Clinica Chimica Acta 431 (2014) 164–168

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Clinica Chimica Acta

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Annexin A11 in disease

Jiasheng Wang a, Chunmei Guo a, Shuqing Liu b, Houbao Qi a, Yuling Yin b, Rui Liang c,Ming-Zhong Sun a,⁎, Frederick T. Greenaway d

a Department of Biotechnology, Dalian Medical University, Dalian 116044, Chinab Department of Biochemistry, Dalian Medical University, Dalian 116044, Chinac Department of General Surgery, The Second Hospital of Dalian Medical University, Dalian 116027, Chinad Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA

⁎ Corresponding author at: Department of Biotechnol9 West Section Lvshun Southern Road, Dalian 116044, Ch

E-mail address: mxs288@gmail.com (M.-Z. Sun).

http://dx.doi.org/10.1016/j.cca.2014.01.0310009-8981/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 November 2013Received in revised form 16 January 2014Accepted 18 January 2014Available online 6 February 2014

Keywords:Anxa11FunctionDiseaseCancer

Ubiquitously expressed in many cell types, annexin A11 (Anxa11) is a member of the multigene family of Ca2+-regulated phospholipid-dependent and membrane-binding annexin proteins. Studies have shown that Anxa11plays an important role in cell division, Ca2+ signaling, vesicle trafficking and apoptosis. The deregulation andmutation of Anxa11 are involved in systemic autoimmune diseases, sarcoidosis and the development,chemoresistance and recurrence of cancers. Malfunction of Anxa11may lead to or enhance the metastasis, inva-sion and drug resistance of cancers through the platelet-derived growth factor receptor (PDGFR) pathway and/orthe mitogen-activated protein kinase (MAPK)/p53 pathway. In a variety of diseases, Anxa11 is most commonlyreported to function through interactions with apoptosis-linked gene-2 protein (ALG-2) and/or calcyclin(S100A6). Although it has been little studied, Anxa11 is a promising biomarker for the diagnosis, treatmentand prognosis of certain diseases. In this review, the associations of Anxa11 with Ca2+-regulated exocytosis, cy-tokinesis, sex differentiation, autoimmune diseases, thrombolysis and cancers are summarized and interpreted.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

1.1. The features of annexins

The annexins are Ca2+-regulated phospholipid-binding proteinswidely expressed in all eukaryotic cells except yeasts [1,2]. The annexinshave been classified into five groups. Group A includes 12 members(annexins A1–A11 and A13; their chromosome locations are summa-rized in Table 1) found in vertebrates (mammalian); Group B, C, D andE annexins are found in non-vertebrates, fungi/molds, plants and pro-tists, respectively [3]. The annexins are cytosolic proteins distributedboth in the cellular cytoplasm and in the nuclear membranes. They ex-hibit diverse biological functions bymediating the interactions betweenproteins in cell membranes with other proteins in cells, in the nuclearmembrane, and in the extracellular matrix [4–7].

The annexins are composed of a conserved C-terminal core and avariable N-terminal domain [7]. The C-terminal core contains four ho-mologous repeat domains except for annexin A6, which has eight.Each annexin repeat contains about 70 amino acid residues, and theserepeats pack into a highlyα-helical diskwith a slight curvature. Ca2+ fa-cilitates the binding of membrane phospholipids to the disk region ofthe annexins and the removal of Ca2+ leads to the unbinding of the

ogy, Dalian Medical University,ina.

phospholipids. The N-terminal domain is located on the concave sideof annexin, on the opposite side to the Ca2+-binding sites [7], and ismore flexible. Different annexins vary considerably in length, aminoacid sequence and hydrophobicity of the N-terminal domain, whichplays an important role in mediating the interaction of annexins withcytoplasmic proteins andmembranes. Its diversity is the principal crite-rion for distinguishing the different annexin subfamilies [7,8]. Variousreports indicate that annexins play significant roles in autoimmune dis-eases, cardiovascular disease and cancers [9–14] and as a consequence,more attention has been recently paid to the relationship betweenannexins and diseases.

1.2. The structural features of Anxa11

Annexin A11 (Anxa11), also named Annexin XI, is a member of thegroup A annexins. The Anxa11 gene is located on human chromosome10q22–q23 and is composed of 15 exons and 14 introns without the5′ flanking region [15]. The N-terminal domain and C-terminal tetradcore repeat region of Anxa11 are encoded by exons 2–5 and 6–15, re-spectively [16]. Anxa11 is encoded by 504 amino acids and has amolec-ular weight of 56 kDa [17]. Anxa11 has three mRNA isoforms a, b and cdue to alternative splicing; the a isoform is the most common one. Al-though all three isoforms of Anxa11 exist in humans, only one Anxa11product is expressed [16]. Anxa11 is also a structural prototype and fun-damental functional model for other members based on the cladogramassay of annexin family.

Table 1The chromosomal locations of human Annexins.

Annexin Chromosome

Annexin A1 (Anxa1) 9q12–q21.2Annexin A2 (Anxa2) 15q21–q22Annexin A3 (Anxa3) 4q13–q22Annexin A4 (Anxa4) 2p13Annexin A5 (Anxa5) 4q28–q32Annexin A6 (Anxa6) 5q32–q34Annexin A7 (Anxa7) 10q21.1–q21.2Annexin A8 (Anxa8) 10q11.22Annexin A9 (Anxa9) 1q21Annexin A10 (Anxa10) 4q33Annexin A11 (Anxa11) 10q22-q23Annexin A13 (Anxa13) 8q24.13

165J. Wang et al. / Clinica Chimica Acta 431 (2014) 164–168

The Anxa11 N-terminal tail, which is important for the nuclear loca-tion and degradation of Anxa11 [18,19], is composed of 219 amino acidresidues and is rich in glycine, tyrosine and proline residues [20]. The C-terminus of Anxa11 contains the homologous tetrad annexin repeatcore and Ca2+ binding sites. The presence of Ca2+ is required for mem-brane binding, thermostability and the tertiary structure of Anxa11.Anxa11 binding with phosphatidylethanolamine, phosphatidylserineandphosphatidic acid is Ca2+-dependent. Binding of Anxa11 to Ca2+ in-creases its melting temperature by ~14 °C and α-helical content by 9%.The presence of Ca2+ affects the tertiary structure of Anxa11 by drivingfour tyrosine residues into a more hydrophobic environment [8]. TheAnxa11 N-terminal tail is critical for interaction with apoptosis-linkedgene-2 protein (ALG-2) and with calcyclin (S100A6) [21,22]. Anxa11can be phosphorylated by mitogen-activated protein kinase (MAPK)and platelet-derived growth factor (PDGF) although the phosphorylat-ed amino acid residues and the reaction mechanisms are not known[23,24]. In this review, we summarize the biological functions ofAnxa11, and its roles and potential action mechanisms of action indiseases.

Fig. 1. The potential biological actionmechanisms of Anxa11.(1) Anxa11may be a factor downsmay bind to S100A6 or ALG-2 to affect exocytosis and differentiation. (3) The interaction betwebalance among Anxa11, S100A6 and p53 may be essential and Anxa11 may act as an inhibitoactivity of p53, and results in cell survival, growth, apoptosis and differentiation and developm

2. The biological functions of Anxa11

2.1. Anxa11 and Ca2+-regulated exocytosis

Ca2+-regulated exocytosis regulates the release of hormones, diges-tive enzymes, immune modulators and neurotransmitters [25–27] andAnxa11 is an important factor in this process.

Several members of the annexin family have been found to beinvolved in vesicle trafficking and exocytosis [28–30]. Sjölin et al. firstdetected Anxa11 fragments in neutrophil-specific granules [31]. Frag-mental or intact Anxa11 protein was also detected in active granulesor insulin granules in a Ca2+-dependent manner by proteomics andIHC assays [32–34]. Although anti-Anxa11 antibody could not alter in-sulin release induced by calcyclin (S100A6) [35] in pancreatic β cells,Iino et al. have demonstrated that anti-Anxa11 antibody can inhibitCa2+-induced insulin release [34] in pancreatic β cells, as shown inFig. 1.

2.2. Anxa11 and cytokinesis

Cytokinesis is the final stage of the cell cycle occurring at the end ofnuclear division [36]. Themidbodyplays an important role in the abscis-sion step of cytokinesis [37]. Anxa11 has been reported to be involved incytokinesis by interrupting the formation of the midbody.

Tomas found Anxa11 to be involved in cell cycle progression [38].During progression of cell cycle, Anxa11 was co-localized with S100A6in the nuclear envelope during the prophase but not in other phasesfor A431 (epidermoid carcinoma) cells, and was finally translocated tothe midbody during cytokinesis [2]. Depletion of Anxa11 inhibitsmidbody formation in A431 cells, which causes the daughter cells tolink together and leads to cell apoptosis due to cytokinesis failure. In ad-dition, Anxa11 co-localizes with mitotic kinesin-like protein CHO1 incytokinesis [2]. How does Anxa11 influence cytokinesis? A mechanismhas been suggested whereby Ca2+-activated tyrosine kinase activatesAnxa11 and the resulting activated-Anxa11 interacts with S100A6

tream fromMAPK that exerts its biological functions as a phosphorylated form. (2) Anxa11en Anxa11 and ALG-2 is possibly involved in or interferes with the caspase pathway. (4) Ar that interferes with interactions between S100A6 and p53, mediates the transcriptionalent of cancer cells.

166 J. Wang et al. / Clinica Chimica Acta 431 (2014) 164–168

leading to microtubule-induced folds that serve an initiating role in nu-clear envelope breakdown [38]. Anxa11 co-localizes with CHO1 in cyto-kinesis [2], suggesting that these two proteins, both indispensable formidbody formation, are in some way functionally linked. Both the sep-arate and joint molecular functions of Anxa11 and CHO1 in cytokinesisare worthy of further study.

2.3. Anxa11 and sex differentiation

Sex differentiation is a complex process of developing a functionaltestis or ovary from a bipotential vertebrate gonad. Sry is the Y-chromosomal gene pivotal in mammalian sex determination [39,40].Anxa11 participates in the sex differentiation processmainly by interac-tion with S100A6.

Anxa11mRNA levels in the developing gonadhave been analyzed bythe whole-mount in situ hybridization (WISH) technique. Sry only ex-presses during 10.5 to 12.5 days post-coitum (dpc), and the expressionof SrymRNA reaches the highest levels at 11.5 dpc [39,41]. Interestingly,following high Sry expression Anxa11 is upregulated inmales but not infemales until 13 dpc, and after 11.5 dpc the differential expression ofAnxa11 between females and males becomes increasingly significant.It becomes increasingly abundant in the developing testis, while it isprogressively lost in the female. IHC revealed that Anxa11 was morehighly expressed in testis cords but not at all in the ovary at 12.5 dpc. In-terestingly, S100A6 expressionwas also detected in proliferating cells ofembryonic testis, supporting a possible interaction of S100A6 withAnxa11 in vivo [39]. The functional mechanism of Anxa11 in sex differ-entiation is illustrated in Fig. 1.

3. The association of Anxa11 with disease

3.1. Anxa11 is associated with autoimmune diseases

Autoimmune diseases (ADs) result from abnormal B/T lymphocyterecognition of self-reactive antigens, which leads to high levels of auto-antibodies in patients causing inflammation and irreversible structuraland functional damage [42,43]. Anxa11 is reported to be associatedwith ADs [44–47].

Using Hela or Raji cell extracts as substrates, anti-Anxa11 antibodieswere detected in the serum of patients with SLE (systemic lupus erythe-matosus), undifferentiated connective tissue disease, rheumatoid ar-thritis and APS (anti-phospholipid togliere antibodies syndrome)[44–47], which indicates the implication of anti-Anxa11 antibodies inADs. But why are anti-Anxa11 antibodies present in the sera of patientswith AD? As yet there is no direct evidence indicating the regulationmechanisms of Anxa11 in AD. Apoptosis has been hypothesized as animportant process for AD, so based on the previously summarized re-sults that Anxa11 can bind to ALG-2 and S100A6, two key factors impli-cated in the stimulation of apoptosis process [48,49], we propose thatapoptosis bodies in the lesions captured by APCs (antigen-presentingcells) produce Anxa11 autoantigens.

Anxa11 has been identified as a new susceptibility locus for sarcoid-osis, a rare genetic AD [50–54]. A common non-synonymous SNP,rs10449550, has been found to be closely associated with the biologicalfunctions of Anxa11. An R230C mutation (Anxa11R230C) located at thefirst annexin repeat of Anxa11 strongly correlated with sarcoidosis.The effect of the mutation of the conserved R230 of Anxa11 in sarcoid-osis was interpreted using annexin V as a study model. Mutation of theconserved Arg 45 localized in the first annexin repeat of annexin V, as inAnxa11, led to greater sensitivity to proteolytic digestion and lowerphospholipid affinity in comparisonwith thewild type [18,55], suggest-ing the likelihood of a similar effect of the conserved Arg residue onAnxa11 function in sarcoidosis. In addition, themRNAandprotein levelsof Anxa11 were significantly downregulated when activating stimuliwere given in two immune cells important in sarcoidosis, CD8+ T andCD19+ B cells [54]. This suggests that genetic instability and mutation

of a key residue in Anxa11 might contribute to the development andprogression of sarcoidosis through immunocytes.

3.2. Anxa11 is associated with thrombolysis

Acute myocardial infarction and ischemic strokes remain two lead-ing causes of death throughout the world. Thrombolytic therapy is animportant treatment for them but certain disadvantages reduce thetherapeutic efficacy of available thrombolytic agents [56,57]. Anxa11has proved to have potential as an adjuvant therapeutic promotingthrombolytic efficiency.

SAK (staphylokinase) is a promising blood clot dissolving agent.However, clinical trials have already proved that it is a good fibrin-specific agent and it has been used as a clotting component in chimeras[56,58]. Chiou et al. found that the fusion protein SAK–Anxa11 pos-sessed a similar efficiency in plasma clot lysis as SAK, but a higherefficiency in retarding clot formation. SAK–Anxa11 dissolved bothplatelet rich plasma (PRP) clots and platelet poor plasma (PPP) clotswith an efficiency similar to SAK, but SAK–Anxa11 showed a strongereffect in dose-dependent extension of clotting time. It has been sug-gested that the long N-terminal tail of Anxa11 probably serves as a nat-ural linker to enable twomoieties to function properly in a complicatedmicroenvironmentwhen they are still linked [14]. Anxa11 could bind toan important pro-coagulant, phosphatidyl-L-serine (PS) [59,60]. Thus,the SAK–Anxa11 chimera could suppress the accumulation of coagula-tion enzyme complexes on platelets and enhance the clot resolvingefficiency.

4. Anxa11 and cancers

4.1. Anxa11 is associated with ovarian cancer recurrence anddrug resistance

Ovarian cancer is the leading cause of death due to gynecological tu-mors [61]. The combination of surgery and chemotherapy is the basictreatment for ovarian cancer, but chemoresistance is always a consider-able treatment problem during chemotherapy [62,63].

Anxa11 is associated with recurrence and chemoresistance of ovari-an cancer. Antibody microarray and immunoblotting results have indi-cated that Anxa11 is down-regulated 3–8 folds in cisplatin-resistantovarian cancer cell lines 2008/C13*5.25, HEY C2 and A2780cis com-pared to cisplatin-sensitive 2008, HEY and A2780 lines, respectively.Consistent with these results, the resistance of 2008 cells to cisplatin in-creased 2.6-fold after Anxa11 knockdown (P b 0.01) [11]. HigherAnxa11 expression levels seemed to prolong the disease-free intervalof patients. IHC analysis showed that Anxa11 levels in recurrent ovariancancers were much lower than in primary ovarian cancers and its levelwas inversely correlated with the recurrence and cisplatin resistance ofovarian cancer [11,63]. These results implicate Anxa11 down-regulationas a characteristic for cisplatin-resistant ovarian cancer cells and a con-tributor to tumor recurrence. Thus Anxa11 may be a potential markerfor chemo-resistance and earlier recurrence in ovarian cancer patients.

P53 is a common transcriptional factor associatedwith cancer devel-opment and chemo-resistance [64]. Anxa11 might influence ovariancancer development and drug resistance by way of p53. Anxa11 canbind to S100A6 in the presence of Ca2+, and S100A6 can bind to p53in a Ca2+-dependent manner, which inhibits the ubiquitylation of p53byMDM2 (p53 tumor suppressor) and enhances p53 transcriptional ac-tivity [65]. Although no direct relationship has yet been reported be-tween advanced ovarian cancer and chemoresistance, both p53 andS100A6 are up-regulated in advanced ovarian cancers [66–68]. There-fore, the dynamic balance among Anxa11, S100A6 and p53 should bestrictly controlled. Anxa11 may act as an inhibitor interfering with theinteraction of S100A6 and p53. Down-regulation of Anxa11 probably in-creases the binding capacity of S100A6 to p53, resulting in enhanced

Fig. 2. The relationship between Anxa11 and cancers.

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p53 transcriptional activation and leading to increased chemoresistanceof ovarian cancers.

4.2. Anxa11 up-regulation enhances the progression of breast cancer

Breast cancer (BC) is the most common cancer in women. It affectsover one million women globally, accounting for over 400,000 deathsannually [69].

Anxa11 up-regulation is positively correlated with the malignancyof BC. Using T7 phage cDNA library assay, Tang et al. revealed that theN-terminal 41–74 amino acid sequence of Anxa11 was positivelyexpressed in 60% of sera of patients with DCIS (ductal carcinoma insitu, the earliest formof BC) and 7% of sera of patientswith IDC (invasiveductal carcinoma) [70]. Furthermore, Anxa11 protein was up-regulatedtwo fold in malignant BC tissues compared with non-malignant BC tis-sues. Interestingly, p53 was also up-regulated 2.15-fold in malignantBC tissues. The basis of why Anxa11 affects BC progression might beconnected with p53 [71]. Anxa11 can be phosphorylated by MAPKand the downstream effectors of MAPK kinase 3 (MKK3) and MAPK ki-nase 7 (MKK7) were also found to be over-expressed in malignant BC[23,72]. Considering the fact that the MKK3/p38 and MKK7/JNK path-ways can regulate p53 transcription activity in BC, the over-expressionof Anxa11 is likely to contribute to p53 activity.

Butwhy can Anxa11 autoantibody or Anxa11 fragments be detectedin sera of invasive BC patients? It has been proposed that overexpres-sion of tumor-associated antigens (TAAs) in specific tissues may leadto a loss of self-tolerance, resulting in the genesis of autoantibodies orfragments [73]. So far no studies of Anxa11 and TAAs have been report-ed, and the role of Anxa11 in BC progression definitely deserves furtherattention.

4.3. Anxa11 is associated with metastasis and drug resistance of colorectalcancer

Colorectal cancer (CRC) is the fourth most common cancer world-wide [74] and is an increasing clinical and public health challenge.CRC metastasis has a great effect on patients' survival. Anxa11 hasbeen reported to be associatedwith CRCmetastasis [75–77], suggestingthat it is of potential use as a diagnostic and prognostic indicator for CRC.

Comparative proteomics and IHC have shown that Anxa11 isoverexpressed in tissues of primary CRC patients compared to normaltissues (P b 0.001). The up-regulation of Anxa11 significantly correlatedwith the presence of advanced tumors but, interestingly, Anxa11 wassignificantly decreased in CRC tissues with lymph node metastasis(LNM) compared to CRC tissues without LNM (P = 0.01). It is clearthat Anxa11 plays an important role in the development andmetastasisof CRC [76] and may have potential as a therapeutic target for CRC.

Mutation of Anxa11 is associatedwith drug-resistance of CRC.Muta-tion of the basic residue, 230Arg, to the polar residue, Cys (Anxa11R230C),was made with SNP rs1049550 resulting in an increase in the overall re-sponse rate of metastatic CRC patients with bevacizuma (an angiogene-sis inhibitor) treatment by ~200% compared with patients with wildtype Anxa11. It has been proposed that Anxa11R230C plays an importantrole in angiogenesis by influencing its chemosensitivity [12,77], and thatits level is associated with CRC chemoresistance. This might be associat-edwith an altered ability of cancer cells to break through the ECM. Com-pared to wild type Anxa11-transfected PKO CRC cells (without Anxa11expression), Anxa11R230C-transfected PKO CRC cells showed reducedMMP-9 expression following treatment with a combined regimen of5-FU, leucovorin, irinotecna and bevacizumab [12]. Like its effect ofAnxa11R230C in sarcoidosis [55], the mutation of Arg230 of Anxa11might exert this action in CRC cells by altering its sensitivity to proteolyt-ic digestion and its phospholipid binding affinity. Thus, although there isno direct evidence so far, we postulate that Anxa11 may interfere withdrug sensitivity in CRC metastasis by way of the MAPK pathway,S100A6 or ALG-2.

5. Conclusion and perspectives

Anxa11 is a Ca2+-regulated phospholipid-binding protein. It playsimportant roles in cytokinesis, apoptosis, sex differentiation, exocytosisand cancers. The dysregulation and mutation of Anxa11 are involved insystemic autoimmune diseases and sarcoidosis, and are associated withthe development, chemoresistance and recurrence of cancers. The evi-dence for and potential mechanisms of action of Anxa11 in diseasehave been summarized and are illustrated in Figs. 1 and 2. In summary,results indicate that Anxa11 is involved in the metastasis, invasion anddrug resistance of cancers through the PDGFR andMAPK/p53 pathways.ALG-2 and/or S100A6 are the molecules most frequently reported to beassociated with Anxa11 in diseases. We suggest that Anxa11 may be auseful indicator for the diagnosis, treatment and prognosis of certaindiseases.

Acknowledgements

This work was supported by grants from the National NaturalScience Foundation of China (81171957; 81272186; 81050010), theDistinguished Young Scholars of Liaoning College and University(LJQ2011094), the Liaoning BaiQianWan Talent Project (2012921015)and the Key Laboratory of the Department of Education of Liaoning(LS2010050).

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