Decreased nuclear expression and increased cytoplasmic expression of ING5 may be linked to tumorigenesis and progression in human head and neck squamous cell carcinoma

Abstract

Purpose: This study aimed to assess the protein level of inhibitor of growth gene 5 (ING5) in head and neck squamous cell carcinoma (HNSCC) and to explore its roles in tumorigenesis and cancer progression.

Methods: ING5 expression was assessed in 172 cases of HNSCC by immunohistochemistry using tissue microarray, and in 3 oral SCC cell lines by immunohistochemistry and Western blot. Expression of ING5 was compared with clinicopathological variables, TUNEL assay staining, and the expression of several tumorigenic markers. In addition, double immunofluorescence labeling was performed in order to analyze the colocalization of ING5 with p300 and p21.

Results: ING5 expression was primarily observed in the nuclei, but was also occasionally found in the cytoplasm of both SCC cell lines and tissue samples of HNSCC. Nuclear expression of ING5 in HNSCC was significantly lower than that of non-cancerous epithelium, and was positively correlated with a well-differentiated status. In contrast, cytoplasmic expression of ING5 was significantly increased in HNSCC, and was inversely correlated with a well-differentiated status and nuclear ING5 expression. In addition, nuclear expression of ING5 was positively correlated with p21 and p300 expression, and with the apoptotic index. In contrast, cytoplasmic expression of ING5 was negatively correlated with the expression of p300, p21, and PCNA. Although no statistical association was found between the expression of nuclear ING5 and mutant p53 in HNSCC, patients with high expression of nuclear ING5 tended to have converse prognoses when grouped according to mutant p53 expression.

Conclusions: Our results suggest that a decrease in nuclear ING5 localization and cytoplasmic translocation are involved in tumorigenesis and tumor differentiation in HNSCC. Nuclear ING5 may modulate the transactivation of target genes, and may promote apoptosis and cell cycle arrest by interacting with the p300 and p21 proteins. ING5 may function as a tumor suppressor gene or oncogene tightly linked with p53 status, and may play an important role in the prognosis of HNSCC patients. Therefore, we propose that ING5 represents a novel potential molecular therapeutic target for HNSCC.

Fig. 1

The expression of ING5 and other tumor-related molecular markers in the cell lines of oral SCC. a Immunohistochemical staining of ING5 and other tumor-related molecular markers: mutant p53, p300, and p21 in the cell lines of oral SCC: HSC-2, B88, and SAS. b Western blot analysis of the protein level of ING5 in cell lysates of nucleus, cytoplasm, and the whole cell. H2AX was used as internal loading control of nuclear protein, and β-actin as the control of cytoplasmic and total protein

Fig. 2

Immunohistochemical staining of ING5 and other tumor-related molecular markers. The immunohistochemical staining of ING5 in whole tissue sections under low magnification (a) (Magnification ×12.5). High-magnification images of the fields indicated by boxes showed that the nuclear expression of ING5 gradually decreased from the adjacent non-cancerous epithelia (b) and dysplastic lesion (c) to cancerous lesion (d). Both nuclear and cytoplasmic expression of ING5 was found in some HNSCC cases (e). However, only high cytoplasmic expression of ING5 was detected in a few HNSCC cases (f). Expression of p300 (g), p21 (h), mutant p53 (i) and PCNA (j), was observed in the nuclei of tumor cells. Apoptotic cells were identified by the TUNEL assay and exhibited brown nuclear staining (k) (Magnification ×400)

Fig. 3

Double immunofluorescence staining of ING5 and p300 or p21 in HNSCC. Staining of ING5 protein is shown in red, and p300 or p21 in green. Colocalization of both proteins is shown in orange in the merged images (Magnification 400×)

Fig. 4

Correlation between ING5 expression and prognosis of HNSCC patients. Kaplan–Meier curves show the cumulative survival rate of HNSCC patients grouped according to nuclear (a) or cytoplasmic (b) ING5 expression. In comparison with the patients with low ING5 nuclear expression, patient survival with high ING5 nuclear expression was higher in the no mutant p53 expression group (c), but conversely lower in the mutant p53 expression group (d); no statistical significance existed