CXCR4–SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion

Abstract

We hypothesized that the CXC chemokine receptor-4 (CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4 ؉ tumor cells into the bone marrow and lymph nodes, which secrete the ␣-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3-and PAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHR gene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4-SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1)

CXCR4 expression reflects tumor progression and regulates motility of bladder cancer cells

“…It has also been shown that CXCL12 enhances MMP2 activity in rhabdomyosarcoma cell lines. 29 Thus, CXCR4 may coordinate cytoskeletal and proteolytic responses that culminate in tumor growth and metastasis.…”

Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer

“…Western blot analysis was performed on protein extracts from cells as described [33][34][35][36][37] after the lung cancer cells were stimulated with PMV or exosomes (30 g/mL) for 5 or 10 min at 37°C. Phosphorylation of serine/threonine kinase AKT and p44/42 mitogen-activated protein kinase (MAPK) was detected by protein immunoblotting using mouse monoclonal 44/42 phospho-specific MAPK antibody and rabbit phospho-specific polyclonal antibodies (all from New England Biolabs, Beverly, MA) for each of the remainder, with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) as secondary antibody, as described.…”

“…Phosphorylation of serine/threonine kinase AKT and p44/42 mitogen-activated protein kinase (MAPK) was detected by protein immunoblotting using mouse monoclonal 44/42 phospho-specific MAPK antibody and rabbit phospho-specific polyclonal antibodies (all from New England Biolabs, Beverly, MA) for each of the remainder, with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) as secondary antibody, as described. [33][34][35][36][37] Equal loading in the lanes was evaluated by stripping the blots and reprobing them with the appropriate monoclonal or polyclonal antibodies: p42/44 anti-MAPK antibody clone 9102 and anti-AKT antibody clone 9272 (Santa Cruz Biotechnology). The membranes were developed with an ECL reagent (Amersham Life Sciences, Little Chalfont, UK), dried and exposed to film (HyperFilm, Amersham).…”

“…33,40,41 The lower chambers contained media supplemented with PMV (30 g/mL) or media only (control). The cells pre-incubated with PMV, or not, were loaded onto the upper compartments (3 ϫ 10 5 cells/chamber) and incubated (at 37°C, 5% CO 2 ) for 48 hr.…”

“…After incubation, the plates were washed 4 times to remove nonadherent cells, and the number of adherent cells was estimated by measuring fluorescence in a spectrofluorimeter as described. 33,42 In vivo model of metastasis Five-to 6-week-old female C57BL/6 mice (National Cancer Institute) were injected intravenously with 10 4 -10 5 LLC cells that had been incubated or not with murine PMV. Incubation was done in 100 L of phosphate buffer saline (PBS) with 0.5% BSA, 1 mM CaCl 2 for 30 min at 37°C and at a PMV concentration of 300 ng/mL.…”