Tumor suppressive functions of WNT5A in rhabdomyosarcoma

Ragab,Nada; Bauer,Julia; Uhmann,Anja; Marx,Alexander; Hahn,Heidi; Simon-Keller,Katja

doi:10.3892/ijo.2022.5392

Tumor suppressive functions of WNT5A in rhabdomyosarcoma

Authors:
- Nada Ragab
- Julia Bauer
- Anja Uhmann
- Alexander Marx
- Heidi Hahn
- Katja Simon-Keller
View Affiliations

Affiliations: Institute of Human Genetics, University Medical Center Göttingen, D‑37073 Göttingen, Germany, Institute of Pathology, University Medical Center Mannheim, University of Heidelberg, D‑68167 Mannheim, Germany
Published online on: July 7, 2022 https://doi.org/10.3892/ijo.2022.5392
Article Number: 102
Copyright: © Ragab et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Rhabdomyosarcoma (RMS) is a highly aggressive soft tissue malignancy that predominantly affects children. The main subtypes are alveolar RMS (ARMS) and embryonal RMS (ERMS) and the two show an impaired muscle differentiation phenotype. One pathway involved in muscle differentiation is WNT signaling. However, the role of this pathway in RMS is far from clear. Our recent data showed that the canonical WNT/β‑Catenin pathway serves a subordinate role in RMS, whereas non‑canonical WNT signaling probably is more important for this tumor entity. The present study investigated the role of WNT5A, which is the major ligand of non‑canonical WNT signaling, in ERMS and ARMS. Gene expression analysis showed that WNT5A was expressed in human RMS samples and that its expression is more pronounced in ERMS. When stably overexpressed in RMS cell lines, WNT5A decreased proliferation and migration of the cells as demonstrated by BrdU incorporation and Transwell migration or scratch assay, respectively. WNT5A also decreased the self‑renewal capacity and the expression of stem cell markers and modulates the levels of muscle differentiation markers as shown by sphere assay and western blot analysis, respectively. Finally, overexpression of WNT5A can destabilize active β‑Catenin of RMS cells. A WNT5A knockdown has opposite effects. Together, the results suggest that WNT5A has tumor suppressive functions in RMS, which accompanies downregulation of β‑Catenin.

View Figures

View References

1	Dagher R and Helman L: Rhabdomyosarcoma: An overview. Oncologist. 4:34–44. 1999. View Article : Google Scholar : PubMed/NCBI
2	Skapek SX, Ferrari A, Gupta AA, Lupo PJ, Butler E, Shipley J, Barr FG and Hawkins DS: Rhabdomyosarcoma. Nat Rev Dis Primers. 5:12019. View Article : Google Scholar : PubMed/NCBI
3	Williamson D, Missiaglia E, de Reyniès A, Pierron G, Thuille B, Palenzuela G, Thway K, Orbach D, Laé M, Fréneaux P, et al: Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol. 28:2151–2158. 2010. View Article : Google Scholar : PubMed/NCBI
4	Hettmer S and Wagers AJ: Muscling in: Uncovering the origins of rhabdomyosarcoma. Nat Med. 16:171–173. 2010. View Article : Google Scholar : PubMed/NCBI
5	Girardi F and Le Grand F: Wnt signaling in skeletal muscle development and regeneration. Prog Mol Biol Transl Sci. 153:157–179. 2018. View Article : Google Scholar : PubMed/NCBI
6	Martinez-Font E, Pérez-Capó M, Vögler O, Martin-Broto J, Alemany R and Obrador-Hevia A: WNT/β-catenin pathway in soft tissue sarcomas: New therapeutic opportunities? Cancers (Basel). 13:55212021. View Article : Google Scholar
7	Nusse R and Clevers H: Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell. 169:985–999. 2017. View Article : Google Scholar : PubMed/NCBI
8	Clevers H and Nusse R: Wnt/beta-catenin signaling and disease. Cell. 149:1192–1205. 2012. View Article : Google Scholar : PubMed/NCBI
9	De A: Wnt/Ca2+ signaling pathway: A brief overview. Acta Biochim Biophys Sin (Shanghai). 43:745–756. 2011. View Article : Google Scholar
10	Thiele S, Rachner TD, Rauner M and Hofbauer LC: WNT5A and its receptors in the bone-cancer dialogue. J Bone Miner Res. 31:1488–1496. 2016. View Article : Google Scholar : PubMed/NCBI
11	Sato A, Yamamoto H, Sakane H, Koyama H and Kikuchi A: Wnt5a regulates distinct signalling pathways by binding to frizzled2. EMBO J. 29:41–54. 2010. View Article : Google Scholar :
12	Bouron-Dal Soglio D, Rougemont AL, Absi R, Giroux LM, Sanchez R, Barrette S and Fournet JC: Beta-catenin mutation does not seem to have an effect on the tumorigenesis of pediatric rhabdomyosarcomas. Pediatr Dev Pathol. 12:371–373. 2009. View Article : Google Scholar : PubMed/NCBI
13	Singh S, Vinson C, Gurley CM, Nolen GT, Beggs ML, Nagarajan R, Wagner EF, Parham DM and Peterson CA: Impaired Wnt signaling in embryonal rhabdomyosarcoma cells from p53/c-fos double mutant mice. Am J Pathol. 177:2055–2066. 2010. View Article : Google Scholar : PubMed/NCBI
14	Annavarapu SR, Cialfi S, Dominici C, Kokai GK, Uccini S, Ceccarelli S, McDowell HP and Helliwell TR: Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma. Lab Invest. 93:1090–1099. 2013. View Article : Google Scholar : PubMed/NCBI
15	Giralt I, Gallo-Oller G, Navarro N, Zarzosa P, Pons G, Magdaleno A, Segura MF, Sábado C, Hladun R, Arango D, et al: Dickkopf-1 inhibition reactivates Wnt/β-catenin signaling in rhabdomyosarcoma, induces myogenic markers in vitro and impairs tumor cell survival in vivo. Int J Mol Sci. 22:129212021. View Article : Google Scholar
16	Nitzki F, Cuvelier N, Dräger J, Schneider A, Braun T and Hahn H: Hedgehog/patched-associated rhabdomyosarcoma formation from delta1-expressing mesodermal cells. Oncogene. 35:2923–2931. 2016. View Article : Google Scholar
17	Ragab N, Viehweger F, Bauer J, Geyer N, Yang M, Seils A, Belharazem D, Brembeck FH, Schildhaus HU, Marx A, et al: Canonical WNT/β-catenin signaling plays a subordinate role in rhabdomyosarcomas. Front Pediatr. 6:3782018. View Article : Google Scholar
18	Surmann-Schmitt C, Widmann N, Dietz U, Saeger B, Eitzinger N, Nakamura Y, Rattel M, Latham R, Hartmann C, von der Mark H, et al: Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis. J Cell Sci. 122:3627–3637. 2009. View Article : Google Scholar : PubMed/NCBI
19	von Maltzahn J, Chang NC, Bentzinger CF and Rudnicki MA: Wnt signaling in myogenesis. Trends Cell Biol. 22:602–609. 2012. View Article : Google Scholar : PubMed/NCBI
20	Kephart JJ, Tiller RG, Crose LE, Slemmons KK, Chen PH, Hinson AR, Bentley RC, Chi JT and Linardic CM: Secreted frizzled-related protein 3 (SFRP3) is required for tumorigenesis of PAX3-FOXO1-positive alveolar rhabdomyosarcoma. Clin Cancer Res. 21:4868–4880. 2015. View Article : Google Scholar : PubMed/NCBI
21	Chen X, Stewart E, Shelat AA, Qu C, Bahrami A, Hatley M, Wu G, Bradley C, McEvoy J, Pappo A, et al: Targeting oxidative stress in embryonal rhabdomyosarcoma. Cancer cell. 24:710–724. 2013. View Article : Google Scholar : PubMed/NCBI
22	Davicioni E, Anderson MJ, Finckenstein FG, Lynch JC, Qualman SJ, Shimada H, Schofield DE, Buckley JD, Meyer WH, Sorensen PH and Triche TJ: Molecular classification of rhabdomyosarcoma-genotypic and phenotypic determinants of diagnosis: A report from the Children's oncology group. Am J Pathol. 174:550–564. 2009. View Article : Google Scholar : PubMed/NCBI
23	Dräger J, Simon-Keller K, Pukrop T, Klemm F, Wilting J, Sticht C, Dittmann K, Schulz M, Leuschner I, Marx A and Hahn H: LEF1 reduces tumor progression and induces myodifferentiation in a subset of rhabdomyosarcoma. Oncotarget. 8:3259–3273. 2017. View Article : Google Scholar :
24	Najdi R, Proffitt K, Sprowl S, Kaur S, Yu J, Covey TM, Virshup DM and Waterman ML: A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities. Differentiation. 84:203–213. 2012. View Article : Google Scholar : PubMed/NCBI
25	Morgenstern JP and Land H: Advanced mammalian gene transfer: High titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587–3596. 1990. View Article : Google Scholar : PubMed/NCBI
26	Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, et al: Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 9:493–501. 2003. View Article : Google Scholar : PubMed/NCBI
27	Czarnek M, Sarad K, Karaś A, Kochan J and Bereta J: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest. Mol Ther Nucleic Acids. 26:711–731. 2021. View Article : Google Scholar : PubMed/NCBI
28	Weng Y, Shi Y, Xia X, Zhou W, Wang H and Wang C: A multi-shRNA vector enhances the silencing efficiency of exogenous and endogenous genes in human cells. Oncol Lett. 13:1553–1562. 2017. View Article : Google Scholar : PubMed/NCBI
29	Walter D, Satheesha S, Albrecht P, Bornhauser BC, D'Alessandro V, Oesch SM, Rehrauer H, Leuschner I, Koscielniak E, Gengler C, et al: CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres. PLoS One. 6:e195062011. View Article : Google Scholar : PubMed/NCBI
30	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
31	Link AJ and LaBaer J: Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb Protoc. 2011:993–994. 2011. View Article : Google Scholar : PubMed/NCBI
32	Simon-Keller K, Paschen A, Hombach AA, Ströbel P, Coindre JM, Eichmüller SB, Vincent A, Gattenlöhner S, Hoppe F, Leuschner I, et al: Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells. Am J Pathol. 182:2121–2131. 2013. View Article : Google Scholar : PubMed/NCBI
33	Tenente IM, Hayes MN, Ignatius MS, McCarthy K, Yohe M, Sindiri S, Gryder B, Oliveira ML, Ramakrishnan A, Tang Q, et al: Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma. Elife. 6:e192142017. View Article : Google Scholar : PubMed/NCBI
34	Deel MD, Slemmons KK, Hinson AR, Genadry KC, Burgess BA, Crose LES, Kuprasertkul N, Oristian KM, Bentley RC and Linardic CM: The transcriptional coactivator TAZ is a potent mediator of alveolar rhabdomyosarcoma tumorigenesis. Clin Cancer Res. 24:2616–2630. 2018. View Article : Google Scholar : PubMed/NCBI
35	Slemmons KK, Crose LES, Riedel S, Sushnitha M, Belyea B and Linardic CM: A novel notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma. Mol Cancer Res. 15:1777–1791. 2017. View Article : Google Scholar : PubMed/NCBI
36	Yamamoto H, Yoo SK, Nishita M, Kikuchi A and Minami Y: Wnt5a modulates glycogen synthase kinase 3 to induce phosphorylation of receptor tyrosine kinase Ror2. Genes Cells. 12:1215–1223. 2007. View Article : Google Scholar : PubMed/NCBI
37	Mikels A, Minami Y and Nusse R: Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling. J Biol Chem. 284:30167–30176. 2009. View Article : Google Scholar : PubMed/NCBI
38	Pedersen EA, Menon R, Bailey KM, Thomas DG, Van Noord RA, Tran J, Wang H, Qu PP, Hoering A, Fearon ER, et al: Activation of Wnt/β-catenin in ewing sarcoma cells antagonizes EWS/ETS function and promotes phenotypic transition to more metastatic cell states. Cancer Res. 76:5040–5053. 2016. View Article : Google Scholar : PubMed/NCBI
39	Wu T, Wang LN, Tang DR and Sun FY: SOST silencing promotes proliferation and invasion and reduces apoptosis of retinoblastoma cells by activating Wnt/β-catenin signaling pathway. Gene Ther. 24:399–407. 2017. View Article : Google Scholar : PubMed/NCBI
40	Mavila N: Thundimadathil J. The emerging roles of cancer stem cells and Wnt/beta-catenin signaling in hepatoblastoma. Cancers (Basel). 11:14062019. View Article : Google Scholar
41	Qin L, Yin YT, Zheng FJ, Peng LX, Yang CF, Bao YN, Liang YY, Li XJ, Xiang YQ, Sun R, et al: WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis. Oncotarget. 6:10239–10252. 2015. View Article : Google Scholar : PubMed/NCBI
42	Liu JJ, Zhang YJ, Xu R, Du J, Hu Z, Yang L, Chen Y, Zhu Y and Gu L: PI3K/Akt-dependent phosphorylation of GSK3β and activation of RhoA regulate Wnt5a-induced gastric cancer cell migration. Cell Signal. 25:447–456. 2013. View Article : Google Scholar
43	Prgomet Z, Axelsson L, Lindberg P and Andersson T: Migration and invasion of oral squamous carcinoma cells is promoted by WNT5A, a regulator of cancer progression. J Oral Pathol Med. 44:776–784. 2015. View Article : Google Scholar
44	Sinnberg T, Levesque MP, Krochmann J, Cheng PF, Ikenberg K, Meraz-Torres F, Niessner H, Garbe C and Busch C: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype. Mol Cancer. 17:592018. View Article : Google Scholar : PubMed/NCBI
45	Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, Bittner M and Trent JM: Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell. 1:279–288. 2002. View Article : Google Scholar : PubMed/NCBI
46	Kremenevskaja N, von Wasielewski R, Rao AS, Schöfl C, Andersson T and Brabant G: Wnt-5a has tumor suppressor activity in thyroid carcinoma. Oncogene. 24:2144–2154. 2005. View Article : Google Scholar : PubMed/NCBI
47	Thiele S, Göbel A, Rachner TD, Fuessel S, Froehner M, Muders MH, Baretton GB, Bernhardt R, Jakob F, Glüer CC, et al: WNT5A has anti-prostate cancer effects in vitro and reduces tumor growth in the skeleton in vivo. J Bone Miner Res. 30:471–480. 2015. View Article : Google Scholar
48	Castell A and Larsson LG: Targeting MYC translation in colorectal cancer. Cancer Discov. 5:701–703. 2015. View Article : Google Scholar : PubMed/NCBI
49	Säfholm A, Tuomela J, Rosenkvist J, Dejmek J, Härkönen P and Andersson T: The Wnt-5a-derived hexapeptide Foxy-5 inhibits breast cancer metastasis in vivo by targeting cell motility. Clin Cancer Res. 14:6556–6563. 2008. View Article : Google Scholar : PubMed/NCBI
50	Blanc E, Roux GL, Bénard J and Raguénez G: Low expression of Wnt-5a gene is associated with high-risk neuroblastoma. Oncogene. 24:1277–1283. 2005. View Article : Google Scholar
51	Cheng R, Sun B, Liu Z, Zhao X, Qi L, Li Y and Gu Q: Wnt5a suppresses colon cancer by inhibiting cell proliferation and epithelial-mesenchymal transition. J Cell Physiol. 229:1908–1917. 2014. View Article : Google Scholar : PubMed/NCBI
52	Wang T, Liu X and Wang J: Up-regulation of Wnt5a inhibits proliferation and migration of hepatocellular carcinoma cells. J Cancer Res Ther. 15:904–908. 2019. View Article : Google Scholar : PubMed/NCBI
53	Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ and Yang Y: Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol. 162:899–908. 2003. View Article : Google Scholar : PubMed/NCBI
54	Mikels AJ and Nusse R: Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol. 4:e1152006. View Article : Google Scholar : PubMed/NCBI
55	Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC, et al: The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells. 8:645–654. 2003. View Article : Google Scholar : PubMed/NCBI
56	Osman J, Bellamkonda K, Liu Q, Andersson T and Sjölander A: The WNT5A agonist foxy5 reduces the number of colonic cancer stem cells in a xenograft mouse model of human colonic cancer. Anticancer Res. 39:1719–1728. 2019. View Article : Google Scholar : PubMed/NCBI
57	Ying J, Li H, Yu J, Ng KM, Poon FF, Wong SC, Chan AT, Sung JJ and Tao Q: WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/beta-catenin signaling, and is frequently methylated in colorectal cancer. Clin Cancer Res. 14:55–61. 2008. View Article : Google Scholar : PubMed/NCBI
58	Nirdé P, Derocq D, Maynadier M, Chambon M, Basile I, Gary-Bobo M and Garcia M: Heat shock cognate 70 protein secretion as a new growth arrest signal for cancer cells. Oncogene. 29:117–127. 2010. View Article : Google Scholar :