
==== Front
Evid Based Complement Alternat Med
Evid Based Complement Alternat Med
ECAM
Evidence-based Complementary and Alternative Medicine : eCAM
1741-427X
1741-4288
Hindawi

10.1155/2022/8748434
Research Article
Evaluation of Annexins Family as Potential Biomarker for Predicting Progression and Prognosis in Clear Renal Cell Carcinoma
Zhao Jiyu 1
https://orcid.org/0000-0002-9226-4809
Chang Luchen 2
Tu Jianping 3
https://orcid.org/0000-0002-4317-9595
Sun Bei sunpei003@sina.com
4
https://orcid.org/0000-0003-4734-3900
Wei Xi weixi@tmu.edu.cn
2
1Department of Urology, ChuiYangLiu Hospital Affiliated to Tsinghua University, 100021 Beijing, China
2Department of Diagnostic and Therapeutic Ultrasonography, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, 300060 Tianjin, China
3Department of Urology, The Third Hospital of Xiamen, 361199 Xiamen, Fujian, China
4Department of Outpatient Office, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, 300060 Tianjin, China
Academic Editor: Vijaya Anand

2022
8 6 2022
8 6 2022
2022 87484347 11 2021
23 4 2022
26 4 2022
Copyright © 2022 Jiyu Zhao et al.
2022
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background

Annexins family (ANXAs), as a Ca2+-dependent phospholipid-binding protein superfamily, participates in a wide variety of biological activities and has been reported to be dysregulated in numerous types of human cancers. Evidence from cell lines and human tissues indicates that ANAXs are involved in kidney clear renal cell carcinoma (KIRC) tumorigenesis. However, their prognostic value and expression pattern associated with KIRC remain to be elucidated.

Methods

We visited public databases, including ONCOMINE, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan–Meier plotter, cBioPortal, and GeneMANIA, to conduct comprehensive bioinformatics analysis and tried to detect basic relationships between each Annexins family member and KIRC.

Results

We found that the expression level of ANXA1/2/4/5/6/7/8/13 in clear renal cell carcinoma tissue was higher than that in the kidney tissue, while the expression level of ANXA3/9/11 in the former was lower than that in the latter. The expression level of ANXA7/8/13 is related to the stage of the tumour. Survival analysis using the Kaplan–Meier plotter database showed that a high transcription level of ANXA2/5/8/10 is related to a low overall survival rate (OS) in predicting KIRC patients. In contrast, high ANXA3/4/7/9/11/13 levels are associated with a high OS in these patients.

Conclusions

Our study implies that ANXA4/8/13 are potential targets of precision therapy for patients with KIRC and that ANXA2/5/8/10 are new biomarkers for the prognosis of KIRC.

National Natural Science Foundation of China#81771852 Tianjin Science and Technology Program#18ZXZNSY00300 Tianjin Health Research Project#ZD20018 Tianjin Research Innovation Project for Postgraduate Students#2020YJSS178
==== Body
pmc1. Introduction

Renal cell cancer (RCC) is one of the most common malignancies, and approximately 73,820 patients were diagnosed in 2019 [1]. The majority of RCC patients have kidney clear renal cell carcinoma (KIRC) histology (75–80%), and the other common histological types include papillary, chromophobe, hereditary leiomyomatosis-associated RCC, and collecting duct carcinoma. These are called “nonclear cell cancers” [2]. Despite advancements in diagnostic methods and operative techniques that have allowed for more effective treatment of KIRC, the 5-year overall survival rate of metastatic KIRC remains less than 20% [1]. Therefore, it is necessary to investigate molecular markers to refine prognostic prediction and identify potential treatment targets.

Annexins are a Ca2+ dependent, phospholipid-binding protein superfamily, with members expressed in the zoology and botany cell. Annexins, including 12 family members, play a major role in the regulation of a broad range of physiological processes linked to cellular membranes [3]. Previous studies have revealed that the aberrant expression of ANXAs is frequently observed in various types of cancer, including cervical cancer [4], bladder cancer [5, 6], breast cancer [7–9], gastric cancer [10], lung cancer [11, 12], oral squamous cell carcinoma [13], hepatocellular carcinoma [14, 15], and cholangiocarcinoma [16]. ANXAs may function as either oncogenes or suppressors depending on tumour biology.

ANXA1/2/3/4/5/7 have been reported to be dysregulated in KIRC [17–20]. However, the majority of these studies only focused on the changes in expression levels and a few prognostic data available. The expression patterns, potential biological functions, and prognostic value of Annexins in KIRC have yet to be fully elucidated.

To the best of our knowledge, no bioinformatics analysis of Annexins profiles has been applied to investigate KIRC. The use of large-scale DNA/RNA sequencing has undergone revolutionary development and has become an integral part of biomedical research. In the present study, we performed a collective analysis of thousands of gene expression or variation in copy-number analyses published online to investigate the expression of Annexin family members in the database to determine its clinical value in KIRC.

2. Materials and Methods

2.1. Ethics Statement

This study was approved by the Academic Committee of ChuiYangLiu Hospital affiliated with Tsinghua University and performed in accordance with the ethical principles expressed in the Declaration of Helsinki. All data were collected from published literature.

2.2. ONCOMINE Analysis

The transcription levels of ANXAs in different cancers were analysed using the online tumour microarray database ONCOMINE (https://www.oncomine.org/). Comparison of ANXAs mRNA expression levels between tumour and normal samples was performed using Student's t-test. The P value and fold change were used as cut-off points at 0.0001 and 2, respectively.

2.3. Gene Expression Profiling Interactive Analysis (GEPIA) Dataset

We used the online database Gene Expression Profiling Interactive Analysis (GEPIA, https://gepia.cancer-pku.cn/) to analyse ANXAs mRNA sequencing expression data [21]. GEPIA could provide mRNA differential expression analysis according to cancer stage and patient survival data using one-way ANOVA. The analysis setting is |log2(FC)| ≥ 1 and P ≤ 0.01.

2.4. The Kaplan–Meier Plotter

We examined the prognostic value (OS and RFS) of ANXA mRNA expression in KIRC using the Kaplan–Meier plotter database [22] (https://www.kmplot.com/). We only selected the JetSet best probe set and automatically chose the best cut-off to perform the analysis.

2.5. cBioPortal

We examined the mutations and putative copy number alterations (CNA) of ANXAs in KIRC using cBioPortal (https://www.cbioportal.org/), an online database on 30 different cancers collected from The Cancer Genome Atlas [23]. A total of 317 KIRC cases with pathology reports were selected in our study. The online tools and data sources are provided by cBioPortal.

2.6. GeneMANIA

To explore interactive functional associations among ANXAs, we used GeneMANIA (https://www.genem/ania.org) to create an interactive functional-association network. The online tools and data sources are provided by GeneMANIA.

3. Results

3.1. Transcriptional Levels of ANXAs in Patients with KIRC

Twelve ANXA factors have been found in mammalian cells. We used the ONCOMINE database to compare the transcription levels of ANXA in cancer with those in normal samples (Figure 1). The mRNA expression levels of ANXA4 were significantly upregulated in patients with KIRC in twelve datasets. In Higgins's dataset [24], ANXA4 was overexpressed compared with that in normal samples in KIRC, with a fold change of 3.662. In Gumz's dataset [25], ANXA4 was also overexpressed in KIRC with a fold change of 13.931. The transcription levels of ANXA4 in KIRC are higher than those in normal tissues in Yusenko [26] and Jones's datasets [27], and their fold changes are 4.763 and 2.598, respectively (Table 1).

Higgins [24] showed another mRNA expression factor with increased expression; that is, ANXA2 has a fold change of 2.358 in patients with kidney renal clear cell carcinoma compared with that in patients with normal kidney tissues. ANXA2 overexpression is also found in kidney renal clear cell carcinoma, with a fold change of 2.444 in Gumz's dataset [25] and 2.383 in Jones's dataset [27] (Table 1).

The mRNA expression levels of ANXA1 and ANXA7 were upregulated in patients with KIRC. The transcription level of ANXA1 in KIRC is higher than that in kidney tissues, and their fold changes are 4.853, 5.348, and 3.26 in Yusenko [26], Gumz [25], and Beroukhim's datasets [28], respectively. In Jones's datasets [27], the mRNA expression of ANXA7 in KIRC increased by 2.565-fold.

3.2. Relationship between the mRNA Levels of ANXAs and the Clinicopathological Characteristics of Patients with KIRC

Using the GEPIA (Gene Expression Profiling Interactive Analysis) dataset (https://gepia.cancer-pku.cn/), we compared the mRNA expression of ANXAs factors between KIRC and kidney tissues. The results indicated that the expression levels of ANXA1, ANXA2, ANXA4, ANXA5, ANXA6, ANXA8, and ANXA13 were higher in KIRC tissues than in kidney tissues, whereas the expression levels of ANXA3, ANXA7, ANXA9, and ANXA11 were lower in the former than in the latter (Figure 2). We also analysed the expression of ANXAs with tumour stage for KIRC. The ANXA7, ANXA8, and ANXA13 groups significantly varied, whereas the ANXA1, ANXA2, ANXA3, ANXA4, ANXA5, ANXA6, ANXA9, ANXA10, and ANXA11 groups did not significantly differ (Figure 3).

3.3. Association of the Increased and Decreased mRNA Expression of ANXAs with the Improved Prognosis of Patients with KIRC

We further explored the critical efficiency of ANXAs in the survival of KIRC patients. We applied the Kaplan–Meier plotter tool using a publicly available dataset (https://kmplot.com/analysis/index.php?p=service&cancer=pancancer_rnaseq) to analyse 530 KIRC patients for ANXAs correlation between mRNA levels and survival. Analysis by Kaplan–Meier curve and log-rank test revealed that increased levels of ANXA2/5/8/10 mRNA and decreased levels of ANXA3/4/7/9/11/13 mRNA in KIRC patients were significantly associated with decreased overall survival (OS) (P < 0.05) (Figure 4). KIRC patients with higher levels of ANXA1/4/5/6 mRNA or lower levels of ANXA10 mRNA were predicted to have lower relapse-free survival (RFS) (P < 0.05) (Figure 5).

3.4. Amplification, Deletion, Mutation, and Fusion of ANXAs in KIRC

Genetic variations of Annexins in 317 cases retrieved from four studies, including 35 cases from Dana-Farber Cancer Institute [29] (DFCI), 98 cases from Beijing Genomics Institute [30] (BGI), 78 cases from Cancer Research UK London Research Institute [31] (IRC), and 106 cases from the University of Tokyo [32] (Utokyo), were analysed using the cBioPortal database (Figure 6). We found varying degrees of genetic variation among the 12 ANXA family members, of which ANXA6 has the highest incidence of genetic variation. Most genetic variations in Annexins were amplifications, although ANXA3, ANXA4, and ANXA10 had higher probabilities of mutation events. Deep deletions were found in ANXA5 and ANXA10. In addition, no gene fusion events were found in the four datasets.

3.5. Correlations between ANXAs and Construction of a Gene-Gene Interaction Network

The constructed functional network based on the gene function predictions of the 12 ANXAs through the GeneMANIA database is shown in Figure 7. The 12 central nodes representing ANXAs were surrounded by genes that were strongly correlated with physical interactions, coexpression, predictions, colocalization, and genetic interactions.

We established a gene-gene interaction network of 12 Annexin genes and analysed their functions through the GeneMANIA database (Figure 7). The top 5 genes representing the most relevant ones with the ANXAs included STXBP2 (syntaxin binding protein 2), RACK1 (receptor for activated C kinase 1), S100A10 (S100 calcium binding protein A10), NDRG1 (N-myc downstream regulated 1), and HARS (histidyl-tRNA synthetase). STXBP2 was correlated with ANXA3 in terms of colocalization and physically interacted with ANXA3 and ANXA11. RACK1 physically interacted with ANXA2. S100A10 was correlated with ANXA2 in terms of physical interactions, colocalized with ANXA5, and coexpressed with ANXA11, ANXA2, ANXA6, ANXA7, ANXA8, ANXA9, ANXA10, and ANXA11. NDRG1 physically interacted with ANXA5 and was coexpressed with ANXA3, ANXA4, and ANXA6. In addition, HARS was correlated with ANXA5 in terms of physical interactions and correlated with ANXA6 in terms of coexpression.

We also found that ANXAs had the greatest correlation with calcium-dependent phospholipid binding. Additionally, they were also correlated with specific granules, phospholipid binding, S100 protein binding, lipase inhibitor activity, secretory granules, regulation of vesicle-mediated transport, postGolgi vesicle-mediated transport, calcium-dependent protein binding, and enzyme-inhibitor activity.

4. Discussion

ANAXs, as a Ca2+-dependent, phospholipid-binding protein superfamily, have been reported in numerous types of human cancer and have participated in a wide variety of biological activities, such as tumorigenesis, progression, and resistance to chemotherapeutic agents [3, 33]. However, a further comprehensive bioinformatics analysis of ANAXs in KIRC has yet to be performed. In the present study, we analysed the relationship between the expression of different ANAX factors and prognoses (OS and RFS) of KIRC for the first time. We hope that our findings may help create a foundation to better understand and improve current therapies and prognostic accuracy for patients with KIRC.

ANXA1 is known as an anti-inflammatory protein but is recognized to have a broader role in tumour cell biology beyond inflammation alone. ANXA1, located on human chromosome 9q21.13, is the first characterized member of the superfamily [12]. Transposition of ANXA1, which was found in oesophageal cancer, could affect the activities of arachidonic acid metabolism [34]. However, it is more likely a double-edged sword due to its numerous and sometimes opposite functions. Yamanoi et al. [18] found that knockdown of ANXA1 inhibits the proliferation, migration, invasion, and adhesion of kidney carcinoma cells. We first used the Oncomine database to reveal that ANXA1 mRNA expression levels were significantly higher in KIRC tissues than in normal tissues. However, its expression did not correlate with specific clinical features in KIRC. Using the Kaplan–Meier plotter, we estimated the prognostic value of ANXA1 in patients with KIRC. Patients with KIRC with high mRNA levels of ANXA1 were predicted to have a lower RFS.

The ANXA2 monomer exists in the cell cytoplasm, and the heterotetramer complexed with S100A10, which exists on cell membranes. Sadashiv et al. [35] reported that moderate immune expression of ANXA2 was found in the proximal convoluted tubules, which were considered the origin of KIRC. Yang et al. [20] demonstrated that the migration and invasion abilities of tumour cells were suppressed by silencing ANXA2 expression, whereas tumour cell proliferation was not affected. In our study, the expression of ANXA2 in KIRC tissues was higher than that in normal tissues. We also demonstrated that high ANXA2 expression was significantly correlated with poor OS in patients with KIRC. However, its expression was not correlated with tumour stage.

Bianchi et al. [36] cultured primary cells from human renal cell carcinoma and observed two spliced isoforms of ANXA3. However, one spliced isoform of ANXA3 was downregulated in KIRC, and the other was upregulated. The total ANXA3 protein was downregulated in KIRC cultures based on microarray analysis. In our report, we demonstrated that the expression of ANXA3 was not significantly different between tumour and normal tissues, which seemed inconsistent with Bianchi et al. However, we found that high ANXA3 expression was significantly correlated with favourable OS in patients with KIRC.

A data-independent acquisition-mass spectrometry proteomic approach demonstrated that ANXA4 expression levels are higher in KIRC than those in normal tissues [37]. In our research, we proved for the first time that ANXA4 mRNA expression was higher in KIRC tissues than in normal tissues, but this expression did not correlate with tumour progression stage. Additionally, low ANXA4 expression was remarkably associated with poor OS in KIRC patients. This study suggests that ANXA4 acts as a tumour suppressor gene.

ANXA5 was upregulated in KIRC, and high expression was associated with a higher clinical stage and histological grade [19]. We found a similar result in our study; that is, the expression of ANXA5 in KIRC tissues was higher than that in normal tissues. Higher ANXA5 expression was significantly related to poor OS in patients with KIRC.

ANXA6 is highly expressed in a variety of tumours, such as acute myeloid leukaemia [38], bladder cancer [39], and breast carcinoma [40]. However, its expression and prognostic role in KIRC have not been reported. In the present study, we confirmed that ANXA6 expression was higher in KIRC tissues than that in normal tissues, but this expression was not related to the stage of the tumour. Higher ANXA6 expression was significantly correlated with poor RFS in patients with KIRC. Moreover, ANXA6 displayed the highest incidence rate of genetic variations among the super family members, which might be related to kidney tumour progression.

ANXA7 GTPase is considered a tumour suppressor frequently inactivated by genomic alterations at 10q21 in a variety of human malignancies, including KIRC [41]. In this report, we demonstrated that the expression of ANXA7 in KIRC tissues was higher than that in normal tissues in Jones's dataset [27], but we also obtained the opposite result from the GEPIA dataset. It was discovered that high-stage KIRC exhibited significantly reduced ANAX7 expression. Moreover, decreased ANXA7 mRNA levels were significantly associated with lower OS, which conformed to its role as a tumour suppressor.

ANXA8 is highly expressed in patients with several malignancies, such as ovarian cancer [42] and bladder cancer [43]. Harumi et al. [44] found that cotransfection with an expression vector for ANXA8 and a reporter gene vector containing the HIF-1α promoter enhanced the activity of the HIF-1α promoter. It might play a role in calcium fluctuation-mediated HIF-1α transcriptional activation in pancreatic cancer. However, ANXA8 expression and its prognostic role in KIRC have not been reported. In this report, we found that the expression of ANXA8 in KIRC tissues was higher than that in normal tissues, and high-stage KIRC exhibited significantly increased ANAX8 expression. Increased ANXA8 mRNA was significantly associated with poor OS.

ANXA9 and ANXA11 expression and prognostic roles in KIRC have not been reported. Yu et al. [45] demonstrated that ANXA9 promoted the invasion and metastasis of colorectal cancer and predicted poor prognosis. ANXA9 showed high expression in head and neck squamous cell carcinomas and was associated with the tumour differentiation grade. Hua et al. [46] found that ANXA11 participated in gastric cancer proliferation, migration, and invasion via the AKT/GSK-3β pathway. However, in our report, we demonstrated that the expression of ANXA9 and ANXA11 in KIRC tissues was lower than that in normal tissues, and the expression did not correlate with tumour stage. Decreased ANXA9 and ANXA11 mRNA levels were significantly associated with poor OS.

It has been proven that abnormal expression of ANXA10 plays a key role in the generation, progression, and prognosis of tumours [47–49], although its functional role remains to be clarified and has not been reported in KIRC. The decreased expression of ANXA10 probably participates in malignant progression and poor prognosis [50]. Overexpression of ANXA10 could promote apoptosis of hepatocellular carcinoma cells [51]. However, the results of the present study confirmed that high ANXA10 expression was significantly associated with poor OS. The expression of ANXA10 was not associated with clinical stage.

ANXA13 is the latest ANXA member to be identified. There are limited studies available that focus on ANXA13 in cancer. ANXA13 increases cell growth and invasion. It also portends lymph node metastasis and poor prognosis in human lung adenocarcinoma patients [52]. ANXA13 was identified as a regulator of chemotherapy resistance because ectopic overexpression of ANXA13 could increase the sensitivity of malignant breast cancer cells to rapamycin [53]. In the present study, the expression level of ANXA13 was higher in KIRC tissues than in kidney tissues, and high-stage KIRC exhibited significantly decreased ANXA13 expression. Moreover, the decreased ANXA13 mRNA level was significantly associated with poor OS.

Cancer cells carry different mutations, which leads to a wide variety of clinical manifestations. Many genes show variations in copy-number alterations and may be associated with recurrence and death [54]. In the present study, we found gene alterations in 9.32% of the Annexin family in the database, including an amplification rate of 7.77% and a mutation rate of 1.44%. The incidence rate of genetic variations in the ANXA6 gene was up to 7.0%, whereas ANXA3, ANXA4, and ANXA10 had higher probabilities of mutation events. However, our findings showed that KIRC has a relatively low alteration rate in ANXAs compared with other cancers [42, 55–57].

We adopted the GeneMANIA database to construct a gene-gene interaction network that clarified the mechanisms of function of ANXAs in kidney cancer. The results showed that 20 genes, including STXBP2, RACK1, S100A10, NDRG1, and HARS, were enriched in this network based on their functions associated with physical interactions, coexpression, colocalization, pathways, and genetic interactions. It has been demonstrated that RACK1 could regulate ANXA2 phosphorylation to make it involved in the invasion and metastasis of drug-resistant carcinoma cells. [58] NDRG1, like ANXAs involved in plasma membrane repair, has been recognized as a suppressor of carcinoma by decreasing EMT-associated protein expression [59]. Aberrant expression of these interacting genes is related to the tumorigenesis and progression of tumours, but these interactions with ANXAs in kidney cancer are intriguing and still need substantial experimental confirmation. It has been demonstrated that S100A10, located in the plasma membrane, can unite with ANXA2 to form a heterotetramer composed of two subunits, S100A10 and ANXA2. The heterotetramer could activate the plasminogen activation pathway, playing a key role in cellular repair and further promoting degradation of the extracellular matrix to increase the invasion capability of carcinoma cells [60].

5. Conclusion

In the present study, the expression of ANXAs was systemically analysed to evaluate their clinical and prognostic value in KIRC, which provided an important molecular biological basis for understanding the complex development of KIRC. The results indicated that ANXA1/2/4/7/13 may be potential therapeutic targets for KIRC treatment, whereas ANXA2/5/8/10 may be potential prognostic biomarkers of KIRC. The results of the present study introduced ANXA4/8/13 as good candidates for future experimental works.

Acknowledgments

This study has received funding from the National Natural Science Foundation of China (#81771852), the Tianjin Major Science and Technology Project of Artificial Intelligence (#18ZXZNSY00300), Tianjin Health Research Project (#ZD20018), and Tianjin Research Innovation Project for Postgraduate Students (#2020YJSS178).

Abbreviations

ANXAs: Annexins family

KIRC: Kidney clear renal cell carcinoma

GEPIA: Gene expression profiling interactive analysis

OS: Overall survival

RCC: Renal cell cancer

CNA: Copy-number alterations

RFS: Relapse-free survival

STXBP2: Syntaxin binding protein 2

RACK1: Receptor for activated C kinase 1

S100A10: S100 calcium binding protein A10

NDRG1: N-myc downstream regulated 1

HARS: Histidyl-tRNA synthetase.

Data Availability

The datasets generated during and/or analysed during the current study are available in the ONCOMINE database (https://www.oncomine.org/), Gene Expression Profiling Interactive Analysis (GEPIA, https://gepia.cancer-pku.cn/), the Kaplan–Meier plotter database, and cBioPortal (https://www.cbioportal.org/).

Additional Points

A preprint has been published in research square https://www.researchsquare.com/article/rs-837226/v1, “Evaluation of Annexins Family as Potential Biomarker for Predicting Progression and Prognosis in Clear Renal Cell Carcinoma [61],” but it is not published or under consideration by any journal.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors' Contributions

Jiyu Zhao and Luchen Chang contributed equally to this work.

Figure 1 The transcription levels of ANXAs in different types of cancers (ONCOMINE). The threshold was set to following parameters: fold change = 2 and P-value = 0.0001. The cell number indicates the number of datasets that meets the thresholds. The color red or blue directly indicates up- or downregulation, respectively.

Figure 2 The mRNA expression levels of ANXAs in clear renal cell carcinoma and normal kidney tissues (GEPIA). The plots show mRNA expression of Annexins in kidney tumour (red plot) and the corresponding expression in normal tissues (green plot).

Figure 3 Correlation analysis of the ANXAs expression and clinical stages in clear renal cell carcinoma (GEPIA). Spearman's correlation analysis between gene and clinical stages is based on the entire clinical stages.

Figure 4 Prognostic values of ANXAs in clear renal cell carcinoma (overall survival in Kaplan–Meier plotter). The P-values were calculated using the log-rank test.

Figure 5 Prognostic values of ANXAs in clear renal cell carcinoma (relapse-free survival in Kaplan–Meier plotter). The P-values were calculated using the log-rank test.

Figure 6 Analyses of genetic variations in ANXAs in clear renal cell carcinoma (cBioPortal). (a) OncoPrint visual summary of variations on a query of Annexin family members and overview of the analyses of genetic variations in ANXAs. (b) Analyses of genetic variations in Annexin family members reported in different studies. (c) Analyses of genetic variations in Annexin family members respectively in different studies.

Figure 7 Gene-gene interaction network among Annexin family members (GeneMANIA). Each node represents a gene. The node size represents the strength of interactions. The connection lines represent the types of gene-gene interactions, and the line color represents the types of interactions. The node color represents the possible functions of respective genes.

Table 1 The ANXA expression between different types of renal clear cell carcinoma and kidney tissues.

GENE	Cancer tissue vs normal tissue	Fold change	t-test	P-value	Dataset	
ANXA1	Renal clear cell carcinoma vs kidney	4.853	11.741	1.18E − 12	Yusenko et al.	
Renal clear cell carcinoma vs kidney	5.348	11.22	7.71E − 10	Gumz et al.	
Renal clear cell carcinoma vs kidney	3.26	6.477	4.53E − 06	Beroukhim et al.	
ANXA2	Renal clear cell carcinoma vs kidney	2.358	10.276	3.49E − 10	Higgins et al.	
Renal clear cell carcinoma vs kidney	2.444	9.038	2.17E − 08	Gumz et al.	
Renal clear cell carcinoma vs kidney	2.383	10.586	9.99E − 14	Jones et al.	
ANXA3	NA	NA	NA	NA	NA	
ANXA4	Renal clear cell carcinoma vs kidney	3.662	12.691	2.99E − 12	Higgins et al.	
Renal clear cell carcinoma vs kidney	3.577	13.931	2.23E − 11	Gumz et al.	
Renal clear cell carcinoma vs kidney	4.763	9.538	5.94E − 08	Yusenko et al.	
Renal clear cell carcinoma vs kidney	2.598	8.071	1.12E − 09	Jones et al.	
ANXA5	NA	NA	NA	NA	NA	
ANXA6	NA	NA	NA	NA	NA	
ANXA7	Renal clear cell carcinoma vs kidney	2.565	7.775	5.98E − 07	Jones et al.	
ANXA8	NA	NA	NA	NA	NA	
ANXA9	NA	NA	NA	NA	NA	
ANXA10	NA	NA	NA	NA	NA	
ANXA11	NA	NA	NA	NA	NA	
ANXA13	NA	NA	NA	NA	NA
==== Refs
1 Siegel R. L. Miller K. D. Jemal A. Cancer statistics, 2020 CA: A Cancer Journal for Clinicians 2020 70 1 7 30 10.3322/caac.21590 31912902
2 Moch H. Cubilla A. L. Humphrey P. A. Reuter V. E. Ulbright T. M. The 2016 WHO classification of tumours of the urinary system and male genital organs-Part A: renal, penile, and testicular tumours European Urology 2016 70 1 93 105 10.1016/j.eururo.2016.02.029 2-s2.0-84971574471 26935559
3 Xi Y. Ju R. Wang Y. Roles of Annexin A protein family in autophagy regulation and therapy Biomedicine & Pharmacotherapy 2020 130 110591 10.1016/j.biopha.2020.110591
4 Buttarelli M. Babini G. Raspaglio G. A combined ANXA2-NDRG1-STAT1 gene signature predicts response to chemoradiotherapy in cervical cancer Journal of Experimental & Clinical Cancer Research 2019 38 1 p. 279 10.1186/s13046-019-1268-y 2-s2.0-85068858237
5 Yao X. Qi X. Wang Y. Identification and validation of an annexin-related prognostic signature and therapeutic targets for bladder cancer: integrative analysis Biology 2022 11 2 p. 259 10.3390/biology11020259
6 Wei W.-S. Chen X. Guo L.-Y. TRIM65 supports bladder urothelial carcinoma cell aggressiveness by promoting ANXA2 ubiquitination and degradation Cancer Letters 2018 435 10 22 10.1016/j.canlet.2018.07.036 2-s2.0-85050860679 30075204
7 Okano M. Oshi M. Butash A. L. Triple-negative breast cancer with high levels of annexin A1 expression is associated with mast cell infiltration, inflammation, and angiogenesis International Journal of Molecular Sciences 2019 20 17 p. 4197 10.3390/ijms20174197 2-s2.0-85071631459 31461932
8 Zhu S. Li Y. Wang Y. Efficacy of neoadjuvant chemotherapy and Annexin A3 expression in breast cancer Journal of BUON: Official Journal of the Balkan Union of Oncology 2019 24 522 528
9 Zóia M. A. P. Azevedo F. V. P. Vecchi L. Inhibition of triple-negative breast cancer cell aggressiveness by cathepsin D blockage: role of annexin A1 International Journal of Molecular Sciences 2019 20 6 1337 1420 10.3390/ijms20061337 2-s2.0-85063272708 30884823
10 Zhao R.-R. Mao X.-R. Wang X.-F. Zheng Y. Wang Y. P. Zhou Y. N. Role of annexin A family in tumorigenesis and chemoresistance of gastric cancer Neoplasma 2022 69 2 251 263 10.4149/neo_2021_210629n872 35068160
11 Wang L. Li X. Ren Y. Cancer-associated fibroblasts contribute to cisplatin resistance by modulating ANXA3 in lung cancer cells Cancer Science 2019 110 5 1609 1620 10.1111/cas.13998 2-s2.0-85065238951 30868675
12 Yumura M. Nagano T. Jimbo N. Annexin A10 expression as a novel prognostic marker in lung adenocarcinoma Anticancer Research 2022 42 3 1289 1294 10.21873/anticanres.15595 35220218
13 Zhu D.-W. Sun W.-W. Zhao T.-C. Zhong L.-P. Zhang Z.-Y. The effect and mechanism of ANXA1 on TPF chemosensitivity in oral squamous cell carcinoma Shanghai Journal of Stomatology, 2019 28 225 230 31489406
14 Tang L. Liu J.-X. Zhang Z.-J. High expression of Anxa2 and Stat3 promote progression of hepatocellular carcinoma and predict poor prognosis Pathology, Research & Practice 2019 215 6 152386 10.1016/j.prp.2019.03.015 2-s2.0-85063434218
15 Wei X. Wang M. Wang X. Prediction of cervical lymph node metastases in papillary thyroid microcarcinoma by sonographic features of the primary site Cancer biology & medicine 2019 16 3 587 594 10.20892/j.issn.2095-3941.2018.0310 2-s2.0-85073097500 31565487
16 Shao Y.-Y. Kuo H.-Y. Jeng Y.-M. Association of annexin A10 expression with poor prognosis of intrahepatic cholangiocarcinoma BMC Cancer 2022 22 1 p. 219 10.1186/s12885-022-09288-8 35227227
17 Baba M. Editorial comment to annexin A1 expression is correlated with malignant potential of renal cell carcinoma International Journal of Urology 2019 26 2 p. 291 10.1111/iju.13899 2-s2.0-85060034903
18 Yamanoi M. Yamanoi K. Fujii C. Fukuda M. N. Nakayama J. Annexin A1 expression is correlated with malignant potential of renal cell carcinoma International Journal of Urology 2019 26 2 284 290 10.1111/iju.13869 2-s2.0-85057983365 30506742
19 Tang J. Qin Z. Han P. High Annexin A5 expression promotes tumor progression and poor prognosis in renal cell carcinoma International Journal of Oncology 2017 50 5 1839 1847 10.3892/ijo.2017.3942 2-s2.0-85018516412 28393205
20 Yang S.-F. Hsu H.-L. Chao T.-K. Hsiao C. J. Lin Y. F. Cheng C. W. Annexin A2 in renal cell carcinoma: expression, function, and prognostic significance Urologic Oncology: Seminars and Original Investigations 2015 33 1 10.1016/j.urolonc.2014.08.015 2-s2.0-84922632417
21 Tang Z. Li C. Kang B. Gao G. Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses Nucleic Acids Research 2017 45 W1 W98 W102 10.1093/nar/gkx247 2-s2.0-85023171204 28407145
22 Gyorffy B. Lánczky A. Szállási Z. Implementing an online tool for genome-wide validation of survival-associated biomarkers in ovarian-cancer using microarray data from 1287 patients Endocrine-Related Cancer 2012 19 2 197 208 10.1530/erc-11-0329 2-s2.0-84860903681 22277193
23 Gao J. Aksoy B. A. Dogrusoz U. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal Science Signaling 2013 6 269 10.1126/scisignal.2004088 2-s2.0-84875740314
24 Higgins J. P. T. Shinghal R. Gill H. Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray American Journal of Pathology 2003 162 3 925 932 10.1016/s0002-9440(10)63887-4 2-s2.0-0344127560 12598325
25 Gumz M. L. Zou H. Kreinest P. A. Secreted frizzled-related protein 1 loss contributes to tumor phenotype of clear cell renal cell carcinoma Clinical Cancer Research 2007 13 16 4740 4749 10.1158/1078-0432.ccr-07-0143 2-s2.0-34548071005 17699851
26 Yusenko M. V. Kuiper R. P. Boethe T. Ljungberg B. Van Kessel A. G. Kovacs G. High-resolution DNA copy number and gene expression analyses distinguish chromophobe renal cell carcinomas and renal oncocytomas BMC Cancer 2009 9 1 p. 152 10.1186/1471-2407-9-152 2-s2.0-66749104378
27 Jones J. Otu H. Spentzos D. Gene signatures of progression and metastasis in renal cell cancer Clinical Cancer Research 2005 11 16 5730 5739 10.1158/1078-0432.ccr-04-2225 2-s2.0-23844524767 16115910
28 Beroukhim R. Brunet J.-P. Di Napoli A. Patterns of gene expression and copy-number alterations in von-hippel lindau disease-associated and sporadic clear cell carcinoma of the kidney Cancer Research 2009 69 11 4674 4681 10.1158/0008-5472.can-09-0146 2-s2.0-66349100709 19470766
29 Miao D. Margolis C. A. Gao W. Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma Science 2018 359 801 806 10.1126/science.aan5951 2-s2.0-85040072272 29301960
30 Guo G. Gui Y. Gao S. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma Nature Genetics 2011 44 1 17 19 10.1038/ng.1014 2-s2.0-84655176646 22138691
31 Gerlinger M. Horswell S. Larkin J. Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing Nature Genetics 2014 46 3 225 233 10.1038/ng.2891 2-s2.0-84895876130 24487277
32 Sato Y. Yoshizato T. Shiraishi Y. Integrated molecular analysis of clear-cell renal cell carcinoma Nature Genetics 2013 45 860 867 10.1038/ng.2699 2-s2.0-84880967722 23797736
33 Zhang H. Ma H. Yang X. Cell fusion-related proteins and signaling pathways, and their roles in the development and progression of cancer Frontiers in Cell and Developmental Biology 2022 9 10.3389/fcell.2021.809668
34 Wang K. L. Wu T.-T. Resetkova E. Expression of annexin A1 in esophageal and esophagogastric junction adenocarcinomas: association with poor outcome Clinical Cancer Research 2006 12 15 4598 4604 10.1158/1078-0432.ccr-06-0483 2-s2.0-33748028823 16899607
35 Sadashiv R. Bannur B. M. Shetty P. Differential expression pattern of annexin A2 during nephrogenesis and kidney carcinoma Romanian Journal of Morphology and Embryology 2019 60 895 904 31912102
36 Bianchi C. Bombelli S. Raimondo F. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of annexin A3 American Journal of Pathology 2010 176 4 1660 1670 10.2353/ajpath.2010.090402 2-s2.0-77950554417 20167856
37 Song Y. Zhong L. Zhou J. Data-independent acquisition-based quantitative proteomic analysis reveals potential biomarkers of kidney cancer Proteomics—Clinical Applications 2017 11 11-12 1700066 10.1002/prca.201700066 2-s2.0-85038365469
38 Niu Y. Yang X. Chen Y. Distinct prognostic values of annexin family members expression in acute myeloid leukemia Clinical & Translational Oncology 2019 21 1186 1196 30694461
39 Zhu J. Wang H. Ma T. Identification of immune-related genes as prognostic factors in bladder cancer Scientific Reports 2020 10 1 10.1038/s41598-020-76688-w
40 Williams S. D. Sakwe A. M. Reduced expression of annexin A6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells Cancers 2022 14 5 p. 1108 10.3390/cancers14051108
41 Leighton X. Eidelman O. Jozwik C. Pollard H. B. Srivastava M. ANXA7-GTPase as tumor suppressor: mechanisms and therapeutic opportunities Methods in Molecular Biology 2017 1513 23 35 10.1007/978-1-4939-6539-7_3 2-s2.0-84994213346 27807828
42 Zhu L. Gou R. Guo Q. Wang J. Liu Q. Lin B. High expression and potential synergy of human epididymis protein 4 and Annexin A8 promote progression and predict poor prognosis in epithelial ovarian cancer American Journal of Tourism Research 2020 12 7 4017 4030
43 Wu W. Yang Y. Yao F. AAV-mediated in vivo genome editing in vascular endothelial cells Methods 2021 194 12 17 10.1016/j.ymeth.2020.12.001 33309782
44 Harumi H. Tatemichi M. Nakadate T. Involvement of annexin A8 in the properties of pancreatic cancer Molecular Carcinogenesis 2014 53 3 181 191 10.1002/mc.21961 2-s2.0-84894275130 23001853
45 Yu S. Bian H. Gao X. Gui L. Annexin A9 promotes invasion and metastasis of colorectal cancer and predicts poor prognosis International Journal of Molecular Medicine 2018 41 2185 2192 10.3892/ijmm.2018.3432 2-s2.0-85041574996 29393380
46 Hua K. Li Y. Zhao Q. Fan L. Tan B. Gu J. Downregulation of annexin A11 (ANXA11) inhibits cell proliferation, invasion, and migration via the AKT/GSK-3β pathway in gastric cancer Medical Science Monitor 2018 24 149 160 10.12659/msm.905372 2-s2.0-85040563355 29306955
47 Kim J. Kim M. A. Jee C. D. Jung E. J. Kim W. H. Reduced expression and homozygous deletion of annexin A10 in gastric carcinoma International Journal of Cancer 2009 125 8 1842 1850 10.1002/ijc.24541 2-s2.0-70349258301 19582876
48 van der Heijden A. G. Mengual L. Lozano J. J. A five-gene expression signature to predict progression in T1G3 bladder cancer European Journal of Cancer 2016 64 127 136 27414486
49 Zhu J. Wu J. Pei X. Tan Z. Shi J. Lubman D. M. Annexin A10 is a candidate marker associated with the progression of pancreatic precursor lesions to adenocarcinoma PLoS One 2017 12 4 e0175039 10.1371/journal.pone.0175039 2-s2.0-85016648017
50 Liu S.-H. Lin C.-Y. Peng S.-Y. Down-regulation of annexin A10 in hepatocellular carcinoma is associated with vascular invasion, early recurrence, and poor prognosis in synergy with p53 mutation American Journal of Pathology 2002 160 1831 1837 10.1016/S0002-9440(10)61129-7 2-s2.0-0036092297 12000734
51 Liu X. Peng X. Hu Z. Effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2 Journal of Huazhong University of Science and Technology—Medical sciences 2012 32 5 669 674 10.1007/s11596-012-1015-5 2-s2.0-84870563571
52 Xue G. Zhang C. Zheng G. Zhang L. Bi J. Annexin A13 predicts poor prognosis for lung adenocarcinoma patients and accelerates the proliferation and migration of lung adenocarcinoma cells by modulating epithelial-mesenchymal transition Fundamental & Clinical Pharmacology 2020 34 6 687 696 10.1111/fcp.12555 32145097
53 Reiske H. Sui B. Ung-Medoff H. Identification of annexin A13 as a regulator of chemotherapy resistance using random homozygous gene perturbation Analytical, Quantitative Cytology and Histology 2010 32 61 69
54 Cortés-Ciriano I. Gulhan D. C. Lee J. J.-K. Melloni G. E. M. Peter J. P. Computational analysis of cancer genome sequencing data Nature Reviews Genetics 2021 23
55 Mobarra N. Shafiee A. Rad S. M. A. H. Overexpression of microRNA-16 declines cellular growth, proliferation and induces apoptosis in human breast cancer cells In Vitro Cellular & Developmental Biology—Animal 2015 51 6 604 611 10.1007/s11626-015-9872-4 2-s2.0-84930820120 25672252
56 Mirsaeidi M. Gidfar S. Vu A. Schraufnagel D. Annexins family: insights into their functions and potential role in pathogenesis of sarcoidosis Journal of Translational Medicine 2016 14 1 p. 89 10.1186/s12967-016-0843-7 2-s2.0-85007524035
57 Zhuang C. Wang P. Sun T. Zheng L. Ming L. Expression levels and prognostic values of annexins in liver cancer Oncology Letters 2019 18 6657 6669 10.3892/ol.2019.11025 31807177
58 Fan Y. Si W. Ji W. Rack1 mediates tyrosine phosphorylation of Anxa2 by Src and promotes invasion and metastasis in drug-resistant breast cancer cells Breast Cancer Research 2019 21 1 p. 66 10.1186/s13058-019-1147-7 2-s2.0-85066470367
59 Menezes S. V. Fouani L. Huang M. L. H. The metastasis suppressor, NDRG1, attenuates oncogenic TGF-β and NF-κB signaling to enhance membrane E-cadherin expression in pancreatic cancer cells Carcinogenesis 2019 40 805 818 10.1093/carcin/bgy178 2-s2.0-85062659782 30561520
60 Noye T. M. Lokman N. A. Oehler M. K. Ricciardelli C. S100A10 and cancer hallmarks: structure, functions, and its emerging role in ovarian cancer International Journal of Molecular Sciences 2018 19 12 p. 4122 10.3390/ijms19124122 2-s2.0-85058911999
61 Zhao J. Y. Chang L. C. Tu J. P. Sun B. Wei X. Evaluation of annexins family as potential biomarker for predicting progression and prognosis in clear renal cell carcinoma Research Square 2021
