Targeting Nodal/TGF-β pathway to inhibit invasion and growth of retinoblastoma

Abstract

Purpose : To identify pathways promoting retinoblastoma invasion and growth.

Methods : RNA-seq was performed on ten snap frozen retinoblastoma specimens, including five invasive (4 retrolaminar, 1 intralaminar) and five non-invasive cases (4 prelaminar, 1 no optic nerve invasion). Nodal/TGF-β pathway components were analysed by Western blot and qPCR in WERI Rb1, Y79, RB143, HSJD-RBT-1, HSJD-RBT-2, and HSJD-RBVS-10 retinoblastoma lines. ALK signaling was suppressed using ACVR1C (ALK7) receptor shRNA and SB505124, an inhibitor of ALK4/5/7 receptors. Invasion, growth, proliferation were measured by transwell invasion, CCK8 (Cell Counting kit 8), and Ki67 assays, respectively. Apoptosis was measured by cleaved PARP immunoblotting and cleaved caspase 3 immunofluorescence assay. Longitudinal intravital imaging was used to quantify invasion of GFP-tagged retinoblastoma cells in an orthotopic model in zebrafish.

Results : We found three-fold increase in mRNA levels of ACVR1C, a type I receptor of the TGF-β family, in all invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of Nodal/TGF-β signaling, was observed in most of the invasive cases. To further evaluate the role of Nodal/TGF-β pathway in retinoblastoma invasion we performed a transwell assay and observed two to three-fold increase in ALK7 mRNA in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed at varying degrees in six retinoblastoma lines, and evidence of SMAD2 signaling was present in all six lines. The ALK inhibitor SB505124 potently repressed invasion and growth in a dose-dependent manner in Y79 and WERI Rb1. Genetic downregulation of ALK7 in Y79 also decreased proliferation, survival, invasion, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. In an orthotopic model of retinoblastoma in zebrafish, a 40% decrease in tumor spread was noted (p=0.0026), as measured by the minimum bounding sphere (MBS) volume, when larvae were treated with 3µM of SB505124 for 4 days after intravitreal injection of Y79-GFP cells, as compared to DMSO.

Conclusions : Our data support a role for Nodal/TGF-β pathway in promoting invasion and overall growth in retinoblastoma. Thus, inhibition of this pathway may be effective in treating both local growth and invasive spread of retinoblastoma.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.