A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers

Abstract

Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-beta (TGF-beta) in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-beta receptor kinase inhibitors (TRKI) are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-beta, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-beta-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-beta, including TGF-beta-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-beta, whereas it reduces colony formation by cells that are growth-promoted by TGF-beta. However, SB-431542 has no effect on a cell line that failed to respond to TGF-beta. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-beta-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-beta.

Figure 1

Effect of SB-431542 in blocking TGF-β signaling. (A) Immunoprecipitation. 293T, FET, and A549 cells were treated with 12.5 ng/ml TGF-β1 in the presence of SB-431542 (SB) for 1 hour. Cell lysates were subjected to immunoprecipitation with anti-Smad2 and anti-Smad3 polyclonal antibodies, and the immunoprecipitates were analyzed by Western blot analysis with anti-Smad4 antibody (top). Cell lysates were subjected to Western blot analysis with anti-phospho Smad2, anti-Smad2, anti-Smad3, and anti-Smad4 antibodies (bottom section). An equal amount of protein loading was verified by Western blot analysis with anti-β-actin monoclonal antibody. Each experiment was repeated four times. (B) Nuclear translocation of Smad proteins was analyzed by separating cytoplasmic (C) and nuclear (N) fractions after treating A549 cells with TGF-β and/or SB-431542 for either 30 or 90 minutes. Smad proteins were detected by Western blot analysis using antibodies against Smad2, Smad3, and Smad4. Complete fractionations of cytoplasmic and nuclear proteins were verified by Western blot analysis with antibodies against Rho-GDI and PARP. (C) Blockade of TGF-β-induced nuclear import of Smad2 and Smad3 by SB-431542 in HaCaT cells as detected by immunofluorescence. Cells were serum-starved and treated with TGF-β in the presence of SB-431542 for 1 hour and processed for immunofluorescence using either anti-Smad2 or anti-Smad3 polyclonal antibodies. Fluorescence was visualized by antirabbit antibody conjugated to Cy3 using a Zeiss Axioplan fluorescence microscope. Nuclei of the same cells were stained with DAPI. Each experiment was repeated four times.

Figure 2

Effect of SB-431542 in blocking TGF-β-induced reporter gene activation. (A) Reporter assay. FET cells were transiently transfected with CMV-β-gal and p3TP-Lux or (CAGA)9MLP-Luc reporter plasmids. Cells were then treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (0.5, 3, and 10 µM) for 22 hours. Luciferase activity was normalized to β-gal activity, and the relative luciferase activity was expressed as the mean ± SD of triplicate measurements. (B and C) Reporter assay. HepG2 cells were transiently transfected with CMV-β-gal and p21-Luc (B) or PAI-1-Luc (C) plasmids. Cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (0.5, 2, and 10 µM) for 22 hours. Cell lysates were analyzed for both luciferase and β-gal activities as mentioned above. Each experiment was repeated four times.

Figure 3

SB-431542 blocks TGF-β-induced PAI-1, fibronectin (FN), and p21 CIP1 expression. (A) Mv1Lu cells were serum-starved for 16 hours and treated with 5 ng/ml TGF-β1 in the presence of SB-431542 for 6 hours. Cell lysates were analyzed by Western blot analysis with anti-PAI-1 and anti-FN antibodies. (B) RIE cells were treated as above and cell lysates were analyzed by Western blot analysis with anti-PAI-1 (Torrey Pines Biolabs, Inc., Houston, TX) and anti-p21 (Santa Cruz Biotechnology, Inc.) antibodies. An equal amount of protein loading was verified by Western blot analysis with anti-β-actin antibody. Each experiment was repeated three times.

Figure 4

SB-431542 inhibits TGF-β-induced growth suppression and apoptosis. (A) [³H]thymidine incorporation assay. FET cells were treated with TGF-β1 in the presence of SB-431542 for 26 hours and then treated for an additional 2 hours with [³H]thymidine. Cells were fixed in TCA, washed, and lysed in 0.2 N NaOH. Radioactivity incorporated into TCA-insoluble [³H]thymidine was measured by scintillation counting. Individual data points are the mean ± SD of triplicate determinations. (B) Cell counting assay. Mv1Lu cells were cultured in DMEM containing 10% FBS and treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for a total of 6 days. Cells were counted every day and the cell numbers are plotted. Individual data points are the mean ± SD of triplicate determinations. (C) FACScan analysis. FET, RIE, and Mv1Lu cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 for 35 hours. Cells were collected and evaluated for DNA content by flow cytometric analyses. Results are expressed as the mean ± SD of three replicate values of the percentage of cells in different phases of the cell cycle. (D) Quantitative cell death ELISA. HepG2 cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 for 24 hours. Cell lysates were analyzed by cell death ELISA as described in the Materials and Methods section. An individual data point is a representative of the mean ± SD of three individual measurements. Each experiment is repeated three times. (E) DNA laddering. HepG2 cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (2 µM) in serum-free medium for 46 hours. Cells were lysed and fragmented DNA was isolated from the cell lysates. Each DNA was loaded onto 1.6% agarose gel and visualized by staining with ethidium bromide.

Figure 4

SB-431542 inhibits TGF-β-induced growth suppression and apoptosis. (A) [³H]thymidine incorporation assay. FET cells were treated with TGF-β1 in the presence of SB-431542 for 26 hours and then treated for an additional 2 hours with [³H]thymidine. Cells were fixed in TCA, washed, and lysed in 0.2 N NaOH. Radioactivity incorporated into TCA-insoluble [³H]thymidine was measured by scintillation counting. Individual data points are the mean ± SD of triplicate determinations. (B) Cell counting assay. Mv1Lu cells were cultured in DMEM containing 10% FBS and treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for a total of 6 days. Cells were counted every day and the cell numbers are plotted. Individual data points are the mean ± SD of triplicate determinations. (C) FACScan analysis. FET, RIE, and Mv1Lu cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 for 35 hours. Cells were collected and evaluated for DNA content by flow cytometric analyses. Results are expressed as the mean ± SD of three replicate values of the percentage of cells in different phases of the cell cycle. (D) Quantitative cell death ELISA. HepG2 cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 for 24 hours. Cell lysates were analyzed by cell death ELISA as described in the Materials and Methods section. An individual data point is a representative of the mean ± SD of three individual measurements. Each experiment is repeated three times. (E) DNA laddering. HepG2 cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (2 µM) in serum-free medium for 46 hours. Cells were lysed and fragmented DNA was isolated from the cell lysates. Each DNA was loaded onto 1.6% agarose gel and visualized by staining with ethidium bromide.

Figure 5

SB-431542 abrogates TGF-β-induced EMT in both NMuMG and PANC-1 cells. (A) NMuMG cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for 24 hours. Cells were stained with anti-E-cadherin and anti-ZO1 antibodies, and visualized by antimouse antibody conjugated to Cy3. Phase contrast images were recorded at x200 magnification. (B) Cell lysates were prepared from NMuMG cells as treated above, and were analyzed by Western blot analysis with anti-E-cadherin, anti-ZO1, and anti-β-catenin antibodies. An equal amount of protein loading was verified by Western blot analysis with anti-β-actin antibody. (C) PANC-1 cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for 60 hours as described before, fixed, and incubated with anti-E-cadherin antibody, and visualized by antimouse antibody conjugated to Cy3 as mentioned above. Phase contrast images were recorded at x200 magnification. (D) PANC-1 cells were treated with TGF-β1 in the presence of SB-431542 for 24, 48, and 60 hours. Cell lysates were analyzed by Western blot analysis with anti-E-cadherin antibody. An equal amount of protein loading was verified by Western blot analysis with anti-β-actin antibody. Each experiment was repeated three times with similar results.

Figure 6

SB-431542 abrogates TGF-β-induced migration, invasion, wound healing, and COX-2 expression. (A) Migration assay. A549 cells were treated with TGF-β, and SB-431542 cells were allowed to migrate through a 8-µM pore in transwells. Cells that migrated through the pores were stained and counted. The data were represented as the mean ± SD of three independent wells. (B and C) Invasion assays. A549 cells were treated as above and allowed to pass through collagen-coated (B) and Matrigel-coated (C) membrane in transwells. Cells that invaded through the filter were stained and counted. Individual data point is represented as the mean ± SD of three independent wells. (D) Wound healing. Wounded MDA-MB-231 cells were treated with 3 ng/ml TGF-β1 for 30 hours in the presence of SB-431542. Phase contrast images were shown. All of these experiments were repeated four times. (E) Growth inhibition of MDA-MB-231 cells by TGF-β was determined by cell counting (left) and thymidine incorporation (right) assays as described before. (F) Effect of SB-431542 on TGF-β-induced COX-2 expression. Oncogenic Ras-inducible rat intestinal epithelial (RIE-iRas) cells were treated with 5 mM IPTG and/or 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for 24 hours. Cell lysates were analyzed by Western blot analysis with anti-COX-2 (Santa Cruz Biotechnology) and anti-Pan-Ras (Oncogene Research Products) antibodies. An equal amount of protein loading was verified by Western blot analysis with anti-β-actin antibody. This experiment was repeated three times with similar results.

Figure 7

SB-431542 blocks TGF-β-induced VEGF secretion. A549 (A) and HT29 (B) cells were treated with 5 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for 24 hours. Supernatant media were analyzed for VEGF secretion by ELISA. (C) ELISA for TGF-β1. A549, VMRC-LCD, and HT29 cells express similar amounts of TGF-β1. Cells were treated with SB-431542 for 24 hours. Supernatant media were activated and used for TGF-β1 assay. Individual data point in either VEGF or TGF-β assay is a representative of the mean ± SD of three individual measurements. Each experiment is repeated at least three times.

Figure 8

Differential effects of SB-431542 on anchorage-independent growth of different cell lines. (A, C, and E) Soft agarose assay. A549, VMRC-LCD, and HT29 cells were plated and treated with SB-431542 (10 µM) for 2 weeks. Pictures of colonies grown on soft agarose were shown. (G) Colonies were counted by automated colony counter and the data are represented as the mean ± SD of three individual plates. (B, D, and F) Cell counting assay. A549 (B), VMRC-LCD (D), and HT29 (F) cells were treated with 7 ng/ml TGF-β1 in the presence of SB-431542 (10 µM) for a total of 6 days. Cells were counted and the results are plotted. Individual data points are the mean ± SD of triplicate determinations. Each experiment was repeated three times.

Figure 9

Blockade of tumor-permissive effects of TGF-β by TRKIs represents a potential therapeutic approach. Genetic changes that are associated with colorectal cancer progression are shown [31] and used as an example. TGF-β has biphasic effects during tumorigenesis. It plays a tumor-suppressive role by its ability to inhibit growth, induce apoptosis, and inhibit genomic instability in normal epithelium and in the early stage of tumor progression. During cancer progression, high levels of TGF-β promote tumor growth in an autocrine and/or paracrine manner by inducing angiogenesis, invasion, and metastasis. The mechanism by which TGF-β promotes tumor progression represents a novel potential application of these inhibitors as therapeutic agents in advanced human cancers.