Laboratory Investigation (2013) 93, 1090–1099 & 2013 USCAP, Inc All rights reserved 0023-6837/13 Characterization of Wnt/b-catenin signaling in rhabdomyosarcoma Srinivas R Annavarapu1,8,9, Samantha Cialfi2,9, Carlo Dominici2,3, George K Kokai4, Stefania Uccini5, Simona Ceccarelli6, Heather P McDowell2,3,7 and Timothy R Helliwell1 Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. b-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/b-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed b-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of b-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including b-catenin, glycogen synthase kinase-3b, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of b-catenin, stabilization of the active cytosolic form and nuclear translocation of b-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/b-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target. Laboratory Investigation (2013) 93, 1090–1099; doi:10.1038/labinvest.2013.97; published online 2 September 2013 KEYWORDS: myogenic differentiation; proliferation; rhabdomyosarcoma; Wnt signaling Rhabdomyosarcoma (RMS) is the most common soft- understood. The histological diagnosis for RMS is based on tissue sarcoma of adolescence and childhood, and accounts immunohistochemical expression of myogenin and MyoD1, for 5% of malignant paediatric tumours.1–3 Compared with both of which are known downstream products of Wnt embryonal RMS, alveolar RMS has a higher frequency in signaling in muscle.5 older children, is more aggressive, is more frequently Wnt signaling is an evolutionally conserved pathway that is associated with bone marrow metastases and has a operative in the skeletal development of invertebrates and significantly worse outcome.2,4 Most alveolar RMSs have vertebrates. The multifunctional nuclear transcription factor t(2;13) or t(1;13) translocations, involving the PAX3–FKHR b-catenin is the major effector of the Wnt pathway and and PAX7–FKHR fusion genes, respectively. The fusion gene is involved in cell transformation.6,7 Wnt signaling is product appears to deregulate the differentiation of muscle also actively involved in myogenesis5,8–11 and embryonic progenitor cells through mechanisms that are incompletely somite patterning.12,13 The Wnt pathway is crucial in cell 1Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, UK; 2Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Rome, Italy; 3School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool, UK; 4Department of Paediatric Histopathology, Alder Hey Children’s NHS Foundation Trust, Liverpool, UK; 5Department of Clinical and Molecular Medicine, Sapienza University, Rome, Italy; 6Department of Experimental Medicine, Sapienza University, Rome, Italy and 7Department of Paediatric Oncology, Alder Hey Children’s NHS Foundation Trust, Liverpool, UK Correspondence: Dr TR Helliwell, MD, FRCPath, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Duncan Building, Daulby Street, Liverpool, L69 3GA, UK. E-mail: [email protected] 8Current address: Department of Cellular Pathology, Royal Victoria Infirmary, Newcastle-upon-Tyne, NE1 4LP, UK. 9These authors contributed equally to this work. Received 6 April 2013; revised 6 July 2013; accepted 7 July 2013 1090 Laboratory Investigation | Volume 93 October 2013 | www.laboratoryinvestigation.org Wnt signaling in rhabdomyosarcoma SR Annavarapu et al proliferation, cell migration, polarity and cell death.14,15 calf serum, glutamine, 100 mg/ml streptomycin and 100 U/ml Dysregulation of Wnt/b-catenin signaling facilitates cancer penicillin. invasion and metastases in various tumours.16,17 Inactive Wnt signaling results in cytoplasmic b-catenin Tissue Microarray and Immunohistochemistry associating with a multimolecular complex containing casein Tissue microarrays (TMAs) containing a total of 136 samples kinase a, glycogen synthase kinase-3b (GSK-3b), axin 15,16 from 34 RMS cases (4 samples per case containing viable and adenomatosis polyposis coli (APC) protein. This tumour cells) and 16 samples from 8 controls was assembled complex promotes phosphorylation of b-catenin leading in duplicate using eight 1 mm-deep cores. Ten cases were to its ubiquitination and subsequent degradation by the unsuitable for TMA because the blocks were very thin proteasome.15,16,18 Wnt signaling is activated when Wnt (o1 mm) (n ¼ 10); these cases were assessed on full-face ligands bind to Frizzled receptors and co-receptors on sections. The primary antibody against b-catenin (1:200, the cellular surface. Active Wnt signaling promotes the monoclonal mouse anti-human b-catenin and clone downstream signaling cascade and the activation of b-catenin-1; DAKO, UK) was applied to 4 mm sections and disheveled (Dsh) protein, which in turn inactivates GSK- 14–18 biotinylated secondary antibodies used for the catalyzed 3b. This allows b-catenin to translocate into the nucleus signal amplification technique (Amersham, UK). b-Catenin where it binds to T-cell factor/lymphoid-enhancing factor expression was evaluated in a blind manner by two (TCF/LEF), leading to activation of target genes including 19 20,21 22 23,24 25 independent observers (TH and SA). Positive cases had at c-Myc, cyclin D1, c-Jun, Slug and Cox2. In least four representative cores, each with at least 10% of neo- addition, TCF/LEF/b-catenin complex may cooperate with plastic cells expressing b-catenin. The intensity of expression factors activated by other signaling pathways to modulate was scored as 0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, 3 ¼ cellular remodeling. Indeed, many of these genes, such as strong, and the pattern of expression defined as cytoplasmic/ cyclin D1 and c-Myc, have crucial roles in cell growth, membranous or nuclear (Figure 1). Cases with tumour het- proliferation and differentiation, and are deregulated in many 26–29 erogeneity showing equal numbers of positive and negative types of cancer. cores were considered positive only if at least three cores Given the role of Wnt signaling in embryonal myogenesis showed 2 þ expression. If the expression was weak (1 þ ), the and tumourigenesis, this study aimed to characterize the Wnt case was considered negative for b-catenin. Cases assessed on pathway in RMS using biopsy material and in vitro studies of full-face sections (n ¼ 10) were regarded as positive only if at the presence and activity of components of the pathway. least 10% cells showed moderate or strong expression. MATERIALS AND METHODS Clinical Material Protein Extraction and Western Blot Analysis Fifty-four RMS samples were obtained from the histo- Protein extracts were obtained from cells using standard lysis pathology archives of Alder Hey Children’s NHS Foundation buffer (for details, see Supplementary methods). For western Trust (n ¼ 40, 1991–2009) and ‘Policlinico Umberto I’ Hos- blot analysis, equal amounts of proteins were resolved by pital, Sapienza University, Rome, Italy (n ¼ 14; 2005–2009). SDS-PAGE and transferred onto a PVDF membrane (Milli- Approval from the ethical and research committees of Alder pore, Billerica, MA, USA) using standard procedures (for Hey Children’s NHS Foundation Trust and ‘Policlinico antibodies and other reagents, see Supplementary methods). Umberto I’ Hospital was obtained. All patients or their All experiments were carried out at least three times. parents gave informed consent. The minimum follow-up period of the patients was 5 years. Forty-four cases out of 54 Phosphorylation Status of b-Catenin in Response to had sufficient tissue to be included. Patient data and tumour Wnt3a Stimulation characteristics are presented in the supporting information Time course experiments (see Supplementary Information, Supplementary Table S1). b-Catenin in its inactive state is phosphorylated by GSK-3b The diagnosis of RMS was made by the characteristic in cooperation with a degradation complex and is tagged for morphological pattern (embryonal, alveolar, not otherwise proteasomal degradation.15,16 Inactivation of GSK-3b allows specified) and immunohistochemical nuclear expression of active b-catenin to accumulate within