Overexpression of TRIM24 Stimulates Proliferation and Glucose Metabolism of Head and Neck Squamous Cell Carcinoma

Abstract

TRIM24 (Tripartite Motif Containing 24) is a recently identified oncogene overexpressed in various cancers. However, the molecular mechanism of TRIM24 in the progression of head and neck squamous cell carcinoma (HNSCC) remains ambiguous. In the present study, we analyzed the expression pattern of TRIM24 in 100 HNSCC tissues and found that TRIM24 was overexpressed in 43/100 HNSCC cases. Significant association was found between TRIM24 overexpression and tumor-node-metastasis (TNM) stage (p = 0.0034) and T stage (p = 0.0048). Furthermore, we overexpressed and knocked down TRIM24 in Detroit 562 and FaDu cell lines, respectively. TRIM24 overexpression promoted proliferation, colony formation, and invasion, while TRIM24 depletion inhibited proliferation, colony formation, and invasion. Further studies showed that TRIM24 facilitated cell cycle transition and upregulated cyclin D1 and p-Rb. In addition, we found that GLUT3, a key protein involved in regulating glucose metabolism, was altered in HNSCC cells overexpressing TRIM24. We demonstrated that TRIM24 overexpression increased glucose uptake ATP production. Overexpression of TRIM24 increases cell sensitivity to glucose deprivation in Detroit cells. Depleting TRIM24 in FaDu cells demonstrated the opposite results. We also showed that TRIM24 could bind to the promoter region of cyclin D1. In conclusion, TRIM24 is upregulated in HNSCC and promotes HNSCC cell growth and invasion through modulation of cell cycle, glucose metabolism, and GLUT3, making TRIM24 a potential oncoprotein in HNSCC.

Figure 1

Expression of TRIM24 protein in head and neck squamous cell carcinoma tissues. (a) Negative nuclear staining of TRIM24 in normal laryngeal squamous epithelium. (b) Negative TRIM24 expression in a case of well-differentiated HNSCC. (c) Moderate TRIM24 expression in a case of HNSCC. (d) Strong nuclear staining of TRIM24 in poor-differentiated carcinoma. Magnification 400x.

Figure 2

Efficiency of TRIM24 overexpression and depletion. (a) Protein and mRNA expression of TRIM24 in HNSCC cell lines KB, FaDu, and Detroit 562. (b) Western blot and real-time RT-PCR analysis showed that TRIM24 plasmid transfection upregulated its mRNA and protein expression, while TRIM24 siRNA downregulated its mRNA and protein levels.

Figure 3

TRIM24 regulates cell proliferation, colony formation, and invasion. (a) CCK-8 assay showed that TRIM24 overexpression promoted while its depletion inhibited cell growth rate. (b) Colony formation assay was performed in cells transfected with TRIM24 siRNA and plasmid. An increase of colony number was found in cells transfected with plasmid. A decrease in colony formation was seen in the groups with siRNA treatment in comparison with the controls. (c) TRIM24 overexpression greatly facilitated cell invasion, while TRIM24 depletion remarkably blocked invasion.

Figure 4

TRIM24 regulates cell cycle distribution and protein levels of cyclin D1, p-Rb, and GLUT3. (a) TRIM24 depletion reduced S phase percentage in FaDu cell line while its overexpression upregulated S phase percentage in Detroit 562 cell line. (b) Western blot analysis revealed that knockdown of TRIM24 downregulated while TRIM24 transfection increased the protein levels of cyclin D1, p-Rb, Rb, and GLUT3. TRIM24 downregulated while TRIM24 transfection increased the mRNA level of GLUT3. ^∗p < 0.05.

Figure 5

TRIM24 increases glucose metabolism in HNSCC. (a) TRIM24 depletion in FaDu cells reduced glucose consumption while TRIM24 overexpression increased glucose consumption in Detroit 562 cells. (b) TRIM24 depletion reduced ATP production in FaDu cells while TRIM24 overexpression upregulated ATP production in Detroit 562 cells. (c) Cell growth was examined after 3 days of glucose deprivation. With glucose deprivation, the cell viability of control cells was higher than that of TRIM24 overexpressed Detroit 562 cells and lower than TRIM24 depleted FaDu cells. (d) ChIP assay was performed in FaDu and Detroit 562 cells. Data were presented as fold enrichment of the TRIM24 antibody signal versus the control IgG, calculated using the comparative Ct method. ^∗Statistical significance.