Expression of CXCR4 and CXCR7 Receptors for Stromal Derived Factor-1 (SDF- 1) Is Differently Regulated in Human Rhabdomyosarcomas (RMS): The Biological Consequences
Authors: Tarnowski Maciek , Maksym Radoslaw , Reca Ryan , Ratajczak Mariusz Z , Kucia Magdalena 
Published: April 2020

Abstract
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates bone marrow (BM). RMS is divided in two sub-types, alveolar-RMS (ARMS) and embryonal-RMS (ERMS). The ARMS subtype is often associated with a more aggressive phenotype, poorer clinical prognosis, and expression of Pax3-FKHR and Pax7-FKHR fusion proteins that function as potent transcriptional activators with enhanced activity as compared to normal Pax3 or Pax7 transcription factors. We reported that the stromal derived factor-1 (SDF-1)-CXCR4 receptor axis plays a crucial role in RMS metastasis to BM (Blood2002;100:2597, Cancer Res2003;63:7926, Cancer Res.2007;67:213). After the recent identification of CXCR7, a new receptor for SDF-1, we became interested in its potential role in metastases of RMS cells. We found that CXCR7 was expressed and functional on all 10 human RMS cell lines investigated in this study. Furthermore, we noticed that while CXCR4 was more involved in regulating RMS chemotaxis, CXCR7 primarily regulated adhesion of RMS cells. Based on this observation, we assessed the mechanisms that regulate expression of these receptors. We noticed that CXCR4 is highly expressed on the more metastatic ARMS while CXCR7 is expressed at higher levels on the less metastatic ERMS cell lines. However, both receptors express several hypoxia-inducible factor (HIF)-1a binding sites, expression of CXCR4 was not affected by exposure to hypoxia, and expression of CXCR7 was even paradoxically downregulated in hypoxic conditions. We also observed that ERMS cells transduced with Pax3-FKHR, a gene typical for ARMS fusion that enhances RMS metastatic behavior, highly upregulated expression of CXCR4. In contrast, expression of CXCR7 was again down-regulated. To learn more on the role of Pax-3-FKHR in the expression of CXCR4, we performed a functional analysis of promoter fragments subcloned into luciferase vector. To our surprise, expression of CXCR4 did not depend on direct binding of Pax3-FKHR to classical Pax3 binding sites in promoter sequences but on direct protein-to-protein interaction with nuclear respiratory factor (NRF)-1 transcription factor. These results suggest that the Pax3-FKHR-NRF-1 complex binds to the NRF-1 binding site and aberrantly activates transcription. In conclusion, both of the SDF-1 binding receptors (CXCR4 and CXCR7) are differently regulated in RMS cells. Since CXCR7 is more important in adhesion and becomes downregulated during hypoxia, this may give an advantage for SDF-1 to signal through CXCR4 receptor to activate pathways critical for chemotactic responses. As a biological consequence, this may increase the metastatic potential of RMS by “mobilizing” RMS cells from the primary tumor to migrate and metastasize. In addition, the novel transcriptional mechanism for the Pax3-FKHR-NRF-1 complex in regulating CXCR4 receptors in RMS cells has been identified.