PDGFRβ Signaling Cooperates with β-Catenin to Modulate c-Abl and Biologic Behavior of Desmoid-Type Fibromatosis

Abstract

Purpose: This study sought to identify β-catenin targets that regulate desmoid oncogenesis and determine whether external signaling pathways, particularly those inhibited by sorafenib (e.g., PDGFRβ), affect these targets to alter natural history or treatment response in patients.

Experimental design: In vitro experiments utilized primary desmoid cell lines to examine regulation of β-catenin targets. Relevance of results was assessed in vivo using Alliance trial A091105 correlative biopsies.

Results: CTNNB1 knockdown inhibited hypoxia-regulated gene expression in vitro and reduced levels of HIF1α protein. ChIP-seq identified ABL1 as a β-catenin transcriptional target that modulated HIF1α and desmoid cell proliferation. Abrogation of either CTNNB1 or HIF1A inhibited desmoid cell-induced VEGFR2 phosphorylation and tube formation in endothelial cell co-cultures. Sorafenib inhibited this activity directly but also reduced HIF1α protein expression and c-Abl activity while inhibiting PDGFRβ signaling in desmoid cells. Conversely, c-Abl activity and desmoid cell proliferation were positively regulated by PDGF-BB. Reduction in PDGFRβ and c-Abl phosphorylation was commonly observed in biopsy samples from patients after treatment with sorafenib; markers of PDGFRβ/c-Abl pathway activation in baseline samples were associated with tumor progression in patients on the placebo arm and response to sorafenib in patients receiving treatment.

Conclusions: The β-catenin transcriptional target ABL1 is necessary for proliferation and maintenance of HIF1α in desmoid cells. Regulation of c-Abl activity by PDGF signaling and targeted therapies modulates desmoid cell proliferation, thereby suggesting a reason for variable biologic behavior between tumors, a mechanism for sorafenib activity in desmoids, and markers predictive of outcome in patients.

Figure 1.

Desmoid cells promote endothelial cell tube formation by activating VEGFR2 in a HIF1A- and CTNNB1-dependent manner. A and B, Effect of CTNNB1 knockdown on (A) HIF1α protein and (B) mRNA expression assessed by dual luciferase assay. C and D, Effect of knockdown of (C) HIF1A or (D) CTNNB1 in DES9525 or DES8163 desmoid cells on co-culture–induced HUVEC tube formation. Representative photos show HUVECs following co-culture with DES9525 cells transduced as indicated; quantitated results average triplicate experiments. E and F, Effect of knockdown of (E) CTNNB1 or (F) HIF1A in desmoid cells on phosphorylation of VEGFR2 in HUVEC cells treated with desmoid-conditioned media for the indicated times; MC indicates nonconditioned media control. G and H, Effect of HIF1α overexpression on (G) HIF1α protein expression and (H) representative trial of co-culture–induced HUVEC tube formation in desmoid cells in which CTNNB1 is knocked down. Control for HIF1α overexpression is red fluorescent protein (RFP, Ctrl; *, P < 0.05).

Figure 2.

Sorafenib inhibits HIF1-mediated paracrine VEGF signaling. Effect of sorafenib on (A) the number of HUVEC tubes formed following co-culture with desmoid cells, (B) VEGFR2 phosphorylation in HUVEC cells treated with desmoid-conditioned media (+, desmoids grown in the presence of sorafenib; +*, sorafenib added to conditioned media after it was added to HUVEC cultures), (C) HIF1α expression and Akt phosphorylation, and (D) HIF1-mediated transcription as defined by a luciferase reporter assay (*, P < 0.05).

Figure 3.

PDGF signaling is a sorafenib target that regulates HIF1α accumulation, enhances paracrine effects, and increases proliferation. A, Sorafenib (40 μmol/L) decreases phosphorylation of PDGFRβ as detected by receptor tyrosine kinase array. B, Immunoblot analysis of activation of PDGFRβ signaling pathway components and HIF1α expression in desmoid cells treated with sorafenib in the presence and absence of PDGF-BB. C–E, Effect of PDGF-BB on (C) relative HIF1α transcriptional activity assessed by luciferase reporter assay (20 ng/mL PDGF-BB), relative rate of HUVEC tube formation in desmoid co-cultures, and (D) desmoid cell proliferation as assessed by DNA quantification. E, Relative proliferation of desmoid cells grown in the presence or absence of PDGF-BB (20 ng/mL) at varying concentrations of sorafenib normalized to that in cultures without the drug at 72 hours after treatment. All quantitated results average triplicate experiments (*, P < 0.05; **, P < 0.005).

Figure 4.

β-Catenin transcriptional target ABL1 is necessary for cell proliferation and HIF1α accumulation in desmoid cells. A and B, Effect of CTNNB1 knockdown on (A) relative transcript levels of ABL1 (detected by RT-PCR) and (B) expression of c-Abl and activation of its canonical phosphorylation targets. C–E, Effect of ABL1 knockdown on (C) activation of c-Abl phosphorylation targets, (D) proliferation (as measured by DNA content) of desmoid cells (average of triplicate experiment; *, P < 0.05; **, P < 0.005) and (E) VEGFR2 phosphorylation detected by immunoblot. F and G, Effect of (F) PDGF-BB and (G) sorafenib on activation of canonical PDGFRβ and c-ABL pathway components as measured by immunoblot.

Figure 5.

Inhibition of PDGFRβ/c-Abl signaling may correlate with response to sorafenib in desmoid patients. A, Activation of PDGFRβ, c-Abl, and Akt and expression of HIF1α and β-catenin as detected by immunoblot in baseline (B) and post-treatment (T) biopsies in individual patients treated on the placebo and sorafenib arms of Alliance A091105. Best response (partial response, stable or progressive disease) as assayed by RECIST criteria or clinical progression, CTNNB1 mutations and their variant allele frequencies (VAF; identified by RNA-seq) are annotated. Samples with no mutations detected are annotated (-). B, PFS defined by clinical progression or radiographic increase in size (>15% above baseline) in patients and stratified according to high (≥50% of desmoid cells) or low (<50% of cells positive) levels of nuclear EGR1 staining on IHC of baseline samples obtained from patients in the placebo arm of the trial. C, Percent change in tumor size after four to five cycles of sorafenib in patients on Alliance A091105. Tumors with ≥90% cells staining for nuclear EGR1 are designated as EGR1 high as opposed to low.