Pirfenidone suppressed triple‐negative breast cancer metastasis by inhibiting the activity of the TGF‐β/SMAD pathway

Abstract Among breast cancer patients, metastases are the leading cause of death. Despite decades of effort, little progress has been made to improve the treatment of breast cancer metastases, especially triple‐negative breast cancer (TNBC). The extracellular matrix plays an important role in tumour growth and metastasis by causing its deposition, remodelling, and signalling. As we know, the process of fibrosis results in excessive amounts of extracellular matrix being deposited within the cells. So, it will be interesting to study if the use of anti‐fibrotic drugs in combination with conventional chemotherapy drugs can produce synergistic antitumor effects. In this study, we assessed the efficacy of Pirfenidone (PFD), an FDA‐approved medication for the treatment of idiopathic pulmonary fibrosis, on TNBC cells as well as its anti‐tumour effects in xenograft tumour model. PFD inhibited in a dose‐dependent manner breast cancer cell proliferation, migration, and invasion, while promoted their apoptosis in vitro. PFD also suppressed TGF‐β‐induced activation of Smad signalling pathway and expression level of EMT‐inducing transcription factors (e.g. SNAI2, TWIST1, ZEB1) as well as the mesenchymal genes such as VIMENTIN and N‐Cadherin. On the contrary, the expression level of epithelial marker gene E‐Cadherin was up‐regulated in the presence of PFD. In vivo, PFD alone exerted a milder but significant anti‐tumour effect than the chemotherapy drug nanoparticle albumin‐bound paclitaxel (nab‐PTX) did in the breast cancer xenograft mouse model. Interestingly, PFD synergistically boosted the cancer‐killing effect of nab‐PTX. Furthermore, Our data suggest that PFD suppressed breast cancer metastasis by inhibiting the activity of the TGFβ/SMAD pathway.


| INTRODUC TI ON
Every year, there are about 1 million women worldwide are diagnosed with breast cancer, among which 15%-20% patients are estimated to be the triple-negative phenotype. 1,2 Triple-negative breast cancer (TNBC) carries a high risk of early metastasis and has a poorer prognosis than other breast cancer subtypes. 3 Therefore, one of the major goals of breast cancer research is to prevent metastasis of breast tumours. Therefore, TNBC patients are unlikely to benefit greatly from current therapies, and new therapies are urgently needed. Additionally, it is not easy to treat TNBC due to the chemo-resistant nature of TNBC. Therefore, exploring the underlying mechanisms of TNBC progression is needed for novel therapies.
Breast tumours are usually detected by manual palpation because they are more rigid than the surrounding normal tissue. This increased in tissue stiffness or matrix stiffness plays an important role in tumour progression. Organized collagen fibre arrangement is a surrogate marker for increasing matrix stiffness in the tumour microenvironment and is associated with breast tumour progression. [4][5][6] On a stiffened matrix similar to breast tumours, cells lose apical-base polarity, form weaker junctions, and invade through the basement membrane. 7 It's well known that tumour microenvironment influences tumorigenesis, cancer progression, and metastasis. 8,9 Because the metabolisms of tumour cells are more vigorous than normal cells, during the progression, breast cancer is characterized by stiffening of the cell-extracellular matrix (ECM) due to excessive deposition and cross-linking of collagen, which greatly affects tumour behaviour and fate. 10 As cancer progresses, intercellular communication and ECM interactions are closely linked to the development of metastatic and localized tumour microenvironments (TMEs) that allow metastasis to occur. 11 Breast cancer development is supported by increased collagen deposition, which provides both physical and biochemical signals that promote tumour growth and invasion. 12 The increased extracellular matrix stiffness of breast cancer promotes epithelial to mesenchymal cell transformation, leading to cell infiltration and metastasis. 13 From a biomechanics and mechanobiology perspective, there is a strong correlation between stiffening of the TME and activation of epithelial-to-mesenchymal transition pathways, tumour growth, and increased malignancy. [14][15][16][17] Therefore, it is crucial to understand whether the progression and metastasis of breast cancer can be regulated by reducing the degree of extracellular matrix fibrosis.
Pirfenidone (PFD), a pyridone derivative with anti-fibrotic and anti-inflammatory properties, was discovered and extensively tested for the treatment of lung fibrosis. 18 Since the process of metastasis is accompanied by the fibrosis of tumour tissue, so if reversing the degree of tumour tissue fibrosis can reverse tumour metastasis is deserved consideration. The mechanism of PFD is partially due to its inhibition on production/activity of transforming growth factor β (TGFβ). 19,20 On one hand, TGFβ is such a potent driver of the transition that it has been frequently used as a positive control in epithelial-mesenchymal transition (EMT) -inducing studies. 21 On the other hand, EMT is a developmentally conserved biological process by which a polarized epithelial cell loses its attachment on basement membrane while acquiring capacities of migration and invasion. As a result of the reprograming, the epithelial cell is endowed with a mesenchymal phenotype. 22,23 In addition to its essential contribution to embryonic development and tissue repair, evidence shows that EMT is also involved in tumorigenesis. 24,25 Tumour cells undergone EMT acquire the properties of invasion, infiltrate into the surrounding stroma and form a microenvironment facilitating tumour growth and metastasis. 26 The process also directly confers cancer cells apoptosis-resisting property to chemotherapy and drugs. 27 In view of the strong EMT-inducing effect of TGFβ, a drug suppressing TGFβ signalling pathway could be a potential strategy treating EMTcaused metastasis in breast cancer.
Here, we hypothesized that the combination of anti-fibrotic drug with commonly adopted chemotherapy may produce synergistic effects. Therefore, we aimed to investigate the efficacy and safety of PFD combined with nab-PTX as the treatment of TNBC. The results showed that the expression of TGFβ is overall higher in breast cancer tissues than that of in healthy controls and negatively associated with disease free survival (DSF) of the disease. In vitro, experiments showed that PFD induced breast cancer cell MDA-MB-231 apoptosis and inhibited their proliferation, migration, and invasion. These breast cancer prevention effects of PFD were partially attributed to its dose-dependent suppression on both TGFβ expression and TGFβ-stimulated Smad signalling pathways. Such anti-cancer function of PFD was further validated in vivo in mouse xenograft breast cancer model. The results from our study highlighted the potential medical value of PFD as a suppressor of EMT-mediated breast cancer metastasis.
breast cancer xenograft mouse model. Interestingly, PFD synergistically boosted the cancer-killing effect of nab-PTX. Furthermore, Our data suggest that PFD suppressed breast cancer metastasis by inhibiting the activity of the TGFβ/SMAD pathway.

K E Y W O R D S
breast cancer, EMT, metastasis, Pirfenidone, TGFβ 2 | MATERIAL S AND ME THODS

| Materials
Pirfenidone was purchased from Sigma Aldrich and resuspended in DMSO per manufacturers recommendation. Type-I and type-II TGFβ receptors antagonist LY2109761 were purchased from Med Chem Express. PTX nanoparticle for injection (albumin bound) (Aiyue®) was produced by Jiangsu Hengrui Pharmaceutical Co., Ltd.
Fetal bovine serum (FBS) was purchased from ExCell Biotech Co., Ltd.Matrigel® was obtained from Corning.

| Cell culture and treatments
Breast cancer cell lines MDA-MB-231 were purchased from the Chinese Academy of Sciences, and cultured in L15 (Zhong Qiao Xin Zhou) medium supplemented with 10% Fetal Bovine Serum (FBS) and penicillin (100 IU/ml) /streptomycin (100 μg/ml) (Gibco) in a humidified incubator at 37°C. For testing the effect of PFD on TGFβ-induced activation of Smad signalling pathway and expression of EMT-related genes, the breast cancer cells were pre-treated with 5 ng/ml TGFβ (Millipore Sigma) for 24 h, followed by addition of PFD (4 mM) or LY2109761 (2 μM) for another 24 h.     were pre-coated with Matrigel (300 μg/ml, 100 μl per chamber) (BD Biosciences) followed by performing the afore-described procedure for the migration assay.

| Western blot assay
Total protein from breast cancer cells and tumours were extracted using RIPA cell lysis solution (Solarbio) supplemented with protease inhibitor cocktail (Sigma) and quantified using BCA protein assay kit. Quantification of the protein bands were performed using ImageJ software (https://imagej.nih.gov/ij).

| Total RNA extraction and quantitative Real-Time PCR
total RNA from breast cancer cells was isolated using TRIzol™ Reagent (Life Technologies) according to the manual. Single-stranded cDNA was synthesized using a Fastquant reverse kit (Tiangen). Quantitative RT-PCR analysis was performed using the SYBR Green (TaKaLa) on Bio-Rad CFX qPCR instrument. The primers used were listed in Table 1.

| Immunofluorescence
MDA-MB-231 cells were fixed by 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking for non-specific binding sites with 5% BSA, the cells were stained with anti-TGFβ primary antibody (1:200; proteintech, Cat No:21898) overnight at 4°C. On next day, cells were washed with PBS, and the primary antibody was removed, followed by fluorescence secondary antibody.

| Immunohistochemistry
Tumours were fixed with 4% paraformaldehyde and embedded with paraffin followed by sectioned into 5 μm sections. The sections were deparaffinized, rehydrated, and heated in citrate buffers of pH 6 for antigen retrieval. After blocking with normal goat serum, the sections were incubated overnight with primary antibodies against E-Cadherin (1:30; Abcam, AB181296), Vimentin

| Enzyme-linked immunosorbent assay (ELISA)
Collagen Iα, Collagen III, and TGF-β1 were measured using a double antibody sandwich avidin-biotin complex ELISA. To measure the concentrations, ELISA kits for Collagen Iα, Collagen III, and TGF-β1(Elabscience, Houston) were used according to the manufacturer's instructions. The absorbance values of the samples were measured at 450 nm using an ELISA plate reader. The Collagen Iα, Collagen III, and TGF-β1 concentration was positively proportionate to the absorbance values; therefore, the Collagen Iα, Collagen III, and TGF-β1 concentrations in the samples were calculated using a standard curve that was plotted as absorbance versus Collagen Iα, Collagen III, and TGF-β1 concentration. All samples and standards were measured in duplicate.

| Data acquisition and re-analysis
Human TGFβ expression levels in different tumour types from TCGA database compared to adjacent normal tissues were analysed and visualized in Tumour Immune Estimation Resource (TIMER; https://cistr ome.shiny apps.io/timer/). 28 Association between expression levels TA B L E 1 Primers used for qRT-PCR in this study.

| Statistical analysis
Data are presented as the mean ± SD of three independent experiments. Statistical differences between treatment and control groups were via one-way anova test using Prism GraphPad Prism software. A p Value less than 0.05 was considered statistically significant.

| TGFβ expression is increased in breast cancer and negatively associated with DFS
Metastasis or stage IV cancer is at a stage when tumour cells spread from the original site to surrounding and distant parts of the body. Metastasis is estimated to account for approximately 90% of cancer deaths. 32 One of the well acknowledged causes of metastasis is EMT through which cancer cells acquire stem cell-like properties and exhibit enhanced apoptosis and chemotherapeutic resistances. 27,33,34 As TGFβ is known as a potent EMT driver, 21 we confirmed the association of expression of TGFβ and EMT-related genes with breast cancer carcinogenesis and metastasis in TCGA pan-cancer dataset. Indeed, TGFβ expression level is elevated in overall breast cancer tissues in comparison to the corresponding normal control ( Figure 1A). More importantly, amplification of TGFβ gene is the most frequent type of mutation associated with breast invasive carcinoma ( Figure 1B), which is in keeping with the fact that TGFβ is a strong inducer of EMT. 21 We also observed a close association between metastasis and the increased expression of numerous EMT-related genes across pan-cancer, including different subtypes of breast invasive carcinomas ( Figure 1C). Finally, an estimation of the effect of TGFβ expression on DFS of overall breast cancer patients by Kaplan-Meier method showed that a TGFβ mRNA expression level over median level is significantly (p < 0.05) associated with more reduced DFS than the patients with lower level of TGFβ ( Figure 1D).

| PFD inhibits MDA-MB-231 cells proliferation, migration, and promotes their apoptosis in vitro
PFD is an antifibrotic drug for treatment of idiopathic pulmonary fibrosis. 18 Previous study demonstrated that it exhibits a strong inhibitory effect on TNBC tumour growth induced by cancerassociated fibroblasts (CAFs). 35 At the beginning of this study, we the abrogated autocrine TGFβ signalling. 37 In addition, evidence from a previous study showed that PFD attenuates TGFβ-induced fibroblast activity and restores fibroblast-mediated collagen gel contraction and migration. 19 Therefore, we examined the effect of PFD on Collagen Iα and Collagen III production in breast cancer cells. We found that the generation of CollagenIα and Collagen III was significantly reduced after PFD administration ( Figure.

| PFD represses TGFβ -induced EMT in breast cancer cells
Activation of Smad signalling is essential in mediating TGFβinduced EMT. 21

| PFD inhibits xenografted tumour growth in vivo
We continued to evaluate the anti-tumour effect of PFD in vivo.
Immunocompromised BALB/c nude mice were given a subaxillary

| DISCUSS ION
Although treatable, breast cancer is difficult to cure when it has spread to surrounding or remote parts of the body, such as bones, lungs, brain, or liver. A recent statistic from the American Cancer Society estimates that the five-year survival rate of metastatic breast cancer is 29% for women. 1 Recently, tumour fibrosis and inflammation have been increasingly recognized as important factors which influence tumour progression and metastasis. Overexpression of TGFβ ligands in cancers at late stage has been reported to be positively associated with the aggressive and metastatic phenotype. 42,43 We noticed that one of the mechanisms of the anti-fibrotic drug, Pirfenidone, for treating idiopathic pulmonary fibrosis is its inhibitory effect on TGFβ signalling pathway. 44 Accordingly, we hypothesized that it might be used for counteracting the pro-progression role of TGFβ in breast cancer.
Indeed, evidence from the earlier studies shows that PFD enables Concomitantly, the synergistical effect of PFD on the anti-tumour efficacy of doxorubicin is also via depletion of CAFs. 35 Notably, data from these studies demonstrated that although PFD synergistically inhibited primary tumour growth and lung metastasis in combination with doxorubicin in TNBC xenograft mouse model, little or no effect by this anti-fibrotic drug monotherapy was observed on primary tumour growth and lung metastasis. 35 We think During tumour progression, cell proliferation and fibrosis take into account the stiffness and aggressiveness of the tumour as well as lymphatic invasion. While EMT or fibrosis or EMT combined with fibrosis appears to contribute to tumour metastasis, the exact contribution is unknown. Solid tumours are highly heterogeneous and consist of not only cancer cells but also many other TME resident cells, including fibroblasts, endothelial cells, epithelial cells, and infiltrating immune cells. One of the novel aspects of our current study is that unlike the existing studies aiming on the role of PFD in non-cancer resident cells, e.g., suppressing collagen production by CAFs, 35,45 we had focused on exploring its direct actions on tumour cells. It is well known that TGFβ is overproduced in almost all the advance human tumours and is positively associated with tumour growth, invasion, and metastasis. 47,48 The autocrine TGFβ signalling has been demonstrated as essential in promoting survival of MCF-7 cells but not untransformed human mammary epithelial cells through activating Erk but inhibiting p38 signalling pathways. 37 Our data showed that PFD treatment represses autocrine TGFβ produc- Nevertheless, we are excited to see PFD synergistically boosted cancer-killing effect of nab-PTX in vivo. This could be partially due to its strong repression on TGFβ expression as well as TGFβinduced EMT. TGFβ is known as a potent inducer of EMT which is a driving force of cancer metastasis. 33 Mechanistically, TGFβ induces EMT through phosphorylating Smad2 and Smad3. 49,50 The activated Smads induce subsequently the expression of transcriptional repressors, including SNAI2, ZEB1/2 and TWIST etc. 21 These are well-established E-Cadherin repressors through inducing hypermethylation and histone deacetylation at the promoter. 51 Along with the loss of E-Cadherin, the mesenchymal phenotype signature genes such as N-Cadherin and VIM are up-regulated instead. Such epithelial phenotype to mesenchymal phenotype conversion liberates the tight adherent junction-confined tumour cells and facilitates their free migration to the surrounding tissues. 33 We saw TGFβ-activated Smad signalling pathway and most of the EMT-inducing transcription factors as well as the epithelial and mesenchymal signature genes were significantly suppressed or reversed by PFD, which was a more pronounced phenotype than direct killing of tumour cells.

AUTH O R CO NTR I B UTI O N S
qin dai Luo: Conceptualization (equal); data curation (equal); writing -original draft (equal). lin xian Zeng: Data curation (equal); formal analysis (equal). ling shu Zhang: Methodology (equal); resources F I G U R E 8 Mechanistic diagram of PFD actions on TGFβ-induced EMT program TGFβ binds to a tetrameric complex of TβRI and TβRII on cell membrane, which consequently phosphorylates Smad2 and Smad3 proteins. Smad2 and Smad3 proteins are then dimerized and combined further with Smad4. The resultant heterotrimer translocates into the nucleus and activates expression of EMT-inducing transcription factors, including SNAI2, ZEB1, TWIST1, etc. PFD strongly suppresses TGFβ-induced phosphorylation of Smad-2 and -3 and thereby inhibits the downstream EMT program. Meanwhile, PFD also represses TGFβ transcription and thus reduces the autocrine TGFβ in breast cancer cells. The elements of the diagram were adopted from library of icons in Reactome. 52 (equal). hong dao Li: Investigation (equal); methodology (equal). mei zhi Cheng: Investigation (equal); methodology (equal). yun Wang: Project administration (equal); resources (equal). hua jin Long: Project administration (equal); supervision (equal). quan zu Hu: Funding acquisition (equal); project administration (equal); supervision (equal). qi shi Long: Investigation (equal); resources (equal).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.