| PURPOSE: To study the involvement of the chemokine receptor CXCR4 in the outgrowth of colon carcinoma metastases, to test its involvement in other carcinomas and melanomas, and to study the possible role of other chemokine receptors in tumor growth. BACKGROUND: CXCR4 is the receptor for the chemokine SDF-1. We have shown that CXCR4 is essential for metastasis of a T-lymphoma, as SDF-1 stimulates invasion of lymphoma cells. Importantly, CXCR4 is also expressed in carcinomas and anti-CXCR4 antibodies were recently shown to inhibit metastasis of breast carcinoma. This was also ascribed to a role of CXCR4 in invasion. We also found that a block of CXCR4 function prevented metastasis in a colon carcinoma. However, this must be due to a role in tumor growth rather than invasion, for the following reasons: (1) Gi proteins are required for SDF-1-induced invasion, but blocking Gi had no effect on metastasis, and (2) CXCR4 levels are low on cultured tumor cells but increase strongly in vivo, after the metastasis has been established. Whereas SDF-1-induced Gi-dependent migration signals are rapid and transient, long-term exposure to SDF-1 results in persistent signals. These signals are involved in survival and proliferation and in blood vessel formation in the embryo, and in part independent of Gi proteins. We propose that these signals play a crucial role in the survival and growth of a micrometastasis. CXCR4-blocking drugs have been developed for AIDS therapy. Therefore, it is highly relevant to explore their potential utility and mechanism of action in impairing tumor growth. PLAN OF INVESTIGATION: (1) Metastatic growth of GFP- and luciferase-transfected CT26 murine colon carcinoma cells will be monitored using RT-PCR of GFP and in vivo imaging of luciferase activity, and GFP fluorescence and immune histochemistry on tissue sections. This will be correlated with CXCR4 levels, stromal cell influx and vascularization (collaboration with Dr. D. de Jong, Department of Pathology), to define the time window in which CXCR4 inhibition impairs growth, and reveal clues as to the underlying mechanisms. CXCR4 and SDF-1 will be constitutively expressed in CT26 cells, and effects on metastatic growth assessed. Finally, we will test whether the CXCR4 inhibitor AMD3100 blocks metastasis of these cells. (2) Our 'intrakine' approach will be used to knock down CXCR4 function, and novel retroviral siRNA technology to block CXCR4 expression in carcinoma (human HT29 colon, mouse TA3/St mammary, human PC3 prostate) and melanoma (mouse B16-F10, human BLM) cell lines, and effects on lung and liver metastasis will be assessed. This will indicate how general the CXCR4 effect is. (3) Microarray analysis of SDF-1-treated, CXCR4-transfected and CXCR4-blocked cells, in which Gi is inhibited by pertussis toxin, should reveal potentially critical changes in transcription. We will use specific inhibitors to define the signal pathways involved. To determine the relevance in vivo, these pathways and selected target genes will be suppressed by transfection of dominant-negative mutants or retroviral siRNA, and effects on metastasis assessed. (4) Next to CXCR4, we will test the role of CXCR5 in metastatic growth. We detected CXCR5 on colon and breast carcinoma, and found that CXCR5 is also strongly upregulated in vivo. CXCR5 binds to BCA-1 which is highly expressed in liver. Using similar approaches as for CXCR4, we will determine a possible role for CXCR5 in CT26 colon carcinoma metastasis. POTENTIAL RESULTS: This project will define the role of CXCR4 and potentially of other chemokine receptors in development of carcinoma and melanoma metastases, and may provide a basis for the use of CXCR4 inhibitors in cancer therapy. |
