Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas

Abstract

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.

Figure 1

Panels A and B: Expression of CXCR4 and CXCR7 on human ARMS and ERMS cell lines. Flow cytometry was performed for detection of CXCR4 and CXCR7. The experiment was repeated three times with similar results. A representative study is shown. Panels C and D: SDF-1 and I-TAC activate intracellular signaling in human RMS cell lines. Phosphorylation of MAPK p42/44 and AKT in human RMS cell lines stimulated by SDF-1 (300 ng/mL for 5 min) and I-TAC (100 ng/mL for 5 min). The experiment was repeated three times with similar results. A representative study is shown.

Figure 1

Panels A and B: Expression of CXCR4 and CXCR7 on human ARMS and ERMS cell lines. Flow cytometry was performed for detection of CXCR4 and CXCR7. The experiment was repeated three times with similar results. A representative study is shown. Panels C and D: SDF-1 and I-TAC activate intracellular signaling in human RMS cell lines. Phosphorylation of MAPK p42/44 and AKT in human RMS cell lines stimulated by SDF-1 (300 ng/mL for 5 min) and I-TAC (100 ng/mL for 5 min). The experiment was repeated three times with similar results. A representative study is shown.

Figure 2. Effect of I-TAC on the motility and the adhesiveness of RMS cell lines

Panel A: The composite trajectories of RD, RH18, RH28, and RH30 cells migrating without (control) or with the addition of 100 ng/mL (I-TAC) are shown in circular diagrams drawn with the initial point of each trajectory at the origin of the plot. Panel B: Chemotaxis of RMS cells across Transwell membranes covered with gelatin to SDF-1 or I-TAC gradient. Gray bars show chemotaxis to control medium (no SDF-1 or I-TAC in upper and lower chambers), white bars show chemotaxis to SDF-1 (300 ng/mL) present in lower chamber, and black bars show chemotaxis to I-TAC (100 ng/mL) present in lower chamber. Data from 5 separate experiments are pooled together. * P<0,05. Panels C and D: Adhesion of human RMS cells to fibronectin(C) and to HUVECs (D). RMS were not stimulated (control; gray columns) or stimulated with SDF-1 (white columns) or I-TAC (black columns). Data from 4 separate experiments are pooled together. * p<0,05.

Figure 2. Effect of I-TAC on the motility and the adhesiveness of RMS cell lines

Panel A: The composite trajectories of RD, RH18, RH28, and RH30 cells migrating without (control) or with the addition of 100 ng/mL (I-TAC) are shown in circular diagrams drawn with the initial point of each trajectory at the origin of the plot. Panel B: Chemotaxis of RMS cells across Transwell membranes covered with gelatin to SDF-1 or I-TAC gradient. Gray bars show chemotaxis to control medium (no SDF-1 or I-TAC in upper and lower chambers), white bars show chemotaxis to SDF-1 (300 ng/mL) present in lower chamber, and black bars show chemotaxis to I-TAC (100 ng/mL) present in lower chamber. Data from 5 separate experiments are pooled together. * P<0,05. Panels C and D: Adhesion of human RMS cells to fibronectin(C) and to HUVECs (D). RMS were not stimulated (control; gray columns) or stimulated with SDF-1 (white columns) or I-TAC (black columns). Data from 4 separate experiments are pooled together. * p<0,05.

Figure 3. Effect of T140 and AMD3100 on SDF-1 and I-TAC -dependent chemotaxis and adhesion of RMS cells

Representative study of chemotaxis for ARMS (panel A) cells and ERMS (panel B) to medium alone (control) or medium with SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). Before chemotaxis, cells were pre-incubated 1 hr with T140 or AMD3100. Data from 3 separate experiments are pooled together. * p<0.0001. Representative study of adhesion to fibronectin of ARMS (panel A) cells and ERMS (panel B) in presence of SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). Before adhesion, cells were pre-incubated 5 min with T140 or AMD3100. Data from 3 separate experiments are pooled together. * p<0.0001.

Figure 4. Effect of SDF-1 and I-TAC on RMS cells with over-expression and knockdown of CXCR7

Panel A: shows expression of CXCR4 and CXCR7 on RH30 cells with over-expression of CXCR7. The experiment was repeated three times with similar results. A representative study is shown. Panel B shows chemotaxis of RH30, RH30 cells transfected with empty vector, and RH30 cells with over-expression of CXCR7 toward gradient of SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). Data from 3 separate experiments are pooled together. Panel C shows expression of CXCR4 and CXCR7 on RH18 cells with knock-down of CXCR7. The experiment was repeated three times with similar results. A representative study is shown. Panel D shows chemotaxis of RH18, RH18 scrambled, and RH18 cells with knock-down of CXCR7 toward gradient of SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). Data from 3 separate experiments are pooled together.

Figure 5. Effect of PTX on CXCR7 signaling

Panel A: Effect of PTX on chemotaxis of RH30 cells with over-expression of CXCR7 toward gradient of SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). Data from 3 separate experiments are pooled together. * p<0.0001. Panel B: Effect of PTX on internalization of CXCR4 and CXCR7 induced by SDF-1 (1000 ng/ml) and I-TAC (500 ng/ml). Data from 3 separate experiments are pooled together. * p<0.0001. Panel C: Effect of PTX on phosphorylation of MAPK p42/44 and AKT induced in response to SDF-1 (300 ng/ml) and I-TAC (100 ng/ml). The experiment was repeated twice with similar results. A representative study is shown.