Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Expression Characteristics of SODD and ALG-2 as Possible Biomarkers for Evaluating Lymphatic Metastasis Potential of Hepatocarcinoma in a Mouse Model.

Annexin A7 has been confirmed in our previous research to be an important factor in lymph node metastasis (LNM) of hepatocellular carcinoma (HCC). SODD and ALG-2 are the binding proteins of Annexin A7 and can work in protein complexes. The present study was carried out with the constructed cell lines in mouse model of metastasis for further elaboration of possible mechanisms and identification of associated genes in the LNM of HCC. This experiment used inbred Chinese 615 mice, as well as Hca-F and Hca-P cells. Quantification of the relative messenger RNA (mRNA) expression of SODD and ALG-2 was realized by using qRT-PCR. Quantification of the protein expressions of SODD and ALG-2 was achieved by using western blot. Experimental mice (n=160) (6-8weeks old, 18-22g, SCXK [LIAO] 2008-0002) were randomly classified into four groups equally, which were separately inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells. Serum levels of SODD and ALG-2 were measured by ELISA. Immunohistochemical analysis of SODD and ALG-2 was further conducted. Tumor LNM-related factors of SODD and ALG-2 showed the same tendency in their expression correspondingly with the up-regulated expression of Annexin A7. Our experiment further explored the roles of SODD and ALG-2 based on Annexin A7 up-regulation vectors construction and the establishment of corresponding controls in vivo. Furthermore, the mouse model of primary tumors was constructed by injecting Hca-F, FAnxa7-upregulated and Hca-P, PAnxa7-upregulated cells into the mouse footpad. Mice were sacrificed at the designated time points for detecting SODD and ALG-2 expression in tumor tissue and serum samples. Collectively, our work indicates SODD in tumors and in serum and ALG-2 in serum are valuable in evaluating LNM in mice with HCC.

AnnexinA7 promotes epithelial-mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and metastasis is the major cause of the high mortality of HCC. In this study, we identified that AnnexinA7 (ANXA7) and Sorcin (SRI) are overexpressed and interacting proteins in HCC tissues and cells. In vitro functional investigations revealed that the interaction between ANXA7 and SRI regulated epithelial-mesenchymal transition (EMT), and then affected migration, invasion, and proliferation in HCC cells. Furthermore overexpression/knockdown of ANXA7 was remarkably effective in promoting/inhibiting tumorigenicity and EMT in vivo. Altogether, our study unveiled a mechanism that ANXA7 promotes EMT by interacting with SRI and further contributes to the aggressiveness in HCC, which provides a novel potential therapeutic target for preventing recurrence and metastasis in HCC.

ANXA7 Regulates Platelet Lipid Metabolism and Ca2+ Release in Arterial Thrombosis.

[Figure: see text].

Promoting TTC4 and HSP70 interaction and translocation of annexin A7 to lysosome inhibits apoptosis in vascular endothelial cells.

We previously demonstrated that Tetraticopeptide 4 (TTC4) inhibited apoptosis in vascular endothelial cells (VEC) deprived of serum and fibroblast growth factor 2 (FGF-2). In this study, we aimed to resolve the mechanism of TTC4 inhibiting VEC apoptosis. TTC4, predicted as a HSP70 co-chaperone protein, may regulate the fate of cells by affecting the activity of HSP70, however, there is no experimental evidence showing the interaction of TTC4 and HSP70. Using Co-immunoprecipitation (Co-IP), we demonstrated that TTC4 interacted with HSP70. If HSP70 was knockdown, TTC4 no longer suppressed apoptosis. Furthermore, we found ABO, an inhibitor of annexin A7 (ANXA7) GTPase, could promote the interaction of TTC4 and HSP70 and the translocation of ANXA7 to lysosome. At the same time, ABO inhibited the interaction of HSP70 and ANXA7. Moreover, Akt, as a downstream effector of HSP70 was upregulated, and ANXA7 translocating to lysosome protected the stability of lysosomal membrane. Here, we discovered a special mechanism by which TTC4 inhibited apoptosis via HSP70 in VECs. On the one hand, increasing TTC4 and HSP70 interaction upregulated Akt that inhibited apoptosis. On the other hand, decreasing HSP70 and ANXA7 interaction promoted the translocation of ANXA7 to lysosome, which inhibited apoptosis through protecting the lysosomal membrane stability.

ANXA7 promotes the cell cycle, proliferation and cell adhesion-mediated drug resistance of multiple myeloma cells by up-regulating CDC5L.

This study aimed to investigate whether annexin A7 (ANXA7) could promote the cell cycle, proliferation and cell adhesion-mediated drug resistance (CAM-DR) of multiple myeloma (MM) cells by up-regulating cell division cycle 5-like (CDC5L). As a result, ANXA7 expression was increased in the serum of MM patients and the expression of ANXA7 and CDC5L was also increased in MM cell lines. ANXA7 overexpression promoted the proliferation and cycle of U266 and RPMI8226 cells. The expression of proliferation cell nuclear antigen (PCNA), KI67, cyclin dependent kinase 1 (CDK1) and cyclinB1 in transfected cells was consistent with the changes of proliferation and cell cycle. In co-culture system of BMSC cells and MM cells, expression of CD44, ICAM1 and VCAM1 in MM cells was increased, which was further increased by ANXA7 overexpression. Bortezomib could increase the apoptosis of U266 and RPMI8226 cells. In co-culture system of BMSC cells and MM cells, the promotion effects of bortezomib on apoptosis of MM cells was decreased, which was further suppressed by ANXA7 overexpression. The above effects exerted by ANXA7 overexpression could be reversed by ANXA7 interference. Moreover, ANXA7 was proved to be combined with CDC5L. CDC5L interference could inhibit the promotion effects of ANXA7 overexpression on proliferation and cell cycle and inhibition effects of ANXA7 overexpression on apoptosis of MM cells treated with bortezomib in co-culture system. In conclusion, ANXA7 could promote the cell cycle, proliferation and CAM-DR of MM cells by up-regulating CDC5L.

Knockdown of annexin VII enhances nasopharyngeal carcinoma cell radiosensitivity in vivo and in vitro.

BACKGROUND: Radioresistance leads to treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, enhancing the radiosensitivity of NPC cells would likely increase the effectiveness of radiotherapy. Annexin VII (Annexin A7, ANXA7) might be a tumor promoter in NPC but its functions in radiosensitivity remain unclear. METHODS: NPC cell lines CNE2-shANXA7 and CNE2-pLKO.1 were generated and CNE2-shANXA7 nude mice xenograft tumor models were established. The main effects and molecular mechanisms of ANXA7 knockdown in NPC radiosensitivity were studied in vitro and in vivo by analyzing cell viability, clonogenicity, apoptosis, cell cycle distribution, tumor radioresponse and immunohistochemistry assay. RESULTS: ANXA7 knockdown revealed potentially enhanced NPC cell radiosensitivity via apoptosis and increased the cell number at the G2/M phase. In the xenograft model, NPC cells with ANXA7 knockdown were dramatically sensitive to irradiation and tumor growth was significantly suppressed. Compared to CNE2-pLKO.1 xenografts, CNE2-shANXA7 showed more γ-H2AX foci and less phospho-DNA PKcs. CONCLUSIONS: ANXA7 knockdown increased the radiosensitivity of NPC by enhancing apoptosis, modulating the cell cycle distribution into more radiosensitive phases, promoting DNA damage, and inhibiting repair. We showed that decreased ANXA7 levels enhanced radiosensitivity and provided insights into the therapeutic targets for NPC radiotherapy.

MROH7-TTC4 read-through lncRNA suppresses vascular endothelial cell apoptosis and is upregulated by inhibition of ANXA7 GTPase activity.

Apoptosis of vascular endothelial cells (VEC) is the main form of vascular injury that is closely linked to numerous cardiovascular diseases. Therefore, it is important to find new factors that can suppress VEC apoptosis. By using long noncoding RNA (lncRNA) microarray analysis, we found a new read-through lncRNA, MROH7-TTC4, which acted as an apoptosis inhibitor in VECs. Furthermore, by using the inhibitor (ABO) of annexin A7 (ANXA7) GTPase, we discovered that ANXA7 translocated into nucleus and interacted with 5'→3' exoribonuclease (XRN2). The decreased XRN2 phosphorylation induced by ANXA7 GTPase activity inhibition, promoted MROH7-TTC4 expression. Moreover, T-cell intracellular antigen-1 (TIA1), a binding protein of MROH7-TTC4, processed it into MROH7 and TTC4 that could inhibit VEC apoptosis. Here, we conclude that inhibiting ANXA7 GTPase activity promotes the interaction of ANXA7 and XRN2 in nucleus, which regulates the read-through transcription of MROH7-TTC4, and TIA1 is responsible for the process of MROH7-TTC4 that inhibits apoptosis through MROH7 and TTC4.

ANXA7 regulates trophoblast proliferation and apoptosis in preeclampsia.

PROBLEM: Preeclampsia (PE) is a unique gestational disorder leading to maternal and neonatal morbidity and mortality. AnnexinA7 (ANXA7) is a calcium-dependent phospholipid-binding protein that promotes membrane fusion during exocytosis. However, the function of ANXA7 in placental trophoblast is poorly understood. The present study aimed to investigate a possible association between ANXA7 and human trophoblast apoptosis. METHODS: We collected human placental tissues from patients with PE and normal pregnant women to elucidate the expression level of ANXA7. The ANXA7-knockdown and ANXA7-overexpressing HTR8/SVneo cells were utilized for studying the function of ANXA7 in trophoblast. The proliferation and apoptosis levels of trophoblast were examined with Western blot assay, flow cytometry, Cell Counting Kit-8 assay, and immunohistochemistry. RESULTS: ANXA7 expression was significantly lower in placentas from patients with PE patients compared with that in from normal pregnant controls. Knockdown of ANXA7 induced cell apoptosis and inhibited cell proliferation in HTR-8 via by downregulating BCL2 protein levels. Overexpression of ANXA7 reduced apoptosis and promoted HTR8 proliferation. Further analyses showed that ANXA7 knockdown inhibited the activation of the JAK1/STAT3 pathway in HTR-8 cells. CONCLUSION: Our findings revealed a new regulatory pathway of ANXA7/JAK1/STAT3 in trophoblast apoptosis in preeclampsia, suggesting that ANXA7 is a potential therapeutic target for preeclampsia.

Annexin A7 is required for ESCRT III-mediated plasma membrane repair.

The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca2+ - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca2+ flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.

Suppressive effect of an analog of the antimicrobial peptide of LL­37 on colon cancer cells via exosome­encapsulated miRNAs.

Antimicrobial peptides (AMPs) are multifunctional factors with an important role in the innate immune system. Our previous studies revealed that the human cathelicidin LL­37 and its analog, FF/CAP18, limit the proliferation of colon cancer cell lines. In the present study, the exosomes released by HCT116 cells treated with FF/CAP18 were analyzed. After the treatment, exosomes were isolated from the culture supernatant by ultrafiltration and using the miRCURY™ Exosome Isolation Kit. Membrane vesicles 40­100­nm expressing CD63 and CD81 were identified before and after FF/CAP18 treatment. Exosome concentration in the culture supernatant was increased after treatment with FF/CAP18. Exosomes formed in HCT116 cells treated with FF/CAP18 induced growth suppression of the cells in a dose­dependent manner. By contrast, the exosomes formed in non­treated HCT116 cells did not affect cell viability. Microarray analysis of miRNA expression indicated that FF/CAP18 treatment induced increases in the expression of three miRNAs (miR­584­5p, miR­1202 and miR­3162­5p) in both HCT116 cells and exosomes. These results suggest that FF/CAP18 treatment increases exosome formation, and that exosome­encapsulated miRNAs suppress HCT116 cell proliferation. Exosomal miRNAs are considered to be involved in the dissemination of cell signals to control local cellular microenvironments. The present findings suggest that FF/CAP18 regulates cancer growth by modulating cell­to­cell communication. AMPs localize in the cytoplasm of cancer cells and enhance the expression of growth­suppressing miRNAs. These miRNAs are also transported to other cancer cells via exosomes. Therefore, transportation of these miRNAs has the potential to suppress cancer growth. AMPs exert their effects directly by targeting cancer cells and indirectly via exosomes.

High co-expression of the SDF1/CXCR4 axis in hepatocarcinoma cells is regulated by AnnexinA7 in vitro and in vivo.

BACKGROUND: SDF1/CXCR4 and AnnexinA7 play important roles in many physiological and pathological conditions, but the molecular association between them in cancer cells has not been studied thus far. METHODS: The expression changes of SDF1/CXCR4 were detected by gene transcriptome sequencing, qRT-PCR, Western blotting, cytoimmunofluorescence and immunohistochemistry in mouse hepatocarcinoma F/P cells, AnnexinA7 downregulated expression F (FA7DOWN) cells, AnnexinA7 overexpression P (PA7UP) cells, AnnexinA7 unrelated sequence F (FSHUS) cells, empty vector P (PNCEV) cells and normal liver cells in vitro and in vivo. RESULTS: SDF1 and CXCR4 were co-expressed in hepatocarcinoma cells. SDF1 was localized mainly in the cytoplasm of cells, while CXCR4 was mainly localized in the cell membrane. Both in vitro and in vivo, expression levels of SDF1/CXCR4 in F and P cells were higher than in normal liver cells, and expression levels of SDF1/CXCR4 in F cells with high lymphatic metastatic potential were higher than those in P cells with low lymphatic metastatic potential. Expression of SDF1 was higher than that of CXCR4 in P cells and normal liver cells, while expression of CXCR4 was higher than that of SDF1 in F cells. Expression levels of SDF1/CXCR4 were completely consistent with AnnexinA7 regulation. After the AnnexinA7 gene was downregulated or upregulated, expression levels of SDF1/CXCR4 in FA7DOWN/PA7UP cells were lower or higher than those in FSHUS/PNCEV cells. Furthermore, CXCR4 was more sensitively modulated by AnnexinA7 regulation than SDF1. CONCLUSIONS: High co-expression of SDF1/CXCR4 is a molecular characteristic of hepatocarcinoma cells, especially those with high lymphatic metastatic potential. AnnexinA7 positively regulates expression levels of SDF1/CXCR4, in particular CXCR4, and AnnexinA7 is a functional regulator of SDF1/CXCR4.

Human sperm proteins identified by 2-dimensional electrophoresis and mass spectrometry and their relevance to a transcriptomic analysis.

The aim of this study was to identify and analyse human sperm proteins from normozoospermic men using 2-dimensional electrophoresis (2-DE) and mass spectrometry (MS). We identified 73 different sperm proteins, including two less characterized human sperm proteins, Annexin A7 (ANXA7) and c14orf105. Bioinformatic analysis of detected sperm proteins revealed new carbohydrate and lipid metabolic pathways, which supply energy to motile sperm. A comparison of our data with available mRNA microarray data from the human testis allows for validation of identified sperm proteins and aids in the recognition of their physiological pathways.

Critical role for Annexin A7 in secondary brain injury mediated by its phosphorylation after experimental intracerebral hemorrhage in rats.

Glutamate excitotoxicity has been implicated in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Synaptosome associated protein 23 (SNAP23) and SNAP25 are respectively participate in presynaptic glutamate release and postsynaptic glutamate receptor (NMDA receptor) trafficking, both of which are essential for glutamate-mediated excitatory toxicity. SNAP23 and SNAP25 exhibit high homology and SNAP23 has been shown to interact with Annexin A7 (ANXA7). This study was to examine the role of ANXA7 in ICH-induced neuronal damage. A collagenase ICH model was performed in adult male Sprague Dawley rats. First, a possible relationship between ANXA7 and ICH pathology was confirmed by an increase in the protein and mRNA level of ANXA7 in the brain tissue around hematoma of ICH rats and the rescue effects of ANXA7 knockdown in vivo on neuronal death, blood-brain barrier damage, brain edema, neurobehavioral deficient, and inflammatory response. In addition, the rescue effect of ANXA7 knockdown on neurobehavioral deficient was also verified in rat autologous blood injection ICH model. Second, we found that ICH significantly increased the phosphorylation ratio of ANXA7 at the threonine residues mainly in neurons. Finally, based on site-specific mutagenesis, we identified that ANXA7 phosphorylation at threonine 286 is required for its interaction with SNAP25 at presynaptic axon terminal and SNAP23 at postsynaptic axon terminal. Collectively, our findings suggest that ANXA7 contributed to SBI at least partially through regulating glutamate toxicity after ICH. Selective inhibition of ANXA7 phosphorylation may be a novel approach to ameliorate ICH-induced SBI.

SEC-induced activation of ANXA7 GTPase suppresses prostate cancer metastasis.

Annexin A7 (ANXA7) is a suppressor of tumorigenesis and metastasis in prostate cancer. Activated ANXA7 GTPase promotes prostate cancer cell apoptosis. However, the role and underlying mechanism of ANXA7 GTPase in prostate cancer metastasis have not been established. RKIP is a metastatic suppressor and downregulated in prostate cancer metastases. The binding of RKIP and its target proteins could inhibit the activation of its interactive partners. However, the effect of RKIP on ANXA7 GTPase activation is not clear. Here, we report that activation of ANXA7 GTPase by a small molecule SEC ((S)-ethyl 1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3- (4-methoxyphenyl)-1H-pyrazole-5-carboxylate) effectively inhibited prostate cancer metastasis. Mechanistically, activated ANXA7 promoted AMPK phosphorylation, leading to decreased mTORC1 activity, suppressed STAT3 nuclear translocation, and downregulation of pro-metastatic genes, including CCL2, APLN, and IL6ST. Conversely, RKIP interacted with ANXA7 and impaired activation of ANXA7 GTPase by SEC and its downstream signaling pathway. Notably, SEC treatment suppressed metastasis of prostate cancer cells in in vivo orthotopic analysis. Together, our findings provide a novel insight into how metastasis of prostate cancer with low RKIP expression is suppressed by SEC-induced activation of ANXA7 GTPase via the AMPK/mTORC1/STAT3 signaling pathway.

Ezrin expression is altered in mice lymphatic metastatic hepatocellular carcinoma and subcellular fractions upon Annexin 7 modulation in-vitro.

Ezrin and Annexin seven (A7) have been suggested to be involved in several roles in cancers metastasis. However, the role of Ezrin and the effect of A7 on Ezrin expression in lymphatic metastatic hepatocellular carcinoma (LNM-HCC) have not been extensively explored yet. This study reports expression of Ezrin in high lymphatic metastasis (Hca-F >70%) and low metastatic metastasis (Hca-P <30%) HCC cell lines, and the effect of A7 on Ezrin expression. Real-Time PCR, Western blot, Subcellular fractionation, Immunocytochemistry and Immunofluorescence were used to investigate Ezrin expression in addition to migration and invasion behaviors of A7 up-regulated Hca-F cells, A7 down-regulated Hca-P and in their respective negative control (NC) cells. Ezrin expression was higher in high LNM-HCC than low LNM-HCC (p=0.0046). Cell fractionation analysis reveals that Ezrin was highly present in the cytoplasm, nucleus and cytoskeleton of NC-Hca-F cells. However, Ezrin was highly observed in the cell membrane, nucleus and cytoskeleton of NC-Hca-P cells. A7 up-regulation in Hca-F suppressed Ezrin expression (p=0.0248), but increase the migration and invasion, whereas Ezrin was mainly located in the cytoplasm and nucleus fractions. Down-regulation of A7 in Hca-P cells, enhanced Ezrin expression (p<0.0001) in the cytoplasm and nucleus fractions, and suppressed migration and invasion. In conclusion, Ezrin may play a role in LNM-HCC and might be inversely associated with A7 expression. The subcellular localization of Ezrin and A7 was varied according to the metastatic levels. Ezrin may thus be a potential diagnostic and/or prognostic biomarker for HCC.

ANXA7-GTPase as Tumor Suppressor: Mechanisms and Therapeutic Opportunities.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity at 10q, are commonly observed in most human tumors, including prostate, breast, and kidney cancers. The ANXA7-GTPase is a tumor suppressor, which is frequently inactivated by genomic alterations at 10q21. In the last few years, considerable amounts of data have accumulated describing inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase. ANXA7-GTPase contains a calcium binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, emerging evidence suggests that ANXA7-GTPase is a critical factor associated with the metastatic state of several cancers and can be used as a risk biomarker for HER2 negative breast cancer patients. Cross talk between ANXA7, PTEN, and EGFR leads to constitutive activation of PI3K-AKT signaling, a central pathway of tumor cell survival and proliferation. This review focuses on the recent progress in understanding the tumor suppressor functions of ANXA7-GTPase emphasizing the role of this gene in Ca2+ metabolism, and exploring opportunities for function as an example of a calcium binding GTPase acting as a tumor suppressor and opportunities for ANXA7-GTPase gene cancer therapy.

47kDa isoform of Annexin A7 affecting the apoptosis of mouse hepatocarcinoma cells line.

Our previous studies have shown that Annexin A7 is associated with the cell proliferation and apoptosis. Both the isoforms 47kDa and 51kDa of Annexin A7 are expressed in liver cancer cells. This study aimed at elucidating which isoform of Annexin A7 affects the cell apoptosis and how to affect in the hepatocarcinoma cell line. The results showed that Annexin A7 co-immunoprecipitated with Bcl2 and co-located in the cytoplasm and the mitochondria. Therein, the regulated 47kDa isoform of Annexin A7 altered the expression of Bcl2 and the mitochondrial membrane potential. These results indicate that 47kDa isoform of Annexin A7 could associated with the apoptosis of Hca-P cells through its interaction with Bcl2.

[Expression of annexin A7 and its clinical significance in gastric carcinoma].

OBJECTIVE: To investigate the expression of annexin A7 (ANXA7) in the differentiation and lymphatic metastasis of gastric cancer (GC), and to investigate the relationship between ANXA7 and biological characteristics of GC. METHODS: The clinicopathological data of 124 patients with gastric cancer who underwent surgical treatment in our hospital were retrospectively reviewed and analyzed. Immunohistochemical staining and Western blot were performed to analyze the expression of ANXA 7 in primary GC tissues. Logistic regression analysis was conducted to evaluate the association between ANXA7 expression level and differentiation of the GC. RESULTS: A total of 124 GC patients were enrolled in this study, and the expression rate of ANXA7 was 65.3% in the GC. The survival rate of ANXA7-positive patients was significantly lower than that in the patients with negative expression (P<0.001). The results of Cox regression analysis showed that the positive expression of ANXA7, submucosal confinement and pathological stage of GC were associated with poor clinical outcomes. The ratio of pixel density value of primary GC tissues with lymph node metastasis was significantly higher than those in the tissues without lymph node metastasis (0.51±0.07 vs. 0.39±0.06, P<0.001). ROC analysis showed a high area under the curve for the ratio of pixel density value of annexin A7 in the primary GC tissues. At a cut-off level of >0.419, the ratio of pixel density value of ANXA7 exhibited a sensitivity of 91.2% and a specificity of 72.7% for detecting lymph node metastasis of GC. CONCLUSION: High annexin A7 expression is associated with poor differentiation of gastric cancer, and it may become a predictor for lymphatic metastasis of GC.

In vivo and in vitro effect of hepatocarcinoma lymph node metastasis by upregulation of Annexin A7 and relevant mechanisms.

We unveiled the association of Annexin A7 with vascular endothelial growth factor-C (VEGF-C) and the effect of upregulation of Annexin A7 in Hca-F and Hca-P cells on inhibiting hepatocarcinoma (HCC) lymph node metastasis (LNM) in vitro and in vivo. A total of 200 inbred 615 mice were randomly divided into four equal groups inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells, respectively. The primary tumor, popliteal, inguinal, and iliac lymph nodes were prepared for immunohistochemical (IHC) staining, real-time quantitative polymerase chain reaction (qRT-PCR) analysis, Western blot, and hematoxylin-eosin (H&E) staining. There was over 50 % increase both in the number of FAnxa7-upregulated and PAnxa7-upregulated cells migrated through the filter compared to their controls (FAnxa7-control, Hca-F and PAnxa7-control, Hca-P). However, no significant differences were noted in invasion ability between them (all P > 0.05). Tumor lymph vessels were significantly reduced in FAnxa7-upregulated and PAnxa7-upregulated tumors when compared with Hca-F and Hca-P tumors (all P < 0.05). Blood vessel density did not differ significantly between FAnxa7-upregulated and PAnxa7-upregulated tumors and Hca-F and Hca-P tumors. Enzyme-linked immunosorbent assay (ELISA) for VEGF-C showed that upregulating Annexin A7 decreased VEGF-C secretion in FAnxa7-upregulated and PAnxa7-upregulated cells (P < 0.05). The IHC staining result showed that the level of serum Annexin A7 was found to be statistically higher in all experimental groups than that in the control group (P < 0.05). The present results indicated that alterations in serum Annexin A7 expression may be of prognostic relevance in HCC lymphatic metastasis.