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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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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