CX3CR1 is expressed by prostate epithelial cells and androgens regulate the levels of CX3CL1/fractalkine in the bone marrow: potential role in prostate cancer bone tropism - PubMed (original) (raw)

CX3CR1 is expressed by prostate epithelial cells and androgens regulate the levels of CX3CL1/fractalkine in the bone marrow: potential role in prostate cancer bone tropism

Whitney L Jamieson et al. Cancer Res. 2008.

Abstract

We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide--an antagonist of the androgen receptor--as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma.

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Figures

Figure 1

Figure 1

Expression of CX3CR1 in prostate glands from human donors. Normal and malignant prostate glands stain either negative (A) or show different staining intensities and distributions for CX3CR1 (B and C). As previously reported by others (34), smooth muscle cells were consistently found positive for CX3CR1 (white arrow). CX3CR1 expression was also detected in tissue cores of skeletal metastases obtained from subjects affected by prostate adenocarcinoma (D). Original magnification, ×200.

Figure 2

Figure 2

Detection of both cell-bound and soluble forms of fractalkine in human bone marrow. A, normal bone marrow stains positive for fractalkine expression, showing a staining diffused throughout the entire marrow stromal architecture. B, cells of the bone marrow obtained from aspirates of three human donors express the full-length and cell-bound forms of fractalkine when analyzed by Western blotting. A recombinant, full-length form of fractalkine was loaded along with the cell lysates in the same gel and used as a positive control (top). The supernatants of bone marrow aspirates from five different human donors were analyzed by ELISA and tested positive for the soluble form of fractalkine (bottom). C, when distinct human bone marrow cell types were analyzed by Western blotting, they were all found positive for the full-length form of fractalkine, with the lowest expression levels detected in osteoblasts (NHOst), the highest in BSC and hMSCs showing intermediate levels. D, the location of fractalkine at the plasma membrane level was confirmed by fluorescence confocal microscopy (white arrows) in the hMSC shown; *, a possible intracellular storage for the chemokine (26). Original magnification, ×600.

Figure 3

Figure 3

The cleavage of fractalkine from the cell surface is regulated by the androgen receptor. hMSCs from bone marrow were exposed to 100 nmol/L of dihydrotestosterone for 1 hour with or without previous 30-minute incubations with 1 μmol/L of nilutamide. The dramatic decrease in full-length fractalkine induced by 100 nmol/L of dihydrotestosterone was completely blocked by nilutamide, whereas nilutamide alone did not significantly affect the levels of cell-bound fractalkine. A and B, results are representative of five separate experiments (*, P < 0.05). An ELISA-based assay was used to detect the soluble form of fractalkine in hMSC supernatants and shows that dihydrotestosterone induces the cleavage of the membrane-bound chemokine into its soluble form. C, three separate experiments were conducted using supernatants of hMSC cells obtained from different donors and assessed in duplicate (*, P < 0.05). D, subsequently, fractalkine at the cell surface is restored in a time-dependent fashion (top). The broad-spectrum MMP inhibitor GM6001 partially blocked the decrease in cell-bound fractalkine induced by dihydrotestosterone, confirming that the reduction in this chemokine level is a consequence of cleavage from the cells and that MMPs are involved in this process (bottom). For Western blotting analysis, a recombinant and full-length form of fractalkine was loaded along with the cell lysates in the same gel and used as a positive control.

Figure 4

Figure 4

Expression of fractalkine by HBME cells and lack of effect of dihydrotestosterone on its cleavage. A, confocal microscopy analysis shows that fractalkine is expressed on the plasma membrane of HBME cells. The exposure of these cells to 100 nmol/L of dihydrotestosterone did not affect the levels of plasma membrane fractalkine, even after prolonged treatment. B, the results are representative of five separate experiments. C, the lack of reduction in fractalkine at the cell surface corresponds to the absence of dihydrotestosterone-induced cleavage of the cell-bound chemokine into its soluble form, as established by an ELISA assay performed using supernatants of HBME cells derived from five separate experiments and assessed in duplicate. D, the premature and mature forms of ADAM-17/TACE protease were detected in hMSC and HBME cells by Western blotting (top). The hMSC express significantly higher levels of the mature ADAM-17 as compared with the HBME cells, which show almost exclusive expression of the premature form of the protease (bottom).

Figure 5

Figure 5

Possible roles of fractalkine and its receptor CX3CR1 in the adhesion of circulating prostate cancer cells to the bone marrow endothelium and their extravasation. A, the cell-bound fractalkine expressed on the surface of HBME cells directly functions as an adhesion molecule and allows CX3CR1-bearing cells to adhere to the vascular wall of bone marrow sinusoids. B, adherent cancer cells migrate into the marrow stroma following the concentration gradient of soluble fractalkine, produced and released by cells of the bone microenvironment.

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