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. 2020 Nov 6:10:560572.
doi: 10.3389/fonc.2020.560572. eCollection 2020.

Cyclin G2 Inhibits Oral Squamous Cell Carcinoma Growth and Metastasis by Binding to IGFBP3 and Regulating the FAK-SRC-STAT Signaling Pathway

Danning Wang  1 , Jinlan Gao  1 , Chenyang Zhao  1 , Sen Li  1 , Di Zhang  1 , Xiaoyu Hou  1 , Xinbin Zhuang  1 , Qi Liu  1 , Yang Luo  1
Affiliations

Cyclin G2 Inhibits Oral Squamous Cell Carcinoma Growth and Metastasis by Binding to IGFBP3 and Regulating the FAK-SRC-STAT Signaling Pathway

Danning Wang et al. Front Oncol. .

Abstract

The cell cycle protein cyclin G2 is considered a tumor suppressor. However, its regulatory effects and potential mechanisms in oral cancers are not well understood. This study aimed to investigate the effect of cyclin G2 on oral squamous cell carcinoma (OSCC). The data from 80 patients with OSCC were utilized to predict the abnormal expression of cyclin G2. The proliferation and metastasis were determined by a cell counting Kit-8 assay, flow cytometry, a wound-healing assay, and a cell invasion assay. The expression of key proteins and genes associated with the cyclin G2 signaling pathways was determined by western blotting and real-time PCR, respectively. The orthotopic nude mice model was established by a mouth injection of SCC9 cells overexpressing cyclin G2. We showed that the low level of cyclin G2 in OSCC, which is negatively correlated with clinical staging, was a negative prognostic factor for the disease. We also found that cyclin G2 inhibited the proliferation, metastasis, and blocked the cell cycle at G1/S of OSCC cells, suggesting that cyclin G2 has an inhibitory effect in OSCC. Mechanistically, cyclin G2 inhibited the growth and metastasis of OSCC by binding to insulin-like growth factor binding protein 3 (IGFBP3) and regulating the focal adhesion kinase (FAK) -SRC-STAT signal transduction pathway. Cyclin G2 competed with integrin to bind to IGFBP3; the binding between integrin and IGFBP3 was reduced after cyclin G2 overexpression, thereby inhibiting the phosphorylation of FAK and SRC. These results showed that cyclin G2 inhibited the progression of OSCC by interacting with IGFBP3 and that it may be a new target for OSCC treatment.

Keywords: c-Src; cell cycle protein cyclin G2; focal adhesion kinase; insulin-like growth factor binding protein 3; integrin; oral squamous cell carcinoma; tumor suppressor.

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Figures

Figure 1
Figure 1
Cyclin G2 expression negatively correlates with clinical stage in OSCC. (A) The expression of cyclin G2 in OSCC at the different clinical stages by an immunohistochemical analysis. Scale bar = 100 µm. (B) Immunohistochemical scores of cyclin G2 at the different clinical stages. **p < 0.01 vs. Normal group.
Figure 2
Figure 2
Cyclin G2 inhibits the proliferation and metastasis of OSCC cells and blocks the cell cycle at G1/S. (A) The expression of cyclin G2 protein and mRNA levels in NC groups and cyclin G2 overexpressing groups in SCC-9 and Cal 27 cell lines. (B) Absorbance of the SCC-9 and Cal 27 cells in the CCK8 cell proliferation experiment. (C, D) Flow cytometry detection of the cell cycle in the SCC-9 and Cal 27 cells. (E, F) Representative photos and quantification of the wounded areas after wounding the SCC-9 and Cal 27 cells. Scale bar = 100 µm. (G, H) Analysis of the invasion and quantification of the SCC-9 and Cal 27 cells. Scale bar = 100 µm. *p < 0.05, **p < 0.01 vs. vector.
Figure 3
Figure 3
Cyclin G2 inhibits the growth and metastasis of OSCC in vivo. (A) The tumor in the rear of the tongue and the swollen lymph nodes after 42 days. (B) Overall images of the tumors and lymph nodes. (C, D) Tumor volumes and weights in the cyclin G2-overexpressing and negative control groups. (E) Hematoxylin and eosin staining of the tumor in the rear of the tongue and the swollen lymph nodes. (F) Immunohistochemical staining of Ki67 in the oral tumors and lymph nodes. Scale bar = 100 µm. **p < 0.01 vs. vector.
Figure 4
Figure 4
Cyclin G2 may inhibit the binding between IGFBP3 and integrin by interacting with IGFBP3. (A) Co-IP analysis of the interaction between cyclin G2 and IGFBP3 in SCC-9 cells. (B) Cyclin G2 and IGFBP3 co-localized in the cytoplasm of the SCC-9 cells. (C) The combination effects of IGFBP3 and integrin were reduced after cyclin G2 overexpression. **p < 0.01, ***p < 0.001 vs. vector.
Figure 5
Figure 5
Cyclin G2 inhibits the FAK-SRC-STAT pathway in vitro and in vivo. (A) The protein expressions along with quantification of FAK, P-FAK, SRC, P-SRC, STAT3, P-STAT3, Bcl-2, c-Myc, and MMP9 in the FAK-SRC-STAT pathway. (B–D) The relative mRNA expressions of Bcl-2, c-Myc and MMP9. (E) Analysis of MMP9 secretion and its quantification in SCC-9 cells. (F, G) Immunohistochemical staining and analysis of the expression of cyclin G2, p-FAK, p-SRC and p-STAT3. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vector.
Figure 6
Figure 6
Model for cyclin G2 inhibiting the growth and metastasis of OSCC by binding to IGFBP3 and regulating the FAK-SRC-STAT signal pathway. Cyclin G2 interacts with IGFBP3 and inhibits the binding between integrin and IGFBP3 and the phosphorylation of FAK. FAK dephosphorylation inhibits the FAK-SRC-STAT signal pathway, thereby inhibiting tumorigenesis.
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