ATP7A as a prognostic biomarker and potential therapeutic target in gastric cancer.

Am J Transl Res 2025;17(1):512-527 www.ajtr.org /ISSN:1943-8141/AJTR0161812 Original Article ATP7A as a prognostic biomarker and potential therapeutic target in gastric cancer Zhongmei Shi1,2, Zhiyun Mao1, Ming Cui1, Dongjin Xu2, Yan Wang3, Rongrong Jing1 Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, Jiangsu, China; 2Department of Laboratory Medicine, Dongtai Hospital of Traditional Chinese Medicine, Dongtai 224200, Jiangsu, China; 3Department of Pathology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China 1 Received November 7, 2024; Accepted December 20, 2024; Epub January 15, 2025; Published January 30, 2025 Abstract: Objectives: To investigate the roles of Cu transporter ATPase copper transporting alpha (ATP7A) in gastric cancer (GC) progression and prognosis. Methods: ATP7A expression was investigated using databases, immunohistochemistry (IHC) and qPCR in tumor tissues and GC cell lines. Diagnostic and prognostic value of ATP7A was assessed by Receiver Operating Characteristic (ROC) and Kaplan-Meier curve, respectively. The roles of ATP7A were explored using protein-protein interaction (PPI), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA), ssGSEA algorithm and Tumor Immune Estimation Resource (TIMER) databases. Subsequently, the effects of ATP7A were evaluated by Cell Counting Kit-8 (CCK-8), colony formation, and transwell assays. Results: ATP7A overexpression was associated with a higher IHC score and a larger area under the ROC curve (0.746). Elevated ATP7A expression correlated with shorter survival time, greater invasion depth of GC lesions, advanced pathological stages, and older age in GC patients. Comprehensive analysis revealed that ATP7A was involved in copper ion transport, transition metal ion homeostasis, cellular transition metal ion homeostasis, and copper ion homeostasis. Additionally, ATP7A was linked to key signaling pathways, including Hedgehog, Wnt/β-catenin, and Notch, along with the top 10 hub genes. Furthermore, ATP7A played a role in immune infiltration, influencing T cells, dendritic cells, B cells, macrophages, and neutrophils, as well as the expression of immune checkpoints such as Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), Programmed Cell Death Protein 1 Ligand 1 (PD-L1), T-Cell Immunoglobulin, and Mucin Domain-Containing Protein 3 (TIM-3). Experimental validation demonstrated that silencing ATP7A suppressed GC cell proliferation, colony formation, migration, and invasion. Conclusion: ATP7A promoted GC progression and acted as a promising prognostic target for the treatment of GC. Keywords: Gastric cancer, ATP7A, cell proliferation, cell migration, cell invasion Introduction Gastric cancer (GC) is one of the most prevalent malignancies worldwide. According to global cancer statistics, 968,000 new cases of GC and 660,000 deaths were reported in 2022 [1]. Due to the subtle symptoms of early-stage GC, most patients are diagnosed at advanced stages. Unfortunately, despite standard treatments such as surgery, chemotherapy, and radiotherapy, the 5-year survival rate for advanced GC remains only 36.2% [2]. In recent years, promising new approaches, including immunotherapy, have been explored for GC patients [3]. However, due to the complexity of the tumor immune microenvironment, there is a lack of reliable biomarkers for accurate diagnosis and management to effectively guide immunotherapy in GC. Therefore, the urgent identification of novel molecular targets is critical to improving the diagnosis and treatment of GC. Copper is a redox-active metal ion essential for maintaining human homeostasis. In 2022, Tsvetkov et al. identified a novel pattern of cell death termed cuproptosis, which is induced by copper and associated with mitochondrial respiration [4]. Unlike other known forms of cell death, cuproptosis relies on the intracellular accumulation of copper ions. These ions bind https://doi.org/10.62347/UYMP7222 ATP7A in gastric cancer: biomarker and therapeutic target directly to lipoylated enzymes, disrupting the tricarboxylic acid (TCA) cycle, leading to the aggregation and dysregulation of TCA cycle proteins, ultimately triggering cell death. Cuproptosis has been implicated in various cancers [5-7]. This discovery opens new avenues for the prevention and treatment of GC. In our study, we identified 19 cuproptosis-related genes, including ATPase copper transporting alpha (ATP7A) [4, 8]. ATP7A, localized in the cellular Golgi apparatus, is a P-type ATPase enzyme responsible for heavy metal transport [9]. Beyond its role in copper transmembrane transport, ATP7A plays a critical role in cancer pathogenesis [10]. Notably, ATP7A enhances tumor invasiveness by supplying large quantities of copper ions to oncogenic enzymes across various tumor types [11, 12]. However, the role of ATP7A in the clinical characteristics of GC and its impact on patient prognosis remain unclear. The primary objective of this study is to investigate the expression pattern, clinical significance, and biological functions of ATP7A in GC. We aim to determine whether ATP7A can serve as a prognostic biomarker and a potential therapeutic target for GC patients. Meanwhile, the functional regulations of ATP7A were examined through a series of comprehensive analyses, including Gene Ontology (GO), protein-protein interaction (PPI), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Tumor Immune Estimation Resource (TIMER) databases. Furthermore, the roles of ATP7A in GC cell growth, migration, and invasion were verified through in vitro experiments, with the hope of providing a reference for future GC research and clinical treatment. The research flowchart is shown in Figure 1. Materials and methods Data acquisition and preprocessing Data acquisition and preprocessing: All pancancer data, including tumor samples from various cancer types (n = 18,102), were obtained from the UCSC Xena website (https:// xenabrowser.net/datapages/). This comprehensive dataset facilitated a broad analysis of gene expression patterns across different malignancies. Differentially expressed gene data were derived from RNA-seq information in The Cancer Genome Atlas (TCGA)-Stomach Adenocarcinoma (STAD) project in level 3 HTSeq513 Counts format [13]. The series matrix file of GSE54129 was retrieved from the Gene Expression Omnibus database. Notably, RNA-seq data in FPKM format were converted to Transcripts Per Million (TPM) format and subjected to log2 transformation for further analysis. Tissue samples Two GC tissue microarrays (ZL-stmA961 and ZL-stmA962) were obtained from Shanghai Wellbio Technology Co., Ltd. (Shanghai, China). Additionally, the tissue microarray (HstmA180su20) was provided by Shanghai Outdo Biotech Company. The final analysis included 152 GC tissues and 117 adja (...truncated)