PERSPECTIVE For reprint orders, please contact: [email protected]

On the contribution of S100A10 and annexin A2 to plasminogen activation and oncogenesis: an enduring ambiguity Moamen Bydoun1 & David M Waisman*,1,2

ABSTRACT Plasminogen receptors are becoming increasingly relevant in regulating many diseases such as cancer, stroke and inflammation. However, controversy has emerged concerning the putative role of some receptors, in particular annexin A2, in binding plasminogen. Several reports failed to account for the effects of annexin A2 on the stability and conformation of its binding partner S100A10. This has created an enduring ambiguity as to the actual function of annexin A2 in plasmin regulation. Supported by a long line of evidence, we conclude that S100A10, and not annexin A2, is the primary plasminogen receptor within the annexin A2-S100A10 complex and contributes to the plasmin-mediated effects that were originally ascribed to annexin A2. A plethora of studies have documented the importance of plasminogen receptors in cell surface regulation of plasmin production using both physiological and pathological models [1,2] . A canonical plasminogen receptor possesses a carboxyl-terminal lysine residue that directly binds the kringle domain(s) of plasminogen. This binding induces the localization of plasminogen with plasminogen activators, namely the urokinase plasminogen activator (uPA) and the tissue plasminogen activator (tPA). These activators, by themselves, have a limited capacity to activate plasminogen and hence require a receptor to bind plasminogen. The formation of a plasminogen-receptor complex induces a conformational change in plasminogen from a compact poorly activatable to a more open structure. In its open conformation, plasminogen is readily acted upon by the plasminogen activators. The active plasmin is generated in proximity with plasminogen receptors and acts as a multifunctional protease to cleave extracellular matrix components, release trapped growth factors within the matrix, activate pro-uPA into active uPA (through a positive feedback loop), activate other proteases predominantly pro-matrix metalloproteinases (pro-MMPs) into active MMPs and act as a signaltransducing ligand. This diversity of plasmin function renders the plasminogen activation system crucial in not only maintaining normal body physiology but also, when deregulated, in promoting the pathology of some diseases. The heterotetrameric complex (AIIt) formed by annexin A2 and S100A10 is a classic example of how plasminogen receptors are implicated in pathological conditions such as inflammation, stroke and cancer [3] . However, the contributions of the annexin A2 subunit to these conditions have been ambiguous primarily because a significant number of published studies have not taken into consideration that manipulating cellular levels of annexin A2 inevitably affects cellular S100A10 levels. This has much created confusion within the literature. It is now well established that the loss of annexin A2 results in concomitant loss of S100A10 [4,5] . This is critically important because unlike annexin

KEYWORDS 

• ambiguity • annexin A2 • cancer • fibrinolysis • heterotetramer • invasion • plasmin • plasminogen • S100A10

Department of Pathology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, PO Box 1500, Halifax, Nova Scotia, B3H 4R2, Canada 2 Department of Biochemistry & Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, PO Box 1500, Halifax, Nova Scotia, B3H 4R2, Canada *Author for correspondence: Tel.: +1 902 494 1803; Fax: +1 902 494 1355; [email protected] 1

10.2217/FON.14.163 © 2014 Future Medicine Ltd

Future Oncol. (2014) 10(15), 2469–2479

part of

ISSN 1479-6694

2469

Perspective  Bydoun & Waisman A2, S100A10 is a well-characterized plasminogen receptor. Since most studies utilize cells depleted of annexin A2 (using short hairpin RNA), it has been difficult to ascribe the observed decline in plasminogen activation to annexin A2 depletion. This is further supported by direct evidence, which demonstrates that annexin A2 does not bind plasminogen or tPA [6] . In fact, it is now well accepted that S100A10 is the only/primary plasminogen receptor within the heterotetramer and is therefore the main regulator of plasminogen activation [4–5,7–11] . Therefore, the purpose of this review is to disambiguate the postulated function of annexin A2 as a plasminogen receptor. Upon rigorous analysis of published data, we conclude that annexin A2 is not a plasminogen or tPA receptor and that reports that suggest a role for annexin A2 in cancer cell invasion or fibrinolysis, based on annexin A2 depletion experiments, are likely inaccurate. The necessity of C-terminal lysines for plasminogen binding: why is annexin A2 not a plasminogen receptor? In 1994, Hajjar et al. purified a 40 kDa protein from human placental extracts by PAGE and showed that it bound plasminogen. The protein was then identified as the calcium-dependent phospholipid-binding protein, annexin A2 (sometimes referred to in the literature as p36). Since the ability of the purified protein to bind plasminogen was compromised by carboxypeptidase B, Hajjar et al. concluded that it possessed a carboxyl-terminal lysine, which participated in plasminogen binding. However, since fulllength annexin A2 possesses a carboxyl-terminal aspartic acid residue (Asp-338) and not a lysine residue, Hajjar et al. postulated that cell surface annexin A2 was proteolytically cleaved at the Lys307-Arg-308 bond through a post-translational event, which exposed a new carboxyl-terminal lysine residue (Lys-307) [12] . It therefore became entrenched in the literature that the extracellular surface form of annexin A2 was the protein that was truncated at Lys-307. However, cleaved annexin A2 has never been detected on cells that are actively engaged in plasmin generation [4–5,13] . It is also now well established that the extracellular form of annexin A2 is full-length (Ser-1-Asp-338), which does not possess a carboxyl-terminal lysine (Table 1) [13] . In addition, it is an enduring ambiguity how the entirety of the annexin A2 preparation isolated

2470

Future Oncol. (2014) 10(15)

by Hajjar et al. would be proteolytically cleaved since only 4–10% of the total cellular annexin A2 is present on the cell surface [12] . Therefore, it would be expected that the extracellular protease responsible for processing annexin A2 would process the extracellular pool only. Since 90% of the purified tissue protein was of intracellular origin and at best 10% of extracellular origin and cleaved at the cell surface by a theoretical protease, it is difficult to rationalize how their preparation could be homogenous (reported as a single band on SDS-PAGE gel). Moreover, the cleavage of annexin A2 at Lys-307 results in a truncated protein that can be easily distinguished from the intact protein on a SDS-PAGE [4] . It is therefore more likely that the purified protein, regardless of whether it was extracellular or intracellular, was completely proteolyzed during purification and consisted of an N-terminal fragment and truncated annexin A2 with a carboxyl-terminal lysine residue to which plasminogen could bind. Cesarman et al. also reported that their purified annexin A2 used was not cleaved by plasmin [14] . However, it is now well accepted that the amino-terminal region of intact annexin A2 is a direct target for many proteases including trypsin, chymotrypsin [16–18] and importantly, plasmin (at Lys-27) [8,19] . For that reason, standard purification methods of annexin A2 from recombinant or tissue sources require extensive use of protease inhibitors as it is readily cleaved resulting in the release of several well-defined amino-terminal fragments [20] . It is then unclear how the 40 kDa annexin A2 protein, purified by Hajjar et al. and Cesarman et al., bound plasminogen. Therefore, the annexin A2 used in these 1994 studies was likely proteolyzed during ­purification and hence was physiologically irrelevant. Subsequent studies by our group have identified that annexin A2 exists as part of a heterotetrameric complex with S100A10 [(Annexin A2)2-(S100A10)2] at the surface of endothelial cells [5] , macrophages [10] and several cancer cell lines [21] . In 2003, we characterized how the carboxyl-terminal lysine of the S100A10 component directly bound plasminogen and accounted for the stimulation of tPA-dependent plasminogen activation [6] , a role that was originally ascribed to annexin A2 [14] . Despite such characterization, later studies by several laboratories reported that loss of plasminogen binding and activation ensued from depletion of cellular annexin A2 [14] (reviewed in [3]). However, with recent reports showing that depletion of annexin A2 results in

future science group

S100A10: plasminogen activation & oncogenesis 

Perspective

Table 1. Summary of PROS and CONS of the functions of annexin A2 as a plasminogen and tissue plasminogen activator binding protein. Pros

Cons

Plasminogen Annexin A2 purified from human placenta binds plasminogen [12,14]

Tissue annexin A2 does not possess a C-terminal Lysine. It should not be affected by carboxypeptidase B   Plasminogen binding by purified annexin A2 was initiated by carboxypeptidase B [12,14]. The purified annexin A2 was then degraded during purification Knockdown or knockout of annexin A2 reduces plasminogen binding Knockdown or knockout of annexin A2 affects expression of the and plasmin generation plasminogen receptor S100A10 as well as other proteins/genes (Figure 1)

tPA Annexin A2 purified from human placenta binds tPA by forming a disulfide bond (through Cys-8)       Annexin A2 is the major target for homocysteine   Homocysteine blocks the fibrinolytic activity of annexin A2

Binding of tPA is blocked by agents that mimic the C-terminal lysines of cell surface tPA receptors tPA reversibly binds C-terminal lysines Disulfide bonds are irreversible tPA binding to Cys-8 cannot occur physiologically Annexin A2 was present in the unbound fraction of a tPA affinity resin Cys-8 of annexin A2 is a reactive thiol that is redox active Genetic mouse models of hyperhomocysteinemia have high blood levels of homocysteine but normal rates of fibrinolysis

The pros represent postulated functions of annexin A2 based on earlier work by Hajjar et al. [15]. The cons describe most of the validated functions of annexin A2 as corrected in the literature. tPA: Tissue plasminogen activator.

the concomitant loss of S100A10 [22] through ubiquitin-mediated proteasomal degradation [23] , it has become inaccurate to attribute plasminogen binding to annexin A2 (Table 1) . This was later elucidated by a series of findings where the depletion of S100A10 dramatically affected cellular plasminogen binding and plasmin generation (discussed later) and has further supported the possibility that most if not all of the reports supporting a role for annexin A2 as a plasminogen receptor were misinterpreted. Consequently, the postulated role of annexin A2 as a plasminogen receptor has become an enduring ambiguity. The irreversibility of disulfide bonds: why is annexin A2 not a tPA receptor? The pioneering work of Lindsey Miles, Jordi Felez and Edward Plow defined the physiological rule book for the regulation of plasmin generation at the cell surface [24] . Using nine different cell types, they demonstrated that tPA and plasminogen competed for the same cell surface binding sites. In fact, Felez et al. showed that tPA could block more than 95% of the plasminogen binding to cells [24] . The addition of the lysine mimetic ε-aminocaproic acid or removal of lysine residues from cell surface proteins with carboxypeptidase B similarly affected both tPA

future science group

and plasminogen binding to the cell surface. Therefore, it was concluded that carboxyl-terminal lysines of cell surface proteins provide a common and reversible binding site for both tPA and plasminogen [24,25] . Since plasminogen and tPA have similar binding sites, another enduring ambiguity manifested itself as whether annexin A2 is a tPA-binding protein and whether an interaction between annexin A2 and tPA occurs under physiological conditions. Contradicting with Felez et al. and others [26–28] , reports by Hajjar et al. indicated that annexin A2 bound tPA and plasminogen at distinct sites and that t-PA did not compete with plasminogen for binding to annexin A2. The reported Kd for the tPA binding to purified annexin A2 was 25 nM [12] , which was approximately 30-fold higher than the binding of tPA to endothelial cells [25] . The postulated site of binding was a specific cysteine residue on annexin A2, namely Cys-8, which formed a disulfide bond with tPA [12] . Since disulfide bonds are covalent and irreversible, it was therefore not possible that plasminogen could compete with tPA for a common binding site on annexin A2. It is now well established that the kringle-2 domain of tPA reversibly binds to lysine residues of surface proteins [26] and this interaction does not

www.futuremedicine.com

2471

Perspective  Bydoun & Waisman involve forming disulfide bonds as it was previously documented by Hajjar et al. [15,29] . In fact, a recent study has verified that tPA binding to the endothelial cell surface does not co-localize with annexin A2, which indicates that annexin A2 is not a tPA receptor on the surface of these cells (Table 1) [27] . A follow-up study by Hajjar et al. showed that Cys-8 of annexin A2 forms a disulfide bond with homocysteine and it competed with tPA [30] . This was particularly difficult to understand because homocysteine does not form a disulfide bond with thiol groups unless the thiol is oxidized and present as a sulfenic group (-SOH) or as a mixed disulfide (-S-S-R) [31] . Therefore, it is likely that the Cys-8 thiol of annexin A2 used in these studies was oxidized. The reduced form of annexin A2 has been shown not to bind tPA [6] . Annexin A2 can only form a disulfide bond with a free thiol group. However, the only free thiol group is in the EGF domain of tPA, which does not participate in mediating tPA binding to the cell surface [28] . Therefore, it remains unclear how homocysteine targets Cys-8 of annexin A2 and accounts for decrease in fibrinolytic activity as it was documented by Hajjar et al. [30] . Not surprisingly, a comparison of wild-type mice with a genetically-engineered hyperhomocysteinemic mice, which exhibited high levels of homocysteine in their blood, failed to show any difference in fibrinolytic rates [32] , indicating that either homocysteine is not targeting annexin A2 in this model or homocysteinylated annexin A2 does not affect the in vivo rates of fibrinolysis. It is then unlikely that this putative interaction of annexin A2 with tPA or homocysteine is of any physiological significance. S100A10 binds plasminogen and stimulates tPA-dependent activation of plasminogen: why is S100A10 the only plasminogen receptor within the tetramer? Our group used multiple approaches to study the mechanism by which AIIt participates in plasmin regulation. In one approach, plasminogen was modified in which the serine of the plasmin catalytic site was replaced by a cysteine, which was subsequently labeled with fluorescein. Fluorescein acts as a reporter group and its quenching is indicative of conformational changes. We observed that purified AIIt binding induced a large conformational change in [Glu]-plasminogen (S741C-fluorescein) with a Kd of 1.26 μM [13] .

2472

Future Oncol. (2014) 10(15)

Interestingly, purified monomeric annexin A2 did not produce a conformational change in the [Glu]-plasminogen [13] , suggesting that either annexin A2 did not bind plasminogen or that the interaction of plasminogen with annexin A2 was mechanistically distinct than with AIIt. We then compared the ability of annexin A2 and AIIt to stimulate tPA-dependent plasminogen activation. The complex stimulated a 300fold increase in plasminogen activation in the presence of tPA compared with an approximate sixfold stimulation by monomeric annexin A2. Furthermore, the complex produced a 90-fold increase in the catalytic efficiency (Kcat/K m) of t-PA for [Glu]-plasminogen [8,33] . Surface plasmon resonance analysis showed that AIIt bound both tPA (Kd, 0.68 μM) and plasminogen (Kd, 0.11 μM), while monomeric annexin A2 failed to do so [6] . Collectively, these studies were the first to determine the affinity of the AIIt complex for plasminogen and to define the mechanism by which the complex stimulated tPA-dependent conversion of plasminogen into plasmin. In order to address the discrepancy between the ability of annexin A2 and the complex to bind plasminogen and promote tPA-dependent plasminogen activation, we expressed human annexin A2 and S100A10 in bacteria. Recombinant S100A10 stimulated a 46-fold increase in tPAdependent plasmin generation compared with a twofold increase by recombinant annexin A2 and 77-fold increase by recombinant AIIt [6,33] . This suggested that within the complex, S100A10 was the key plasminogen-binding protein and that the binding of annexin A2 to S100A10 stimulated S100A10-dependent plasmin generation. To rule out the possibility that annexin A2 may require S100A10 binding to be able to bind plasminogen, we compared the activity of recombinant AIIt formed by the combination of wildtype annexin A2 with either wild-type S100A10 or a mutant S100A10 (p11ΔKK), which lacks two C-terminal lysines. We observed that the mutant complex possessed about 12% of the activity of the nonmutated complex, which emphasizes the importance of the two C-terminal lysines of S100A10 in plasminogen binding. Not surprisingly, carboxypeptidase B treatment cleaved the terminal lysines of the native [(Annexin A2)2, (S100A10)2] and resulted in an 80% loss of activity by the complex [34] . Noteworthy, it was reported that S100A10 is also a substrate for other carboxypeptidases, including TAFI and carboxypeptidase N [34] . We then compared the

future science group

S100A10: plasminogen activation & oncogenesis  activity of recombinant AIIt with a truncated AIIt in which wild-type S100A10 was combined with a peptide to the first 15 amino acids of the N-terminus of annexin A2. Since this region of annexin A2 encompasses the S100A10 binding site, the mutant was unable to induce a conformational change in S100A10 and hence dramatically reduced its plasmin-generating activity [35] . Collectively, these reports confirm that fulllength annexin A2 is not a plasminogen receptor and that the plasmin-regulatory function of the tetramer is primarily contributed by the S100A10 subunits (Table 1) . Hence, S100A10 emerges as the sole plasminogen receptor within the AIIt complex, which brings into question the validity of previous studies that attributed the reduction in plasminogen activation to the loss of annexin A2 with disregard to S100A10 involvement (discussed next). Misinterpreting the individual contribution of annexin A2 to malignancy: how much can we rely on annexin A2 depletion studies? Multiple reports have demonstrated that depletion of annexin A2 in cancer cells resulted in loss of plasmin generation and reduction of tumor burden, invasiveness and/or metastasis [36,37] (also reviewed in [3] ). This experimental depletion approach may not be adequate or sufficient to establish a role of annexin A2 in tumorigenesis as these studies have disregarded the individual contribution of annexin A2 and S100A10 to plasmin regulation. Consequently, they have been unsuccessful in characterizing a well-defined role for annexin A2 in plasmin generation during oncogenesis. An increasing number of laboratories have come to realize that annexin A2 depletion experiments are not direct proof of a link between annexin A2-dependent plasmin generation and oncogenesis. Rather, considering the tenuous link between annexin A2 and plasmin regulation, the fact that annexin A2 is not a plasminogen receptor and annexin A2-dependent loss of S100A10 reduces plasmin production, it is far more likely that the reported link between annexin A2-dependent plasmin regulation and oncogenesis is actually misinterpreted and can be more accurately attributed to S100A10-dependent plasmin regulation. This has been further supported by experiments in which selective depletion of S100A10 decreases plasmin production and mitigates oncogenesis (discussed in V) (Table 1) .

future science group

Perspective

Since annexin A2 associates with some signal-transducing receptors [3,19] , it is plausible that knockdown of annexin A2 might affect the transcription of downstream genes. In fact, multiple studies did not take into account the possible involvement of other genes whose expression is dramatically affected by annexin A2 depletion. Gou et al. identified 61 genes in type II alveolar cells that were deregulated by the shRNA knockdown of annexin A2 [38] . These genes are involved in several physiological processes such as membrane fusion, cell adhesion and lipid metabolism. Through a comprehensive screen of the literature and the Gene Ontology database, we stratified these genes (with ≥twofold change) based on their roles in cancer progression, especially in terms of cell survival, proliferation, invasion and metastasis (Figure 1) . For instance, SP4, PSTI, ID1 and KRT19, which are well-established oncogenes, are found to be downregulated. In contrast, many of the upregulated genes have been identified as tumor suppressors (p55CDC and DCX). Increased expression of oncogenes and/or decreased expression of tumor suppressors can directly contribute to the regression of tumors seen in ANXA2-knockout mouse models [38] . In addition, Hou et al. revealed that ANXA2 -/L5178Y mouse lymphoma cells have reduced protein expression of S100A10, plasmin and fascin, all of which are key regulators of cancer cell invasiveness [17] . Interestingly and rather intriguingly, S100A10 mRNA levels were also reduced in these cells, which suggests that annexin A2 (or a protein that is affected by ANXA2 knockout) is an activator of S100A10 gene transcription. Further investigation is required to delineate a mechanism of annexin A2-mediated regulation of S100A10 gene expression. Noteworthy, the mRNA levels of PLG (plasminogen), the inactive form of plasmin, were not affected. Nonetheless, based on the aforementioned empirical evidence, it is safe to conclude that the reduction in plasmin generation in ANXA2 -/- L5178Y cells is primarily due to the loss of the plasminogen receptor S100A10. Consistent with its function in actin binding and association with lipid rafts (extensively reviewed in [3]), annexin A2 is also involved in intestinal cell migration during injury/wound repair. Using a loss of function approach, Rankin et al. showed that annexin A2 depletion by shRNA reduces internalization of β1 integrin and causes increased phosphorylation of paxillin,

www.futuremedicine.com

2473

Perspective  Bydoun & Waisman and metas asion tas I nv is

FN1

S100A10

S100A6

Tenascin C

PSTI

Paxillin

TM4SF3

WAVE2

FABP1

RAC1

ID1

β 1 integrin

KRT19

PRDX6 EGFR

P55CDC MAP17 EDN3

RAB14 PS20

Proliferation

Apoptosis

TNF-α

SP4 SFTPC DCX S100A1

Black: Change in mRNA level Red: Change in protein level/activity only

Figure 1. Venn diagram representing the mRNA and protein changes of genes affected by annexin A2 depletion. The genes highlighted in black are affected at the transcript level while genes highlighted in red represent those whose the level or activity of their encoded proteins are affected. Upward-facing arrows reflect an upregulation while downward-facing arrows represent a downregulation.

which maintains cellular adhesion and prevents migration [39] . Since β1 integrin internalization has been linked to epithelial cell migration and cancer cell invasiveness [40] , an experimental complication arises concerning the link between annexin A2 and cell migration. More specifically, the loss of migration and invasion observed in annexin A2-depleted cells may result from annexin A2-dependent changes in β1 integrin and paxillin, and not necessarily through annexin A2-dependent changes in plasmin generation.

2474

Future Oncol. (2014) 10(15)

More recently, De Graauw et al. reported that annexin A2 depletion delays the endocytosis of the EGF receptor and consequently dampens EGF-dependent signaling [41] . These pathways are key in modulating the formation of metastases [41,42] . Oskarsson et al. identified that Tenascin C, a glycoprotein that helps establish pre-metastatic niches, interacted with annexin A2 and its function was also affected by annexin A2 depletion [43] . Zhao et al. also revealed that the activity of WAVE2 and RAC1, two key proteins that

future science group

S100A10: plasminogen activation & oncogenesis  sustain the invasive mesenchymal phenotype of cancer cells, is diminished upon the depletion of annexin A2 [44] . Annexin A2 depletion in monocytes and colon cells affects TNF-α shedding [45] , which is a known regulator of inflammation and malignant transformation (Figure 1) . Together, these reports emphasize that there is a discrepancy between the cell surface plasmin-regulatory function and the intracellular functions of annexin A2, which must be addressed and accounted for before drawing conclusions. Unless these effects of annexin A2 depletion are carefully controlled in model systems, the use of annexin A2-depleted cells or the ANXA2-knockout mouse cannot be simply interpreted. The emergence of S100A10 as a regulator of cancer progression: is the evidence sufficient to claim the plasmin-mediated oncogenesis of the heterotetramer? Since annexin A2 protects intracellular S100A10 from ubiquitin-mediated proteasomal degradation and acts as an anchor for cell surface S100A10 [22] , it is likely that any reported oncogenic stimulation of annexin A2 [46] also serves to increase S100A10 levels [5] . This may have a direct impact on cell surface plasmin generation and proteolytic activity of cancer cells and renders distinguishing the individual contribution of annexin A2 and S100A10 difficult. Various cancer models have attributed plasmin-mediated cancer cell invasion to annexin A2 while totally disregarding any potential role of S100A10 [3,36–37] . These studies must be re-examined in light of earlier findings, which validate the lack of plasminogen-binding capability of annexin A2 and the recent reports supporting S100A10 as the sole plasminogen receptor within the heterotetramer. Extensive evidence from our group has shown that S100A10 plays a central role in recruiting and binding plasminogen as well as stimulating its conversion to plasmin, which promotes the acquisition of an invasive phenotype [8,13,33] . S100A10 is crucial in generating plasmin and promoting the invasiveness of HT1080 fibrosarcoma [21] and Colo 222 colorectal [47] human cancer cells. Consequently, the depletion of S100A10 (by shRNA) in these cells or treatment with carboxypeptidase B dramatically reduced plasmin production and in vitro invasiveness [10,21,47] . The loss of S100A10 also reduced the metastatic burden in lungs of mice injected with HT1080 cells [21] . Intriguingly, Colo 222 cells do not express annexin A2 on their cell surface and hence

future science group

Perspective

plasminogen binding and activation were reliant on cell surface S100A10, but were independent of annexin A2 [47] . Recently, we demonstrated that S100A10 also regulates the infiltration of tumor-associated macrophages into tumor sites and is hence crucial for growth of xenografted mouse Lewis lung carcinoma (LLC) cells [7,10] . As a result, LLC tumors failed to grow in S100A10null mice and were less angiogenic due to failure of recruitment of these macrophages [7] , a contribution that was originally accredited to annexin A2 [48] . Importantly, tumor growth was recovered when knockout macrophages were intratumorally injected (and not intravenously) into LLC tumors of S100A10-null mice [7] , suggesting that S100A10 was required for the infiltration step through blood vessel walls. However, our studies with acute promyelocytic leukemia (APL) were a classic example of how the roles of annexin A2 and S100A10 in potentiating APL-associated fibrinolysis were disambiguated. It is known that APL patients suffer from severe bleeding symptoms primarily caused by a hyperfibrinolytic vasculature and low platelet counts. Hyperfibrinolysis is mediated through an accelerated capability to generate plasmin, which breaks down fibrin clots. It has been reported that APL patients express higher levels of annexin A2 when compared with other leukemia patients [49] . Annexin A2 depletion in leukemic NB4 cells, a model human cell line used to study APL, causes a 60% decrease in their plasmin-generating capacity [50] . However, in a recent report by O’Connell et al., we showed that S100A10 depletion in NB4 cells resulted in a 70% decrease in the plasminogen-binding and plasmin-generating capabilities of these cells. The ability to degrade fibrin was also hampered in S100A10-depleted cells. In addition, induced expression of PML-RARA, the oncogene responsible for APL, upregulated the expression of cell surface S100A10 (and annexin A2) in the myeloid precursor PR9 cells, which provided an explanation of why APL patients present with increased plasmin-mediated fibrinolysis. Knockdown of S100A10 in PML-RARA-expressing cells hindered plasminogen binding and activation [9] . Moreover, all-trans-retinoic-acid, a standardized treatment for APL patients, downregulated annexin A2 protein levels, which was associated with amelioration of APL-associated bleeding [51] , an event that was later correctly linked to S100A10 downregulation [9] . The study by O’Connell et al. has identified the mechanism of action by which all-trans-retinoic-acid reduces plasmin-mediated fibrinolysis.

www.futuremedicine.com

2475

Perspective  Bydoun & Waisman S100A10-null mice have impaired fibrinolysis and accumulate fibrin deposits in their tissues, which signify the importance of S100A10 in fibrin clot dissolution [11] . Collectively, our work clearly indicated that S100A10, and not annexin A2, is the actual regulator of plasmin-mediated fibrinolysis and that PML-RARA-mediated S100A10 upregulation is likely the contributor for the bleeding symptoms in APL patients. Both annexin A2 & S100A10 mediate the production of plasmin as well as its selfdestruction In addition to its well-characterized activity degrading substrates such as fibrin, laminin and fibronectin, plasmin can also utilize itself as a substrate, a reaction called autoproteolysis. We have demonstrated that in addition to its role as a regulator of plasmin production, AIIt also stimulates plasmin autoproteolysis [52] . This selfdestruct mechanism is thought to be important in preventing collateral tissue damage caused by the accumulation of plasmin in the tissues. The involvement of AIIt in plasmin production and destruction suggests that AIIt could produce a transient pulse of plasmin at the cell surface. Angiostatin, a 38 kDa fragment of the autoproteolysed plasmin (Lys78–Ala440) was originally identified in the urine of mice bearing LLC (reviewed in [52]). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases [53] . Importantly, AIIt catalyzed the reduction of the Cys462-Cys541 disulfide, which allowed the release of angiostatin from the plasminogen molecule. Mutagenesis of annexin A2 (Cys334Ser) and S100A10 (Cys61Ser or Cys82Ser) inactivated the plasmin reductase activity of the isolated subunits of AIIt, suggesting that specific cysteine residues of each subunit participated in the plasmin reductase activity of AIIt [53] . Recently, it was established from the crystal structure of plasminogen that the Cys462–Cys541 disulfide is located in a domain that becomes partially disordered after cleavage of Lys77–Lys78 [54] , thus supporting our model. Conclusion Despite the advances that support the involvement of the AIIt complex in both physiological and pathological conditions, the role(s) that annexin A2 plays in the complex remains elusive. This is primarily due to the failure of researchers

2476

Future Oncol. (2014) 10(15)

to acknowledge that the loss of annexin A2 results in multiple changes in the expression of other proteins. As a result, the ANXA2knockout mouse model is no longer sufficient as a model system to elucidate the plasmin-regulatory functions of annexin A2. Our studies with multiple cell types showcase how annexin A2 depletion studies can be misinterpreted and how S100A10 is a key regulator of cell surface plasmin generation. Future perspective The enduring ambiguity concerning annexin A2 function as a plasminogen receptor continues to generate confusion in the literature. Hopefully, by expounding this ambiguity, we will stimulate a more rigorous approach to the study of annexin A2 function. Given the evidence that we presented in this review, the contribution of S100A10 to plasmin generation and cancer invasion must always be addressed in annexin A2 depletion studies. Future studies must always take into account the complex relationship that exists between S100A10 and annexin A2 and how it affects the function of either component. Moreover, future animal models must address the issue of bystander effects of standard gene knockouts. More specifically, the removal of a gene not only creates a negative selection environment during embryonic development, but also a functional vacancy, which affects genes/proteins whose expression/activity is dependent on the deleted gene. As a result, current in vivo knockout animal models are inducible (by tetracycline or Cre recombinase), some of which are tissue-specific. These models will avoid the negative selection and the bystander effects, which result from the permanent absence of a gene d­uring pre- and postnatal development. Financial & competing interests disclosure M Bydoun is supported through the Cancer Research Training Program (CRTP) administered by the Beatrice Hunter Cancer Research Institute (BHCRI) and funded by the Canadian Institute of Health Research (CIHR), Terry Fox Research Institute (TFRI), Cancer Care Nova Scotia, Dalhousie Medical Research Foundation (DMRF) and the Canadian Cancer Society Nova Scotia Division. 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.

future science group

S100A10: plasminogen activation & oncogenesis 

Perspective

EXECUTIVE SUMMARY The necessity of C-terminal lysines for plasminogen binding: why is annexin A2 not a plasminogen receptor? ●●

Only full-length annexin A2 is present on the cell surface.

●●

Annexin A2 lacks a C-terminal lysine, which is required for plasminogen binding.

Why is annexin A2 not a tissue plasminogen activator receptor? ●●

Tissue plasminogen activator (tPA) competes with plasminogen for reversible binding to C-terminal lysines of surface receptors, in vivo. tPA forms an irreversible disulfide bond with cysteine-8 of annexin A2, in vitro. Therefore, annexin A2-tPA binding does not occur physiologically.

Why is S100A10 the only plasminogen receptor within the heterotetramer? ●●

Unlike annexin A2, S100A10 has two C-terminal lysines that bind tPA and plasminogen.

●●

Treatment of the heterotetramer with carboxypeptidase B removes the C-terminal lysines of S100A10 and blocks tPA and plasminogen binding and plasmin generation by the heterotetramer.

The enduring ambiguity: can we rely on annexin A2 depletion studies? ●●

The bystander effects of gene knockdowns or knockouts obviate in vivo functional analysis. Annexin A2 depletion affects multiple genes and proteins including a well-established plasminogen receptor, S100A10.

●●

Thus, the loss in cell surface plasmin production observed in annexin A2 depletion studies is misinterpreted and is likely due to loss of S100A10.

Is the evidence sufficient to claim that S100A10 is a key regulator of cell surface plasmin generation? ●●

S100A10 regulates 50–90% of the plasmin generation of most cells.

●●

S100A10 overexpression is responsible for the life-threatening hyperfibrinolysis observed in acute promyelocytic leukemia.

●●

S100A10 is essential and sufficient for macrophage migration to tumor sites, a rate-limiting step in tumor progression.

●●

S100A10 is required for the growth, invasiveness and metastasis of many cancer cells.

Conclusion ●●

S100A10 and not annexin A2 is a key regulator of cell surface plasmin generation and plays an important role in hemostasis, inflammation and oncogenesis.

●●

The use of annexin A2 knockdown and depletion models to demonstrate the function of annexin A2 should be abandoned.

Future perspective ●●

Future studies of the physiological function of annexin A2 must utilize expression of domain-swapped annexin A2.

References

•

Papers of special note have been highlighted as: • of interest; •• of considerable interest

Extensive review on annexin A2 structure and functions.

4

Madureira PA, O’connell PA, Surette AP, Miller VA, Waisman DM. The biochemistry and regulation of S100A10: a multifunctional plasminogen receptor involved in oncogenesis. J. Biomed. Biotechnol. 2012, 353687 (2012).

•

Extensive review on S100A10 structure and functions.

5

Madureira PA, Surette AP, Phipps KD, Taboski MA, Miller VA, Waisman DM. The role of the annexin A2 heterotetramer in vascular fibrinolysis. Blood 118(18), 4789–4797 (2011).

1

Miles LA, Plow EF, Waisman DM, Parmer RJ. Plasminogen receptors. J. Biomed. Biotechnol. 2012, 130735 (2012).

2

Godier A, Hunt BJ. Plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease. J. Thromb. Haemost. 11(1), 26–34 (2013).

3

Bharadwaj A, Bydoun M, Holloway R, Waisman D. Annexin A2 heterotetramer: structure and function. Int. J. Mol. Sci. 14(3), 6259–6305 (2013).

future science group

6

Macleod TJ, Kwon M, Filipenko NR, Waisman DM. Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin. J. Biol. Chem. 278(28), 25577–25584 (2003).

7

Phipps KD, Surette AP, O’connell PA, Waisman DM. Plasminogen receptor S100A10 is essential for the migration of tumor-promoting macrophages into tumor sites. Cancer Res. 71(21), 6676–6683 (2011).

•

Demonstrates that S100A10 is required for the infiltration of macrophages into tumor

www.futuremedicine.com

2477

Perspective  Bydoun & Waisman sites to maintain growth, a role that was orginally attributed to annexin A2. 8

•

9

Kassam G, Le BH, Choi KS et al. The p11 subunit of the annexin II tetramer plays a key role in the stimulation of t-PA-dependent plasminogen activation. Biochemistry 37(48), 16958–16966 (1998).

O’connell PA, Madureira PA, Berman JN, Liwski RS, Waisman DM. Regulation of S100A10 by the PML-RAR-alpha oncoprotein. Blood 117(15), 4095–4105 (2011).

18 Gould KL, Woodgett JR, Isacke CM, Hunter

Svenningsson P, Waisman DM. S100A10 regulates plasminogen-dependent macrophage invasion. Blood 116(7), 1136–1146 (2010). 11 Surette AP, Madureira PA, Phipps KD, Miller

VA, Svenningsson P, Waisman DM. Regulation of fibrinolysis by S100A10 in vivo. Blood 118(11), 3172–3181 (2011). 12 Hajjar KA, Jacovina AT, Chacko J. An

endothelial cell receptor for plasminogen/ tissue plasminogen activator. I. Identity with annexin II. J. Biol. Chem. 269(33), 21191–21197 (1994). Study where the quality of the purified annexin A2 is questioned. The purified product bound plasminogen because it was probably proteolyzed, which exposed a C-terminal lysine.

13 Kwon M, Macleod TJ, Zhang Y, Waisman

DM. S100A10, annexin A2, and annexin a2 heterotetramer as candidate plasminogen receptors. Front. Biosci. 10, 300–325 (2005). 14 Cesarman GM, Guevara CA, Hajjar KA. An

endothelial cell receptor for plasminogen/ tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. J. Biol. Chem. 269(33), 21198–21203 (1994). •

binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J. Biol. Chem. 261(16), 7247–7252 (1986). 17 Hou Y, Yang L, Mou M et al. Annexin A2

10 O’connell PA, Surette AP, Liwski RS,

Study where the quality of the purified annexin A2 is questioned. The purified product bound plasminogen because it was probably proteolyzed, which exposed a C-terminal lysine.

15 Hajjar KA, Mauri L, Jacovina AT et al. Tissue

plasminogen activator binding to the

2478

26 Bizik J, Stephens RW, Grofova M, Vaheri A.

Binding of tissue-type plasminogen activator to human melanoma cells. J. Cell. Biochem. 51(3), 326–335 (1993).

16 Glenney J. Phospholipid-dependent Ca2+

Demonstrates, for the first time, that S100A10 (p11) is essential for AIIt-mediated tissue plasminogen activator-dependent activation of plasminogen.

•• Demonstrates that S100A10 expression is regulated by the oncoprotein PML-RAR-α and is a direct target of all-trans-retinoicacid. This paper showed that S100A10, and not annexin A2, is the actual regulator of plasmin-mediated fibrinolysis in acute promyelocytic leukemia.

•

annexin II tail domain. Direct modulation by homocysteine. J. Biol. Chem. 273(16), 9987–9993 (1998).

regulates the levels of plasmin, S100A10 and Fascin inL5178Y cells. Cancer Invest. 26(8), 809–815 (2008). T. The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo. Mol. Cell. Biol. 6(7), 2738–2744 (1986).

27 Suzuki Y, Mogami H, Ihara H, Urano T.

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells. Blood 113(2), 470–478 (2009). 28 Novokhatny VV, Ingham KC, Medved LV.

Domain structure and domain-domain interactions of recombinant tissue plasminogen activator. J. Biol. Chem. 266(20), 12994–13002 (1991). 29 Roda O, Valero ML, Peiro S, Andreu D, Real

FX, Navarro P. New insights into the tPA-annexin A2 interaction. Is annexin A2 CYS8 the sole requirement for this association? J. Biol. Chem. 278(8), 5702–5709 (2003).

19 Laumonnier Y, Syrovets T, Burysek L,

Simmet T. Identification of the annexin A2 heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes. Blood 107(8), 3342–3349 (2006).

30 Hajjar KA, Jacovina AT. Modulation of

annexin II by homocysteine: implications for atherothrombosis. J. Invest. Med. 46(8), 364–369 (1998).

20 Drust DS, Creutz CE. Aggregation of

chromaffin granules by calpactin at micromolar levels of calcium. Nature 331(6151), 88–91 (1988).

31 Sengupta S, Wehbe C, Majors AK, Ketterer

ME, Dibello PM, Jacobsen DW. Relative roles of albumin and ceruloplasmin in the formation of homocystine, homocysteinecysteine-mixed disulfide, and cystine in circulation. J. Biol. Chem. 276(50), 46896–46904 (2001).

21 Choi KS, Fogg DK, Yoon CS, Waisman DM.

p11 regulates extracellular plasmin production and invasiveness of HT1080 fibrosarcoma cells. FASEB J. 17(2), 235–246 (2003). 22 He KL, Deora AB, Xiong H et al. Endothelial

cell annexin A2 regulates polyubiquitination and degradation of its binding partner S100A10/p11. J. Biol. Chem. 283(28), 19192–19200 (2008). 23 Puisieux A, Ji J, Ozturk M. Annexin II

up-regulates cellular levels of p11 protein by a post-translational mechanisms. Biochem. J. 313(Pt 1), 51–55 (1996). 24 Felez J, Chanquia CJ, Fabregas P, Plow EF,

Miles LA. Competition between plasminogen and tissue plasminogen activator for cellular binding sites. Blood 82(8), 2433–2441 (1993). •• Key report that characterized how tissue plasminogen activator and plasminogen competed for binding to C-terminal lysines of surface receptors. 25 Felez J, Chanquia CJ, Levin EG, Miles LA,

Plow EF. Binding of tissue plasminogen activator to human monocytes and monocytoid cells. Blood 78(9), 2318–2327 (1991).  •• Key report that characterized how tissue plasminogen activator and plasminogen competed for binding to C-terminal lysines of surface receptors. 

Future Oncol. (2014) 10(15)

32 Dayal S, Chauhan AK, Jensen M et al.

Paradoxical absence of a prothrombotic phenotype in a mouse model of severe hyperhomocysteinemia. Blood 119(13), 3176–3183 (2012). •

Provides an insight into how hyperhomocysteinemic mice have normal rates of fibrinolysis, which indicated that either annexin A2 is not involved in fibrinolysis of this model or that homocysteine does not target annexin A2.

33 Kassam G, Choi KS, Ghuman J et al. The

role of annexin II tetramer in the activation of plasminogen. J. Biol. Chem. 273(8), 4790–4799 (1998). 34 Fogg DK, Bridges DE, Cheung KK et al.

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase. Biochemistry 41(15), 4953–4961 (2002). 35 Jones PG, Moore GJ, Waisman DM. A

nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling. J. Biol. Chem. 267(20), 13993–13997 (1992).

future science group

S100A10: plasminogen activation & oncogenesis  36 Sharma MC, Sharma M. The role of annexin

II in angiogenesis and tumor progression: a potential therapeutic target. Curr. Pharm. Design 13(35), 3568–3575 (2007). 37 Zheng L, Foley K, Huang L et al. Tyrosine 23

phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer. PLoS One 6(4), e19390 (2011). 38 Gou D, Mishra A, Weng T et al. Annexin A2

interactions with Rab14 in alveolar type II cells. J. Biol. Chem. 283(19), 13156–13164 (2008). •• Demonstrates that annexin A2 depletion results in changes in expression of 61 genes. 39 Rankin CR, Hilgarth RS, Leoni G et al.

Annexin A2 regulates beta1 integrin internalization and intestinal epithelial cell migration. J. Biol. Chem. 288(21), 15229–15239 (2013). 40 Barkan D, Chambers AF. Beta1-integrin: a

potential therapeutic target in the battle against cancer recurrence. Clin. Cancer Res. 17(23), 7219–7223 (2011). 41 De Graauw M, Cao L, Winkel L et al.

Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation. Oncogene 33(20), 2610–2619 (2013). 42 Normanno N, De Luca A, Bianco C et al.

Epidermal growth factor receptor (EGFR)

future science group

signaling in cancer. Gene 366(1), 2–16 (2006). 43 Oskarsson T, Acharyya S, Zhang XH et al.

Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs. Nat. Med. 17(7), 867–874 (2011). 44 Zhao P, Zhang W, Wang SJ et al. HAb18G/

CD147 promotes cell motility by regulating annexin II-activated RhoA and Rac1 signaling pathways in hepatocellular carcinoma cells. Hepatology 54(6), 2012–2024 (2011). 45 Tsukamoto H, Tanida S, Ozeki K et al.

Annexin A2 regulates a disintegrin and metalloproteinase 17-mediated ectodomain shedding of pro-tumor necrosis factor-alpha in monocytes and colon epithelial cells. Inflamm. Bowel Dis. 19(7), 1365–1373 (2013). 46 Madureira PA, Hill R, Miller VA,

Giacomantonio C, Lee PW, Waisman DM. Annexin A2 is a novel cellular redox regulatory protein involved in tumorigenesis. Oncotarget 2(12), 1075–1093 (2011). 47 Zhang L, Fogg DK, Waisman DM. RNA

interference-mediated silencing of the S100A10 gene attenuates plasmin generation and invasiveness of Colo 222 colorectal cancer cells. J. Biol. Chem. 279(3), 2053–2062 (2004). 48 Sharma M, Ownbey RT, Sharma MC. Breast

Perspective

migration and neoangiogenesis via tPA dependent plasmin generation. Exp. Mol. Pathol. 88(2), 278–286 (2010). 49 Menell JS, Cesarman GM, Jacovina AT,

Mclaughlin MA, Lev EA, Hajjar KA. Annexin II and bleeding in acute promyelocytic leukemia. N. Engl J. Med.340(13), 994–1004 (1999). 50 Liu Y, Wang Z, Jiang M et al. The expression

of annexin II and its role in the fibrinolytic activity in acute promyelocytic leukemia. Leuk. Res. 35(7), 879–884 (2011). 51 Xie Y, Wang ZY, Zhang W, Dai L, Bai X.

The fibrinolytic activity in leukemic cell lines and its alteration on all-trans retinoic acid treatment. Zhonghua Xue Ye Xue Za Zhi 27(9), 588–592 (2006). 52 Fitzpatrick SL, Kassam G, Choi KS, Kang

HM, Fogg DK, Waisman DM. Regulation of plasmin activity by annexin II tetramer. Biochemisty 39(5), 1021–1028 (2000). 53 Kassam G, Kwon M, Yoon CS et al.

Purification and characterization of A61. An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors. J. Biol. Chem. 276(12), 8924–8933 (2001). 54 Law RH, Caradoc-Davies T, Cowieson N

et al. The x-ray crystal structure of full-length human plasminogen. Cell Rep. 1(3), 185–190 (2012).

cancer cell surface annexin II induces cell

www.futuremedicine.com

2479