissue and Bone, 4th ed. WHO Press, 2013. [10] C.D. Fletcher, The evolving classification of soft tissue tumours — an update based on the new 2013 WHO classification, Histopathology 64 (2014) 2–11. [11] C. Fisher, Immunohistochemistry in diagnosis of soft tissue tumours, Histopathology 58 (2011) 1001–1012. [12] C. Fisher, Dataset for Histopathology Reporting of Soft Tissue Sarcomas, in: T.R.C.o. Pathologists (Ed.), 2014. [13] M.S. Lawrence, P. Stojanov, C.H. Mermel, J.T. Robinson, L.A. Garraway, T.R. Golub, M. Meyerson, S.B. Gabriel, E.S. Lander, G. Getz, Discovery and saturation analysis of cancer genes across 21 tumour types, Nature 505 (2014) 495–501. [14] B. Vogelstein, N. Papadopoulos, V.E. Velculescu, S. Zhou, L.A. Diaz Jr., K.W. Kinzler, Cancer genome landscapes, Science 339 (2013) 1546–1558. [15] C.R. Antonescu, F. Le Loarer, J.M. Mosquera, A. Sboner, L. Zhang, C.L. Chen, H.W. Chen, N. Pathan, T. Krausz, B.C. Dickson, I. Weinreb, M.A. Rubin, M. Hameed, C.D. Fletcher, Novel YAP1-TFE3 fusion defines a distinct subset of epithelioid hemangioendothelioma, Genes Chromosom. Cancer 52 (2013) 775–784. [16] C. Errani, L. Zhang, Y.S. Sung, M. Hajdu, S. Singer, R.G. Maki, J.H. Healey, C.R. Antonescu, A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites, Genes Chromosom. Cancer 50 (2011) 644–653. [17] Z. Helias-Rodzewicz, G. Perot, F. Chibon, C. Ferreira, P. Lagarde, P. Terrier, J.M. Coindre, A. Aurias, YAP1 and VGLL3, encoding two cofactors of TEAD transcription factors, are amplified and overexpressed in a subset of soft tissue sarcomas, Genes Chromosom. Cancer 49 (2010) 1161–1171. [18] A.M. Tremblay, E. Missiaglia, G.G. Galli, S. Hettmer, R. Urcia, M. Carrara, R.N. Judson, K. Thway, G. Nadal, J.L. Selfe, G. Murray, R.A. Calogero, C. De Bari, P.S. Zammit, M. Delorenzi, A.J. Wagers, J. Shipley, H. Wackerhage, F.D. Camargo, The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation, Cancer Cell 26 (2014) 273–287. [19] K.H. Hallor, R. Sciot, J. Staaf, M. Heidenblad, A. Rydholm, H.C. Bauer, K. Astrom, H.A. Domanski, J.M. Meis, L.G. Kindblom, I. Panagopoulos, N. Mandahl, F. Mertens, Two genetic pathways, t(1;10) and amplification of 3p11-12, in myxoinflammatory fibroblastic sarcoma, haemosiderotic fibrolipomatous tumour, and morphologically similar lesions, J. Pathol. 217 (2009) 716–727. [20] K. Gambaro, M.C. Quinn, P.M. Wojnarowicz, S.L. Arcand, M. de Ladurantaye, V. Barres, J.S. Ripeau, A.M. Killary, E.C. Davis, J. Lavoie, D.M. Provencher, A.M. MesMasson, M. Chevrette, P.N. Tonin, VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer, Mol. Oncol. 7 (2013) 513–530. [21] D.M. Parham, D.A. Ellison, Rhabdomyosarcomas in adults and children: an update, Arch. Pathol. Lab. Med. 130 (2006) 1454–1465. [22] D. Van Mater, L. Ano, J.M. Blum, M.T. Webster, W. Huang, N. Williams, Y. Ma, D.M. Cardona, C.M. Fan, D.G. Kirsch, Acute tissue injury activates satellite cells and

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