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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.

Simple Summary Cancer cachexia is a systemic inflammatory disease characterized by the loss of muscle and fat and occurs in 50–80% of cancer patients. In cancer cachexia, the tumor tissues interact with other tissues and organs using secretory factors. Differentiated immune cells from hematopoietic stem cells (HSCs) of the bone marrow contribute to the systemic inflammation and may be affected by these intertissue interactions. However, the significant changes that occur in the bone marrow and the underlying mechanisms are still unclear. Here, we investigated the effects of cancer cachexia on bone and stem cells that reside in the bone marrow using a lung cancer cachexia animal model. Cancer cachexia induces bone loss and impairs the properties of the bone marrow mesenchymal stem cells via JAK/STAT and glucocorticoid signaling. Our findings provide new insights for developing a novel therapeutic strategy for cancer cachexia. Abstract Cancer cachexia is a multifactorial systemic inflammation disease caused by complex interactions between the tumor and host tissues via soluble factors. However, whether cancer cachexia affects the bone marrow, in particular the hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), remains unclear. Here, we investigated the bone marrow and bone in a cancer cachexia animal model generated by transplanting Lewis lung carcinoma cells. The number of bone marrow mononuclear cells (BM-MNCs) started to significantly decrease in the cancer cachectic animal model prior to the discernable loss of muscle and fat. This decrease in BM-MNCs was associated with myeloid skewing in the circulation and the expansion of hematopoietic progenitors in the bone marrow. Bone loss occurred in the cancer cachexia animal model and accompanied the decrease in the bone marrow MSCs that play important roles in both supporting HSCs and maintaining bone homeostasis. Glucocorticoid signaling mediated the decrease in bone marrow MSCs in the cancer cachectic environment. The cancer cachexia environment also skewed the differentiation of the bone marrow MSCs toward adipogenic fate via JAK/STAT as well as glucocorticoid signaling. Our results suggest that the bone loss induced in cancer cachexia is associated with the depletion and the impaired differentiation capacity of the bone marrow MSCs.

Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.

Preservation of pancreatic β-cells is required for the development of therapies for type 1 and type 2 diabetes (T1D and T2D, respectively). Our previous study demonstrated that substance P (SP) preserves β-cell populations in mice with streptozotocin-induced T1D. Here, we demonstrated that chronic systemic treatment with SP restored the mass of β-cells both in nonobese diabetic (NOD) mice with T1D or db/db mice with T2D. SP delayed the onset of T1D in NOD mice via immune modulation. SP inhibited immune infiltration into islets and the salivary glands of NOD mice. In db/db mice, SP treatment rescued glucose intolerance. Moreover, SP inhibited apoptosis, as well as the activation of pancreatic stellate cells in pancreatic islets of db/db mice. SP downregulated the number of α-smooth muscle actin (α-SMA) expressing cells in db/db pancreatic islets. Cleaved-caspase-3 expression was reduced in islets of SP-treated db/db mice compared to that in the control. Therefore, these results suggested that SP may preserve pancreatic β-cells through immune modulation and protection from the stimulated activation of pancreatic stellate cells and apoptosis in T1D and T2D, respectively.

Wound healing is essential for the survival and tissue homeostasis of unicellular and multicellular organisms. The current study demonstrated that the neuropeptide substance P (SP) accelerated the wound healing process, particularly in the skin. Subcutaneous treatment of SP accelerated wound closing, increased the population of α-smooth muscle actin positive myofibroblasts, and increased extracellular matrix deposition at the wound site. Moreover, SP treatment enhances angiogenesis without a local increase in the expression levels of vascular endothelial growth factor and stromal cell-derived factor-1. Importantly, SP treatment increased both the population of circulating endothelial progenitor cells in the peripheral blood and in CD31 positive cells in Matrigel plugs. The tube forming potential of endothelial cells was also enhanced by SP treatment. The results suggested that the subcutaneous injection of SP accelerated the wound healing in the skin via better reconstitution of blood vessels, which possibly followed an increase in the systemic mobilization of endothelial progenitor cells and a more effective assembly of endothelial cells into tubes.

Transforming growth factor-beta (TGF-β) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-β are unclear. In this study, we showed that TGF-β activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-β type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-β-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-β were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-β-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-β. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-β was mediated through N-cadherin and noncanonical TGF-β signals.

Dermal fibroblasts play essential roles in wound healing and their dysfunction has been shown to be associated with impaired wound healing in diabetes. In the present study, we aimed at investigating whether Yes-associated protein (YAP), a mediator of mechanotransduction in dermal fibroblasts, is associated with impaired wound healing in diabetic mice. Compared with that in the control, the rate of wound contraction was decreased twofold in db/db type 2 diabetic mice (db/db mice). To mimic diabetic pathological condition, dermal fibroblasts were cultured under high glucose conditions (25.5 mM glucose). Further, dermal fibroblast-mediated contraction of wound was evaluated by in vitro collagen gel contraction assay. Dermal fibroblasts cultured under hyperglycemic condition showed impaired gel contraction and mitochondrial dysfunction, compared to the cells cultured under normoglycemic conditions (5.5 mM glucose). Importantly, compared with the normal dermal fibroblasts, diabetic db/db dermal fibroblasts expressed lower levels of growth factors and cytokines that enhance wound healing, such as insulin-like growth factor-1, stromal cell-derived factor-1, connective tissue growth factor, and transforming growth factor-β (TGF-β). The quantity of YAP mRNA was also lower in diabetic db/db dermal fibroblasts, compared with that in the control fibroblasts. These results indicate that impaired wound healing in diabetics is associated with the dysfunction of dermal fibroblasts, including downregulation of YAP, which plays essential roles in extracellular matrix remodeling and TGF-β-mediated wound healing.