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www.nature.com/npjbcancer All rights reserved 2374-4677/15 ARTICLE OPEN Activin-A signaling promotes epithelial–mesenchymal transition, invasion, and metastatic growth of breast cancer 1 1 2 1 Mohsin Bashir , Surekha Damineni , Geetashree Mukherjee and Paturu Kondaiah BACKGROUND: Activins belong to the transforming growth factor-β (TGF-β) superfamily of cytokines. Although the role of TGF-β in cancer progression has been highly advocated, the role of activin signaling in cancer is not well known. However, overexpression of activin-A has been observed in several cancers. AIMS: The gene expression proﬁle indicated higher expression of Activin-A in breast tumors. Hence the aim of this study was to evaluate the status and role of Activin signaling pathway in these tumors. METHODS: Microarray analysis was performed to reveal gene expression changes in breast tumors. The results were validated by quantitative PCR and immunohistochemical analysis in two independent sets of normal and tumor samples. Further, correlation of activin expression with survival and distant metastasis was performed to evaluate its possible role in tumor progression. We used recombinant activin-A, inhibitors, overexpression, and knockdown strategies both in vitro and in vivo, to understand the mechanism underlying the protumorigenic role of this signaling pathway. RESULTS: We report that activin-A signaling is hyperactivated in breast cancers as indicated by higher activin-A, phosphoSMAD2, and phosphoSMAD3 levels in advanced breast cancers. Bone morphogenetic proteins and molecules involved in this signaling pathway were downregulated, suggesting its suppression in breast cancers. Activin-A expression correlates inversely with survival and metastasis in advanced breast cancers. Further, activin-A promotes anchorage-independent growth, epithelial–mesenchymal transition, invasion, angiogenesis, and stemness of breast cancer cells. We show that activin-A-induced phenotype is mediated by SMAD signaling pathway. In addition, activin-A expression affects the tumor-forming ability and metastatic colonization of cancer cells in nude mice. CONCLUSIONS: These results suggest that activin-A has a critical role in breast cancer progression and, hence, targeting this pathway can be a valuable strategy in treating breast cancer patients. npj Breast Cancer (2015) 1, 15007; doi:10.1038/npjbcancer.2015.7; published online 12 August 2015 INTRODUCTION has also been advocated. Activin-A has been shown to induce a protumorigenic phenotype by facilitating tumor cell–tumor micro- Activins are members of the transforming growth factor-β (TGF-β) environment interactions, leading to increased levels of cytokine superfamily of cytokines, which includes TGF-β, activins, nodal, production and cell motility. inhibins, growth and differentiation factors (GDFs), and bone 1,2 The role of activins in breast cancer progression is not well morphogenetic proteins (BMPs). Activin-A binds to type II studied. Earlier, downregulation of activin signaling in breast transmembrane serine–threonine kinase receptor (ActRIIA or tumors has been reported. On the contrary, increased serum ActRIIB), which in turn activates type I receptor (ActRIB), leading level of activin has been reported in women with breast cancers. to phosphorylation of SMAD2/SMAD3. On phosphorylation, We performed global differential expression of genes in breast SMAD2/3 forms a complex with SMAD4, which then translocates cancers with respect to normal tissue samples. The data revealed to the nucleus. In the nucleus, Smad2/3/4 complex along with INHBA to be one of the highly upregulated genes with no other co-factors regulate the expression of a large number of appreciable change in the expression of any TGF-β isoforms. It is genes. Activins were initially shown to have an important role in important to note that TGF-β, which has been implicated in the gonadal function. Subsequently, they have been shown to have 5 6 progression and metastatic spread of breast cancers, also an important role in gonadal function, embryonic development, 7 8 functions through the same set of downstream effectors, Smad2 pancreatic function, bone formation, mammary gland develop- 9 10 11 and 3. Hence, it becomes important to evaluate the role of activin- ment, cell proliferation, maintenance of embryonic stem cells, A in breast cancer progression. In this study we show that activin- and immune response. A signaling pathway is activated in breast cancers and provide Activin-A has been shown to be overexpressed in metastatic 13 14 15 data that suggests its active role in breast cancer progression. prostate cancer, stage 4 colorectal cancers, lung cancer, 16 17 hepatocellular carcinomas, and pancreatic cancers. Activin signaling has also been shown to promote aggressiveness of MATERIALS AND METHODS esophageal squamous cell carcinoma cells and skin tumori- genesis. Interestingly, inhibin-α (activin antagonist)-deﬁcient Reagents mice have been shown to develop gonadal and adrenal cortical Recombinant human activin-A (338-AC-010) and activin-A antibody tumors. The involvement of activin signaling in cancer cachexia (AF338) were purchased from R&D Systems (Minneapolis, MN, USA); 1 2 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India and Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore, India. Correspondence: P Kondaiah (firstname.lastname@example.org) Received 27 January 2015; revised 25 March 2015; accepted 4 May 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Activin-A promotes breast cancer M Bashir et al phosphoSMAD2 (3101 and 9510) from Cell Signaling Technology (Boston, (both from ATCC) were seeded for 12 and 18 days, respectively. The MA, USA); SMAD3 (1735), E-cadherin (1702), N-cadherin (2019), and α- experiments were performed in triplicates for three independent times. smooth muscle actin (1184-1) from Epitomics (CA, USA); Vimentin (V2258) and ﬂuorescein isothiocyanate-conjugated phalloidin (P5282) from Sigma Reporter assay (St Louis, MO, USA); phosphoSMAD3 (ab52903) and BMP2 (ab14933) from Matrix metalloproteinase-2 (MMP2) and VEGF reporter constructs were Abcam (Cambridge, MA, USA); vascular endothelial growth factor-A (VEGF-A) gifts from Etty Benveniste, University of Alabama, and Gail C. Fraizer, Kent (M7273) from Dako (Denmark); and PE-CD44 (560533)/PE-cy7 CD24 (555428) State University, respectively. Luciferase reporter assays were carried out from BD (NJ, USA). The antibodies were used at a dilution of 1:100 or 1:200 for using promega kit (E4030, Madison, WI, USA) following the manufacturer’s immunohistochemistry and most of the antibodies were used at a dilution of protocol. Renilla luciferase under a TK promoter was used as an internal 1:1,000 for western blotting. Small hairpin RNA against activin-A is from control. The experiment was performed two independent times in Dharmacon (Lafayette, CO, USA) and small hairpin RNA for SMAD3 was a kind triplicates. gift from Dr Lalage Wakeﬁeld. INHBA was overexpressed in mammalian expression pcDNA3.1 vector. The Student’s t-test was conducted to calculate the P-value for individual experiments and the median values plotted. Migration/invasion assay For scratch assay, cells were cultured in a six-well plate till a conﬂuent Tissue samples and immunohistochemistry monolayer was formed. A scratch was made through the center of the well with a tip and images were recorded before and after activin-A treatment. Normal and tumor tissue samples were procured after obtaining informed Transwell migration and invasion assays were performed following the consent of patients who underwent surgery at the Kidwai Memorial manufacturer’s protocol, using BD BioCoat Matrigel (NJ, USA) invasion/ Institute of Oncology, Bangalore. The study has been approved by the control chambers. The experiments were performed in triplicates and Institute Ethical Committee of Kidwai Memorial Institute of Oncology. repeated two independent times. The images were recorded under a Details about tissue samples and expression analysis are listed with the microscope with a ﬁxed camera (×2 magniﬁcation) and cells were counted Gene Expression Omnibus accession number GSE40206. All the patient from several random ﬁelds. details including histopathology data for expression of estrogen receptor, progesterone receptor, Her2/neu, tumor stage/grade, size, and lymph Zymography node status were documented for each case. The normal tissue samples were taken from non-tumor-bearing individuals, whereas all the tumor Equal number of cells was seeded and the supernatant was collected after 24 h of serum starvation (stable clones) or activin-A treatment (parental tissues belonged to grade III category. Staining was performed using the cells). The sample was given a spin and equal volume was loaded into a standard method of deparafﬁnization and gradual rehydration, followed by non-denaturing gelatin containing gel (0.1%). The gel was incubated for 8– heat-mediated antigen retrieval in tris-EDTA or citrate buffer. The sections 10 h followed by Coomassie staining. The images were recorded using a were further treated with 3% hydrogen peroxide in methanol to block Uvipro platinum gel imaging system (Cambridge, UK). endogenous peroxidase activity. Primary antibody incubation was done at room temperature for 2 h followed by incubation with an appropriate horseradish peroxidase-conjugated secondary antibody for 30 min. Detec- Mice experiments 5 5 tion was carried out using supersensitive polymer-horseradish peroxidase For tumor xenograft growth, 5 × 10 of MDA-MB-231 and 20 × 10 MCF-7 immunohistochemistry/diaminobenzidine detection system. The staining for cells were injected into the ﬂank region of 4- to 5-week-old female nude different proteins was performed using the same set of tumor and normal mice. The animals were allocated by the animal facility and 10 animals samples. Analysis was performed by the pathologist determining the were placed in each group randomly. Tumor formation was followed for 8 staining intensity of each section (with minimum of 10% cells positive) and and 10 weeks, respectively. The tumors were excised and the weight of the grading from 0 (no staining) to 3+ (highest intensity). The list of primers tumors was plotted. To study metastatic spread of tumor cells, 20 × 10 used in the study is provided in Supplementary Table S1. cells were injected into the tail vein of nude mice (5 animals each group). Mice were killed after 10 weeks of injection and various organs were analyzed for nodule formation. The size measurement was carried out on a Proliferation and soft agar assay Gatan Microscopy Suite (Gatan, Pleasanton, CA, USA), using a line scan Proliferation of MCF-7 and MDA-MB-231 cells (from ATCC, Manassas, VA, that gives the number of pixels in a particular direction. The t-test was USA) was assayed using bromodeoxyuridine cell proliferation assay kit performed using Graphpad prism software (La Jolla, CA, USA) to evaluate (Calbiochem, San Diego, CA, USA) following the manufacturer’s protocol the statistical signiﬁcance of the data and a P-value above 0.05 was (24 h). For soft agar assay, 3,000 and 5,000 cells of MDA-MB-231 and MCF-7 considered signiﬁcant. All the experiments were performed in accordance Table 1. qPCR analysis of normal and breast tumor samples shows that components and regulators of activin-A signaling pathway are deregulated in breast tumors Gene Function Median fold change P-value INHBA A member of TGFβ superfamily of cytokines, activating SMAD2/3 signaling pathway 11.31 o0.0001 INHA Binds to ACTRII and acts as a negative regulator of activin signaling No change — ACVR2A Cell surface serine–threonine kinase receptor of activin signaling 2.44 0.0007 SMAD2 Acts as a mediator of activin signaling pathway 2.02 0.0453 FST Binds directly to activin and prevents its binding to the receptor − 1.81 0.0050 TGFBR3 Acts as a co-receptor for inhibin binding to ACTRII − 6.49 o0.0001 IGSF1 Binds to activin directly and prevents its binding to the receptor − 2.75 0.0001 IGSF10 Binds to activin directly and prevents its binding to the receptor − 25.63 o0.0001 BMP2 A member of the TGF-β superfamily of cytokines, activating SMAD1/5 signaling pathway − 5.2 0.0003 BMP3 Binds ACTRIIB, however, has no deﬁned type I receptor − 5.81 0.0254 BMP6 A member of the TGF-β superfamily of cytokines, activating SMAD1/5 signaling pathway − 3.07 0.0012 RGMA Acts as a co-receptor in BMP signaling − 8.11 o0.0001 SMAD1 Acts as a signal transducer of BMP signaling − 2.60 0.0217 ZNF521 Acts as a co-transcription factor in association with SMAD1 − 2.65 o0.0001 GDF10 Also known as BMP3B and binds to ACTRII with no deﬁned type I receptor − 13.36 o0.0001 GREMLIN1 Binds to BMPs directly and prevents their binding to the receptor 2.25 0.0060 TWSG1 Forms a ternary complex with BMPs along with chordin 2.12 0.0019 Abbreviations: BMP, bone morphogenetic protein; qPCR, quantitative PCR; TGF-β, transforming growth factor-β. npj Breast Cancer (2015) 15007 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Activin-A promotes breast cancer M Bashir et al with the institutional guidelines established for the Animal Facility at IISc This differential expression of the INHBA was not dependent on and the study has approval from the Animal Ethics Committee of IISc. the category of breast tumors such as estrogen receptor, progesterone receptor, or Her2 status. We used an independent set of 15 normal (from non-cancer individuals) and 30 breast RESULTS cancer samples (grade 3), to analyze the expression of various Activin-A signaling is active in breast cancer components of activin-A signaling pathway. As summarized in Microarray study done in our laboratory revealed INHBA and Table 1 and shown in Figure 1a, we observed upregulation of various other genes involved in the activin signaling pathway to various components of activin-A signaling pathway including be differentially expressed in breast cancers (invasive ductal INHBA, SMAD2, and activin type II receptor. Interestingly, some of carcinoma) compared with normal tissue samples, suggesting the negative regulators of activin signaling pathway such as activation of this signaling pathway (Supplementary Figure S1). follistatin, β-glycan, IGSF1, and IGSF10 showed downregulation in INHBA (p<0.0001) SMAD2 (p=0.0453) FST (p=0.0050) BMP2 (p=0.003) 6 4 1.0 4 0.5 4 2 0.0 2 0 –0.5 –2 –1.0 0 –2 –4 –1.5 –6 –2 –4 –2.0 Normal Tumor Normal Tumor Normal Tumor Normal Tumor (i) Activin-A (p<0.0001) (ii) pSMAD2 (p=0.0034) (iii) pSMAD3 (p<0.0001) (iv) BMP2 (p<0.0001) 2.5 4 4 4 2.0 3 3 1.5 2 2 1.0 1 1 0.5 0 0 0 Normal Tumor Normal Tumor Normal Tumor Normal Tumor Figure 1. Expression of activin and correlation with breast tumor progression. (a) Quantitative PCR analyses of INHBA, SMAD2, FST, and BMP2 expression in breast tumors compared with that in normal breast tissues. It is worth noting the signiﬁcant increase in the expression of INHBA (activin-A) and Smad2. (b) Immunohistochemistry of activin-A (i), pSMAD2 (ii), pSMAD3 (iii), and BMP2 (iv) in normal and breast tumor sections. Breast tumors show higher levels of activin-A, pSMAD2, and pSMAD3, whereas normal samples have higher levels of BMP2 compared with tumor samples. Each graph below shows the intensity score of individual normal and tumor sample on a scale of 0 to 3. The statistical signiﬁcance is indicated in the representative graph. (c) GOBO gene set analysis shows that INHBA expression inversely correlates with overall survival (OS) of high-grade breast cancer patients (ii). In addition, INHBA expression correlates inversely with the distant metastasis-free survival (DMSF) of breast cancer patients (iii and iv). (d) GOBO box plot expression analysis shows that expression levels of negative regulators of activin signaling pathway, FST and TGFβR3, decrease progressively from grade 1 to grade 3. © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007 Intensity value Log Fold change Tumor Normal Intensity value Log Fold change Log Fold change Intensity value Intensity value Log Fold change 2 Activin-A promotes breast cancer M Bashir et al All tumors ; Gene=INHBA Grase3 tumors ; Gene=INHBA All tumors ; Gene=INHBA Grase3 tumors ; Gene=INHBA (i) (ii) (iii) (iv) 100 100 100 90 90 90 80 80 80 80 70 70 70 60 60 60 50 50 50 50 p=0.35749 p=0.04868 p=0.01937 p=0.00737 40 40 40 40 30 30 30 20 20 20 10 10 10 [–4.16,–0.278] [–4.16,–0.278] [–4.16,–0.278] [–4.16,–0.278] [–0.278,0.278] [–0.278,0.278] [–0.278,0.278] [–0.278,0.278] 0 0 [0.450,2.778] 0 [0.450,2.778] [0.450,2.778] [0.450,2.778] 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Years Years Years Years All tumors : INHBA : Grade, p=0.08558 All tumors : FST : Grade, p<0.00001 All tumors : TGFBR3 : Grade, p<0.00001 239 677 495 239 677 495 239 677 495 1 1 –1 –1 –2 –2 –1 –3 –3 –2 –4 –4 12 3 1 2 3 1 2 3 INHBA FST TGFBR3 Figure 1. Continued. breast tumors compared with normal samples. Furthermore, phosphoSMAD2 and phosphoSMAD3 levels compared with normal although TGF-β1 expression was upregulated, TGFβRII showed a tissues (Figure 1b, ii and iii). In addition, in the same set of tumors, very signiﬁcant downregulation in tumors, compared with normal BMP2 staining showed a reduced expression pattern (Figure 1b, iv), tissues (Supplementary Figure S2). We also analyzed various compared with normal tissues. We also analyzed the expression of available breast cancer gene expression data sets (oncomine.org). inhibin in some normal and breast tumor samples (data not shown) In accordance with our study, analysis of these data sets shows and found that most of the tumor samples have very low levels of that activin-A signaling components are frequently deregulated in inhibin compared with normal tissues. This suggests that over- breast cancers (Supplementary Table S2). TGF-β/activin signaling expression of INHBA results in reduced inhibin expression, possibly has been shown to be opposed by BMP signaling pathways in due to INHβA homo-dimerization. In conclusion, our data shows 1,25,26 development and disease. In congruent with this, BMP that breast tumors have higher levels of activin-A and low levels of isoforms 2, 3, and 6, and various other genes involved in BMP various BMPs, suggesting activation of activin signaling pathway in signaling such as SMAD1, ZNF521, RGMA, and Gremlin1 were found these tumors. to be downregulated. To conﬁrm our results, we performed Further, we also analyzed various publicly available breast immunohistochemistry with another set of 13 normal and 29 cancer clinical data sets (co.bmc.lu.se/gobo/gsa.pl). As represented tumor samples. As shown in Figure 1b (i), most of the tumors have in Figure 1c (ii), the analysis shows that INHBA expression is higher levels of activin-A compared with normals tissues. Activation negatively correlated with the overall survival of grade 3 breast of activin signalling results in phosphorylation of SMAD2 and cancer patients. However, there was no signiﬁcant correlation SMAD3. In good correlation, breast tumors showed increased between INHBA expression and overall patient survival, when all npj Breast Cancer (2015) 15007 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited OS (%) Log2 Expression OS (%) DMSF (%) DMSF (%) Activin-A promotes breast cancer M Bashir et al 1.5 2.0 1.0 1.5 0.8 1.5 1.0 1.0 0.6 1.0 0.4 0.5 0.5 0.5 0.2 0.0 0.0 0.0 0.0 Control Act-A Control Overexpression Control Act-A Control Knockdown (i) (ii) (iii) (iv) MCF-7 MDA-MB-231 (p=0.02) (p=0.005) (p=0.002) 1.5 2.0 1.5 1.5 1.5 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 Control Act-A Control Overexpression Control Act-A Control Knockdown (i) (ii) (iii) (iv) MCF-7 MDA-MB-231 Figure 2. Activin-A promotes anchorage-independent growth but not proliferation of breast cancer cells. (a) Bromodeoxyuridine (BrdU) incorporation assay following activin-A treatment of MCF7 (i) and MDA-MB-231 (iii) cells. Overexpression of INHBA in MCF7 cells (ii) and knockdown of INHBA by small hairpin RNA (shRNA) in MDA-MB-231 cells (iv). MCF-7 cells but not MDA-MB-231 cells show mild inhibition in proliferation on activin-A treatment. (b) Treatment of MCF-7 cells with activin-A results in reduced colony growth (i), but MCF-7 clones overexpressing activin-A show higher colony-forming ability (ii). Treatment of MDA-MB-231 cells with activin-A in soft agar does not show any effect (iii) and stable knockdown of activin-A in MDA-MB-231 cells results in a signiﬁcant decrease in colony formation (iv). the grades are taken together (Figure 1c, i). Further, the analysis of activin-A signaling on proliferation and anchorage-independent revealed that INHBA expression correlates negatively with the growth of these cell lines in monolayer and anchorage- distant metastasis-free survival of breast cancer patients independent culture conditions. On activin-A treatment MCF-7 (Figure 1c, iii and iv). Interestingly, expression of two important cells showed a decrease, whereas MDA-MB-231 cells showed no change in proliferation as assessed by bromodeoxyuridine negative regulators of activin signaling pathway, FST and TGFBR3, incorporation assay (Figure 2a, i and iii). We cloned and is progressively reduced from grade 1 to grade 3 (Figure 1d). This overexpressed INHBA (activin-A) in MCF-7 and knocked down its suggests that activin signaling may progressively increase from expression using small hairpin RNA in MDA-MB-231 cells grade 1 to grade 3, even without any signiﬁcant change in the (Supplementary Figure S7). Stable overexpression or knockdown expression of INHBA. On the other hand, expression of FST and of activin-A in MCF-7 and MDA-MB-231 cells, respectively, did not TGFBR3 is positively correlated with the distant metastasis-free affect their proliferation (Figure 2a, ii and iv). In contrast to activin- survival of breast cancer patients (Supplementary Figure S3). All A treatment, which resulted in a decrease in proliferation of MCF-7 these results suggest that activin-A signaling pathway has an cells, activin A-expressing clones may represent cell populations important role in the metastasis and, hence, survival of breast that have overcome the growth inhibitory action of activin-A. In cancer patients. addition, given the heterogeneous nature of cell lines, there may be a subpopulation that is refractory to growth inhibition by Activin-A promotes anchorage-independent growth of breast activin. It is likely to be that during the process of selection, cells cancer cells that are sensitive to growth inhibition by activin get eliminated In order to understand the role of activin-A in breast tumors, we and cells refractory to the growth inhibitory signals of activin-A used MCF-7 and MDA-MB-231 cells as models to study the get selected, which mimics the actual tumor progression. response of these cell lines to activin-A. Both these cell lines are Interestingly, even treatment of activin-A-overexpressing MCF-7 responsive to activin-A treatment (addition of recombinant cells with TGF-β did not result in any inhibition in their human activin-A) as determined by an increase in phosphoSMAD2 proliferation (Supplementary Figure S8). On activin-A treatment, levels (Supplementary Figure S6). Although these two cell lines MCF-7 cells showed a decrease, whereas there was no signiﬁcant represent different categories of breast tumors, one being change in number of colonies formed by MDA-MB-231 cells, in estrogen receptor positive and the other being triple negative, soft agar (Figure 2b, i and iii). MDA-MB-231 cells are considered to high low there was no difference in the expression of activin in these be aggressive with enriched CD44 and CD24 population. tumors, as mentioned in the previous section. Most importantly, Hence, addition of Activin-A may not result in any further increase we chose these cells based on their INHBA expression levels for in number of colonies. Intrestingly, MCF-7 cells overexpressing overexpression and knockdown studies. We evaluated the effect activin-A showed an increase, whereas its knockdown showed a © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007 Absorbance 450-595 Fold change Absorbance 450-595 Fold change Absorbance 450-595 Fold change Fold change Absorbance 450-595 Activin-A promotes breast cancer M Bashir et al Control Control Act-A Control Overexpression Act-A Control Knockdown N-Cad E-Cad N-Cad Vim Vim α-SMA α-SMA β-Actin β-Actin (i) (ii) (iii) (iv) E-cadherin α-SMA Stress fibre Figure 3. Activin regulates epithelial–mesenchymal transition (EMT) markers in breast cancer cells. (a) Western blot analyses showing activin-A treatment (i) or its stable overexpression (ii) in MCF7 cells, or activin A treatment (iii) or knockdown (iv) in MDA-MB-231 cells regulates EMT markers (it is noteworthy that MDA-MB-231 cells do not express E-cadherin). (b) Confocal microscope images of activin-A-treated MCF7 cells show cytoskeletal changes marked by decreased E-cadherin, increased α-smooth muscle actin (SMA) and stress ﬁbre formation (phalloidin- ﬂuorescein isothiocyanate staining). Activin-A promotes migration and invasion of MDA-MB-231 breast decrease in the number of colonies formed by MCF-7 and MDA- cancer cells MB-231 cells, respectively (Figure 2b, ii and iv). In conclusion, activin-A promotes anchorage-independent growth of cancer cells EMT has been associated with migratory and invasive behavior of in a context-dependent manner. cancer cells. Hence, we studied whether activin-A has any effect on migratory and invasive behavior of MDA-MB-231 cells. Wound- healing (scratch) assay showed that activin-A treatment of MDA- Activin-A induces epithelial–mesenchymal transition in breast cancer cells MB-231 cells promotes their migration, whereas knockdown of activin-A results in decreased migration of these cells (Figure 4a, i Epithelial–mesenchymal transition (EMT), a process in which and ii). We also performed transwell migration assay and observed epithelial cells lose some of their characteristics and acquire that treatment of activin-A increases, whereas stable knockdown properties of mesenchymal cells, has been proposed to have a of activin-A decreases migration of MDA-MB-231 cells (Figure 4b, i pivotal role in invasion/metastasis of cancer cells. Unlike TGF-β, and ii). To assess whether activin-A affects the invasive behavior of the role of activin-A in this regard is unknown. Western blot MDA-MB-231 cells, we performed Matrigel invasion assay and analysis shows that treatment or overexpression of activin-A leads observed that treatment with activin-A increases, whereas knock- to a decrease in the expression of E-cadherin and an increase in down of activin-A reduces invasive potential of MDA-MB-231 cells the expression of various mesenchymal markers in MCF-7 cells (Figure 4c, i and ii). Next, we performed zymography to analyze (Figure 3a, i and ii). Interestingly, we could also observe a MMP2 and MMP9 activity. The results show that activin-A mesenchymal phenotype in activin-A-overexpressing MCF-7 cells treatment increases, whereas its knockdown decreases MMP2 (Supplementary Figure S9). EMT is also marked by stress ﬁber formation associated with changes in the cytoskeleton. Confocal activity in MDA-MB-231 cells (Figure 4d, i and ii). Further, we used microscopy indicated that treatment with activin-A leads to Luciferase-conjugated MMP2 promoter and assayed its inducibility by activin-A. Activin-A treatment of HEK-293T cells transfected downregulation of E-cadherin, induction of α-smooth muscle actin, and stress ﬁbre formation in MCF-7 cells (Figure 3b). Similar with the reporter construct showed a signiﬁcant increase in the results were observed on treatment or knockdown of activin-A in promoter activity (Figure 4e). These results suggest that activin-A MDA-MB-231 cells (Figure 3a, iii and iv). In conclusion, similar to can activate transcription of MMP2 and promote migration and TGF-β, activin-A also is an inducer of EMT in cancer cells. invasion of breast cancer cells. npj Breast Cancer (2015) 15007 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Act-A Control Activin-A promotes breast cancer M Bashir et al Many recent studies have shown that SMAD3 has an impor- half a million activin-A knockdown MDA-MB-231 and control cells 28,29 tant role in TGF-β-induced EMT, migration, and invasion. (optimal cell number generally used is two million) subcuta- Hence, we used a speciﬁc inhibitor (SIS3) and small hairpin neously in nude mice. Although 7 out of 10 animals formed RNA-mediated stable knockdown of SMAD3 in MDA-MB-231 tumors in control mice, only 3 out of 10 animals injected with cells. The results show that ablation of SMAD3 activity leads to activin-A knockdown cells could lead to tumor formation blockade of activin-A-induced EMT and invasion in these cells (Figure 5a, ii). Signiﬁcant differences in the weight of the tumors (Figure 4f–i). Together, these results suggest that activin-A- were also observed. We also performed immunohistochemistry on induced phenotype is dependent on SMAD3 signaling in MDA- the tumors formed by MCF7 cells (Figure 5b). We observed that MB-231 cells. MCF7 overexpressing activin-A tumors have higher Ki-67 percen- tage (~80%) as compared with control tumors (~50%). In addition, Activin-A promotes tumorigenicity of breast cancer cells staining for various EMT markers conﬁrmed the mesenchymal state of tumors formed by activin-A-overexpressing MCF-7 cells. The process of tumor formation is highly complex. TGF-β signaling Recruitment of blood vessels or de-novo formation of blood has been shown to have an important role in the establishment of 30–32 vessels can inﬂuence the tumor growth in vivo. Hence, we tumors in vivo. Hence, we wanted to investigate how activin- investigated the expression of VEGF, which is known to promote A would affect the tumor-forming ability of cancer cells in vivo. angiogenesis. Activin-A treatment or its stable overexpression in We injected activin-A-overexpressing MCF7 cells in the ﬂank of MCF-7 cells induced VEGF expression (Figure 5c, i and ii). Further, immunocompromised mice and followed till the tumors reached we performed Luciferase reporter assay to study the regulation of to a prominent size. Our results show that activin-A- overexpressing MCF-7 cells have better tumor-forming ability in VEGF promoter by activin-A. Activin-A treatment of HEK-293T comparison with control cells (Figure 5a, i). We also injected only transfected with Luciferase-conjugated VEGF promoter showed (i) Control Act-A (0 hr) Control Act-A (24 hr) (ii) Control Knockdown (0 hr) Control Knockdown (24 hr) (p=0.003) (p=0.005) (p=0.02) (p=0.01) 4 2.5 1.5 1.5 2.0 1.0 1.0 1.5 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Control Act-A Control Knockdown Control Act-A Control Knockdown (i) (ii) (i) (ii) Figure 4. Activin promotes migration and invasion of breast cancer cells. (a and b) Activin-A treatment (i) increases, whereas its stable knockdown (ii) decreases migration of MDA-MB-231 cells as shown by the scratch assay and transwell migration assay, respectively. (c) Matrigel invasion assay shows that activin-A treatment (i) increases, whereas its knockdown (ii) decreases invasion of MDA-MB-231 cells. (d) Zymography using MDA-MB-231 cell supernatant shows that activin-A treatment increases (i) and its knockdown decreases (ii) active matrix metalloproteinase-2 (MMP2) levels. (e) Luciferase reporter assay in HEK 293T cells shows that activin-A regulates MMP2 promoter activity. (f and g) Activin-A regulation of epithelial–mesenchymal transition (EMT) markers is inhibited using SMAD3 inhibitor or by stable knockdown of SMAD3 using small hairpin RNA (shRNA) in MDA-MB-231 cells. (h and i) Activin-A-induced increase in invasion in MDA-MB-231 cells is abrogated in the presence of SMAD3 inhibitor or stable knockdown of SMAD3. © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007 Fold Change Fold Change Fold Change Fold Change Activin-A promotes breast cancer M Bashir et al Control Act-A Control Knockdown (i) (ii) Control Act-A Act-A – + – + Act-A – + – + SIS3 inh –– ++ SMAD3 shRNA –– ++ N-Cad N-Cad Vim Vim α-SMA α-SMA p-SMAD3 SMAD3 β-Actin β-Actin 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Act-A – + – + Act-A – + – + SIS3 inh – – ++ shSMAD3 – – ++ Figure 4. Continued. that activin-A induces activity of VEGF promoter signiﬁcantly enrichment of breast cancer stem-like cells (Figure 5f, i). In (Figure 5d). We also performed tail vein injections with activin-A- addition, treatment of MCF-7 and MDA-MB-231 cells with activin-A overexpressing MCF-7 cells. Even though we did not ﬁnd a leads to an increase in expression of several stemness markers signiﬁcant difference in the number of nodules formed, we (Figure 5f, ii). Taken together, these results suggest that activin-A observed that activin-A-overexpressing cells formed much bigger has multiple effects on tumor establishment and progression nodules as compared with the control cells (Figure 5e, iii and iv). in vivo. This suggests that growth of colonized MCF-7 cells is promoted by activin-A expression. Tumor-forming ability of cancer cells and DISCUSSION aggressiveness of various cancers has been associated with the 33,34 presence of cells having stem-like phenotype. Activin-A has Activin-A, a member of the TGF-β superfamily, binds to its cognate been shown to regulate expression of various stemness markers receptor and activates SMAD2/3 signaling pathway. However, 35,36 such as Nanog, Sox2, and Oct4 in various cells. Hence, to unlike TGF-β, the role of activin in cancer is not well known. Here investigate whether activin-A expression affects stemness of we show that high-grade breast tumors have activation of activin- high breast cancer cells, we analyzed population of CD44 and A signaling pathway. Interestingly, we did not observe much low CD24 cells. Fluorescence-activated cell sorting analysis of signiﬁcant change in the expression of TGF-β ligands. We also activin-A-overexpressing MCF-7 cells and activin-A knockdown demonstrate that many components and regulators of activin- MDA-MB-231 cells shows that activin-A expression leads to signaling pathway are deregulated, favoring the activation of this npj Breast Cancer (2015) 15007 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Fold Change Fold Change RLU Activin-A promotes breast cancer M Bashir et al signaling pathway in these tumors. Our results show that breast towards a factor is modulated by the presence or the absence of tumors have higher phosphoSMAD2 and phosphoSMAD3 levels as many other molecules, which is further governed by cellular compared with normals, which shows that this signaling pathway context. Here we show that BMPs and various components, as well is active in these tumors. Cellular response towards various as regulators, of this signaling pathway are deregulated, favoring environmental stimuli is a highly complex process. Response suppression of BMP signaling in breast tumors. In addition, breast 0.4 0.4 (p= 0.0048) (p= 0.0698) 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 Control Overexpression Control Knockdown (i) (ii) α-SMA H&E Ki-67 E-Cad VEGF *** Control Act-A Control Overexpression VEGF-A β-Actin (i) (ii) Control Act-A Figure 5. Activin promotes tumorigenicity of breast cancer cells in immunocompromised mice. (a) Stable overexpression of activin-A in MCF-7 enhances (i), whereas stable knockdown of activin-A in MDA-MB-231 cells (ii) reduces their tumor-forming ability in nude mice. Shown below are the representative images of the tumors formed s.c. (b) Immunohistochemical analysis of MCF-7-overexpressing tumors shows EMT-like changes and higher ki-67 index. (c) Treatment of MCF-7 cells with activin-A (i) or overexpression of activin-A in MCF-7 cells (ii) results in increased levels of vascular endothelial growth factor-A (VEGF-A). (d) Luciferase reporter assay in HEK 293T cells shows that activin-A regulates VEGF promoter activity. (e) Tail vein injection of activin-A-overexpressing MCF-7 cells shows better tumor-forming ability in the livers of nude mice (i). Normal is shown here as the reference from an animal without injection of any cells. The panel below (ii) shows the hematoxylin and eosin (H&E) staining of the liver tissue sections. The graph (iii) and (iv) shows number and size of nodules formed in the liver per animal. high/ low (f) CD44 CD24 ﬂuorescence-activated cell sorting (FACS) analysis of activin-A-overexpressing or knockdown cells shows that activin-A inﬂuences stemness of breast cancer cells (i). Quantitative PCR analysis shows that treatment of MCF-7 or MDA-MB-231 cells with recombinant activin-A induces various markers of stemness (ii). © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007 Overexpression Control Tumor weight (gm) Tumor weight (gm) RLU Activin-A promotes breast cancer M Bashir et al (i) Normal Vector Overexpression Tumor Normal (ii) Normal Tumor Normal Vector Overexpression p=0.07 p=0.01 15 40 (iii) (iv) 20 Control Overexpression Control Overexpression Figure 5. Continued. in majority (~98%) of the breast cancers. tumors revealed signiﬁcant downregulation of SMAD1, which acts With the disease as a downstream mediator of the BMP signaling pathway. progression, these cells may become refractory to the growth Downregulation of SMAD1 in breast tumor cells will abrogate inhibitory effect of activin-A signaling, as exempliﬁed in prostate any BMP signaling (locally or at a distant site) in these cells. BMP cancer. Activin has been shown to inhibit proliferation of LNCaP signaling pathway has been shown to have an antagonistic role to and DU145 (low- and moderate-grade PCa) but not PC3 cell line TGF-β/activin signaling in various physiological and pathological (high grade). Interestingly, circulating levels of activin-A were 37,38 conditions. In addition, recent studies have shown that BMP demonstrated to increase signiﬁcantly in metastatic prostate and 39 41,42 signaling has a tumor-suppressive function in cancers. Hence, breast cancers. In addition, SMAD deletions are observed only hyperactivation of activin-A signaling and loss of BMP signaling in 1–2% of breast cancers. In other words, this signaling pathway may have a critical role in the clinical outcome of breast cancers. remains intact in most of the breast cancers. Given the abundant We show that INHBA expression correlates negatively with the expression of activin-A in tumor cells and the presence of an intact overall survival of high-grade breast tumors and metastasis-free signaling pathway, it is reasonable to believe that these cells have survival of breast cancer patients. In agreement with these results, acquired resistance to the growth inhibitory effect of activin-A. FST and TGFBR3 expression confers a good prognosis for breast Moreover, it is quite comprehensible that SMADs may then have cancers. It suggests that activin-A signaling has an important role an important role in tumor progression. Although TGF-β is well in the dissemination of breast cancer cells and, hence, may known to induce EMT in various cell lines, not much is known determine the outcome of the disease. We show that over- about activin-A in this regard. EMT is considered to be a expression or knockdown of activin-A affects anchorage- prerequisite for tumor cells to migrate and invade into neighbor- independent growth of breast cancer cells. We also show that ing tissues, and hence lead to metastasis. In recent times, even activin-A induces EMT and promotes migration and invasion in nodal, another member of the TGF-β superfamily that signals breast cancer cells. Primarily, the SMAD2/3 signaling pathway through the activin receptors has been reported to promote inhibits proliferation of normal epithelial cells and, hence, is cancer progression. Our results demonstrate that activin-A- considered to have a tumor-suppressor function. Similar to TGF-β, induced expression of EMT markers and invasion is SMAD3 proliferation of normal or tumor cells in the earlier stages of tumor dependent. SMAD3 has been shown to promote growth of breast development may be inhibited by activin-A. However, it is cancer cells in nude mice. However, activin-A may also activate interesting to note that this signaling pathway still remains intact non-SMAD signaling pathways, which may contribute to its pro- npj Breast Cancer (2015) 15007 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Number of nodules/animal Average nodule size/animal Activin-A promotes breast cancer M Bashir et al Control Overexpression CD44 4 4 10 10 CD24 3 3 10 10 2 2 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 CD24 Control Knockdown 4 4 10 10 80 CD44 3 3 10 10 2 2 10 10 CD24 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 CD24 (i) 4 4 3 3 2 2 1 1 0 0 MCF-7 MDA-MB-231 (ii) Figure 5. Continued. tumorigenic actions. We observed that activin affects tumor known to have an important role in maintenance of stem cell 11 36 formation and colonization of tumor cells in nude mice. The data phenotype and stemness. In addition, EMT has been shown suggest that activin-A expression affects the establishment of to induce drug resistance and stem cell-like phenotype in cancer tumors, which is a very complex process. Establishment of tumors cells. It has also been shown that tumors that are more or metastasis is also inﬂuenced by interaction between tumor cells aggressive are less differentiated and vice versa. We show that and the microenvironment. Hence, factors that can modulate the activin-A expression affects the stemness of breast cancer cells tumor microenvironment can have a key role in cancer progres- and hence may contribute to the aggressiveness of the disease. sion. In this context, activin may also be important in the Our study highlights the importance of activin signaling in the establishment of metastases from disseminated cells by modifying progression of breast tumors. Administration of a circulating the tumor niche. One of the factors known to be important for dominant-negative type II TGF-β receptor in mice has been shown tumor growth is VEGF, which leads to recruitment of blood to prevent metastasis of breast tumors. However, our study vessels, hence providing nourishment to the tumor cells. As we emphasizes the role of activins and the expression pattern of have demonstrated here, activin-A induces VEGF expression and these ligands should be considered from a clinical perspective. It is may inﬂuence the proliferation of the cancer cells in vivo. It may, to possible that different tumors may use either TGF-β or activin in a some extent, also explain how activin overexpression promotes context-dependent manner. Hence, it is important to carefully proliferation of tumor cells in vivo. It will be interesting to evaluate examine the expression of these ligands while designing how activin-A expression would affect the response of breast strategies to block their actions. Although most studies in the cancer patients to various conventional treatments. Activin-A is past have focussed on the role of TGF-β, this study emphasizes © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007 Control Act- O/E Control Act- O/E Snail Control Slug Act- KD Twist Sox2 Control Nanog Act- O/E Oct4 Snail Slug Twist Sox2 Nanog Oct4 MDA-MB-231 MCF-7 CD44 CD44 Fold Change Fold Change Percent cells Percent cells Activin-A promotes breast cancer M Bashir et al activin-A’s role in the progression of breast tumors. Interestingly, 13 Kang H-Y, Huang H-Y, Hsieh C-Y, Li C-F, Shyr C-R, Tsai M-Y et al. Activin a enhances prostate cancer cell migration through activation of androgen receptor and is many other cancers have been reported to have increased overexpressed in metastatic prostate cancer. J Bone Min Res 2009; 24: 1180–1193. expression of activins, suggesting that activation of the activin 14 Wildi S, Kleeff J, Maruyama H, Maurer C, Buchler M, Korc M. Overexpression of signaling pathway may be widely involved in carcinogenesis. As activin A in stage IV colorectal cancer. Gut 2001; 49:409–417. activins have multiple roles in physiological context, their role in 15 Seder CW, Hartojo W, Lin L, Silvers AL, Wang Z, Thomas DG et al. Upregulated cancer may be equally diverse. Importantly, our study emphasizes INHBA expression may promote cell proliferation and is associated with poor the role of SMAD pathway in the progression of breast tumors and survival in lung adenocarcinoma1. Neoplasia 2009; 11: 388–396. targeting this pathway may be a useful strategy in the treatment 16 Deli A, Kreidl E, Santifaller S, Trotter B, Seir K, Berger W et al. Activins and activin antagonists in hepatocellular carcinoma. World J Gastroenterol 2008; 14: of breast cancers. 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Cell- extrinsic consequences of epithelial stress: activation of protumorigenic tissue phenotypes. Breast Cancer Res 2012; 14: R155. COMPETING INTERESTS 23 Jeruss JS, Sturgis CD, Rademaker AW, Woodruff TK. Down-regulation of activin, The authors declare no conﬂict of interest. activin receptors, and Smads in high-grade breast cancer. Cancer Res 2003; 63: 3783–3790. 24 Reis FM, Luisi S, Carneiro MM, Cobellis L, Federico M, Camargos AF et al. Activin, FUNDING inhibin and the human breast. Mol Cell Endocrinol 2004; 225:77–82. Funding for this work was from the Department of Biotechnology, Government of 25 Candia AF, Watabe T, Hawley SH, Onichtchouk D, Zhang Y, Derynck R et al. India, by partnership program and infrastructure supports. MRDG receives Cellular interpretation of multiple TGF-beta signals: intracellular antagonism infrastructure support from DST FIST and UGC-SAP programs. between activin/BVg1 and BMP-2/4 signaling mediated by Smads. 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Cell Res 2009; 19:89–102. by/4.0/ Supplemental Information accompanies the paper on the npj Breast Cancer website (http://www.nature.com/npjbcancer) © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15007
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