Endoplasmic reticulum aminopeptidase 2 involvement in metastasis of oral cavity squamous cell carcinoma discovered by proteome profiling of primary cancer cells

Abstract

Oral cavity squamous cell carcinoma (OSCC) is a leading cause of cancer-related deaths worldwide and associated with poor prognosis and mortality. Discovery of proteins that can improve OSCC treatment is needed. Using comparative proteome profiling of primary cells derived from OSCC and adjacent noncancerous epithelium, endoplasmic reticulum aminopeptidases 2 (ERAP2) has been identified as an OSCC-associated protein. Compared with the adjacent normal tissues, ERAP2 levels were determined to be significantly elevated in OSCC tissues using quantitative real-time PCR and immunohistochemistry. Importantly, overexpression of ERAP2 was associated with positive N stage, advanced overall stage, positive perineural invasion, and tumor depth (P = 0.041, 0.015, 0.010, and 0.032, respectively). The overall survival rates of patients without and with the ERAP2 overexpression were 71.9% and 56.0%, respectively (P = 0.029). Furthermore, knockdown of ERAP2 inhibited the migration and invasion abilities of OSCC cells. Our results collectively show that ERAP2 overexpression is associated with the cervical metastasis and poorer prognosis of OSCC.

Keywords: ERAP2; OSCC; metastasis; prognosis; proteome.

Figure 1. Overexpression of ERAP2 in OSCC tissues

(A) Box and whisker plots showing ERAP2 mRNA transcript levels in the 40 paired normal and tumor tissues, as assessed by quantitative real-time RT-PCR. ERAP2 was highly overexpressed in OSCC tissues. (Box, the range of the middle 50% of ERAP2 level; line inside box, median; whiskers, minimal to maximal levels) (B) Western blot analysis of ERAP2 expression was shown in five OSCC cell lines and three paired tumor (T) and noncancerous (N) tissues. The β-actin signal was used as a loading control. (C) Immunohistochemical staining of ERAP2 in pericancerous adjacent normal epithelia (adjacent normal), and tumor tissues from two representative cases (scale bar = 100 μm). Expression patterns (brown staining) of ERAP2 expression indicate that this protein is localized in the tumor cell cytoplasm and membrane. (D) Statistical analysis for immunohistochemical scores of ERAP2 in 157 paired samples revealed higher ERAP2 expression levels in tumor cells than non-tumor normal epithelia (157.1 ± 79.04 vs. 5.0 ± 20.73, P < 0.001). The P value is obtained with paired t test.

Figure 2. Association of high ERAP2 expression with poorer prognosis of patient survival

(A) Kaplan-Meier plot for overall survival (OS) indicated that the 5-year OS rates for patient subgroups stratified by ERAP2 expression were 71. 9% vs. 56.0%, respectively (P = 0.029). (B) Five-year disease-free survival (DFS) rates for patients stratified by ERAP2 expression were 73.8% vs. 60.0%, respectively (P = 0.037).

Figure 3. Involvement of ERAP2 in viability, migration, and invasion of OSCC cells

(A) SCC4 cells were transfected with control siRNA (Scr Ctrl) and ERAP2 siRNA (siERAP2), respectively. After 48 h, protein extracts (20 μg) were prepared and ERAP2 was detected with Western blot. (B) Quantitative analysis of the MTT assay. Data are presented as mean values obtained from three independent experiments. Error bars indicated the standard deviation. (C) Representative microphotographs of filters obtained from the 16 h transwell migration (D) and 24 h invasion (E) assays. Original magnification: 200×. The migration (D) and invasion (E) assays were performed using SCC4 cells in which ERAP2 was eliminated.