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Co-treatment with vactosertib, a novel, orally bioavailable activin receptor-like kinase 5 inhibitor, suppresses radiotherapy-induced epithelial-to-mesenchymal transition, cancer cell stemness, and lung metastasis of breast cancer

Jiwon Choi
Jiwon Choi
, 
Jiyoung Park
Jiyoung Park
, 
Ilyoung Cho
Ilyoung Cho
 e 
Yhunyhong Sheen
Yhunyhong Sheen
   | 07 apr 2022
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Article Category: Research Article
Pubblicato online: 07 apr 2022
Pagine: 185 - 197
Ricevuto: 26 set 2021
Accettato: 28 gen 2022
DOI: https://doi.org/10.2478/raon-2022-0012
© 2022 Jiwon Choi, Jiyoung Park, Ilyoung Cho, Yhunyhong Sheen, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Breast cancer recurrence and metastasis after radiotherapy is related to the upregulation of the TGF-β signaling and vactosertib inhibits breast cancer metastasis to the lung. (A) Snapshot of gene set enrichment analysis (GSEA) results based on gene-expression profile from the Kreike dataset. The GSEA results showing as enrichment plots suggested that breast cancer recurrence after radiotherapy was significantly correlated with upregulation of the TGF-β pathway and epithelial-to-mesenchymal transition-related gene sets. (B) Scheme of the experimental mouse model of breast cancer for radiotherapy (4T1-Luc allografted BALB/c syngeneic mice) and GSEA results based on the gene expression profile from the microarray analysis of the breast cancer mouse model. Mice were injected with 4T1-Luc cells and then divided randomly into two groups (control group and radiotherapy group) when the tumor volume reached 70–100 mm3. The control group received no treatment and the radiotherapy group received whole body irradiation with 4 Gy/day for three days. At 14 days after the first day of radiotherapy, mice were euthanized and breast tumor samples were obtained from each group to perform the microarray analysis. GSEA results based on gene expression profile from the microarray analysis showed that TGF-β signaling– related genes were considerably upregulated in the radiotherapy group. (C) Changes in tumor weight in the radiation group and in the radiation and vactosertib combination therapy group compared to the control group. The weight of the tumor in each group was calculated as the relative organ weight to the mouse body weight. (D) Vactosertib inhibited breast cancer metastasis to the lung. Inhibition of lung metastasis by vactosertib was evaluated by the number of metastatic nodules in lung using India ink staining.
Breast cancer recurrence and metastasis after radiotherapy is related to the upregulation of the TGF-β signaling and vactosertib inhibits breast cancer metastasis to the lung. (A) Snapshot of gene set enrichment analysis (GSEA) results based on gene-expression profile from the Kreike dataset. The GSEA results showing as enrichment plots suggested that breast cancer recurrence after radiotherapy was significantly correlated with upregulation of the TGF-β pathway and epithelial-to-mesenchymal transition-related gene sets. (B) Scheme of the experimental mouse model of breast cancer for radiotherapy (4T1-Luc allografted BALB/c syngeneic mice) and GSEA results based on the gene expression profile from the microarray analysis of the breast cancer mouse model. Mice were injected with 4T1-Luc cells and then divided randomly into two groups (control group and radiotherapy group) when the tumor volume reached 70–100 mm3. The control group received no treatment and the radiotherapy group received whole body irradiation with 4 Gy/day for three days. At 14 days after the first day of radiotherapy, mice were euthanized and breast tumor samples were obtained from each group to perform the microarray analysis. GSEA results based on gene expression profile from the microarray analysis showed that TGF-β signaling– related genes were considerably upregulated in the radiotherapy group. (C) Changes in tumor weight in the radiation group and in the radiation and vactosertib combination therapy group compared to the control group. The weight of the tumor in each group was calculated as the relative organ weight to the mouse body weight. (D) Vactosertib inhibited breast cancer metastasis to the lung. Inhibition of lung metastasis by vactosertib was evaluated by the number of metastatic nodules in lung using India ink staining.

Figure 2

Vactosertib inhibits TGF-β/Smad signaling. (A) Fluorescence immunohistochemistry assay of p-SMAD2/3 in irradiated primary tumors and lung of 4T1-Luc allografted BALB/c syngeneic mice. Protein expression of p-SMAD2/3 was detected by fluorescence immunohistochemistry (magnification: 20×, scale bar: 50 μm). In confocal images, bright green fluorescence indicated p-SMAD2/3. Representative images are shown from three independent experiments. (B) Protein expression of p-SMAD2/3 was detected in 4T1-Luc cells by western blot analysis. Protein expression was normalized by that of GAPDH.
Vactosertib inhibits TGF-β/Smad signaling. (A) Fluorescence immunohistochemistry assay of p-SMAD2/3 in irradiated primary tumors and lung of 4T1-Luc allografted BALB/c syngeneic mice. Protein expression of p-SMAD2/3 was detected by fluorescence immunohistochemistry (magnification: 20×, scale bar: 50 μm). In confocal images, bright green fluorescence indicated p-SMAD2/3. Representative images are shown from three independent experiments. (B) Protein expression of p-SMAD2/3 was detected in 4T1-Luc cells by western blot analysis. Protein expression was normalized by that of GAPDH.

Figure 3

Vactosertib attenuates radiation - induced EMT and breast cancer cell migration. (A) Protein expression of VIMENTIN, FIBRONECTIN, SNAIL, and N-CADHERIN in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice with or without co-treatment with vactosertib. For quantification, the intensity of each marker band was normalized by that of β-ACTIN. The graph shows protein expression as a measure of fold change from the relative value of the control group. (B) Fluorescence immunohistochemistry assay of VIMENTIN, FIBRONECTIN, and SNAIL in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice. Protein expression of each marker was detected by fluorescence microscopy (magnification: 20×, scale bar: 50 μm). In confocal images, bright green fluorescence indicated VIMENTIN, FIBRONECTIN, and SNAIL. Representative images are shown from three independent experiments. (C) Protein expression of SLUG, TWIST, and E-CADHERIN in breast cancer cell lines (4T1-Luc and MDA-MB-231) was detected by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (D) Effect of vactosertib on the motility of breast cancer cells. The motility of 4T1-Luc cells was measured by wound healing assay. The left panels show the phase contrast microscopic images (magnification: 10×, scale bar: 100 μm) at the start point (0 h) and the end point (20 h). Representative images are shown from three independent experiments. The right graph shows the wound closure as a measure of fold change from the relative value of the control group.
Vactosertib attenuates radiation - induced EMT and breast cancer cell migration. (A) Protein expression of VIMENTIN, FIBRONECTIN, SNAIL, and N-CADHERIN in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice with or without co-treatment with vactosertib. For quantification, the intensity of each marker band was normalized by that of β-ACTIN. The graph shows protein expression as a measure of fold change from the relative value of the control group. (B) Fluorescence immunohistochemistry assay of VIMENTIN, FIBRONECTIN, and SNAIL in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice. Protein expression of each marker was detected by fluorescence microscopy (magnification: 20×, scale bar: 50 μm). In confocal images, bright green fluorescence indicated VIMENTIN, FIBRONECTIN, and SNAIL. Representative images are shown from three independent experiments. (C) Protein expression of SLUG, TWIST, and E-CADHERIN in breast cancer cell lines (4T1-Luc and MDA-MB-231) was detected by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (D) Effect of vactosertib on the motility of breast cancer cells. The motility of 4T1-Luc cells was measured by wound healing assay. The left panels show the phase contrast microscopic images (magnification: 10×, scale bar: 100 μm) at the start point (0 h) and the end point (20 h). Representative images are shown from three independent experiments. The right graph shows the wound closure as a measure of fold change from the relative value of the control group.

Figure 4

Vactosertib weakens radiation – induced breast cancer stem cell properties. (A) Fluorescence immunohistochemistry assay of NANOG in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice. In confocal images, green fluorescence indicates NANOG (magnification: 20×, scale bar: 50 μm). Representative images are shown from three independent experiments. (B) Protein expression of cancer stem cell markers (NANOG, OCT4, KLF4, SCA1) in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice with or without co-treatment with vactosertib. For quantification, protein expression was normalized by that of β-ACTIN. The graph shows the protein expression of each marker as a measure of fold change from the relative value of the control group. (C) Relative mRNA expression of pluripotent stem cell regulators (Nanog, Oct4, Sox2, c-Myc and Klf4) in 4T1-Luc cells. The mRNA expression level was normalized by that of Ppia mRNA. (D) Protein expression of pluripotent stem cell regulators (NANOG, OCT4, SOX2, C-MYC and KLF4) in 4T1-Luc cells was determined by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (E) Radiation-induced mammosphere formation ability in breast cancer cell lines (4T1-Luc and MDA-MB-231). Breast cancer cells were irradiated with 10 Gy with or without 30-min pretreatment with vactosertib (100 nM). After 1, 2, 6, and 24 h of incubation, cells were reseeded in ultra-low attachment dishes and cultured for seven days. The left panels show the phase contrast microscopic images of spheres after seven days of culture in the ultra-low attachment condition (magnification: 5x, scale bar: 200 μm). Representative images are shown from three independent experiments. The right graph shows the mammosphere forming efficiency (MSFE) based on the number of spheres (> 50 μm) from 24 h–incubated cells.
Vactosertib weakens radiation – induced breast cancer stem cell properties. (A) Fluorescence immunohistochemistry assay of NANOG in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice. In confocal images, green fluorescence indicates NANOG (magnification: 20×, scale bar: 50 μm). Representative images are shown from three independent experiments. (B) Protein expression of cancer stem cell markers (NANOG, OCT4, KLF4, SCA1) in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice with or without co-treatment with vactosertib. For quantification, protein expression was normalized by that of β-ACTIN. The graph shows the protein expression of each marker as a measure of fold change from the relative value of the control group. (C) Relative mRNA expression of pluripotent stem cell regulators (Nanog, Oct4, Sox2, c-Myc and Klf4) in 4T1-Luc cells. The mRNA expression level was normalized by that of Ppia mRNA. (D) Protein expression of pluripotent stem cell regulators (NANOG, OCT4, SOX2, C-MYC and KLF4) in 4T1-Luc cells was determined by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (E) Radiation-induced mammosphere formation ability in breast cancer cell lines (4T1-Luc and MDA-MB-231). Breast cancer cells were irradiated with 10 Gy with or without 30-min pretreatment with vactosertib (100 nM). After 1, 2, 6, and 24 h of incubation, cells were reseeded in ultra-low attachment dishes and cultured for seven days. The left panels show the phase contrast microscopic images of spheres after seven days of culture in the ultra-low attachment condition (magnification: 5x, scale bar: 200 μm). Representative images are shown from three independent experiments. The right graph shows the mammosphere forming efficiency (MSFE) based on the number of spheres (> 50 μm) from 24 h–incubated cells.

Figure 5

Vactosertib suppresses ROS stress generated by radiotherapy. (A) Protein and mRNA expression of NOX4 in breast cancer cell lines (4T1-Luc and MDA-MB-231). Protein expression of NOX4 in 4T1-Luc and MDA-MB-231 cells were analyzed by western blot analysis. Protein expression was normalized by that of GAPDH. The mRNA expression level of Nox4 in 4T1-Luc cells was analyzed by quantitative reverse transcription¬–polymerase chain reaction. The mRNA expression level of Nox4 was normalized by that of Ppia mRNA. (B) Protein expression of 4HNE in 4T1-Luc and MDA-MB-231 cells was determined by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (C) Summary plot of radiation-induced epithelial-to-mesenchymal transition (EMT), breast cancer stem cells, and ROS stress generation mediated by TGF-β signaling. TGF-β-induced EMT and CSC properties promote metastasis to distant organs and cancer recurrence. An ALK5 inhibitor, vactosertib, suppresses TGF-β-induced EMT, CSC properties, and ROS stress generation. Vactosertib may be an attractive strategy for prevention of cancer metastasis and recurrence in breast cancer patients undergoing radiotherapy.
Vactosertib suppresses ROS stress generated by radiotherapy. (A) Protein and mRNA expression of NOX4 in breast cancer cell lines (4T1-Luc and MDA-MB-231). Protein expression of NOX4 in 4T1-Luc and MDA-MB-231 cells were analyzed by western blot analysis. Protein expression was normalized by that of GAPDH. The mRNA expression level of Nox4 in 4T1-Luc cells was analyzed by quantitative reverse transcription¬–polymerase chain reaction. The mRNA expression level of Nox4 was normalized by that of Ppia mRNA. (B) Protein expression of 4HNE in 4T1-Luc and MDA-MB-231 cells was determined by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (C) Summary plot of radiation-induced epithelial-to-mesenchymal transition (EMT), breast cancer stem cells, and ROS stress generation mediated by TGF-β signaling. TGF-β-induced EMT and CSC properties promote metastasis to distant organs and cancer recurrence. An ALK5 inhibitor, vactosertib, suppresses TGF-β-induced EMT, CSC properties, and ROS stress generation. Vactosertib may be an attractive strategy for prevention of cancer metastasis and recurrence in breast cancer patients undergoing radiotherapy.
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