Expression of the CXCR4-CXCL12 axis has been reported to coordinate events critical to tumor development and/or progression in (solid) tumors of different histological origin. The present study demonstrates an (expression level-dependent) negative prognostic impact of CXCR4 protein expression in therapy-naïve EWS and points to a role for the CXCR4-CXCL12 axis in promotion of EWS cell growth. CXCL12-dependent modulation of tumor cell proliferation and survival (under suboptimal conditions) has been observed in several tumor types, including ovarian carcinoma, small cell lung cancer and prostate cancer. Here, we demonstrate positive correlations between CXCR4 expression levels in therapy-naïve EWS and tumor volume at diagnosis. Moreover, and consistent with previous gene expression results[15, 24], we show expression of CXCL12 protein by most EWS tumors (65%) and, explicitly, within the tumor microenvironment of virtually all (>95%) EWS cases. Combined, these observations may reflect the existence of auto- and/ or paracrine growth stimulatory loops, mediated by the CXCR4-CXCL12 axis. Indeed, in vitro functional analyses demonstrate CXCL12-induced proliferation of EWS cell lines expressing substantial levels of CXCR4, which could be inhibited by CXCR4-antagonist AMD3100. Addition of AMD3100 alone did not interfere with spontaneous cell proliferation, suggesting a predominant role for paracrine (stroma-derived CXCL12) rather than autocrine (tumor cell-derived CXCL12) signalling. No correlations were observed with other established prognostic factors in Ewing sarcoma. Due to limited sample size, multivariate analysis to assess CXCR4 expression as independent prognostic factor in Ewing sarcoma could not be performed. Moreover, due to the nature of this (bone) tumor, attempts to establish primary tumor cell cultures from therapy-naive biopsies for evaluation of CXCL12-induced proliferation have so far been unsuccessful. Nuclear localization of CXCR4 has been described, and demonstrated to correlate with disease progression, in several distinct cancer types[25, 26]. Within our series of therapy-naive and metastatic Ewing sarcoma, however, no nuclear accumulation of CXCR4 has been observed.
Recently, CXCR4 gene expression was reported to associate with both EWS and osteosarcoma metastases[15, 27]. Although we previously observed a correlation between CXCR4 gene expression and disease extension/ metastatic disease at diagnosis (unpublished results), the current study does not shown any correlation between CXCR4 protein expression and occurrence of metastatic disease. Moreover, metastatic EWS lesions demonstrated significantly lower CXCR4 protein expression levels as compared to (corresponding) therapy-naïve tumors. Reduced expression of CXCR4 in metastatic lesions as compared to corresponding primary tumors has been reported in breast carcinoma, and hypothesized to be due to CXCL12-induced internalization and degradation and/or lower microenvironmental HIF-1α levels. With regard to EWS, no significant differences in CXCL12 protein expression levels (in neither tumor nor stromal areas) were observed between therapy-naïve and metastatic lesions (data not shown). Moreover, although no data exist on HIF-1α expression in metastatic EWS lesions, our in vitro and in vivo analyses revealed no effect of hypoxia on CXCR4 expression nor a correlation between HIF-1α and CXCR4 expression levels (Figure3B and Figure3C, respectively). An alternative explanation for the observed reduced expression of CXCR4 in metastatic as compared to therapy-naïve EWS lesions might be that the CXCR4-CXCL12 axis is essential for retention of EWS cells within the primary tumor site, as has been described for CD34+ hematopoietic stem cells and leukemic cells within the hematopoietic microenvironment and, more recently, for osteosarcoma. Hypothetically, reduced expression of CXCR4 might result in preferential metastasizing of individual cells, provided that alternative growth factors are present. Whether the apparent discrepancy in correlation of CXCR4 gene transcript () and CXCR4 protein expression (current study) with metastatic disease in EWS reflects true biological differences (e.g. differences at the mRNA level are not reflected at the protein level (or vice versa), due to post-transcriptional and/or -translational regulation) or are attributable to technical differences (e.g. different samples and/or sensitivity and dynamic ranges of the methods used for mRNA transcript and protein analysis) is not known. Based on our results, we delineate a role for the CXCR4-CXCL12 axis in promotion of EWS cell growth rather than its metastatic potential.
Hypoxia is a common phenomenon in (large and/or fast-growing) solid tumors, which is associated with therapy-resistance and represents an independent prognostic indicator of poor outcome. HIF-1α, being the best characterized inducer of gene transcription in hypoxic cells, is overexpressed in various cancer types including EWS[18, 20, 30], and a key role for this protein in hypoxic induction of CXCR4 has been described[3, 31]. Although the observed positive correlation between CXCR4 expression in therapy-naïve EWS and tumor volume at diagnosis might have been indicative for hypoxia-induced HIF1α-dependent CXCR4 activation, our analyses did not provide support for a contribution of hypoxia to CXCR4 expression in this tumor. In addition to the observed lack of correlation between HIF-1α and CXCR4 protein expression within individual tumor samples, culture of cell lines under hypoxic conditions did not affect CXCR4 surface expression. These observations are in line with results previously obtained by Aryee et al., demonstrating a lack of change in CXCR4 pathway genes upon hypoxic exposure.
Until rather recently, CXCR4 and CXCL12 were considered exclusive partners. However, a second CXCL12-binding chemokine receptor, CXCR7, was identified and demonstrated to be involved in progression of several cancer types, including (pediatric) sarcomas[29, 32, 33]. In Ewing sarcoma, CXCR7 gene expression was recently reported to associate with patient survival. As yet, no data exist on CXCR7 protein expression in Ewing sarcoma. Our preliminary results point to limited expression of this chemokine receptor in Ewing sarcoma cell lines (CXCR7 surface expression, as assessed by flow cytometry, in 1/10 cell lines). In vivo s tudies, using Ewing sarcoma samples or either murine or human (xenografted) tumor models may provide further insight into the role of the CXCR7-CXCR4-CXCL12 axis in Ewing sarcoma.
Hitherto, our results indicate that the CXCR4/CXCL12 axis is frequently expressed in EWS and affects tumor progression and patient survival by promoting cell growth. Successful inhibition of EWS proliferation by AMD3100, one of several CXCR4-specific antagonists that are currently being evaluated for treatment of patients with both hematological and solid tumors indicates that disruption of the CXCR4-CXCL12 axis may indeed interfere with EWS progression. Integration of strategies that target CXCR4 signaling into either first- or second-line treatment regimens may represent a promising treatment option for patients with EWS.