Interruption of nuclear localization of ATBF1 during the histopathologic progression of head and neck squamous cell carcinoma

Abstract

Background: The AT-motif binding factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers, including head and neck squamous cell carcinomas (HNSCCs).

Methods: ATBF1 expression and localization were examined in 5 cell lines and 197 clinical specimens of HNSCC, and correlated with pathologic and clinical characteristics.

Results: ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p = .0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p < .0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.

Conclusions: Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.

Figure 1. ATBF1 protein detected by immunohistochemical staining in primary tumors of head and neck cancer

A & B, highly heterogeneous and mainly nuclear staining of ATBF1 in squamous epithelial cells of simple hyperplasia. C & D, positive nuclear staining and slightly increased cytoplasmic staining of ATBF1 in low-grade dysplasia. E & F, significantly increased cytoplasmic ATBF1 staining in high-grade dysplasia. G & H, strong cytoplasmic but reduced or absent nuclear staining of ATBF1 in invasive tumors. Panels at the right (B, D, F, and H) are higher magnification views of the panels at the left (A, C, E, and G respectively). All scale bars are 50 μm.

Figure 2. Correlation between cytoplasmic ATBF1 and worse overall survival and disease free survival as determined by Kaplan and Meier analysis

Patients were divided into two groups based on the median weight index of cytoplasmic ATBF1 (high and low ATBF1), and the percentage of patients was plotted against overall survival (in years) (A) or disease-free survival (B). Higher levels of cytoplasmic ATBF1 significantly correlate with shorter overall survival (p=0.0386) and disease-free survival (p=0.0253).

Figure 3. Expression and subcellular localization of ATBF1 in HNSCC cell lines

A. ATBF1 mRNA level determined by semi-quantitative RT-PCR in five cell lines. B. ATBF1 protein level in the same five cell lines detected by western blotting. ACTB served as the internal control in both A and B. C. Subcellular localization of ATBF1 detected by immunofluorescent staining. Nuclei were counterstained with DAPI. Endogenous ATBF1 was notably detected in the cytoplasm of the Tu212 and Tu177 cancer cell lines. Scale bars are 10 μm.