Non-canonical WNT6/WNT10A signal factor expression in EBV+ post-transplant smooth muscle tumors

Non-canonical WNT6/WNT10A signal factor expression in EBV+ post-transplant smooth muscle tumors Post-transplant smooth muscle tumors (PTSMTs) are rare mesenchymal neoplasms which occur after solid organ or haematopoietic stem cell transplantation. PTSMT typically consist of Epstein–Barr-virus (EBV )+ smooth muscle-like cells and show an intermediate malignancy. Their main occurrences are visceral organs, especially the liver, but intrac- ranial appearances are described and associated with a poor prognosis. EBV drives the growth of PTSMT; however, the underlying molecular mechanisms still remain unclear. Gene expression analysis of a set of morphologically similar tumors (leiomyomas, leiomyosarcomas, angioleiomyomas and endothelial haemangiomas) from patients without immunosuppression or EBV-association was performed. Our findings indicate that PTSMT’s growth is driven by two factors of the wingless-type protein family: WNT6 and WNT10A. We are first to report that in PTSMTs, a non-canonical activation of WNT, independent of beta-catenin, drives tumor cell proliferation via MTOR/AKT1, MYC and Cyclin D2. Keywords: PTSMT, Post-transplant smooth muscle tumors, Angioleiomyomas, EBV, WNT patients [5]) but represent an important clinical and radi- Background ological differential diagnosis in immune-compromised Tumors with predominant or partial smooth muscle dif- patients, particularly to post-transplant lymphoprolifera- ferentiation make up a broad spectrum of mesenchymal tive diseases (PTLD). In PTSMT, the site of manifesta- neoplasms. Most of these tumors are based on sponta- tion determines the patients’ outcome [2]. In particular, neous mutations of a mesenchymal stem cell. A rare and cerebral PTSMT has a poor prognosis, while histological virus-associated entity are the post-transplant smooth features (mitotic rate, cellular atypia, and necrosis) have muscle tumors (PTSMT) [1, 2]. These tumors are typi - no prognostic impact [1]. cally positive for the Epstein–Barr virus (EBV) and can Epstein–Barr virus is specialized to infect B cells and manifest themselves at any time and in any organ after almost all adults harbor a small population of EBV+ non- transplantation [1]. Similar neoplasms can also occur neoplastic B cells, which are controlled by T cell homeo- in any other immunosuppressive condition, in particu- stasis. It is not known how EBV enters non-B cell tissues, lar after infection with human immunodeficiency virus such as mesenchymal cells. It is thought that the PTSMT (HIV; HIV-SMT) or congenital immune defects (CI- cell of origin is derived from a perivascular non-endothe- SMT) [1, 3, 4]. lial mesenchymal smooth muscle stem cell. EBV+ Post-transplant smooth muscle tumors are rare in PTSMT have a type III-like latency with expression regard to the total population (< 1% of transplanted of EBV protein EBNA (Epstein–Barr nuclear antigen) but often lack EBV latent membrane protein 1 (LMP1) expression [6]. The tumorigenic role of EBV in smooth *Correspondence: Teiken.Kristin@mh-hannover.de Kristin Teiken and Mark Kuehnel contributed equally to this work muscle neoplasms is not clear because particularly HIV- Institute of Pathology, Hannover Medical School (MHH), SMT can be EBV negative, indicating that the virus is not Carl-Neuberg-Str. 1, 30625 Hannover, Germany Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 2 of 8 absolutely necessary for aberrant smooth muscle prolif- uncertain and aberrant myogenous venous wall cells eration [1]. are still under discussion as the cells of origin [2, 8]. In our previous works, we found a molecular microenvi- Before performing molecular analysis, the angioleio- ronment which is not related to EBV infection, but rather myomas were histologically sub grouped according to to smooth muscle differentiation [5 ]. In addition, EBV is the old but current standard of Morimoto [9] (13/28 able to induce neoangiogenesis, but we found only minor capillary solid, 7/28 venous, 5/28 cavernous, 3/28 changes on the transcriptional level, including increased mixed capillary-cavernous). All samples of forma- levels of angiopoietin 2 (ANGPT2) [7]. Defining driver lin fixed and paraffin-embedded (FFPE) tumors were mutations in PTSMT are still not known. However, we selected from the archive of the Institutes of Pathology and others found up-regulation of MYC proto-oncogene in Muenster (12/28 angioleiomyomas) and Hannover transcripts, protein expression of phosphorylated mecha- (all other samples). nistic target of rapamycin kinase (MTOR) and phospho- rylated AKT serine/threonine kinase 1 (AKT1) signaling Transcript expression analysis factors [2, 6]. Samples contained > 80% tumor tissue and RNA was In the present analyses we wanted to asses if and which extracted and purified with our established mineral oil transcript profile of mesenchymal stem cell and signaling method and RNeasy Mini Kit (Qiagen, Hilden, Ger- factors differ between PTSMT and other neoplasms with many) via Ma xwell system (Promega cooperation, smooth muscle differentiation. Mannheim, Germany). RNA was quantified by Qubit 2.0 fluorometer (Life Technologies/Thermo Fisher Sci - Methods entific, Waltham, MA, USA). Transcript expression Tumor samples analysis was performed via NanoString nCounter sys- EBV+ PTSMTs comprised six tumors from five patients; tem. NanoString uses digital fluorescent reporters for one patient had a tumor in the spleen and another in a gene detection and enables analysis of small amount of cerebral sinus. We previously reported the clinical and RNA while minimizing background signals. We used histopathological characteristics of four of these patients the prefabricated stem cell panel (includes 193 target [2]. The fifth patient was a female (age: 61  years) who genes and six endogenous control genes) as described developed a PTSMT in the liver 5  years after kidney in the manufacturer’s protocol (NanoString Technolo- transplantation (tumor diameter 3.1 cm, 10% Ki67, posi- gies, Seattle, WA, USA). Endogenous control genes were tive for EBV in situ hybridization (EBER), smooth muscle glucuronidase beta (GUSB), glyceraldehyde-3-phosphate actin, caldesmon and desmin). dehydrogenase (GAPDH), clathrin heavy chain (CLTC), For control purposes, the following tumors were hypoxanthine phosphoribosyltransferase 1 (HPRT1), analyzed (Table  1, Fig.  1): (i) smooth muscle tumors phosphoglycerate kinase 1 (PGK1) and tubulin beta class (nine visceral leiomyomas and four central venous I (TUBB). G1–2 leiomyosarcomas)  and (ii) mixed smooth mus- cle and vascular tumors (28 with different histological Immunohistochemistry subtypes of angioleiomyomas). Additionally, we added Deparaffinized and rehydrated FFPE tissue sections (iii) a vascular tumor control group (five endothelial (1–2  μm) were processed in an automated staining sys- haemangiomas), as the actual origin of PTSMT is still tem (Benchmark ULTRA, Ventana Medical Systems, Inc., Table 1 Sample set and clinical data Entities PTSMT Leiomyoma (LM) Leiomyosarcoma (LMS) Angioleiomyoma Endothelial haeman- (ALM) gioma (EHA) Number of n = 6 (5 patients) n = 9 n = 4 n = 28 n = 5 tumors Age (median, 11.5 (6–61) 60 (29–71) 63 (45–71) 60 (23–79) 48 (32–58) range) Gender ♀ 100% ♀ 89% ♀ 75% ♀ 64% ♀ 60% ♂ 0% ♂ 11% ♂ 25% ♂ 36% ♂ 40% Tumor localisation Kidney (n = 2) Stomach (n = 3) Pulmonary artery (n = 2) Lower extremities Lower extremities (n = 3) Lung (n = 1) Kidney (n = 2) Right atrium (n = 1) (n = 13) Head–Neck (n = 1) Colon (n = 1) Skin (n = 2) Adrenal gland vein Upper extremities (n = 2) Mediastinal (n = 1) Spleen (n = 1) Esophagus (n = 1) (n = 1) Head–Neck (n = 3) Confluens sinuum (n = 1) Mesenterium (n = 1) Genital ♀ (n = 2) Skin/soft tissue (n = 8) Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 3 of 8 Fig. 1 Histological appearance of the five different entities. a Post transplant smooth muscle tumor (haematoxylin–eosin stain (HE stain, 100×) with inserted positive EBV-in situ hybridization (EBER), b visceral leiomyoma (HE stain, 100×), c visceral leiomyosarcoma of the central venous tract (right atrium, HE stain, 100×) with prominent atypia and increased level of mitosis figures, d endothelial haemangioma (HE stain, 100×) with promi- nent vessels, e angioleiomyoma (HE stain, 50×), in particular venous subtype with prominently walled vessels (arrows) Tucson, AZ, USA). A monoclonal anti-beta-catenin anti- comparisons. P values < 0.05 were considered as statisti- body was used. cally significant. Heatmaps were generated using R. Data analysis Results Raw data of transcript target gene expression were ana- Increased WNT6/WNT10A levels in PTSMT lyzed by nSolver software (NanoString Technologies) Transcript analysis showed significant differential and set in relation to the average of endogenous control expression of several genes among the different types of genes (geometric mean of controls as level of relative smooth muscle tumors (Table  2, Additional file  1: Fig- gene expression: reference gene index). Further statisti- ure S1). Several factors were increased in only one type cal analysis for comparison of different tumor groups of tumor. PTSMT showed increased Wnt family mem- was performed with Prism 5.0 (Graph Pad Software, ber 6 (WNT6) and WNT10A levels while all other types San Diego, CA, USA) by using the non-parametric of tumors showed no different expression or low levels Mann–Whitney test for two group comparison and the of these two WNT factors (Fig.  2, Table  2). WNT6 and Krustal–Wallis test and post hoc Dunn-test for multiple WNT10A are encoded as a gene cluster on chromosomal Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 4 of 8 Table 2 Significant differentially regulated genes in PTSMTs and leiomyosarcomas Regulation Gene Median PTSMT Median ALM Median LM Median LMS Median EHA Upregulated in PTSMT WNT6 0.39301 0.00961 0.00439 0.01074 0.01285 WNT10A 0.12282 0.00545 0.00482 0.00670 0.03415 MYC 0.48877 0.13366 0.11219 0.24502 0.50480 Cyclin D2 2.89498 0.54088 1.02805 0.44413 0.32572 Downregulated in PTSMT WNT9B 0.00192 0.00416 0.00193 0.00951 0.01138 GAS1 0.02407 0.05434 0.19764 0.28556 0.30450 PRKD1 0.07241 0.21601 0.22282 0.15769 0.12693 FGFR1 0.36842 0.80905 1.72532 0.93143 0.48040 Beta-catenin 1.57460 2.09349 2.49687 2.23053 1.47197 Upregulated in leiomyosarcomas compared to PTSMT WNT9B 0.00192 0.00416 0.00193 0.00951 0.01138 GAS1 0.02407 0.05434 0.19764 0.28556 0.30450 DHH 0.00297 0.01077 0.00831 0.00259 0.04553 LFNG 0.23836 0.77146 0.22185 0.76354 2.24482 MFNG 0.05390 0.07672 0.05256 0.05478 0.50480 PRKACA 0.85552 1.53877 1.51137 1.44838 1.70591 IGF 0.04295 0.12047 0.11971 0.16677 0.67152 segment 2q35. In individual PTSMT cases, both WNT glycogen synthase kinase 3 beta (GSK-3beta), Cyclin D1 factors were co-expressed at similar levels. (CCND1) as well as the two small GTPase ROH family In addition to WNT6/WNT10A, cell cycle G1/S tran- members ras homolog family member A (ROHA) and sition regulator Cyclin D2 (CCND2) was increased. As cell division cycle 42 (CDC42). These findings indicate could be expected from our previous results on uter- that other canonical and non-canonical WNT pathways ine leiomyomas and PTSMT [2], MYC proto-oncogene, are not activated and affirm our theory. Therefore, the bHLH transcription factor (MYC) transcripts were sig- downstream effectors of the elevated WNT6/WNT10A nificantly higher in PTSMT than in non-uterine vis - levels remain unclear. ceral leiomyomas (Fig.  2). These three factors are not encoded on 2q35 or any other chromosome 2 segment, Different gene expression profiles in PTSMT but on chromosome 8 (MYC: 8q24) and chromosome 12 and leiomyosarcomas (CCND2: 12p13). There were significant differences between visceral leio - WNT9B (17q21.32), S phase entry blocker growth myomas, angioleiomyomas and haemangiomas (Addi- arrest specific 1 (GAS1) and serine/threonine pro - tional file 1: Figure S1). tein kinase D1 (PRKD1) were expressed at low levels in In comparison to all other tumors, leiomyosarcomas PTSMT while the other analyzed tumor types showed showed several increased gene expression levels (Table 2). higher levels, in particular, leiomyosarcomas. Similar to The following expression levels were significantly higher our previous results [7], fibroblast growth factor recep - in leiomyosarcomas than in PTSMT (Table  2): WNT9B, tor 1 (FGFR1) levels are lower in PTSMT as compared to GAS1, signaling factor desert hedgehog (DHH), the two leiomyomas. Notch signaling pathway factors LFNG O-fucosylpeptide WNT signaling-related transcript levels of catenin 3-beta-N-acetylglucosaminyltransferase provided (LFNG) beta 1 (CTNNB1) were not increased in PTSMT (Fig.  2, and MFNG O-fucosylpeptide 3-beta-N-acetylglucosami- Table  2). Beta-catenin protein expression was evalu- nyltransferase (MFNG), protein kinase cAMP-activated ated by immunohistochemistry in PTSMT. All of them catalytic subunit alpha (PRKACA) and insulin-like growth showed no nuclear beta-catenin protein localisation. factor 1 (IGF1). Roland et al. [10] showed that leiomyosar- Compared to the other tumor types, several other canon- comas also express the IGF receptor which indicates par- ical and non-canonical WNT signaling pathway factors acrine activation. were not significantly deregulated in PTSMT, in particu - lar, frizzled class receptors (FZD), WNT1-8, WNT9A, Histopathological subgroups of angioleiomyomas show no WNT10B, WNT11 and WNT16, beta-catenin-related molecularpathological correlation transcription factor 7 (TCF7), WNT signaling path- There are three distinct histopathological subtypes of way regulator adenomatous-polyposis-coli gen (APC), angioleiomyomas which were originally described on Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 5 of 8 WNT6 WNT10A ab p=ns p=ns p=0.0004 p=ns 0.8 p=<0.0001 1.5 p=ns p=0.0211 p=ns 0.6 1.0 0.4 0.5 0.2 0.0 0.0 Cyclin D2 MYC cd p=0.0021 p=ns 1.5 6 p=ns p=ns p=0.0087 p=ns p=0.0496 p=0.0024 1.0 4 0.5 2 0.0 0 Canonical, beta-catenin ef Beta-Catenin dependent WNT-pathway p=ns p=ns p=ns p=0.0008 Cyclin- 2 APC Beta- D2 Carcinogenesis GSK-3beta Catenin myc Axin Fig. 2 a–e Significant upregulated gene members of the WNT signaling pathway in PTSMT. Note that non-significant (p = ns) differences between PTSMT and other tumors regarding WNT6 and WNT10A expression are related to non-detactable transcripts in several ALM, LMS and EHA (a, b). f Beta-catenin is bound and inactivated in a complex formation containing adenomatous polyposis coli gene (APC), glycogen synthase kinase 3 beta (GSK-3beta) and axin-1 (Axin). In the classical, beta-catenin-dependent WNT signaling pathway, members of WNT family bind to the frizzled class receptors (FZD) whereas beta-catenin is set free and initiates carcinogenesis via activation of MYC and Cyclin D2 FZD WNT PTSMT ALM LM LMS EHA PTSMT PTSMT ALM ALM LM LM LMS LMS EHA EHA PTSMT PTSMT ALM ALM LM LM LMS LMS EHA EHA relative expression relative expression relative expression relative expression relative expression Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 6 of 8 the basis of histopathological features by Morimoto in WNT signaling is often characterized by alternative 1973 [9]. Regarding the genes under investigation, manly signaling without cytoplasmic stabilization of soluble these subgroups showed the same molecularpathological beta-catenin [17–19]. These include the WNT/calcium characteristics. Only six genes show significant differ - pathway and the planar cell polarity (PCP). In those alter- ences between the angioleiomyoma types (ISL1, NCSTN, native pathways, other WNT factors than WNT6 and TCF7, WNT10A, WNT11, WNT7A) but their relative WNT10A are usually involved [17–19]. In contrast to expression levels were very low, which indicates no bio- PTSMT, it has been shown in renal cell carcinoma that logical relevance (< 0.4 compared to standardized refer- WNT10A is involved in canonical WNT/beta-catenin ence gene index). signaling [20]. In the murine kidney, Wnt6 is also related to canonical Wnt/beta-catenin signaling [21]. In mes- Discussion enchymal cells expression of WNT6, WNT10A and Post-transplant smooth muscle tumor share morpho- WNT10B can be associated with osteoblastogenesis and logical similarities with leiomyomas and low-grade leio- inhibition of adipogenesis but not with smooth mus- myosarcomas and only show rarely high-grade atypia. cle differentiation [22]. WNT6 is involved in differen - One of the main differences is the EBV association. Our tiation of non-smooth muscle myogenic cells, e.g. in the previous molecular analysis showed no clear relationship heart, and proliferation of stromal cells in the placenta between EBV infection of tumor cells and (de)regulation [23–26]. There are reports which imply a special relation - of gene expression. One reason could be that PTSMT ship between WNT/beta catenin signaling and CCND2 usually do not express LMP1, a viral protein which is in glioma cells [27] but not in smooth muscle cells. At associated with manipulation of the host cell cycle and least in cardiomyocytes, there are evidences of a MYC- cytokine expression. In one of the very first analyses dependent activation of CCND2 [28]. In insulin sensitive which addressed the question of specific signaling path - pancreatic beta cells, activated MTOR leads to CCND2- way deregulation in PTSMT, Ong et al. [6] demonstrated associated proliferation [29, 30]. Therefore, these signal - that these tumors express phosphorylated MTOR and ing factors could also be linked in PTSMT. Based on the phosphorylated AKT1 proteins and that Ras association finding that MTOR signaling is activated, Sirolimus has domain family member 1 (RASSF1) was hypermethyl- been used for targeted therapy in PTSMT [31]. ated. Activated MTOR/AKT1 signaling, RASSF1 hyper- methylation and MYC expression can also be found in a Conclusions subset of leiomyosarcomas [6, 11, 12]. We could confirm In summary, we are the first to report that PTSMT cells our previous result that PTSMT have higher expression may harbor a unique variant of a non-canonical WNT levels of MYC than uterine and non-uterine leiomyo- pathway, which is based on WNT6/WNT10A, and which mas. In our current analysis we found that this differ - may induce cell proliferation and differentiation via ence regarding MYC was not significant in comparison MTOR/AKT1, MYC and CCND2. to leiomyosarcomas. Remarkably, for the very first time, Additional file we could show that PTSMT are characterized by WNT6/ WNT10A regulation while all other tumor entities under Additional file 1: Figure S1. Heatmap of all investigated stem cell investigation showed almost no expression of these two genes shows differences between the five entities. PTSMT: post-transplant factors. There are no reports on a particular WNT6 smooth muscle tumors, ALM: angioleiomyomas and their histomor- or WNT10A expression in EBV infected cells [13, 14]. phological subtypes, LM: leiomyomas, EHA: endothelial haemangiomas and LMS: leiomyosarcomas of the central venous tract. Colors encode Therefore, it is unlikely that EBV is the main cause of significance level of pairwise group comparison. The abbreviations of the this WNT6/WNT10A expression. The only other known five entities stand for the corresponding significantly different regulated tumor entity characterized by WNT6/WNT10A overex- group. ns: p > 0.05, *: p ≤ 0.05, **: ≤0.01, ***: ≤0.001. pression are colorectal carcinomas and it is assumed that their mode of proliferation is linked to differential beta- Abbreviations catenin expression—as opposed to PTSMTs [15]. AKT1: serin/threonin kinase 1; ANGPT2: angiopoietin 2; APS: adenomatous- Both genes are encoded as a cluster on segment 2q35. polyposis-coli gen; CCND1: Cyclin-D1; CCND2: Cyclin-D2; CDC42: cell division cycle 42; CI-SMT: congenital immune defects-smooth muscle tumors; CLTC: Co-expression of these two factors indicates a co-reg- clathrin heavy chain; CTNNB1: beta-catenin 1; DHH: desert hedgehog; EBER: ulation of the respective promotors. The fact that many Epstein–Barr virus; EBNA: Epstein–Barr nuclear antigen; EBV: Epstein–Barr canonical WNT signaling factors were not increased virus-encoded small RNA; FFPE: formalin fixed and paraffin-embedded; FGFR1: fibroblast growth factor receptor 1; FZD: frizzled class receptor; GAPDH: and that we found no nuclear beta-catenin indicates glyceraldehyde-3-phosphate dehydrogenase; GAS1: S phase entry blocker activation of the non-canonical WNT pathway. Similar growth arrest specific 1; GSK-3beta: glycogen synthase kinase 3 beta; GUSB: to PTSMT, leiomyosarcomas usually show no aberrant glucuronidase beta; HE: hematoxylin and eosin; HIV: human immunodefi- ciency virus; HIV-SMT: human immunodeficiency virus smooth muscle tumors; nuclear beta-catenin expression [10, 16]. Non-canonical Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 7 of 8 HPRT1: hypoxanthine phosphoribosyltransferase 1; IGF: insulin like growth 3. Issarachaikul R, Shuangshoti S, Suankratay C. Epstein–Barr virus-asso- factor 1; ISL1: insulin gene enhancer protein; LFNG: LFNG O-fucosylpeptide ciated smooth muscle tumors in AIDS patients: a largest case (series). 3-beta-N-acetylglucosaminyltransferase provided; LMP1: latent membrane Intern Med. 2014;53(20):2391–6. protein 1; MFNG: MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltrans- 4. Schober T, Magg T, Laschinger M, Rohlfs M, Linhares ND, Puchalka J, ferase; MTOR: rapamycin kinase; NCSTN: nicastrin; PCP: planar cell polarity; Weisser T, Fehlner K, Mautner J, Walz C, Hussein K, Jaeger G, Kammer B, PGK1: phosphoglycerate kinase 1; PRKACA: protein kinase cAMP-activated Schmid I, Bahia M, Pena SD, Behrends U, Belohradsky BH, Klein C, Hauck F. catalytic subunit alpha; PRKD1: serine/threonine protein kinase D1; PTLD: post- A human immunodeficiency syndrome caused by mutations in CARMIL2. transplant lymphoproliferative disease; PTSMT: post-transplant smooth muscle Nat Commun. 2017;8:14209. tumor; RASSF1: Ras associated domain family member 1; ROHA: GTPase ROH 5. Jonigk D, Izykowski N, Maegel L, Schormann E, Maecker-Kolhoff B, family members ras homolog family member A; TCF7: beta-catenin-related Laenger F, Kreipe H, Hussein K. MicroRNA expression in Epstein–Barr transcription factor 7; TUBB: tubulin beta class 1; WNT6: WNT family gene virus-associated post-transplant smooth muscle tumours is related to member 6; WNT10A: WNT family gene member 10A. leiomyomatous phenotype. Clin Sarcoma Res. 2013;3(1):9. 6. Ong KW, Teo M, Lee V, Ong D, Lee A, Tan CS, Vathsala A, Toh HC. Expres- Authors’ contributions sion of EBV latent antigens, mammalian target of rapamycin, and tumor Histomorphology (KT, DJ, KH, FL, JR, HK), molecular analysis (KT, MK, DJ ), data suppression genes in EBV-positive smooth muscle tumors: clinical and collection, analysis of data and manuscript preparation (MK, KT, DJ, KH). All therapeutic implications. Clin Cancer Res. 2009;15(17):5350–8. authors read and approved the final manuscript. 7. Jonigk D, Izykowski N, Maegel L, Schormann E, Ludewig B, Kreipe H, Hussein K. Tumour angiogenesis in Epstein–Barr virus-associated post- Author details transplant smooth muscle tumours. 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Non-canonical WNT6/WNT10A signal factor expression in EBV+ post-transplant smooth muscle tumors

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Biomedicine; Cancer Research; Oncology; Surgical Oncology
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Abstract

Post-transplant smooth muscle tumors (PTSMTs) are rare mesenchymal neoplasms which occur after solid organ or haematopoietic stem cell transplantation. PTSMT typically consist of Epstein–Barr-virus (EBV )+ smooth muscle-like cells and show an intermediate malignancy. Their main occurrences are visceral organs, especially the liver, but intrac- ranial appearances are described and associated with a poor prognosis. EBV drives the growth of PTSMT; however, the underlying molecular mechanisms still remain unclear. Gene expression analysis of a set of morphologically similar tumors (leiomyomas, leiomyosarcomas, angioleiomyomas and endothelial haemangiomas) from patients without immunosuppression or EBV-association was performed. Our findings indicate that PTSMT’s growth is driven by two factors of the wingless-type protein family: WNT6 and WNT10A. We are first to report that in PTSMTs, a non-canonical activation of WNT, independent of beta-catenin, drives tumor cell proliferation via MTOR/AKT1, MYC and Cyclin D2. Keywords: PTSMT, Post-transplant smooth muscle tumors, Angioleiomyomas, EBV, WNT patients [5]) but represent an important clinical and radi- Background ological differential diagnosis in immune-compromised Tumors with predominant or partial smooth muscle dif- patients, particularly to post-transplant lymphoprolifera- ferentiation make up a broad spectrum of mesenchymal tive diseases (PTLD). In PTSMT, the site of manifesta- neoplasms. Most of these tumors are based on sponta- tion determines the patients’ outcome [2]. In particular, neous mutations of a mesenchymal stem cell. A rare and cerebral PTSMT has a poor prognosis, while histological virus-associated entity are the post-transplant smooth features (mitotic rate, cellular atypia, and necrosis) have muscle tumors (PTSMT) [1, 2]. These tumors are typi - no prognostic impact [1]. cally positive for the Epstein–Barr virus (EBV) and can Epstein–Barr virus is specialized to infect B cells and manifest themselves at any time and in any organ after almost all adults harbor a small population of EBV+ non- transplantation [1]. Similar neoplasms can also occur neoplastic B cells, which are controlled by T cell homeo- in any other immunosuppressive condition, in particu- stasis. It is not known how EBV enters non-B cell tissues, lar after infection with human immunodeficiency virus such as mesenchymal cells. It is thought that the PTSMT (HIV; HIV-SMT) or congenital immune defects (CI- cell of origin is derived from a perivascular non-endothe- SMT) [1, 3, 4]. lial mesenchymal smooth muscle stem cell. EBV+ Post-transplant smooth muscle tumors are rare in PTSMT have a type III-like latency with expression regard to the total population (< 1% of transplanted of EBV protein EBNA (Epstein–Barr nuclear antigen) but often lack EBV latent membrane protein 1 (LMP1) expression [6]. The tumorigenic role of EBV in smooth *Correspondence: Teiken.Kristin@mh-hannover.de Kristin Teiken and Mark Kuehnel contributed equally to this work muscle neoplasms is not clear because particularly HIV- Institute of Pathology, Hannover Medical School (MHH), SMT can be EBV negative, indicating that the virus is not Carl-Neuberg-Str. 1, 30625 Hannover, Germany Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 2 of 8 absolutely necessary for aberrant smooth muscle prolif- uncertain and aberrant myogenous venous wall cells eration [1]. are still under discussion as the cells of origin [2, 8]. In our previous works, we found a molecular microenvi- Before performing molecular analysis, the angioleio- ronment which is not related to EBV infection, but rather myomas were histologically sub grouped according to to smooth muscle differentiation [5 ]. In addition, EBV is the old but current standard of Morimoto [9] (13/28 able to induce neoangiogenesis, but we found only minor capillary solid, 7/28 venous, 5/28 cavernous, 3/28 changes on the transcriptional level, including increased mixed capillary-cavernous). All samples of forma- levels of angiopoietin 2 (ANGPT2) [7]. Defining driver lin fixed and paraffin-embedded (FFPE) tumors were mutations in PTSMT are still not known. However, we selected from the archive of the Institutes of Pathology and others found up-regulation of MYC proto-oncogene in Muenster (12/28 angioleiomyomas) and Hannover transcripts, protein expression of phosphorylated mecha- (all other samples). nistic target of rapamycin kinase (MTOR) and phospho- rylated AKT serine/threonine kinase 1 (AKT1) signaling Transcript expression analysis factors [2, 6]. Samples contained > 80% tumor tissue and RNA was In the present analyses we wanted to asses if and which extracted and purified with our established mineral oil transcript profile of mesenchymal stem cell and signaling method and RNeasy Mini Kit (Qiagen, Hilden, Ger- factors differ between PTSMT and other neoplasms with many) via Ma xwell system (Promega cooperation, smooth muscle differentiation. Mannheim, Germany). RNA was quantified by Qubit 2.0 fluorometer (Life Technologies/Thermo Fisher Sci - Methods entific, Waltham, MA, USA). Transcript expression Tumor samples analysis was performed via NanoString nCounter sys- EBV+ PTSMTs comprised six tumors from five patients; tem. NanoString uses digital fluorescent reporters for one patient had a tumor in the spleen and another in a gene detection and enables analysis of small amount of cerebral sinus. We previously reported the clinical and RNA while minimizing background signals. We used histopathological characteristics of four of these patients the prefabricated stem cell panel (includes 193 target [2]. The fifth patient was a female (age: 61  years) who genes and six endogenous control genes) as described developed a PTSMT in the liver 5  years after kidney in the manufacturer’s protocol (NanoString Technolo- transplantation (tumor diameter 3.1 cm, 10% Ki67, posi- gies, Seattle, WA, USA). Endogenous control genes were tive for EBV in situ hybridization (EBER), smooth muscle glucuronidase beta (GUSB), glyceraldehyde-3-phosphate actin, caldesmon and desmin). dehydrogenase (GAPDH), clathrin heavy chain (CLTC), For control purposes, the following tumors were hypoxanthine phosphoribosyltransferase 1 (HPRT1), analyzed (Table  1, Fig.  1): (i) smooth muscle tumors phosphoglycerate kinase 1 (PGK1) and tubulin beta class (nine visceral leiomyomas and four central venous I (TUBB). G1–2 leiomyosarcomas)  and (ii) mixed smooth mus- cle and vascular tumors (28 with different histological Immunohistochemistry subtypes of angioleiomyomas). Additionally, we added Deparaffinized and rehydrated FFPE tissue sections (iii) a vascular tumor control group (five endothelial (1–2  μm) were processed in an automated staining sys- haemangiomas), as the actual origin of PTSMT is still tem (Benchmark ULTRA, Ventana Medical Systems, Inc., Table 1 Sample set and clinical data Entities PTSMT Leiomyoma (LM) Leiomyosarcoma (LMS) Angioleiomyoma Endothelial haeman- (ALM) gioma (EHA) Number of n = 6 (5 patients) n = 9 n = 4 n = 28 n = 5 tumors Age (median, 11.5 (6–61) 60 (29–71) 63 (45–71) 60 (23–79) 48 (32–58) range) Gender ♀ 100% ♀ 89% ♀ 75% ♀ 64% ♀ 60% ♂ 0% ♂ 11% ♂ 25% ♂ 36% ♂ 40% Tumor localisation Kidney (n = 2) Stomach (n = 3) Pulmonary artery (n = 2) Lower extremities Lower extremities (n = 3) Lung (n = 1) Kidney (n = 2) Right atrium (n = 1) (n = 13) Head–Neck (n = 1) Colon (n = 1) Skin (n = 2) Adrenal gland vein Upper extremities (n = 2) Mediastinal (n = 1) Spleen (n = 1) Esophagus (n = 1) (n = 1) Head–Neck (n = 3) Confluens sinuum (n = 1) Mesenterium (n = 1) Genital ♀ (n = 2) Skin/soft tissue (n = 8) Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 3 of 8 Fig. 1 Histological appearance of the five different entities. a Post transplant smooth muscle tumor (haematoxylin–eosin stain (HE stain, 100×) with inserted positive EBV-in situ hybridization (EBER), b visceral leiomyoma (HE stain, 100×), c visceral leiomyosarcoma of the central venous tract (right atrium, HE stain, 100×) with prominent atypia and increased level of mitosis figures, d endothelial haemangioma (HE stain, 100×) with promi- nent vessels, e angioleiomyoma (HE stain, 50×), in particular venous subtype with prominently walled vessels (arrows) Tucson, AZ, USA). A monoclonal anti-beta-catenin anti- comparisons. P values < 0.05 were considered as statisti- body was used. cally significant. Heatmaps were generated using R. Data analysis Results Raw data of transcript target gene expression were ana- Increased WNT6/WNT10A levels in PTSMT lyzed by nSolver software (NanoString Technologies) Transcript analysis showed significant differential and set in relation to the average of endogenous control expression of several genes among the different types of genes (geometric mean of controls as level of relative smooth muscle tumors (Table  2, Additional file  1: Fig- gene expression: reference gene index). Further statisti- ure S1). Several factors were increased in only one type cal analysis for comparison of different tumor groups of tumor. PTSMT showed increased Wnt family mem- was performed with Prism 5.0 (Graph Pad Software, ber 6 (WNT6) and WNT10A levels while all other types San Diego, CA, USA) by using the non-parametric of tumors showed no different expression or low levels Mann–Whitney test for two group comparison and the of these two WNT factors (Fig.  2, Table  2). WNT6 and Krustal–Wallis test and post hoc Dunn-test for multiple WNT10A are encoded as a gene cluster on chromosomal Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 4 of 8 Table 2 Significant differentially regulated genes in PTSMTs and leiomyosarcomas Regulation Gene Median PTSMT Median ALM Median LM Median LMS Median EHA Upregulated in PTSMT WNT6 0.39301 0.00961 0.00439 0.01074 0.01285 WNT10A 0.12282 0.00545 0.00482 0.00670 0.03415 MYC 0.48877 0.13366 0.11219 0.24502 0.50480 Cyclin D2 2.89498 0.54088 1.02805 0.44413 0.32572 Downregulated in PTSMT WNT9B 0.00192 0.00416 0.00193 0.00951 0.01138 GAS1 0.02407 0.05434 0.19764 0.28556 0.30450 PRKD1 0.07241 0.21601 0.22282 0.15769 0.12693 FGFR1 0.36842 0.80905 1.72532 0.93143 0.48040 Beta-catenin 1.57460 2.09349 2.49687 2.23053 1.47197 Upregulated in leiomyosarcomas compared to PTSMT WNT9B 0.00192 0.00416 0.00193 0.00951 0.01138 GAS1 0.02407 0.05434 0.19764 0.28556 0.30450 DHH 0.00297 0.01077 0.00831 0.00259 0.04553 LFNG 0.23836 0.77146 0.22185 0.76354 2.24482 MFNG 0.05390 0.07672 0.05256 0.05478 0.50480 PRKACA 0.85552 1.53877 1.51137 1.44838 1.70591 IGF 0.04295 0.12047 0.11971 0.16677 0.67152 segment 2q35. In individual PTSMT cases, both WNT glycogen synthase kinase 3 beta (GSK-3beta), Cyclin D1 factors were co-expressed at similar levels. (CCND1) as well as the two small GTPase ROH family In addition to WNT6/WNT10A, cell cycle G1/S tran- members ras homolog family member A (ROHA) and sition regulator Cyclin D2 (CCND2) was increased. As cell division cycle 42 (CDC42). These findings indicate could be expected from our previous results on uter- that other canonical and non-canonical WNT pathways ine leiomyomas and PTSMT [2], MYC proto-oncogene, are not activated and affirm our theory. Therefore, the bHLH transcription factor (MYC) transcripts were sig- downstream effectors of the elevated WNT6/WNT10A nificantly higher in PTSMT than in non-uterine vis - levels remain unclear. ceral leiomyomas (Fig.  2). These three factors are not encoded on 2q35 or any other chromosome 2 segment, Different gene expression profiles in PTSMT but on chromosome 8 (MYC: 8q24) and chromosome 12 and leiomyosarcomas (CCND2: 12p13). There were significant differences between visceral leio - WNT9B (17q21.32), S phase entry blocker growth myomas, angioleiomyomas and haemangiomas (Addi- arrest specific 1 (GAS1) and serine/threonine pro - tional file 1: Figure S1). tein kinase D1 (PRKD1) were expressed at low levels in In comparison to all other tumors, leiomyosarcomas PTSMT while the other analyzed tumor types showed showed several increased gene expression levels (Table 2). higher levels, in particular, leiomyosarcomas. Similar to The following expression levels were significantly higher our previous results [7], fibroblast growth factor recep - in leiomyosarcomas than in PTSMT (Table  2): WNT9B, tor 1 (FGFR1) levels are lower in PTSMT as compared to GAS1, signaling factor desert hedgehog (DHH), the two leiomyomas. Notch signaling pathway factors LFNG O-fucosylpeptide WNT signaling-related transcript levels of catenin 3-beta-N-acetylglucosaminyltransferase provided (LFNG) beta 1 (CTNNB1) were not increased in PTSMT (Fig.  2, and MFNG O-fucosylpeptide 3-beta-N-acetylglucosami- Table  2). Beta-catenin protein expression was evalu- nyltransferase (MFNG), protein kinase cAMP-activated ated by immunohistochemistry in PTSMT. All of them catalytic subunit alpha (PRKACA) and insulin-like growth showed no nuclear beta-catenin protein localisation. factor 1 (IGF1). Roland et al. [10] showed that leiomyosar- Compared to the other tumor types, several other canon- comas also express the IGF receptor which indicates par- ical and non-canonical WNT signaling pathway factors acrine activation. were not significantly deregulated in PTSMT, in particu - lar, frizzled class receptors (FZD), WNT1-8, WNT9A, Histopathological subgroups of angioleiomyomas show no WNT10B, WNT11 and WNT16, beta-catenin-related molecularpathological correlation transcription factor 7 (TCF7), WNT signaling path- There are three distinct histopathological subtypes of way regulator adenomatous-polyposis-coli gen (APC), angioleiomyomas which were originally described on Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 5 of 8 WNT6 WNT10A ab p=ns p=ns p=0.0004 p=ns 0.8 p=<0.0001 1.5 p=ns p=0.0211 p=ns 0.6 1.0 0.4 0.5 0.2 0.0 0.0 Cyclin D2 MYC cd p=0.0021 p=ns 1.5 6 p=ns p=ns p=0.0087 p=ns p=0.0496 p=0.0024 1.0 4 0.5 2 0.0 0 Canonical, beta-catenin ef Beta-Catenin dependent WNT-pathway p=ns p=ns p=ns p=0.0008 Cyclin- 2 APC Beta- D2 Carcinogenesis GSK-3beta Catenin myc Axin Fig. 2 a–e Significant upregulated gene members of the WNT signaling pathway in PTSMT. Note that non-significant (p = ns) differences between PTSMT and other tumors regarding WNT6 and WNT10A expression are related to non-detactable transcripts in several ALM, LMS and EHA (a, b). f Beta-catenin is bound and inactivated in a complex formation containing adenomatous polyposis coli gene (APC), glycogen synthase kinase 3 beta (GSK-3beta) and axin-1 (Axin). In the classical, beta-catenin-dependent WNT signaling pathway, members of WNT family bind to the frizzled class receptors (FZD) whereas beta-catenin is set free and initiates carcinogenesis via activation of MYC and Cyclin D2 FZD WNT PTSMT ALM LM LMS EHA PTSMT PTSMT ALM ALM LM LM LMS LMS EHA EHA PTSMT PTSMT ALM ALM LM LM LMS LMS EHA EHA relative expression relative expression relative expression relative expression relative expression Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 6 of 8 the basis of histopathological features by Morimoto in WNT signaling is often characterized by alternative 1973 [9]. Regarding the genes under investigation, manly signaling without cytoplasmic stabilization of soluble these subgroups showed the same molecularpathological beta-catenin [17–19]. These include the WNT/calcium characteristics. Only six genes show significant differ - pathway and the planar cell polarity (PCP). In those alter- ences between the angioleiomyoma types (ISL1, NCSTN, native pathways, other WNT factors than WNT6 and TCF7, WNT10A, WNT11, WNT7A) but their relative WNT10A are usually involved [17–19]. In contrast to expression levels were very low, which indicates no bio- PTSMT, it has been shown in renal cell carcinoma that logical relevance (< 0.4 compared to standardized refer- WNT10A is involved in canonical WNT/beta-catenin ence gene index). signaling [20]. In the murine kidney, Wnt6 is also related to canonical Wnt/beta-catenin signaling [21]. In mes- Discussion enchymal cells expression of WNT6, WNT10A and Post-transplant smooth muscle tumor share morpho- WNT10B can be associated with osteoblastogenesis and logical similarities with leiomyomas and low-grade leio- inhibition of adipogenesis but not with smooth mus- myosarcomas and only show rarely high-grade atypia. cle differentiation [22]. WNT6 is involved in differen - One of the main differences is the EBV association. Our tiation of non-smooth muscle myogenic cells, e.g. in the previous molecular analysis showed no clear relationship heart, and proliferation of stromal cells in the placenta between EBV infection of tumor cells and (de)regulation [23–26]. There are reports which imply a special relation - of gene expression. One reason could be that PTSMT ship between WNT/beta catenin signaling and CCND2 usually do not express LMP1, a viral protein which is in glioma cells [27] but not in smooth muscle cells. At associated with manipulation of the host cell cycle and least in cardiomyocytes, there are evidences of a MYC- cytokine expression. In one of the very first analyses dependent activation of CCND2 [28]. In insulin sensitive which addressed the question of specific signaling path - pancreatic beta cells, activated MTOR leads to CCND2- way deregulation in PTSMT, Ong et al. [6] demonstrated associated proliferation [29, 30]. Therefore, these signal - that these tumors express phosphorylated MTOR and ing factors could also be linked in PTSMT. Based on the phosphorylated AKT1 proteins and that Ras association finding that MTOR signaling is activated, Sirolimus has domain family member 1 (RASSF1) was hypermethyl- been used for targeted therapy in PTSMT [31]. ated. Activated MTOR/AKT1 signaling, RASSF1 hyper- methylation and MYC expression can also be found in a Conclusions subset of leiomyosarcomas [6, 11, 12]. We could confirm In summary, we are the first to report that PTSMT cells our previous result that PTSMT have higher expression may harbor a unique variant of a non-canonical WNT levels of MYC than uterine and non-uterine leiomyo- pathway, which is based on WNT6/WNT10A, and which mas. In our current analysis we found that this differ - may induce cell proliferation and differentiation via ence regarding MYC was not significant in comparison MTOR/AKT1, MYC and CCND2. to leiomyosarcomas. Remarkably, for the very first time, Additional file we could show that PTSMT are characterized by WNT6/ WNT10A regulation while all other tumor entities under Additional file 1: Figure S1. Heatmap of all investigated stem cell investigation showed almost no expression of these two genes shows differences between the five entities. PTSMT: post-transplant factors. There are no reports on a particular WNT6 smooth muscle tumors, ALM: angioleiomyomas and their histomor- or WNT10A expression in EBV infected cells [13, 14]. phological subtypes, LM: leiomyomas, EHA: endothelial haemangiomas and LMS: leiomyosarcomas of the central venous tract. Colors encode Therefore, it is unlikely that EBV is the main cause of significance level of pairwise group comparison. The abbreviations of the this WNT6/WNT10A expression. The only other known five entities stand for the corresponding significantly different regulated tumor entity characterized by WNT6/WNT10A overex- group. ns: p > 0.05, *: p ≤ 0.05, **: ≤0.01, ***: ≤0.001. pression are colorectal carcinomas and it is assumed that their mode of proliferation is linked to differential beta- Abbreviations catenin expression—as opposed to PTSMTs [15]. AKT1: serin/threonin kinase 1; ANGPT2: angiopoietin 2; APS: adenomatous- Both genes are encoded as a cluster on segment 2q35. polyposis-coli gen; CCND1: Cyclin-D1; CCND2: Cyclin-D2; CDC42: cell division cycle 42; CI-SMT: congenital immune defects-smooth muscle tumors; CLTC: Co-expression of these two factors indicates a co-reg- clathrin heavy chain; CTNNB1: beta-catenin 1; DHH: desert hedgehog; EBER: ulation of the respective promotors. The fact that many Epstein–Barr virus; EBNA: Epstein–Barr nuclear antigen; EBV: Epstein–Barr canonical WNT signaling factors were not increased virus-encoded small RNA; FFPE: formalin fixed and paraffin-embedded; FGFR1: fibroblast growth factor receptor 1; FZD: frizzled class receptor; GAPDH: and that we found no nuclear beta-catenin indicates glyceraldehyde-3-phosphate dehydrogenase; GAS1: S phase entry blocker activation of the non-canonical WNT pathway. Similar growth arrest specific 1; GSK-3beta: glycogen synthase kinase 3 beta; GUSB: to PTSMT, leiomyosarcomas usually show no aberrant glucuronidase beta; HE: hematoxylin and eosin; HIV: human immunodefi- ciency virus; HIV-SMT: human immunodeficiency virus smooth muscle tumors; nuclear beta-catenin expression [10, 16]. Non-canonical Teiken et al. Clin Sarcoma Res (2018) 8:10 Page 7 of 8 HPRT1: hypoxanthine phosphoribosyltransferase 1; IGF: insulin like growth 3. Issarachaikul R, Shuangshoti S, Suankratay C. Epstein–Barr virus-asso- factor 1; ISL1: insulin gene enhancer protein; LFNG: LFNG O-fucosylpeptide ciated smooth muscle tumors in AIDS patients: a largest case (series). 3-beta-N-acetylglucosaminyltransferase provided; LMP1: latent membrane Intern Med. 2014;53(20):2391–6. protein 1; MFNG: MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltrans- 4. Schober T, Magg T, Laschinger M, Rohlfs M, Linhares ND, Puchalka J, ferase; MTOR: rapamycin kinase; NCSTN: nicastrin; PCP: planar cell polarity; Weisser T, Fehlner K, Mautner J, Walz C, Hussein K, Jaeger G, Kammer B, PGK1: phosphoglycerate kinase 1; PRKACA: protein kinase cAMP-activated Schmid I, Bahia M, Pena SD, Behrends U, Belohradsky BH, Klein C, Hauck F. catalytic subunit alpha; PRKD1: serine/threonine protein kinase D1; PTLD: post- A human immunodeficiency syndrome caused by mutations in CARMIL2. transplant lymphoproliferative disease; PTSMT: post-transplant smooth muscle Nat Commun. 2017;8:14209. tumor; RASSF1: Ras associated domain family member 1; ROHA: GTPase ROH 5. Jonigk D, Izykowski N, Maegel L, Schormann E, Maecker-Kolhoff B, family members ras homolog family member A; TCF7: beta-catenin-related Laenger F, Kreipe H, Hussein K. MicroRNA expression in Epstein–Barr transcription factor 7; TUBB: tubulin beta class 1; WNT6: WNT family gene virus-associated post-transplant smooth muscle tumours is related to member 6; WNT10A: WNT family gene member 10A. leiomyomatous phenotype. Clin Sarcoma Res. 2013;3(1):9. 6. Ong KW, Teo M, Lee V, Ong D, Lee A, Tan CS, Vathsala A, Toh HC. Expres- Authors’ contributions sion of EBV latent antigens, mammalian target of rapamycin, and tumor Histomorphology (KT, DJ, KH, FL, JR, HK), molecular analysis (KT, MK, DJ ), data suppression genes in EBV-positive smooth muscle tumors: clinical and collection, analysis of data and manuscript preparation (MK, KT, DJ, KH). All therapeutic implications. Clin Cancer Res. 2009;15(17):5350–8. authors read and approved the final manuscript. 7. Jonigk D, Izykowski N, Maegel L, Schormann E, Ludewig B, Kreipe H, Hussein K. Tumour angiogenesis in Epstein–Barr virus-associated post- Author details transplant smooth muscle tumours. Clin Sarcoma Res. 2014;4(1):1. Institute of Pathology, Hannover Medical School (MHH), Carl-Neuberg-Str. 8. Chaves NJ, Kotsimbos TC, Warren MA, et al. Cranial leiomyosarcoma in an 1, 30625 Hannover, Germany. Institute of Pathology, University of Muenster, Epstein–Barr virus (EBV )-mismatched lung transplant recipient. J Heart Domagkstraße 17, 48149 Muenster, Germany. Lung Transplant. 2007;26:753–5. 9. Morimoto N. Angiomyoma (vascular leiomyoma): a clinicopathological Acknowledgements study. Med J Kagoshima Univ. 1973;24:663–83. This work was supported by the Collaborative Research Centre 738 (CRC 738), 10. Roland CL, Boland GM, Demicco EG, Lusby K, Ingram D, May CD, Kivlin in particular Danny Jonigk and Florian Laenger. The authors would like to CM, Watson K, Al Sannaa GA, Wang WL, Ravi V, Pollock RE, Lev D, Cormier thank A. Hori for patiently translating the original angioleiomyoma paper by JN, Hunt KK, Feig BW, Lazar AJ, Torres KE. 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Biochem Biophys Res Commun. 2001;283(4):798–805. 16. Gogou PN, Batistatou A, Pakos EE, Apostolikas N, Stefanou D, Tsekeris PG. Funding Expression of E-cadherin, beta-catenin and topoisomerase II alpha in The project was funded by the German Research Foundation as part of Col- leiomyosarcomas. Clin Transl Oncol. 2009;11(8):548–51. laborative Research Centre 738 (CRC738, Danny Jonigk, Florian Laenger). 17. Cuppens T, Tuyaerts S, Amant F. Potential therapeutic targets in uterine sarcomas. Sarcoma. 2015;2015:243298. 18. Kim JH, Park SY, Jun Y, Kim JY, Nam JS. Roles of Wnt target genes in the Publisher’s Note journey of cancer stem cells. Int J Mol Sci. 2017;18(8):1604. https://doi. Springer Nature remains neutral with regard to jurisdictional claims in pub- org/10.3390/ijms18081604. lished maps and institutional affiliations. 19. Sedgwick AE, D’Souza-Schorey C. Wnt signaling in cell motility and invasion: drawing parallels between development and cancer. 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Redox regulation of Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

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Clinical Sarcoma ResearchSpringer Journals

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