UNLABELLED BACKGROUND Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a... Show moreUNLABELLED BACKGROUND Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. METHODS To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. RESULTS Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p < 0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p = 0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p = 0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression. CONCLUSIONS Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma. Show less
Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast... Show moreHypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast and osteoblast function, we studied angiopoietin-like 4 (ANGPTL4), the top HIF target gene in an Illumina HumanWG-6 v3.0 48k array of normoxic vs. hypoxic osteoclasts differentiated from human CD14(+) monocytes (14.3-fold induction, P<0.0004). ANGPTL4 mRNA and protein were induced by 24 h at 2% O-2 in human primary osteoclasts, monocytes, and osteoblasts. ANGPTL4 protein was observed by immunofluorescence in osteoclasts and osteoblasts in vivo. Normoxic inducers of HIF (CoCl2, desferrioxamine, and L-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability. Isoform-specific HIF-1 alpha small interfering RNA ablated hypoxic induction of ANGPTL4 and of resorption, which was rescued by addition of exogenous ANGPTL4 (P<0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization. These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1 alpha deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.-Knowles, H. J., Cleton-Jansen, A.-M., Korsching, E., and Athanasou, N.A. Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4. FASEB J. 24, 4648-4659 (2010). www.fasebj.org Show less
