Clinical Significance and the Role of Guanylate-Binding Protein 5 in Oral Squamous Cell Carcinoma

Abstract

Guanylate binding protein 5 (GBP5) is the interferon (IFN)-inducible subfamily of guanosine triphosphatases (GTPases) and is involved in pathogen defense. However, the role played by GBP5 in cancer development, especially in oral squamous cell carcinoma (OSCC), is still unknown. Herein, next-generation sequencing analysis showed that the gene expression levels of GBP5 were significantly higher in OSCC tissues compared with those found in corresponding tumor adjacent normal tissues (CTAN) from two pairs of OSCC patients. Higher gene expression levels of GBP5 were also found in tumor tissues of 23 buccal mucosal squamous cell carcinoma (BMSCC)/14 tongue squamous cell carcinoma (TSCC) patients and 30 oral cancer patients from The Cancer Genome Atlas (TCGA) database compared with those in CTAN tissues. Immunohistochemical results showed that protein expression levels of GBP5 were also higher in the tumor tissues of 353 OSCC patients including 117 BMSCC, 187 TSCC, and 49 lip squamous cell carcinoma patients. Moreover, TCGA database analysis indicated that high gene expression levels of GBP5 were associated with poor overall survival in oral cancer patients with moderate/poor cell differentiation, and associated with poor disease-free survival in oral cancer patients with moderate/poor cell differentiation and lymph node metastasis. Furthermore, GBP5-knockdowned cells exhibited decreased cell growth, arrest at G1 phase, and decreased invasion/migration. The gene expression of markers for epithelial-mesenchymal transition and cancer stemness was also reduced in GBP5-silenced oral cancer cells. Taken together, GBP5 might be a potential biomarker and therapeutic target for OSCC patients, especially for those with poor cell differentiation and lymph node metastasis.

Keywords: guanylate binding protein 5; malignancy; oral squamous cell carcinoma; prognosis.

Figure 1

Comparison of GBP5 expression between tumor and adjacent normal tissues in oral cancer patients. (A) Relative fold-changes of GBP5 RPKM in CTAN tissues and tumor tissues in two paired OSCC patients by NGS. (B) Comparison of GBP5 gene expression between tumor tissues and CTAN tissues from 23 paired BMSCC and 14 paired TSCC patients by RT–PCR. (C) RPKM values of GBP5 between 29 normal tissues and 303 tumor tissues in oral cancer patients from TCGA based RNA seq. (D) GBP5 IHC scores in CTAN tissues and tumor tissues: 182 BMSCC, 245 TSCC, and 72 LSCC. Data on the whole cohort of 499 patients are also shown (N: CTAN tissues; T: tumor tissues). A value of p < 0.05 was considered significant (*** p < 0.001).

Figure 2

Effects of GBP5 in cell growth of OSCC cells. (A) Gene expression levels of GBP5 in transient GBP5–silenced SAS and TW2.6 cells by RT-PCR. All cells were transfected with 10 nM scramble siRNA (siCtrl) or GBP5 siRNA (siGBP5) for 72 h. (B) Protein expression levels of GBP5 in transient GBP5–silenced SAS and TW2.6 cells by Western blotting. Beta-actin was used as loading control (ACTB). All cells were transfected with 10 nM scramble siRNA (-) or GBP5 siRNA (+) for 72 h. (C) Colony formation of transient GBP5–silenced SAS and TW2.6 cells. After two weeks, these cells were fixed and stained with crystal violet to count the number of colonies. All cells were transfected with 10 nM scramble siRNA (siCtrl) or GBP5 siRNA (siGBP5) for 72 h. (D) Formation of tumorspheres and tumorsphere viability of transient GBP5–silenced SAS and TW2.6 cells. Tumorsphere viability was measured by the 3D CellTiter Glo assay. All cells were transfected with 10 nM scramble siRNA (siCtrl) or GBP5 siRNA (siGBP5) for 72 h. All quantitative results are calculated as the mean ± SEM from three independent experiments. A value of p < 0.05 was considered significant (** p < 0.01, * p < 0.05).

Figure 3

Effects of GBP5 in cell cycle progression of OSCC cells. (A) The stable GBP5-silenced TW1.5 cells harboring scrambled shRNA (shCtrl) or shRNA against GBP5 (shGBP5) were seeded for 24 h and harvested for cell cycle distributions by flow cytometry. All fixed cells were stained with propidium iodide to examine the proportions of different cell cycle phases using flow cytometry. FlowJo software was used to analyze the flow cytometry data. (B) Gene expression levels of p21 and p27 in GBP5-silenced SAS and TW2.6 cells. All cells were transfected with 10 nM scramble siRNA (siCtrl) and GBP5 siRNA (siGBP5) for 72 h. All quantitative results are calculated as the mean ± SEM from three independent experiments. A value of p < 0.05 was considered significant (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 4

Effects of GBP5 in cell invasion and migration of OSCC cells. (A) Invasion of GBP5–silenced SAS and TW1.5 cells. (B) Migration of GBP5-silenced SAS and TW1.5 cells. (C) Gene expression levels of EMT-related markers in GBP5-silenced SAS and TW1.5 cells by RT-PCR. Transient GBP5–silenced cells were cells transiently transfected with 10 nM scramble siRNA (siCtrl) or GBP5 siRNA (siGBP5) for 72 h. Stable GBP5-silenced cells were cells transfected with scrambled shRNA (shCtrl) or shRNA against GBP5 (shGBP5). All quantitative results are calculated as the mean ± SEM from three independent experiments. A value of p < 0.05 was considered significant (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 5

Effects of GBP5 in cancer stemness of OSCC cells. Gene expression levels of cancer stemness-related markers in GBP5-silenced (A) SAS and (B) TW1.5 cells by RT-PCR. All cells were transiently transfected with 10 nM scramble siRNA (siCtrl) or GBP5 siRNA (siGBP5) for 72 h. All quantitative results are calculated as the mean ± SEM from three independent experiments. A value of p < 0.05 was considered significant (*** p < 0.001, ** p < 0.01, * p < 0.05).