CCT4 Suppression Inhibits Tumor Growth in Hepatocellular Carcinoma by Interacting with Cdc20
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, ranking as the fourth most common cause of cancer-related death. In the United States, HCC is projected to become the third leading cause of cancer-related mortality by 2030. The disease exhibits high heterogeneity, with variations in incidence and outcomes based on race, region, gender, and age. Despite advancements in treatment, the prognosis for HCC remains poor, necessitating the identification of novel therapeutic targets and a deeper understanding of the molecular mechanisms driving tumor progression.
One promising area of research involves the chaperonin containing t-complex (CCT) proteins, which play a critical role in protein folding and cellular homeostasis. The CCT protein family consists of eight paralogous subunits, including CCT4, which has been implicated in various cancers. However, the specific role of CCT4 in HCC pathogenesis remains poorly understood. This study aims to elucidate the mechanisms by which CCT4 contributes to HCC progression, with a particular focus on its interaction with the cell cycle regulator Cdc20.
CCT4 Expression in HCC Tissues and Its Prognostic Significance
The study began by analyzing the expression of CCT4 in HCC tissues using data from The Cancer Genome Atlas (TCGA) via the UALCAN platform. The results revealed that CCT4 expression was significantly higher in HCC tumor tissues compared to normal tissues. Furthermore, high CCT4 expression was associated with poor overall survival in HCC patients, suggesting that CCT4 may serve as a prognostic marker for the disease.
To validate these findings, the researchers examined CCT4 expression in 15 pairs of HCC tumor tissues and adjacent normal tissues using western blot (WB) analysis. Consistent with the TCGA data, CCT4 protein levels were significantly elevated in tumor tissues compared to normal tissues. These results underscore the potential role of CCT4 in HCC progression and its clinical relevance as a biomarker.
CCT4 Knockdown Inhibits HCC Cell Proliferation and Induces Apoptosis
To investigate the functional role of CCT4 in HCC, the researchers used lentivirus-mediated short-hairpin RNA (shRNA) to knock down CCT4 expression in two HCC cell lines, Huh7 and Hep3b. Quantitative polymerase chain reaction (qPCR) and WB analysis confirmed that CCT4 mRNA and protein levels were significantly reduced in both cell lines following shRNA transfection.
The impact of CCT4 knockdown on cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. The CCK-8 assay demonstrated that CCT4 suppression significantly inhibited cell proliferation in both Huh7 and Hep3b cells. Similarly, the EdU assay revealed a marked reduction in the EdU-positive rate, indicating decreased DNA synthesis and cell proliferation in CCT4-depleted cells.
To evaluate the effect of CCT4 on apoptosis, the researchers performed flow cytometry (FCM) analysis using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. The results showed that CCT4 knockdown significantly increased the apoptosis rate in both Huh7 and Hep3b cells. These findings suggest that CCT4 plays a crucial role in promoting HCC cell survival and proliferation.
Mechanisms of CCT4 in Regulating Tumor Growth
To explore the molecular mechanisms underlying CCT4’s role in HCC, the researchers investigated its interaction with Cdc20, a key regulator of the anaphase-promoting complex/cyclosome (APC/C). Co-immunoprecipitation (co-IP) assays confirmed that CCT4 interacts with Cdc20 in Huh7 cells. This interaction is significant because Cdc20 is essential for the proper progression of the cell cycle, particularly during mitosis, by regulating the degradation of specific substrates such as securin and Bim.
Securin is an inhibitor of separase, a protease required for sister chromatid separation during mitosis. The researchers found that CCT4 knockdown led to the accumulation of securin, which in turn downregulated cyclin D1, a protein critical for cell cycle progression. The downregulation of cyclin D1 likely contributes to the observed inhibition of cell proliferation in CCT4-depleted cells.
In addition to its role in cell cycle regulation, CCT4 also influences apoptosis through its interaction with Cdc20. The study found that CCT4 knockdown resulted in the accumulation of Bim, a pro-apoptotic protein. Bim promotes apoptosis by inhibiting B-cell lymphoma-2 (Bcl-2) and activating caspase 9, a key effector of the apoptotic pathway. The upregulation of Bim and the subsequent activation of caspase 9 in CCT4-depleted cells further explain the increased apoptosis observed in these cells.
Discussion and Implications
The findings of this study highlight the critical role of CCT4 in HCC pathogenesis. By interacting with Cdc20, CCT4 regulates both cell cycle progression and apoptosis, two fundamental processes in cancer development. The upregulation of CCT4 in HCC tissues and its association with poor prognosis suggest that CCT4 may serve as a valuable biomarker for the disease. Moreover, the inhibition of CCT4 expression significantly reduces HCC cell proliferation and induces apoptosis, indicating that CCT4 could be a potential therapeutic target for HCC treatment.
The interaction between CCT4 and Cdc20 is particularly intriguing, as it provides a mechanistic link between CCT4 and the regulation of the APC/C complex. The APC/C complex is essential for the proper progression of the cell cycle, and its dysregulation is a hallmark of cancer. By promoting the dissociation of the mitotic checkpoint complex (MCC) from APC/C, CCT4 enhances the activity of APC/CCdc20, leading to the degradation of key substrates such as securin and Bim. This dual role of CCT4 in cell cycle regulation and apoptosis underscores its importance in maintaining cellular homeostasis and its potential as a therapeutic target.
The study also sheds light on the broader role of CCT proteins in cancer. Previous research has implicated other CCT subunits, such as CCT3 and CCT8, in various cancers, including HCC. The findings of this study add to the growing body of evidence that CCT proteins play a critical role in cancer progression and may serve as potential targets for cancer therapy.
Conclusion
In conclusion, this study demonstrates that CCT4 is upregulated in HCC tissues and is associated with poor prognosis. CCT4 promotes HCC cell proliferation and survival by interacting with Cdc20 and regulating the activity of the APC/C complex. The inhibition of CCT4 expression significantly reduces cell proliferation and induces apoptosis in HCC cells, highlighting its potential as a therapeutic target. Further research is needed to explore the clinical applications of targeting CCT4 in HCC treatment and to fully elucidate the molecular mechanisms underlying its role in cancer progression.
doi.org/10.1097/CM9.0000000000001851
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