LncRNA HOXD-AS1 promotes oral squamous cell carcinoma by sponging miR-203a-5p.

OBJECTIVE: In the present study, we aimed to explore lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) expression in oral squamous cell carcinoma (OSCC) tissues, its biological roles, and the underlying potential mechanisms in OSCC progression. MATERIALS AND METHODS: HOXD-AS1 expression in paired OSCC and non-tumor tissues from 60 OSCC patients was determined by RT-qPCR. The effects of HOXD-AS1 and miR-203a-5p overexpression on expression of Annexin A4, a validated target of miR-203a-5p, were analyzed by RT-qPCR and Western blot. The roles of HOXD-AS1, miR-203a-5p, and Annexin A4 in the invasion and migration of OSCC cells were analyzed by Transwell assays. RESULTS: HOXD-AS1 overexpression in OSCC predicted poor survival. HOXD-AS1 was predicted to interact with miR-203a-5p, but its expression was not significantly correlated with miR-203a-5p. HOXD-AS1 overexpression increased Annexin A4 expression, while miR-203a-5p overexpression decreased Annexin A4 expression in OSCC cells. Transwell assays showed that invasion and migration of OSCC cells were enhanced by HOXD-AS1 and Annexin A4 overexpression but were reduced by miR-203a-5p overexpression. In addition, miR-203a-5p overexpression suppressed the role of HOXD-AS1 in cell movement and Annexin A4 expression. CONCLUSIONS: HOXD-AS1 may upregulate Annexin A4 by sponging miR-203a-5p in OSCC to promote cancer cell invasion and migration.

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

Circular RNA circ_0000592 elevates ANXA4 expression via sponging miR-1179 to facilitate tumor progression in gastric cancer.

Increasing evidence indicated that dysregulated circular RNAs were implicated in the progression of multiple malignancies. However, the function of circ_0000592 in gastric cancer (GC) progression and its associated mechanism remain poorly understood. Quantitative real-time PCR and Western blot assay were performed to detect RNA and protein expression. Cell proliferation, migration and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine staining assay, Transwell migration assay and Transwell invasion assay, respectively. The glucose/lactate assay kit was used to assess the rates of glucose consumption and lactate production. The interaction between microRNA-1179 (miR-1179) and circ_0000592 or Annexin A4 (ANXA4) was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Xenograft tumor model was established to investigate the effect of circ_0000592 on tumor growth in vivo. Circ_0000592 expression was elevated in GC tissues and cells. Circ_0000592 knockdown hampered cell proliferation, migration, invasion and glycolysis of GC cells. MiR-1179 was a direct target of circ_0000592, and circ_0000592 silencing-mediated effects in GC cells were partly reversed by the knockdown of miR-1179. MiR-1179 interacted with the 3' untranslated region (3'UTR) of ANXA4. Circ_0000592 silencing reduced ANXA4 expression partly by upregulating miR-1179 in GC cells. ANXA4 overexpression partly overturned circ_0000592 knockdown-induced effects in GC cells. Circ_0000592 depletion markedly suppressed xenograft tumor growth in vivo. Circ_0000592 contributed to GC progression through regulating miR-1179/ANXA4 axis, which provided novel potential biomarkers and therapeutic targets for GC treatment.

Annexin A4 N-terminal peptide inhibits adenylyl cyclase 5 and limits ß-adrenoceptor-mediated prolongation of cardiac action potential.

Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over ß-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, ß-adrenoceptor (ßAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

GSK3ß-Ikaros-ANXA4 signaling inhibits high-glucose-induced fibroblast migration.

Previous studies showed that the activation of Wnt signaling reduced high glucose (HG)-mediated fibroblast damage, but the molecular basis for this phenomenon remains elusive. This study aimed to analyze the level of phosphorylation of GSK3ß Ser9 (pGSK3ß Ser9) during HG damage. Moreover, the phosphomimic form of pGSK3ß Ser9 was expressed to analyze its effect on cell migration via the phosphorylation of Ikaros. The results revealed that HG treatment significantly reduced the pGSK3ß Ser9 level. The overexpression of GSK3ß Ser9D and GSK3ß Ser9A accelerated and inhibited fibroblast cell migration, respectively. P110α knockdown or treatment with SP600125, an inhibitor of JNK, also reduced the pGSK3ß Ser9 level under HG condition. Treatment with SP600125 inhibited the migration of fibroblasts, but not in GSK3ß Ser9D-expressing cells. Further, yeast two-hybrid screening and biochemical analysis identified that GSK3ß interacted and phosphorylated Ikaros at Ser391. Besides, GSK3ß Ser9D, but not GSK3ß Ser9A, activated Ikaros Ser391 phosphorylation. Expressing Ikaros or ß-catenin significantly promoted cell migration, suggesting that GSK3ß modulated cell migration partially via the activation of Ikaros besides ß-catenin signaling under HG condition. The expression of the phosphomimic form of Ikaros Ser391D resulted in a significant increase in the extent of cell migration compared with Ikaros under HG condition. Moreover, the Ikaros Ser391D DNA-binding affinity toward the ANXA4 promoter increased, and ANXA4 suppression promoted cell migration. In conclusion, the results of this study provided a new regulatory mechanism by which GSK3ß negatively regulated human skin fibroblast cell migration.

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia.

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

Targeting pathogenic postischemic self-recognition by natural IgM to protect against posttransplantation cardiac reperfusion injury.

BACKGROUND: Natural IgM antibodies represent a class of innate pattern recognition receptors that recognize danger-associated molecular patterns expressed on stressed or dying cells. They play important roles in tissue homeostasis by disposing of prenecrotic cells and suppressing inflammation. However, ischemic insult leads to a pathogenic level of IgM binding and complement activation, resulting in inflammation and injury. We investigate the role of self-reactive IgM in the unique setting of transplantation where the donor organ undergoes both cold and warm ischemia and global ischemic insult. METHODS AND RESULTS: By transplanting hearts from wild-type donor mice into antibody-deficient mice reconstituted with specific self-reactive IgM monoclonal antibodies, we identified neoepitopes expressed after transplantation and demonstrated a key role for IgM recognition of these epitopes in graft injury. With this information, we developed and characterized a therapeutic strategy that exploited the postischemia recognition system of natural antibodies. On the basis of neoepitope identification, we constructed an anti-annexin IV single-chain antibody (scFv) and an scFv linked to Crry, an inhibitor of C3 activation (scFv-Crry). In an allograft transplantation model in which recipients contain a full natural antibody repertoire, both constructs blocked graft IgM binding and complement activation and significantly reduced graft inflammation and injury. Furthermore, scFv-Crry specifically targeted to the transplanted heart and, unlike complement deficiency, did not affect immunity to infection, an important consideration for immunosuppressed transplant recipients. CONCLUSIONS: We identified pathophysiologically important epitopes expressed within the heart after transplantation and described a novel translatable strategy for targeted complement inhibition that has several advantages over currently available approaches.

Overexpression of annexin A4 indicates poor prognosis and promotes tumor metastasis of hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) after surgical resection remains unsatisfactory for the majority of HCC patients who developed early recurrence or metastasis. There is still a lack of reliable biomarkers that can be used to predict the possibility of recurrence/metastasis in HCC patients after operation. In the current study, annexin A4, a calcium-dependent phospholipid-binding protein, has been found to be significantly elevated in HCC patients with early recurrence/metastasis, and had a strong correlation with portal vein tumor thrombosis (p = 0.03) and advanced BCLC stage (p = 0.002). Cox proportional hazards regression analysis revealed that annexin A4 was an independent prognostic predictor for both early recurrence/metastasis (HR = 1.519, p = 0.032) and overall survival (HR = 1.827, p = 0.009) after surgical resection. Meanwhile, Kaplan-Meier analysis showed that Patients with high-expression levels of annexin A4 had higher recurrence rate and shorter overall survival than those with low expression (log-rank test, p < 0.001). Furthermore, in vitro studies have demonstrated that overexpression of annexin A4 facilitated HCC cell migration and invasion via regulating epithelial-mesenchymal transition (EMT). In conclusion, annexin A4 has played important roles in the progression of HCC, and might act as a potential prognostic biomarker for HCC.

Prognostic significance of annexin A2 and annexin A4 expression in patients with cervical cancer.

BACKGROUND: The annexins (ANXs) have diverse roles in tumor development and progression, however, their clinical significance in cervical cancer has not been elucidated. The present study was to investigate the clinical significance of annexin A2 (ANXA2) and annexin A4 (ANXA4) expression in cervical cancer. METHODS: ANXA2 and ANXA4 immunohistochemical staining were performed on a cervical cancer tissue microarray consisting of 46 normal cervical epithelium samples and 336 cervical cancer cases and compared the data with clinicopathological variables, including the survival of cervical cancer patients. RESULTS: ANXA2 expression was lower in cancer tissue (p = 0.002), whereas ANXA4 staining increased significantly in cancer tissues (p < 0.001). ANXA2 expression was more prominent in squamous cell carcinoma (p < 0.001), whereas ANXA4 was more highly expressed in adeno/adenosquamous carcinoma (p < 0.001). ANXA2 overexpression was positively correlated with advanced cancer phenotypes, whereas ANXA4 expression was associated with resistance to radiation with or without chemotherapy (p = 0.029). Notably, high ANXA2 and ANXA4 expression was significantly associated with shorter disease-free survival (p = 0.004 and p = 0.033, respectively). Multivariate analysis indicated that ANXA2+ (HR = 2.72, p = 0.003) and ANXA2+/ANXA4+ (HR = 2.69, p = 0.039) are independent prognostic factors of disease-free survival in cervical cancer. Furthermore, a random survival forest model using combined ANXA2, ANXA4, and clinical variables resulted in improved predictive power (mean C-index, 0.76) compared to that of clinical-variable-only models (mean C-index, 0.70) (p = 0.006). CONCLUSIONS: These findings indicate that detecting ANXA2 and ANXA4 expression may aid the evaluation of cervical carcinoma prognosis.

Quantitative proteomic analysis for novel biomarkers of buccal squamous cell carcinoma arising in background of oral submucous fibrosis.

BACKGROUND: In South and Southeast Asian, the majority of buccal squamous cell carcinoma (BSCC) can arise from oral submucous fibrosis (OSF). BSCCs develop in OSF that are often not completely resected, causing local relapse. The aim of our study was to find candidate protein biomarkers to detect OSF and predict prognosis in BSCCs by quantitative proteomics approaches. METHODS: We compared normal oral mucosa (NBM) and paired biopsies of BSCC and OSF by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) to discover proteins with differential expression. Gene Ontology and KEGG networks were analyzed. The prognostic value of biomarkers was evaluated in 94 BSCCs accompanied with OSF. Significant associations were assessed by Kaplan-Meier survival and Cox-proportional hazards analysis. RESULTS: In total 30 proteins were identified with significantly different expression (false discovery rate < 0.05) among three tissues. Two consistently upregulated proteins, ANXA4 and FLNA, were validated. The disease-free survival was negatively associated with the expression of ANXA4 (hazard ratio, 3.4; P = 0.000), FLNA (hazard ratio, 2.1; P = 0.000) and their combination (hazard ratio, 8.8; P = 0.002) in BSCCs. CONCLUSION: The present study indicates that iTRAQ quantitative proteomics analysis for tissues of BSCC and OSF is a reliable strategy. A significantly up-regulated ANXA4 and FLNA could be not only candidate biomarkers for BSCC prognosis but also potential targets for its therapy.

Annexin A4 is a novel direct regulator of adenylyl cyclase type 5.

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on ß-adrenoceptor (ß-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the ß-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to ß-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

Identification of Annexin A4 as a hepatopancreas factor involved in liver cell survival.

To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing ß-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic ß-cell apoptosis and cancer cell chemoresistance.

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

Clinicopathological heterogeneity in ovarian clear cell adenocarcinoma: a study on individual therapy practice.

Ovarian clear cell adenocarcinoma (CCA) has been believed to be a lethal histological subtype of an epithelial ovarian adenocarcinoma (EOA); its precursor has been assumed to be endometriosis. However, it has been reported that CCAs occasionally exhibit different clinical behaviors, suggesting that CCAs might not belong to a single category. We focused on CCAs combined with other histological types of EOAs; we re-evaluated the pathology of 46 CCAs and divided them into two subgroups: 35 CCAs alone (pure-type CCAs); and 11 CCAs with other histological types, endometrioid adenocarcinomas (EAs) or/and serous adenocarcinomas (SAs) (mixed-type CCAs). Immunohistochemical analysis for expression of ARID1A, p53, PTEN, Annexin 4, hepatocyte nuclear factor-1ß (HNF-1ß), and WT-1 was employed. We identified that patients with endometriosis were younger than those without endometriosis in pure-type CCAs (P < 0.005). In mixed-type CCAs, the immunohistochemical-staining patterns revealed internal transition of each histological component. In pure-type CCAs, expressions of ARID1A and p53 were mutually altered, and altered expression of p53 was associated with worse prognosis than that of ARID1A (P < 0.001). Our results provide evidence that CCAs would have clinicopathological heterogeneity, determining the patient's prognosis. Furthermore, immunohistochemical analysis may shed light on the selection of appropriate treatment, including chemotherapy.

[Pathologic features of fallopian tubal fimbriae in patients with endometrial serous carcinoma].

OBJECTIVE: To discuss the potential relationship between endometrial serous carcinoma (ESC) and tubal epithelial lesions by pathologic examination of fallopian tubes with ESC. METHODS: A total of 30 cases of typical ESC were reexamined and chosen by the pathologist. In each case, bilateral fallopian tubes were submitted to examination of pathologic morphology and immunostaining for p53, annexin IV (ANX-IV), human epidermal growth factor receptor 2 (HER2)/neu, and high-mobility group protein A2 (HMGA2). RESULTS: Fallopian tubal epithelial lesions were found in 15 cases, including 9 cases tubal serous carcinoma, 2 cases serous tubal intraepithelial carcinoma (STIC) and 2 cases epithelial hyperplasia. Both sides of tubal serous carcinoma and STIC were found in 1 case. The results showed the positive expression for p53 in 26 (87%) out of 30 endometrial malignant specimens tissues and 9 (30%) tubal tissues samples (P > 0.05). Twenty-five (83%) endometrial malignant specimens tissues and 6 (20%) tubal tissues samples showed the positive expression of ANX-IV. Twenty-one (70%) endometrial malignant tissues and 7 (23%) tubal tissues showed the positive expression of HER2/neu. Twenty-five (83%) endometrial malignant tissues and 6 (20%) tubal tissues showed the positive expression of HMGA2. While, there were significant differences among the expression of three proteins between endometrium and the fallopian tube site (all P < 0.05). CONCLUSIONS: STIC may be associated with the occurrence of ESC. The expression of p53 was positively correlated between the fallopian tube and the endometrium. ANX-IV, HER2/neu and HMGA2 were extensively expressed in ESC.

Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A.

Although platinum drugs are often used for the chemotherapy of human cancers, platinum resistance is a major issue and may preclude their use in some cases. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to carboplatin through increased extracellular efflux of the drug. However, the precise mechanisms underlying that chemoresistance and the relationship of Anx A4 to platinum resistance in vivo remain unclear. In this report, the in vitro mechanism of platinum resistance induced by Anx A4 was investigated in endometrial carcinoma cells (HEC1 cells) with low expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, HEC1 control cells were compared with Anx A4-overexpressing HEC1 cells in xenografted mice. Significantly greater chemoresistance to cisplatin was observed in vivo in Anx A4-overexpressing xenografted mice. Immunofluorescence analysis revealed that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became colocalized with ATP7A, a copper transporter also well known as a mechanism of platinum efflux. ATP7A expression suppressed by small interfering RNA had no effect on HEC1 control cells in terms of chemosensitivity to platinum drugs. However, suppression of ATP7A in Anx A4-overexpressing platinum-resistant cells improved chemosensitivity to platinum drugs (but not to 5-fluorouracil) to a level comparable to that of control cells. These results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A.

Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.