Figure 6 shows the effects of hypoxia and CXCR4 stimulation with SDF 1 or CXCR4 blockade with AMD3100 on MMP1 mRNA expression and secreted active MMP1 protein. Hypoxia increased MMP1 mRNA expression 9 fold which was further increased to 23 fold by SDF1 stimulation. There was no effect of SDF1 or AMD3100 during normoxia on MMP1 mRNA things level. AMD3100 blocked the SDF1 mediated increase in MMP1 mRNA during hypoxia. Similarly, hypoxia and SDF1 increased active MMP1 in conditioned media of cells cultured in hypoxia. AMD3100 had no effect during hypoxia with out SDF1. AMD3100 in the presence of SDF1 had a similar effect as the MMP inhibitor O phenanthroline. Downstream effects of hypoxia Inhibitors,Modulators,Libraries and CXCR4/SDF 1 are mediated through ERK signaling In order to assess the role of MAP kinases in CXCR4/ SDF1 signaling, time course analysis of MAP kinase expression after SDF1 exposure was performed.

SDF1 stimulation Inhibitors,Modulators,Libraries during hypoxia transiently increased phos phorylated ERK which reached a peak at 10 minutes. The increase in phosphorylated ERK could be inhibited by MEK inhibitor U0126. There was less effect of SDF1 on phosphorylated JNK and no effect on p38. SDF1 stimulation during hypoxia also increased MMP1 protein expression. Both the CXCR4 inhibitor AMD3100, the ERK inhibitor U0126, and ERK1/2 siRNA inhibited MMP1 protein expression. The SDF1 mediated increase in cell invasion during hypoxia was also inhibited by U0126 and ERK1/2 siRNA, but not by the other MAP kinase inhibitors SP600125 and SB203580. Discussion A better understanding of the mechanisms underlying invasive behavior of a cancer is an important first step in developing improved treatment strategies.

This study provides the first indication that CXCR4 is regulated by hypoxia and specifically HIF 1a in chondrosarcoma cells. We also show that increased CXCR4 signaling regulates expression of MMP1, a factor known to be involved with chondrosarcoma metastasis and a marker for poor prognosis. Overexpression of CXCR4 has Inhibitors,Modulators,Libraries been reported in a variety of tumors, primarily carcinoma. In carcinoma, CXCR4 expression mediates metastasis to bone, which has relatively high Inhibitors,Modulators,Libraries levels of SDF1. In chon drosarcoma, it is possible that local SDF1 stimulates local tumor growth in a paracrine manner, Inhibitors,Modulators,Libraries and for those cells which gain access to the circulation, may also partially account for the tendency of these tumors to develop lung metastases, since the lung also contains high levels of SDF1. Factors such as MMP1 mediate local migration out of the microenvironment, ie stroma for carcinoma and bone for chondrosarcoma, and into the circulation. Factors furthermore such as CXCR4 mediate homing and growth at distant sites. Within sarcoma, CXCR4 expression has been detected in osteosarcoma and recently in chondrosarcoma.

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