Background
Ovarian cancer is one of the most common malignant tumors in the female reproductive system. The GLOBOCAN 2018 estimates indicated that ovarian cancer causes approximately 180,000 deaths worldwide each year, with the greatest mortality rate among malignant gynecological tumors [
1]. Over 70% of ovarian cancer patients are diagnosed at late stages due to the absence of early typical clinical symptoms and effective diagnostic methods [
2]. Despite gradual improvements in surgery and targeted therapeutic drugs, these therapeutic methods still fail to yield a desirable progression-free survival (PFS) in patients with ovarian cancer, and the subsequent treatment of recurrent ovarian cancer still faces challenges [
3]. Hence, it has become a current research trend to investigate the mechanisms underlying the tumorigenesis and progression of ovarian cancer and to search for tumor biomarkers with high sensitivity and specificity.
Annexins comprise a Ca
2+-dependent, phospholipid-binding protein superfamily of 12 members (A1–A11, and A13) that share a conserved core domain containing a Ca
2+-binding site. In eukaryotic cells, Annexins are mainly involved in various biological activities associated with membrane transport and membrane surfaces, such as vesicle trafficking, signal transduction, cell proliferation, cell differentiation, and apoptosis [
4]. Recent findings have revealed abnormal expression levels of Annexins in tumors and their involvement in various biological processes, such as the tumorigenesis and progression, as well as chemoresistance in tumors [
5]. Recent studies have shown that ANXA2 is highly expressed in ovarian cancer [
6] and that ANXA3 and ANXA4 are involved in cisplatin resistance in ovarian cancer [
7,
8]. However, the roles and mechanisms of most Annexins remain unclear. ANXA8 is a member of the Annexin superfamily and was first discovered in complementary DNA libraries generated from human placentas [
9]. Previous studies confirmed that ANXA8 is upregulated in numerous types of malignant tumors, such as acute promyelocytic leukemia [
10], cholangiocarcinoma [
11], breast cancer [
12], and pancreatic cancer [
13], but its relevant mechanisms have been rarely reported, and its roles in ovarian cancer have not yet been elucidated. We conducted a preliminary screen for differentially expressed genes in three ovarian cancer cell lines and found that
ANXA8 was upregulated in cell lines with greater malignancy and drug resistance [
14].
Here, we searched for mRNA expression levels of Annexins between ovarian cancer and normal ovarian tissues using the Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) databases, analyzed the prognostic value of each Annexin family member in ovarian cancer using the Kaplan–Meier plotter database, and constructed a gene–gene interaction network for Annexins in order to explore their mechanisms of function. We further analyzed ANXA8, which was significantly correlated with the prognosis of patients with ovarian serous tumors. ANXA8 was subjected to gene set enrichment analysis (GSEA) using The Cancer Genome Atlas (TCGA) database to explore its biological functions and relevant pathways. Correlation between ANXA8 and the immune system were analyzed using the TISIDB database. In addition, ANXA8 expression in ovarian cancer was evaluated and validated using clinical samples. Our study was aimed at exploring the clinical significance of ANXA8 and providing a theoretical basis for the early diagnosis, prognostic judgments, and targeted therapy of ovarian cancer.
Discussion
Annexins belong to a multigene superfamily of Ca2+-regulated, phospholipid-binding proteins that are dysregulated in various malignant tumors and are involved in various biological processes, such as signal transduction, proliferation, and apoptosis of tumor cells. However, the roles of Annexin family members in malignant tumors, especially ovarian cancer, have rarely been studied, and their mechanisms of function have yet to be elucidated. In this study, we investigated the expression, prognostic value, genetic variations, and potential roles of Annexin family members in ovarian cancer for the first time. ANXA8, which displayed the greatest correlation with the prognosis of patients with ovarian cancer, as well as the closest association with its tumorigenesis and progression, was selected for subsequent in-depth investigations of its biological processes and signaling pathways, as well as its correlations with immunity. We also validated the expression of ANXA8 in ovarian cancer by performing IHC assays and analyzed relationships between its expression with clinicopathological parameters, survival, and prognosis in patients with ovarian cancer.
Our research group aimed to study correlations between Annexin family members and ovarian cancer. The results suggested that ANXA2 and ANXA4 were correlated with ovarian cancer tumorigenesis and progression. We confirmed that ANXA2 was highly expressed in ovarian cancer, especially serous and mucinous cystadenocarcinoma, and that its high expression was closely associated with lymph node metastasis. ANXA2 and human epididymis protein 4 (HE4) are interacting proteins, and the binding between them can activate various signaling pathways, such as the MAPK and FOCAL pathways, thereby promoting the invasion, metastasis, and epithelial-mesenchymal transition (EMT) of ovarian cancer cells [
27]. Our results also showed that ANXA4 was highly expressed in ovarian cancer, and in particular, it displayed the highest expression level in ovarian clear cell carcinoma. Binding between ANXA4 and Vimentin promotes the proliferation, invasion, and migration of ovarian clear cell carcinoma, and inhibits cell apoptosis by activating the ERK and NF-kB pathways. Both ANXA2 and ANXA4 are Lewis y-modified proteins whose biological functions are further enhanced by glycosylation [
28,
29]. The above results are consistent with the results reported by Mogami et al. [
30]. In addition, Madureira PA et al. illustrated that two molecules of ANXA2 bind to a dimer of the protein S100A10 to form a heterotetramer, also known as AIIt. The ANXA2 subunit helps to stabilize and anchor S100A10 to the plasma membrane, and AIIt activates plasminogen via tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (uPA). This in turn increases the production of plasmin, leading to the activation of metalloproteinases (MMPs) and the degradation of extracellular matrix (ECM) proteins, thereby promoting tumor progression and chemoresistance [
31,
32]. Liu et al. found that ovarian cancer proliferation and invasion can be suppressed by inhibiting ANXA2 via β-catenin/EMT [
33]. Cua et al. presented evidence revealing that the monoclonal antibody 2448 can be used to monitor the EMT of ovarian and breast cancers by targeting a unique glycan epitope on ANXA2 [
34]. These findings complemented our preliminary studies. Both the Oncomine and GEPIA database analyses showed that
ANXA2 mRNA expression level was significantly upregulated in ovarian cancer. The TCGA database mainly focused on ANXA4 expression level in ovarian serous cystadenocarcinoma, which may have led to a discrepancy with our results that showed the highest expression level of ANXA4 in ovarian clear cell carcinoma. Prognostic analysis suggested that
ANXA2 and
ANXA4 mRNA upregulation was significantly correlated with poor OS in patients with ovarian serous tumors. Therefore, we believe that ANXA2 and ANXA4 are closely associated with the tumorigenesis and progression of ovarian cancer.
In addition, our preliminary prediction based on microarray data suggested that
ANXA8 may be closely associated with ovarian cancer tumorigenesis and progression. Screening for differentially expressed genes in three groups of ovarian cancer cell lines with different degrees of malignancy showed that
ANXA8 was highly expressed in cell lines with greater degrees of malignancy and drug resistance, and its expression pattern was consistent with that of α1,2-fucosyltransferase [
14]. It was recently confirmed that ANXA8 induced HIF-1α transcription via calcium signaling pathways in pancreatic cancer, and can be regulated by zinc-finger transcription factor ZIC2 to inhibit apoptosis [
13,
35]. The bioinformatics prediction performed by Rossetti et al. suggested that all-trans retinoic acid receptor alpha participates in the morphogenesis of breast cancer by directly targeting the promoter region of
ANXA8 or targeting the 3′-untranslated region of ANXA8 mRNA via miR-342 [
12]. However, no report has demonstrated a correlation between ANXA8 expression and ovarian cancer.
The correlation between ANXA8 expression and ovarian cancer was further validated using various databases. The Oncomine database showed that ANXA8 mRNA expression level was significantly upregulated in ovarian cancer, and the TISIDB database showed that ANXA8 mRNA expression increased significantly with advanced FIGO stages. The results of the prognostic analysis showed that, among Annexin family members, the upregulation of ANXA8 mRNA expression displayed the greatest correlation with poor OS and PFS in patients with ovarian serous tumors. In addition, ANXA8 showed an even more significant correlation with prognosis in patients with poor differentiation, advanced FIGO stages, and TP53 mutations. Hence, we speculate that ANXA8 is extremely important for evaluating the survival and prognosis in patients with ovarian cancer and may serve as an indicator for TP53 mutations in patients with ovarian cancer.
The genes co-expressed with
ANXA8 identified using the cBioPortal database were subjected to functional and pathway enrichment analyses, and the results indicated that they are mainly involved in biological processes, such as cell migration, cell adhesion, angiogenesis, and inflammatory responses, as well as in regulating various cancer related signaling pathways, such as PI3K-Akt, focal adhesion, and proteoglycans in cancer, thereby affecting the tumorigenesis and progression of ovarian cancer. It has been reported that ANXA8 affects the formation of CD63/VEGFR2/β1 integrin complexes and promotes the VEGF-A-mediated human umbilical vein endothelial cell sprouting [
36]. EGF-mediated FOXO4 phosphorylation in cholangiocarcinoma leads to the transcriptional inhibition of
ANXA8, FAK downregulation, and alteration of F-actin kinetics [
11]. Therefore, we infer that
ANXA8 and its co-expressed genes jointly regulate ovarian cancer tumorigenesis and progression through a complex regulatory network.
In recent years, increasing attention has been paid to the treatment of tumors via immunotherapy, which targets cancer by activating the body’s own immune system. Recent findings have suggested that ovarian cancer is an immunogenic tumor and, thus, developing vaccines, adoptive T cell therapy, and immunomodulators has broad prospects for reducing mortality rates in patients with ovarian cancer [
37]. In this study, we assessed the correlation between
ANXA8 and the immune system via the TISIDB database, and the results showed that
ANXA8 had the greatest correlation with lymphocytes (such as Th1, MDSC, Tcm_CD4, Neutrophil), immunoinhibitors (such as CSF1R, HAVCR2, KDR, and CD244), immunostimulators (such as C10orf54, TNFSF9, CXCL12, and CD70), and MHC molecules (such as HLA-DQA1, HLA-DPB1, HLA-DQB1, and HLA-DPA1). The epigenetic silencing of Th1-type chemokines is a novel mechanism of immune evasion in tumors, and selective epigenetic reprogramming can enhance the clinical efficacy of treatments against ovarian cancer [
38]. As the most notable biomarker in tumor research in the past decade,membrane-bound PD-L1 in ovarian cancer cells can be induced by soluble inflammatory factors derived from tumor associated macrophage (TAM), thus leading to immune escape [
39]. Dual blockade of CXCL12-CXCR4 and PD-1-PD-L1 pathways have been found to inhibit ovarian tumor growth and prevent immunosuppression [
40]. In addition, osteopontin overexpression upregulates PD-L1 expression in hepatocellular carcinoma cells by activating the CSF1–CSF1R pathway in macrophages, and blockage of CSF1/CSF1R prevents TAM trafficking. Hence, CSF1R inhibitors can be used to synergistically enhance the therapeutic efficacy of PD-L1 antibodies [
41]. Therefore,
ANXA8, which is associated with these immune molecules, may provide a new target for studying the immune evasion of ovarian cancer cells and can potentially serve as a immunotherapeutic target for ovarian cancer.
IHC assays demonstrated that ANXA8 was found mainly in the cell membrane and cytoplasm, and was highly expressed in epithelial ovarian cancer. In addition, its expression level was correlated with FIGO stages. Patients with high ANXA8 expression and advanced FIGO stages had a relatively poor prognosis. COX regression model analysis suggested that high ANXA8 expression was an independent risk factor affecting the survival and prognosis in patients with epithelial ovarian cancer. Therefore, ANXA8 has the greatest correlation with prognosis in patients with ovarian cancer and is extremely closely related to ovarian cancer tumorigenesis and progression. ANXA8 is a high candidate as a biomarker for early diagnosis, immunotherapy, and prognostic judgments of ovarian cancer.
In addition, correlations between other members of the Annexin family and ovarian cancer were validated using these databases. The results indicated that they were also correlated with ovarian cancer tumorigenesis and progression.
Previous studies confirmed that some members of the Annexin family are associated with chemoresistance in ovarian cancer. ANXA3 was upregulated in platinum-resistant ovarian cancer [
7], whereas ANXA1 and ANXA11 expression was negatively correlated with paclitaxel and cisplatin resistance in ovarian cancer, respectively [
42,
43]. The Oncomine database showed that
ANXA1 mRNA expression was downregulated in ovarian cancer, while both of the databases showed that
ANXA3 and
ANXA11 mRNA expression levels were significantly upregulated in ovarian cancer, among which the upregulation of
ANXA11 mRNA displayed a significant correlation with poor PFS in patients with ovarian serous tumors.
There are relatively few studies on other members of the Annexin family in ovarian cancer, but it has been demonstrated that they are dysregulated in other malignant tumors, such as breast cancer, gastric cancer, colorectal cancer, and pancreatic cancer [
44‐
49]. The results of our database analyses showed that the mRNA expression levels of
ANXA5, ANXA6,
ANXA7 and
ANXA13 were downregulated.
ANXA7 mRNA downregulation was significantly correlated with poor PFS in patients with ovarian serous tumors, and
ANXA9 mRNA upregulation was significantly correlated with poor OS and PFS in patients with ovarian serous tumors.
Cancer cells carry different types of mutations that can lead to uncontrolled cell replication. Copy number alterations are ubiquitous in cancer, and are associated with cancer outcomes such as recurrence and death [
50]. Although some studies have suggested that copy number variations correlate with mRNA expression [
51], we found that gene amplification of Annexin family and high mRNA expression were not significantly associated. However, we found gene alteration in 49.74% of Annexin family in the TCGA database, including an amplification rate of 43.22%, and a deep deletion rate of 5.32%. The incidence rate of genetic variations in the
ANXA13 gene was up to 34.65%, whereas the incidence rate of amplifications in the
ANXA9 gene was up to 12.01%. Therefore, genetic variations in
ANXA9 and
ANXA13 genes may be correlated with ovarian cancer tumorigenesis and progression. In addition,
ANXA2,
ANXA4,
ANXA8,
ANXA9, and
ANXA11, for which mRNA upregulation were significantly associated with poor OS or PFS, had a higher incidence rate of amplifications than deep deletions. Therefore, increased amplifications of Annexin family may be associated with poor prognosis in ovarian cancer.
To clarify the mechanisms of function of Annexins in ovarian cancer, we constructed a gene–gene interaction network using the GeneMANIA database. The results suggested that Annexins interact intensively with other genes, such as
STXBP2,
NDRG1,
STX4,
RACK1, and
ACTA1. It has been demonstrated that
NDRG1 interacts with the Wnt co-receptor, LRP6, to inhibit the EMT mediated by the Wnt-β-catenin signaling pathway [
52]. The interaction between STX4 and Muncl18c facilitates invadopodium formation in tumor cells [
53]. RACK1 promotes the phosphorylation of Akt and MAPK, as well as the proliferation, migration, and invasion of ovarian cancer cells [
54]. It has also been confirmed that RACK1 and ANXA7 are interacting proteins that participate in liver cancer metastasis [
55]. Therefore, these interacting genes are closely associated with the tumorigenesis and progression of tumors, but their interactions with Annexins in ovarian cancer still require further experimental validations. Our functional analyses indicated that Annexins were correlated with calcium-dependent phospholipid binding and S100 protein binding. Jaiswal et al. demonstrated that S100A11 and ANXA2 form a complex at the site of injury which facilitates local remodeling of the actin cytoskeleton and excision of damaged cell membrane [
56]. Hatoum et al. identified differential regulation of the annexin/S100A family by activation of p14ARF-p53-p21 in breast cancer cells, and demonstrated that ANXA1, ANXA2, ANXA4, ANXA6, and ANXA9 were upregulated, with a high expression of ANXA9 functioning as a predictor for poor OS after endocrine therapy in estrogen receptor-positive (ER+) patients [
57].
Therefore, Annexins may be closely associated with and play important roles in ovarian cancer. However, functional mechanisms of Annexins still require further experimental validations, owing to differences found among databases, limited sample sizes, and few relevant experimental studies.