S100A10 is a member of the S100 family of proteins Citation[1] and was originally identified intracellularly in a complex with annexin A2 in many different cells and tissues. Studies from our laboratory have established that S100A10 also exists on the cell surface primarily as part of the annexin A2 heterotetramer (AIIt). AIIt has been shown to play a significant role in plasmin generation, which contributes to a number of disease processes, inflammatory cell recruitment and endothelial cell invasion Citation[2–6]. Controversially, it has been proposed that annexin A2 is the key subunit for plasmin generation in these systems Citation[7]. However, our laboratory has published a series of papers extensively characterizing the role that S100A10 plays in plasmin generation, which indicate that S100A10, and not annexin A2, is the protein responsible for regulating plasmin generation at the cell surface. We propose, rather, that annexin A2 serves to tether the complex to the cell membrane through its phospholipid binding sites and, by doing so, allows S100A10 binding to plasminogen, thereby facilitating its activation Citation[8–11]. Here we critically review the literature that proves that S100A10 is the plasminogen-binding subunit of the AIIt and is responsible for plasmin generation in oncogenesis.
In order for a protein to be considered a plasminogen receptor it must be demonstrated to bind plasminogen. Reports from three laboratories have established that intact annexin A2 does not bind plasminogen Citation[11–13], but it has been proposed that proteolytic cleavage at Lys-307 exposes a carboxy-terminal lysine, thus forming a plasminogen-binding site on annexin A2 Citation[13,14]. However, the proteinase involved in this cleavage event has not been identified and, importantly, cleavage of annexin A2 by plasmin has been ruled out Citation[14]. Furthermore, we have demonstrated that only intact annexin A2, and not a proteolytically processed annexin A2, is present on the surface of thioglycollate-elicited macrophages, suggesting that annexin A2 is not cleaved in vivo and is not involved in plasminogen binding and plasmin generation Citation[2]. Similarly, our laboratory has demonstrated that proteolyzed annexin A2 is not present on the surface of HT1080 fibrosarcoma cells that are actively engaged in plasmin generation Citation[15]. Surface plasmon resonance studies have demonstrated that S100A10 binds plasminogen at its carboxyl-terminal lysine and this binding event is essential for plasmin activation at the cell surface Citation[11]. Removal of the carboxy-terminal lysines of S100A10 by genetic manipulation or by carboxypeptidase B attenuates plasminogen and tPA binding Citation[16]. Binding of plasminogen to plasminogen receptors is known to promote an open, activation-susceptible conformation. Studies using plasminogen that was fluorescein isothiocyanate labeled at its active site showed that addition of either AIIt or S100A10 alone, but not annexin A2 alone, resulted in quenching of the fluorescence of plasminogen, suggesting that S100A10 promoted an open, activatable conformation of plasminogen Citation[8].
In order to establish the function of a protein as a plasminogen receptor it must be demonstrated that loss of this protein from the cell surface results in a loss of cellular plasmin generation. Numerous articles have been published demonstrating that knockdown of annexin A2 resulted in decreased plasmin generation, matrix remodeling and a dramatic loss in directed migration Citation[7,14,17,18]. However, annexin A2 knockdown results in concomitant loss of S100A10 Citation[3], so it is difficult to attribute these effects to annexin A2 or S100A10. Das et al. demonstrated that cleavage of annexin A2 by trypsin was necessary to activate plasminogen binding and plasmin generation was blocked by an inhibitory antibody to annexin A2 Citation[12]. Surprisingly, this inhibitory antibody reacted with the last ten amino acids of the carboxyl-terminus of annexin A2, a region of annexin A2 that should have been cleaved according to the Hajjar model Citation[12]. The cleavage of annexin A2 by a trypsin-like protease has not been demonstrated in vivo. By contrast, we have demonstrated that loss of S100A10 in colorectal and fibrosarcoma cells had no effect on cell surface levels of annexin A2, but plasmin generation was decreased by 75–90%. Surprisingly, our analysis revealed that the cell surface annexin A2 was intact and had not been proteolyzed, despite the fact that the cells were rapidly generating plasmin Citation[19,20]. More recently we showed that, compared with wild-type macrophages, macrophages from S100A10-null mice display a 45% reduction in plasmin generation in vitro and also show a significant loss of invasive capabilities both in vivo and in vitroCitation[2]. Therefore, although a role for S100A10 as a plasminogen receptor is supported by a large body of data, the role for annexin A2 as a plasminogen receptor has yet to be rigorously established.
Our laboratory has reported in multiple publications the importance of S100A10 as a plasminogen receptor in oncogenesis. Plasmin has long been established as an important protease involved in cancer cell migration and invasion and several studies from our laboratory have shown that S100A10 plays an important role in tumor invasion and metastasis. Choi et al. demonstrated that injection of S100A10-depleted HT1080 fibrosarcoma cells into mice resulted in a threefold decrease in the number of metastatic foci in the lungs compared with mice injected with control HT1080 cells, and overexpression of S100A10 in the HT1080 cells resulted in a 16-fold increase in the number of lung metastases in these mice Citation[20]. Furthermore, S100A10-depleted HT1080 cells produced dramatically smaller tumors in severe combined immunodeficiency mice compared with those produced by the vector control HT1080 cells. These results show that metastasis of HT1080 tumor cells is significantly impaired by decreased expression of S100A10. Another study from our laboratory demonstrated that S100A10-depleted CCL-222 colorectal cancer cells displayed dramatic reductions in plasminogen binding, plasmin generation and basement membrane degradation Citation[19]. Of particular interest to this study was the fact that CCL-222 cells do not express annexin A2 on the cell surface. This suggests that the proteolytic capabilities of these cells, in terms of regulation of plasmin, were independent of annexin A2 and largely dependent on S100A10.
We have demonstrated that S100A10 plays a significant role in mediating plasmin generation at the surface of macrophages Citation[2]. This has implications not only in immune cell function but also in cancer progression. Tumor-associated macrophages (TAMs) represent a prominent component of the inflammatory cell population of solid tumors and the density of these TAMs within a tumor correlates with a poor prognosis Citation[21]. It has been hypothesized that macrophages mobilize a number of cell surface plasminogen receptors to generate plasmin, thereby facilitating proteolysis of basement membrane and extracellular matrices. We demonstrated that S100A10 plays a significant role in tumor growth due to the decreased proteolytic capability of tumor-associated cells, specifically TAMs Citation[4]. Lewis lung carcinoma cells were injected into wild-type and S100A10-null mice, tumor growth rates were measured, and it was found that tumors from S100A10-null mice were ten-times smaller than those from their wild-type counterparts. Further analysis revealed that macrophage infiltration into the tumor was severely impaired in the S100A10-null mice, resulting in decreased tumor size and volume. Interestingly, peritoneal injection of wild-type macrophages into S100A10-null mice prior to injection of Lewis lung carcinoma cells resulted in tumor growth rates and tumor sizes that were comparable with those in wild-type mice.
Acute promyelocytic leukemia (APL) is characterized by disseminated intravascular coagulation and excessive fibrinolysis, which is thought to be a result of the increased production of plasmin. This excessive production of plasmin can result in internal hemorrhaging that is often fatal. Previous work has shown that annexin A2 levels are elevated in APL and annexin A2 protein levels have been implicated as the cause for the excessive fibrinolysis that is associated with the disease. It was observed that, when APL cells isolated from human patients were treated with all-trans retinoic acid (ATRA), expression of cell surface annexin A2 was lost and in vitro treatment of the (15;17)-positive human APL cell line, NB4, with ATRA significantly reduced both the cellular expression of annexin A2 and plasmin generation Citation[18]. However, since annexin A2 knockdown results in loss of S100A10, it is difficult to be certain whether these effects are a result of loss of annexin A2 or S100A10. We demonstrated that S100A10 plays a significant role in regulating the generation of plasmin at the surface of APL cells using NB4 cells as a model system for studying plasminogen receptors of leukemic promyelocytes Citation[5]. Depletion of S100A10 by RNAi resulted in a 70% loss in plasminogen binding, a 64% loss in plasmin generation by the NB4 cells and 60% fewer NB4 cells migrating through a fibrin barrier. This established the importance of S100A10 in plasmin generation in promyelocytic leukemia cells. Furthermore, we treated NB4 cells with ATRA, which resulted in a rapid loss of S100A10, concomitant with a 60% loss in plasminogen binding, 40% loss in plasmin activity and a 60% loss in migration through a fibrin barrier. The complete loss of cellular levels of S100A10 after ATRA treatment presents the possibility that the remission of hemorrhagic complications and oncogenic phenotype of the leukemic promyelocytes observed during treatment of APL patients with ATRA could be due to the ATRA-mediated loss of S100A10 from the surface of the leukemic promyelocytes.
These data strengthen our hypothesis that annexin A2 anchors S100A10 to the cell surface and, in doing so, allows S100A10 to play a prominent role in the activation of plasminogen in angiogenesis and oncogenesis.
Financial & competing interests disclosure
This work was supported by grants from the Canadian Cancer Society Research Institute and Canadian Institutes of Health Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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References
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