N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells

Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor β (TGF-β), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration. Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-β was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells. Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-β increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-β-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-β pathways, such as extracellular signal‐regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-β. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-β type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells. Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-β canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-β via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.

TGF-β ligands play important roles in the regulation of tumor initiation, progression, and metastasis [24] and the plasma level of TGF-β1 ligand increases in patients with breast cancer and decreases after surgical removal of the tumor [25]. Different responses of breast tumor cells to TGF-β have been reported [26]. TGF-β signaling in tumor cells enhances tumorigenesis by stimulating the epithelialmesenchymal transition (EMT), invasion, and metastasis of breast tumor cells [27][28][29]. Moreover, the activation of TGF-β signaling is associated with the induction of the cancer stem cell phenotype in breast tumor cells [30][31][32][33]. However, the loss of TGF-β signaling in tumor cells is associated with an increase in both lung metastasis and infiltration of bone marrow-derived myeloid cells that enhance tumor cell invasion and metastasis [34,35]. TGF-β signaling has also been shown to be intrinsically important in non-tumor cells in the breast tumor microenvironment [26,27]. This TGF-β-mediated regulation of tumor microenvironment is critical for the identification of TGF-β as a tumor suppressor or promoter [26,27]. For example, TGF-β activates CAFs to stimulate the synthesis and secretion of growth factors, cytokines, and ECM molecules that collectively form a tumor-promoting microenvironment [27]. However, it is still unclear whether breast tumor cell-derived TGF-β ligands modulate the recruitment of BM-MSCs into breast tumor.
BM-MSCs are multipotent adult stem cells [36,37] that contribute to the maintenance of bone homeostasis [38] and formation of a niche for hematopoietic stem cells inside the bone marrow [39,40]. BM-MSCs also participate in the repair and regeneration of injured peripheral tissues [41]. BM-MSCs are partially derived from the neural crest. Cells from the neural crest that forms during the early embryonic development in vertebrates are very motile to migrate to their final destination where they differentiate into a variety of cells [42,43]. Similar to the neural crest cells, BM-MSCs are migratory. They can migrate into injured tissues to participate in regeneration [41,44]. BM-MSCs migrate in response to various molecules, including stromal cell-derived factor-1 (SDF-1) and TGF-β, secreted by injured tissues [45][46][47]. In our previous studies, we demonstrated that BM-MSCs migrate in response to TGF-β1 in a manner dependent on neural cadherin (N-cadherin), a cell-cell adhesion molecule [48,49].
Cadherins are a large superfamily of transmembrane proteins, which include N-cadherin, epithelial cadherin (E-cadherin), osteoblast cadherin (OB-cadherin), and vascular endothelial cadherin (VE-cadherin) [50]. They mediate intercellular adhesion to neighboring cells via calcium-dependent hemophilic and cadherin type-specific interactions in the extracellular domain [50]. β-Catenin and p120 interact with the cytoplasmic domain of cadherins and are physically and functionally connected with the actin cytoskeletal system through ɑ-catenin to form adherens junctions [50]. Cadherin-mediated cell-cell adhesion controls important biological processes, such as cell sorting, cell rearrangement, and cell movement, as well as intercellular interactions between neighboring cells [50]. E-cadherin and N-cadherin play important roles in the collective directional migration of different cell types [51][52][53]. It remains to be determined whether N-cadherin present on BM-MSCs mediates the collective migration of BM-MSCs toward the tumor microenvironment in a cell-cell adhesion-dependent manner, although we previously demonstrated that N-cadherin mediates the migration of BM-MSCs to TGF-β. In the present study, we examined the migration of BM-MSCs toward breast tumor cells expressing TGF-β. TGF-β increased the expression of N-cadherin on the borders between BM-MSCs in a Smad4-dependent manner. N-cadherin-dependent cell-cell adhesion is required for the migration of BM-MSCs in response to conditions mimicking those prevalent in breast tumor.

Cell lines and cell culture
Human BM-MSCs were obtained from Lonza (Basel, Switzerland) and cultured in Mesenchymal Stem Cell Growth Medium (Lonza). TrypLE (Invitrogen, Carlsbad, CA, USA) was used for subculture, and the medium was changed every 3 days. For all experiments, BM-MSCs between passages 5 and 7 were used. Dulbecco's Modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with heat-inactivated fetal bovine serum (FBS; Invitrogen), 1% L-glutamine (Invitrogen), and 1% penicillin and streptomycin (P/S; Invitrogen) was used for migration experiments using BM-MSCs. MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MDA-MB-231 cells were cultured in DMEM/high glucose (DMEM/high, GE Healthcare Life Sciences), supplemented with 10% FBS and 1% P/S. MCF7 cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). MCF7 cells were cultured in DMEM:Nutrient Mixture F-12 (DMEM/F-12; 1:1; Invitrogen), supplemented with 10% FBS and 1% P/S. All the cells were maintained at 37 °C in a humidified incubator containing 5% CO2.

Preparation of bone marrow cell suspension and colony-forming unit fibroblast (CFU-F) assay
All animal experiments were approved by the Animal Experiment Ethics Committee of Kyung Hee Hospital Medical Center (KHMC-IACUC-2019-014) and performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines. Eight-week-old male C57BL/6 mice (DBL, Seoul, South Korea) were used in all the animal experiments. Femurs and tibias were resected and crushed with a pestle. The crushed bones were washed twice using a Hanks' balanced salt solution supplemented with 2% FBS (Invitrogen) and 1% P/S (Invitrogen). The bone fragments were collected and incubated for 1 h at 37 °C in DMEM (Invitrogen) containing 0.2% collagenase (Wako Chemicals USA, Richmond, VA, USA) and 1% P/S (Invitrogen) with occasional agitation. The suspension was filtered through a 40-μm cell strainer (BD Biosciences, San Jose, CA, USA) to remove debris. The cell suspension was centrifuged at 300 × g for 10 min at 4 °C. The cell pellet, thus obtained, was washed with Minimum Essential Medium Eagle Alpha Modification (α-MEM; Sigma) containing 20% FBS (Invitrogen), 1% L-glutamine (Invitrogen), 5% pyruvate (Invitrogen), and 1% P/S (Invitrogen). The cells were then seeded in 24-well plates at a density of 1.5 × 10 4 cells/well using the medium used for washing the cell pellet. After 10 days, cell colonies were fixed with 4% paraformaldehyde (PFA; 3M Science, Saint Paul, MN, USA) in phosphate-buffered saline (PBS) for 10 min on ice after washing three times with ice-cold PBS. Immunocytochemistry for N-cadherin was performed using a primary antibody for N-cadherin (1:30, Invitrogen) according to standard protocols.

Conditioned medium
MDA-MB-231 and MCF7 cells were cultured in DMEM/high supplemented with 10% FBS and 1% P/S and DMEM/F-12 (1:1) supplemented with 10% FBS and 1% P/S, respectively. After culturing to confluence, the cells were rinsed with PBS and incubated with DMEM/high supplemented with 1% P/S for 3 days. To prepare conditioned medium (CM) for the control, DMEM/high supplemented with 1% P/S was incubated for 3 days under cell-free conditions. Conditioned media from MDA-MB-231 (MDA CM), MCF7 (MCF7 CM), or the control (CON CM) were filtered through a 0.2-μm filter (Corning, NY, USA), aliquoted, and stored at -80 °C before use.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the SuperScript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed using the Power SYBR Green PCR Master Mix (Invitrogen). Human ribosomal protein S9 gene (RPS9) was used as an endogenous control. The primers used to detect the expression levels of RPS9, N-cadherin, OB-cadherin, Smad4, TGF-β1, TGFBR1, and TGFBR2 were as

Immunocytochemistry
BM-MSCs were plated at a density of 1 × 10 4 cells/cm 2 on type 1 collagen (Nitta Gelatin Inc., Osaka, Japan)-coated cover slips in 24-well plates and cultured in the culture medium for 24 h. Thereafter, the medium was replaced with serum-free DMEM supplemented with 1% L-glutamine and 1% P/S and the cells were cultured for another 18 h. The cells were then treated with TGF-β for 24 h, and subsequently fixed with 4% PFA (Sigma) in PBS for 10 min on ice. After fixation, immunocytochemistry was performed according to standard protocols. The fixed cells were washed with PBS three times and permeabilized with 0.2% Triton-X 100 (Sigma) in PBS for 5 min at 25 °C. The cells were again washed three times with 0.1% Triton-X 100 in PBS and blocked with 5% skimmed milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) prepared in 0.1% Triton-X 100, for 30 min at 25 °C. After blocking, the cells were incubated for 1 h 30 min with the following primary antibodies: anti-N-cadherin (1:30, Invitrogen) and anti-β-catenin (1:100, Bethyl Laboratories, Montgomery, TX, USA). Next, the cells were washed three times with 1% skimmed milk in 0.1% Triton-X 100 and incubated with secondary antibodies for 30 min at 25 °C. If necessary, actin was stained with Alexa Fluor 546-tagged phalloidin (1:1,000, Invitrogen). After incubation with secondary antibody, the cells were washed three times with PBS, and nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) for 10 min. The cells were then mounted using Fluoromount-G solution (SouthernBiotech, Birmingham, AL, USA). Images were taken using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence intensity of N-cadherin-positive structures at the borders between BM-MSCs was measured using the ImageJ software (NIH).

Transwell migration assay
BM-MSCs were incubated with DMEM supplemented with 2% FBS, 1% L-glutamine, and 1% P/S for 18 h. Millicell culture plate inserts (8 μm pore size; EMD Millipore) were coated with Type I collagen (5 μg/mL; Nitta Gelatin NA Inc.). The serum-starved BM-MSCs were seeded at a density of 2 × 10 4 cells/cm 2 in the upper chamber of each insert using DMEM supplemented with 2% FBS, 1% L-glutamine, and 1% P/S and incubated for 6 h. Thereafter, the media in both the lower and upper chamber of each insert were replaced with serum-free DMEM supplemented with 1% L-glutamine and 1% P/S. TGF-β (10 ng/mL) or solvent was added to the lower chamber of each well. If necessary, cells were treated with SB505124 (500 nM) or EGTA (4 mM) for 30 min prior to treatment with TGF-β. Minimum Essential Medium (Invitrogen), and sterile ultrapure distilled water. Three microliters of the cellcollagen mixture was solidified in 12-well plates and incubated with serum-free DMEM supplemented with 1% L-glutamine and 1% P/S for 6 h. The media were then changed with the CM from MDA-MB-231 or the control. The migration of BM-MSCs was observed after 24 h. The migration of BM-MSCs was determined by dividing the area occupied by cells migrated from each gel drop by the area occupied by non-migrated cells in the outer edge corresponding to 10% of each drop prior to adding the CM. Images were captured using a Nikon ECLIPSE TS 100 inverted microscope (Nikon Instruments Inc., Melville, NY, USA), and the area was measured using the ImageJ software (NIH). A cell-collagen mixture containing BM-MSCs transfected with N-cadherin siRNA and BM-MSCs transfected with the control siRNA was used to evaluate the effects of N-cadherin on cell-cell adhesion between migrating BM-MSCs. BM-MSCs transfected with the control siRNA were stained with calcein AM before mixing. The cellcollagen mixture was solidified on a μ-dish 35 mm, low Grid-500 (ibidi, Munich, Germany). Migrating cells on the surface of 13 randomly selected areas were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss) to analyze the number of migrating control BM-MSCs and migrating N-cadherin knockdown BM-MSCs.

Statistical analysis
Quantitative data are expressed as the mean or mean ± SD. Statistical significance was analyzed using the Student's t-test. The GraphPad version 6.07 (GraphPad Software, Inc. San Diego, CA, USA) was used for statistical analyses. A value of P < 0.05 was considered significant.

TGF-β-induced migration of BM-MSCs is associated with the increase in N-cadherin
We first investigated whether MSCs isolated from adult mouse bone marrow express N-cadherin. A suspension of bone marrow cells was prepared by treating crushed long bones of adult mice with collagenase, and the cells were allowed to form colonies. All the colony-forming MSCs expressed N-cadherin on the border between the cells inside the colonies ( Figure 1A). It is likely that N-cadherin may contribute to the formation of cell-cell adhesion-like structures between BM-MSCs. In a previous study, we demonstrated that N-cadherin mediates the migration of human BM-MSCs in response to TGF-β [48]. We examined whether the expression of N-cadherin is regulated by TGF-β. TGF-β increased the expression of N-cadherin at both the protein and mRNA levels in BM-MSCs as well as the phosphorylation of Smad2/3; however, SB505124, an inhibitor of TGF-β type 1 receptor, inhibited the TGF-β-induced increase in N-cadherin expression and phosphorylation of Smad2/3 ( Figure 1B-D). Interestingly, TGF-β induced only a slight increase in the expression of OB-cadherin, another major cadherin in BM-MSCs, compared with the expression of N-cadherin ( Figure  S1A-B). Therefore, N-cadherin may be a major cadherin that is induced in BM-MSCs by TGF-β. N-cadherin, induced by TGF-β, was concentrated and localized at the border between BM-MSCs, and β-catenin co-localized with N-cadherin at the borders ( Figure 1E-F). These results suggest that TGF-β-induced N-cadherin may be concentrated at the borders between BM-MSCs and may contribute to the formation of cell-cell adhesion-like structures of BM-MSCs, which are similar to adherens junctions of epithelial and endothelial cells. Importantly, TGF-β induced the migration of BM-MSCs via the TGF-β type 1 receptor-mediated signaling. TGF-β increased the migration of BM-MSCs, but SB505124 inhibited their migration toward TGF-β ( Figure 1G-H).

N-cadherin is required for the migration of BM-MSCs in response to TGF-β
Next, we performed siRNA-mediated knockdown of N-cadherin to investigate whether the upregulation of N-cadherin by TGF-β results in the migration of BM-MSCs to TGF-β. Compared with the control siRNA, transfection with N-cadherin siRNA decreased the expression levels of N-cadherin at the protein and mRNA levels (Figure 2A-B). The knockdown of N-cadherin downregulated the migration of BM-MSCs toward TGF-β ( Figure 2C-D). EGTA, a high-affinity chelator of calcium ions, which are essential for homophilic trans-interactions of N-cadherin, also downregulated the migration of BM-MSCs toward TGF-β ( Figure 2E-F). These results suggest that the intercellular interaction of BM-MSCs, mediated by N-cadherin, upregulated by TGF-β, is required for their migration toward TGF-β.

TGF-β enhances the expression of N-cadherin in BM-MSCs via Smad4
We investigated the mechanisms underlying the increase in the expression of N-cadherin in BM-MSCs in response to TGF-β. TGF-β activates non-canonical signaling pathways, including Akt, ERKs, and p38, as well as canonical signaling, as shown in our previous study [48]. TGF-β induced Smad 2/3 phosphorylation in BM-MSCs as early as 10 min post-treatment, and the highest phosphorylation level was observed at 30 min post-treatment, followed by a gradual decrease in the level of Smad 2/3 phosphorylation at later time points ( Figure 3A). The phosphorylation of Akt, ERK, and p38 was upregulated in BM-MSCs in response to TGF-β ( Figure 3A). TGF-β gradually increased the expression of N-cadherin ( Figure 3A). The TGFβ-mediated canonical signaling is dependent on Smad4 [21]. siRNA-mediated knockdown of Smad4 significantly inhibited the increase in the expression of N-cadherin induced by TGF-β (Figure 3B-E). However, this TGF-β-induced increase was slightly affected by inhibitors of TGF-β non-canonical signaling. Pretreatment with LY294002 (a specific inhibitor of phosphoinositol 3-kinase, upstream of Akt), PD98059 (a specific inhibitor of mitogenactivated protein kinase kinase, upstream of ERKs), or SB203580 (a specific inhibitor of p38) did not inhibit the increase in the expression of N-cadherin in BM-MSCs ( Figure 3F-H). Importantly, TGFβ-mediated migration of BM-MSCs was reduced by the knockdown of Smad4, to a similar extent as in the case of N-cadherin knockdown (Figure 3I-J). Therefore, these results suggest that N-cadherin increases in response to TGF-β via Smad4 and mediates the migration of BM-MSCs toward TGF-β.

N-cadherin mediates the migration of BM-MSCs in response to breast tumor cells
The expression of TGF-β was seen to be higher in human breast cancer tissue than in the normal tissue ( Figure 4A). There are conflicting reports about the tumor suppressor and tumor-promoting functions of TGF-β in breast cancer [24,31,[54][55][56][57][58] Figure 4G). SB431542, another inhibitor of TGF-β type 1 receptor, also decreased N-cadherin upregulation induced by MDA CM (Figure S2A-B). siRNA-mediated knockdown of TGF-β type 1 inhibited the phosphorylation of Smad2/3 in BM-MSCs in response to TGF-β ( Figure 4H-I)

N-cadherin mediates cell-cell adhesion required for the collective migration of BM-MSCs in response to breast tumor cells
Next, we examined the role of N-cadherin in the migration of BM-MSCs toward breast cancer cells. Cell migration was analyzed using a 3D cell migration assay [60,61] to examine whether BM-MSCs collectively migrate in response to breast cancer cells and maintain cell-cell adhesion during migration ( Figure 6A). Indeed, BM-MSCs migrated collectively and maintained cell-cell adhesion. The collective migration of BM-MSCs increased in response to MDA CM treatment compared with that of the control, while they maintained cell-cell adhesion (Figure 6B-D). However, the knockdown of N-cadherin downregulated the migration of BM-MSCs ( Figure  6E), suggesting that N-cadherin may contribute to cell-cell adhesion, which is required for the migration of BM-MSCs. The results of immunocytochemistry showed that N-cadherin and β-catenin co-localized at the cell-cell adhesion sites, the borders between the migrating BM-MSCs, in response to MDA CM ( Figure  6F). N-cadherin on the borders was linked to actin filaments in the cytoplasm ( Figure 6F). A 3D cell migration assay was performed using a mixture of control BM-MSCs expressing N-cadherin and BM-MSCs in which N-cadherin was knocked down. A lesser number of cells in which N-cadherin was knocked down migrated in response to MDA CM than the control cells with intact expression of N-cadherin ( Figure 6G-H). Although some populations of BM-MSCs in which N-cadherin was knocked down still migrated in response to MDA CM, more than 60% of such BM-MSCs showed cell-cell adhesion-free migration. In contrast, less than 30% of the migrating cells expressing N-cadherin showed cell-cell adhesion-free migration ( Figure 6I). Overall, these results suggest that N-cadherin is required for cell-cell adhesion for mediating the collective migration of BM-MSCs toward breast tumor cells.

Discussion
Cadherins are transmembrane adhesive molecules of adhesion junctions that control cell-cell adhesion, cell sorting, tissue dynamics, cellular metabolism, and cellular migration, and interact with various signal transducer proteins. N-cadherin is a critical regulator of collective migration of cells. However, it remains unclear whether N-cadherin contributes to cell-cell adhesion-dependent collective migration of BM-MSCs in response to various signals, including tumor-derived factors, such as TGF-β. In a previous study, we demonstrated that N-cadherin mediates the migration of BM-MSCs toward TGF-β.
In the present study, we demonstrate that the expression of N-cadherin in BM-MSCs increases in a manner dependent on TGF-β canonical signaling (Smad4-dependent manner), and that N-cadherindependent cell-cell adhesion is required for the migration of BM-MSCs in vitro in response to TGF-β and breast tumor cells expressing TGF-β.
Adherens junctions, the dynamic cooperative cell-cell adhesion complexes, are required for the collective migration of mesenchymal cells, including BM-MSCs [62][63][64]. The cell-cell adhesion mediated by adherens junctions consisting of cadherins mechanically links one cell to another and regulates their motility and protrusive behavior so as to mediate coordinated migration of mesenchymal cell cohorts. The coordinated collective migration of mesenchymal cells, such as the migration of the neural crest and head mesendoderm, has been found in embryonic development [62,64]. We demonstrate that cell-cell adhesion consisting of N-cadherin mediates the collective migration of BM-MSCs to MDA CM, an experimental condition simulating the tumor microenvironment derived by TGF-β-secreting breast tumor cells, but the mechanism(s) by which N-cadherin-dependent cell-cell adhesion mediates the collective migration of BM-MSCs remains unknown. N-cadherin-dependent cell-cell adhesion is required for the collective chemotactic migration of neural crest cells toward stromal cell-derived factor 1 (SDF-1). For migration, N-cadherin at the cell-cell contacts inhibits Rac1 activity and protrusion and in turn enhances the Rac1 activity and protrusion at the free edge [64]. N-cadherin-dependent intercellular interaction also mediates the collective migration of glial cells [65] and of hepatocyte growth factor-treated MDCK cells [66] via the regulation of focal adhesion and dynamic regulation of the interaction between N-cadherin and ɑ-catenin, respectively. Further experiments are required to identify the mechanisms by which N-cadherin-dependent cell-cell adhesion mediates the collective migration of BM-MSCs toward breast tumors.
BM-MSCs express both OB-cadherin and N-cadherin. TGF-β and CM from breast tumor cells expressing TGF-β increased the expression of N-cadherin, but not that of OB-cadherin. As reported in our previous study, N-cadherin mediates the response of murine bone marrow-derived mesenchymal stem cell-like ST2 cells to TGF-β, although the expression level of N-cadherin in ST2 cells is not affected by TGF-β treatment [48]. Therefore, further experiments are required to determine whether the migration of BM-MSCs is also regulated in an OB-cadherin-dependent manner. It has been found that the expression level of N-cadherin is tightly regulated for the directional invasive migration of tumor glial cells [65]. Low levels of N-cadherin in glial cells cause less directional migration of the cells and accompany the formation of smaller focal adhesion points. Therefore, it is important to unravel the mechanisms underlying the increase in the expression of N-cadherin in BM-MSCs induced by TGF-β. TGF-β ligands intrinsically transduce canonical signaling in BM-MSCs in a manner dependent on Smad4 and non-canonical signaling independently of Smad4. Both TGF-β canonical and non-canonical signaling pathways were found to be required for the migration of BM-MSCs (Figure 4 and unpublished) [48]. There was a significant increase in the expression of N-cadherin mRNA 3 h after TGF-β treatment of BM-MSCs (unpublished). The knockdown of Smad4 inhibited TGF-β-induced increase in the expression of N-cadherin in BM-MSCs. In contrast, inhibitors of TGF-β non-canonical signaling pathways, such as those involving Akt, ERKs, and p38, did not appear to affect the expression of N-cadherin in BM-MSCs in response to TGF-β. However, further investigations might be necessary to examine whether the inhibitors downregulate the respective non-canonical signaling pathways. Importantly, TGF-β enhances the expression of N-cadherin in lung cancer cells by binding the Smad 2/3 complex to the N-cadherin promoter [67]. The knockdown of Smad4 downregulated the expression of N-cadherin at mRNA and protein levels in TGF-β-treated BM-MSCs. Therefore, Smad protein complexes containing Smad4 may activate the promoter of N-cadherin in BM-MSCs to increase the expression of N-cadherin in response to TGF-β. It is also necessary to investigate whether canonical and/or non-canonical TGF-β signaling mediates the increase in N-cadherin in BM-MSCs in response to breast tumor-derived TGF-β. Further experiments are required to investigate these possibilities.
In this study, we introduce a promising novel strategy for re-engineering the breast tumor microenvironment. Homophilic trans-interactions of N-cadherin can be targeted to inhibit the recruitment of BM-MSCs into the breast tumor microenvironment that promotes metastasis and invasion of breast tumor cells and angiogenesis.