Topoisomerase II Alpha Promotes Gallbladder Cancer Proliferation and Metastasis Through Activating Phosphatidylinositol 3-Kinase/Protein Kinase B/Mammalian Target of Rapamycin Signaling Pathway
Gallbladder cancer (GBC) is a highly aggressive malignancy with a poor prognosis, often diagnosed at advanced stages when surgical resection is no longer feasible. The lack of effective therapeutic targets and the resistance to conventional chemotherapy and radiotherapy highlight the urgent need to explore the molecular mechanisms underlying GBC progression. In this context, the role of topoisomerase II alpha (TOP2A) in GBC has been investigated, revealing its critical involvement in tumor proliferation, metastasis, and activation of key signaling pathways.
TOP2A is a nuclear enzyme that plays a pivotal role in DNA replication and transcription by altering the topological states of DNA. It has been implicated in the tumorigenesis of various cancers, including colon and pancreatic cancer. However, its biological role in GBC remained unexplored until this study. The findings demonstrate that TOP2A is significantly up-regulated in GBC tissues and is associated with poor prognosis, advanced tumor stages, and lymph node metastasis. Mechanistically, TOP2A promotes GBC progression by activating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, making it a potential therapeutic target for GBC.
The study began with a bioinformatics analysis using the Gene Expression Profiling Interactive Analysis (GEPIA) database, which revealed that TOP2A expression is notably increased in GBC tissues compared to adjacent normal tissues. This up-regulation was further confirmed through quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) in 45 paired GBC and non-tumor tissue samples. The results showed that TOP2A expression was significantly higher in GBC tissues (12.62 vs. 0.34 in normal tissues). Moreover, high TOP2A expression was correlated with advanced tumor-node-metastasis (TNM) stages, the presence of lymph node metastasis, and poor overall survival in GBC patients.
To investigate the functional role of TOP2A in GBC, in vitro experiments were conducted using GBC cell lines (NOZ, SGC-996, GBC-SD, and OCUG). TOP2A expression was found to be elevated in these cell lines compared to the human renal epithelial cell line 293T. Knockdown of TOP2A using small interfering RNA (siRNA) significantly inhibited cell proliferation, as demonstrated by cell counting kit-8 (CCK-8) assays. Conversely, overexpression of TOP2A enhanced the proliferative capacity of GBC cells. These findings suggest that TOP2A acts as a tumor activator in GBC by promoting cell proliferation.
The study also explored the role of TOP2A in GBC metastasis through transwell migration and invasion assays. Knockdown of TOP2A in NOZ and SGC-996 cells resulted in a significant reduction in their migratory and invasive abilities. Conversely, overexpression of TOP2A in GBC-SD cells enhanced these capabilities. These results indicate that TOP2A facilitates the metastatic potential of GBC cells.
Epithelial-mesenchymal transition (EMT) is a critical process in tumor progression and metastasis, characterized by the loss of epithelial markers and the acquisition of mesenchymal traits. To determine whether TOP2A is involved in EMT, the expression of key EMT-related markers was assessed. Western blotting revealed that TOP2A knockdown up-regulated the expression of tight junction protein 1 (ZO-1) and E-cadherin, while down-regulating N-cadherin, vimentin, and snail. These findings suggest that TOP2A promotes the EMT process in GBC, thereby enhancing tumor cell migration and invasion.
The PI3K/Akt/mTOR signaling pathway is a well-known regulator of cell growth, proliferation, and survival, and its hyperactivation is frequently observed in various cancers. Given its importance in GBC progression, the study investigated whether TOP2A exerts its effects through this pathway. Western blotting results showed that TOP2A knockdown significantly reduced the phosphorylation levels of PI3K, Akt, and mTOR, without affecting their total protein levels. Rescue experiments using the PI3K inhibitor LY294002 further confirmed that TOP2A promotes GBC progression by activating the PI3K/Akt/mTOR pathway. Specifically, LY294002 reversed the enhanced proliferation induced by TOP2A overexpression in NOZ cells.
To validate the in vitro findings, an in vivo xenograft model was established using nude mice. Stable knockdown of TOP2A in NOZ cells was achieved using lentiviral vectors, and these cells were injected into the left axilla of the mice. Tumor volume and weight were significantly lower in the TOP2A knockdown group compared to the control group. IHC analysis of the xenograft tumors revealed that TOP2A silencing decreased the expression of TOP2A, Ki-67, proliferating cell nuclear antigen (PCNA), N-cadherin, vimentin, phospho-Akt, and phospho-mTOR. These in vivo results corroborated the in vitro findings, demonstrating that TOP2A promotes GBC tumor growth and metastasis through the PI3K/Akt/mTOR pathway.
In summary, this study provides compelling evidence that TOP2A is a critical regulator of GBC progression. Its up-regulation in GBC tissues is associated with advanced tumor stages, lymph node metastasis, and poor prognosis. Functionally, TOP2A promotes GBC cell proliferation, migration, invasion, and EMT by activating the PI3K/Akt/mTOR signaling pathway. These findings highlight the potential of TOP2A as a novel prognostic biomarker and therapeutic target for GBC. Future studies should focus on developing TOP2A inhibitors and evaluating their efficacy in preclinical and clinical settings to improve the outcomes of GBC patients.
doi.org/10.1097/CM9.0000000000001075
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