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

N-[[4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline (vactosertib) was synthesized by Dr. D. K. Kim (Ewha Womans University, Seoul, Korea).

Gene-expression raw data of breast cancer patients from the Kreike dataset were downloaded from ‘R2: Genomics Analysis and Visualization Platform (https://r2.amc.nl)’.²³ Gene Set Enrichment Analysis (GSEA) was performed to evaluate correlation between radiation response gene sets and recurrence of breast cancer.

The 4T1-Luc mouse breast cancer cells and MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection. The 4T1-Luc cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) (GenDEPOT, Katy, TX, USA) containing hydroxyethyl piperazineethanesulfonic acid (HEPES) sodium salt (Sigma-Aldrich, St. Louis, MO, USA), sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 5% fetal bovine serum (FBS) (GenDEPOT, Katy, TX, USA), and 100X penicillin-streptomycin solution (GenDEPOT, Katy, TX, USA). MDA-MB-231 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco, Grand Island, NY, USA) containing HEPES sodium salt, sodium bicarbonate, sodium pyruvate solution (Gibco, Grand Island, NY, USA), 5% FBS, and 100X penicillin-streptomycin solution. Cells were grown at 37°C in 5% CO². Cells were seeded in culture plates and maintained with serum-reduced (0.2% FBS) medium for 16 h for starvation. Cells were pretreated with vactosertib (100 nM) for 30 min and then irradiated with 10 Gy. After 24 h of incubation, in vitro assays were conducted.

All experimental procedures were approved by the Animal Care Committee of Ewha Womans University and complied with the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, WA, USA). Female BALB/c mice were purchased from Central Lab Animal Inc. (Korea). Four to five mice were housed per cage at 21°C with 50% humidity and a 12-h light cycle. A total of 4 X 10⁴ 4T1-Luc cells were injected into the fourth mammary fat pad of female BALB/c mice. When the tumor volume reached 70-100 mm³, mice were randomly divided into three groups: control, radiation, and vactosertib + radiation group (n = 8/group). The control group received no treatment, the radiation group received whole body irradiation with 4 Gy/day for three days, and the vactosertib + radiation group was treated with vactosertib 2.5 mg/kg p.o. for two weeks and concurrently received whole body irradiation 4 Gy/day for three consecutive days.

Mice were sacrificed, and 15% India ink (Hardy Diagnostics) in PBS was injected into the lung through the bronchi using a 1 ml syringe. After washing the stained lungs, the number of metastatic nodules on the surface of the left lobe of the lung was counted.

Total RNA from breast cancer pieces (100 mm³) was isolated using Trizol solution (Invitrogen, Carlsbad, CA, USA) and purified using a RNeasy column (Qiagen, Chatsworth, CA, USA). Aliquots of the RNA samples from each group of mice were pooled (n=8). Experiments were performed on Affymetrix GeneChip Mouse Gene 2.0 ST arrays that cover 26,515 genes (RefSeq-Entrez gene count; Affymetrix, Santa Clara, CA, USA). Arrays were scanned using a GeneChip™ Scanner 3000 7G controlled by GeneChip^® Operating Software (GCOS, Affymetrix). Scanned data were analyzed with GeneChip^® Operating and Expression Console Software (Affymetrix) and GenPlex v3.0 (ISTECH, Goyang, Korea). The signal intensity of each gene from different arrays was normalized according to the total intensity of all genes in each array to compare the results of separate hybridization experiments. Normalized signals of each array were compared to determine the fold induction or reduction in the gene expression between samples.

For detecting p-SMAD2/3, SNAIL, VIMENTIN, FIBRONECTIN, NANOG, OCT4, SOX2, c-MYC and KLF4 by fluorescence immunohistochemistry assay, formalin-fixed and paraffin-embedded sections of mouse tissues were dewaxed in an OTTIX bath (Diapath, Martinengo, Italy) and blocked with solution containing 5% BSA (GenDEPOT, Katy, TX, USA) and 0.1% Triton 100X (Merk, Darmstadt, Germany). Slides were incubated with a primary antibody mixture of anti-p-SMAD2/3 mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SNAIL rabbit IgG (Cell Signaling, Beverly,

MA, USA) and anti-VIMENTIN mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); a mixture of anti-FIBRONECTIN mouse IgG (BD Biosciences, San Diego, CA, USA), anti-NANOG rabbit IgG (Abcam, Cambridge, MA, USA) and anti-OCT4 rabbit IgG (Abcam, Cambridge, MA, USA); or a mixture of anti-SOX2 rabbit IgG (Abcam, Cambridge, MA, USA), anti-c-MYC mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-KLF4 rabbit IgG (Abcam, Cambridge, MA, USA). A mixture of Alexa 488-conjugated anti-rabbit IgG (Cell Signaling, Beverly, MA, USA) F(ab’) fragments were used for visualization of SNAIL, NANOG, OCT4, SOX2 and KLF4 proteins. A mixture of Alexa 488-conjugated anti-mouse IgG (Cell Signaling, Beverly, MA, USA) F(ab’) fragments were used for visualization of p-SMAD2/3, VIMENTIN, FIBRONECTIN, and c-MYC proteins. Slides were counterstained with 4’6-diamidino-2-phenylindole (DAPI). Fluorescence was visualized using an Axio observer inverted microscope (Carl Zeiss, Jena, Germany).

Mouse tissues and cultured cells were homogenized in RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM Na³VO⁴, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche, Mannheim, Germany)] for 30 min on ice. Lysates were centrifuged at 13,000 rpm and 4°C for 10 min. The protein content of the lysates was determined by the BCA protein assay kit (Thermofisher Scientific, Waltham, MA, USA). Proteins (5 – 10 μg) were separated by 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose (Whatman, Middlesex, UK) or polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin (BSA) (GenDEPOT, Katy, TX, USA) or 5% skim milk (BD Biosciences, San Diego, CA, USA) and incubated at room temperature for 1 h with specific primary antibodies, as listed in Supplementary Table A.1. Following washes in 1X Tris-buffered saline including 0.1% Tween^®20 detergent, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 30 min. Proteins were detected by an enhanced chemiluminescence kit (ATTO, Tokyo, Japan). Band intensities were analyzed using a LAS-3000 densitometer (Fujifilm, Tokyo, Japan).

Cells were seeded in 6 cm plates and incubated for 24 h for attachment. Cells were incubated with serum-reduced (0.2% FBS) medium for 16 h for starvation and irradiated with 10 Gy with or without 30-min pretreatment with 100 nM vactosertib. After 1, 2, 6, and 24 h of incubation, we harvested the adherent cells with trypsin-EDTA and reseeded the cells in ultra-low attachment dishes. Cells were incubated in DMEM/F-12 (1:1) (GenDEPOT, Katy, TX, USA) supplemented with B-27™ Supplement (50X) (Gibco Laboratories, Grand Island, NY, USA), 10 ng/ml bFGF (Invitrogen, Carlsbad, CA, USA), and 10 ng/ml EGF (Sigma-Aldrich, St. Louis, MO, USA). To measure mammosphere-forming efficiency (MSFE), the number of spheres (> 50 μm) was counted after one week. MSFE was determined as the number of spheres divided by the original number of cells seeded and is presented as %.

The 4T1-Luc cells were seeded in 6 cm culture plates and incubated for 24 h for attachment. Cells were incubated with serum-reduced (0.2% FBS) medium for 16 h for starvation and irradiated with 10 Gy with or without 30-min pretreatment with vactosertib (100 nM). After 24 h of incubation, 2.1 X 10⁴ cells were seeded in each well of a cell culture insert (Ibidi, Munich, Germany) in a 35 mm plate. When cells reached 80% confluence, the insert was removed and 1 mL medium containing 0.2% FBS was added. After 20 h of incubation, the wound area was observed using an Axio observer inverted microscope (Carl Zeiss, Jena, Germany). The closure of the wound area was calculated as a percentage of the initial wound area.

Blood from each group of mice was collected after 2 weeks treatment of vactosertib. The collected blood was centrifuged at 1,500 rpm and 4°C for 15 min and the supernatant was discarded. The cell pellet was resuspended in extraction solution (0.1 M ethylene-diamine-tetraacetic acid (EDTA), 0.2 M NaCl, 0.05 M Tris-HCl (pH 8.0), 0.5% SDS, 50 mg/ mL RNase). Proteolytic enzyme K (100 mg/mL, EM Science, Gibbstown, NJ, USA) was mixed with the suspension and the suspension was incubated for 2–3 h at 55°C. Phenol/chloroform (1:1) was mixed with the suspension and the sample was centrifuged at 1,500 rpm at room temperature (RT) for 5 min. The supernatant was transferred into a new tube and chloroform/isoamyl alcohol (24:1) was added; the mixture was centrifuged at 1,500 rpm at RT for 5 min. The supernatant was transferred into a new tube and mixed with 3 M sodium acetate (pH 5.2) and ethanol; the mixture was shaken until the DNA precipitated. A glass pipette that had been formed into a loop over a Bunsen burner was used to capture the DNA. The DNA was washed in 70% ethanol and dried by vacuum. DNA was dissolved in ddH²O to measure the purity and then analyzed by quantitative reverse transcription - polymerase chain reaction (qRT-PCR).

Total RNA was isolated from mouse tissues and cultured cells using Ambion’s TRizol reagent. cD-NAs were synthesized from 1 μg of total RNA using M-MLV reverse transcriptase (BIONEER, Oakland, CA, USA) and random primers (Promega, Madison, WI, USA). Synthesized cDNAs were amplified for PCR using SYBR Green real-time quantitative RT-PCR reagent (Applied Biosystems, Foster City, CA, USA). The primers are listed in Supplementary Table A.2. To amplify cDNA a Step-One Real-time PCR system (Applied Biosystems, Foster City, CA, USA) was used.

Data are expressed as mean ± standard error of the mean (SEM). Data represent means of three independent experiments performed in triplicate. Statistical values were determined by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. Asterisks are used to indicate statistical significance (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively).

We first performed an in silico analysis to examine the possible mechanism for radiotherapy-induced TGF-β signaling promoting recurrence in breast cancer. Kreike et al. constructed a gene expression profile predictive for local recurrence after breast-conserving surgery with postoperative radiotherapy from 343 breast cancer patients.²³ We analyzed the gene expression signature in the breast cancer patients with local recurrence after radiotherapy compared with that of patients without local recurrence by GSEA. The GSEA results showed that breast cancer recurrence after radiotherapy was significantly correlated with upregulation of the TGF-β pathway and EMT (Figure 1A). This data suggest a mechanism by which radiotherapy-induced TGF-β signaling contributed cancer recurrence and metastasis via EMT and cancer cell stemness.

Figure 1

To investigate the anti-metastatic effect of vactosertib, we established a 4T1-Luc allografted BALB/c syngeneic mouse model. GSEA of microarray data of the breast cancer mouse model revealed that TGF-β signaling–related genes were upregulated when the breast cancer model mice were irradiated (Figure 1B). The radiation dose (4 Gy X 3) we set significantly reduced the weight of tumor in mice, and the administered dose of vactosertib (2.5 mg/kg) had no effect on other organs (Figure 1C, Supplementary Figure A). Radiomonotherapy in the 4T1-Luc allografted mouse model considerably increased the metastatic nodules in lung and circulating tumor cell DNA compared with the control group. However, the combination treatment of vactosertib with radiation significantly relieved the risk of lung metastasis compared with radiomonotherapy (Figure 1D). These results suggest that vactosertib combined with radiation may be an effective breast cancer treatment by destroying breast cancer cells and suppressing lung metastasis.

We next examined whether the ALK5 inhibitor vactosertib blocked radiation-induced TGF-β signaling by detecting the phosphorylation of SMAD2/3 both in vivo (primary tumors of irradiated 4T1-Luc allografted BALB/c syngeneic mice) and in vitro (4T1-Luc cells). p-SMAD2/3 expression in the primary tumor as measured by fluorescence immunohistochemistry (IHC) assay was induced by irradiation and subsequently reduced by co-treatment of vactosertib (Figure 2A). In line with the in vivo data, co-treatment of vactosertib significantly reduced the level of p-SMAD2/3 in irradiated 4T1-Luc cells (Figure 2B). Since breast cancer spreads preferentially to the lung²⁴, we also investigated the effect of vactosertib in lung tissue of the breast cancer mouse model. The expression of p-SMAD2/3 in lung tissue was elevated after irradiation but co-treatment of vactosertib attenuated this effect. However, the ALK5 inhibitory effect of vactosertib was lower in lung tissue than in the primary tumor (Figure 2A).

Figure 2

Considering that EMT is a significant factor of cancer metastasis, we analyzed EMT markers in vitro and in vivo. During EMT, upregulation of Smad2/3 induces the expressions of mesenchymal markers, such as the intermediate filament protein Vimentin and the extracellular matrix component Fibronectin.^4,10 SNAIL acts as a key factor in developing oncogenic EMT and enhancing stem-like properties by suppressing epithelial genes, including E-cadherin and activating mesenchymal genes, including N-cadherin.¹¹ Therefore, we conducted western blot analysis and fluorescence IHC assay to examine whether vactosertib inhibits the induction of VIMENTIN, FIBRONECTIN, SNAIL, and N-CADHERIN by radiation in vivo. We found that the protein levels of VIMENTIN, FIBRONECTIN, SNAIL, and N-CADHERIN increased in irradiated primary tumors of 4T1-Luc allografted mice but this increase was significantly abolished by co-treatment of vactosertib (Figure 3A). Consistent with the western blot results, the protein levels of VIMENTIN, FIBRONECTIN, and SNAIL in fluorescence IHC analysis were induced by radiation in primary tumors of 4T1-Luc allografted mice but they were reduced with co-treatment of vactosertib with radiation (Figure 3B).

Figure 3

We also conducted in vitro experiments in several breast cancer cell lines to examine the effect of radiation and vactosertib on EMT markers. In 4T1-Luc cells, radiation up-regulated the mRNA expression of Vimentin, Fibronectin, Snail, and N-cadherin but co-treatment of vactosertib considerably reduced this effect (Supplementary Figure B1). In line with the result in 4T1-Luc cells, the protein levels of the EMT markers increased in irradiated MDA-MB-231 cells and decreased by co-treatment of vactosertib (Supplementary Figure B2). In addition to SNAIL, SLUG and TWIST are also EMT-associated transcription factors that repress the expression of E-cadherin and stimulate the expression of N-cadherin.¹¹ Therefore, we examined the protein levels of SLUG and TWIST to investigate whether vactosertib blocks the transcriptional activators in developing EMT. In vitro, radiation up-regulated the protein levels of SLUG and TWIST but co-treatment of vactosertib reduced their expression both in 4T1-Luc and MDA-MB-231 cells (Figure 3C). Furthermore, we compared the protein level of E-CADHERIN to examine whether vactosertib relieves the radiation-induced mesenchymal traits. In contrast to results with the EMT-associated transcription factors (SLUG, TWIST), the protein level of E-CADHERIN in irradiated 4T1-Luc and MDA-MB-231 cells was decreased compared with that in the control group but it was recovered to a level similar to the control group with co-treatment of vactosertib (Figure 3C).

We next examined the motility of the irradiated breast cancer cells with or without vactosertib treatment by wound healing assay. The wound closure in irradiated 4T1-Luc cells was significantly increased compared with that in non-irradiated 4T1-Luc cells; however, this increase was abolished in irradiated 4T1-Luc cells under the treatment of vactosertib (Figure 3D). These results suggest that radiotherapy promotes metastasis and invasion of cancer cells by inducing mesenchymal traits in vivo, and vactosertib may be a promising candidate for an anti-metastatic drug by reducing radiotherapy-induced EMT.

Radiotherapy can cause metastasis and invasion of cancer cells by promoting EMT and CSC properties, which is mediated by TGF-β signaling.⁴ Cancer cells undergoing EMT obtain CSC properties and spread to distant organs.^6,9 Therefore, we investigated whether radiation up-regulates the pluripotent stem cell regulators that play a significant role in the self-renewal of CSCs and whether co-treatment with vactosertib down-regulates these factors. The protein levels of pluripotent stem cell regulators, such as NANOG and Yamanaka’s factors (octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Cellular myelocytomatosis oncogene (C-MYC), Kruppel-like factor 4 (KLF4)), were increased in irradiated primary tumor of 4T1-Luc allografted mouse but decreased by co-treatment with vactosertib as shown by fluorescence IHC analysis (Figure 4A, Supplementary Figure C1). Furthermore, western blot analysis showed that the protein levels of NANOG, OCT4, KLF4, and SCA1 (Stem cell antigen 1) were significantly elevated in irradiated primary tumors of 4T1-Luc allografted mice but co-treatment of vactosertib alleviated these elevations (Figure 4B). Consistent results with in vivo data were reproduced in the in vitro assays conducted in 4T1-Luc and MDA-MB-231 cells. Radiation induced the mRNA expression of Nanog and Yamanaka’s factors (Oct4, Sox2, c-Myc, Klf4), whereas co-treatment of vactosertib with radiation reduced the mRNA expression of pluripotent stem cell regulators in 4T1-Luc cells (Figure 4C). In addition, the protein levels of the markers increased by radiation and decreased by combinatorial treatment of vactosertib in 4T1-Luc cells and MDA-MB-231 cells as shown by western blot analysis (Figure 4D, Supplementary Figure C2). We also conducted sphere forming assays in breast cancer cell lines to examine whether radiation induces CSC properties such as mammosphere forming ability and if co-treatment of vactosertib abolishes this effect. The results showed that radiation considerably increased mammosphere forming efficiency (MSFE) but vactosertib weakened radiation-induced MSFE in both 4T1-Luc and MDA-MB-231 cells (Figure 4E). Taken together, these results indicate that vactosertib attenuates radiation-induced CSC properties in vitro and in vivo by repressing the pluripotent stem cell regulators.

Figure 4

Radiotherapy damages cancer cells through generating ROS. ROS is also a mediator of radiotherapy-induced EMT with the acquisition of cell stemness via stimulation of transcription factors including SNAIL, Zinc finger E-box-binding homeobox (ZEB), and TWIST that are promoted by various signaling pathways including the TGF-β pathway.⁶ Therefore, we conducted in vitro experiments and examined several ROS markers to investigate whether vactosertib obstructs oxidative stress generated by radiation. NOX4, one of NADPH oxidases (NOX family members), is activated by the Smad-dependent TGF-β pathway and produces ROS.²⁵ Western blot confirmed that the protein level of NOX4 was increased in 4T1-Luc and MDA-MB-231 cells after irradiation but it decreased significantly by co-treatment of vactosertib. The mRNA expression of Nox4 also showed a similar pattern in irradiated 4T1-Luc cells in qRT-PCR analysis (Figure 5A). Another biomarker of oxidative stress, 4-hydroxy-2,3-nonenal (4-HNE), is a product of lipid peroxidation.¹⁹ 4-HNE is one of the most powerful reactive aldehydes and stimulates the TGF-β pathway.²⁶ We observed that the elevated protein level of 4-HNE in irradiated breast cancer cell lines decreased by concomitant treatment with vactosertib in western blot analysis (Figure 5B). These data suggest that vactosertib may mitigate radiotherapy-induced EMT and cancer cell stemness by inhibiting ROS stress, which is also generated by radiotherapy.

Figure 5