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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 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.

  3. 3.

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

  4. 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.

  5. 5.

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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 17.

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

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.

  24. 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.

  25. 25.

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

  26. 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.

  27. 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.

  28. 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.

  29. 29.

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

  30. 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.

  31. 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.

  32. 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.

  33. 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.

  34. 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.

  35. 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.

  36. 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.

  37. 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.

  38. 38.

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

  39. 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.

  40. 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.

  41. 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.

  42. 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.

  43. 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.

  44. 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.

  45. 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.

  46. 46.

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

  47. 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.

  48. 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.

  49. 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.

  50. 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.

  51. 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.

  52. 52.

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

  53. 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.

  54. 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.

  55. 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.

  56. 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.

  57. 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.

  58. 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.

  59. 59.

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

Download references

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).

Author information

Author notes

    • Hind Alkatan

    Present address: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia

Affiliations

  1. Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA

    • Laura Asnaghi
    • , Nolan Key
    • , Joshua Choi
    •  & Charles G. Eberhart
  2. Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA

    • David T. White
    • , Deepak P. Edward
    • , Christopher G. Hurtado
    • , Grace Y. Lee
    • , Jeff S. Mumm
    •  & Charles G. Eberhart
  3. King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia

    • Alka Mahale
    • , Hind Alkatan
    • , Deepak P. Edward
    • , Sahar M. Elkhamary
    • , Saleh Al-Mesfer
    • , Azza Maktabi
    •  & Leen Abu Safieh
  4. University of Illinois Eye and Ear Infirmary, Chicago, IL, USA

    • Deepak P. Edward
  5. Department of Diagnostic Radiology, Mansoura Faculty of Medicine, Mansoura, Egypt

    • Sahar M. Elkhamary
  6. Institut de Recerca Sant Joan de Deu, Barcelona, Spain

    • Angel M. Carcaboso
  7. Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA

    • Charles G. Eberhart

Authors

  1. Search for Laura Asnaghi in:

  2. Search for David T. White in:

  3. Search for Nolan Key in:

  4. Search for Joshua Choi in:

  5. Search for Alka Mahale in:

  6. Search for Hind Alkatan in:

  7. Search for Deepak P. Edward in:

  8. Search for Sahar M. Elkhamary in:

  9. Search for Saleh Al-Mesfer in:

  10. Search for Azza Maktabi in:

  11. Search for Christopher G. Hurtado in:

  12. Search for Grace Y. Lee in:

  13. Search for Angel M. Carcaboso in:

  14. Search for Jeff S. Mumm in:

  15. Search for Leen Abu Safieh in:

  16. Search for Charles G. Eberhart in:

Conflict of interest

The Authors declare no conflict of interest.

Corresponding authors

Correspondence to Leen Abu Safieh or Charles G. Eberhart.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

Issue Date

DOI

https://doi.org/10.1038/s41388-018-0543-2