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ACVR1C/SMAD2 signaling promotes invasion and growth in retinoblastoma

Abstract

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-β family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.

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References

  1. Honavar SG, Singh AD. Management of advanced retinoblastoma. Ophthalmol Clin North Am. 2005;18:65–73.

    Article  Google Scholar 

  2. Chantada GL, Qaddoumi I, Canturk S, Khetan V, Ma Z, Kimani K, et al. Strategies to manage retinoblastoma in developing countries. Pediatr Blood Cancer. 2011;56:341–8.

    Article  Google Scholar 

  3. Villegas VM, Hess DJ, Wildner A, Gold AS, Murray TG. Retinoblastoma. Curr Opin Ophthalmol. 2013;24:581–8.

    Article  Google Scholar 

  4. Bondestam J, Huotari MA, Morén A, Ustinov J, Kaivo-Oja N, Kallio J, et al. cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine kinase receptor ALK7 (ACVR1C). Cytogenet Cell Genet. 2001;95:157–62.

    Article  CAS  Google Scholar 

  5. Shi Y, Massagué J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113:685–700.

    Article  CAS  Google Scholar 

  6. Loomans HA, Andl CD. Activin receptor-like kinases: a diverse family playing an important role in cancer. Am J Cancer Res. 2016;6:2431–47.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Tsuchida K, Nakatani M, Hitachi K, Uezumi A, Sunada Y, Ageta H, et al. Activin signaling as an emerging target for therapeutic interventions. Cell Commun Signal. 2009;7:15.

    Article  Google Scholar 

  8. Peng L, Yuan XQ, Zhang CY, Ye F, Zhou HF, Li WL, et al. High TGF-β1 expression predicts poor disease prognosis in hepatocellular carcinoma patients. Oncotarget. 2017;8:34387–97.

    PubMed  PubMed Central  Google Scholar 

  9. Lonardo E, Hermann PC, Mueller MT, Huber S, Balic A, Miranda-Lorenzo I, et al. Nodal/Activin signaling drives self-renewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy. Cell Stem Cell. 2011;9:433–46.

    Article  CAS  Google Scholar 

  10. Lawrence MG, Margaryan NV, Loessner D, Collins A, Kerr KM, Turner M, et al. Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells. Prostate. 2011;71:1198–209.

    Article  CAS  Google Scholar 

  11. Kirsammer G, Strizzi L, Margaryan NV, Gilgur A, Hyser M, Atkinson J, et al. Nodal signaling promotes a tumorigenic phenotype in human breast cancer. Semin Cancer Biol. 2014;29:40–50.

    Article  CAS  Google Scholar 

  12. Bashir M, Damineni S, Mukherjee G, Kondaiah P. Activin-A signaling promotes epithelial mesenchymal transition, invasion, and metastatic growth of breast cancer. NPJ Breast Cancer. 2015;1:15007.

    Article  CAS  Google Scholar 

  13. Condro MC, Matynia A, Foster NN, Ago Y, Rajbhandari AK, Van C, et al. High-resolution characterization of a PACAP-EGFP transgenic mouse model for mapping PACAP- expressing neurons. J Comp Neurol. 2016;524:3827–48.

    Article  CAS  Google Scholar 

  14. Morini M, Astigiano S, Gitton Y, Emionite L, Mirisola V, Levi G, et al. Mutually exclusive expression of DLX2 and DLX5/6 is associated with the metastatic potential of the human breast cancer cell line MDA-MB-231. BMC Cancer. 2010;10:649.

    Article  CAS  Google Scholar 

  15. Chung IC, Chen LC, Chung AK, Chao M, Huang HY, Hsueh C, et al. Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma. BMC Cancer. 2014;14:348.

    Article  Google Scholar 

  16. Lv FZ, Wang JL, Wu Y, Chen HF, Shen XY. Knockdown of MMP12 inhibits the growth and invasion of lung adenocarcinoma cells. Int J Immunopathol Pharmacol. 2015;28:77–84.

    Article  CAS  Google Scholar 

  17. White CD, Brown MD, Sacks DB. IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett. 2009;583:1817–24.

    Article  CAS  Google Scholar 

  18. Yang Y, Zhao W, Xu QW, Wang XS, Zhang Y, Zhang J. IQGAP3 promotes EGFR-ERK signaling and the growth and metastasis of lung cancer cells. PLoS One. 2014;9:e97578.

    Article  Google Scholar 

  19. Schubert FR, Sobreira DR, Janousek RG, Alvares LE, Dietrich S. Dact genes are chordate specific regulators at the intersection of Wnt and Tgf-β signaling pathways. BMC Evol Biol. 2014;14:157.

    Article  Google Scholar 

  20. Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, et al. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci USA. 2008;105:4329–34.

    Article  CAS  Google Scholar 

  21. Tanaka K, Imoto I, Inoue J, Kozaki K, Tsuda H, Shimada Y, et al. Frequent methylation- associated silencing of a candidate tumor-suppressor, CRABP1, in esophageal squamous-cell carcinoma. Oncogene. 2007;26:6456–68.

    Article  CAS  Google Scholar 

  22. Liu Y, Hu H, Liang M, Xiong Y, Li K, Chen M, et al. Regulated differentiation of WERI- Rb-1 cells into retinal neuron-like cells. Int J Mol Med. 2017;40:1172–84.

    Article  CAS  Google Scholar 

  23. DaCosta Byfield S, Major C, Laping NJ, Roberts AB. SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2004;65:744–52.

    Article  Google Scholar 

  24. Leal-Leal CA, Rivera-Luna R, Flores-Rojo M, Juárez-Echenique JC, Ordaz JC, Amador-Zarco J. Survival in extra-orbital metastatic retinoblastoma:treatment results. Clin Transl Oncol. 2006;8:39–44.

    Article  Google Scholar 

  25. Busch M, Papior D, Stephan H, Dünker N. Characterization of etoposide- and cisplatin-chemoresistant retinoblastoma cell lines. Oncol Rep. 2018;39:160–72.

    CAS  PubMed  Google Scholar 

  26. Badhu B, Sah SP, Thakur SK, Dulal S, Kumar S, Sood A, et al. Clinical presentation of retinoblastoma in Eastern Nepal. Clin Exp Ophthalmol. 2005;33:386–9.

    Article  Google Scholar 

  27. Chawla B, Hasan F, Seth R, Pathy S, Pattebahadur R, Sharma S, et al. Multimodal therapy for stage III retinoblastoma (International Retinoblastoma Staging System): A Prospective Comparative Study. Ophthalmology. 2016;123:1933–9.

    Article  Google Scholar 

  28. James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 2005;132:1273–82.

    Article  CAS  Google Scholar 

  29. Massagué J. TGFbeta in Cancer. Cell. 2008;134:215–30.

    Article  Google Scholar 

  30. Topczewska JM, Postovit LM, Margaryan NV, Sam A, Hess AR, Wheaton WW, et al. Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness. Nat Med. 2006;12:925–32.

    Article  CAS  Google Scholar 

  31. Strizzi L, Postovit LM, Margaryan NV, Lipavsky A, Gadiot J, Blank C, et al. Nodal as a biomarker for melanoma progression and a new therapeutic target for clinical intervention. Expert Rev Dermatol. 2009;4:67–78.

    Article  CAS  Google Scholar 

  32. Hu T, Su F, Jiang W, Dart DA. Overexpression of Activin receptor-like kinase 7 in breast cancer cells is associated with decreased cell growth and adhesion. Anticancer Res. 2017;37:3441–51.

    CAS  PubMed  Google Scholar 

  33. Pacifici M, Shore EM. Common mutations in ALK2/ACVR1, a multi-faceted receptor, have roles in distinct pediatric musculoskeletal and neural orphan disorders. Cytokine Growth Factor Rev. 2016;27:93–104.

    Article  CAS  Google Scholar 

  34. Gong W, Sun B, Zhao X, Zhang D, Sun J, Liu T, et al. Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3 pathway. Oncotarget. 2016;7:70152–67.

    PubMed  PubMed Central  Google Scholar 

  35. Yoshinaga K, Inoue H, Utsunomiya T, Sonoda H, Masuda T, Mimori K, et al. N-cadherin is regulated by activin A and associated with tumor aggressiveness in esophageal carcinoma. Clin Cancer Res. 2004;10:5702–7.

    Article  CAS  Google Scholar 

  36. Schmierer B, Schuster MK, Shkumatava A, Kuchler K. Activin A signaling induces Smad2, but not Smad3, requiring protein kinase A activity in granulosa cells from the avian ovary. J Biol Chem. 2003;278:21197–203.

    Article  CAS  Google Scholar 

  37. Liu C, Gaça MD, Swenson ES, Vellucci VF, Reiss M, Wells RG. Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. J Biol Chem. 2003;278:11721–8.

    Article  CAS  Google Scholar 

  38. Kalluri R, Weinberg RA. The basics of epithelial–mesenchymal transition. J Clin Investig. 2009;119:1420–8.

    Article  CAS  Google Scholar 

  39. Gregory PA, Bracken CP, Smith E, Bert AG, Wright JA, Roslan S, et al. An autocrine TGF-β/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition. Mol Biol Cell. 2011;22:1686–98.

    Article  CAS  Google Scholar 

  40. Joseph JV, Conroy S, Tomar T, Eggens-Meijer E, Bhat K, Copray S, et al. TGF-β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion. Cell Death Dis. 2014;5:e1443.

    Article  CAS  Google Scholar 

  41. Arima Y, Hayashi H, Sasaki M, Hosonaga M, Goto TM, Chiyoda T, et al. Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer. J Biol Chem. 2012;287:7896–906.

    Article  CAS  Google Scholar 

  42. Liu Y, Sánchez-Tilló E, Lu X, Huang L, Clem B, Telang S, et al. Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression. J Biol Chem. 2013;288:11572–80.

    Article  CAS  Google Scholar 

  43. Bertacchi M, Lupo G, Pandolfini L, Casarosa S, D’Onofrio M, Pedersen RA, et al. Activin/Nodal signaling supports retinal progenitor specification in a narrow time window during pluripotent stem cell neuralization. Stem Cell Rep. 2015;5:532–45.

    Article  Google Scholar 

  44. Sakami S, Etter P, Reh TA. Activin signaling limits the competence for retinal regeneration from the pigmented epithelium. Mech Dev. 2008;125:106–16.

    Article  CAS  Google Scholar 

  45. Kanno C, Kashiwagi Y, Horie K, Inomata M, Yamamoto T, Kitanaka C, et al. Activin inhibits cell growth and induces differentiation in human retinoblastoma Y79 cells. Curr Eye Res. 2009;34:652–9.

    Article  CAS  Google Scholar 

  46. Gray PC, Vale W. Cripto/GRP78 modulation of the TGF-β pathway in development and oncogenesis. FEBS Lett. 2012;586:1836–45.

    Article  CAS  Google Scholar 

  47. McFall RC, Sery TW, Makadon M. Characterization of a new continuous cell line derived from a human retinoblastoma. Cancer Res. 1977;37:1003–10.

    CAS  PubMed  Google Scholar 

  48. Reid TW, Albert DM, Rabson AS, Russell P, Craft J, Chu EW, et al. Characteristics of an established cell line of retinoblastoma. J Natl Cancer Inst. 1974;53:347–60.

    Article  CAS  Google Scholar 

  49. Theodoropoulou S, Kolovou PE, Morizane Y, Kayama M, Nicolaou F, Miller JW, et al. Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase. FASEB J. 2010;24:2620–30.

    Article  CAS  Google Scholar 

  50. Pascual-Pasto G, Olaciregui NG, Vila-Ubach M, Paco S, Monterrubio C, Rodriguez E, et al. Preclinical platform of retinoblastoma xenografts recapitulating human disease and molecular markers of dissemination. Cancer Lett. 2016;380:10–19.

    Article  CAS  Google Scholar 

  51. Asnaghi L, Tripathy A, Yang Q, Kaur H, Hanaford A, Yu W, et al. Targeting Notch signaling as a novel therapy for Retinoblastoma. Oncotarget. 2016;7:70028–44.

    Article  Google Scholar 

  52. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–26.

    Article  CAS  Google Scholar 

  53. Weingart MF, Roth JJ, Hutt-Cabezas M, Busse TM, Kaur H, Price A, et al. Disrupting LIN28 in atypical teratoid rhabdoid tumors reveals the importance of the mitogen activated protein kinase pathway as a therapeutic target. Oncotarget. 2015;6:3165–77.

    Article  Google Scholar 

  54. Teng Y, Xie X, Walker S, White DT, Mumm JS, Cowell JK. Evaluating human cancer cell metastasis in zebrafish. BMC Cancer. 2013;13:453.

    Article  Google Scholar 

  55. Chen X, Wang J, Cao Z, Hosaka K, Jensen L, Yang H, et al. Invasiveness and metastasis of retinoblastoma in an orthotopic zebrafish tumor model. Sci Rep. 2015;5:10351.

    Article  CAS  Google Scholar 

  56. Asnaghi L, Handa JT, Merbs SL, Harbour JW, Eberhart CG. A role for Jag2 in promoting uveal melanoma dissemination and growth. Invest Ophthalmol Vis Sci. 2013;54:295–306.

    Article  CAS  Google Scholar 

  57. Hamilton L, Astell KR, Velikova G, Sieger D. A zebrafish live imaging model reveals differential responses of microglia toward glioblastoma cells in vivo. Zebrafish. 2016;13:523–34.

    Article  CAS  Google Scholar 

  58. White DT, Sengupta S, Saxena MT, Xu Q, Hanes J, Ding D, et al. Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina. Proc Natl Acad Sci USA. 2017;14:E3719–E3728.

    Article  Google Scholar 

  59. Wickham H ggplot2 - Elegant Graphics for Data Analysis. 2nd Ed Springer International Publishing, Cham, Switzerland, 2016.

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Acknowledgements

We thank Jennifer Meyers for her technical assistance in the RNA-seq, Dr. Wayne Yu for pathway analysis, Dr. Michael Goggins for providing PANC-1 cells, and Urvi Patel for technical support. RNA-seq analysis was conducted at The Sidney Kimmel Cancer Center, Next Generation Sequencing Core at the Johns Hopkins University. This study was supported by King Khaled Eye Specialist Hospital (KKESH)—Wilmer Eye Institute (WEI) Collaborative Research Grant, by the NIH Grant R21CA229919, the core grant EY001765 and by The Jenny Fund. AMC acknowledges funding from ISCIII-FEDER (CP13/00189) and MINECO (Retos program; Cure4RB project RTC-2015-4319-1).

Funding

King Khaled Eye Specialist Hospital (KKESH)—Wilmer Eye Institute (WEI) Collaborative Research Grant, NIH Grant R21CA229919, the core grant EY001765, and The Jenny Fund. AMC acknowledges funding from ISCIII-FEDER (CP13/00189) and MINECO (Retos program; Cure4RB project RTC-2015-4319-1).

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Correspondence to Leen Abu Safieh or Charles G. Eberhart.

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Asnaghi, L., White, D.T., Key, N. et al. ACVR1C/SMAD2 signaling promotes invasion and growth in retinoblastoma. Oncogene 38, 2056–2075 (2019). https://doi.org/10.1038/s41388-018-0543-2

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