Translational implications of targeting annexin A2: From membrane repair to muscular dystrophy, cardiovascular disease and cancer

Abstract

Annexin A2 (A2) contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca²⁺-regulated phospholipid-binding annexins. Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links membrane with the cytoskeleton, calcium-dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post-translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro-tumorigenic activities.

Graphical Abstract

Annexin A2 is a key repair protein that works with S100A10 and other S100 proteins to execute its membrane repair and extracellular roles.

Annexin A2 is a therapeutic target because loss of annexin A2 function enhances cellular degeneration, which exacerbates muscular dystrophy and cardiovascular disease.

Annexin A2-mediated protection is hijacked by cancer cells to enhance survival and metastasis.

Introduction

Maintaining plasma membrane integrity is essential for cell survival and homeostasis. However, cell membrane disruption occurs frequently due to exercise, pore-forming toxins, and other damaging agents in mammalian tissues. Skeletal and cardiac muscle, epithelia, and the endothelium are especially prone to damage. Damaged cells die without membrane repair, contributing to tissue degeneration¹. For example, failure to repair skeletal and cardiac muscles causes muscular dystrophy and cardiomyopathy². Similarly, endothelial injuries cause increased vascular permeability, loss of homeostasis, inflammation, and complications of vascular diseases³. On the other hand, membrane repair is detrimental to the host when cancer cells use it for protection, survival, and metastasis. Several mechanisms promote membrane repair, including microvesicle shedding, patch repair, and clogging¹. One family of repair proteins implicated in all of these mechanisms is the annexin family. Annexins are Ca²⁺- and phospholipid-binding proteins that interact with other proteins to facilitate membrane repair⁴. Annexins connect membrane repair to other cellular functions, allowing the host to act on information about membrane integrity. One key annexin is annexin A2. Annexin A2 is important because its deficiency enhances cellular degeneration, exacerbates muscular dystrophy and cardiomyopathy, and reduces cancer. This review provides an overview of annexin A2 and the translational implications of annexin A2-mediated membrane repair in muscular dystrophy, cardiomyopathy, and cancer.

Annexin A2 is an annexin protein family member with important intracellular and extracellular roles

Annexin A2 is one of 12 mammalian annexins, annexin A1-A11 and A13, that bind to the membrane when intracellular Ca²⁺ is elevated (reviewed in^4,5). Annexins have a variable N-terminal domain and a conserved C-terminal core domain. The C-terminal domain comprises four tandem, calcium-binding repeats of ⁓70 amino acids, except for annexin A6, which has eight repeats due to a duplication of this domain⁵. The annexin core houses calcium-binding sites that help switch annexins from their soluble cytosolic forms to membrane-bound forms⁵. Elevated levels of intracellular Ca²⁺ let annexins bind to the membrane. Upon membrane binding, annexins interact with other proteins to seal membranes during membrane repair, control membrane domain organization, membrane-cytoskeleton linkages, exocytosis, and apoptosis⁵. Thus, annexins use membrane binding to drive multiple intracellular processes.

Even though annexins have a high percentage of sequence and structural similarity, different relative affinities for lipids and Ca²⁺ provides stepwise binding of annexins to the membrane. At steady state, annexins are cytoplasmic⁵. Following the influx of Ca²⁺ into the cytoplasm annexins bind to membrane lipids, including phosphatidylserine (PS)⁵. To study annexin membrane binding, calcium ionophores or membrane damage is used. Certain annexins translocate earlier following damage, while others act later after damage. However, there is controversy over the hierarchy of annexin binding to the membrane during membrane repair^6–9, which is distinct from the Ca²⁺ affinity and predicted ability of annexins to bind to PS¹⁰ (Table 1). Notable differences between studies that examined annexin recruitment in response to Ca²⁺ ionophores, damage via laser wounding and damage from pore-forming toxins^6–11 center on the timing of annexin A1 and annexin A2 recruitment. With one reported exception, annexin A1 requires more Ca²⁺ than its PS binding predicts (Table 1). Annexin A2 is proposed to be either the first or last annexin on the membrane (Table 1). Notably, laser wounding assays observed the late recruitment of annexin A2^7,9. Annexin A2 recruitment was flipped from fast to slow in response to pore-forming toxins when MEK was inhibited¹¹. This suggests these discrepancies can be attributed to the type of signal or damage driving translocation, and/or the need for additional signals, like dephosphorylation, to drive membrane binding.

Table 1:

Annexin recruitment order to damaged membranes by model system.

Recruitment order (first to last)	Source of membrane damage/ Ca2+ concentration	Organism/Model (cells/tissues)	References
A2, A1, A6, A4, A5	Increasing Ca2+ concentration required for PS binding	Human tissues (placenta, liver, spleen)	10
A2, A4, A6, A1, A5	Increasing Ca2+ concentration using ionomycin	Murine neuroblastoma (N1E-115)	6
A6, A11a, A2a, A1a	Laser wounding	Zebrafish myofibers	7
A1, A6/A5, A4, A2	Laser wounding	Human LHCN-M2 skeletal muscle cells	9
A2, A6, A11, A4, A1, A5, A7	Pneumolysin	Human HEK cells	8
A2, A6, A1	Streptolysin, perfringolysin, intermedilysin	Human HeLa cells	11

In addition to intracellular roles like repair, annexins can be released from cells. Once extracellular, annexins promote extracellular matrix degradation, angiogenesis, plasmin generation, cell-cell adhesion, and bone mineralization. Most extracellular roles are annexin-specific, though there is redundancy in some of these functions. One example of redundancy is the presence of annexins A2, A5, and A6 in the matrix vesicles that drive mineralization in bones and in arteries. Annexins contribute to mineralization from matrix vesicles by linking Ca²⁺ to the vesicle membrane¹². Annexin A2 and annexin A6 act extracellularly in both skeletal and extra-skeletal (e.g. arterial) mineralization by generating bioactive matrix vesicles and mineralizing type 1 collagen via Ca²⁺ uptake¹³. Thus, annexins released extracellularly can cooperate to promote outcomes like bone mineralization, or calcification of arteries.

Annexin A2 also has unique extracellular roles. Annexin A2 contributes to plasminogen activation. Annexin A2 can heterotetramerize with the calcium-binding protein S100A10. Annexin A2 binding stabilizes S100A10, preventing its rapid degradation¹⁴. This stable complex creates an annexin A2/S100A10 heterotetramer (A2tet), associated with both the inner and outer leaflets of the plasma membrane¹⁴. On the outer leaflet, annexin A2 stimulates S100A10 function, activating plasminogen to plasmin¹⁴. Extracellular annexin A2 on the surface of endothelial cells further binds tissue-type plasminogen activator (tPA), which allows for localized activation of plasminogen to plasmin, promoting angiogenesis¹⁴. Finally, A2tet facilitates the cell homing and heterotypic cell-cell adhesion of hematopoietic stem cells to osteoblasts and vascular endothelial cells¹⁴. Thus, annexin A2 can activate the plasmin system to drive angiogenesis.

In addition to the unique extracellular roles, annexin A2 contributes to many intracellular processes necessary for human health. Annexin A2 promotes membrane repair and forms cholesterol-containing platforms crucial for endosomal carrier vesicle/multivesicular body formation, thus regulating the start of the degradation pathway⁵. In the nucleus, monomeric annexin A2 can transport specific transcription factors and mRNA, while annexin A2 nuclear translocation protects against DNA damage¹⁵. A2tet links the actin cytoskeleton to the membrane¹⁴. A2tet further enables pH-dependent membrane binding¹⁶. Thus, annexin A2 mediates many intracellular functions in addition to repair.

Annexin A2 functions are regulated by many post-translational modifications including oxidation, acetylation, phosphorylation, ubiquitination, and SUMOylation. Many sites of post-translational modification are on the annexin A2 N-terminus. The accessibility of the entire N-terminus to post-translational modifications relies on hydrophobic interactions from Val3, Ile6, Leu7, and Leu-10¹⁴, though it can also be proteolytically cleaved¹⁶. In cancer cells, Cys8 oxidation of annexin A2 reduces reactive oxygen species (ROS) induced damage/death during tumorigenesis¹⁵. Phosphorylation of annexin A2 at Tyr23 by insulin receptor kinase or Src family tyrosine kinases enables A2tet interaction with the Rho-ROCK1/2 pathway and initiates actin/cytoskeletal remodeling upon Ca²⁺ influx^17,18. Annexin A2 Tyr23 phosphorylation also promotes viral infection because blockade of Tyr23 phosphorylation by Src kinase activity interferes with pseudorabies replication¹⁹. Along with tyrosine phosphorylation, two serines in the N-terminus of annexin A2, Ser11 and Ser25, can be phosphorylated. Ser25 is phosphorylated by protein kinase C (PKC)²⁰, which exposes Ser11. Ser11 can be phosphorylated by protein kinase A, or calcium/calmodulin-dependent protein kinase²¹. Serine phosphorylation promotes annexin A2 recruitment to secretory granules, and Ser11 phosphorylation inhibits its association with S100A10^20,21. In PC12 cells, Ser25 phosphorylation and ubiquitination/SUMOylation of annexin A2 sequesters non-polysomal mRNAs in the perinuclear region in an inactive but transport competent form²¹. The ubiquitination/SUMOylation of phosphorylated annexin A2 suggests crosstalk between these modifications may regulate annexin A2 localization and function²¹. Overall, Tyr23, Ser11, and Ser25 phosphorylation are critical regulators of annexin A2 functional dynamics, cellular location, and function, including membrane repair.

Contribution of Annexin A2 to membrane repair

Many cellular functions of annexin A2 spring from its contribution to membrane repair. Annexin A2 contributes to repair in many cell types including human skeletal muscle cells, vascular endothelial cells, epithelial cells, MCF-7, and HeLa cells^5,9,11,22,23. During repair, annexin A2 can be detected on both interior and exterior plasma membrane, where its concentration is proportional to the amount of repair^22,23. Like other annexins, annexin A2 is involved in several different repair responses, including clogging, patch formation, and microvesicle shedding^5–9,11,24 (Fig 1). Membrane clogging is the repair of the membrane by physically blocking the disruption. Patch formation is repair of the membrane through the hetero- and homotypic fusion of internal vesicles, especially endosomes and lysosomes, with the plasma membrane. Microvesicle shedding is the sequestration and shedding of damaged membranes from the cell. Annexin A2 contributes to membrane repair by physically forming part of the barrier, while also coordinating protein recruitment, phospholipid interaction, and actin regulation (Fig 1). At the lesion site, annexin A2 interacts with other annexins, including annexin A1, annexin A5 and annexin A6, to form protein caps that clog and seal off membrane perforations²⁴. Annexin A2 contributes to patch repair by facilitating protein-protein interactions and lipid accumulation to join vesicles. This was shown in giant unilamellar vesicles, where annexin A1 and annexin A2 localized to cross-linked membranes, enabling membrane cross-lining or fusion²⁵. Annexin A2, like annexin A1, is a free-edge membrane crosslinker that interacts with other annexins and repair proteins during plasma membrane repair²⁵. This function helps annexin A2 clog membranes and contribute to patch repair. Annexin A2 can interact with BIN1, EHD2, phosphatidylinositol-4,5-bisphosphate, and phosphatidylserine to physically blockade the site of membrane injury⁵. These functions are consistent with findings that annexin A2 is an early responder to membrane damage site following Ca²⁺ flux^6,8.

Figure 1. Annexin A2 promotes membrane repair.

During membrane perforation, annexin A2 senses Ca²⁺ flux. Annexin A2-S100A10 tetramers can be recruited to the membrane. Once recruited, annexin A2 tetramers complex with actin and facilitate recruitment of other repairs proteins, such as annexins (A1, A4, A5 or A6), and dysferlin to promote physical blockade of the pore by proteins (clogging), internal vesicles (patch repair), or sequestration and shedding of the damaged membrane (microvesicular shedding) using lipid and/or ESCRT-III mediated mechanisms. The figure was created using BioRender.

At the injury site, A2tet and other annexin A2-S100 complexes contribute to repair. Interestingly, annexin A2 can interact with other S100 proteins besides S100A10 to promote repair. Annexin A2 tetramers with S100A11 are implicated in resealing the plasma membrane in actin-dependent processes⁵ (Fig 2). Annexin A2 interacts with S100A13, which could further impact repair²⁶. It is likely that annexin A2 interacts with additional S100A proteins. Since each S100A protein has similar structure, but distinct cellular functions, interacting with multiple S100A proteins could enable annexin A2 to coordinate repair with multiple cellular functions. The mechanisms by which annexin A2 interacts with specific S100A proteins remains unknown. Annexin A2 may act in parallel to other repair mechanisms, such as Anoctamin 5 (TMEM16E). Annexin A2 rescues the failure of Anoctamin 5 (TMEM16E)-mediated membrane repair²⁷. While annexin A6 recruitment was TMEM16E-dependent, annexin A2 recruitment was enhanced when TMEM16E was blocked²⁷. Overall, annexin A2 helps coordinate a protein network for physical blockade of membrane lesions.

Figure 2. Annexin A2 protects organisms from muscular dystrophy and cardiovascular diseases.

(1a or 1b) Upon membrane injury and Ca²⁺ influx, (2a and 2b) intracellular annexin A2 binds and stabilizes S100A10 to form annexin A2/S100A10 tetramers (A2tet) via Ser1 acetylation. A2tet facilitates repair in (3a) skeletal and (3b) cardiac muscles. Two alternative pathways may occur, described for peripheral muscles or cardiomyocytes. (4a) In muscle cells, extracellular annexin A2 can activate TLR4 signaling to trigger inflammation by via pro-inflammatory cytokines. Active TLR4 activates protein kinase C (PKC). (5a) Activated PKC phosphorylates annexin A2 at Ser25, exposing Ser11 to phosphorylation. Ser11 is phosphorylated by cyclic AMP/protein kinase A (cAMP/PKA) or calcium/calmodulin-dependent protein kinase (CaMK). Ser11/Ser25 phosphorylation dissociates intracellular A2tet, allowing (6a) ubiquitination of S100A10 and/or (7a) SUMOylation of annexin A2. These post-translational modifications target S100A10 for degradation, and reduce annexin A2 function. (4b) Another intracellular pathway occurs from Src activation. (5b) Activated Src can phosphorylate annexin A2 at Tyr23 and release A2tet into the extracellular matrix. (6b) A2tet then binds to tPA and plasminogen, generating plasmin, leading to vascular fibrinolysis and angiogenesis. These functions promote cardiovascular health, while mineralization of arteries is detrimental to cardiovascular health. The figure was created using BioRender.

Annexin A2 supports patch repair of the sarcolemma. A2tet interacts with actin, which provides a scaffold for the recruitment of other membrane repair proteins, including the patch repair protein dysferlin^24,28 (Fig 1). Dysferlin positive vesicles fuse with the membrane during repair²⁹, supporting dysferlin cross-linking membranes to promote patch repair. However, dysferlin is also present on the plasma membrane, so this could be the source of dysferlin during repair instead. Dysferlin may cooperate with annexin A6 to recruit annexin A2 to the site of repair⁷. Other groups suggest that annexin A2 recruits dysferlin to the membrane, which drives dysferlin accumulation in the plasma membrane²⁸. Differences in dysferlin trafficking could be dependent on the type of injury.

One alternative explanation for the discrepancy in order of annexin A2 vs dysferlin recruitment could be the amount of cholesterol in the membrane. Accumulation of both annexin A2 and dysferlin at the damage site depends on cholesterol^28,30. Cholesterol-binding is proposed to be a core property of annexins³¹, so under cholesterol-high conditions, annexins might drive dysferlin recruitment, whereas under cholesterol low conditions, dysferlin could drive annexin recruitment. Annexin binding to cholesterol is consistent with the finding that both annexin A2 and annexin A6 translocate to detergent-resistant sites of the smooth muscle cell plasmalemma in a Ca²⁺-dependent manner, with annexin A2 promoting the association of lipid raft microdomains with Ca^{2+ 32}. However, one argument against cholesterol binding as a core annexin property is that it is the unique N-terminus of annexin A2 that mediates the interaction with cholesterol³⁰, instead of the shared annexin core.

In addition to other modes of repair, annexin A2 is a downstream effector during microvesicle shedding. Microvesicle shedding can occur by ESCRT-dependent³³, lipid-dependent, or MAP kinase-dependent¹¹ mechanisms. During MAP kinase dependent microvesicle shedding, Mixed Lineage Kinase 3 activates MEK, which leads to non-canonical, ERK-independent activation of annexin A2¹¹ (Fig 1). Annexin A2 is not shed to the same extent as annexin A1 or annexin A6¹¹. Blockade of MEK activation reduced the extent of microvesicle shedding, and switched annexin A2 from binding the membrane faster than annexin A1 and annexin A6 to much slower than annexin A1 or annexin A6¹¹. These data suggest that in addition to Ca²⁺, phosphorylation or another post-translational modification controls annexin A2 access to the membrane during repair. Interestingly, PKC was not the responsible kinase¹¹. Differences in kinase activation could underly the difference in recruitment order for annexins observed by different groups^7–9,11 (Table 1). Thus, annexin A2 is involved in shedding.

Finally, annexins participate in endosomal and lysosomal repair. Annexin A1 and annexin A2 are recruited to a subset of damaged lysosomes in a Ca²⁺-dependent manner to limit lysosomal leakage³⁴. This repair was independent of ESCRT-mediated endolysosomal repair³⁴. These findings are not surprising given the role of annexin A2 in plasma membrane repair, and endosomal trafficking⁵. Overall, annexin A2 promotes membrane repair, and transmits information about membrane damage to other key signaling pathways.

Annexin A2 in muscular dystrophy

Annexin A2 contributions to membrane repair account for its complex roles in diseases like muscular dystrophy. On one hand, the only annexin mutation associated with muscular dystrophy to date is a dominant active mutation in annexin A11³⁵. On the other hand, annexin A2 and annexin A1 are elevated in Limb Girdle Muscular Dystrophy (LGMD) types 2B and 2I and Miyoshi myopathy³⁶. Annexin A2 is active during membrane repair in muscles³⁰. Further evidence that annexin A2 contributes to muscle health is the annexin A2 translocation between the membrane and cytosol during muscle contraction, due in part to Ca²⁺ influx³⁰. Finally, loss of annexin A2 reduces myofiber repair³⁷. However, simultaneous loss of dysferlin mitigated the pathogenic phenotype of annexin A2 knockout³⁷. This suggests that annexin A2 promotes membrane repair during muscular dystrophy, but also transduces pathogenic signals about the membrane during membrane damage.

Annexin A2 may transduce pathogenic signals about the membrane when it is extracellular. For example, during muscular dystrophy, extracellular annexin A2 promotes inflammation by activating TLR4³⁸ (Fig 2). Extracellular annexin A2 is implicated in the expansion of fibro/adipogenic progenitors that degrade muscle tissue³⁷. This could create a negative feedback loop. Loss of dysferlin initiates muscle damage. During repair, annexin A2 is secreted. Extracellular annexin A2 stimulates inflammation and activates fibro/adipogenic progenitors, which degrades muscle tissue. Loss of muscle tissue plus the continued absence of dysferlin leads to more damage, continuing the cycle. To compensate, annexin A2 is elevated, leading to an increase in extracellular annexin A2. This in turn exacerbates muscular dystrophy due to extracellular signaling of annexin A2. However, annexin A2 signaling can be dirupted. Annexin A2 can be cleaved by calpains in myofibers during contraction³⁰. This regulation may help limit its ability to transduce pathogenic signals during normal myofiber contraction. Activation of TLR4 can activate PKC³⁹. Activated PKC phosphorylates annexin A2 at Ser25 which dissociates intracellular A2tet, degrading S100A10²¹. Phosphorylated annexin A2 may undergo further post-translational modifications such as ubiquitination, SUMOylation or acetylation, which controls annexin A2 function and regulation (Fig 2). Thus, the intracellular and extracellular roles of annexin A2 may explain its complex roles in muscular dystrophy.

Annexin A2 in cardiovascular diseases

As may be expected for a protein involved in muscle homeostasis, annexin A2 is important for heart health. Annexin A2, along with other annexins, are upregulated in the failing human heart². At the organ level, one response to tissue damage is the proliferative tissue repair response, where cells proliferate to correct tissue damage. The proliferative tissue repair response is regulated by intracellular annexin A2 membrane recruitment via the Hippo pathway. Hippo activation controls organ size, including cardiac hypertrophy, and regenerative responses⁴⁰. Cytosolic annexin A2 does not block Yes-Associated Protein 1(YAP), but when membrane-bound, annexin A2 recruits kinases that inhibit YAP⁴¹, which limits stemness and proliferative responses. While this mechanism is proposed to help sense cell density in tissues because annexin A2 associates with adherens junctions⁴¹, this also provides a mechanism that could tie organ homeostasis to cytoplasmic pH¹⁶ and membrane repair.

During loss of membrane integrity, annexin A2 is released extracellularly, where it can modify coronary artery disease, heart failure, hypertension, stroke, and atherosclerosis by acting in key pathways, including tissue remodeling, angiogenesis, and thrombosis (Fig 2). Lack of repair and blood vessel regulation can lead to myocardial infarction and venous thromboembolism⁴². Higher serum annexin A2 concentrations are observed in diabetic cardiomyopathy patients, implying that circulating annexin A2 may be a biomarker for diastolic cardiac function and early diabetic cardiomyopathy diagnosis⁴³. Meanwhile, annexin A2 promotes tissue remodeling via its interactions with extracellular matrix proteins. For example, annexin A2 binds to extracellular collagen and promotes cell-cell adhesion, which helps during post-injury remodeling of the myocardial interstitium to prevent perivascular cardiac fibrosis⁴⁴. On the other hand, interaction of annexin A2-laden matrix vesicles contributes to calcifying type I collagen¹³. Since annexin A2 contributes to calcification by matrix vesicles¹², annexin A2 can be beneficial for bone mineralization, but deleterious for arterial calcification. Annexin A2 further regulates vascular integrity, inflammation, inflammasome activation, leukocyte recruitment, and their successive release of inflammatory mediators⁴⁵ (Fig 2). Overall, annexin A2 plays a complex and multifaceted role in cardiovascular physiology and pathology, making it a subject of ongoing research and potential therapeutic targeting.

These targets include annexin A2 involvement in angiogenesis, fibrinolysis, thrombosis, and lipid metabolism. Due to its association with S100A10, annexin A2 is an anti-thrombotic protein involved in fibrin homeostasis⁴⁵ and angiogenesis¹⁴. When normal levels of annexin A2 are expressed, processes such as cell membrane repair, angiogenesis, and fibrinolysis are functional. In cases of ischemic disease, A2tet stimulates the conversion of plasminogen into plasmin by binding to tPA¹⁴ (Fig 2). Binding to tPA activates plasminogen, which breaks down fibrin, removing thrombi in the vessels and allowing for new vessel formation as necessary⁴⁵ (Fig 2). Increased thrombotic occlusion is observed in the carotid artery of annexin A2-deficient mice after experimental injury⁴⁵. Similarly, annexin A2 deficiency lowers S100A10 levels⁴⁵. Silencing of annexin A2 upregulates caspases and increases apoptosis in human umbilical vein endothelial cells, suggesting a crucial role of annexin A2 in the development of atherosclerosis⁴⁶. Further, annexin A2 deficiency is linked to higher levels of circulating plasma low-density lipoprotein cholesterol (LDL-C)⁴⁷. However, the impact of annexin A2 deficiency in atherosclerosis remains debated. For instance, endothelial cells with disturbed blood flow due to oscillatory stress accelerated the dephosphorylation of annexin A2, allowing integrin α5 to bind to the annexin A2 C-terminal domain, which activates integrin α5β1, and leads to atherosclerosis progression⁴⁸. Further studies in other cell types associated with atherosclerosis are needed to determine the impact of annexin A2 post-translational modifications in cardiovascular disease. Overall, reducing injury and required repair to endothelial cells may help reduce the pathologic effects of annexin A2 while preserving its beneficial roles.

Annexin A2 in Cancer

The benefits of annexin A2 discussed so far—promoting membrane repair and degrading extracellular matrix (ECM) to promote angiogenesis—can enhance cancer growth and spread. Annexin A2 overexpression is associated with adverse outcomes in many cancers^49,50. Annexin A2 confers resistance to treatment for cancer cells via effective membrane repair (Fig 3). During membrane repair, annexin A2 is released into the extracellular space, stabilizing S100A10 for plasminogen receptor, resulting in plasmin production, ECM degradation, and cancer metastasis (Fig 3). Thus, blocking annexin A2 represents a cancer target.

Figure 3. Annexin A2 promotes cancer.

Overexpression of annexin A2 confers resistance to membrane stress caused by pore forming toxins, radiation, and reactive oxidative species by phosphorylating annexin A2. Phosphorylation of annexin A2 by a tyrosine kinase may allow for recruitment to the membrane for repair. This enables annexin A2 to detoxify oxidative damage and prevent chemotoxic agent penetration into the cancer cell. Annexin A2 further promotes survival in cancer cells by neutralizing p53, blocking the p53-dependent arrest of the cell cycle at stage G2. This permits rapid division, proliferation and failure of apoptosis in cancer cells. Finally, elevated extracellular annexin A2 cleaves plasminogen, promotes extracellular matrix degradation, giving cancer cells both blood supply and escape from the primary tumor. These functions promote tumor metastasis. The figure was created using BioRender.

Overexpression of annexin A2 aids tumor cells in survival, invasion, proliferation, and migration. Cancer cells use annexin A2 for membrane repair like noncancerous cells⁵¹. For example, in the MCF7 breast cancer line, annexin A2 and S100A11 interacts with the actin cytoskeleton to promote plasma membrane repair⁵. Membrane repair helps cells resist destruction by immune pore-forming toxins, including perforin and the membrane attack complex. Annexin A2 further provides chemoresistance by acting as an antioxidant that eliminates reactive oxidative species responsible for oxidative stress and damage (Fig 3)¹⁴. Oxidation of Cys8 on annexin A2 detoxifies H₂O₂ to H₂O, protecting the cancer cell from oxidative stress¹⁴.

Along with survival, cancer cells overexpress annexin A2 to aid growth and proliferation. When overexpressed, annexin A2 inhibits p53. Blockade of p53 prevents G2 cell cycle arrest, enabling cancer cells to rapidly divide and proliferate⁵² (Fig 3). Knockdown of annexin A2 relieves the JNK/c-Jun-mediated repression of p53 transcription⁵². Thus, annexin A2 promotes cell growth via p53 inhibition.

Finally, extracellular annexin A2 enhances tumor metastasis. Annexin A2 is associated with metastasis in triple negative breast cancer cells⁵³. To spread beyond the primary tumor, tumor cells must degrade the extracellular matrix. Enhanced extracellular matrix degradation can be driven by plasminogen activation by extracellular A2tet⁵⁰ (Fig 3). Extracellular Hsp90α increases annexin A2 at the cell surface that upregulates this cascade of events associated with ECM degradation⁵⁴. ECM degradation exposes new sites in the ECM that enable cancer cell attachment and metastasis⁵⁴. Thus, annexin A2 overexpression promotes tumor spread.

Therapeutic potential of Annexin A2

Due to the wide range of diseases in which annexin A2 is involved, targeting annexin A2 is expected to reduce multiple diseases. One application is the addition of recombinant annexin A2 to reduce cardiovascular disease. Annexin A2 improved ischemic stroke treatment in mouse models’ hearts⁵⁵. Annexin A2 amplified tPA and reduced the effective thrombolytic dose of tPA, resulting in less hemorrhage and brain infarction⁵⁵. Treatment with recombinant annexin A2 also reduced thrombus formation in rat middle cerebral arteries⁵⁶. Similarly, annexin A2 treatment reduced detrimental neurological outcomes, including neurological cell death and neuroinflammation after traumatic brain injury⁵⁷. In contrast, inhibition of annexin A2-mediated inflammation may be a novel therapeutic avenue for treating muscle loss in dysferlinopathy³⁷. Thus, annexin A2 is a potential therapeutic for treating muscular dystrophy and cardiovascular diseases.

A2 represents a potential biomarker and therapeutic target to reduce cancer metastasis. The rationale is that high annexin A2 levels will promote increased vascularization and repair while reducing apoptosis. Annexin A2 is proposed as a predictive biomarker for chemotherapy responsiveness in aggressive triple-negative breast cancer cells⁵⁰. Esophageal cancer patients with high annexin A2 in extracellular vesicles were more responsive to neoadjuvant chemotherapy⁵⁸. This suggests annexin A2 serves as a potential biomarker for future cancer treatment. Mechanistically, annexin A2 can bind to ICAM-3 on dendritic cells to suppress DC-SIGN signaling, enhancing tumor immune escape⁵⁸. Anti-annexin A2 antibodies suppressed metastasis in a mouse model of pancreatic ductal adenocarcinoma¹⁸. Antibodies targeting the annexin A2 N-terminus prevent the production of plasmin and, therefore, block the growth and expansion of cancer cells. This suggests that annexin A2 represents a cancer target.

The challenge of using annexin A2 as a therapeutic treatment for cancer is the selectivity to only target cancer cells or targeting specific regions on annexin A2 to prevent full knockdown. The versatile nature of annexin A2 provides the protein with beneficial functions until hijacked. Knocking down the whole protein could be detrimental to normal growth, proliferation, and repair of noncancerous cells. Since overexpressed annexin A2 is linked to the multidrug resistance and resilience of cancer cells, annexin A2 can serve as a therapeutic target to downregulate the inhibition of apoptosis, and the promotion of growth and repair taking place by aberrantly expressed annexin A2⁵⁰. As a result, partial knockdown of annexin A2, or targeting annexin A2 interactions may be needed to preserve beneficial annexin A2 function, while blocking detrimental functions of annexin A2. Consequently, post-translational modifications of annexin A2 have been targeted. Angiostatin blocks the binding site of plasminogen on annexin A2, which inhibits the conversion of plasminogen into plasmin⁵⁹. Tyrosine kinases that phosphorylate annexin A2 represent another target for controlling overexpressed annexin A2 in cancer cells (Fig. 3).

Demystifying the complex molecular mechanisms driving the balance between intracellular and extracellular functions of annexin A2 and their possible feedback on annexin A2 roles is needed. For instance, identifying the specific post-translational modifications, such as essential phosphorylation sites regulating the recruitment of annexin A2 during membrane repair and disease pathogenesis, represent therapeutic targets. However, further studies are needed to optimize these annexin A2 therapeutic regimens. Future research is needed to better understand the molecular mechanism and balance needed to optimize the therapeutic potentials of intracellular/endogenous and extracellular annexin A2 in muscular dystrophy, cardiomyopathy, and other annexin A2-associated diseases.

Concluding Remarks

Intracellular annexin A2 promotes plasma membrane repair by interacting and coordinating with other repair proteins, which is essential for maintaining cellular integrity and preventing cell death. During membrane damage and repair, annexin A2 escapes into the extracellular space, where it modifies many diseases via extracellular matrix degradation, vascular fibrinolysis, angiogenesis, skeletal mineralization, and cell-cell adhesion (Fig 4). A better understanding of how annexin A2 interacts with membrane repair proteins, and its regulation by post-translational modifications, will enable selective targeting of annexin A2 functions. This understanding will help future studies focused on optimizing the beneficial role of annexin A2 to reduce muscular dystrophy and cardiovascular diseases, without enhancing pro-tumorigenic activities.

Figure 4. Annexin A2 connects repair to many downstream signaling pathways.

Annexin A2 is a key membrane repair protein that works with S100A10 and other S100 proteins to execute its membrane repair and extracellular roles. Annexin A2 is a therapeutic target because loss of annexin A2 function enhances cellular degeneration, which exacerbates muscular dystrophy and cardiovascular disease. Annexin A2-mediated protection is hijacked by cancer cells to enhance survival and metastasis. The figure was created using BioRender.

Data Availability Statement

No primary data were generated to produce this review article