Ectodomain shedding of TβRIII is required for TβRIII-mediated suppression of TGF-β signaling and breast cancer migration and invasion

Abstract

The type III transforming growth factor β (TGF-β) receptor (TβRIII), also known as betaglycan, is the most abundantly expressed TGF-β receptor. TβRIII suppresses breast cancer progression by inhibiting migration, invasion, metastasis, and angiogenesis. TβRIII binds TGF-β ligands, with membrane-bound TβRIII presenting ligand to enhance TGF-β signaling. However, TβRIII can also undergo ectodomain shedding, releasing soluble TβRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TβRIII on TGF-β signaling and breast cancer biology, we defined TβRIII mutants with impaired (ΔShed-TβRIII) or enhanced ectodomain shedding (SS-TβRIII). Inhibiting ectodomain shedding of TβRIII increased TGF-β responsiveness and abrogated TβRIII's ability to inhibit breast cancer cell migration and invasion. Conversely, expressing SS-TβRIII, which increased soluble TβRIII production, decreased TGF-β signaling and increased TβRIII-mediated inhibition of breast cancer cell migration and invasion. Of importance, SS-TβRIII-mediated increases in soluble TβRIII production also reduced breast cancer metastasis in vivo. Taken together, these studies suggest that the ratio of soluble TβRIII to membrane-bound TβRIII is an important determinant for regulation of TβRIII- and TGF-β-mediated signaling and biology.

FIGURE 1:

Mutations in the juxtamembrane domain of TβRIII alter ectodomain shedding. (A) Binding and cross-linking of transiently transfected, HA-tagged WT-TβRIII in COS7 cells. After [¹²⁵I]TGF-β1 binding and cross-linking, cell lysate and conditioned medium were immunoprecipitated with an antibody against HA. (B) Schematic of TβRIII with the amino acid sequence of the juxtamembrane domain. (C) Western blots of WT-TβRIII and indicated NAAIRS mutants transiently transfected in COS7 cells. EV, empty vector. β-Actin was used as a loading control. (D) Binding and cross-linking of COS7 cells transiently transfected with the indicated constructs. Cells were grown in full growth medium for 20 h. After [¹²⁵I]TGF-β1 binding and cross-linking, cell lysates and conditioned medium were immunoprecipitated with an antibody against HA. Asterisk denotes the mutants that were further used in these studies. Representative images from two independent experiments. (E) Quantification of D. Densitometric analysis was performed in ImageJ, and the ratio of soluble/cell-surface TβRIII was determined. (F) ELISA analysis of soluble TβRIII from COS7 cells transiently transfected with the indicated constructs. Media were conditioned for 24 h. Concentration of soluble TβRIII was determined from a standard curve. Soluble TβRIII levels were then normalized to TβRIII expression determined via Western blotting from control lysates. Data are from two independent experiments and shown as mean ± SEM normalized to WT-TβRIII.

FIGURE 2:

A single–amino acid substitution at M742 significantly inhibits ectodomain shedding. (A) Western blot showing expression of WT-TβRIII and M9 alanine point mutants transiently transfected in COS7 cells. β-Actin was used as a loading control. (B) Binding and cross-linking of COS7 cells transiently transfected with the indicated constructs. Cells were grown in full growth medium for 20 h. After [¹²⁵I]TGF-β1 binding and cross-linking, cell lysates and conditioned medium were immunoprecipitated with an antibody against HA. Representative images from two independent experiments. (C) Quantification of B. Densitometric analysis was performed in ImageJ, and the ratio of soluble/cell-surface TβRIII was determined. (D) ELISA analysis of soluble TβRIII from COS7 cells transiently transfected with the indicated constructs. Media were conditioned for 24 h. Concentration of soluble TβRIII was determined from a standard curve. Soluble TβRIII levels were then normalized to TβRIII expression determined via Western blotting from control lysates. Representative data from two independent experiments. (E) Binding and cross-linking of monoclonal stable lentiviral MDA-MB-231 cell lines made with EV, WT-TβRIII (WT), ΔShed-TβRIII (ΔS) (M9 mutant), or Super-Shed TβRIII (SS; M13 mutant). After [¹²⁵I]TGF-β1 binding and cross-linking, cell lysates and conditioned medium were immunoprecipitated with an antibody against the extracellular domain of TβRIII. β-Actin was used as a loading control. Representative data from three independent experiments. (F) ELISA analysis of soluble TβRIII from stable MDA-MB-231 cell lines. Media were conditioned for 24 h. Concentration of soluble TβRIII was determined from a standard curve of known amounts. Soluble TβRIII levels were then normalized to β-actin expression determined via Western blotting from control lysates. Data are from three independent experiments and shown as mean ± SEM. One-way ANOVA: p < 0.0001. Tukey's multiple-comparisons tests: *p < 0.05; **p < 0.001; ***p < 0.0001.

FIGURE 3:

Effects of altered TβRIII ectodomain shedding on TGF-β signaling. (A, B) Lentiviral stable MDA-MB-231 cell lines expressing EV, WT-TβRIII, ΔShed-TβRIII, or Super-Shed TβRIII were plated in full serum medium and allowed to condition for 20 h before treatment with the indicated concentrations of TGF-β1 for 30 min. Western blot analysis was performed with the indicated antibodies. Total Smad2, Smad3, and β-actin were used as loading controls. Quantification of densitometric analysis shown as ratio of phosphorylated Smad2/β-actin. Data are representative of at least three independent experiments. (C) Lentiviral stable MDA-MB-231 cell lines expressing EV, WT-TβRIII, ΔShed-TβRIII. or Super-Shed TβRIII were treated with reduced serum medium (1% FBS) that had been preconditioned from the corresponding cell line for 20 h. Cells were serum starved for 6 h before treatment with the indicated concentrations of TGF-β1 for 30 min. Western blot analysis was performed with the indicated antibodies. Total Akt and β-actin were used as loading controls. Quantification of densitometric analysis is shown as ratio of phosphorylated Akt/β-actin. Data are representative of at least three independent experiments. (D) Stable MDA-MB-231 cell lines were transfected with a pE2.1-responsive luciferase construct and a Renilla construct. The next day, cells were treated with serum-free medium that had been preconditioned from the corresponding cell line for 24 h and 50 pM TGF-β1. Cells were treated for 24 h. Results from four independent experiments are shown as pE2.1/Renilla activity and normalized to ligand untreated condition of each cell line. Two-way ANOVA, p < 0.001. *One-sample t test, p < 0.05; ^#two-tailed t-test, p < 0.05, relative to ΔS + TGF-β1.

FIGURE 4:

TβRIII ectodomain shedding regulates the kinetics and magnitude of TGF-β signaling in MDA-MB-231 cells. (A) Lentiviral stable MDA-MB-231 cell lines expressing EV, WT-TβRIII, ΔShed-TβRIII, or Super-Shed TβRIII were plated in full serum medium and allowed to condition for 20 h before treatment with 50 pM of TGF-β1 for the indicated times. Western blot analysis was performed with indicated antibodies. Total Smad2 and β-actin were used as loading controls. Quantification of densitometric analysis is shown below as levels of phosphorylated Smad2/β-actin. Representative data from four independent experiments. (B) Summary of time-course experiment data. Densitometric analysis of phosphorylated Smad2/β-actin. Data for at least independent experiments for each time point shown as mean ± SEM. One-way ANOVA p < 0.05 for 15-m, 30-m, 1-h, 2-h, 3-h, 4-h, 5-h, and 6-h time points. (C) Integrated signaling over 6-h time course. Densitometric analysis of phosphorylated Smad2/β-actin of each experiment plotted as line graphs, with area under the curve calculated for each cell line. Data from four independent experiments shown as mean ± SEM. One-way ANOVA, p < 0.05. *Two-tailed t-test, p < 0.05.

FIGURE 5:

Effects of altered TRIII ectodomain shedding on TGF-β–mediated migration and invasion. Stable MDA-MB-231 cell lines were plated in medium that had been preconditioned from the corresponding cell line for 24 h in (A, B) fibronectin-coated Transwell chambers or (C, D) Matrigel-coated Transwell chambers in either the absence (UnT) or presence of 50 pM TGF-β1. Cells were allowed to migrate or invade for 24 h. (A) Representative images of migrated cells. (B) Summary of four experiments. Data normalized to EV UnT and shown as mean ± SEM. Two-way ANOVA for cell line and treatment, p < 0.05. Tukey's multiple-comparisons tests: ^#p < 0.05 relative to EV UnT; *p < 0.05 relative to EV + TGF-β1; ^†p < 0.05 relative to ΔS UnT. (C) Representative images of invaded cells. (D) Summary of five experiments. Data normalized to EV UnT and shown as mean ± SEM. Two-way ANOVA for interaction, cell line, and treatment, p < 0.05. Tukey's multiple-comparisons tests: ^#p < 0.05 relative to EV UnT; *p < 0.05 relative to EV + TGF-β1; ^†p < 0.05 relative to ΔS UnT.

FIGURE 6:

Effects of TβRIII ectodomain shedding on MDA-MB-231-4175 metastatic growth. MDA-MB-231-4175 cells expressing EV (N = 13), WT-TβRIII (N = 14), or SS-TβRIII (N = 10) were injected into 6-wk-old athymic mice via tail-vein injection. (A) Initial and weekly bioluminescence of lung metastatic lesions expressed as photon flux of mice after initial injection. Kruskal–Wallis analysis: *p < 0.05, **p < 0.01. (B) Kaplan–Meier survival curve of mice injected with cells expressing EV, WT-TβRIII, or SS-TβRIII. Log-rank Mantel–Cox test: p < 0.01. (C) Representative images of bioluminescence scans of each cohort of mice at each time point. (D) Schematic summary of the effects of altered ratios of soluble and cell-surface TβRIII on TGF-β–mediated signaling and biology in the context of breast cancer.