Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.
References
[1]
Loeb DM, Thornton K, Shokek O (2008) Pediatric soft tissue sarcomas. Surg Clin North Am 88: : 615–627, vii.
[2]
Parham DM, Ellison DA (2006) Rhabdomyosarcomas in adults and children: an update. Arch Pathol Lab Med 130: 1454–1465.
[3]
Missiaglia E, Williamson D, Chisholm J, Wirapati P, Pierron G, et al. (2012) PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification. J Clin Oncol 30: 1670–1677. doi: 10.1200/jco.2011.38.5591
[4]
Tapscott SJ, Thayer MJ, Weintraub H (1993) Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis. Science 259: 1450–1453. doi: 10.1126/science.8383879
[5]
Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284: 770–776. doi: 10.1126/science.284.5415.770
[6]
Kopan R (2002) Notch: a membrane-bound transcription factor. J Cell Sci 115: 1095–1097.
[7]
Iso T, Kedes L, Hamamori Y (2003) HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 194: 237–255. doi: 10.1002/jcp.10208
[8]
Conboy IM, Conboy MJ, Smythe GM, Rando TA (2003) Notch-mediated restoration of regenerative potential to aged muscle. Science 302: 1575–1577. doi: 10.1126/science.1087573
[9]
Sun H, Li L, Vercherat C, Gulbagci NT, Acharjee S, et al. (2007) Stra13 regulates satellite cell activation by antagonizing Notch signaling. J Cell Biol 177: 647–657. doi: 10.1083/jcb.200609007
[10]
Kuroda K, Tani S, Tamura K, Minoguchi S, Kurooka H, et al. (1999) Delta-induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J Biol Chem 274: 7238–7244. doi: 10.1074/jbc.274.11.7238
[11]
Buas MF, Kabak S, Kadesch T (2010) The Notch effector Hey1 associates with myogenic target genes to repress myogenesis. J Biol Chem 285: 1249–1258. doi: 10.1074/jbc.m109.046441
[12]
Wen Y, Bi P, Liu W, Asakura A, Keller C, et al. (2012) Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells. Mol Cell Biol 32: 2300–2311. doi: 10.1128/mcb.06753-11
[13]
Gagan J, Dey BK, Layer R, Yan Z, Dutta A (2012) Notch3 and Mef2c proteins are mutually antagonistic via Mkp1 protein and miR-1/206 microRNAs in differentiating myoblasts. J Biol Chem 287: 40360–40370. doi: 10.1074/jbc.m112.378414
[14]
Vasyutina E, Lenhard DC, Wende H, Erdmann B, Epstein JA, et al. (2007) RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells. Proc Natl Acad Sci U S A 104: 4443–4448. doi: 10.1073/pnas.0610647104
[15]
Kitzmann M, Bonnieu A, Duret C, Vernus B, Barro M, et al. (2006) Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells. J Cell Physiol 208: 538–548. doi: 10.1002/jcp.20688
[16]
Dahlqvist C, Blokzijl A, Chapman G, Falk A, Dannaeus K, et al. (2003) Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development 130: 6089–6099. doi: 10.1242/dev.00834
[17]
Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321: 1095–1100. doi: 10.1126/science.1155998
[18]
Roma J, Masia A, Reventos J, Sanchez de Toledo J, Gallego S (2011) Notch pathway inhibition significantly reduces rhabdomyosarcoma invasiveness and mobility in vitro. Clin Cancer Res 17: 505–513. doi: 10.1158/1078-0432.ccr-10-0166
[19]
Raimondi L, Ciarapica R, De Salvo M, Verginelli F, Gueguen M, et al. (2012) Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo. Cell Death Differ 19: 871–881. doi: 10.1038/cdd.2011.171
[20]
Nagao H, Setoguchi T, Kitamoto S, Ishidou Y, Nagano S, et al. (2012) RBPJ is a novel target for rhabdomyosarcoma therapy. PLoS One 7: e39268. doi: 10.1371/journal.pone.0039268
[21]
Belyea BC, Naini S, Bentley RC, Linardic CM (2012) Inhibition of the Notch-Hey1 axis blocks embryonal rhabdomyosarcoma tumorigenesis. Clin Cancer Res 17: 7324–7336. doi: 10.1158/1078-0432.ccr-11-1004
[22]
Wilson EM, Rotwein P (2007) Selective control of skeletal muscle differentiation by Akt1. J Biol Chem 282: 5106–5110. doi: 10.1074/jbc.c600315200
[23]
Clement N, Gueguen M, Glorian M, Blaise R, Andreani M, et al. (2007) Notch3 and IL-1beta exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-kappaB drives crosstalk. J Cell Sci 120: 3352–3361. doi: 10.1242/jcs.007872
[24]
Graziani I, Eliasz S, De Marco MA, Chen Y, Pass HI, et al. (2008) Opposite effects of Notch-1 and Notch-2 on mesothelioma cell survival under hypoxia are exerted through the Akt pathway. Cancer Res 68: 9678–9685. doi: 10.1158/0008-5472.can-08-0969
[25]
Yamaguchi N, Oyama T, Ito E, Satoh H, Azuma S, et al. (2008) NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells. Cancer Res 68: 1881–1888. doi: 10.1158/0008-5472.can-07-1597
[26]
Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, et al. (2009) Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 113: 1689–1698. doi: 10.1182/blood-2008-03-147967
[27]
Giovannini C, Gramantieri L, Chieco P, Minguzzi M, Lago F, et al. (2009) Selective ablation of Notch3 in HCC enhances doxorubicin's death promoting effect by a p53 dependent mechanism. J Hepatol 50: 969–979. doi: 10.1016/j.jhep.2008.12.032
[28]
Hoemann CD, Beaulieu N, Girard L, Rebai N, Jolicoeur P (2000) Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice. Mol Cell Biol 20: 3831–3842. doi: 10.1128/mcb.20.11.3831-3842.2000
[29]
Bellavia D, Campese AF, Alesse E, Vacca A, Felli MP, et al. (2000) Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice. Embo J 19: 3337–3348. doi: 10.1093/emboj/19.13.3337
[30]
Weng AP, Ferrando AA, Lee W, Morris JPt, Silverman LB, et al. (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306: 269–271. doi: 10.1126/science.1102160
[31]
Thompson BJ, Buonamici S, Sulis ML, Palomero T, Vilimas T, et al. (2007) The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia. J Exp Med 204: 1825–1835. doi: 10.1084/jem.20070872
[32]
Ciarapica R, Annibali D, Raimondi L, Savino M, Nasi S, et al. (2009) Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation. Oncogene 28: 1881–1891. doi: 10.1038/onc.2009.56
[33]
Golde TE, Koo EH, Felsenstein KM, Osborne BA, Miele L (2013) gamma-Secretase inhibitors and modulators. Biochim Biophys Acta 1828: 2898–2907. doi: 10.1016/j.bbamem.2013.06.005
[34]
Koch U, Radtke F (2010) Notch signaling in solid tumors. Curr Top Dev Biol 92: 411–455. doi: 10.1016/s0070-2153(10)92013-9
[35]
Ranganathan P, Weaver KL, Capobianco AJ (2011) Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 11: 338–351. doi: 10.1038/nrc3035
[36]
Davicioni E, Anderson JR, Buckley JD, Meyer WH, Triche TJ (2010) Gene expression profiling for survival prediction in pediatric rhabdomyosarcomas: a report from the children's oncology group. J Clin Oncol 28: 1240–1246. doi: 10.1200/jco.2008.21.1268
[37]
Williamson D, Missiaglia E, de Reynies A, Pierron G, Thuille B, et al. (2010) Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol 28: 2151–2158. doi: 10.1200/jco.2009.26.3814
[38]
Miele L, Golde T, Osborne B (2006) Notch signaling in cancer. Curr Mol Med 6: 905–918. doi: 10.2174/156652406779010830
[39]
Cao L, Yu Y, Bilke S, Walker RL, Mayeenuddin LH, et al. (2010) Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res 70: 6497–6508. doi: 10.1158/0008-5472.can-10-0582
[40]
Ramirez-Peinado S, Alcazar-Limones F, Lagares-Tena L, El Mjiyad N, Caro-Maldonado A, et al. (2011) 2-deoxyglucose induces Noxa-dependent apoptosis in alveolar rhabdomyosarcoma. Cancer Res 71: 6796–6806. doi: 10.1158/0008-5472.can-11-0759
[41]
Lee MH, Jothi M, Gudkov AV, Mal AK (2011) Histone methyltransferase KMT1A restrains entry of alveolar rhabdomyosarcoma cells into a myogenic differentiated state. Cancer Res 71: 3921–3931. doi: 10.1158/0008-5472.can-10-3358
[42]
Crose LE, Etheridge KT, Chen C, Belyea B, Talbot LJ, et al. (2012) FGFR4 blockade exerts distinct antitumorigenic effects in human embryonal versus alveolar rhabdomyosarcoma. Clin Cancer Res 18: 3780–3790. doi: 10.1158/1078-0432.ccr-10-3063
