Osteopontin has pleotropic roles in tumor cell biology. Although it is involved in angiogenesis, apoptosis, anchorage-independent cell growth, and metastasis, the mechanisms accounting for these functions have not been entirely explained.4
Many studies have demonstrated the effect of osteopontin in tumor growth and metastasis, with different types of solid tumors and sites of metastasis.5
However, these reports have focused on the effect of endogenous osteopontin. Because osteopontin presumably functions at several sites in the multi-step 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 gain insight on 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
Our findings show that osteopontin has marked and reproducible pro-metastatic effects on human melanoma and sarcoma cells. Osteopontin often induced a doubling in experimental lung metastasis that was ultimately 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
Although the lung colony assay is indeed not a bone fide metastasis model and the findings might 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. Our functional results indicate 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 pro-metastatic effects of osteopontin require an intact RGD motif, an observation indicating that a molecular interaction with integrins is critical for enhancement of lung colonization. Although this possibility could perhaps have been anticipated because the RGD domain in osteopontin contributes to lymphatic metastases of breast cancer5
, 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 (i.e., 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 this 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 (i.e., 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. As an additional positive control, a polymeric form of fibronectin blocked metastasis (data not shown), an effect that has been previously reported.18
Small RGD-containing peptides also exhibited a mild inhibitory effect on experimental metastasis; this result is consistent with several earlier reports showing that soluble RGD-peptides and RGD-containing snake venom proteins can reduce experimental metastasis.36
Therefore, osteopontin behaves quite differently from other RGD-containing molecules. Although the reason for this functional distinction is unclear, one possibility is that CD44 also functions as a receptor for osteopontin in our model.31
Both CD44 and αv
integrins are expressed on the tumor cells used in our studies, and we have confirmed that the isoforms v6/7 and v9 of CD44 that have been shown to bind osteopontin35
are expressed in osteosarcoma cells (unpublished observations). However, our findings in in vitro, in vivo, and ex vivo assays consistently indicate that the RGD motif is required for the pro-metastatic 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 pro-metastatic effects of osteopontin are due to its direct effects on the host nude mice. First, 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 led to enhanced binding to vascular endothelium. These in vitro events are host-independent and were osteopontin-specific. Thus, despite evidence that host-related factors can also influence metastasis43
, 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 non-mutually exclusive possibility that locally produced osteopontin (by host or tumor cell) also affects either tumor or host cells after the tumor cells exit the bloodstream and establish metastases at distant sites; such 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
Thus, we evaluated c-Src activation in cancer cells after exposure to osteopontin by determination of the state of phosphorylation of 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, data indicating 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 the previous studies were carried out with immobilized osteopontin and adherent cells45
, or in different experimental settings.44
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 show that rapid changes in signaling take place after tumor cells interact with osteopontin. This result is further supported by the fact that addition of soluble osteopontin to adherent cells causes reduction of 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 but one might 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--while limited and restricted in scope relative to in vivo settings--might simulate at least some of the early general events that may occur when tumor cells encounter an intact vascular endothelium. Based on all our in vitro results, one might surmise that osteopontin induces similar cytoskeletal arrangements in tumor cells that occur during T-cell activation28
, when interaction with APCs involves the formation of large areas of intimate cell-membrane contact. Faure et al. has 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 through rapid inactivation of ezrin-radixin-moesin proteins via a Vav1-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, we show that osteopontin has marked pro-metastatic effects in experimental models of metastasis. We also show that the RGD motif in osteopontin is functional and required for the effects. Finally, in non-adherent cells we observed Src dephosphorylation, and enhanced tumor cell binding to the vascular endothelium that was completely blocked by phosphatase inhibitors. This osteopontin-dependent biological phenomenon might be a molecular mechanism for tumor cells in suspension. If so, 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 two types of osteopontin-mediated sequential effects: (i) an initial rapid Src dephosphorylation accompanied by an increase in the compliance of the cell membrane while in circulation, and (ii) a later Src phosphorylation after cells attach to a metastasis site. If confirmed, these mechanistic data might lead to novel anti-metastatic strategies in human cancer.