We hypothesized that the CXC chemokine receptor-4 (CXCR4)–stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4+ tumor cells into the bone marrow and lymph nodes, which secrete the α-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3- andPAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHRgene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4–SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1) phosphorylation of mitogen-activated protein kinase p42/44; (2) locomotion; (3) directional chemotaxis across membranes covered by laminin, fibronectin, or Matrigel; (4) adhesion to laminin, fibronectin, and endothelial cells; and (5) increased MMP-2 and diminished tissue inhibitors of metalloproteinases secretion. The small-molecule CXCR4-specific inhibitor, T140, effectively blocked the in vitro responses of RMS cells to SDF-1. On the basis of these observations we suggest that the CXCR4–SDF-1 axis may play an important role in tumor spread and metastasis of RMS cells to bone marrow and that molecular strategies aimed at inhibiting this axis could thus prove to be useful therapeutic measures.

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, α-chemokine stromal-derived factor 1 (SDF-1), is secreted in muscle tissue. These cell lines responded to SDF-1 stimulation by chemotaxis, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT serine-threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34+ hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4+ satellite cells just as they chemoattract CD34+ cells. Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34+ cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle- or bone marrow-derived fibroblasts. Based on these observations, we postulate that the SDF-1-CXCR4 axis is involved in chemoattracting circulating CXCR4+ muscle stem/progenitor and circulating CXCR4+ hematopoietic CD34+ cells to both muscle and bone marrow tissues. Thus, it appears that tissue-specific stem cells circulating in peripheral blood could compete for SDF-1+ niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.

The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74-dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); 1328–43. ©2010 AACR.

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2^nd and 3^rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

We have demonstrated that the α‐chemokine stromal‐derived factor (SDF)‐1‐CXCR4 axis plays an important role in rhabdomyosarcoma (RMS) metastasis. With the recent description of CXCR7, a new receptor for SDF‐1 that also binds the interferon‐inducible T‐cell α chemoattractant (ITAC) chemokine, we became interested in the role of the CXCR7‐SDF‐1/ITAC axis in RMS progression. To address this issue, we evaluated 6 highly metastatic alveolar (A)RMS and 3 less metastatic embryonal (E)RMS cell lines and found that all these cell lines express CXCR7. Although CXCR4 was expressed at a much higher level by highly metastatic ARMS lines, CXCR7 was present at a high level on ERMS lines. We also noticed that CXCR7 expression on RMS cells was downregulated in hypoxic conditions. More importantly, the CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF‐1 as evidenced by mitogen‐activated protein kinase (MAPK)p42/44 and AKT phosphorylation as well as CXCR7 internalization, chemotaxis, cell motility and adhesion assays. Similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Moreover, CXCR7 ⁺ RMS cells responded to SDF‐1 and I‐TAC in the presence of CXCR4 antagonists (T140, AMD3100). Furthermore, while intravenous injection of RMS cells with overexpressed CXCR7 resulted in increased seeding efficiency of tumor cells to bone marrow, CXCR7 downregulation showed the opposite effect. In conclusion, the CXCR7‐SDF‐1/ITAC axis is involved in the progression of RMS; targeting of the CXCR4‐SDF‐1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy for inhibiting SDF‐1‐mediated prometastatic responses of RMS cells.