Dynamic and Nuclear expression of Pdgfrα and Igf1r in Alveolar … · 3 Since Pdgfrα and Igf1r are reported to act in concert for resistance to Igf1r small molecule inhibition in
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Dynamic and Nuclear expression of Pdgfrα and Igf1r in Alveolar Rhabdomyosarcoma
Running Title: Nuclear Receptor Tyrosine Kinases in Rhabdomyosarcoma
Elaine T. Huang2, Martin W. Goros6, Joel E. Michalek4, Shuyu Wang7,
Atiya Mansoor8, Brian J. Druker1,9, Amy J. Wagers3*, Jeffrey W. Tyner1*, Charles Keller2*
1Knight Cancer Institute, Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR 97239 USA
2Pediatric Cancer Biology Program, Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239 USA
5Greehey Children’s Cancer Research Institute and 6Department of Epidemiology & Biostatistics,
University of Texas Health Science Center, San Antonio, TX 78229 USA
3Howard Hughes Medical Institute, Department of Stem Cell and Regenerative Biology, Harvard
University, Harvard Stem Cell Institute, and Joslin Diabetes Center, 7 Divinity Ave., Cambridge, MA
02138 USA 4Department of Pediatric Oncology, Dana Farber Cancer Institute and Division of Pediatric Hematology/ Oncology, Children’s Hospital, 7Harvard University, Boston, MA 02115 USA
8Department of Pathology, Oregon Health & Science University, Portland, OR 97239 USA
9Howard Hughes Medical Institute, Portland, OR 97239 USA
†Howard Hughes Medical Institute Medical Research Fellows Program, 4000 Jones Bridge Rd., Chevy Chase, MD 20815 USA # contributed equally *Correspondence: Charles Keller, 3181 SW Sam Jackson Park Rd, MC-L321, Portland, OR 97239. Phone: (503) 494-1210, Fax (503) 418-5044, [email protected], and Jeffrey W. Tyner, 3181 SW Sam Jackson Park Rd., MC-L592, Portland, OR 97239. Phone: (503) 346-0603; Fax: (503) 494-3688, [email protected]; Amy J.Wagers, 7 Divinity Avenue, Cambridge, MA 02215, Phone: (617)309-2590, Fax: (617)309-2593, [email protected]
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
1. Arndt CA, Crist WM. Common musculoskeletal tumors of childhood and adolescence. N Engl J Med. 1999;341:342-52. 2. Breneman JC, Lyden E, Pappo AS, Link MP, Anderson JR, Parham DM, et al. Prognostic factors and clinical outcomes in children and adolescents with metastatic rhabdomyosarcoma--a report from the Intergroup Rhabdomyosarcoma Study IV. J Clin Oncol. 2003;21:78-84. 3. Pappo AS, Shapiro DN, Crist WM. Rhabdomyosarcoma. Biology and treatment. Pediatric clinics of North America. 1997;44:953-72. 4. Keller C, Arenkiel BR, Coffin CM, El-Bardeesy N, DePinho RA, Capecchi MR. Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function. Genes Dev. 2004;18:2614-26. 5. Nishijo K, Chen QR, Zhang L, McCleish AT, Rodriguez A, Cho MJ, et al. Credentialing a preclinical mouse model of alveolar rhabdomyosarcoma. Cancer research. 2009;69:2902-11. 6. Abraham J, Prajapati SI, Nishijo K, Schaffer BS, Taniguchi E, Kilcoyne A, et al. Evasion mechanisms to Igf1r inhibition in rhabdomyosarcoma. Mol Cancer Ther. 2011;10:697-707. 7. Taniguchi E, Nishijo K, McCleish AT, Michalek JE, Grayson MH, Infante AJ, et al. PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma. Oncogene. 2008;27:6550-60. 8. Armistead PM, Salganick J, Roh JS, Steinert DM, Patel S, Munsell M, et al. Expression of receptor tyrosine kinases and apoptotic molecules in rhabdomyosarcoma: correlation with overall survival in 105 patients. Cancer. 2007;110:2293-303. 9. Cen L, Arnoczky KJ, Hsieh FC, Lin HJ, Qualman SJ, Yu S, et al. Phosphorylation profiles of protein kinases in alveolar and embryonal rhabdomyosarcoma. Mod Pathol. 2007;20:936-46. 10. Kim ES, Herbst RS, Wistuba, II, Lee JJ, Blumenschein GR, Jr., Tsao A, et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 2011;1:44-53. 11. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883-92. 12. Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:16281-6. 13. Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer research. 2007;67:6796-805. 14. Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T, et al. CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. The Journal of clinical investigation. 2008;118:2111-20. 15. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature. 2004;432:396-401. 16. Gupta PB, Fillmore CM, Jiang G, Shapira SD, Tao K, Kuperwasser C, et al. Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. Cell. 2011;146:633-44. 17. Aleksic T, Chitnis MM, Perestenko OV, Gao S, Thomas PH, Turner GD, et al. Type 1 insulin-like growth factor receptor translocates to the nucleus of human tumor cells. Cancer Res. 2010;70:6412-9. 18. Ettenberg SA, Magnifico A, Cuello M, Nau MM, Rubinstein YR, Yarden Y, et al. Cbl-b-dependent coordinated degradation of the epidermal growth factor receptor signaling complex. The Journal of biological chemistry. 2001;276:27677-84.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
19. Johnston CL, Cox HC, Gomm JJ, Coombes RC. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. The Journal of biological chemistry. 1995;270:30643-50. 20. Liao HJ, Carpenter G. Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression. Molecular biology of the cell. 2007;18:1064-72. 21. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nature cell biology. 2001;3:802-8. 22. Sardi SP, Murtie J, Koirala S, Patten BA, Corfas G. Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain. Cell. 2006;127:185-97. 23. Duncan JS, Whittle MC, Nakamura K, Abell AN, Midland AA, Zawistowski JS, et al. Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell. 2012;149:307-21. 24. Huang F, Hurlburt W, Greer A, Reeves KA, Hillerman S, Chang H, et al. Differential mechanisms of acquired resistance to insulin-like growth factor-i receptor antibody therapy or to a small-molecule inhibitor, BMS-754807, in a human rhabdomyosarcoma model. Cancer Res. 2010;70:7221-31. 25. Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A. 2011;108:7950-5. 26. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pancreatic cancer stem cells. Cancer research. 2007;67:1030-7. 27. Le Roy C, Wrana JL. Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol. 2005;6:112-26. 28. Wang Y, Pennock SD, Chen X, Kazlauskas A, Wang Z. Platelet-derived growth factor receptor-mediated signal transduction from endosomes. The Journal of biological chemistry. 2004;279:8038-46. 29. Wu A, Chen J, Baserga R. Nuclear insulin receptor substrate-1 activates promoters of cell cycle progression genes. Oncogene. 2008;27:397-403. 30. Parker A, Cheville JC, Lohse C, Cerhan JR, Blute ML. Expression of insulin-like growth factor I receptor and survival in patients with clear cell renal cell carcinoma. The Journal of urology. 2003;170:420-4. 31. Mukherjee S, Tessema M, Wandinger-Ness A. Vesicular trafficking of tyrosine kinase receptors and associated proteins in the regulation of signaling and vascular function. Circulation research. 2006;98:743-56. 32. Sehat B, Tofigh A, Lin Y, Trocme E, Liljedahl U, Lagergren J, et al. SUMOylation mediates the nuclear translocation and signaling of the IGF-1 receptor. Sci Signal. 2010;3:ra10. 33. Deng H, Lin Y, Badin M, Vasilcanu D, Stromberg T, Jernberg-Wiklund H, et al. Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9. Biochem Biophys Res Commun. 2011;404:667-71. 34. Lo HW. Nuclear mode of the EGFR signaling network: biology, prognostic value, and therapeutic implications. Discov Med. 2010;10:44-51.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Figure 1. Murine aRMS isolated by Pdgfrα and Igf1r expression. (A) U23674 (murine aRMS) cell expression profile by FACS of Pdgfrα (APC) and Igf1r (Cy3.5) gated with respect to isotype and secondary only control. (B) Purity of isolated cells by FACS shown. Isolated populations according to cells positive and negative for each receptor designated as hi and lo, respectively. (C) Phase contrast microscopy of sorted populations demonstrating differential morphology of cells grown in culture 7 days post-sort. Scale bar: 200 µm.
Figure 2. Murine aRMS demonstrate dynamic expression of Pdgfrα and Igf1r by FACS in a temporal manner. (A) FACS profile of Pdgfrα and Igf1r expression in U23674 cells at day of sort (Day 0), and 2 and 14 days post sort. Day 7 was similar to Day 14.
Figure 3. Functional Pdgfrα and Igf1r expression post-sort. (A) Immunoblot of sorted populations grown in culture for 1 week post-sort, demonstrating phosphorylated as well as total Pdgfrα, Igf1r and Erk 1/2. (B) Representative data showing mean fluorescence intensity (MFI) of U23674 sorted cells 7 days post sort indicating surface expression of Pdgfrα and Igf1r.
Figure 4. Cells sorted for Igf1r establish more aggressive tumors in vivo. (A) Tumors established from murine aRMS cells sorted for Pdgfrα and Igf1r show a relative dynamic and static expression of each receptor, respectively, reflective of cells in vitro. (B) PdgfrαhiIgf1rlo sorted cells demonstrate a significantly slower growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to PdgfrαloIgf1rhi sorted cells. (C) PdgfrαhiIgf1rhi sorted cells grow at a significantly faster growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to PdgfrαloIgf1rlo sorted cells. (D) Tumor establishment or growth rate of unsorted U23674 cells in vivo do not differ from PdgfrαloIgf1rlo sorted cells.
Figure 5. Nuclear localization of Pdgfrα and Igf1r correlates with cells sorted by FACS for surface expression of the respective receptor. (A) Representative confocal images of U23674 sorted populations showing localization of Pdgfrα and Igf1r (40x) with inset box enlarging image of a single cell to appreciate localization. Scale bar: 100 µm. Larger magnifications of insets are given in Supplemental Figure S3.
Figure 6. Igf1r is present in soluble nuclear fraction of sorted cells and is associated with anchorage-independent colony formation ability. (A) Cytosolic and nuclear fractions of sorted U23674 cells show cells sorted by Igf1rhi express higher levels of nuclear Igf1r. (B) Cells with higher nuclear Igf1r, show a significantly further decrease in colony formation ability compared to control (non-specific siRNA) when Igf1r is silenced by siRNA. * indicated p <0.05 compared to non-specific siRNA by student’s t-test.
Figure 7. IGF1R localization in human aRMS. (A) Human aRMS sections with chiefly cytosolic IGF1R expression. (B) Images of aRMS sections demonstrating both cytosolic (left) and nuclear (right) IGF1R expression. Scale bar: 100 μM.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598