The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.
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Ognjanovic S, Linabery AM, Charbonneau B, Ross JA. Trends in childhood rhabdomyosarcoma incidence and survival in the United States, 1975–2005. Cancer. 2009;115:4218–26.
Taylor AC, Shu L, Danks MK, Poquette CA, Shetty S, Thayer MJ, et al. P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines. Med Pediatr Oncol. 2000;35:96–103.
Stratton MR, Fisher C, Gusterson BA, Cooper CS. Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction. Cancer Res. 1989;49:6324–7.
Chen L, Shern JF, Wei JS, Yohe ME, Song YK, Hurd L, et al. Clonality and evolutionary history of rhabdomyosarcoma. PLoS Genet. 2015;11:e1005075.
Chen X, Stewart E, Shelat AA, Qu C, Bahrami A, Hatley M, et al. Targeting oxidative stress in embryonal rhabdomyosarcoma. Cancer Cell. 2013;24:710–24.
Taylor JGT, Cheuk AT, Tsang PS, et al. Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models. J Clin Invest. 2009;119:3395–407.
Stewart E, McEvoy J, Wang H, Chen X, Honnell V, Ocarz M, et al. Identification of therapeutic targets in rhabdomyosarcoma through integrated genomic, epigenomic, and proteomic analyses. Cancer Cell. 2018;34:411–26.e19.
Shern JF, Chen L, Chmielecki J, Wei JS, Patidar R, Rosenberg M, et al. Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors. Cancer Discov. 2014;4:216–31.
Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, et al. PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group. J Clin Oncol. 2002;20:2672–9.
Rudzinski ER, Anderson JR, Chi YY, Gastier-Foster JM, Astbury C, Barr FG, et al. Histology, fusion status, and outcome in metastatic rhabdomyosarcoma: A report from the Children’s Oncology Group. Pediatr Blood Cancer. 2017;64: https://doi.org/10.1002/pbc.26645.
Cao L, Yu Y, Bilke S, Walker RL, Mayeenuddin LH, Azorsa DO, et al. Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res. 2010;70:6497–508.
Gryder BE, Yohe ME, Chou HC, Zhang X, Marques J, Wachtel M, et al. PAX3-FOXO1 establishes myogenic super enhancers and confers BET bromodomain vulnerability. Cancer Disco. 2017;7:884–99.
Otabe O, Kikuchi K, Tsuchiya K, Katsumi Y, Yagyu S, Miyachi M, et al. MET/ERK2 pathway regulates the motility of human alveolar rhabdomyosarcoma cells. Oncol Rep. 2017;37:98–104.
Taulli R, Scuoppo C, Bersani F, Accornero P, Forni PE, Miretti S, et al. Validation of met as a therapeutic target in alveolar and embryonal rhabdomyosarcoma. Cancer Res. 2006;66:4742–9.
Schoffski P, Adkins D, Blay JY, Gil T, Elias AD, Rutkowski P, et al. An open-label, phase 2 study evaluating the efficacy and safety of the anti-IGF-1R antibody cixutumumab in patients with previously treated advanced or metastatic soft-tissue sarcoma or Ewing family of tumours. Eur J Cancer. 2013;49:3219–28.
Pappo AS, Vassal G, Crowley JJ, Bolejack V, Hogendoorn PC, Chugh R, et al. A phase 2 trial of R1507, a monoclonal antibody to the insulin-like growth factor-1 receptor (IGF-1R), in patients with recurrent or refractory rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and other soft tissue sarcomas: results of a Sarcoma Alliance for Research Through Collaboration study. Cancer. 2014;120:2448–56.
Schoffski P, Wozniak A, Leahy MG, Aamdal S, Rutkowski P, Bauer S, et al. The tyrosine kinase inhibitor crizotinib does not have clinically meaningful activity in heavily pre-treated patients with advanced alveolar rhabdomyosarcoma with FOXO rearrangement: European Organisation for Research and Treatment of Cancer phase 2 trial 90101 ‘CREATE’. Eur J Cancer. 2018;94:156–67.
Davicioni E, Finckenstein FG, Shahbazian V, Buckley JD, Triche TJ, Anderson MJ. Identification of a PAX-FKHR gene expression signature that defines molecular classes and determines the prognosis of alveolar rhabdomyosarcomas. Cancer Res. 2006;66:6936–46.
Williamson D, Missiaglia E, de Reynies A, Pierron G, Thuille B, Palenzuela G, et al. Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol. 2010;28:2151–8.
Lae M, Ahn EH, Mercado GE, Chuai S, Edgar M, Pawel BR, et al. Global gene expression profiling of PAX-FKHR fusion-positive alveolar and PAX-FKHR fusion-negative embryonal rhabdomyosarcomas. J Pathol. 2007;212:143–51.
Wachtel M, Dettling M, Koscielniak E, Stegmaier S, Treuner J, Simon-Klingenstein K, et al. Gene expression signatures identify rhabdomyosarcoma subtypes and detect a novel t(2;2)(q35;p23) translocation fusing PAX3 to NCOA1. Cancer Res. 2004;64:5539–45.
Mercado GE, Xia SJ, Zhang C, Ahn EH, Gustafson DM, Lae M, et al. Identification of PAX3-FKHR-regulated genes differentially expressed between alveolar and embryonal rhabdomyosarcoma: focus on MYCN as a biologically relevant target. Genes Chromosomes Cancer. 2008;47:510–20.
Mahlapuu M, Ormestad M, Enerback S, Carlsson P. The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development. 2001;128:155–66.
Kalinichenko VV, Zhou Y, Bhattacharyya D, Kim W, Shin B, Bambal K, et al. Haploinsufficiency of the mouse Forkhead Box f1 gene causes defects in gall bladder development. J Biol Chem. 2002;277:12369–74.
Ustiyan V, Bolte C, Zhang Y, Han L, Xu Y, Yutzey KE, et al. FOXF1 transcription factor promotes lung morphogenesis by inducing cellular proliferation in fetal lung mesenchyme. Dev Biol. 2018;443:50–63.
Ren X, Ustiyan V, Guo M, Wang G, Bolte C, Zhang Y, et al. Postnatal alveologenesis depends on FOXF1 signaling in c-KIT(+) endothelial progenitor cells. Am J Respir Crit Care Med. 2019;200:1164–76.
Hoggatt AM, Kim JR, Ustiyan V, Ren X, Kalin TV, Kalinichenko VV, et al. The transcription factor Foxf1 binds to serum response factor and myocardin to regulate gene transcription in visceral smooth muscle cells. J Biol Chem. 2013;288:28477–87.
Hinson AR, Jones R, Crose LE, Belyea BC, Barr FG, Linardic CM. Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: utility and pitfalls. Front Oncol. 2013;3:183.
Gryder BE, Wachtel M, Chang K, El Demerdash O, Aboreden NG, Mohammed W, et al. Miswired enhancer logic drives a cancer of the muscle lineage. iScience. 2020;23:101103.
Ren X, Zhang Y, Snyder J, Cross ER, Shah TA, Kalin TV, et al. Forkhead box M1 transcription factor is required for macrophage recruitment during liver repair. Mol Cell Biol. 2010;30:5381–93.
Cai Y, Balli D, Ustiyan V, Fulford L, Hiller A, Misetic V, et al. Foxm1 expression in prostate epithelial cells is essential for prostate carcinogenesis. J Biol Chem. 2013;288:22527–41.
Ustiyan V, Wert SE, Ikegami M, Wang IC, Kalin TV, Whitsett JA, et al. Foxm1 transcription factor is critical for proliferation and differentiation of Clara cells during development of conducting airways. Dev Biol. 2012;370:198–212.
Black M, Milewski D, Le T, Ren X, Xu Y, Kalinichenko VV, et al. FOXF1 inhibits pulmonary fibrosis by preventing CDH2-CDH11 cadherin switch in myofibroblasts. Cell Rep. 2018;23:442–58.
Black M, Arumugam P, Shukla S, Pradhan A, Ustiyan V, Milewski D, et al. FOXM1 nuclear transcription factor translocates into mitochondria and inhibits oxidative phosphorylation. Mol Biol Cell. 2020;31:1411–24.
Goda C, Balli D, Black M, Milewski D, Le T, Ustiyan V, et al. Loss of FOXM1 in macrophages promotes pulmonary fibrosis by activating p38 MAPK signaling pathway. PLoS Genet. 2020;16:e1008692.
Sun L, Ren X, Wang IC, Pradhan A, Zhang Y, Flood HM, et al. The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice. Sci Signal. 2017;10:eaai8583
Bolte C, Flood HM, Ren X, Jagannathan S, Barski A, Kalin TV, et al. FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy. Sci Rep. 2017;7:10690.
Vallabh S, Kartashov AV, Barski A. Analysis of ChIP-Seq and RNA-Seq data with BioWardrobe. Methods Mol Biol. 2018;1783:343–60.
Kartashov AV, Barski A. BioWardrobe: an integrated platform for analysis of epigenomics and transcriptomics data. Genome Biol. 2015;16:158.
Machanick P, Bailey TL. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics. 2011;27:1696–7.
Shao Z, Zhang Y, Yuan GC, Orkin SH, Waxman DJ. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets. Genome Biol. 2012;13:R16.
Gryder BE, Wu L, Woldemichael GM, Pomella S, Quinn TR, Park PMC, et al. Chemical genomics reveals histone deacetylases are required for core regulatory transcription. Nat Commun. 2019;10:3004.
Tenente IM, Hayes MN, Ignatius MS, McCarthy K, Yohe M, Sindiri S, et al. Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma. Elife. 2017;6:e19214.
Gryder BE, Khan J, Stanton BZ. Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP). Nat Protoc. 2020;15:1209–36.
Robinson JT, Turner D, Durand NC, Thorvaldsdottir H, Mesirov JP, Aiden EL. Juicebox.js provides a cloud-based visualization system for Hi-C data. Cell Syst. 2018;6:256–8 e1.
Milewski D, Balli D, Ustiyan V, Le T, Dienemann H, Warth A, et al. FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas. PLoS Genet. 2017;13:e1007097.
Bolte C, Ren X, Tomley T, Ustiyan V, Pradhan A, Hoggatt A, et al. Forkhead box F2 regulation of platelet-derived growth factor and myocardin/serum response factor signaling is essential for intestinal development. J Biol Chem. 2015;290:7563–75.
Cheng X-H, Black M, Ustiyan V, Le T, Fulford L, Sridharan A, et al. SPDEF inhibits prostate carcinogenesis by disrupting a positive feedback loop in regulation of the Foxm1 oncogene. PLoS Genet. 2014;10:e1004656.
Kalin TV, Meliton L, Meliton AY, Zhu X, Whitsett JA, Kalinichenko VV. Pulmonary mastocytosis and enhanced lung inflammation in mice heterozygous null for the Foxf1 gene. Am J Respir Cell Mol Biol. 2008;39:390–9.
Gryder BE, Pomella S, Sayers C, Wu XS, Song Y, Chiarella AM, et al. Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma. Nat Genet. 2019;51:1714–22.
Szafranski P, Dharmadhikari AV, Wambach JA, Towe CT, White FV, Grady RM, et al. Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins. Am J Med Genet A. 2014;164A:2013–9.
Thakore PI, D’Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods. 2015;12:1143–9.
Alaggio R, Zhang L, Sung YS, Huang SC, Chen CL, Bisogno G, et al. A molecular study of pediatric spindle and sclerosing rhabdomyosarcoma: identification of novel and recurrent VGLL2-related fusions in infantile cases. Am J Surg Pathol. 2016;40:224–35.
Mehner C, Miller E, Khauv D, Nassar A, Oberg AL, Bamlet WR, et al. Tumor cell-derived MMP3 orchestrates Rac1b and tissue alterations that promote pancreatic adenocarcinoma. Mol Cancer Res. 2014;12:1430–9.
Ebauer M, Wachtel M, Niggli FK, Schafer BW. Comparative expression profiling identifies an in vivo target gene signature with TFAP2B as a mediator of the survival function of PAX3/FKHR. Oncogene. 2007;26:7267–81.
Hakozaki M, Hojo H, Tajino T, Yamada H, Kikuchi S, Kikuta A, et al. Therapeutic cytodifferentiation in alveolar rhabdomyosarcoma without genetic change of the PAX3-FKHR chimeric fusion gene: a case study. Hum Cell. 2013;26:149–54.
Smith LM, Anderson JR, Coffin CM. Cytodifferentiation and clinical outcome after chemotherapy and radiation therapy for rhabdomyosarcoma (RMS). Med Pediatr Oncol. 2002;38:398–404.
Jothi M, Nishijo K, Keller C, Mal AK. AKT and PAX3-FKHR cooperation enforces myogenic differentiation blockade in alveolar rhabdomyosarcoma cell. Cell Cycle. 2012;11:895–908.
Skrzypek K, Kusienicka A, Trzyna E, Szewczyk B, Ulman A, Konieczny P, et al. SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function. Cell Death Dis. 2018;9:643.
Feng Y, Ngu H, Alford SK, Ward M, Yin F, Longmore GD. alpha-actinin1 and 4 tyrosine phosphorylation is critical for stress fiber establishment, maintenance and focal adhesion maturation. Exp Cell Res. 2013;319:1124–35.
Davicioni E, Anderson MJ, Finckenstein FG, Lynch JC, Qualman SJ, Shimada H, et al. Molecular classification of rhabdomyosarcoma–genotypic and phenotypic determinants of diagnosis: a report from the Children’s Oncology Group. Am J Pathol. 2009;174:550–64.
Williamson D, Lu YJ, Gordon T, Sciot R, Kelsey A, Fisher C, et al. Relationship between MYCN copy number and expression in rhabdomyosarcomas and correlation with adverse prognosis in the alveolar subtype. J Clin Oncol. 2005;23:880–8.
Cairns J, Fridley BL, Jenkins GD, Zhuang Y, Yu J, Wang L. Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation. EMBO Rep. 2018;19:e44767.
Cai Y, Bolte C, Le T, Goda C, Xu Y, Kalin TV, et al. FOXF1 maintains endothelial barrier function and prevents edema after lung injury. Sci Signal. 2016;9:ra40.
Ren X, Ustiyan V, Pradhan A, Cai Y, Havrilak JA, Bolte CS, et al. FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells. Circ Res. 2014;115:709–20.
Pradhan A, Dunn A, Ustiyan V, Bolte C, Wang G, Whitsett JA, et al. The S52F FOXF1 mutation inhibits STAT3 signaling and causes alveolar capillary dysplasia. Am J Respir Crit Care Med. 2019;200:1045–56.
Wang S, Yan S, Zhu S, Zhao Y, Yan J, Xiao Z, et al. FOXF1 induces epithelial-mesenchymal transition in colorectal cancer metastasis by transcriptionally activating SNAI1. Neoplasia. 2018;20:996–1007.
Nilsson G, Kannius-Janson M. Forkhead Box F1 promotes breast cancer cell migration by upregulating lysyl oxidase and suppressing Smad2/3 signaling. BMC Cancer. 2016;16:142.
Lo PK, Lee JS, Liang X, Han L, Mori T, Fackler MJ, et al. Epigenetic inactivation of the potential tumor suppressor gene FOXF1 in breast cancer. Cancer Res. 2010;70:6047–58.
Milewski D, Pradhan A, Wang X, Cai Y, Le T, Turpin B, et al. FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21(Cip1) CDK inhibitor. Oncogene. 2017;36:850–62.
d’Amore ES, Tollot M, Stracca-Pansa V, Menegon A, Meli S, Carli M, et al. Therapy associated differentiation in rhabdomyosarcomas. Mod Pathol. 1994;7:69–75.
Kikuchi K, Tsuchiya K, Otabe O, Gotoh T, Tamura S, Katsumi Y, et al. Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma. Biochem Biophys Res Commun. 2008;365:568–74.
Iland HJ, Bradstock K, Supple SG, Catalano A, Collins M, Hertzberg M, et al. All-trans-retinoic acid, idarubicin, and IV arsenic trioxide as initial therapy in acute promyelocytic leukemia (APML4). Blood. 2012;120:1570–80. quiz 752
Zhang Y, Guan DX, Shi J, Gao H, Li JJ, Zhao JS, et al. All-trans retinoic acid potentiates the chemotherapeutic effect of cisplatin by inducing differentiation of tumor initiating cells in liver cancer. J Hepatol. 2013;59:1255–63.
Brewster AM, Lee JJ, Clayman GL, Clifford JL, Reyes MJ, Zhou X, et al. Randomized trial of adjuvant 13-cis-retinoic acid and interferon alfa for patients with aggressive skin squamous cell carcinoma. J Clin Oncol. 2007;25:1974–8.
Rekhi B, Gupta C, Chinnaswamy G, Qureshi S, Vora T, Khanna N, et al. Clinicopathologic features of 300 rhabdomyosarcomas with emphasis upon differential expression of skeletal muscle specific markers in the various subtypes: a single institutional experience. Ann Diagn Pathol. 2018;36:50–60.
Liu QC, Zha XH, Faralli H, Yin H, Louis-Jeune C, Perdiguero E, et al. Comparative expression profiling identifies differential roles for Myogenin and p38alpha MAPK signaling in myogenesis. J Mol Cell Biol. 2012;4:386–97.
Dias P, Chen B, Dilday B, Palmer H, Hosoi H, Singh S, et al. Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am J Pathol. 2000;156:399–408.
Zhao Z, Yin Y, Zhang J, Qi J, Zhang D, Ma Y, et al. Spindle cell/sclerosing rhabdomyosarcoma: case series from a single institution emphasizing morphology, immunohistochemistry and follow-up. Int J Clin Exp Pathol. 2015;8:13814–20.
Sun W, Chatterjee B, Wang Y, Stevenson HS, Edelman DC, Meltzer PS, et al. Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. Mod Pathol. 2015;28:1214–24.
We thank Tien Le for technical assistance. This work was supported by the NIH grants R01 CA142724 (TVK), R01 HL132849 (TVK), R01 HL141174 (VVK).
Conflict of interest
The work of TVK is supported by NIH (R01CA142724, R01HL132849), the work of VVK is supported by NIH (R01HL141174). AB is a co-founder of Datirium, LLC, the developer of SciDAP data analysis platform. Other authors declare no conflict of interest.
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Milewski, D., Shukla, S., Gryder, B.E. et al. FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma. Oncogene 40, 2182–2199 (2021). https://doi.org/10.1038/s41388-021-01694-9
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