Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin

Authors


Abstract

BACKGROUND:

Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis.

METHODS:

Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis.

RESULTS:

In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site.

CONCLUSIONS:

The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies. Cancer 2009. © 2009 American Cancer Society.

Osteopontin (for reviews, see Denhart et al, Furger et al, Rangaswami et al, and Weber1-4) plays a central role in the metastatic potential of both human and experimental tumors.5-7 Clinically, elevated levels of circulating osteopontin in cancer patients correlate with increased metastasis and poor prognosis for many solid tumors, documented most notably in malignant tumors of the breast,6, 8 prostate,9, 10 liver,11 and head and neck.12 Since the original discovery of this matricellular protein by Oldberg et al,13 the precise extracellular and intracellular mechanisms through which osteopontin promotes metastasis and correlates with poor prognosis in cancer patients have remained unresolved, although there is a recognized dependence on the arginine-glycine-aspartic acid (RGD) motif.5

Osteopontin (also termed secreted phosphoprotein-1, urinary stone protein, and early T-lymphocyte activation-1) is an acidic, secreted, noncollagenous matricellular protein with cytokine- and chemokine-like functions.1, 14 It was isolated first as a major bone sialoprotein containing an RGD motif.13 Early work established that osteopontin binds to αvβ3 integrin on osteoclasts; later, it became clear that osteopontin also recognizes several other members of the αv and β1 families of integrins (reviewed in reports by Denhart et al, Furger et al, Rangaswami et al, and Weber1-4). Such a broad integrin-binding profile indicates that osteopontin mediates cell adhesion, proliferation, migration, and survival. Therefore, it is likely to be relevant in the context of tumor progression, angiogenesis, and metastasis.

Herein, we have developed functional assays in vitro, in tumor cells, in mouse models, and ex vivo with patient-derived samples to gain mechanistic insight into the role of soluble osteopontin in metastasis. Together, the results indicate the existence of an RGD-dependent, osteopontin-triggered activation cascade involving the cellular homolog of the Rous sarcoma virus (c-Src) oncogene pathway in nonadherent tumor cells. Because we also have demonstrated that human cancer cells express functional osteopontin cell-surface receptors regardless of tumor type, these findings have potential implications for therapeutic intervention.

MATERIALS AND METHODS

Cloning and Production of Wild-Type Osteopontin and Mutant Osteopontin-Arginine-Glycine-Glutamic Acid

Total RNA was isolated from cultured human KS1767 Kaposi sarcoma cells with Trizol (Invitrogen, Carlsbad, Calif). After DNase (Promega, Madison, Wis) treatment, 1 μg of total RNA was transcribed with oligo dT primers and SuperScript III reverse transcriptase (Invitrogen). One microliter of combinational DNA was amplified with forward primer 5′-ACTCGGATCCATGAGAATTGCAGTGATTTGCTTT-3′ and reverse primer 5′-TTTGCGGCCGCTTAATTGACCTCAGAAGATG CACTATCT-3′ containing BamHI and NotI restriction sites (nucleotides shown in italics). After digestion with BamHI and NotI (Roche, Basel, Switzerland), polymerase chain reaction products were purified by electrophoresis and cloned into pGEX-6P-1 expression vector (GE Healthcare, Piscataway, NJ). Glutathione S-transferase (GST) and osteopontin with arginine-glycine-glutamic acid (RGE) was generated by amplification of the cloned osteopontin with the following primers: 5-prime end forward primer 5′-CTCGGATCCATGAGAATTGCAGTGATTTGCTTT-3′ and reverse primer 5′-AAACCACACTTTCACCTCGGCCATCATATGTGTCT-3′, and 3-prime end forward primer 5′-ATGGCCGAGGTGA AAGTGTGGTTTATGGACTGAGGT-3′ and reverse primer 5′-TTTGCGGCCGCTTAATTGACCTCAGAA GATGCACTATCT-3′ that generate 5′ and 3′ ends containing the aspartic acid to glutamic acid (Asp→Glu) mutation. The corresponding nucleotide for this single point mutation is shown in boldface. The ends were purified, mixed in 1:1 molar ratio, and used to generate a full-length mutant osteopontin-RGE. The amplification primers used were the same as those for wild-type osteopontin. The integrity of constructs was verified by DNA sequencing and by restriction enzyme mapping.

Purified or Recombinant Proteins and Synthetic Peptides

The GST-osteopontin (wild-type or mutant) constructs were used to transform Escherichia coli strain BL21 (Novagen, Madison, Wis). Recombinant proteins were produced as described previously.15 Briefly, cells were grown to an optical density of approximately 1.0, harvested, resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100 and protease inhibitor cocktail (Roche), and lysed by sonication. The lysate was cleared by centrifugation, and the GST proteins were bound to glutathione-Sepharose (GE Healthcare). After 3 washing steps, the proteins were eluted with reduced glutathione (Sigma, St. Louis, Mo).

Fibronectin was purified by affinity chromatography on gelatin-Sepharose.16 A polymeric form of fibronectin (superfibronectin) was generated as described previously.17, 18 Fibrinogen was purchased from Enzyme Research Laboratories (South Bend, Ind). Soluble peptides were synthesized and cycled (if necessary) at AnaSpec (San Jose, Calif) to our specifications.

Cell Culture

Cell culture reagents were purchased from Invitrogen unless indicated otherwise. Human tumor cell lines C8161 (melanoma) and KRIB (osteosarcoma) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, vitamins, nonessential amino acids, and antibiotics. Cells were grown to approximately 80% confluence at the time of use. After detachment with PBS containing 2.5 mM ethylenediaminetetraacetic acid, cells were washed 3 times with DMEM, counted, and resuspended in DMEM before subsequent treatments. Cell viability was monitored before and after treatments by counting of Trypan blue-excluding cells.

In Vivo Assay

All animal experimentation was reviewed and approved by each respective Institutional Animal Care and Use Committee at the Burnham Institute and at the University of Texas M. D. Anderson Cancer Center. Two-month-old nude mice (Harlan Sprague-Dawley) were anesthetized with Avertin (0.015 mL/g) and were injected intravenously (tail vein) with 106 tumor cells, which were treated previously with 200 μL DMEM containing either 1 μg/mL or 10 μg/mL of GST-osteopontin for 10 minutes at room temperature. The RGE mutant of GST-osteopontin was used in the same manner and at the same concentrations. Linear glycine-arginine-glycine-aspartic acid-serine-proline (GRGDSP) peptide and RGD-4C peptide were used at 1 mg/mL (n = 10 for each group). These experiments were repeated 3 times.

Metastases were monitored during the study by the killing of sentinel tumor-bearing mice from different cohorts at different time points (12-16 weeks) and by determination of metastatic tumor loads.18 In selected cohorts, the number of metastatic foci was counted under a dissecting microscope, and a histologic examination was performed after tissue fixation. Sentinel tumor-bearing mice served to estimate tumor burden before the termination of each cohort. Experiments were terminated when the animals displayed signs of discomfort or weight loss. Actuarial survival was demonstrated with Kaplan-Meier plots.19

Actin, Integrin, and CD44 Visualization

C8161 and KRIB cells were cultured on CultureWell cover glasses (Invitrogen). After several washes, the cells were stimulated with GST-osteopontin or GST-osteopontin-RGE for 10 minutes. Cells were washed 3 times with PBS, fixed with PBS containing 2% paraformaldehyde, rendered permeable with 0.2% Triton X-100 for 5 minutes, and stained with AlexaFluor488 phalloidin (Invitrogen), anti-integrin αv (Chemicon, Temecula, Calif), and anti-CD44 (clone IM7; AbCam, Cambridge, Mass) antibodies. These experiments were repeated 3 times in duplicate.

Cell Signaling

The cells were stimulated for 1 minute, 10 minutes, 20 minutes, 30 minutes, and 60 minutes in suspension; then, they were centrifuged and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, and 0.25% sodium deoxycholate, pH 7.4) containing complete protease inhibitors (Roche) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF; Sigma). Lysates (30 μg per lane) were resolved on sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with mouse antiphosphotyrosine (clone 4G10) and rabbit antiphospho-Src (Tyr-418) antibodies (Upstate, Temecula, Calif). Total Src was detected with rabbit anti-Src immunoglobulin G (Cell Signaling Technology, Danvers, Mass). These experiments were repeated 3 times.

Ex Vivo Cell Binding Assay

For analysis of the binding tumor cells to vascular endothelium, aortas were dissected from mice and cut open longitudinally; then, they were attached on 4% agarose with staples. Next, 105 tumor cells were treated in suspension for 10 minutes with osteopontin, with osteopontin-RGE, with osteopontin and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF; inhibitors were administered 2 minutes before osteopontin), or with DMEM. The cells were allowed to attach to the aortas (n = 3 for each group) for 10 minutes, then they were gently washed 3 times with PBS and fixed with 2% paraformaldehyde. The number of cells bound to the vascular endothelium was determined under the light microscope. This experiment was repeated twice.

Statistical Analysis

Because there were no substantially skewed data points and metastatic foci were distributed evenly throughout the lung, the statistical significance of the differences in the extent of metastases between tumor-bearing mice cohorts initially was determined by a t test. However, to expand statistical data to support the significance of our findings, we performed additional nonparametric tests (ie, Kruskal-Wallis and Wilcoxon). In addition, summary statistics, such as mean, median, and standard deviation, were calculated for lung weight and metastatic foci in the lung. Scatter plots were generated based on the results, and a bar was used on the plots to denote the mean for each group. Because of the number of samples in each cohort, nonparametric tests were carried out based on the ranks of each variable. Dunnett methodology was applied to adjust for the inflated type I error rate generated from multiple comparisons. This method provides a better power in comparisons between a single control and each of the treatment groups while holding the maximum experiment-wise error rate to a level that does not exceed the stated type I error rate. All of the statistical methods that we used supported our conclusions.

RESULTS

Human Melanoma and Sarcoma Cells Express Osteopontin Receptors

We selected well established cell lines that are of human origin, from different solid tumor pathologic types (ie, melanomas and sarcomas), and with wide metastatic capability. C8161 (human melanoma) and KRIB (human osteosarcoma) cells contain the principal known osteopontin receptors that include αvβ3 integrin, αvβ5 integrin, and CD44. The CD44 receptor mediates attachment, homing, and aggregation of immune system cells.4 Immunofluorescence revealed that all human tumor cell types studied expressed αv integrins and CD44 (Fig. 1). Flow cytometric analysis confirmed high expression of these osteopontin receptors (data not shown).

Figure 1.

Osteopontin receptors are expressed on human cancer cells. Cultured human C8161 melanoma and KRIB osteosarcoma cells were immunostained for 2 of the major osteopontin receptors: αv integrins and CD44. Abundant, punctuate αv integrin immunoreactivity was detected in all cell types. The same cells also displayed CD44 immunoreactivity. Negative staining controls (ctrl) with irrelevant isotype control plus secondary antibodies displayed no immunoreactivity. Scale bar = 20 μm.

Exogenous Soluble Osteopontin Increases Experimental Metastasis in Mouse Models in an RGD-Dependent Manner

We reasoned that osteopontin likely functions at several sites in the multistep metastatic cascade (eg, the primary tumor, the circulation, and/or the metastasis site). Relatively little is known about the role of circulating osteopontin. Therefore, we asked whether exogenous administration of osteopontin—which presumably would mimic what cancer cells encounter in the bloodstream—might have an effect on tumor cell adhesion, spreading, and metastasis. After confirming that tumor cells exhibited osteopontin surface receptors, we performed experimental metastasis assays in vivo. Tumor cell burden in the lungs was determined by measuring lung mass, a methodology we validated previously as a suitable surrogate.18 Treatment of cancer cells with recombinant osteopontin enhanced colony formation in the lungs (Fig. 2A). Treatment of C8161 melanoma cells with recombinant osteopontin was associated with a significant increase in lung weight (P < .01) (Fig. 2B) relative to negative controls that included mice treated with vehicle (DMEM) and control proteins. To exclude the possibility that the effect of exogenous osteopontin was restricted to a specific melanoma cell line, we treated the KRIB human osteosarcoma cell line in a manner identical to that of the C8161 cells. The metastatic enhancement effect of osteopontin on sarcoma cells was similar to that observed with melanoma cells. Consistently, osteopontin increased experimental lung metastasis of osteosarcoma cells in compared with negative controls (P < .01) (Fig. 2C).

Figure 2.

Tumor cell exposure to exogenous osteopontin (OPN) increases experimental metastasis that depends on the arginine-glycine-aspartic acid (RGD) motif. (A) Typical, gross appearance of lungs after intravenous administration of tumor cells. Arrows indicate tumor foci in the lungs. Scale bar = 0.5 cm. (B) C8161 melanoma cells were preincubated for 10 minutes with Dulbecco modified Eagle medium (DMEM), glutathione S-transferase (GST), or GST-OPN and subsequently injected intravenously into the tail vein of nude mice (106 cells per mouse). After 12 to 16 weeks, the animals were killed, and the lung weights were measured.18 The lung weight increased significantly (P < .01; asterisks) in nude mice that received human melanoma cells preincubated in GST-OPN compared with DMEM- or GST-treated cells. (C) Lung weight in mice that received human KRIB osteosarcoma cells pretreated for 10 minutes with GST or GST-OPN. The average normal lung weight (0.175 g) is indicated by the line. A significant increase in lung metastasis was observed when tumor cells were treated with GST-OPN compared with GST (P < .01). (D,E) Tumor cells were preincubated for 10 minutes with either GST-OPN-arginine-glycine-glutamic acid (RGE) or GST-OPN. The RGE mutation, which disrupts the integrin-binding RGD domain in OPN, abolished its effects on tumor cells, as observed in the statistically significant difference observed in lung weights of mice that received C8161 melanoma cells (P < .001) or KRIB osteosarcoma cells (P < .001). The average normal lung weight (0.175 g) is indicated by the line. (F) In a related experiment, mice received KRIB osteosarcoma cells that had been treated with either GST-OPN or GST-OPN-RGE before to administration. The survival of tumor-bearing mice was monitored, and the experiment was terminated at Day 80, as indicated.

After establishing that exogenous osteopontin increased experimental metastasis formation, we asked whether this action was integrin-mediated. It is well established that the tripeptide RGD is an essential integrin recognition site. Thus, to address this question, we replaced aspartic acid with glutamic acid (Asp→Glu) in the RGD motif of recombinant osteopontin (GST-osteopontin-RGE mutant protein) and treated human melanoma and osteosarcoma cells with it as we did with wild-type GST-osteopontin. Mutant GST-osteopontin-RGE treatment did not increase tumor metastasis to the lungs of either cell line (P < .001 for each) relative to tumor cells that were treated with vehicle only (Fig. 2D,E; and data not shown). This effect was reflected in the actuarial survival of the tumor-bearing mice. Indeed, mice that received osteosarcoma cells preincubated with wild-type osteopontin had a greatly reduced actuarial survival relative to mice that received cells preincubated with mutant osteopontin-RGE (Fig. 2F).

Enhancement of Experimental Metastasis Is Specific for Osteopontin

Because RGD is present in other adhesive extracellular proteins that bind to αv integrins, it is possible that the prometastatic effect of osteopontin might merely represent a nonspecific effect of any RGD motif in circulating proteins. To exclude this possibility, we compared the effects of 2 RGD-containing, circulating control proteins (fibronectin and fibrinogen) with the effects of osteopontin on experimental metastasis. Neither of these RGD-containing circulating proteins had detectable effects on lung colonization of C8161 melanoma cells (Fig. 3A). Finally, to analyze the role of soluble RGD-containing synthetic peptides in experimental metastasis formation, we also treated melanoma cells with the ‘classic’ linear RGD peptide (sequence GRGDSP20, 21) or with the double-cyclic RGD-4C peptide (sequence ACDCRGDCFCG), which binds selectively to αv integrins.22-26 In contrast to osteopontin, treatment with RGD synthetic peptides was associated only with a mild inhibitory effect on lung colonization (Fig. 3B). These data indicate that the osteopontin-mediated enhancement of experimental metastasis is relatively broad to different tumor cells but is specific to osteopontin.

Figure 3.

The arginine-glycine-aspartic acid (RGD)-dependent, prometastatic effect is specific for osteopontin (OPN). (A) C8161 melanoma cells were preincubated for 10 minutes in fibrinogen and fibronectin, both of which contain the integrin-binding RGD motif, or in glutathione S-transferase (GST)-OPN. Treatment with GST-OPN significantly increased metastasis formation compared with fibrinogen (P < .05; asterisks) and fibronectin (P < .05). The average normal lung weight (0.175 g) is indicated by the line. (B) In a similar experiment, mice received C8161 melanoma cells treated with Dulbecco modified Eagle medium (DMEM), 1 mM linear glycine-arginine-glycine-aspartic acid-serine-proline Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide, 1 mM double-cyclic RGD-4C peptide, or GST-OPN. The cohort that received cells treated with GST-OPN exhibited a significant increase in lung metastasis relative to the other groups (P < .001). Bars indicate mean ± standard deviation.

Soluble Osteopontin Modulates the Actin Cytoskeleton of Tumor Cells

Effective antigen-presenting cell (APC) interaction requires decreased cell rigidity, which is achieved by disengagement of the cortical actin cytoskeleton from the membrane.27 To evaluate whether osteopontin would similarly induce relaxation of the actin cytoskeleton to facilitate binding of tumor cells to the endothelium, we stained adherent human cancer cells with Alexa Fluor 488-conjugated phalloidin to reveal cytoskeletal changes after osteopontin stimulation. Consistently, treatment with osteopontin for only 10 minutes markedly altered the actin fiber network and reduced the density of transcytoplasmic actin cables relative to those in cells treated with osteopontin-RGE (Fig. 4). These results indicate that cytoskeletal configuration could be relevant to tumor/endothelial cell functional interactions, as observed in other cell types, such as certain specialized T-lymphocytes.27, 28

Figure 4.

Osteopontin (OPN) reduces actin fibers in adherent tumor cells. Adherent KRIB osteosarcoma cells were treated with glutathione S-transferase (GST)-OPN or with GST-OPN-arginine-glycine-glutamic acid (RGE) for 10 minutes; then, the cells were fixed and stained for cytoskeletal actin. Tumor cells that were treated with GST-OPN displayed a reduced actin network with a decrease in transcytoplasmic actin cables compared with cells that were treated with GST-OPN-RGE (arrows). Scale bar = 20 μm.

Osteopontin Activates Tumor Cells Through an Src-Mediated Signal Transduction Pathway

Osteopontin activates several intracellular signaling molecules after ligand-receptor binding to cell surface integrins.3 One such molecule is c-Src, which is a member of the nonreceptor protein tyrosine kinase family that plays a central role in signaling downstream from integrins that regulate actin dynamics.29 Therefore, we investigated the activation of c-Src in osteosarcoma cells after osteopontin stimulation.30 Surprisingly, we observed that rapid dephosphorylation of c-Src at Tyr-418 occurred when cells in suspension were stimulated with osteopontin for only 1 minute. When cells were treated with several protein or peptide controls (eg, mutant osteopontin-RGE, GST, or RGD-4C peptide) or were left unstimulated, such dephosphorylation did not occur (Fig. 5A). Moreover, this result also was observed when osteosarcoma lysates were analyzed for general tyrosine phosphorylation. Several proteins (predominantly between 50 and 98 kDa) were identified that were dephosphorylated rapidly after the exposure of cells to osteopontin compared with proteins after the exposure of cells to osteopontin-RGE or compared with proteins from unstimulated cells (Fig. 5B).

Figure 5.

Osteopontin (OPN) treatment is associated with dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src) oncogene in detached tumor cells. (A) KRIB osteosarcoma cells were detached from tissue culture substrates. Some of the cells were allowed to adhere to the culture dish, whereas others were treated for 1 minute with glutathione S-transferase (GST)-OPN, GST-OPN plus arginine-glycine-glutamic acid (RGE), GST, the arginine-glycine-aspartic acid (RGD)-4C peptide, or Dulbecco modified Eagle medium (DMEM) only. The cells subsequently were lysed, and 30 μg of the lysate were analyzed for the phosphorylation of tyrosine 418 (Tyr-418) c-Src. Adhesion of the cells to plastic induced the transient activation of c-Src. Treatment of the cells with GST-OPN induced dephosphorylation of c-Src. Analysis of total Src confirmed equal loading of the proteins. (B) Analysis of tyrosine phosphorylation of the same lysates (30 μg) revealed general dephosphorylation of tyrosine in proteins (range, 50- to 98-kDa proteins). The arrow indicates c-Src; cellular actin (40 kDa) was detected at the same time to ensure equal loading of the proteins.

Osteopontin Enhances Tumor Cell Binding to Vascular Endothelium

To determine whether the specific and rapid dephosphorylation of proteins that we observed had biologic relevance, we analyzed the binding of sarcoma cells to vascular endothelium after exposure to osteopontin but in the presence of phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). Indeed, osteopontin enhanced tumor cell binding to vascular endothelium, and phosphatase inhibitors completely abolished this binding (Fig. 6A,B), data indicating that the interaction of osteopontin with disseminated tumor cells leading to a rapid dephosphorylation of proteins, including c-Src, has a significant impact on circulating tumor cell behavior. This effect also was observed in vivo: The hearts of the mice contained more tumors after they were injected with cells that were treated with osteopontin relative to the cells that were treated with DMEM (Fig. 6C).

Figure 6.

The promotion of tumor cell binding to vascular endothelium by osteopontin (OPN). (A,B) KRIB osteosarcoma cells were treated with Dulbecco modified Eagle medium (DMEM), with glutathione S-transferase (GST)-OPN, with GST-OPN plus arginine-glycine-glutamic acid (RGE), or with phosphatase inhibitors before GST-OPN and subsequently were incubated with the endothelium of the dorsal aorta. Relative to the GST-OPN-RGE and phosphatase inhibition groups, GST-OPN significantly increased cell binding (arrows) to the aorta (P < .05; asterisks). Scale bar = 20 μm in A; in B, bars indicate mean ± standard deviation. (C) The number of tumor foci was increased in nude mice that received human sarcoma cells previously incubated with GST-OPN compared with DMEM-treated cells.

DISCUSSION

Osteopontin has pleotropic roles in tumor cell biology. Although it is involved in angiogenesis, apoptosis, anchorage-independent cell growth, and metastasis, the mechanisms that account for these functions have not been entirely explained.4 Many studies have demonstrated the effect of osteopontin in tumor growth and metastasis in different types of solid tumors and sites of metastasis.5-7, 31-34 However, those reports focused on the effect of endogenous osteopontin. Because osteopontin presumably functions at several sites in the multistep metastatic cascade, altering the level of endogenous expression of osteopontin does not necessarily elucidate the role of circulating osteopontin in metastasis. We reasoned that direct stimulation of tumor cells with exogenous osteopontin would minimize systemic effects and provide insight into whether circulating osteopontin affects the metastasis of disseminated tumor cells. We decided to use recombinant osteopontin, because cells adhere to recombinant and native osteopontin by similar mechanisms.15, 35 Our findings indicate that osteopontin has marked and reproducible, prometastatic effects on human melanoma and sarcoma cells. Osteopontin often induced a doubling in experimental lung metastasis that ultimately was reflected in a severely reduced actuarial survival rate of tumor-bearing mice. This result is particularly relevant, because cancer patients with increased serum osteopontin also have more metastases and poor overall survival.2-4, 6, 8-12 Although the lung colony assay indeed is not a bone fide metastasis model, and the findings may not apply generally to the metastasis of tumors in vivo, our functional results suggest that circulating osteopontin is not only a correlative biomarker for metastatic potential but also enhances the capacity of circulating tumor cells to establish metastases. Thus, the studies presented here provide a previously unrecognized function for circulating osteopontin.

The prometastatic effects of osteopontin require an intact RGD motif, an observation indicating that a molecular interaction with integrins is critical for the enhancement of lung colonization. Although this possibility may have been anticipated because the RGD domain in osteopontin contributes to lymphatic metastases of breast cancer,5 our work provides novel insights for this finding and for the role of osteopontin in metastatic activity. First, soluble exogenous osteopontin appears to function in a manner similar to that of endogenous osteopontin. Second, cells do not need to be adherent (ie, they can be in circulation) to respond to osteopontin. Third, our results demonstrate that osteopontin promotes RGD-dependent endothelial adherence of disseminated tumor cells and, thus, explain the early advantage of osteopontin to metastatic spread. Fourth, a previous report on the functional role of RGD used an osteopontin construct in which the entire RGD sequence was deleted5; in contrast, in the current work, we used the classic negative control (RGE) in this setting: a single point mutation (Asp→Glu) is less likely to generate unpredictable structural effects (ie, steric hindrance) within the osteopontin molecule.

Finally, the effects of osteopontin on lung colonization contrast with the effects of other (control) RGD-containing proteins and peptides; serum proteins, such as soluble fibrinogen and fibronectin, have little or no discernible effects. For an additional positive control, a polymeric form of fibronectin blocked metastasis (data not shown), an effect that was reported previously.18 Small RGD-containing peptides also exhibited a mild inhibitory effect on experimental metastasis; this result is consistent with several earlier reports in which it was demonstrated that soluble RGD peptides and RGD-containing snake venom proteins can reduce experimental metastasis.36-38 Therefore, osteopontin behaves quite differently from other RGD-containing molecules. Although the reason for this functional distinction is unclear, a possibility is that CD44 also functions as a receptor for osteopontin in our model.31, 39-41 Both CD44 and αv integrins are expressed on the tumor cells that were used in our studies, and we have confirmed that the isoforms v6 of 7 and v9 of CD44 that have demonstrated the ability to bind osteopontin35, 42 are expressed in osteosarcoma cells (unpublished observations). However, our findings in in vitro, in vivo, and ex vivo assays consistently indicated that the RGD motif is required for the prometastatic function of osteopontin. Whether or not other receptors are involved and how they cooperate with αv (and perhaps other) integrins in the interaction between cancer cells and osteopontin remain to be determined.

It is possible, although unlikely, that the prometastatic effects of osteopontin are caused by its direct effects on the host nude mice. First, the mixture of tumor cells and osteopontin results in an approximately 10-fold dilution of osteopontin (with a total blood volume in mice of approximately 2 mL). This dilution reduces the concentration of osteopontin below integrin-binding levels. Second, attached tumor cells responded to exogenous soluble osteopontin with a reduction in cytoskeletal actin fibers and, in suspension, with a rapid inactivation of c-Src that leads to enhanced binding to vascular endothelium. These in vitro events are host-independent and were osteopontin-specific. Thus, despite evidence that host-related factors also can influence metastasis,43 the in vitro data presented here clearly indicate that osteopontin directly affects tumor cells (the ‘seed’) rather than the lung microenvironment (the ‘soil’). However, our results do not exclude the nonmutually exclusive possibility that locally produced osteopontin (by host or tumor cells) also affects either tumor or host cells after the tumor cells exit the bloodstream and establish metastases at distant sites; this possibility can be addressed in further studies.

The oncogene Src is important in the regulation of cytoskeletal structure and actin dynamics. Osteopontin affects osteoclast migration and function; notably, osteoclasts are extremely motile, bone-resorbing cells with high rates of cytoskeletal turnover29 through c-Src.44 Thus, we evaluated c-Src activation in cancer cells after exposure to osteopontin by determining the state of phosphorylation in the conserved residue Tyr-418 within the activation loop. Phosphorylation of c-Src at that site enhances kinase activity.30 Unexpectedly, osteopontin induced rapid dephosphorylation of Tyr-418, indicating the inactivation of c-Src after osteopontin exposure. This new observation is in striking contrast to earlier reports describing the effects of osteopontin in the context of c-Src activation. However, all previous studies were carried out with immobilized osteopontin and adherent cells45 or in different experimental settings.44, 46 Indeed, we were able to confirm similar c-Src activation through Tyr-418 phosphorylation when cells were allowed to adhere to plastic dishes. Arguably, the analysis of c-Src phosphorylation in a cell suspension is more relevant, because circulating tumor cells encounter osteopontin in plasma. Our experiments demonstrate that rapid changes in signaling take place after tumor cells interact with osteopontin. This result is supported further by the finding that the addition of soluble osteopontin to adherent cells causes a reduction in actin stress fibers. In addition, exposure to osteopontin induced tumor cell binding to vascular endothelium, which was abolished by phosphatase inhibitors. Aortic metastasis rarely (if ever) occurs in patients, although it is reasonable to speculate that the strong blood flow accompanied by shear forces within the aortic circulation could retard or even prevent metastasis. Thus, we believe that this ‘reductionist’ aortic assay in vitro—although it is limited and restricted in scope relative to in vivo settings—may simulate at least some of the early general events that can occur when tumor cells encounter an intact vascular endothelium. On the basis of all of our in vitro results, we surmise that osteopontin may induce similar cytoskeletal arrangements in tumor cells that occur during T-cell activation,28 when interaction with APCs involves the formation of large areas of intimate cell-membrane contact. Faure et al reported that disanchoring of the cortical actin cytoskeleton from the plasma membrane decreases cellular rigidity and leads to more efficient T cell-APC complex formation; such disanchoring was achieved by the rapid inactivation of ezrin-radixin-moesin proteins through a vav 1 guanine nucleotide exchange factor (Vav1)-ras-related C3 botulinum toxin substrate 1 (Rac1) pathway.27 This type of cell relaxation could allow clustering of adhesion molecules on the cell membrane and, thus, facilitate tumor cell binding and penetration of the vascular endothelium. Although our results support this hypothesis, extensive further experimentation will be required to confirm and expand this interpretation.

In summary, the current results demonstrate that osteopontin has marked prometastatic effects in experimental models of metastasis. We also demonstrated that the RGD motif in osteopontin is functional and is required for the effects. Finally, in nonadherent cells, we observed Src dephosphorylation and enhanced tumor cell binding to the vascular endothelium that was blocked completely by phosphatase inhibitors. This osteopontin-dependent biologic phenomenon may be a molecular mechanism for tumor cells in suspension. If so, then osteopontin could affect Src regulation and the release of adhesion molecules from the cytoskeleton, a reaction allowing their rearrangement on the cell membrane that facilitates enhanced cell binding to vascular endothelium. Presumably, tumor cells undergo 2 types of osteopontin-mediated sequential effects: 1) an initial, rapid Src dephosphorylation accompanied by an increase in the compliance of the cell membrane while in circulation; and 2) a later Src phosphorylation after cells attach to a metastasis site. If these mechanistic data are confirmed, then they may lead to novel antimetastatic strategies in human cancer.

Acknowledgements

We thank Dr. Emmanuel Dias-Neto for comments on the manuscript and Dr. Amin Hajitou for technical assistance.

Conflict of Interest Disclosures

Supported by grants CA69306 (J.W.S.), CA30199 (R.P. and J.W.S.), and CA90270 (R.P. and W.A.) from the National Institutes of Health; grant DAMD 17-98-1-8041 from the Department of Defense (R.P.); and grants from the California Breast Cancer Research Program (J.W.S. and E.C.K.L.). The Army Breast Cancer Research Program (D.D.H.) provided additional support. W.A. and R.P. received support from the Gillson-Longenbaugh Foundation. J.M. received support from the Helsingin Sanomat Centennial Foundation, the Emil Aaltonen Foundation, the Research and Science Foundation of Farmos, and the Maud Kuistila Memorial Foundation.

Ancillary