title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options.
abstract: Patients with translocation-positive alveolar rhabdomyosarcoma (ARMS), an aggressive childhood tumor primarily characterized by the PAX3-FOXO1 oncogenic fusion protein, have a poor prognosis because of lack of therapies that specifically target ARMS tumors. This fact highlights the need for novel pharmaceutical interventions. Posttranslational modifications such as phosphorylation are becoming attractive biological targets for the development of such interventions. Along these lines, we demonstrated that PAX3-FOXO1 is phosphorylated at three specific sites and that its pattern of phosphorylation is altered relative to wild-type Pax3 throughout early myogenesis and in ARMS tumor cells. However, little work has been performed examining the effect of directly inhibiting phosphorylation at these sites on ARMS development. To address this gap in knowledge, we used small molecule inhibitors or mutational analysis to specifically inhibit phosphorylation of PAX3-FOXO1 to investigate how altering phosphorylation of the oncogenic fusion protein affects ARMS phenotypes. We found that inhibiting the phosphorylation of PAX3-FOXO1 at Ser201 significantly reduced migration, invasion and proliferation in two independent ARMS tumor cell lines. Further, we found that inhibition of phosphorylation at Ser205 also decreased proliferation and anchorage-independent growth. Consistent with these in vitro results, we demonstrate for the first time that PAX3-FOXO1 is phosphorylated at Ser201 and Ser205 in a primary tumor sample and in tumor cells actively invading the surrounding normal tissue. This report is the first to demonstrate that the direct inhibition of PAX3-FOXO1 phosphorylation reduces ARMS tumor phenotypes in vitro and that these phosphorylation events are present in primary human ARMS tumors and invading tumor cells. These results identify phosphorylation of PAX3-FOXO1, especially at Ser201, as a novel biological target that can be explored as a promising avenue for ARMS therapies.
authors.affiliation: Louisiana State University Health Sciences Center, Department of Genetics, New Orleans, LA, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Loupe
authors.affiliation: Louisiana State University Health Sciences Center, Department of Genetics, New Orleans, LA, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Miller
authors.affiliation: Department of Pathology, Children's Hospital, New Orleans, LA, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Ruffin
authors.affiliation: Department of Pathology, Children's Hospital, New Orleans, LA, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Stark
authors.affiliation: Louisiana State University Health Sciences Center, Department of Genetics, New Orleans, LA, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Hollenbach
references.citation: Cancer. 2009 Sep 15;115(18):4218-26
references.pmid: 19536876
references.citation: Biochim Biophys Acta. 2007 Oct;1770(10 ):1450-8
references.pmid: 17698292
references.citation: Int J Gynecol Cancer. 2013 Feb;23 (2):361-6
references.pmid: 23295942
references.citation: Protein Sci. 2008 Nov;17 (11):1979-86
references.pmid: 18708529
references.citation: Biochem Biophys Res Commun. 2010 Jan 1;391(1):1049-55
references.pmid: 19995556
references.citation: J Virol. 1998 Jan;72(1):339-48
references.pmid: 9420232
references.citation: Curr Opin Oncol. 2011 Jul;23(4):352-60
references.pmid: 21519259
references.citation: Cancer Res. 2008 May 15;68(10 ):3767-76
references.pmid: 18483260
references.citation: Mod Pathol. 2001 May;14(5):506-14
references.pmid: 11353062
references.citation: Cancer. 2005 Apr 1;103(7):1475-83
references.pmid: 15712283
references.citation: Oncogene. 2001 Sep 10;20(40):5736-46
references.pmid: 11607823
references.citation: Cancer Lett. 2009 Jan 18;273(2):194-200
references.pmid: 18606491
references.citation: Mol Cell Biol. 1998 Jul;18(7):4118-30
references.pmid: 9632796
references.citation: Nat Genet. 1993 Nov;5(3):230-5
references.pmid: 8275086
references.citation: Oncogene. 2013 Apr 11;32(15):1876-87
references.pmid: 22710718
references.citation: Mol Cell Biol. 1999 Jan;19(1):594-601
references.pmid: 9858583
references.citation: EMBO J. 1999 Jul 1;18(13):3702-11
references.pmid: 10393185
references.citation: Circulation. 2004 Mar 16;109(10):1196-205
references.pmid: 15023894
references.citation: Cell Death Dis. 2014 Feb 27;5:e1094
references.pmid: 24577092
references.citation: J Biol Chem. 2003 Nov 14;278(46):45937-45
references.pmid: 12928438
references.citation: Trends Pharmacol Sci. 2003 Sep;24(9):441-3
references.pmid: 12967765
references.citation: Biochem Biophys Res Commun. 2008 Jan 18;365(3):568-74
references.pmid: 18022385
references.citation: Cancer Res. 1993 Nov 1;53(21):5108-12
references.pmid: 8221646
references.citation: J Clin Oncol. 2002 Jun 1;20(11):2672-9
references.pmid: 12039929
references.citation: Cancer Lett. 2008 Oct 18;270(1):10-8
references.pmid: 18457914
references.citation: Pediatr Blood Cancer. 2014 Mar;61(3):452-6
references.pmid: 23956055
references.citation: Oxid Med Cell Longev. 2014;2014:241864
references.pmid: 25002914
references.citation: Cancer Res. 1987 Aug 15;47(16):4501-7
references.pmid: 3607778
references.citation: Nat Rev Cancer. 2009 Jan;9(1):28-39
references.pmid: 19104514
references.citation: Cancer Res. 1998 Aug 15;58(16):3542-6
references.pmid: 9721857
references.citation: Biochem Biophys Res Commun. 2012 Nov 9;428(1):24-30
references.pmid: 23058914
references.citation: Indian J Med Paediatr Oncol. 2013 Jan;34(1):38-41
references.pmid: 23878487
references.citation: J Clin Oncol. 2006 Aug 20;24(24):3844-51
references.pmid: 16921036
references.citation: Biochemistry. 2009 Dec 15;48(49):11786-95
references.pmid: 19904978
references.citation: Biochem Pharmacol. 2010 Sep 1;80(5):739-47
references.pmid: 20361944
references.citation: J Neurooncol. 2010 Dec;100(3):385-95
references.pmid: 20467784
references.citation: Pigment Cell Melanoma Res. 2014 Sep;27(5):846-8
references.pmid: 24903325
references.citation: Growth Horm IGF Res. 2001 Oct;11(5):289-97
references.pmid: 11735247
references.citation: Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5349-54
references.pmid: 24706870
references.citation: Ann Oncol. 2012 Sep;23 Suppl 10:x167-9
references.pmid: 22987955
references.citation: Prostate. 2007 Jun 15;67(9):976-88
references.pmid: 17440966
references.citation: Biochem J. 2007 Dec 15;408(3):297-315
references.pmid: 17850214
references.citation: Mol Cell Biol. 1995 Mar;15(3):1522-35
references.pmid: 7862145
references.citation: Front Oncol. 2013 Jul 17;3:183
references.pmid: 23882450
references.citation: J Pediatr Surg. 1995 Nov;30(11):1607-8
references.pmid: 8583337
references.citation: J Biol Chem. 2012 Jan 6;287(2):893-904
references.pmid: 22102280
references.citation: Int J Biochem Cell Biol. 2011 Jun;43(6):936-45
references.pmid: 21440083