Overexpression of FBI-1 protein in ovarian tumors and cancer cell lines
The differential expression of FBI-1 in the various categories of ovarian tumors was shown in Figure , and its association with clinical-pathological parameters was summarized in Table . All 10 benign cystadenomas (3 serous and 7 mucinous) were completely negative for FBI-1 staining, while 15 out of 19 (78.9%) borderline tumors (7 serous and 12 mucinous) showed weak and focal immunoreactivity (score ≤9). Among the 111 ovarian cancers, 82 (73.8%) cases displayed obvious overexpression of FBI-1 at a high level (score ≥12) both in the cytoplasm and nuclei. Significant higher expression of FBI-1 was found in borderline tumors (P < 0.001) and cancers (P < 0.001) when compared with benign counterparts. Expression of FBI-1 in cancers was also significantly higher than that in borderline tumors (P < 0.001). However, there was no significant difference among the four major histological types of ovarian cancers (Table ).
Figure 1 FBI-1 was overexpressed in ovarian tumors and correlated with prognosis of patients. A, Representative images of FBI-1 immunoreactivity in different ovarian tumor subtypes and malignant ascites (20×) (bottom right; 200×). B, mRNA expression (more ...)
Association analysis between FBI-1 expression and the clinicopathological features of ovarian cancers
Western blot analysis showed that FBI-1 expression was up-regulated in 11 out of the 13 (84.6%) ovarian cancer cell lines (OVCAR3, OC316, DOV13, ES2, OVCA 420, SKOV-3, TOV21G, TOV112 D, SW626, 2780 S, and 2780CP) when compared with two nontumorigenic immortalized human ovarian surface epithelial cell (HOSE) lines (Figure ).
Figure 2 FBI-1 expression was up-regulated in most of the ovarian cancer cell lines when compared with normal ovarian epithelial cell lines HOSE6-3 and HOSE11-12. A, protein expression and B, mRNA level of FBI-1 as determined by western blot and qPCR respectively. (more ...)
Gene amplification contributes to the overexpression of FBI-1 mRNA
In a cohort of 36 pairs of clinical frozen samples, the mRNA level of FBI-1 was found to be significantly up-regulated in ovarian cancers when compared with corresponding non-tumor counterparts (P = 0.005) by qPCR (Figure , left upper panel). Ten of the 13 (76.2%) ovarian cancer cell lines also displayed up-regulation of FBI-1 at mRNA level when compared with the average of HOSE6-3 and HOSE11-12 (Figure ), although a heterogeneous expression profile was observed.
To evaluate the mechanisms underlying the increase in FBI-1 expression, genomic DNA copy number of FBI-1 was further evaluated in these 36 pairs of patient samples using qPCR (Figure , right upper panel). Remarkably, 31 out of 36 (86%) cancers displayed gene amplification of FBI-1 when compared with the corresponding non-tumor counterparts (Figure , lower panel). Among the 31 amplified cases, 19 (61%) showed elevated mRNA levels. In the 5 non-amplified cases, increased mRNA expression of FBI-1 was only found in one case (20%). Spearman's rho test demonstrated that elevated RNA transcription of FBI-1 was closely correlated with its gene amplification (P = 0.037), suggesting that gene amplification was an important mechanism leading to the overexpression of FBI-1 in ovarian cancers.
FBI-1 overexpression was significantly associated with aggressive tumor behavior and poor outcome
In addition to primary cancer samples, 63 metastatic foci in the lymph node, ligament, gut, and uterine serosa derived from advanced ovarian cancers and 17 ascitic samples were also used for evaluating FBI-1 protein expression by immunohistochemistry. Significant up-regulation of FBI-1 expression was detected in metastatic foci (P = 0.036) and malignant ascites (P = 0.021) (Figure ), compared with primary malignancies (Table ). Moreover, higher FBI-1 expression was also found to be closely associated with advanced stage (stage II-IV) (P = 0.012) (Table ), poor overall survival (P = 0.032) and disease-free survival (P = 0.016) (Figure ). Nevertheless, there was no significant correlation between FBI-1 immunoreactivity and histological grade (P = 0.151) or chemosensitivity (P = 0.246). Although increased FBI-1 significantly correlated with survival as mentioned above, multivariable analysis showed that expression of FBI-1 was not an independent predictor of overall survival (95% CI = 0.971-1.089, P = 0.344), or disease-free survival (95% CI = 0.896-1.080, P = 0.729). Together, our findings indicated that FBI-1 may affect the prognosis of patients with ovarian cancer via its effect on cancer progression.
FBI-1 promoted cell migration and invasion of ovarian cancer with up-regulation of MT1-MMP
To further assess the function of FBI-1 on aggressive behavior of ovarian cancer cells, OVCA 420 (harboring wild-type p53
) and SKOV-3 (possessing a single nucleotide deletion at point 267 at codon 90 of p53
, which blocks p53 expression [23
]) cells were transiently transfected with pEGFP-FBI-1, followed by performing cell migration and invasion assays. Among the various ovarian cancer cell lines, OVCA 420 and SKOV-3 showed average levels of FBI-1 expression.
Up to 7 and 10-fold increase in cell migration were observed in OVCA 420 and SKOV-3 cells (both P < 0.001) (Figure , upper panel) after transfection of pEGFP-FBI-1. About 3-fold increase in cell invasion was also observed in OVCA 420 (P = 0.007) and SKOV-3 (P = 0.023) cells (Figure , lower panel). Next, the mRNA and protein expressions of FBI-1, MT1-MMP, MMP-2 and TIMP-2 were investigated. qPCR analysis showed that along with the induced FBI-1 mRNA expression, MT1-MMP, but not MMP-2 or TIMP-2, mRNA expression was increased by 3 to 6 fold after transfection of pEGFP-FBI-1 (Figure , upper panel). Western blot analyses using anti-FBI-1 antibody also showed ectopic expression of exogenous GFP-tagged FBI-1 (106KDa) while no change in endogenous FBI-1 (75KDa) was found. An up-regulation of total and active form of MT1-MMP protein in both OVCA 420 and SKOV-3 cells after transfection of pEGFP-FBI-1 was also demonstrated (Figure , lower panel). No significant change in MMP-2 and TIMP-2 expression was observed (data not shown).
Figure 3 Ectopic overexpression of FBI-1 promoted motility, invasiveness and proliferation of ovarian cancer cells. A, In vitro migration (upper panel) and invasion assays (lower panel) in OVCA 420 (wild-type p53) and SKOV-3 (null p53) cell lines after transfection (more ...)
To test whether MT1-MMP is involved in FBI-1-mediated cell migration and invasion, OVCA 420 and SKOV-3 cells were transiently transfected with pEGFP-FBI-1 and siRNA of MT1-MMP. Transient knockdown of MT1-MMP inhibited both basal and FBI-1-enhanced cell migration and invasion (Figure ; all P values < 0.05).
To further substantiate the notion that FBI-1 conferred to ovarian cancer cell motility and invasiveness, OVCA 420 and SKOV-3 cells with stably knockdown FBI-1 were established and subjected to transwell assays. Consistent with previous results, for both cell lines, a reduction in the number of migrated and invaded cells (p < 0.05) was significantly associated with a decrease in MT1-MMP mRNA and protein levels, in particular the active form of the MT1-MMP (Figure ).
Figure 4 Stable knockdown of FBI-1 with shRNA reduced motility, invasiveness and proliferation of ovarian cancer cells. A, In vitro migration (upper panel) and invasion assays (lower panel) in OVCA 420 and SKOV-3 cell lines after stable knockdown of FBI-1. B, (more ...)
Knockdown of FBI-1 reduced ovarian cancer cell mobility irrespective of p53 status
As OVCA 420 and SKOV-3 have different p53 status, it is likely that FBI-1 may affect the cell migration and invasion in a p53-independent manner. To test this hypothesis, migration and invasion assays were performed on SKOV-3 cells harboring stable knockdown FBI-1 with or without re-induction of wild-type p53. In both stable shFBI-1 cells with or without re-expression of wild-type p53, similar decreases in cell invasion and migration (p < 0.05) were observed, which were again accompanied by a reduction of MT1-MMP expression compared with the negative control (NC) (Figure ). Together, these results suggested that FBI-1 play an important role in ovarian cancer cell invasion, migration, and metastasis, and this function of FBI-1 is mediated at least in part through its regulation of MT1-MMP expression but is independent of p53 status.
Figure 5 Introduction of wild type p53 into SKOV-3 cell lines with stable FBI-1 knockdown (shFBI-1) and negative counterpart (NC) led to A, significant reduction in cell migration and invasion in association with B, reduced expression of FBI-1 (as detected by (more ...)
FBI-1 directly interacts with the promoter of MT1-MMP and enhanced its expression
The ability of FBI-1 to modulate both MT1-MMP mRNA and protein levels suggested that FBI-1 may regulate MT1-MMP expression at transcriptional level. FBI-1 binding sites have recently been characterized [18
], and three putative FBI-1 consensus sequences could be identified within ~450bp upstream of the transcription start site of the MT1-MMP
]. A DNA fragment corresponding to an approximately 600bp of the 5'-flanking sequence of MT1-MMP
was subcloned upstream of a luciferase reporter assay and used for gene promoter analysis. Firstly, we investigated whether FBI-1 would affect MT1-MMP
gene transcription activity in OVCA 420 and SKOV-3 cells after stable FBI-1 knockdown. FBI-1 depletion reduced the MT1-MMP
promoter activity in both cell lines by 3 and 4-fold when compared with the negative controls, respectively (Figure ). Secondly, the pGL3-Basic-MT1-MMP
-Luc reporter was co-transfected with increasing amounts of pEGFP-C3-FBI-1 construct. The result showed that FBI-1 efficiently enhanced MT1-MMP
promoter activity in a dose-dependent manner (Figure ). Yet, FBI-1 expression showed no significant effect on MMP-2
promoter by luciferase reporter assay (data not shown).
Figure 6 Dual luciferase assays demonstrated down-regulation of MT1-MMP transcription in A, OVCA 420 and SKOV-3 with stable shFBI-1 knockdown. Up-regulation of MT1-MMP transcription was found in B, OVCA 420 and SKOV-3 cells after transfection of pEGFP-FBI-1 in (more ...)
In an attempt to further elucidate the induction of MT1-MMP
transcription by FBI-1, a luciferase reporter construct containing 94bp of the human MT1-MMP
promoter (pGL3-Basic-shortMT-MMP-luc) was applied for gene promoter analysis. Theoretically, there is no FBI-1 DNA binding site in this short MT-MMP-luc sequence [18
]. Our result showed that overexpression of FBI-1 could not enhance the transcriptional activity of this short form of MT1-MMP
promoter (data not shown), further supporting the specificity of the transcription regulatory effect of FBI-1 on MT1-MMP
We next examined whether FBI-1 can bind to the endogenous MT1-MMP promoter in vivo using the ChIP assays. Total lysates from parental OVCA 420 and SKOV-3 cells were used as the input samples and positive control, while immunoprecipitated lysates without antibody served as the negative controls. As shown in Figure , FBI-1 bound directly to the promoter region of MT1-MMP gene.
FBI-1 promoted ovarian cancer cell proliferation in a p53-dependent manner
In addition, we also investigated whether FBI-1 could affect cell proliferation using MTT assay. Introduction of FBI-1 into OVCA 420 cells with wild-type p53 resulted in an approximately 2-fold enhancement in cell proliferation (P = 0.003) (Figure ). In contrast, there was no significant effect in SKOV-3 cells which have no p53 expression. Consistent with these findings, stable silencing of FBI-1 in OVCA 420 cells led to a significant decrease in cell proliferation, whereas no significant changes were observed in SKOV-3 cells (Figure ). The results indicated that the FBI-1 may modulate ovarian cancer cell proliferation in a p53-dependent manner.