Targeting all transforming growth factor-β isoforms with an Fc chimeric receptor impairs tumor growth and angiogenesis of oral squamous cell cancer

Abstract

Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-β (TGF-β) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-β comprises three isoforms, TGF-β1, -β2, and -β3, and transduces intracellular signals via TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TβRII (TβRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-β ligands, but several lines of evidence indicate that TβRII-Fc more effectively traps TGF-β1 and TGF-β3 than TGF-β2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-β receptor containing both TβRI and TβRII (TβRI-TβRII-Fc) and found that TβRI-TβRII-Fc trapped all TGF-β isoforms, leading to inhibition of both the TGF-β signal and TGF-β-induced EMT of oral cancer cells, whereas TβRII-Fc failed to trap TGF-β2. Furthermore, we found that TβRI-TβRII-Fc suppresses tumor growth and angiogenesis more effectively than TβRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-β expression. These results suggest that TβRI-TβRII-Fc may be a promising tool for targeting all TGF-β isoforms in the TME.

Keywords: Fc chimeric receptor; Fc receptor; angiogenesis; cancer therapy; epithelial-to-mesenchymal transition (EMT); ligand trap; oral squamous cell cancer; transforming growth factor-β (TGF-β); tumor microenvironment.

Figure 1.

TβRI-TβRII-Fc protein specifically traps all three TGF-β isoforms, resulting in Smad2/3/4 signal inhibition. A, schematic representation of the Fc chimeric receptors used in this study. hIgG-Fc, Fc portion of human IgG; TβRI, extracellular domain of TGF-β type I receptor; TβRII, extracellular domain of TGF-β type II receptor. B, expression of Fc chimeric proteins in HEK293T cells. Conditioned media from HEK293T cells were collected 48 h post-transfection with plasmids expressing various Fc chimeric proteins, and the concentration of each Fc chimeric protein was determined by ELISA. Culture media containing 119 ng of each Fc chimeric protein, respectively, were analyzed by immunoblotting with anti-IgG-Fc antibodies. The bands (black arrowheads) correspond to the expressed ligand traps secreted into the culture media. C, affinity of soluble chimeric receptors toward TGF-β ligands as measured by ELISA. Binding affinity of recombinant Fc chimeric proteins was assayed toward recombinant TGF-β1, TGF-β2, or TGF-β3 ligands. Values represent colorimetric changes of alkaline phosphatase substrate conjugated to anti-human-Fc antibodies and measured at 450–620 nm. D, effect of Fc chimeric proteins on Smad2/3/4 signal activation by various TGF-β isoforms in HEK-Blue TGF-β reporter cells cultured in the absence or presence of medium containing TGF-β1 (Tβ1), TGF-β2 (Tβ2), or TGF-β3 (Tβ3) (1 ng/ml) and conditioned media from HEK293T cells expressing soluble chimeric receptor, Control-Fc, TβRI-Fc, TβRII-Fc, TβRI-TβRII-Fc, or a combination of TβRI-Fc and TβRII-Fc, as described under “Experimental procedures.” The values represent Smad2/3/4 signal activation corresponding to the colorimetric changes of the Quanti-Blue substrate by SEAP at 640 nm. E, inhibitory potential of Fc chimeric proteins toward various TGF-β family members. TGF-β isoforms (1 ng/ml) and activin A (1 ng/ml) were incubated with conditioned media from HEK293T cells containing Control-Fc, TβRI-Fc, TβRII-Fc, TβRI-TβRII-Fc, or SB431542 (10 μm), followed by the measurement of Smad2/3/4 signals activated in HEK-Blue TGF-β reporter cells for 24 h. Samples incubated without soluble Fc chimeric proteins (Control) or containing the TGF-β signal inhibitor, SB431542 (10 μm) were considered as negative and positive controls, respectively. The values are represented as the -fold changes of control samples treated with the indicated ligands and inhibitor. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant.

Figure 2.

TβRI-TβRII-Fc protein suppresses the expression of genes activated by TGF-β signals in SAS oral cancer cells. SAS cells were cultured for 24 h in a mixture of conditioned media from HEK293T cells expressing various chimeric receptors (Control-Fc, TβRI-Fc, TβRII-Fc, or TβRI-TβRII-Fc) and TGF-β isoforms (TGF-β1 (Tβ1), TGF-β2 (Tβ2), or TGF-β3 (Tβ3)) (2 ng/ml) and subjected to qRT-PCR analysis for the expression of TMEPAI (PMEPA1) (A) and PAI-1 (SERPINE1) (B). The negative and positive controls were incubated without soluble Fc chimeric proteins (Control) or with 10 μm SB431542, respectively. All data are normalized to the expression of β-actin (ACTB). Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant.

Figure 3.

EMT program is activated in SAS cells in response to all TGF-β isoforms. SAS cells were cultured in the absence (Ctrl) or presence of TGF-β1 (Tβ1), TGF-β2 (Tβ2), or TGF-β3 (Tβ3) (2 ng/ml) and the TGF-β signal inhibitor, SB431542 (SB; 10 μm) for 72 h, followed by evaluation of EMT by qRT-PCR analyses (A–E), immunoblotting (F), and immunocytochemistry (G). Shown is qRT-PCR analysis for the expression of epithelial cell markers E-cadherin (A) and claudin-1 (B) and mesenchymal markers SM22α (C), vimentin (D), and fibronectin (E) in response to the ligands or inhibitor. All data are normalized to the β-actin expression. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant. F, immunoblot analysis with antibodies specific to epithelial and mesenchymal markers and α-tubulin. G, immunofluorescent images of SAS cells cultured in the presence or absence of ligands or inhibitor, followed by staining for E-cadherin (green), vimentin (red), and nuclei (blue). Representative images of cells cultured under each experimental condition are shown. Scale bar, 50 μm.

Figure 4.

TβRI-TβRII-Fc protein inhibits the EMT of SAS cells. SAS cells were cultured for 72 h in conditioned media from HEK293T cell expressing various Fc chimeric proteins (Control-Fc, TβRI-Fc, TβRII-Fc, or TβRI-TβRII-Fc) in the presence of TGF-β1 (Tβ1), TGF-β2 (Tβ2), or TGF-β3 (Tβ3) (2 ng/ml). The stimulation of EMT was evaluated by qRT-PCR (A–E) and immunocytochemistry (F). Shown is qRT-PCR analysis for the expression of epithelial cell markers E-cadherin (A) and claudin-1 (B) and mesenchymal markers SM22α (C), vimentin (D), and fibronectin (E). All data are normalized to the β-actin expression. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant. F, immunofluorescent images of SAS cells cultured in conditioned media from HEK293T cells expressing various Fc chimeric proteins (Control-Fc, TβRI-Fc, TβRII-Fc, or TβRI-TβRII-Fc) for 72 h in the presence or absence of the ligands. The cells were stained for E-cadherin (green), vimentin (red), and nuclei (blue). Representative images of cells cultured under each experimental condition are shown. Scale bar, 50 μm.

Figure 5.

TβRI-TβRII-Fc protein produced by SAS cells and secreted into the extracellular space can trap endogenous TGF-βs. SAS cells were infected with lentiviruses expressing various ligand traps (Control-Fc, TβRI-Fc, TβRII-Fc, or TβRI-TβRII-Fc). A, expression of each Fc chimeric protein in SAS cells and their secretion into culture media examined by immunoblotting with anti-human IgG-Fc antibody. The same number of cells expressing each Fc chimeric protein was seeded into a 6-well plate and cultured for 48 h. The 84 μg of total proteins (cell lysate) or 50 μl of conditioned medium per lane were loaded onto the gel. The bands (black arrowheads) correspond to the expressed ligand traps secreted into the culture media (left; conditioned medium) or present in the cell lysates (right; cell lysate). α-Tubulin was used as an internal loading control. B, TβRI-TβRII-Fc protein secreted by SAS cells into culture media interacts with endogenous TGF-βs released into the extracellular space. Conditioned media from SAS cells expressing each ligand trap cultured for 48 h were used in a HEK-Blue TGF-β reporter assay as described under “Experimental procedures,” with 10 μm SB431542 used as a positive control for total inhibition of TGF-β signals. The values represent the colorimetric changes of Quanti-Blue substrate measured at 640 nm. C, effects of ligand trap expression on SAS cell proliferation. SAS cells expressing Fc chimeric proteins were grown in 12-well plates for 72 h. Cell proliferation was determined by WST-1 assay. Error bars, S.D. **, p < 0.01; ***, p < 0.001; N.S., not significant.

Figure 6.

TβRI-TβRII-Fc protein inhibits tumor growth in vivo. SAS cells expressing Control-Fc, TβRI-Fc, TβRII-Fc, and TβRI-TβRII-Fc were injected into the left abdominal walls of BALB/c nude mice. Tumor growth was monitored for 8 weeks. Error bars, S.E. *, p < 0.05; ***, p < 0.001; N.S., not significant.

Figure 7.

TβRI-TβRII-Fc protein inhibits blood vessel formation in vivo. SAS cells expressing Control-Fc, TβRI-Fc, TβRII-Fc, and TβRI-TβRII-Fc chimeric proteins were injected into the left abdominal walls of BALB/c nude mice. The tumors were excised 63 days (9 weeks) after the inoculations and examined for vascular density. A, immunostaining for PECAM-1 (red), CD34 (green), and nuclei (blue) of sections obtained from tumors derived from cells expressing Control-Fc, TβRI-Fc, TβRII-Fc, and TβRI-TβRII-Fc proteins, respectively. Scale bar, 100 μm. Negative control represents staining with secondary antibody only. B, level of angiogenesis was quantified. Values represent the PECAM-1–positive areas (%) in the observed fields. Error bars, S.E. *, p < 0.05; **, p < 0.01; N.S., not significant.