MMP-11 promoted the oral cancer migration and Fak/Src activation

Abstract

Matrix metalloproteinase-11 (MMP-11) has been observed in most invasive human carcinomas. The current study investigated the association between the clinicopathological characteristics and MMP-11 expression in oral squamous cell carcinoma (OSCC) patients. Immunohistochemistry (IHC) staining was performed to assess MMP-11 expression in 279 patients with OSCC. In addition, the metastatic effects of the MMP-11 overexpression on the OSCC cells were also investigated. We found that MMP-11 expression was present in 118/279 (42.3%) cases and expression of MMP-11 was associated with higher incidence of lymph node metastasis and worse grade of tumor differentiation. Importantly, OSCC patients with strong expression of MMP-11 had a significantly lower survival rate (p=0.010). Furthermore, MMP-11 overexpression in OSCC cells increased in vitro cell migration. Mechanistically, MMP-11 increased the cell motility of OSCC cells through focal adhesion kinase/Src kinase (FAK/Src) pathway. In conclusion, our results revealed that the MMP-11 expression in OSCC samples can predict the progression, especially lymph node metastasis, and the survival of OSCC patients in Taiwan.

Keywords: FAK/Src pathway; matrix metalloproteinase; metastasis; oral squamous cell carcinoma; survival.

Figure 1. MMP-11 expression in primary oral cancer

Tissue microarrays of primary oral squamous cell carcinomas (OSCCs) were immunohistochemically analyzed for MMP-11. (A and D) no detectable MMP-11 (0). (B and E) weak expression levels (1+). (C and F) strong expression levels (2+). (A-C) low-power field (100x); (D-F) high-power field (200x).

Figure 2. Kaplan-Meier survival curve showing the relation between MMP-11 expression in primary tumors and survival in 279 oral squamous cell carcinoma (OSCC) patients

The overall survival of OSCC patients with positive MMP-11 staining was significantly lower than that of OSCC patients with negative MMP-11 staining (p<0.05, log-rank test).

Figure 3. The relationships between MMP-11 expression and cell migration in TW2.6 OSCC cell lines

(A) The MMP-11 expression of TW2.6 cells transfected with pcDNA3.0-MMP-11 was examined by western blot and RT-PCR. (B-D) Detection of cell migratory abilities by transfection with MMP-11 overexpression vector in TW2.6 cell. Migratory abilities of pcDNA3.0 and pcDNA3.0-MMP-11 cells were evaluated using (B) wound healing assay. The (C) migratory and (D) invasive abilities of pcDNA3.0 and pcDNA3.0-MMP-11 cells were evaluated using Boyden chamber migration and Matrigel invasion assays. Differences are presented as the mean of triplicate experiments compared with control cells. *p < 0.05 compared with control cells.

Figure 4. FAK/Src signaling pathway is involved in MMP-11-promoted cell migration

(A) TW2.6 cells transfected with pcDNA3.0 and pcDNA3.0-MMP-11 overexpression vector for 24 h and the total cell lysates were then subjected to western blot to analyze the phosphorylation of (A) FAK and Src (B) Erk 1/2, JNK1/2 and p38 as described in the Materials and Methods section. (C) TW2.6 cells were treated with a FAK inhibitor (FAKI-14; 10μM) for 24h. FAK phosphorylation was examined by western blot and cell migration was examined by Boyden chamber migration. (D) TW2.6 cells were treated with a Src inhibitor (PPI; 10μM) for 24h. Src phosphorylation was examined by western blot and cell migration was examined by Boyden chamber migration. Data are expressed as the mean ± SEM *p < 0.05 compared with control; #p < 0.05 compared with the pcDNA3.0-MMP-11 overexpression vector group. (E-F) The correlations among mRNA levels of MMP-11 and FAK as well as Src in head and neck squamous cell carcinoma from The Cancer Genome Atlas (TCGA) Data Portal. (E) A significant correlation was found between MMP-11 and FAK (Spearman rank correlation coefficient r =0.2935, p<0.0001). (F) A significant correlation was found between MMP-11 and Src (Spearman rank correlation coefficient r =0.1817, p<0.0001).