Abstract 6168: Beta-catenin and PDGFRB modulate HIF1a in desmoid cells to promote sorafenib-sensitive paracrine signaling pathways
Authors: Hu Jia , Villano Anthony , Connor Rachael , Rozenberg Yuliy , Venkatesh Alankrta , Socci Nicholas , Singer Samuel , Crago Aimee 
Published: June 2022

Abstract
Introduction: Sorafenib has been shown to have clinical benefit in desmoid-type fibromatosis (DF), a mesenchymal neoplasm associated with activating CTNNB1 mutations. This study sought to better understand the mechanisms of sorafenib activity in DF by defining downstream pathways dysregulated by β-catenin.
Methods: Primary DF cell lines (DES9525, 3276, 8163) were developed from surgical specimens and validated by Sanger sequencing of CTNNB1. Gene expression in DF tumors was assessed with U133A arrays and in cell lines using RNA-seq. Lentiviral systems were used to deliver shRNA (or scramble control) and overexpression constructs. Luciferase reporter assay was employed to assess transcription activity. Cell proliferation, protein levels/phosphorylation and gene expression were assessed by CyQuant DNA quantification, immunoblot, and RT-PCR, respectively. Endothelial cell (HUVEC) tube formation was quantitated manually using light microscopy.
Results: Gene set enrichment analysis of DES9525 CTNNB1 knock-down (KD) showed decreased expression of genes commonly associated with hypoxia and angiogenesis. Unsupervised clustering confirmed in vivo relevance as expression levels of hypoxia-associated genes clustered 45 DF tumors separately from normal mesenchymal tissue (fat and muscle). Higher baseline levels of HIF1α were observed in DF cell lines compared to mesenchymal stem cells and abrogation of CTNNB1 reduced cellular levels of HIF1α and HIF transcriptional activity. DF co-culture with endothelial cells induced VEGFR phosphorylation on HUVECs and tube formation; HIF1A KD inhibited VEGFR phosphorylation and reduced tube formation &gt;50% but had no effect on desmoid cell proliferation. Similar inhibition was observed after co-culture of DF CTNNB1 KD cells with endothelial cells (&gt;50% reduction in tube formation, p&lt;0.05), but not after overexpression of HIF1A in CTNNB1 KD cells. Sorafenib inhibited this tube-formation (60%, p&lt;0.05) induced by DF at lower concentrations (1μM) as compared to those required to inhibit DF cell proliferation (IC50 10uM). While sorafenib directly inhibited VEGFR phosphorylation on endothelial cells, it also inhibited phosphorylation of PDGFRB and decreased HIF1α levels in DF cells. Exogenous PDGF-BB conversely increased levels of HIF1α in DF cells and endothelial tube formation (2 times, p&lt;0.05) when added to DF but not to endothelial cells grown in the absence of DF cells.
Conclusion: β-catenin and PDGFRB signaling modulate cellular levels of HIF1α in DF cells to affect endothelial cells via VEGFR. Sorafenib inhibits a PDGFR/β-catenin/HIF1α axis by modulating signaling pathways both on the DF cells themselves as well as RTK targets of sorafenib on stromal cells. Improved understanding of these interactions may allow for more specific therapeutic selection in DF patients.
Citation Format: Jia Hu, Anthony Villano, Rachael Connor, Yuliy Rozenberg, Alankrta Venkatesh, Nicholas Socci, Samuel Singer, Aimee Crago. Beta-catenin and PDGFRB modulate HIF1a in desmoid cells to promote sorafenib-sensitive paracrine signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6168.